WO2002081517A2 - Novel polypeptides and nucleic acids encoded thereby - Google Patents

Novel polypeptides and nucleic acids encoded thereby Download PDF

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Publication number
WO2002081517A2
WO2002081517A2 PCT/US2002/002064 US0202064W WO02081517A2 WO 2002081517 A2 WO2002081517 A2 WO 2002081517A2 US 0202064 W US0202064 W US 0202064W WO 02081517 A2 WO02081517 A2 WO 02081517A2
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Prior art keywords
polypeptide
amino acid
nucleic acid
seq
protein
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PCT/US2002/002064
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French (fr)
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WO2002081517A3 (en
Inventor
Marc F. Decristofaro
Muralidhara Padigaru
Charles Miller
Velizar Tchernev
Haihong Zhong
Mei Zhong
David Anderson
Robert Ballinger
Valerie Gerlach
Kimberly A. Spytek
Luca Rastelli
Ramesh Kekuda
Xiaojia Guo
Bryan Zerhusen
David Andrew
Peter Mezes
Meera Patturajan
Catherine E. Burgess
Andrew Eisen
Adam Wolenc
Jason Baumgartner
Richard A. Shimkets
Vladimir Gusev
Corine A. M. Vernet
Raymond J. Taupier
Carol Pena
Suresh Shenoy
Li Li
Stacie Casman
Ferenc Boldog
Elma Fernandes
Glennda Smithson
Uriel Malyankar
Bruce Taillon
Xiaohong Liu
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Curagen Corporation
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Application filed by Curagen Corporation filed Critical Curagen Corporation
Priority to EP02736481A priority Critical patent/EP1360198A2/en
Priority to CA002435528A priority patent/CA2435528A1/en
Priority claimed from US10/055,877 external-priority patent/US7112668B2/en
Publication of WO2002081517A2 publication Critical patent/WO2002081517A2/en
Priority claimed from US10/453,372 external-priority patent/US20060003323A1/en
Publication of WO2002081517A3 publication Critical patent/WO2002081517A3/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to polynucleotides and the polypeptides encoded by such polynucleotides, as well as vectors, host cells, antibodies and recombinant methods for producing the polypeptides and polynucleotides, as well as methods for using the same.
  • the invention is based in part upon the discovery of nucleic acid sequences encoding novel polypeptides.
  • the novel nucleic acids and polypeptides are referred to herein as NONX, or ⁇ OV1, ⁇ ON2, ⁇ ON3, ⁇ ON4, ⁇ OV5, NON6, ⁇ ON7, ⁇ OV8, NON9, ⁇ ON10, ⁇ ON11, ⁇ ON12, ⁇ ON13, ⁇ ON14, ⁇ ON15, ⁇ ON16, ⁇ ON17, ⁇ ON18, ⁇ OV19, NOV20, NOV21, NOV22, NOV23, NQN24, ⁇ ON25, ⁇ ON26, ⁇ ON27, ⁇ ON28, ⁇ OV29, NON30, ⁇ OV31, NOV32, and NOV33 nucleic acids and polypeptides.
  • NONX nucleic acid or ⁇ OV1, ⁇ ON2, ⁇ ON3, ⁇ ON4, ⁇ OV5, NON6, ⁇ ON7, ⁇ OV8, NON
  • the invention provides an isolated ⁇ ONX nucleic acid molecule encoding a ⁇ ONX polypeptide that includes a nucleic acid sequence that has identity to the nucleic acids disclosed in SEQ ID ⁇ OS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, and 122.
  • the ⁇ ONX nucleic acid molecule will hybridize under stringent conditions to a nucleic acid sequence complementary to a nucleic acid molecule that includes a protein-coding sequence of a ⁇ OVX nucleic acid sequence.
  • the invention also includes an isolated nucleic acid that encodes a ⁇ ONX polypeptide, or a fragment, homolog, analog or derivative thereof.
  • the nucleic acid can encode a polypeptide at least 80% identical to a polypeptide comprising the amino acid sequences of SEQ ID ⁇ OS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, and 123.
  • the nucleic acid can be, for example, a genomic DNA fragment or a cDNA molecule that includes the nucleic acid sequence of any of SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, and 122.
  • an oligonucleotide e.g., an oligonucleotide which includes at least 6 contiguous nucleotides of a NONX nucleic acid (e.g., SEQ ID ⁇ OS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59,
  • a NONX nucleic acid e.g., SEQ ID ⁇ OS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59,
  • NONX polypeptides SEQ ID ⁇ OS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60,
  • the NONX polypeptides include an amino acid sequence that is substantially identical to the amino acid sequence of a human ⁇ ONX polypeptide.
  • the invention also features antibodies that immunoselectively bind to ⁇ OVX polypeptides, or fragments, homologs, analogs or derivatives thereof.
  • the invention includes pharmaceutical compositions that include therapeutically- or prophylactically-effective amounts of a therapeutic and a pharmaceutically- acceptable carrier.
  • the therapeutic can be, e.g., a ⁇ ONX nucleic acid, a ⁇ ONX polypeptide, or an antibody specific for a ⁇ ONX polypeptide.
  • the invention includes, in one or more containers, a therapeutically- or prophylactically-effective amount of this pharmaceutical composition.
  • the invention includes a method of producing a polypeptide by culturing a cell that includes a ⁇ ONX nucleic acid, under conditions allowing for expression of the ⁇ ONX polypeptide encoded by the D ⁇ A. If desired, the ⁇ ONX polypeptide can then be recovered.
  • the invention includes a method of detecting the presence of a ⁇ ONX polypeptide in a sample.
  • a sample is contacted with a compound that selectively binds to the polypeptide under conditions allowing for formation of a complex between the polypeptide and the compound.
  • the complex is detected, if present, thereby identifying the NONX polypeptide within the sample.
  • the invention also includes methods to identify specific cell or tissue types based on their expression of a ⁇ ONX. Also included in the invention is a method of detecting the presence of a ⁇ OVX nucleic acid molecule in a sample by contacting the sample with a ⁇ ONX nucleic acid probe or primer, and detecting whether the nucleic acid probe or primer bound to a ⁇ ONX nucleic acid molecule in the sample.
  • the invention provides a method for modulating the activity of a ⁇ ONX polypeptide by contacting a cell sample that includes the ⁇ ONX polypeptide with a compound that binds to the ⁇ ONX polypeptide in an amount sufficient to modulate the activity of said polypeptide.
  • the compound can be, e.g., a small molecule, such as a nucleic acid, peptide, polypeptide, peptidomimetic, carbohydrate, lipid or other organic (carbon containing) or inorganic molecule, as further described herein.
  • a therapeutic in the manufacture of a medicament for treating or preventing disorders or syndromes including, e.g., trauma, regeneration (in vitro and in vivo), viral/bacterial/parasitic infections, Non Hippel-Lindau (NHL) syndrome, Alzheimer's disease, stroke, tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, cerebral palsy, epilepsy, Lesch- ⁇ yhan syndrome, multiple sclerosis, ataxia- telangiectasia, leukodystrophies, behavioral disorders, addiction, anxiety, pain, actinic keratosis, acne, hair growth diseases, allopecia, pigmentation disorders, endocrine disorders, connective tissue disorders, such as severe neonatal Marfan syndrome, dominant ectopia lentis, familial ascending aortic aneurysm, inflammatory disorders such as osteo- and rheumatoid-arthritis, inflammatory bowel disease, Crohn's
  • the therapeutic can be, e.g., a ⁇ ONX nucleic acid, a ⁇ ONX polypeptide, or a ⁇ ONX- specific antibody, or biologically-active derivatives or fragments thereof.
  • the compositions of the present invention will have efficacy for treatment of patients suffering from the diseases and disorders disclosed above and/or other pathologies and disorders of the like.
  • the polypeptides can be used as immunogens to produce antibodies specific for the invention, and as vaccines. They can also be used to screen for potential agonist and antagonist compounds.
  • a cD ⁇ A encoding ⁇ ONX may be useful in gene therapy, and ⁇ ONX may be useful when administered to a subject in need thereof.
  • the compositions of the present invention will have efficacy for treatment of patients suffering from the diseases and disorders disclosed above and/or other pathologies and disorders of the like.
  • the invention further includes ⁇ a method for screening for a modulator of disorders or syndromes including, e.g., the diseases and disorders disclosed above and/or other pathologies and disorders of the like.
  • the method includes contacting a test compound with a ⁇ ONX polypeptide and determining if the test compound binds to said ⁇ ONX polypeptide. Binding of the test compound to the ⁇ ONX polypeptide indicates the test compound is a modulator of activity, or of latency or predisposition to the aforementioned disorders or syndromes.
  • Also within the scope of the invention is a method for screening for a modulator of activity, or of latency or predisposition to disorders or syndromes including, e.g., the diseases and disorders disclosed above and/or other pathologies and disorders of the like by administering a test compound to a test animal at increased risk for the aforementioned disorders or syndromes.
  • the test animal expresses a recombinant polypeptide encoded by a NONX nucleic acid.
  • Expression or activity of ⁇ ONX polypeptide is then measured in the test animal, as is expression or activity of the protein in a control animal which recombinantly-expresses ⁇ ONX polypeptide and is not at increased risk for the disorder or syndrome.
  • the invention includes a method for determining the presence of or predisposition to a disease associated with altered levels of a ⁇ ONX polypeptide, a ⁇ ONX nucleic acid, or both, in a subject (e.g., a human subject). The method includes measuring the amount of the ⁇ ONX polypeptide in a test sample from the subject and comparing the amount of the polypeptide in the test sample to the amount of the ⁇ ONX polypeptide present in a control sample.
  • an alteration in the level of the ⁇ OVX polypeptide in the test sample as compared to the control sample indicates the presence of or predisposition to a disease in the subject.
  • the predisposition includes, e.g., the diseases and disorders disclosed above and/or other pathologies and disorders of the like.
  • the expression levels of the new polypeptides of the invention can be used in a method to screen for various cancers as well as to determine the stage of cancers.
  • the invention includes a method of treating or preventing a pathological condition associated with a disorder in a mammal by administering to the subject a ⁇ ONX polypeptide, a ⁇ ONX nucleic acid, or a ⁇ OVX-specific antibody to a subject (e.g., a human subject), in an amount sufficient to alleviate or prevent the pathological condition
  • a subject e.g., a human subject
  • the disorder includes, e.g., the diseases and disorders disclosed above and/or other pathologies and disorders of the like.
  • the invention can be used in a method to identity the cellular receptors and downstream effectors of the invention by any one of a number of techniques commonly employed in the art. These include but are not limited to the two-hybrid system, affinity purification, co-precipitation with antibodies or other specific-interacting molecules.
  • NONX nucleic acids and polypeptides are further useful in the generation of antibodies that bind immuno-specifically to the novel ⁇ ONX substances for use in therapeutic or diagnostic methods.
  • These ⁇ ONX antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the "Anti- ⁇ OVX Antibodies" section below.
  • the disclosed ⁇ ONX proteins have multiple hydrophilic regions, each of which can be used as an immunogen. These ⁇ ONX proteins can be used in assay systems for functional analysis of various human disorders, which will help in understanding of pathology of the disease and development of new drug targets for various disorders.
  • the ⁇ ONX nucleic acids and proteins identified here may be useful in potential therapeutic applications implicated in (but not limited to) various pathologies and disorders as indicated below.
  • the potential therapeutic applications for this invention include, but are not limited to: protein therapeutic, small molecule drug target, antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), diagnostic and/or prognostic marker, gene therapy (gene delivery/gene ablation), research tools, tissue regeneration in vivo and in vitro of all tissues and cell types composing (but not limited to) those defined here.
  • the present invention provides novel nucleotides and polypeptides encoded thereby.
  • NONX nucleic acids or “ ⁇ ONX polynucleotides” and the corresponding encoded polypeptides are referred to as “ ⁇ ONX polypeptides” or “ ⁇ ONX proteins.” Unless indicated otherwise, “ ⁇ ONX” is meant to refer to any of the novel sequences disclosed herein. Table A provides a summary of the NONX nucleic acids and their encoded polypeptides.
  • NONX nucleic acids and their encoded polypeptides are useful in a variety of applications and contexts.
  • the various ⁇ ONX nucleic acids and polypeptides according to the invention are useful as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins. Additionally, ⁇ ONX nucleic acids and polypeptides can also be used to identify proteins that are members of the family to which the ⁇ ONX polypeptides belong.
  • ⁇ ON1 is homologous to members of the neurotrophin-6 alpha family of proteins.
  • the ⁇ ON1 nucleic acid and polypeptide, antibodies and related compounds according to the invention may be used to treat immune and nervous system disorders, e.g., proinflammatory disorder, immune disorder, inflammatory disease, septic shock, arthritis, bone pain, or bone deformity.
  • ⁇ ON2 is homologous to members of the guanylate kinase family of proteins.
  • the ⁇ ON2 nucleic acid and polypeptide, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in the mediation of biosynthesis and nucleotide metabolism.
  • the ⁇ ON2 nucleic acid and polypeptide, antibodies and related compounds according to the invention may be used to treat genetic conditions, e.g.,Non Hippel- Lindau (NHL) syndrome, diabetes, or tuberous sclerosis.
  • NPL Non Hippel- Lindau
  • ⁇ ON3 is homologous to members of a family of the 85.6 kDa-like proteins that contain ankyrin domains.
  • ⁇ ON3 nucleic acid and polypeptide, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in the mediation of signal transduction or cell activation.
  • antibodies and related compounds according to the invention may be used to treat genetic conditions, e.g., endometriosis, fertility, adrenoleukodystrophy, congenital adrenal hyperplasia, diabetes, Non Hippel-Lindau (vhl) syndrome , pancreatitis, obesity, hyperparathyroidism, hypoparathyroidism, hyperthyroidism, hypothyroidism, SIDS, xerostomia, scleroderma, hypercalceimia, ulcers, cinhosis, transplantation, inflammatory bowel disease, diverticular disease, hirschsprung's disease, crohn's disease, appendicitis, hemophilia, hypercoagulation, idiopathic thrombocytopenic purpura, autoimmume disease, allergies, immunodeficiencies, graft vesus host, anemia, ataxia-telangiectasia, lymphedema, tonsilitis
  • genetic conditions e.g., endometrios
  • ⁇ ON4 is homologous to members of the mytonic dystrophy kinase-related CDC42- binding kinase family of proteins.
  • the ⁇ ON4 nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat muscle, or cell migration disorders, e.g., myotonic dystrophy, myotonic dystrophy type 2, proximal myotonic myopathy, proximal myotomc dystrophy, neuromuscular diseases associated with cardiomyopathy, multiple endocrine neoplasia type 1(ME ⁇ 1), insulin dependent diabetes mellitus, familial paraganglioma type 2, spinocerebellar ataxia type 5, Bardet-Biedl syndrome, non-hodgkins lymphoma, cancers such as breast cancer, liver, lung, pancrease, and prostate cancers.
  • myotonic dystrophy myotonic dystrophy type 2
  • proximal myotonic myopathy proxi
  • NON5 is homologous tomembers of the SI 00 Calcium binding protein family.
  • the ⁇ ON5 nucleic acid and polypeptide, antibodies and related compounds according to the invention may be used to treat genetic conditions, e.g., various cancers like breast, lung, and colorectal, as well as heart disease such as myocardial ischemia.
  • ⁇ ON6, ⁇ ON16, ⁇ ON18, ⁇ ON31, and ⁇ ON33 are homologous to the olfactory receptor/GPCR-like family of proteins.
  • G-Protein Coupled Receptor GPCRs
  • GPCRs G-Protein Coupled Receptor
  • antibodies and related compounds according to the invention may be used to treat, e.g., developmental diseases, MHC II and III diseases (immune diseases), taste and scent detectability disorders, Burkitt's lymphoma, corticoneurogenic disease, signal transduction pathway disorders, retinal diseases including those involving photoreception, cell growth rate disorders, cell shape disorders, feeding disorders, control of feeding, potential obesity due to over-eating, potential disorders due to starvation (lack of apetite), noninsulin-dependent diabetes mellitus ( ⁇ TDDM1), bacterial, fungal, protozoal and viral infections (particularly infections caused by HIV-1 or HIN-2), pain, cancer (including but not limited to neoplasm, adenocarcinoma, lymphoma, prostate cancer, uterus cancer), anorexia, bulimia, asthma, parkinson's disease, acute heart failure, hypotension, hypertension, urinary retention, osteoporosis, crohn's disease
  • ⁇ ON7 is homologous to members of the carbonate dehydratase/anhydrase family of proteins.
  • the ⁇ ON7 nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat respiratory or CO2 transport disorders, e.g., lung cancer, hypertension, asthma, emphysema, or diabetes.
  • NON8 is homologous to members of the carboxypeptidase family of proteins.
  • the ⁇ ON8 nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat digestive disorders, e.g., xerostomia, hypercalceimia, ulcers, Von Hippel-Lindau (NHL) syndrome, cirrhosis, transplantation, inflammatory bowel disease, diverticular disease, hirschsprung's disease, crohn's disease, appendicitis, stroke, tuberous sclerosis, anxiety, pain, endocrine dysfunctions, nueroprotection, diabetes, obesity, growth and reproductive disorders, myasthenia gravis.
  • digestive disorders e.g., xerostomia, hypercalceimia, ulcers, Von Hippel-Lindau (NHL) syndrome, cirrhosis, transplantation, inflammatory bowel disease, diverticular disease, hirschsprung's disease, crohn's disease, appendicitis, stroke, tuberous sclerosis, anxiety, pain, endocrine dysfunctions, nueroprotection,
  • ⁇ ON9 is homologous to members of the neurotransmitter receptor family of proteins.
  • the ⁇ ON9 nucleic acid and polypeptide, antibodies and related compounds according to the invention may be used to treat, e.g., leukemia, acute nonlymphocytic, spinocerebellar ataxia-1, or neurological disorders.
  • ⁇ ON10 is homologous to members of the proto-oncogene MAF-like family of proteins.
  • the ⁇ ON10 nucleic acid and polypeptide, antibodies and related compounds according to the invention may be used to treat, e.g., anemia, ataxia-telangiectasia, autoimmume disease, cancer, immunodeficiencies, hemophilia, hypercoagulation, idiopathic thrombocytopenic purpura, allergies, transplantation, graft versus host disease (GNHD), lymphaedema, systemic lupus erythematosus, asthma, emphysema, scleroderma, ARDS, diabetes, renal artery stenosis, interstitial nephritis, glomerulonephritis, polycystic kidney disease, systemic lupus erythematosus, renal tubular acidosis, IgA nephropathy, hypercalceimia, or Lesch- ⁇
  • ⁇ ON11 nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat digestive disorders, e.g., diabetes, Non Hippel-Lindau (NHL) syndrome, pancreatitis, obesity, endometriosis, fertility, hemophilia, hypercoagulation, idiopathic thrombocytopenic purpura, autoimmume disease, allergies, immunodeficiencies, transplantation, graft versus host disease (GNHD), lymphaedema, osteoporosis, hypercalceimia, arthritis, ankylosing spondylitis, scoliosis, systemic lupus erythematosus, asthma, emphysema, scleroderma, allergy, ARDS, renal artery stenosis, interstitial nephritis, glomerulonephritis, polycystic kidney disease, systemic lupus erythematosus, renal tubular acidos
  • NON12 is homologous to members of the phosphatase family of proteins.
  • the ⁇ ON12 nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat, e.g., hyperthyroidism, hypothyroidism, endometriosis, fertility, Non Hippel-Lindau (NHL) syndrome, cirrhosis, transplantation, hypogonadism, Alzheimer's disease, stroke, tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, cerebral palsy, epilepsy, Lesch- ⁇ yhan syndrome, multiple sclerosis, ataxia-telangiectasia, leukodystrophies, behavioral disorders, addiction, anxiety, pain, neurodegeneration, endocrine dysfunctions, diabetes, obesity, growth and reproductive disorders, systemic lupus erythematosus, autoimmune disease, asthma, emphysema, scleroderma, allergy, ARDS, renal artery sten
  • ⁇ ON13 is homologous to members of the chloride channel CLC-KA family of proteins.
  • the ⁇ ON13 nucleic acid and polypeptide, antibodies and related compounds according to the invention may be used to treat, e.g., diabetes, autoimmune disease, renal artery stenosis, interstitial nephritis, glomerulonephritis, polycystic kidney disease, systemic lupus erythematosus, renal tubular acidosis, IgA nephropathy, hypercalceimia, or Lesch- ⁇ yhan syndrome.
  • ⁇ ON14 is homologous to members of the mast cell function-associated antigen (MAF A) family of proteins.
  • MAF A mast cell function-associated antigen
  • the ⁇ ON14 nucleic acid and polypeptide, antibodies and related compounds according to the invention may be used to treat, e.g., cancer, autoimmune disease, allergies, immunodeficiencies, transplantation, graft versus host disease (GNHD), or lymphaedema.
  • ⁇ ON15 is homologous to members of the murine epithelial growth factor (MEGF) family of proteins.
  • MEGF murine epithelial growth factor
  • the ⁇ ON15 nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat, e.g., cancer, trauma, bacterial and viral infections, regeneration (in vitro and in vivo), fertility, endometriosis, cardiomyopathy, atherosclerosis, hypertension, congenital heart defects, aortic stenosis, atrial septal defect (ASD), atrioventricular (A-N) canal defect, ductus arteriosus, pulmonary stenosis, subaortic stenosis, ventricular septal defect (NSD), valve diseases, tuberous sclerosis, scleroderma, obesity, transplantation, anemia, bleeding disorders, transplantation, diabetes, autoimmune disease, renal artery stenosis, interstitial nephritis, glomeruloneph
  • NON17 is homologous to members of the monocarboxylate transporter (MCT)-like family of proteins.
  • MCT monocarboxylate transporter
  • the ⁇ ON17 nucleic acids and polypeptides, antibodies and related compounds according to the invention maybe used to treat, e.g., Salla disease, infantile sialic acid storage disease, cystinosis, or streptozotocin-induced diabetes.
  • ⁇ OV19 is homologous to members of the major Duchenne muscular dystrophy (DP71) family of proteins.
  • the NON 19 nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat muscle and nervous system disorders, e.g., Duchenne muscular dystrophy, Becker muscular dystroph, cardiomyopathy, dilated, X- linked, McLeod phenotype, Lesch- ⁇ yhan syndrome, myasthenia gravis.
  • ⁇ ON20 is homologous to members of the GPCR RTA family of proteins.
  • the ⁇ ON20 nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat a wide range of disorders such as cancer, immune disorders, endocrine disorders and other diseases, e.g., developmental diseases; MHCII and III diseases (immune diseases); taste and scent detectability disorders; Burkitt's lymphoma; corticoneurogenic disease; signal transduction pathway disorders; metabolic pathway disorders; retinal diseases including those involving photoreception; cell growth rate disorders; cell shape disorders; metabolic disorders; feeding disorders; control of feeding; the metabolic syndrome X; wasting disorders associated with chronic diseases; obesity; potential obesity due to over-eating or metabolic disturbances; potential disorders due to starvation (lack of appetite); diabetes; noninsulin-dependent diabetes mellitus ( ⁇ IDDM); infectious disease; bacterial, fungal, protozoal and viral infections (particularly infections caused by HIN-1 or HIN-2); pain; cancer (including but not limited
  • hypophosphatemic rickets autosomal dominant (2) Acrocallosal syndrome and dyskinesias, such as Huntington's disease or Gilles de la Tourette syndrome; immune disorders;
  • Adrenoleukodyst ophy Congenital Adrenal Hype ⁇ lasia; Hemophilia; Hypercoagulation;
  • Non Hippel-Lindau (NHL) syndrome Stroke; Tuberous sclerosis; hypercalceimia; Cerebral palsy;
  • Emphysema Scleroderma; ARDS; Renal artery stenosis; Interstitial nephritis;
  • Glomerulonephritis Polycystic kidney disease; Systemic lupus erythematosus; Renal tubular acidosis; IgA nephropathy; Cardiomyopathy; Atherosclerosis; Congenital heart defects; Aortic stenosis ; Atrial septal defect (ASD); Atrioventricular (A-N) canal defect; Ductus arteriosus; Pulmonary stenosis ; Subaortic stenosis; Ventricular septal defect (NSD); valve diseases;
  • Inflammatory bowel disease Diverticular disease; Leukodystrophies; Graft vesus host;
  • Hyperthyroidism Endometriosis; and hematopoietic disorders.
  • ⁇ ON21 is homologous to members of the TFIIIC box B-binding subunit family of proteins.
  • the ⁇ ON21 nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat cancer and viral infections, e.g., TFIIIC box B- binding subunit protein is cleaved and inactivated by the polio virus-encoded 3C protease during poliovirus infection (Shen et al., Mol. Cell. Biol, 16: 4163-71 (1996)).
  • ⁇ ON22 is homologous to members of the nucleoside diphosphate kinase B family of proteins.
  • the ⁇ ON22 nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat cancer, e.g., atherosclerosis, aneurysm, hypertension, fibromuscular dysplasia, stroke, scleroderma, obesity, transplantation, myocardial infarction, embolism, cardiovascular disorders, bypass surgery, fertility disorders, myasthenia gravis, leukodystrophies, pain, neuroprotection, systemic lupus erythematosus, autoimmune disease, asthma, emphysema, scleroderma, allergy, ARDS and other diseases, disorders and conditions of the like.
  • cancer e.g., atherosclerosis, aneurysm, hypertension, fibromuscular dysplasia, stroke, scleroderma, obesity, transplantation, myocardial infarction, emb
  • NON23 is homologous to members of the T-cell family of proteins.
  • the ⁇ ON23 nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to immune disorders, e.g., inflammation, allergies, autoimmune disease, and asthma.
  • ⁇ ON24 is homologous to members of the organic anion transporter (OAT) 3 family of proteins.
  • the ⁇ ON24 nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat a wide range of disorders such as cancer, kidney disorders, immune disorders and other diseases, e.g., Non Hippel-Lindau (NHL) syndrome, Cu ⁇ hosis,Transplantation, Osteoporosis, Hypercalceimia, Artliritis, Ahkylosing spondylitis, Scoliosis, Diabetes, Autoimmune disease, Renal artery stenosis, Interstitial nephritis, Glomerulonephritis, Polycystic kidney disease, Systemic lupus erythematosus, Renal tubular acidosis, IgA nephropathy, Lesch- ⁇ yhan syndrome renal malfunction, nephrotoxicity, disease associated with cytotoxic drug, osteoporosis, osteopetrosis resistance,
  • ⁇ ON25 and ⁇ ON26 are homologous to members of the ficolin family of proteins.
  • nucleic acid or protein therapeutics designed with the protein encoded for by ⁇ ON26 could function as an opsinin to target and eliminate bacteria by complement -mediated destruction.
  • These proteins could be important for the treatment of bacterial septicemia.
  • Ficolins may also have the ability to bind to elastins. Elastins are functionally important for lung alveolar development and inactivation of these proteins can lead to emphysema-like disease.
  • Antibodies against ⁇ ON25 and ⁇ ON26 may prevent tissue destruction mediated by ficolin activity during emphysema, asthma and arthritis.
  • ⁇ ON27 is homologous to members of the peroxisomal Ca + -dependent solute carrier family of proteins.
  • the ⁇ ON27 nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat metabolic disorders, e.g., cancer, trauma, regeneration (in vitro and in vivo), viral/bacterial/parasitic infections, atherosclerosis, aneurysm, hypertension, fibromuscular dysplasia, stroke, scleroderma, obesity, Non Hippel- Lindau (NHL) syndrome, Alzheimer's disease, stroke, tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, cerebral palsy, epilepsy, Lesch- ⁇ yhan syndrome, multiple sclerosis, ataxia-telangiectasia, leukodystrophies, behavioral disorders, addiction, anxiety, pain, diabetes, autoimmune disease, renal artery stenosis, interstitial nephritis, glomerulonephritis, polyc
  • NON28, ⁇ ON29, and ⁇ ON30 are homologous to members of the ⁇ a+/glucose cotransporter family of proteins.
  • the NOV28 nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat metabolic, immune and renal disorders, e.g., metabolic diseases such as diabetes and hypertension, or cancer, trauma, regeneration (in vitro and in vivo), viral/bacterial/parasitic infections, diabetes, autoimmune disease, renal artery stenosis, interstitial nephritis, glomerulonephritis, polycystic kidney disease, systemic lupus erythematosus, renal tubular acidosis, IgA nephropathy, hypercalceimia, Lesch- Nyhan syndrome, cardiomyopathy, atherosclerosis, hypertension, congenital heart defects, aortic stenosis, atrial septal defect (ASD), atrioventricular (A-V) canal defect, duct
  • ⁇ ON32 is homologous to members of the phosphoenolpyruvate carboxykinase family of proteins.
  • the ⁇ ON32 nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat metabolic disorders, e.g., hypoglycemia.
  • ⁇ ONX nucleic acids and proteins of the invention are useful in potential therapeutic applications implicated, for example but not limited to, in various pathologies /disorders as described herein and/or other pathologies/disorders.
  • Potential therapeutic uses for the invention(s) are, for example but not limited to, the following: (i) a protein therapeutic, (ii) a small molecule drug target, (iii) an antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), (iv) a nucleic acid useful in gene therapy (gene delivery/gene ablation), (v) an agent promoting tissue regeneration in vitro and in vivo, and (vi) a biological defense weapon.
  • the disclosed ⁇ ON1 nucleic acid (alternatively referred to herein as CG56181-01) encodes a novel neutrophin-6 alpha-like protein and includes the 796 nucleotide sequence (SEQ ID NO:
  • the novel NOV1 nucleic acid of the invention maps to chromosome 19.
  • the NOVl protein (SEQ ID NO:2) encoded by SEQ TD NO: 1 is 258 amino acid residues in length and is presented using the one-letter amino acid code in Table IB.
  • the SignalP, Psort and/or Hydropathy results indicate that NOVl has a signal peptide and is likely to be localized in the microbody (peroxisome) with a certainty of 0.5952.
  • a NOVl polypeptide is located to the cytoplasm with a certainty of 0.4500, the lysosome (lumen) with a certainty of 0.2100, or the mitochondrial matrix space with a certainty of 0.1000.
  • NOVl nucleic acid sequence of this invention has 762 of 796 bases (95%) identical to a gb:GENBANK-ID:HUMNT4PSG
  • full amino acid sequence of the disclosed protein of the invention has 239 of 258 amino acid residues (92%) identical to, and 244 of 258 amino acid residues (94%) similar to, the 257 amino acid residue ptnr:SWISSPROT-ACC:P34132 protein from Human (NEUROTROPHIN-6 ALPHA (NT-6 ALPHA)).
  • the "E-value” or “Expect” value is a numeric indication of the probability that the aligned sequences could have achieved their similarity to the BLAST query sequence by chance alone, within the database that was searched.
  • the probability that the subject ("Sbjct") retrieved from the IIT BLAST analysis, matched the Query IIT sequence purely by chance is the E value.
  • the Expect value (E) is a parameter that describes the number of hits one can "expect" to see just by chance when searching a database of a particular size. It decreases exponentially with the Score (S) that is assigned to a match between two sequences. Essentially, the E value describes the random background noise that exists for matches between sequences.
  • Blasting is performed against public nucleotide databases such as GenBank databases and the GeneSeq patent database. For example, BLASTX searching is performed against public protein databases, which include GenBank databases, SwissProt, PDB and PIR.
  • the Expect value is used as a convenient way to create a significance threshold for reporting results.
  • the default value used for blasting is typically set to 0.0001.
  • the Expect value is also used instead of the P value (probability) to report the significance of matches.
  • an E value of one assigned to a hit can be interpreted as meaning that in a database of the current size one might expect to see one match with a similar score simply by chance.
  • An E value of zero means that one would not expect to see any matches with a similar score simply by chance. See, e.g., http://www.ncbi.nlm.nih.gov/Education/BLASTinfo/. Occasionally, a string of X's or N's will result from a BLAST search.
  • the NOVl protein of the invention also has homolgy to the proteins shown in the BLASTP data in Table ID.
  • NGF domain 1 of 2 , from 133 to 184 70 . 6 1. 5e-19
  • NGF domain 2 of 2, from 213 to 258 100 . 5 2 . 5e-28
  • NGF HWnSeCkttqtYVRALTmdnnklVgWRflRIDTACVCtLsrKtGrt (SEQ ID NO : 130 )
  • NOVl contains nerve growth factor domains as illustrated in Table IF.
  • the NOVl nucleic acid, and the encoded polypeptide, according to the invention are useful in a variety of applications and contexts.
  • NOVl nucleic acids and polypeptides can be used to identify proteins that are members of the neurotrophin family of proteins.
  • the NOVl nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance NOVl activity or function.
  • the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., development and survival of certain sympathetic and sensory neurons in both the central and peripheral nervous systems. These molecules can be used to treat, e.g., proinflammatory disorder, immune disorder, and inflammatory disease.
  • NOVl nucleic acid and polypeptide according to the invention are useful, inter alia, as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins.
  • the NOVl nucleic acid and polypeptide include structural motifs that are characteristic of proteins belonging to the family of nerve growth factors such as the neurotrophin proteins.
  • Nerve growth factor is the prototype for the neurotrophin family of polypeptides which are essential in the developments and survival of certain sympathetic and sensory neurons in both the central and peripheral nervous systems. NGF was discovered when mouse sarcoma tissue transplants in chicken embryos caused an increase in the size of spinal ganglia.
  • NOVl nucleic acid and polypeptide, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in the mediation of the peripheral and central nervous system.
  • the NOVl nucleic acid and polypeptide, antibodies and related compounds according to the invention may be used to treat immune and nervous system disorders, e.g., proinflammatory disorder, immune disorder, inflammatory disease, septic shock, arthritis, bone pain, or bone deformity.
  • the NOVl nucleic acid and polypeptide are useful for detecting specific cell types. For example, expression analysis has demonstrated that a NOVl nucleic acid is expressed in placenta and uterus.
  • the disclosed NOV2 nucleic acid encodes a novel Guanylate kinase-like protein and includes the 1336 nucleotide sequence (SEQ ID NO:3) shown in Table 2A.
  • the novel NOV2 nucleic acid of the invention maps to chromosome 2. An open reading frame for the mature protein was identified beginning with an ATG initiation codon at nucleotides 3-5, and ending with a TGA stop codon at nucleotides 1326-1328. Putative untranslated regions, if any, are found upstream from the initiation codon and downstream from the termination codon. The start and stop codons are in bold letters.
  • the NOV2 protein (SEQ ID NO:4) encoded by SEQ ID NO:3 is 441 amino acid residues in length and is presented using the one-letter amino acid code in Table 2B.
  • the SignalP, Psort and/or Hydropathy results indicates that NOV2 has a signal peptide and is likely to be localized in the microbody (peroxisome) with a certainty of 0.3000.
  • a NOV2 polypeptide is located to the nucleus with a certainty of 0.3000, the mitochondrial matrix space with a certainty of 0.1000, or the lysosome (lumen) with a certainty of 0.1000.
  • SNP Small nucleotide polymo ⁇ hisms
  • the full amino acid sequence of the disclosed NOV2 protein of the invention has 346 of 441 amino acid residues (78%) identical to, and 380 of 441 amino acid residues (86%) similar to, the 441 amino acid residue ⁇ tnr:SPTREMBL-ACC:Q9QYHl protein from Rattus norvegicus (Rat) (DLG6 ALPHA).
  • the NOV2 protein of the invention also has homolgy to the proteins shown in the BLASTP data in Table 2D.
  • NOV2 contains guanylate kinase domains as illustrated in Table 2F.
  • NOV2 nucleic acid, and the encoded polypeptide, according to the invention are useful in a variety of applications and contexts.
  • NOV2 nucleic acids and polypeptides can be used to identify proteins that are members of the guanylate kinase family of proteins.
  • the NOV2 nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance NOV2 activity or function.
  • the nucleic acids and polypeptides according to the invention maybe used as targets for the identification of small molecules that modulate or inhibit, e.g., cell signaling pathways, cell junction organization, or transmembrane regulation.. These molecules can be used to treat, e.g., Von Hippel-Lindau (VHL) syndrome, diabetes, and tuberous sclerosis.
  • VHL Von Hippel-Lindau
  • the NOV2 nucleic acid and polypeptide according to the invention are useful, ter alia, as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins.
  • the NOV2 nucleic acid and polypeptide include structural motifs that are characteristic of proteins belonging to the family of kinases such as the guanylate kinase proteins.
  • Guanylate kinase is a critical enzyme for biosynthesis of GTP and dGTP, and its role in nucleotide metabolism makes it a target for cancer chemotherapy.
  • the structure of mouse guanylate kinase (gmk) includes an N-terminal ATP binding motif and a neighboring guanylate kinase signature sequence (GKSS).
  • the low molecular mass cytosolic forms of guanylate kinase are implicated in the regulation of the supply of guanine nucleotides to cell signaling pathways, while the related families of high molecular mass and membrane-associated forms of guanylate kinase, such as MAGUK, CASK, SAP102, ZO-1, and MAGI-1, have roles in cell junction organization and transmembrane regulation.
  • the NOV2 nucleic acid and polypeptide, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in the mediation of biosynthesis and nucleotide metabolism. As such the NOV2 nucleic acid and polypeptide, antibodies and related compounds according to the invention may be used to treat genetic conditions, e.g., Von Hippel-Lindau (VHL) syndrome, diabetes, or tuberous sclerosis.
  • VHL Von Hippel-Lindau
  • the NOV2 nucleic acid and polypeptide are useful for detecting specific cell types. For example, expression analysis has demonstrated that a NOV2 nucleic acid is expressed in synovium/synovial membrane.
  • the disclosed NOV3 nucleic acid (alternatively referred to herein as CG53400-01) encodes a novel hypothetical 85.6 kDa-like protem and includes the 3089 nucleotide sequence (SEQ ID NO: 5) shown in Table 3 A.
  • the novel NOV3 nucleic acid of the invention maps to chromosome 12.
  • the NOV3 protein (SEQ ID NO:6) encoded by SEQ ID NO:5 is 993 amino acid residues in length and is presented using the one-letter amino acid code in Table 3B.
  • the SignalP, Psort and/or Hydropathy results indicate that NOV3 has no known signal peptide and is likely to be localized in the mitochondrial matrix space with a certainty of 0.5083.
  • a NOV3 polypeptide is located to the nucleus with a certainty of 0.3000, the mitochondrial inner membrane with a certainty of 0.2317, or the mitochondrial intermembrane space with a certainty of 0.2217.
  • variants of the parent clone NOV3 as shown below in Table 3C. These novel variants were derived by laboratory cloning of cDNA fragments coding for a domain of the full length form of NOV3 (CG53400-01), between residues 596 and 968.
  • NOV3 nucleic acid sequence of this invention has 1552 of 2369 bases (65%) identical to a gb:GENBANK-H):HSM801363
  • the full amino acid sequence of the disclosed NOV3 protein of the invention has 498 of 791 amino acid residues (62%) identical to, and 600 of 791 amino acid residues (75%) similar to, the 791 amino acid residue ptnr:SPTREMBL-ACC:Q9UFA4 protein from Human (HYPOTHETICAL 85.6 KDA PROTEIN).
  • the NOV3 protein of the invention also has homolgy to the proteins shown in the BLASTP data in Table 3E.
  • NOV3 contains ankyrin domains as illustrated in Table 3G.
  • NOV3 nucleic acid, and the encoded polypeptide, according to the invention are useful in a variety of applications and contexts.
  • NOV3 nucleic acids and polypeptides can be used to identify proteins that are members of the ankyrin family of proteins.
  • the NOV3 nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance NOV3 activity or function.
  • the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., red blood cell formation/organization, or signal transduction/cell activation. These molecules can be used to treat, e.g., spherocytosis.
  • the NOV3 nucleic acid and polypeptide according to the invention are useful, inter alia, as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins.
  • the NOV3 nucleic acid and polypeptide include structural motifs that are characteristic of proteins belonging to the family of transmembrane proteins/membrane skeleton proteins such as the ankyrin proteins.
  • Ankyrin is a globular protein (200 kD) that links spectrin and an integral membrane protein (Band III) in the erythrocyte plasma membrane.
  • Ankyrin belongs to a family of closely related polypeptides associated with the plasma membrane of cells in a variety of cell types (e.g.
  • Ankyrin has been shown to underlie membrane proteins including CD44, the voltage-dependent sodium channel, NA+/K+ ATPase and the anion exchanger protein. Functional diversity between members of the ankyrin family is generated by the expression of multiple genes as well as alternative splicing of pre-mRna's. The formation of a direct connection between ankyrin and functionally important transmembrane proteins/membrane skeleton may be one of the earliest events to occur during signal transduction and cell activation.
  • NOV3 nucleic acid and polypeptide, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in the mediation of signal transduction or cell activation.
  • the NOV3 nucleic acid and polypeptide, antibodies and related compounds according to the invention may be used to treat genetic conditions, e.g., endometriosis, fertility, adrenoleukodystrophy, congenital adrenal hyperplasia, diabetes, Von Hippel-Lindau (vhl) syndrome , pancreatitis, obesity, hype ⁇ arathyroidism, hypoparathyroidism, hyperthyroidism, hypothyroidism, SIDS, xerostomia, scleroderma, hypercalceimia, ulcers, cirrhosis, transplantation, inflammatory bowel disease, diverticular disease, hirschsprung's disease, crohn's disease, appendicitis, hemophilia, hypercoagulation, idiopathic thrombocytopenic pu ⁇ ura, autoimmume
  • the NOV3 nucleic acid and polypeptide are useful for detecting specific cell types. For example, expression analysis has demonstrated that a NOV3 nucleic acid is expressed in adrenal gland/suprarenal gland, bone, brain, cartilage, cervix, coronary artery, platelets, kidney, kidney cortex, liver, mammary gland/breast, pancreas, placenta, salivary glands, spleen, synovium/synovial membrane, thymus, cerebral medulla/cerebral white matter, and left cerebellum. Additional utilities for the NOV3 nucleic acid and polypeptide according to the invention are disclosed herein.
  • the NOV4 proteins descibed herein are novel myotonic dystrophy kinase-related CDC-42 binding kinase (MRCK)-like proteins.
  • the NOV4 nucleic acids disclosed herein map to chromosome 1 lql3.
  • Two alternative novel NOV4 nucleic acids and polypeptides are disclosed herein, namely NO V4a andNOV4b.
  • a NOV4 variant is NOV4a (alternatively referred to herein as CG56209-01), which encodes the 3835 nucleotide sequence (SEQ ID NO:7) shown in Table 4A.
  • SEQ ID NO:7 An open reading frame for the mature protein was identified beginning with an ATG codon at nucleotides 98-100 and ending with a TAG codon at nucleotides 3689-3691. Putative untranslated regions, if any, downstream from the termination codon and upstream from the initiation codon are underlined. The start and stop codons are in bold letters.
  • the NOV4a protein (SEQ ID NO:8) encoded by SEQ ID NO:7 is 1197 amino acid residues in length and is presented using the one-letter amino acid code in Table 4B.
  • the SignalP, Psort and/or Hydropathy results indicate that NOV4a has no known signal peptide and is likely to be localized in the nucleus with a certainty of 0.7600.
  • a NOV4a polypeptide is located to the microbody (peroxisome) with a certainty of 0.3114, the lysosome (lumen) with a certainty of 0.1772, or the mitochondrial matrix space with a certainty of 0.1000.
  • NOV4b (alternatively referred to herein as CG56209-02), which includes the 3985 nucleotide sequence (SEQ ID NO:9) shown in Table 4C.
  • SEQ ID NO:9 3985 nucleotide sequence shown in Table 4C.
  • An open reading frame for the mature protein was identified beginning with an ATG codon at nucleotides 98- 100 and ending with a TAG codon at nucleotides 3839-3841. Putative untranslated regions, if any, downstream from the termination codon and upstream from the initiation codon are underlined. The start and stop codons are in bold letters.
  • the NOV4b protein (SEQ ID NO:10) encoded by SEQ ID NO:9 is 1247 amino acid residues in length and is presented using the one-letter amino acid code in Table 4D.
  • the SignalP, Psort and/or Hydropathy results indicate that NOV4b has no known signal peptide and is likely to be localized in the nucleus with a certainty of 0.8800.
  • a NOV4b polypeptide is located to the microbody (peroxisome) with a certainty of 0.3226, the lysosome (lumen) with a certainty of 0.1925, or the mitochondrial matrix space with a certainty of 0.1000.
  • NOV4a or NOV4b any reference to NOV4 is assumed to encompass all variants.
  • a search against the Patp database a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 4E.
  • NOV4a nucleic acid sequence of this invention has 865 of 865 bases (100%) identical to a gb:GENBANK-ro:HSMDPKJN
  • the full amino acid sequence of the disclosed NOV4a protein of the invention has 314 of 572 amino acid residues (54%) identical to, and 418 of 572 amino acid residues (73%) similar to, the 1732 amino acid residue ptnr:SPTREMBL- ACC:O54874 protein from Rattus norvegicus (Rat) (MYTONIC DYSTROPHY KINASE- RELATED CDC42-BINDING KINASE).
  • NOV4 contains protein kinase domains as illustrated in Table 4H.
  • NON4 nucleic acids, and the encoded polypeptides, according to the invention are useful in a variety of applications and contexts.
  • ⁇ ON4 nucleic acids and polypeptides can be used to identify proteins that are members of the protein kinase family of proteins.
  • the ⁇ ON4 nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance ⁇ ON4 activity or function.
  • nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., cytoskeletal reorganization or molecular switch mechanisms. These molecules can be used to treat, e.g., myotonic dystrophy, myotonic dystrophy type 2, proximal myotonic myopathy, or proximal myotonic dystrophy.
  • various ⁇ OV4 nucleic acids and polypeptides according to the invention are useful, ter alia, as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins.
  • the NOV4 nucleic acids and their encoded polypeptides include structural motifs that are characteristic of proteins belonging to the family of protein kinases such as the MRCK protein.
  • MRCK is a Ser/Thr kinase that is highly related to myotonic dystrophy kinase and ROKs.
  • MRCK contains an N-terminal kinase domain, a coiled-coil region, a cysteine-rich domain (CR), a pleckstrin-like domain (PH), and a C-terminal p21 GTPase-binding domain (GBD). Two different MRCK genes are expressed in rat.
  • MRCKa mRNA is enriched in brain and lung, while MRCKb mRNA is expressed in lung and kidney.
  • MRCKa phosphorylates Ser/Thr residues in myelin basic protein, histone HI, and non-muscle myosin regulatory light chain.
  • expression of kinase-dead MRCKa blocks Cdc42-dependent formation of focal complexes and peripheral microspikes, while in PC 12 cells MRCKa may act downstream of Cdc42 and Racl to promote neurite outgrowth.
  • the NOV4 nucleic acids and polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in the mediation of cell migration and differentiation.
  • the NOV4 nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat muscle, or cell migration disorders, e.g., myotonic dystrophy, myotonic dystrophy type 2, proximal myotonic myopathy, proximal myotonic dystrophy, neuromuscular diseases associated with cardiomyopathy, multiple endocrine neoplasia type 1(MEN1), insulin dependent diabetes mellitus, familial paraganglioma type 2, spinocerebellar ataxia type 5, Bardet-Biedl syndrome, non-hodgkins lymphoma, cancers such as breast cancer, liver, lung, pancrease, and prostate cancers.
  • myotonic dystrophy myotonic dystrophy type 2
  • proximal myotonic myopathy proximal myo
  • the NON4 nucleic acids and polypeptides are useful for detecting specific cell types.
  • expression analysis has demonstrated that a ⁇ ON4 nucleic acid is expressed in adrenal gland, bone marrow, brain - amygdala, brain - cerebellum, brain - hippocampus, brain - substantia nigra, brain - thalamus, brain -whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma - Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea, and uterus.
  • the disclosed NOV5 nucleic acid encodes a novel SI 00 calcium binding-like protein and includes the 332 nucleotide sequence (SEQ ID NO: 11) shown in Table 5 A.
  • SEQ ID NO: 11 the 332 nucleotide sequence shown in Table 5 A.
  • SignalP, Psort and/or hydropathy suggest that the SI 00 Calcium Binding Protein-like protein may be localized in the cytoplasm, the protein predicted here is similar to the SI 00 Calcium Binding Protein family, some members of which are secreted. Therefore it is likely that this novel SI 00 Calcium Binding Protein-like protein is available at the same sub-cellular localization and hence accessible to a diagnostic probe and for various therapeutic applications.
  • the NOV5 protein (SEQ ID NO: 12) encoded by SEQ ID NO: 11 is 104 amino acid residues in length and is presented using the one-letter amino acid code in Table 5B.
  • the SignalP, Psort and/or Hydropathy results indicate that NON5 has no known signal peptide and is likely to be localized in the mitochondrial matrix space with a certainty of 0.5964.
  • a ⁇ ON5 polypeptide is located to the mitochondrial inner membrane with a certainty of 0.3037, the mitochondrial intermembrane space with a certainty of 0.3037, or the mitochondrial outer membrane with a certainty of 0.3037.
  • the NOV5 nucleic acid sequence of this invention has 305 of 335 bases (91%) identical to a gb:GENBANK-ID:HUMS100CPl
  • the full amino acid sequence of the disclosed NON5 protein of the invention has 102 of 103 amino acid residues (99%) identical to, and 102 of 103 amino acid residues (99%) similar to, the 104 amino acid residue ptnr:SPTREMBL-ACC:Q9UDP3 protein from Human (WUGSC:H_ ⁇ H0456 ⁇ 16.1 PROTEIN).
  • the NON5 protein of the invention also has homolgy to the proteins shown in the
  • NON5 contains S100 calcium binding domains as illustrated in Table 5F.
  • ⁇ ON5 nucleic acid, and the encoded polypeptide, according to the invention are useful in a variety of applications and contexts.
  • ⁇ ON5 nucleic acids and polypeptides can be used to identify proteins that are members of the S100 family of proteins.
  • the ⁇ OV5 nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance NOV5 activity or function.
  • the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., calcium regulation. These molecules can be used to treat, e.g., various cancers hke breast, lung, or colorectal.
  • the NOV5 nucleic acid and polypeptide according to the invention are useful, inter alia, as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins.
  • the NOV5 nucleic acid and polypeptide include structural motifs that are characteristic of proteins belonging to the family of S100 proteins. S100 proteins are expressed in a cell-type specific manner in higher organisms, including humans, and are involved in the calcium-regulated control of very diverse cellular processes. Proteins of the S100 family belong to the large group of EF-hand calcium-binding proteins.
  • the NON5 nucleic acid and polypeptide, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in the mediation of calcium regulation.
  • ⁇ ON5 nucleic acid and polypeptide antibodies and related compounds according to the invention may be used to treat genetic conditions, e.g., various cancers like breast, lung, and colorectal, as well as heart disease such as myocardial ischemia.
  • the ⁇ ON5 nucleic acid and polypeptide are useful for detecting specific cell types. For example, expression analysis has demonstrated that a ⁇ ON5 nucleic acid is expressed in elevated levels in colorectal cancers compared with that of normal colorectal mucosa, as well as in breast cancer-derived metastatic axillary lymph nodes, but not in normal lymph nodes or breast fibroadenomas.
  • the ⁇ ON5 nucleic acids and polypeptides, antibodies and related compounds accoring to the invention will have diagnostic and therapeutic applications in the detection of cancer, e.g., breast or colorectal cancer.
  • a ⁇ ON5 nucleic acid is also expressed in brain, lung, smooth muscle and keratinocyte tissue.
  • the disclosed NOV6 nucleic acid encodes a novel olfactory receptor-like protein/G-protein coupled receptor (GPCR) protein and includes the 1121 nucleotide sequence (SEQ ID NO: 13) shown in Table 6A.
  • GPCR G-protein coupled receptor
  • An open reading frame for the mature protein was identified beginning with an ATG initiation codon at nucleotides 30-32, and ending with a TAG stop codon at nucleotides 1119- 1121. Putative untranslated regions, if any, are found upstream from the initiation codon and downstream from the termination codon. The start and stop codons are in bold letters.
  • Table 6A NOV6 Nucleotide Sequence (SEQ ID NO:13)
  • the NOV6 protein (SEQ ID NO: 14) encoded by SEQ ID NO: 13 is 363 amino acid residues in length and is presented using the one-letter amino acid code in Table 6B.
  • the SignalP, Psort and/or Hydropathy results indicate that NON6 has a signal peptide and is likely to be localized in the plasma membrane with a certainty of 0.6000.
  • a ⁇ ON6 polypeptide is located to the Golgi body with a certainty of 0.4000, the endoplasmic reticulum (membrane) with a certainty of 0.3000, or the mitochondrial inner membrane with a certainty of 0.0300.
  • the SignalP indicates a likely cleavage site for a ⁇ ON6 polypeptide is between positions 41 and 42, i.e., at the dash in the sequence CFG- ⁇ L.
  • NOV6 nucleic acid sequence of this invention has 723 of 1057 bases (68%) identical to a gb:GENBANK-ID:AF190500
  • LGR7 G protein-coupled receptor 7
  • the full amino acid sequence of the disclosed NOV6 protein of the invention has 203 of 339 amino acid residues (59%) identical to, and 271 of 339 amino acid residues (79%) similar to, the 757 amino acid residue ptnr:TREMBLNEW-ACC:AAG17167 protein from Human (LEUCINE-RICH REPEAT-
  • the NOV6 protein of the invention also has homolgy to the proteins shown in the BLASTP data in Table 6D.
  • SEQ ID NO. 14 NOV6 4. SEQ ID NO. 158 CAC38938
  • NON6 contains 7-transmembrane domains as illustrated in Table 6F.
  • ⁇ ON6 nucleic acid, and the encoded polypeptide, according to the invention are useful in a variety of applications and contexts.
  • ⁇ ON6 nucleic acids and polypeptides can be used to identify proteins that are members of the olfactory receptor family of proteins.
  • the ⁇ ON6 nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance ⁇ ON6 activity or function.
  • the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., cellular recognition, or G-protein-mediated transduction of odorant signals. These molecules can be used to treat, e.g., taste and scent detectability disorders, immune diseases, or signal transduction pathways.
  • the ⁇ ON6 nucleic acid and polypeptide according to the invention are useful, ter alia, as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins.
  • the ⁇ ON6 nucleic acid and polypeptide include structural motifs that are characteristic of proteins belonging to the family of olfactory receptor proteins. Olfactory receptors have great variety, extraordinar specificity, high sensitivity and fast response.
  • the human olfactory epithelium contains two to three thousand distinct olfactory receptors, a class of G-protein coupled receptors.
  • the receptors consist of seven hydrophobic segments that span the cell membrane (trans-membrane domains I-NII), separated by hydrophilic segments that project into the intra- or extra-cellular space.
  • Trans-membrane domains II-NII comprise a hypervariable segment that defines the ligand specificity of the receptor.
  • the ⁇ ON6 nucleic acid and polypeptide, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in the mediation of signal transduction.
  • the ⁇ ON6 nucleic acid and polypeptide, antibodies and related compounds according to the invention may be used to treat, e.g., developmental diseases, MHC II and III diseases (immune diseases), taste and scent detectability disorders, Burkitt's lymphoma, corticoneurogenic disease, signal transduction pathway disorders, retinal diseases including those involving photoreception, cell growth rate disorders, cell shape disorders, feeding disorders, control of feeding, potential obesity due to over-eating, potential disorders due to starvation (lack of apetite), noninsulin-dependent diabetes mellitus ( ⁇ TDDMl), bacterial, fungal, protozoal and viral infections (particularly infections caused by HIN-1 or HIN-2), pain, cancer (including but not limited to neoplasm, adenocarcinoma, lymphoma, prostate cancer, uterus cancer), anorexia
  • the NON6 nucleic acid and polypeptide are useful for detecting specific cell types. For example, expression analysis has demonstrated that a ⁇ ON6 nucleic acid is predominantly expressed in olfactory epithelium and taste receptor cells of the tongue.
  • the ⁇ ON7 proteins descibed herein are novel carbonate dehydratase-like proteins.
  • the ⁇ ON7 nucleic acids disclosed herein map to chromosome 15.
  • Two alternative novel ⁇ ON7 nucleic acids and polypeptides are disclosed herein, namely ⁇ ON7a and ⁇ ON7b.
  • ⁇ OV7a A NON7 variant is ⁇ ON7a (alternatively referred to herein as CG50365-01 ), which encodes the 828 nucleotide sequence (SEQ ID NO: 15) shown in Table 7A.
  • An open reading frame for the mature protein was identified beginning with an ATG codon at nucleotides 16-18 and ending with a TAA codon at nucleotides 802-804. Putative untranslated regions, if any, downstream from the termination codon and upstream from the initiation codon are underlined. The start and stop codons are in bold letters.
  • the NON7a protein (SEQ ID NO: 16) encoded by SEQ TD NO: 15 is 262 amino acid residues in length and is presented using the one-letter amino acid code in Table 7B.
  • the SignalP, Psort and/or Hydropathy results indicate that NOV7a has no known signal peptide and is likely to be localized in the microbody (peroxisome) with a certainty of 0.7480.
  • a NOV7a polypeptide is located to the mitochondrial matrix space with a certainty of 0.1000, the lysosome (lumen) with a certainty of 0.1000, or the endoplasmic reticulum (membrane) with a certainty of 0.1000.
  • a NON7 variant is ⁇ ON7b (alternatively referred to herein as CG50365-02), which includes the 833 nucleotide sequence (SEQ ID NO: 17) shown in Table 7C.
  • SEQ ID NO: 17 An open reading frame for the mature protein was identified beginning with an ATG codon at nucleotides 21-23 and ending with a TAA codon at nucleotides 807-809. Putative untranslated regions, if any, downstream from the termination codon and upstream from the initiation codon are underlined. The start and stop codons are in bold letters.
  • the NON7b protein (SEQ ID ⁇ O:18) encoded by SEQ ID NO:17 is 262 amino acid residues in length and is presented using the one-letter amino acid code in Table 7D.
  • the SignalP, Psort and/or Hydropathy results indicate that NOV7b has no known signal peptide and is likely to be localized in the microbody (peroxisome) with a certainty of 0.7480.
  • a NON7b polypeptide is located to the mitochondrial matrix space with a certainty of 0.1000, the lysosome (lumen) with a certainty of 0.1000, or the endoplasmic reticulum (membrane) with a certainty of 0.1000.
  • any reference to ⁇ ON7 is assumed to encompass all variants.
  • the full amino acid sequence of the disclosed NOV7a protein of the invention has 160 of 257 amino acid residues (62%) identical to, and 197 of 257 amino acid residues (76%) similar to, the 260 amino acid residue ptnr:SWISSPROT-ACC:Q92051 protein from Brachydanio rerio (Zebrafish) (Zebra danio) (CARBONIC ANHYDRASE (EC 4.2.1.1) (CARBONATE DEHYDRATASE)).
  • NOV7b nucleic acid sequence of this invention has 549 of 789 bases (69%) identical to a gb:GENBANK-ID:HSCAIR
  • the full amino acid sequence of the disclosed NOV7b protein of the invention has 156 of 261 amino acid residues (59%) identical to, and 202 of 261 amino acid residues (77%) similar to, the 261 amino acid residue ptnr:pir-id:CRHUl protein from human (carbonate dehydratase (EC 4.2.1.1) I [validated]).
  • NOV7 contains carbonic anhydrase domains as illustrated in Table 7H.
  • NON7 nucleic acids, and the encoded polypeptides, according to the invention are useful in a variety of applications and contexts.
  • ⁇ ON7 nucleic acids and polypeptides can be used to identify proteins that are members of the carbonate dehydratase family of proteins.
  • the ⁇ ON7 nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance ⁇ ON7 activity or function.
  • nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., a variety of biological processes such as respiration, calcification, acid-base balance, bone resorption and the formation of aqueous humor, cerebrospinal fluid, saliva, and gastric acid. These molecules can be used to treat, e.g., hypertension, asthma, or emphysema.
  • ⁇ ON7 nucleic acids and polypeptides according to the invention are useful, ter alia, as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins.
  • the ⁇ ON7 nucleic acids and their encoded polypeptides include structural motifs that are characteristic of proteins belonging to the lyase family such as the carbonic dehydratase protein.
  • Carbonic dehydratase is an enzyme that catalyzes the equilibration of dissolved carbon dioxide and carbonic acid, speeding the movement of carbon dioxide from tissues to blood to alveolar air. It is a zinc metalloenzyme of great physiological importance.
  • ⁇ ON7 nucleic acids and polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in the mediation of CO2 transport.
  • the ⁇ ON7 nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat respiratory or CO2 transport disorders, e.g., lung cancer, hypertension, asthma, emphysema, or diabetes.
  • the ⁇ ON7 nucleic acids and polypeptides are useful for detecting specific cell types. For example, expression analysis has demonstrated that a ⁇ ON7 nucleic acid is expressed in lung.
  • the ⁇ ON8 proteins descibed herein are novel carboxypeptidase-like proteins.
  • the ⁇ ON8 nucleic acids disclosed herein map to chromosome 2.
  • Four alternative novel ⁇ OV8 nucleic acids and polypeptides are disclosed herein, namely NON8a, ⁇ ON8b, ⁇ ON8c and ⁇ ON8d.
  • a NOV8 variant is NOV8a (alternatively referred to herein as CG55794-01), which encodes the 1196 nucleotide sequence (SEQ ID NO: 19) shown in Table 8 A.
  • SEQ ID NO: 19 1196 nucleotide sequence shown in Table 8 A.
  • An open reading frame for the mature protein was identified beginning with an ATG codon at nucleotides 16-18 and ending with a TAA codon at nucleotides 1138-1140. Putative untranslated regions, if any, downstream from the termination codon and upstream from the initiation codon are underlined. The start and stop codons are in bold letters.
  • the NOV8a protein (SEQ ID NO:20) encoded by SEQ ID NO: 19 is 374 amino acid residues in length and is presented using the one-letter amino acid code in Table 8B.
  • the SignalP, Psort and/or Hydropathy results indicate that NOV8a has a signal peptide and is likely to be localized extracellularly at the plasma membrane with a certainty of 0.9190.
  • a NON8a polypeptide is located to the lysosome (membrane) with a certainty of 0.2000, the microbody (peroxisome) with a certainty of 0.1292, or the endoplasmic reticulum (membrane) with a certainty of 0.1000.
  • the SignalP indicates a likely cleavage site for a ⁇ ON8a peptide is between positions 20 and 21, i.e., at the dash in the sequence GLG-YD.
  • Table 8B Encoded NOV8a Protein Sequence (SEQ ID NO:20)
  • a ⁇ ON8 variant is ⁇ ON8b (alternatively referred to herein as CG55794-03), which includes the 1222 nucleotide sequence (SEQ ID ⁇ O:21) shown in Table 8C.
  • SEQ ID ⁇ O:21 1222 nucleotide sequence shown in Table 8C.
  • An open reading frame for the mature protein was identified beginning with an ATG codon at nucleotides 41-43 and ending with a TAA codon at nucleotides 1163-1165. Putative untranslated regions, if any, downstream from the termination codon and upstream from the initiation codon are underlined. The start and stop codons are in bold letters.
  • the NON8b protein (SEQ ID ⁇ O:22) encoded by SEQ ID NO:21 is 347 amino acid residues in length and is presented using the one-letter amino acid code in Table 8D.
  • NON8b has a signal peptide and is likely to be localized extracellularly at the plasma membrane with a certainty of 0.9190.
  • a NON8b polypeptide is located to the lysosome (membrane) with a certainty of 0.2000, the microbody (peroxisome) with a certainty of 0.1345, or the endoplasmic reticulum (membrane) with a certainty of 0.1000.
  • the SignalP indicates a likely cleavage site for a ⁇ ON8b peptide is between positions 20 and 21, i.e., at the dash in the sequence GLG-YD.
  • aNOV8 variant is NON8c (alternatively referred to herein as CG55794-06), which includes the 977 nucleotide sequence (SEQ ID ⁇ O:23) shown in Table 8E.
  • SEQ ID ⁇ O:23 977 nucleotide sequence shown in Table 8E.
  • An open reading frame for the mature protein was identified beginning with an ATG codon at nucleotides 41-43 and ending with a TAG codon at nucleotides 671-673. Putative untranslated regions, if any, downstream from the termination codon and upstream from the initiation codon are underlined. The start and stop codons are in bold letters.
  • the NOV8c protein (SEQ ID NO:24) encoded by SEQ ID NO:23 is 210 amino acid residues in length and is presented using the one-letter amino acid code in Table 8F.
  • the SignalP, Psort and/or Hydropathy results indicate that NON ⁇ c has a signal peptide and is likely to be localized extracellularly at the plasma membrane with a certainty of 0.3700.
  • a ⁇ ON8c polypeptide is located to the microbody (peroxisome) with a certainty of 0.2242, the lysosome (lumen) with a certainty of 0.1900, or the endoplasmic reticulum (membrane) with a certainty of 0.1000.
  • the SignalP indicates a likely cleavage site for a ⁇ ON8c peptide is between positions 20 and 21, i.e., at the dash in the sequence GLG-YD.
  • NON8d a NON8 variant is ⁇ ON8d (alternatively referred to herein as CG55794-07), which includes the 1378 nucleotide sequence (SEQ ID ⁇ O:25) shown in Table 8G.
  • SEQ ID ⁇ O:25 the 1378 nucleotide sequence shown in Table 8G.
  • An open reading frame for the mature protein was identified beginning with an ATG codon at nucleotides 259-261 and ending with a TAA codon at nucleotides 1225-1227. Putative untranslated regions, if any, downstream from the termination codon and upstream from the initiation codon are underlined. The start and stop codons are in bold letters.
  • NOV8d has no known signal peptide and is likely to be localized in the cytoplasm at the endoplasmic reticulum (membrane) with a certainty of 0.8500.
  • a NON8d polypeptide is located to the microbody (peroxisome) with a certainty of 0.4781, the plasma membrane with a certainty of 0.4400, or the mitochondrial inner membrane with a certainty of 0.1000.
  • any reference to ⁇ ON8 is assumed to encompass all variants.
  • NOV8a nucleic acid sequence of this invention has 584 of 914 bases (63%) identical to a gb:GENBANK-ID:AF190274
  • the full amino acid sequence of the disclosed NON8 a protein of the invention has 151 of 325 amino acid residues (46%) identical to, and 219 of 325 amino acid residues (67%) similar to, the 416 amino acid residue ⁇ tnr:SPTREMBL-ACC:Q9PUF2 protein from Bothrops jararaca (Jararaca) (CARBOXYPEPTIDASE HOMOLOG).
  • ⁇ ON8b nucleic acid sequence of this invention has 586 of 914 bases (64%) identical to a gb:GE ⁇ BA ⁇ K-ID:AF190274
  • the full amino acid sequence of the disclosed NOV8b protein of the invention has 152 of 325 amino acid residues (46%) identical to, and 219 of 325 amino acid residues (67%) similar to, the 416 amino acid residue ⁇ tnr:SPTREMBL-ACC:Q9PUF2 protein from Bothrops jararaca (Jararaca) (CARBOXYPEPTIDASE HOMOLOG).
  • NON8c nucleic acid sequence of this invention has 621 of 672 bases (92%) identical to a gb:GE ⁇ BA ⁇ K-ID:AX083139
  • the full amino acid sequence of the disclosed NON8c protein of the invention has 83 of 176 amino acid residues (47%) identical to, and 122 of 176 amino acid residues (69%) similar to, the 422 amino acid residue ptnr:SPTREMBL-ACC:Q9EQN9 protein from Rattus norvegicus (Rat) (PRE-PROCARBOXYPEPTIDASE R).
  • the ⁇ ON8d nucleic acid sequence of this invention has 1073 of 1077 bases (99%) identical to a gb:GE ⁇ BA ⁇ K-ID:AX083139
  • the full amino acid sequence of the disclosed NOV8d protein of the invention has 142 of 283 amino acid residues (50%) identical to, and 199 of 283 amino acid residues (70%) similar to, the 416 amino acid residue ⁇ tnr:SPTREMBL-ACC:Q9PUF2 protein from Bothrops jararaca (Jararaca) (CARBOXYPEPTIDASE HOMOLOG). Additional BLAST results are shown in Table 8 J.
  • Zn_carbOpept domain 1 of 1 , from 50 to 332 384.8 8.7e-112
  • NOV8 contains a zinc carboxypeptidase domain as illustrated in Table 8L.
  • NON8 nucleic acids, and the encoded polypeptides, according to the invention are useful in a variety of applications and contexts.
  • ⁇ ON8 nucleic acids and polypeptides can be used to identify proteins that are members of the carboxypeptidase family of proteins.
  • the ⁇ ON8 nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance ⁇ ON8 activity or function.
  • the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., digestion or hydrolysis of polypeptide chains. These molecules can be used to treat, , e.g., pancreatitis, ulcers, inflammatory bowel disease, diverticular disease, Crohn's disease, appendicitis, or obesity.
  • ⁇ ON8 nucleic acids and polypeptides according to the invention are useful, ter alia, as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins.
  • the ⁇ ON8 nucleic acids and their encoded polypeptides include structural motifs that are characteristic of proteins belonging to the carboxypeptidase family.
  • Carboxypeptidase B, (CPB) like carboxypeptidase A is a pancreatic exopeptidase. Unlike carboxypeptidase A, however, carboxypeptidase B catalyzes the hydrolysis of the peptide bonds involving basic amino acids lysine, arginine and ornithine. This hydrolysis occurs at the C-terminal bond in these polypeptides.
  • the ⁇ ON8 nucleic acids and polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in the mediation of hydrolysis.
  • the ⁇ ON8 nucleic acids and polypeptides, antibodies and related compounds according to the invention maybe used to treat digestive disorders, e.g., xerostomia, hypercalceimia, ulcers, Non Hippel-Lindau (NHL) syndrome, cirrhosis, transplantation, inflammatory bowel disease, diverticular disease, hirschsprung's disease, crohn's disease, appendicitis, stroke, tuberous sclerosis, anxiety, pain, endocrine dysfunctions, nueroprotection, diabetes, obesity, growth and reproductive disorders, myasthenia gravis.
  • digestive disorders e.g., xerostomia, hypercalceimia, ulcers, Non Hippel-Lindau (NHL) syndrome, cirrhosis, transplantation, inflammatory bowel disease, diverticular disease, hirschsprung's disease
  • the NON8 nucleic acids and polypeptides are useful for detecting specific cell types. For example, expression analysis has demonstrated that a ⁇ ON8 nucleic acid is expressed in pooled mammalian tissue, small intestine, and spinal cord.
  • the disclosed ⁇ ON9 nucleic acid (alternatively referred to herein as CG56463-01) encodes a novel neurotransmitter receptor-like protein and includes the 1142 nucleotide sequence (SEQ ID ⁇ O:27) shown in Table 9A.
  • the NON9 nucleic acid disclosed herein maps to chromosome 6p23.
  • NON9 has a signal peptide and is likely to be localized in the plasma membrane with a certainty of 0.6000.
  • a ⁇ ON9 polypeptide is located to the Golgi body with a certainty of 0.4000, the endoplasmic reticulum (membrane) with a certainty of 0.3000, or the mitochondrial inner membrane with a certainty of 0.0300.
  • the SignalP indicates a likely cleavage site for a ⁇ ON9 polypeptide is between positions 47 and 48, i.e., at the dash in the sequence MIA-IL.
  • the ⁇ ON9 nucleic acid sequence of this invention has 601 of 1000 bases (60%) identical to a gb:GE ⁇ BA ⁇ K-ID:AR035954
  • the NOV9 protein of the invention also has homolgy to the proteins shown in the BLASTP data in Table 9D.
  • NOV9 contains 7-transmembrane domains as illustrated in Table 9F.
  • NOV9 nucleic acid, and the encoded polypeptide, according to the invention are useful in a variety of applications and contexts.
  • NOV9 nucleic acids and polypeptides can be used to identify proteins that are members of the neurotransmitter receptor family of proteins.
  • the NOV9 nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance NOV9 activity or function.
  • the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., cellular recognition, or G-protein-mediated transduction of neuromuscular/synaptic signals. These molecules can be used to treat, e.g., neurological disorders, immune diseases, or signal transduction pathways.
  • the NON9 nucleic acid and polypeptide according to the invention are useful, inter alia, as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins.
  • the ⁇ ON9 nucleic acid and polypeptide include structural motifs that are characteristic of proteins belonging to the family of neurotransmitter receptor proteins.
  • Nerve cells are highly specialized for cell-to-cell communication.
  • a number of small molecules called neurofransmitters act as the actual signals, released from one nerve cell only to dock on another cell.
  • neurotransmitter molecule When a neurotransmitter molecule fits into a receptor, it typically opens the gate, allowing ions to travel through the cell's membrane. The ions, in turn, excite the cell. If the receiving cell is a nerve cell, this excitation can lead to release of its own neurofransmitters.
  • the vast majority of neurotransmitter receptors belong to a class of proteins known as the se ⁇ entine receptors. This class exhibits a characteristic transmembrane structure: spaning the cell membrane, seven times.
  • neurotransmitter receptors are subject to ligand-induced desensitization: such that they can become unresponsive upon prolonged exposure to their neurotransmitter.
  • the NOV9 nucleic acid and polypeptide, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in the mediation of signal transduction.
  • the NON9 nucleic acid and polypeptide, antibodies and related compounds according to the invention maybe used to treat, e.g., leukemia, acute nonlymphocytic, spinocerebellar ataxia-1, or neurological disorders.
  • the ⁇ ON9 nucleic acid and polypeptide are useful for detecting specific cell types. For example, expression analysis has demonstrated that a ⁇ ON9 nucleic acid is predominantly expressed in skeletal muscle and selected areas of the brain. Additional utilities for the NON9 nucleic acid and polypeptide according to the invention are disclosed herein.
  • the disclosed ⁇ ON10 nucleic acid (alternatively referred to herein as CG56321-01) encodes a novel proto-oncogene MAF-like protein and includes the 1189 nucleotide sequence (SEQ ID ⁇ O:29) shown in Table 10A.
  • the NON10 nucleic acid disclosed herein maps to chromosome 8.
  • the NOV10 protein (SEQ ID NO:30) encoded by SEQ ID NO:29 is 353 amino acid residues in length and is presented using the one-letter amino acid code in Table 10B.
  • the SignalP, Psort and/or Hydropathy results indicate that NOV10 has no known signal peptide and is likely to be localized in the nucleus with a certainty of 0.7600.
  • a NONIO polypeptide is located to the microbody (peroxisome) with a certainty of 0.5418, the lysosome (lumen) with a certainty of 0.1882, or the mitochondrial matrix space with a certainty of 0.1000.
  • Table 10B Encoded NOV10 Protein Sequence (SEQ ID NO:30)
  • ⁇ ON10 nucleic acid sequence of this invention has 258 of 322 bases (80%) identical to a gb:GE ⁇ BA ⁇ K-ID:AF034693
  • the full amino acid sequence of the disclosed NONIO protein of the invention has 134 of 249 amino acid residues (53%) identical to, and 151 of 249 amino acid residues (60%) similar to, the 311 amino acid residue ptnr:SPTREMBL-ACC:Q90370 protein from Coturnix coturnix japonica (Japanese quail) (MAFB PROTEIN).
  • the NOVl 0 protein of the invention also has homolgy to the proteins shown in the
  • NONIO contains MAF domains as illustrated in Table 10F.
  • ⁇ ON10 nucleic acid, and the encoded polypeptide, according to the invention are useful in a variety of applications and contexts.
  • ⁇ ON10 nucleic acids and polypeptides can be used to identify proteins that are members of the bZip transcription factor family of proteins.
  • the ⁇ ON10 nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance ⁇ ON10 activity or function.
  • the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., gene regulation/expression. These molecules can be used to treat, e.g., autoimmune disorders or antioxidant induction of molecules such as ⁇ QO1, GST,
  • the ⁇ ON10 nucleic acid and polypeptide according to the invention are useful, ter alia, as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins.
  • the ⁇ ON10 nucleic acid and polypeptide include structural motifs that are characteristic of proteins belonging to the family of bZip transcription factor proteins.
  • Transcription factors are proteins that bind to the enhancer or promoter regions and interact such that transcription occurs from only a small group of promoters in any cell. Most transcription factors can bind to specific D ⁇ A sequences, and these trans- regulatory proteins can be grouped together in families based on similarities in structure.
  • transcription factors contain a domain involved in activating the transcription of the gene whose promoter or enhancer it has bound. Usually, this trans-activating domain enables that transcription factor to interact with proteins involved in binding RNA polymerase. This interaction often enhances the efficiency with which the basal transcriptional complex can be built and bind RNA polymerase II.
  • transcription factors There are several families of transcription factors.
  • the bZip transcription factor family of proteins are dimers, each of whose subunits contains a basic DNA-binding domain at the carboxyl end, followed closely by an a helix containing several leucine residues. These leucines are placed in the helix such that they interact with similarly spaced leucine residues on other bZip proteins to form a "leucine zipper" between them, causing dimers to form. This domain is followed by a regulatory domain that can interact with the promoter to stimulate or repress transcription.
  • the NONIO nucleic acid and polypeptide, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in the mediation of gene transcription.
  • antibodies and related compounds according to the invention may be used to treat, e.g., anemia, ataxia-telangiectasia, autoimmume disease, cancer, immunodeficiencies, hemophilia, hypercoagulation, idiopathic thrombocytopenic purpura, allergies, transplantation, graft versus host disease (GNHD), lymphaedema, systemic lupus erythematosus, asthma, emphysema, scleroderma, ARDS, diabetes, renal artery stenosis, interstitial nephritis, glomerulonephritis, polycystic kidney disease, systemic lupus erythematosus, renal tubular acidosis, IgA nephropathy, hypercalceimia, or Lesch- ⁇ yhan syndrome.
  • the ⁇ ON10 nucleic acid and polypeptide are useful for detecting specific cell types. For example, anemia, ataxia-telangiec
  • the NOVl 1 proteins descibed herein are novel lysyl oxidase-like proteins.
  • the NOVl 1 nucleic acids disclosed herein map to chromosome 10.
  • Two alternative novel NOVl 1 nucleic acids and polypeptides are disclosed herein, namely NON1 la and ⁇ ON1 lb.
  • a ⁇ ON11 variant is ⁇ ON1 la (alternatively referred to herein as CG56381-01), which encodes the 2599 nucleotide sequence (SEQ ID ⁇ O:31) shown in Table 1 IA.
  • An open reading frame for the mature protein was identified beginning with an ATG codon at nucleotides 78-80 and ending with a TGA codon at nucleotides 2568-2570. Putative untranslated regions, if any, downstream from the termination codon and upstream from the imtiation codon are underlined. The start and stop codons are in bold letters.
  • the NONl la protein (SEQ ID ⁇ O:32) encoded by SEQ ID NO:31 is 830 amino acid residues in length and is presented using the one-letter amino acid code in Table 1 IB.
  • the SignalP, Psort and/or Hydropathy results indicate that NOVl la has a signal peptide and is likely to be localized in the lysosome (lumen) with a certainty of 0.4247.
  • a NOVl la polypeptide is located extracellularly with a certainty of 0.3700, the microbody (peroxisome) with a certainty of 0.1250, or the endoplasmic reticulum (membrane) with a certainty of 0.1000.
  • the SignalP indicates a likely cleavage site for a NOVl la peptide between positions 24 and 25, i.e., at the dash in the sequence SRP-QS.
  • a ⁇ ON11 variant is ⁇ ON1 lb (alternatively referred to herein as CG56381- 02), which includes the 2592 nucleotide sequence (SEQ ID ⁇ O:33) shown in Table 1 IC.
  • SEQ ID ⁇ O:33 2592 nucleotide sequence shown in Table 1 IC.
  • An open reading frame for the mature protein was identified beginning with an ATG codon at nucleotides 46-48 and ending with a TGA codon at nucleotides 2314-2316. Putative untranslated regions, if any, downstream from the termination codon and upstream from the initiation codon are underlined. The start and stop codons are in bold letters.
  • the NONl lb protein (SEQ ID ⁇ O:34) encoded by SEQ ID NO:33 is 756 amino acid residues in length and is presented using the one-letter amino acid code in Table 1 ID.
  • the SignalP, Psort and/or Hydropathy results indicate that NOVl lb has a signal peptide and is likely to be localized in the lysosome (lumen) with a certainty of 0.4302.
  • a NOVl lb polypeptide is located extracellularly with a certainty of 0.3700, the microbody (peroxisome) with a certainty of 0.1403, or the endoplasmic reticulum (membrane) with a certainty of 0.1000.
  • the SignalP indicates a likely cleavgae site for a NONl lb peptide between positions 24 and 25, i.e., at the dash in the sequence SRP-QS.
  • any reference to ⁇ OV11 is assumed to encompass all variants.
  • a search against the Patp database a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table HE.
  • NOVl la nucleic acid sequence of this invention has 1075 of 1078 bases (99%) identical to a gb:GENBANK-ID:AK025542
  • the full amino acid sequence of the disclosed NONl la protein of the invention has 404 of 705 amino acid residues (57%) identical to, and 523 of 705 amino acid residues (74%) similar to, the 774 amino acid residue ⁇ tnr:SPTREMBL-ACC:Q9Y4K0 protein from Human (LYSYL OXIDASE-RELATED PROTEIN).
  • NOVl lb nucleic acid sequence of this invention has 1070 of 1076 bases (99%) identical to a gb:GENBANK-ID:AK025542
  • the full amino acid sequence of the disclosed NONl lb protein of the invention has 360 of 616 amino acid residues (58%) identical to, and 456 of 616 amino acid residues (74%) similar to, the 895 amino acid residue ptnr:SPTREMBL-ACC:Q9W6Nl protein from Perca flavescens (Yellow perch) (LYSYL OXIDASE RELATED PROTEIN HOMOLOG). Additional BLAST results are shown in Table 1 IF.
  • NONl 1 contains a lysyl oxidase domain as illustrated in Table 1 IH.
  • ⁇ ON11 nucleic acids, and the encoded polypeptides, according to the invention are useful in a variety of applications and contexts.
  • ⁇ ON11 nucleic acids and polypeptides can be used to identify proteins that are members of the copper-dependent amine oxidase family of proteins.
  • the ⁇ ON11 nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance ⁇ ON11 activity or function.
  • the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., crosslinking of extracellular matrix proteins. These molecules can be used to treat, e.g., autoimmune disease, allergies, immunodeficiencies, asthma, psoriasis, acne, or pigmentation disorders.
  • ⁇ ON11 nucleic acids and polypeptides according to the invention are useful, ter alia, as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins.
  • the ⁇ ON11 nucleic acids and their encoded polypeptides include structural motifs that are characteristic of proteins belonging to the lysyl oxidase family.
  • Lysyl oxidase (LOX) is a secreted enzyme that cross-links collagen and elastin, and thus is critical for the integrity of the extracellular matrix, the breakdown of which contributes to cancer invasion and metastasis.
  • LOX is also important to the health of connective tissues and arteries.
  • Lysyl oxidase requires a copper co-factor and therefore its activity can be lowered by a dietary deficiency.
  • the NONl 1 nucleic acids and polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in the mediation of crosslinking and biogenesis of connective tissue matrices.
  • ⁇ ON11 nucleic acids and polypeptides antibodies and related compounds according to the invention may be used to treat digestive disorders, e.g., diabetes, Non Hippel-Lindau (NHL) syndrome, pancreatitis, obesity, endometriosis, fertility, hemophilia, hypercoagulation, idiopathic thrombocytopenic purpura, autoimmume disease, allergies, immunodeficiencies, transplantation, graft versus host disease (GNHD), lymphaedema, osteoporosis, hypercalceimia, arthritis, ankylosing spondylitis, scoliosis, systemic lupus erythematosus, asthma, emphysema, scleroderma, allergy, ARDS, renal artery stenosis, interstitial nephritis, glomerulonephritis, polycystic kidney disease, systemic lupus erythematosus, renal tubular acid
  • the ⁇ ON11 nucleic acids and polypeptides are useful for detecting specific cell types. For example, expression analysis has demonstrated that a ⁇ ON11 nucleic acid is expressed in kidney, lung, lymphoid tissue, mammary gland/breast, ovary, pancreas, testis, uterus, and bone.
  • the ⁇ ON12 proteins descibed herein are novel phosphatase-like proteins.
  • the ⁇ ON12 nucleic acids disclosed herein map to chromosome 17.
  • Two alternative novel ⁇ ON12 nucleic acids and polypeptides are disclosed herein, namely ⁇ ON12a and ⁇ ON12b.
  • ⁇ ON12a A ⁇ ON12 variant is ⁇ ON12a (alternatively referred to herein as CG56436-01), which encodes the 1002 nucleotide sequence (SEQ ID ⁇ O:35) shown in Table 12A.
  • SEQ ID ⁇ O:35 1002 nucleotide sequence
  • the NOV12a protein (SEQ TD NO:36) encoded by SEQ ID NO:35 is 267 amino acid residues in length and is presented using the one-letter amino acid code in Table 12B.
  • the SignalP, Psort and/or Hydropathy results indicate that NON 12a has no known signal peptide and is likely to be localized in the mitochondrial matrix space with a certainty of 0.4728.
  • a ⁇ ON12a polypeptide is located in the microbody (peroxisome) with a certainty of 0.2224, the lysosome (lumen) with a certainty of 0.1905, or the mitochondrial inner membrane with a certainty of 0.1762.
  • NOV12b (alternatively referred to herein as CG56436- 02), which includes the 903 nucleotide sequence (SEQ ID NO:37) shown in Table 12C.
  • SEQ ID NO:37 the 903 nucleotide sequence shown in Table 12C.
  • An open reading frame for the mature protein was identified beginning with an ATG codon at nucleotides 4-6 and ending with a TGA codon at nucleotides 805-807. Putative untranslated regions, if any, downstream from the termination codon and upstream from the initiation codon are underlined. The start and stop codons are in bold letters.
  • Table 12C NON12b Nucleotide Sequence (SEQ ID NO:37)
  • the NOV12b protem (SEQ ID NO:38) encoded by SEQ ID NO:37 is 267 amino acid residues in length and is presented using the one-letter amino acid code in Table 12D.
  • the SignalP, Psort and/or Hydropathy results predict that NOV12b has no known signal peptide and is likely to be localized in the mitochondrial matrix space with a certainty of 0.4728.
  • a NON 12b polypeptide is located in the microbody (peroxisome) with a certainty of 0.2224, the lysosome (lumen) with a certainty of 0.1905, or the mitochondrial inner membrane with a certainty of 0.1762.
  • NOV12a or NOV12b any reference to NOV12 is assumed to encompass all variants.
  • a search against the Patp database a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 12E.
  • NOVl 2a nucleic acid sequence of this invention has 609 of 916 bases (66%) identical to a gb:GENBANK-ID:GGA6529
  • the full amino acid sequence of the disclosed NON12a protein of the invention has 159 of 265 amino acid residues (60%) identical to, and 199 of 265 amino acid residues (75%) similar to, the 268 amino acid residue ⁇ tnr:SPTREMBL-ACC:O73884 protein from Gallus gallus (Chicken) (PUTATIVE PHOSPHATASE).
  • the ⁇ ON12b nucleic acid sequence of this invention has 579 of 865 bases (66%) identical to a gb:GE ⁇ BA ⁇ K-ID:GGA6529
  • the full amino acid sequence of the disclosed NON12b protein of the invention has 159 of 265 amino acid residues (60%) identical to, and 199 of 265 amino acid residues (75%) similar to, the 268 amino acid residue ptnr:SPTREMBL-ACC:O73884 protein from Gallus gallus (Chicken) (PUTATIVE PHOSPHATASE).
  • NOVl2a 267 NOV12b 267 073884 268 Q9D9M5 241 Q9V F0 RASAVAGPTKSPN 306 Q9SU92 245 Q9FZ62 EPIQVPLNLVK- - 279
  • Hyd. nD. apalaaAGvgvamgngg (SEQ ID NO: 198)
  • NOV12 contains a hydrolase domain as illustrated in Table 12H.
  • NON12 nucleic acids, and the encoded polypeptides, according to the invention are useful in a variety of applications and contexts.
  • ⁇ ON12 nucleic acids and polypeptides can be used to identify proteins that are members of the protein phosphatase family of proteins.
  • the ⁇ ON12 nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance ⁇ ON12 activity or function.
  • the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., biological processes that control cell growth and homeostasis.
  • these molecules can be used to treat, e.g., hyper/hypothyroidism, endometriosis, fertility, transplantation, hypogonadism, Alzheimer's disease, Parkinson's disease, neurodegeneration, or grwoth disorders.
  • various ⁇ OV12 nucleic acids and polypeptides according to the invention are useful, ter alia, as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins.
  • the NON 12 nucleic acids and their encoded polypeptides include structural motifs that are characteristic of proteins belonging to the protein phosphatase family. The major protein phosphatases in all cells are highly conserved and widely distributed.
  • NON12 nucleic acids and polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in the mediation of cell signaling/signal transduction.
  • antibodies and related compounds according to the invention may be used to treat, e.g., hyperthyroidism, hypothyroidism, endometriosis, fertility, Non Hippel-Lindau (NHL) syndrome, cirrhosis, transplantation, hypogonadism, Alzheimer's disease, stroke, tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, cerebral palsy, epilepsy, Lesch- ⁇ yhan syndrome, multiple sclerosis, ataxia-telangiectasia, leukodystrophies, behavioral disorders, addiction, anxiety, pain, neurodegeneration, endocrine dysfunctions, diabetes, obesity, growth and reproductive disorders, systemic lupus erythematosus, autoimmune disease, asthma, emphysema, scleroderma, allergy, ARDS, renal artery stenosis, interstitial nephritis, glomeruloneph
  • the ⁇ ON12 nucleic acids and polypeptides are useful for detecting specific cell types. For example, expression analysis has demonstrated that a ⁇ ON12 nucleic acid is expressed in bone marrow, brain, kidney, liver, lung, lung pleura, pituitary gland, placenta, and thyroid.
  • the disclosed ⁇ ON13 nucleic acid (alternatively referred to herein as CG56441-01) encodes a novel chloride channel protein CLC-KA-like protein and includes the 1991 nucleotide sequence (SEQ ID ⁇ O:39) shown in Table 13A.
  • the NON13 protein (SEQ ID ⁇ O:40) encoded by SEQ ID NO:39 is 655 amino acid residues in length and is presented using the one-letter amino acid code in Table 13B.
  • the SignalP, Psort and/or Hydropathy results indicate that NOVl 3 has a signal peptide and is likely to be localized at the plasma membrane with a certainty of 0.6000.
  • a NON13 polypeptide is located to the Golgi body with a certainty of 0.4000, the endoplasmic reticulum (membrane) with a certainty of 0.1882, or the microbody (peroxisome) with a certainty of 0.3000.
  • the SignalP indicates a likely cleavage site for a ⁇ ON13 peptide between positions 66 and 67, i.e., at the dash in the sequence SYA-M ⁇ .
  • NON 13 nucleic acid sequence of this invention has 1768 of 1779 bases (99%) identical to a gb:GE ⁇ BA ⁇ K-TD:HSCLCHPRA
  • the full amino acid sequence of the disclosed NON13 protein of the invention has 578 of 579 amino acid residues (99%) identical to, and 578 of 579 amino acid residues (99%) similar to, the 687 amino acid residue ⁇ tnr:SWISSPROT-ACC:P51800 protein from Human (CHLORIDE CHANNEL PROTEIN CLC-KA (CLC-K1)).
  • the NONl 3 protein of the invention also has homolgy to the proteins shown in the BLASTP data in Table 13D.
  • NON 13 contains CLC domains as illustrated in Table 13F.
  • ⁇ ON13 nucleic acid, and the encoded polypeptide, according to the invention are useful in a variety of applications and contexts.
  • ⁇ ON13 nucleic acids and polypeptides can be used to identify proteins that are members of the voltage-gated chloride chaimel family of proteins.
  • the ⁇ ON13 nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance ⁇ ON13 activity or function.
  • the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., physiological functions such as cell volume regulation, membrane potential stabilization, signal transduction, or transepithelial transport. These molecules can be used to treat, e.g., diseases associated with the kidney such as renal artery stenosis, diabetes, or renal tubular acidosis.
  • the ⁇ ON13 nucleic acid and polypeptide according to the invention are useful, inter alia, as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins.
  • the ⁇ ON13 nucleic acid and polypeptide include structural motifs that are characteristic of proteins belonging to the family of voltage-gated chloride channel proteins. All functionally characterized members of the CLC family transport chloride, some in a voltage-regulated process. These channels serve a variety of physiological functions such as cell volume regulation, membrane potential stabilization, signal transduction, and transepithelial transport.
  • the NONl 3 nucleic acid and polypeptide, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in the mediation of kidney diseases.
  • antibodies and related compounds according to the invention may be used to treat, e.g., diabetes, autoimmune disease, renal artery stenosis, interstitial nephritis, glomerulonephritis, polycystic kidney disease, systemic lupus erythematosus, renal tubular acidosis, IgA nephropathy, hypercalceimia, or Lesch- ⁇ yhan syndrome.
  • the ⁇ ON13 nucleic acid and polypeptide are useful for detecting specific cell types. For example, expression analysis has demonstrated that a ⁇ ON13 nucleic acid is expressed in the kidney.
  • NOV14 nucleic acid (alternatively referred to herein as CG56443-01) encodes a novel mast cell function-associated antigen (MAFA)-like protein and includes the 645 nucleotide sequence (SEQ ID NO:41) shown in Table 14A.
  • the NON14 nucleic acid disclosed herein maps to chromosome 19.
  • NOV14 has a signal peptide and is likely to be localized at the plasma membrane with a certainty of 0.7900.
  • a NOV14 polypeptide is located to the microbody (peroxisome) with a certainty of 0.5804, the Golgi body with a certainty of 0.3000, or the endoplasmic reticulum (membrane) with a certainty of 0.2000.
  • the SignalP indicates a likely cleavage site for a NON14 peptide between positions 57 and 58, i.e:, at the dash in the sequence SLA-LS.

Abstract

Disclosed are novel polypeptides and nucleic acids encoding same. Also disclosed are vectors, host cells, antibodies and recombinant methods for producing the polypeptides and polynucleotides, as well as methods for using same.

Description

NOVEL POLYPEPTIDES AND NUCLEIC ACIDS ENCODED THEREBY
BACKGROUND OF THE INVENTION
The invention relates to polynucleotides and the polypeptides encoded by such polynucleotides, as well as vectors, host cells, antibodies and recombinant methods for producing the polypeptides and polynucleotides, as well as methods for using the same.
SUMMARY OF THE INVENTION
The invention is based in part upon the discovery of nucleic acid sequences encoding novel polypeptides. The novel nucleic acids and polypeptides are referred to herein as NONX, or ΝOV1, ΝON2, ΝON3, ΝON4, ΝOV5, NON6, ΝON7, ΝOV8, NON9, ΝON10, ΝON11, ΝON12, ΝON13, ΝON14, ΝON15, ΝON16, ΝON17, ΝON18, ΝOV19, NOV20, NOV21, NOV22, NOV23, NQN24, ΝON25, ΝON26, ΝON27, ΝON28, ΝOV29, NON30, ΝOV31, NOV32, and NOV33 nucleic acids and polypeptides. These nucleic acids and polypeptides, as well as derivatives, homologs, analogs and fragments thereof, will hereinafter be collectively designated as "NONX" nucleic acid or polypeptide sequences.
In one aspect, the invention provides an isolated ΝONX nucleic acid molecule encoding a ΝONX polypeptide that includes a nucleic acid sequence that has identity to the nucleic acids disclosed in SEQ ID ΝOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, and 122. In some embodiments, the ΝONX nucleic acid molecule will hybridize under stringent conditions to a nucleic acid sequence complementary to a nucleic acid molecule that includes a protein-coding sequence of a ΝOVX nucleic acid sequence. The invention also includes an isolated nucleic acid that encodes a ΝONX polypeptide, or a fragment, homolog, analog or derivative thereof. For example, the nucleic acid can encode a polypeptide at least 80% identical to a polypeptide comprising the amino acid sequences of SEQ ID ΝOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, and 123. The nucleic acid can be, for example, a genomic DNA fragment or a cDNA molecule that includes the nucleic acid sequence of any of SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, and 122. Also included in the invention is an oligonucleotide, e.g., an oligonucleotide which includes at least 6 contiguous nucleotides of a NONX nucleic acid (e.g., SEQ ID ΝOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59,
61, 63, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, and 122) or a complement of said oligonucleotide. Also included in the invention are substantially purified NONX polypeptides (SEQ ID ΝOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60,
62, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109,
111, 113, 115, 117, 119, 121, and 123). hi certain embodiments, the NONX polypeptides include an amino acid sequence that is substantially identical to the amino acid sequence of a human ΝONX polypeptide.
The invention also features antibodies that immunoselectively bind to ΝOVX polypeptides, or fragments, homologs, analogs or derivatives thereof.
In another aspect, the invention includes pharmaceutical compositions that include therapeutically- or prophylactically-effective amounts of a therapeutic and a pharmaceutically- acceptable carrier. The therapeutic can be, e.g., a ΝONX nucleic acid, a ΝONX polypeptide, or an antibody specific for a ΝONX polypeptide. In a further aspect, the invention includes, in one or more containers, a therapeutically- or prophylactically-effective amount of this pharmaceutical composition. hi a further aspect, the invention includes a method of producing a polypeptide by culturing a cell that includes a ΝONX nucleic acid, under conditions allowing for expression of the ΝONX polypeptide encoded by the DΝA. If desired, the ΝONX polypeptide can then be recovered.
In another aspect, the invention includes a method of detecting the presence of a ΝONX polypeptide in a sample. In the method, a sample is contacted with a compound that selectively binds to the polypeptide under conditions allowing for formation of a complex between the polypeptide and the compound. The complex is detected, if present, thereby identifying the NONX polypeptide within the sample.
The invention also includes methods to identify specific cell or tissue types based on their expression of a ΝONX. Also included in the invention is a method of detecting the presence of a ΝOVX nucleic acid molecule in a sample by contacting the sample with a ΝONX nucleic acid probe or primer, and detecting whether the nucleic acid probe or primer bound to a ΝONX nucleic acid molecule in the sample.
In a further aspect, the invention provides a method for modulating the activity of a ΝONX polypeptide by contacting a cell sample that includes the ΝONX polypeptide with a compound that binds to the ΝONX polypeptide in an amount sufficient to modulate the activity of said polypeptide. The compound can be, e.g., a small molecule, such as a nucleic acid, peptide, polypeptide, peptidomimetic, carbohydrate, lipid or other organic (carbon containing) or inorganic molecule, as further described herein. Also within the scope of the invention is the use of a therapeutic in the manufacture of a medicament for treating or preventing disorders or syndromes including, e.g., trauma, regeneration (in vitro and in vivo), viral/bacterial/parasitic infections, Non Hippel-Lindau (NHL) syndrome, Alzheimer's disease, stroke, tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, cerebral palsy, epilepsy, Lesch-Νyhan syndrome, multiple sclerosis, ataxia- telangiectasia, leukodystrophies, behavioral disorders, addiction, anxiety, pain, actinic keratosis, acne, hair growth diseases, allopecia, pigmentation disorders, endocrine disorders, connective tissue disorders, such as severe neonatal Marfan syndrome, dominant ectopia lentis, familial ascending aortic aneurysm, inflammatory disorders such as osteo- and rheumatoid-arthritis, inflammatory bowel disease, Crohn's disease, immunological disorders, AIDS, cancers including but not limited to lung cancer, colon cancer, neoplasm, adenocarcinoma, lymphoma, prostate cancer, uterus cancer, leukemia or pancreatic cancer, blood disorders, asthma, psoriasis, vascular disorders, hypertension, skin disorders, renal disorders including Alport syndrome, immunological disorders, tissue injury, fibrosis disorders, bone diseases, osteogenesis imperfecta, Neurologic diseases, brain and/or autoimmune disorders like encephalomyelitis, neurodegenerative disorders, immune disorders, hematopoietic disorders, muscle disorders, inflammation and wound repair, bacterial, fungal, protozoal and viral infections (particularly infections caused by HIN-1 or HIN-2), pain, acute heart failure, hypotension, hypertension, urinary retention, osteoporosis, angina pectoris, myocardial infarction, ulcers, benign prostatic hypertrophy, arthrogryposis multiplex congenita, keratoconus, scoliosis, pancreatitis, obesity systemic lupus erythematosus, emphysema, scleroderma, allergy, ards, neuroprotection, fertility myasthenia gravis, diabetes, obesity, growth and reproductive disorders, hemophilia, hypercoagulation, immunodeficiencies, graft vesus host, congenital adrenal hyperplasia, endometriosis, xerostomia, ulcers, cirrhosis, transplantation, diverticular disease, hirschsprung's disease, appendicitis, tendinitis, renal artery stenosis, interstitial nephritis, glomerulonephritis, polycystic kidney disease, erythematosus, renal tubular acidosis, IgA nephropathy, anorexia, bulimia, psychotic disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation and dyskinesias, such as Huntington's disease and/or other pathologies and disorders of the like.
The therapeutic can be, e.g., a ΝONX nucleic acid, a ΝONX polypeptide, or a ΝONX- specific antibody, or biologically-active derivatives or fragments thereof. For example, the compositions of the present invention will have efficacy for treatment of patients suffering from the diseases and disorders disclosed above and/or other pathologies and disorders of the like. The polypeptides can be used as immunogens to produce antibodies specific for the invention, and as vaccines. They can also be used to screen for potential agonist and antagonist compounds. For example, a cDΝA encoding ΝONX may be useful in gene therapy, and ΝONX may be useful when administered to a subject in need thereof. By way of non- limiting example, the compositions of the present invention will have efficacy for treatment of patients suffering from the diseases and disorders disclosed above and/or other pathologies and disorders of the like.
The invention further includes ι a method for screening for a modulator of disorders or syndromes including, e.g., the diseases and disorders disclosed above and/or other pathologies and disorders of the like. The method includes contacting a test compound with a ΝONX polypeptide and determining if the test compound binds to said ΝONX polypeptide. Binding of the test compound to the ΝONX polypeptide indicates the test compound is a modulator of activity, or of latency or predisposition to the aforementioned disorders or syndromes. Also within the scope of the invention is a method for screening for a modulator of activity, or of latency or predisposition to disorders or syndromes including, e.g., the diseases and disorders disclosed above and/or other pathologies and disorders of the like by administering a test compound to a test animal at increased risk for the aforementioned disorders or syndromes. The test animal expresses a recombinant polypeptide encoded by a NONX nucleic acid. Expression or activity of ΝONX polypeptide is then measured in the test animal, as is expression or activity of the protein in a control animal which recombinantly-expresses ΝONX polypeptide and is not at increased risk for the disorder or syndrome. Next, the expression of NONX polypeptide in both the test animal and the control animal is compared. A change in the activity of ΝONX polypeptide in the test animal relative to the control animal indicates the test compound is a modulator of latency of the disorder or syndrome. In yet another aspect, the invention includes a method for determining the presence of or predisposition to a disease associated with altered levels of a ΝONX polypeptide, a ΝONX nucleic acid, or both, in a subject (e.g., a human subject). The method includes measuring the amount of the ΝONX polypeptide in a test sample from the subject and comparing the amount of the polypeptide in the test sample to the amount of the ΝONX polypeptide present in a control sample. An alteration in the level of the ΝOVX polypeptide in the test sample as compared to the control sample indicates the presence of or predisposition to a disease in the subject. Preferably, the predisposition includes, e.g., the diseases and disorders disclosed above and/or other pathologies and disorders of the like. Also, the expression levels of the new polypeptides of the invention can be used in a method to screen for various cancers as well as to determine the stage of cancers.
In a further aspect, the invention includes a method of treating or preventing a pathological condition associated with a disorder in a mammal by administering to the subject a ΝONX polypeptide, a ΝONX nucleic acid, or a ΝOVX-specific antibody to a subject (e.g., a human subject), in an amount sufficient to alleviate or prevent the pathological condition, h preferred embodiments, the disorder, includes, e.g., the diseases and disorders disclosed above and/or other pathologies and disorders of the like.
In yet another aspect, the invention can be used in a method to identity the cellular receptors and downstream effectors of the invention by any one of a number of techniques commonly employed in the art. These include but are not limited to the two-hybrid system, affinity purification, co-precipitation with antibodies or other specific-interacting molecules. NONX nucleic acids and polypeptides are further useful in the generation of antibodies that bind immuno-specifically to the novel ΝONX substances for use in therapeutic or diagnostic methods. These ΝONX antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the "Anti-ΝOVX Antibodies" section below. The disclosed ΝONX proteins have multiple hydrophilic regions, each of which can be used as an immunogen. These ΝONX proteins can be used in assay systems for functional analysis of various human disorders, which will help in understanding of pathology of the disease and development of new drug targets for various disorders.
The ΝONX nucleic acids and proteins identified here may be useful in potential therapeutic applications implicated in (but not limited to) various pathologies and disorders as indicated below. The potential therapeutic applications for this invention include, but are not limited to: protein therapeutic, small molecule drug target, antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), diagnostic and/or prognostic marker, gene therapy (gene delivery/gene ablation), research tools, tissue regeneration in vivo and in vitro of all tissues and cell types composing (but not limited to) those defined here.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description and claims.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides novel nucleotides and polypeptides encoded thereby.
Included in the invention are the novel nucleic acid sequences and their encoded polypeptides.
The sequences are collectively referred to herein as "NONX nucleic acids" or "ΝONX polynucleotides" and the corresponding encoded polypeptides are referred to as "ΝONX polypeptides" or "ΝONX proteins." Unless indicated otherwise, "ΝONX" is meant to refer to any of the novel sequences disclosed herein. Table A provides a summary of the NONX nucleic acids and their encoded polypeptides.
TABLE A. Sequences and Corresponding SEQ ID Numbers
Figure imgf000009_0001
Figure imgf000010_0001
NONX nucleic acids and their encoded polypeptides are useful in a variety of applications and contexts. The various ΝONX nucleic acids and polypeptides according to the invention are useful as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins. Additionally, ΝONX nucleic acids and polypeptides can also be used to identify proteins that are members of the family to which the ΝONX polypeptides belong.
ΝON1 is homologous to members of the neurotrophin-6 alpha family of proteins. Thus, the ΝON1 nucleic acid and polypeptide, antibodies and related compounds according to the invention may be used to treat immune and nervous system disorders, e.g., proinflammatory disorder, immune disorder, inflammatory disease, septic shock, arthritis, bone pain, or bone deformity.
ΝON2 is homologous to members of the guanylate kinase family of proteins. Thus, the ΝON2 nucleic acid and polypeptide, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in the mediation of biosynthesis and nucleotide metabolism. As such the ΝON2 nucleic acid and polypeptide, antibodies and related compounds according to the invention may be used to treat genetic conditions, e.g.,Non Hippel- Lindau (NHL) syndrome, diabetes, or tuberous sclerosis.
ΝON3 is homologous to members of a family of the 85.6 kDa-like proteins that contain ankyrin domains. Thus ΝON3 nucleic acid and polypeptide, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in the mediation of signal transduction or cell activation. As such the ΝON3 nucleic acid and polypeptide, antibodies and related compounds according to the invention may be used to treat genetic conditions, e.g., endometriosis, fertility, adrenoleukodystrophy, congenital adrenal hyperplasia, diabetes, Non Hippel-Lindau (vhl) syndrome , pancreatitis, obesity, hyperparathyroidism, hypoparathyroidism, hyperthyroidism, hypothyroidism, SIDS, xerostomia, scleroderma, hypercalceimia, ulcers, cinhosis, transplantation, inflammatory bowel disease, diverticular disease, hirschsprung's disease, crohn's disease, appendicitis, hemophilia, hypercoagulation, idiopathic thrombocytopenic purpura, autoimmume disease, allergies, immunodeficiencies, graft vesus host, anemia, ataxia-telangiectasia, lymphedema, tonsilitis, osteoporosis, hypercalceimia, arthritis, ankylosing spondylitis, scoliosis, tendinitis, muscular dystrophy, lesch-nyhan syndrome, myasthenia gravis, dental disease and infection, cardiomyopathy, atherosclerosis, hypertension, congenital heart defects, aortic stenosis, atrial septal defect (asd), atrioventricular (a-v) canal defect, ductus arteriosus, pulmonary stenosis, subaortic stenosis, ventricular septal defect (vsd), valve diseases, tuberous sclerosis, aneurysm, fibromuscular dysplasia, stroke, bleeding disorders, alzheimer's disease, parkinson's disease, huntington's disease, cerebral palsy, epilepsy, multiple sclerosis, ataxia-telangiectasia, leukodystrophies, behavioral disorders, addiction, anxiety, pain, neuroprotection, endocrine dysfunctions, growth and reproductive disorders, cystitis, incontinence, renal artery stenosis, interstitial nephritis, glomerulonephritis, polycystic kidney disease, systemic lupus erythematosus, renal tubular acidosis, iga nephropathy, or vesicoureteral reflux.
ΝON4 is homologous to members of the mytonic dystrophy kinase-related CDC42- binding kinase family of proteins. Thus, the ΝON4 nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat muscle, or cell migration disorders, e.g., myotonic dystrophy, myotonic dystrophy type 2, proximal myotonic myopathy, proximal myotomc dystrophy, neuromuscular diseases associated with cardiomyopathy, multiple endocrine neoplasia type 1(MEΝ1), insulin dependent diabetes mellitus, familial paraganglioma type 2, spinocerebellar ataxia type 5, Bardet-Biedl syndrome, non-hodgkins lymphoma, cancers such as breast cancer, liver, lung, pancrease, and prostate cancers.
NON5 is homologous tomembers of the SI 00 Calcium binding protein family. Thus, the ΝON5 nucleic acid and polypeptide, antibodies and related compounds according to the invention may be used to treat genetic conditions, e.g., various cancers like breast, lung, and colorectal, as well as heart disease such as myocardial ischemia.
ΝON6, ΝON16, ΝON18, ΝON31, and ΝON33 are homologous to the olfactory receptor/GPCR-like family of proteins. G-Protein Coupled Receptor (GPCRs) have been identified as an extremely large family of protein receptors in a number of species. Thus, the ΝON6 nucleic acid and polypeptide, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in the mediation of signal transduction. As such the ΝON6 nucleic acid and polypeptide, antibodies and related compounds according to the invention may be used to treat, e.g., developmental diseases, MHC II and III diseases (immune diseases), taste and scent detectability disorders, Burkitt's lymphoma, corticoneurogenic disease, signal transduction pathway disorders, retinal diseases including those involving photoreception, cell growth rate disorders, cell shape disorders, feeding disorders, control of feeding, potential obesity due to over-eating, potential disorders due to starvation (lack of apetite), noninsulin-dependent diabetes mellitus (ΝTDDM1), bacterial, fungal, protozoal and viral infections (particularly infections caused by HIV-1 or HIN-2), pain, cancer (including but not limited to neoplasm, adenocarcinoma, lymphoma, prostate cancer, uterus cancer), anorexia, bulimia, asthma, parkinson's disease, acute heart failure, hypotension, hypertension, urinary retention, osteoporosis, crohn's disease, multiple sclerosis, and treatment of albright hereditary ostoeodystrophy, angina pectoris, myocardial infarction, ulcers, asthma, allergies, benign prostatic hypertrophy, and psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation, dentatorubro-pallidoluysian atrophy(DRPLA) hypophosphatemic rickets, autosomal dominant (2) acrocallosal syndrome and dyskinesias, such as huntington's disease or gilles de la tourette syndrome.
ΝON7 is homologous to members of the carbonate dehydratase/anhydrase family of proteins. As such the ΝON7 nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat respiratory or CO2 transport disorders, e.g., lung cancer, hypertension, asthma, emphysema, or diabetes. NON8 is homologous to members of the carboxypeptidase family of proteins. Thus, the ΝON8 nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat digestive disorders, e.g., xerostomia, hypercalceimia, ulcers, Von Hippel-Lindau (NHL) syndrome, cirrhosis, transplantation, inflammatory bowel disease, diverticular disease, hirschsprung's disease, crohn's disease, appendicitis, stroke, tuberous sclerosis, anxiety, pain, endocrine dysfunctions, nueroprotection, diabetes, obesity, growth and reproductive disorders, myasthenia gravis.
ΝON9 is homologous to members of the neurotransmitter receptor family of proteins. Thus, the ΝON9 nucleic acid and polypeptide, antibodies and related compounds according to the invention may be used to treat, e.g., leukemia, acute nonlymphocytic, spinocerebellar ataxia-1, or neurological disorders.
ΝON10 is homologous to members of the proto-oncogene MAF-like family of proteins. Thus, the ΝON10 nucleic acid and polypeptide, antibodies and related compounds according to the invention may be used to treat, e.g., anemia, ataxia-telangiectasia, autoimmume disease, cancer, immunodeficiencies, hemophilia, hypercoagulation, idiopathic thrombocytopenic purpura, allergies, transplantation, graft versus host disease (GNHD), lymphaedema, systemic lupus erythematosus, asthma, emphysema, scleroderma, ARDS, diabetes, renal artery stenosis, interstitial nephritis, glomerulonephritis, polycystic kidney disease, systemic lupus erythematosus, renal tubular acidosis, IgA nephropathy, hypercalceimia, or Lesch-Νyhan syndrome. ΝON11 is homologous to members of the lysyl oxidase family of proteins. Thus, the
ΝON11 nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat digestive disorders, e.g., diabetes, Non Hippel-Lindau (NHL) syndrome, pancreatitis, obesity, endometriosis, fertility, hemophilia, hypercoagulation, idiopathic thrombocytopenic purpura, autoimmume disease, allergies, immunodeficiencies, transplantation, graft versus host disease (GNHD), lymphaedema, osteoporosis, hypercalceimia, arthritis, ankylosing spondylitis, scoliosis, systemic lupus erythematosus, asthma, emphysema, scleroderma, allergy, ARDS, renal artery stenosis, interstitial nephritis, glomerulonephritis, polycystic kidney disease, systemic lupus erythematosus, renal tubular acidosis, IgA nephropathy, Lesch-Νyhan syndrome, psoriasis, actinic keratosis, tuberous sclerosis, acne, hair growth/loss, allopecia, pigmentation disorders, and endocrine disorders. NON12 is homologous to members of the phosphatase family of proteins. Thus, the ΝON12 nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat, e.g., hyperthyroidism, hypothyroidism, endometriosis, fertility, Non Hippel-Lindau (NHL) syndrome, cirrhosis, transplantation, hypogonadism, Alzheimer's disease, stroke, tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, cerebral palsy, epilepsy, Lesch-Νyhan syndrome, multiple sclerosis, ataxia-telangiectasia, leukodystrophies, behavioral disorders, addiction, anxiety, pain, neurodegeneration, endocrine dysfunctions, diabetes, obesity, growth and reproductive disorders, systemic lupus erythematosus, autoimmune disease, asthma, emphysema, scleroderma, allergy, ARDS, renal artery stenosis, interstitial nephritis, glomerulonephritis, polycystic kidney disease, renal tubular acidosis, IgA nephropathy, hemophilia, hypercoagulation, idiopathic thrombocytopenic purpura, immunodeficiencies, and graft versus host disease.
ΝON13 is homologous to members of the chloride channel CLC-KA family of proteins. Thus, the ΝON13 nucleic acid and polypeptide, antibodies and related compounds according to the invention may be used to treat, e.g., diabetes, autoimmune disease, renal artery stenosis, interstitial nephritis, glomerulonephritis, polycystic kidney disease, systemic lupus erythematosus, renal tubular acidosis, IgA nephropathy, hypercalceimia, or Lesch-Νyhan syndrome.
ΝON14 is homologous to members of the mast cell function-associated antigen (MAF A) family of proteins. Thus, the ΝON14 nucleic acid and polypeptide, antibodies and related compounds according to the invention may be used to treat, e.g., cancer, autoimmune disease, allergies, immunodeficiencies, transplantation, graft versus host disease (GNHD), or lymphaedema.
ΝON15 is homologous to members of the murine epithelial growth factor (MEGF) family of proteins. Thus, the ΝON15 nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat, e.g., cancer, trauma, bacterial and viral infections, regeneration (in vitro and in vivo), fertility, endometriosis, cardiomyopathy, atherosclerosis, hypertension, congenital heart defects, aortic stenosis, atrial septal defect (ASD), atrioventricular (A-N) canal defect, ductus arteriosus, pulmonary stenosis, subaortic stenosis, ventricular septal defect (NSD), valve diseases, tuberous sclerosis, scleroderma, obesity, transplantation, anemia, bleeding disorders, transplantation, diabetes, autoimmune disease, renal artery stenosis, interstitial nephritis, glomerulonephritis, polycystic kidney disease, systemic lupus erythematosus, renal tubular acidosis, IgA nephropathy, hypercalceimia, Lesch-Nyhan syndrome, systemic lupus erythematosus, autoimmune disease, asthma, emphysema, allergy, ARDS, von Hippel-Lindau (VHL) syndrome, Alzheimer's disease, stroke, hypercalceimia, Parkinson's disease, Huntington's disease, cerebral palsy, epilepsy, Lesch-Nyhan syndrome, multiple sclerosis, ataxia-telangiectasia, leukodystrophies, behavioral disorders, addiction, anxiety, pain, neurodegeneration, Hirschsprung's disease , Crohn's Disease, and appendicitis.
NON17 is homologous to members of the monocarboxylate transporter (MCT)-like family of proteins. Thus, the ΝON17 nucleic acids and polypeptides, antibodies and related compounds according to the invention maybe used to treat, e.g., Salla disease, infantile sialic acid storage disease, cystinosis, or streptozotocin-induced diabetes.
ΝOV19 is homologous to members of the major Duchenne muscular dystrophy (DP71) family of proteins. Thus, the NON 19 nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat muscle and nervous system disorders, e.g., Duchenne muscular dystrophy, Becker muscular dystroph, cardiomyopathy, dilated, X- linked, McLeod phenotype, Lesch-Νyhan syndrome, myasthenia gravis.
ΝON20 is homologous to members of the GPCR RTA family of proteins. Thus, the ΝON20 nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat a wide range of disorders such as cancer, immune disorders, endocrine disorders and other diseases, e.g., developmental diseases; MHCII and III diseases (immune diseases); taste and scent detectability disorders; Burkitt's lymphoma; corticoneurogenic disease; signal transduction pathway disorders; metabolic pathway disorders; retinal diseases including those involving photoreception; cell growth rate disorders; cell shape disorders; metabolic disorders; feeding disorders; control of feeding; the metabolic syndrome X; wasting disorders associated with chronic diseases; obesity; potential obesity due to over-eating or metabolic disturbances; potential disorders due to starvation (lack of appetite); diabetes; noninsulin-dependent diabetes mellitus (ΝIDDM); infectious disease; bacterial, fungal, protozoal and viral infections (particularly infections caused by HIN-1 or HIN-2); pain; cancer (including but not limited to neoplasm; adenocarcinoma; lymphoma; prostate cancer; uterus cancer); cancer- associated cachexia; anorexia; bulimia; asthma; Parkinson's disease; acute heart failure; hypotension; hypertension; urinary retention; osteoporosis; Crohn's disease; multiple sclerosis;
Albright Hereditary Ostoeodystrophy; angina pectoris; myocardial infarction; ulcers; allergies; benign prostatic hypertrophy; and psychotic and neurological disorders; including anxiety; schizophrenia; manic depression; delirium; dementia; neurodegenerative disorders; Alzheimer's disease; severe mental retardation; Dentatorubro-pallidoluysian atrophy (DRPLA);
Hypophosphatemic rickets; autosomal dominant (2) Acrocallosal syndrome and dyskinesias, such as Huntington's disease or Gilles de la Tourette syndrome; immune disorders;
Adrenoleukodyst ophy; Congenital Adrenal Hypeφlasia; Hemophilia; Hypercoagulation;
Idiopathic thrombocytopenic purpura; autoimmume disease; immunodeficiencies; transplantation;
Non Hippel-Lindau (NHL) syndrome; Stroke; Tuberous sclerosis; hypercalceimia; Cerebral palsy;
Epilepsy; Lesch-Νyhan syndrome; Ataxia-telangiectasia; Leukodystrophies; Behavioral disorders; Addiction; Νeuroprotection; Cirrhosis; Transplantation; Systemic lupus erythematosus;
Emphysema; Scleroderma; ARDS; Renal artery stenosis; Interstitial nephritis;
Glomerulonephritis; Polycystic kidney disease; Systemic lupus erythematosus; Renal tubular acidosis; IgA nephropathy; Cardiomyopathy; Atherosclerosis; Congenital heart defects; Aortic stenosis ; Atrial septal defect (ASD); Atrioventricular (A-N) canal defect; Ductus arteriosus; Pulmonary stenosis ; Subaortic stenosis; Ventricular septal defect (NSD); valve diseases;
Scleroderma; fertility; Pancreatitis; Endocrine dysfunctions; Growth and reproductive disorders;
Inflammatory bowel disease; Diverticular disease; Leukodystrophies; Graft vesus host;
Hyperthyroidism; Endometriosis; and hematopoietic disorders.
ΝON21 is homologous to members of the TFIIIC box B-binding subunit family of proteins. Thus, the ΝON21 nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat cancer and viral infections, e.g., TFIIIC box B- binding subunit protein is cleaved and inactivated by the polio virus-encoded 3C protease during poliovirus infection (Shen et al., Mol. Cell. Biol, 16: 4163-71 (1996)).
ΝON22 is homologous to members of the nucleoside diphosphate kinase B family of proteins. Thus, the ΝON22 nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat cancer, e.g., atherosclerosis, aneurysm, hypertension, fibromuscular dysplasia, stroke, scleroderma, obesity, transplantation, myocardial infarction, embolism, cardiovascular disorders, bypass surgery, fertility disorders, myasthenia gravis, leukodystrophies, pain, neuroprotection, systemic lupus erythematosus, autoimmune disease, asthma, emphysema, scleroderma, allergy, ARDS and other diseases, disorders and conditions of the like. NON23 is homologous to members of the T-cell family of proteins. Thus, the ΝON23 nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to immune disorders, e.g., inflammation, allergies, autoimmune disease, and asthma.
ΝON24 is homologous to members of the organic anion transporter (OAT) 3 family of proteins. Thus, the ΝON24 nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat a wide range of disorders such as cancer, kidney disorders, immune disorders and other diseases, e.g., Non Hippel-Lindau (NHL) syndrome, Cuτhosis,Transplantation, Osteoporosis, Hypercalceimia, Artliritis, Ahkylosing spondylitis, Scoliosis, Diabetes, Autoimmune disease, Renal artery stenosis, Interstitial nephritis, Glomerulonephritis, Polycystic kidney disease, Systemic lupus erythematosus, Renal tubular acidosis, IgA nephropathy, Lesch-Νyhan syndrome renal malfunction, nephrotoxicity, disease associated with cytotoxic drug, osteoporosis, osteopetrosis resistance, liver diseases, and heart diseases.
ΝON25 and ΝON26 are homologous to members of the ficolin family of proteins. Thus, such nucleic acid or protein therapeutics designed with the protein encoded for by ΝON26 could function as an opsinin to target and eliminate bacteria by complement -mediated destruction. These proteins could be important for the treatment of bacterial septicemia. Ficolins may also have the ability to bind to elastins. Elastins are functionally important for lung alveolar development and inactivation of these proteins can lead to emphysema-like disease. Antibodies against ΝON25 and ΝON26 may prevent tissue destruction mediated by ficolin activity during emphysema, asthma and arthritis.
ΝON27 is homologous to members of the peroxisomal Ca +-dependent solute carrier family of proteins. Thus, the ΝON27 nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat metabolic disorders, e.g., cancer, trauma, regeneration (in vitro and in vivo), viral/bacterial/parasitic infections, atherosclerosis, aneurysm, hypertension, fibromuscular dysplasia, stroke, scleroderma, obesity, Non Hippel- Lindau (NHL) syndrome, Alzheimer's disease, stroke, tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, cerebral palsy, epilepsy, Lesch-Νyhan syndrome, multiple sclerosis, ataxia-telangiectasia, leukodystrophies, behavioral disorders, addiction, anxiety, pain, diabetes, autoimmune disease, renal artery stenosis, interstitial nephritis, glomerulonephritis, polycystic kidney disease, systemic lupus erythematosus, renal tubular acidosis, IgA nephropathy, hypercalceimia, muscular dystrophy, Lesch-Nyhan syndrome, and myasthenia gravis.
NON28, ΝON29, and ΝON30 are homologous to members of the Νa+/glucose cotransporter family of proteins. Thus, the NOV28 nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat metabolic, immune and renal disorders, e.g., metabolic diseases such as diabetes and hypertension, or cancer, trauma, regeneration (in vitro and in vivo), viral/bacterial/parasitic infections, diabetes, autoimmune disease, renal artery stenosis, interstitial nephritis, glomerulonephritis, polycystic kidney disease, systemic lupus erythematosus, renal tubular acidosis, IgA nephropathy, hypercalceimia, Lesch- Nyhan syndrome, cardiomyopathy, atherosclerosis, hypertension, congenital heart defects, aortic stenosis, atrial septal defect (ASD), atrioventricular (A-V) canal defect, ductus arteriosus, pulmonary stenosis, subaortic stenosis, ventricular septal defect (NSD), valve diseases, tuberous sclerosis, scleroderma, obesity, transplantation and other diseases, disorders and conditions of the like. ΝON32 is homologous to members of the phosphoenolpyruvate carboxykinase family of proteins. Thus, the ΝON32 nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat metabolic disorders, e.g., hypoglycemia.
The ΝONX nucleic acids and proteins of the invention, therefore, are useful in potential therapeutic applications implicated, for example but not limited to, in various pathologies /disorders as described herein and/or other pathologies/disorders. Potential therapeutic uses for the invention(s) are, for example but not limited to, the following: (i) a protein therapeutic, (ii) a small molecule drug target, (iii) an antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), (iv) a nucleic acid useful in gene therapy (gene delivery/gene ablation), (v) an agent promoting tissue regeneration in vitro and in vivo, and (vi) a biological defense weapon.
ΝOV1
The disclosed ΝON1 nucleic acid (alternatively referred to herein as CG56181-01) encodes a novel neutrophin-6 alpha-like protein and includes the 796 nucleotide sequence (SEQ
ID NO: 1) shown in Table 1 A. The novel NOV1 nucleic acid of the invention maps to chromosome 19.
An open reading frame for the mature protein was identified beginning with an AGC, but no start codon, and ending with a TGA stop codon at nucleotides 775-777. Putative untranslated regions, if any, are found upstream from the initiation codon and downstream from the termination codon. The start and stop codons are in bold letters.
Table IA. NOVl Nucleotide Sequence (SEQ ID NO:l)
AGCAAGGGCTTCCCCATAATCCTGGCAGGCAGGCCTCCCCTGGGGTTTCCAACTTCTGACCCCACTGAAGTGTTT ATCCTCTTCTCTAACCCCAGCCTCCTTTTCCCTGTCTCCATGTGCTCTGAGAGATGCTCTGAGAGATGCTCCCAC TCCCCCAGGCTCCCTCTGCATCCCCCTCATTTTCTTCCTCCCCAGTGTGTCAATGGAGTCCTGGCCCCCACCCTC TCGACATTGTCACCTTTTCCTGATCCAAAGTGGGACCTTCTTTTCCCCCAAGTGGTCCTGTCTAGGGGTGCCGCT GCCGGGCCCCCTCTGGTCTTCCTGCTGCAGACTGGGGCCTTTTGGGAGTCAGCAGGCGCCCGGGCCAACCGCAGC CAGCGTGAGGCGAGCGATGCTTCACCGGCGAGTCATCAGGGTGAGCTGGCCGTGTGCGATGCAGTCAGTGTCTGG GTGACAGATCCCGGGACTGCTGTGGACTTGGTTGTGCTCGAGGTGGAGGTGTTGGGCGAGGTGCCTGCAGCTGTC GGCAGTTCCCTCCACCAACACTTCTTTGTTGCCCACTTCGAGGCCGATAACTCTGAGGAAGGTGGCCCGGGGGTA GGTGGAGGGGCTGCCGCCGGGGTGTGGACCGGGGGGCACTGGGTGTCTGAGTGCAAGGCCAAGCAGTCCTATGTG CGGGCATTGACCGCTGATGCCCAGGGCCGTGTGGACTGGCGATGGATTCAAATTGGCACTGCCTGTGTCTGCACA CTCCTCAGCCGGACTGGCCGGGCCTGAGACCCATGCCCAGGAACTG
The NOVl protein (SEQ ID NO:2) encoded by SEQ TD NO: 1 is 258 amino acid residues in length and is presented using the one-letter amino acid code in Table IB. The SignalP, Psort and/or Hydropathy results indicate that NOVl has a signal peptide and is likely to be localized in the microbody (peroxisome) with a certainty of 0.5952. Alternatively, a NOVl polypeptide is located to the cytoplasm with a certainty of 0.4500, the lysosome (lumen) with a certainty of 0.2100, or the mitochondrial matrix space with a certainty of 0.1000.
Table IB. Encoded NOVl Protein Sequence (SEQ ID NO:2)
SKGFPII AGRPP GFPTSDPTEVFILFSNPSLLFPVSMCSΞRCSERCSHSPRLPLHPPHF PPQCVNGV APTL STLSPFPDPKWD FPQWLSRGAAAGPPLVFLLQTGAF ESAGARANRSQREASDASPASHQGE AVCDAVSV VTDPGTAVDLWLEVEVLGEVPAAVGSSLHQHFFVAHFEADNSEEGGPGVGGGAAAGVWTGGH VSECKAKQSYV RALTADAQGRVD RWIQIGTACVCTLLSRTGRA
A search against the Patp database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table IC.
Table IC. PatP Results for NOVl
Smallest High Sum
Sequences Producing High-Scoring Segment Pairs: Score Prob P (N) patp:AAR22467 Neurotrophic factor 4-gamma - Homo sapiens 1175 3.8e-119 patp:AAR22466 Neurotrophic factor 4-beta - Homo sapiens 1047 1.4e-105 ρatp:AAR22468 Neurotrophic factor 4-delta - Homo sapiens 864 3.4e-86 patp:AAR29735 Human NT-4, encoded by clone 7-2 680 l.le-66 patp:AAR30691 Human neutroρhin-4 678 1.8e-66
In a BLAST search of public sequence databases, it was found, for example, that the NOVl nucleic acid sequence of this invention has 762 of 796 bases (95%) identical to a gb:GENBANK-ID:HUMNT4PSG|acc:M86529.1 mRNA from Human neurotroρhin-4 pseudogene sequence. Further, the full amino acid sequence of the disclosed protein of the invention has 239 of 258 amino acid residues (92%) identical to, and 244 of 258 amino acid residues (94%) similar to, the 257 amino acid residue ptnr:SWISSPROT-ACC:P34132 protein from Human (NEUROTROPHIN-6 ALPHA (NT-6 ALPHA)).
In all BLAST alignments herein, the "E-value" or "Expect" value is a numeric indication of the probability that the aligned sequences could have achieved their similarity to the BLAST query sequence by chance alone, within the database that was searched. For example, the probability that the subject ("Sbjct") retrieved from the IIT BLAST analysis, matched the Query IIT sequence purely by chance is the E value. The Expect value (E) is a parameter that describes the number of hits one can "expect" to see just by chance when searching a database of a particular size. It decreases exponentially with the Score (S) that is assigned to a match between two sequences. Essentially, the E value describes the random background noise that exists for matches between sequences. Blasting is performed against public nucleotide databases such as GenBank databases and the GeneSeq patent database. For example, BLASTX searching is performed against public protein databases, which include GenBank databases, SwissProt, PDB and PIR.
The Expect value is used as a convenient way to create a significance threshold for reporting results. The default value used for blasting is typically set to 0.0001. hi BLAST 2.0, the Expect value is also used instead of the P value (probability) to report the significance of matches. For example, an E value of one assigned to a hit can be interpreted as meaning that in a database of the current size one might expect to see one match with a similar score simply by chance. An E value of zero means that one would not expect to see any matches with a similar score simply by chance. See, e.g., http://www.ncbi.nlm.nih.gov/Education/BLASTinfo/. Occasionally, a string of X's or N's will result from a BLAST search. This is a result of automatic filtering of the query for low-complexity sequence that is performed to prevent artifactual hits. The filter substitutes any low-complexity sequence that it finds with the letter "N" in nucleotide sequence (e.g., "NNNNNNNNNNNNN") or the letter "X" in protein sequences (e.g., "XXXXXXXXX"). Low-complexity regions can result in high scores that reflect compositional bias rather than significant position-by-position alignment. Wootton and Federhen, Methods Enzymol 266:554-571, 1996.
The NOVl protein of the invention also has homolgy to the proteins shown in the BLASTP data in Table ID.
Figure imgf000021_0002
A multiple sequence alignment is given in Table IE, with the NOVl protein of the invention being shown in line 1 in a ClustalW analysis comparing NOV with related protein sequences of Table ID.
Table IE. ClustalW Analysis of NOVl
1. SEQ ID NO.: 2 NOVl 4. SEQ ID NO. 126 AAL35774
2. SEQ TD NO.: 124 Q96K94 5. SEQ ID NO. 127 AAL35775
3. SEQ ID NO.: 125 AAL35776 6. SEQ ID NO. 128 054947
10 20 30 40
Figure imgf000021_0001
Figure imgf000022_0001
190 200 210 220 230 240
NOVl WVTDgG TAVDLJJVLE VJBVLGEVPA V'GS SLH§HFFVAH 187
Q96 94 LGSLgD INLTQJl^LAtlELRbSRLgNDLRDSGATIRlg 202
AAL35776 LVTLHN pJG IS AbEIKDS GETIRTA 193
AAL35774 WTH] fflEpTTFCPHETTAEVTGIPSHTPTpWNGTffiτfiSG TWSMHTEg2J I:PPGKPSKNPTKgl 237
AAL35775 TH] fl P NGTRTSSGDT S^HTE|Il:PPGKPIKNPTK.il 214
054947 QTHKgEITTFYAHETTAEVTETPSYTPAp NGTgTSSEgAWNJIHTVRlPLRKPgRNPTI 235
Figure imgf000022_0002
The presence of identifiable domains in the disclosed NOVl protein was determined by using Pfam and then determining the Interpro number. The results are listed in Table IF with the statistics and domain description. Table IF. Domain Analysis of NOVl
PSSMs Producing Significant Alignments Score E (bits) Value
NGF : domain 1 of 2 , from 133 to 184 70 . 6 1. 5e-19
NGF epvsrRGElSVCDSvSvWVTnDKttAvDirGkeVtVLgeVninngp .
++++++ 11 ++ 111 ++ 1 + 111 i +1 + 1+ +1 + 11++1+++ +++
NOVl SPASHQGELAVCDAVSVWVT-DPGTAVDLWLEVEVLGEVPAAVGSs NGF 1KQYFF (SEQ ID NO: 129)
+ I II
NOVl LHQHFF (SEQ ID NO: 2)
NGF: domain 2 of 2, from 213 to 258 100 . 5 2 . 5e-28
NGF HWnSeCkttqtYVRALTmdnnklVgWRflRIDTACVCtLsrKtGrt (SEQ ID NO : 130 )
1 1 + 1 + 1 +++++ 1 1 1 1 I I ++++++ 1 1 1 + 1 I I 1 1 1 1 + 1 ++++ 1 ++
NOVl HWVSECKAKQSYVRALTADAQGRVDWRWIQIGTACVCTLLSRTGRA (SEQ ID NO : 2 )
Consistent with other known members of the neurotrophin family of proteins, NOVl contains nerve growth factor domains as illustrated in Table IF. The NOVl nucleic acid, and the encoded polypeptide, according to the invention are useful in a variety of applications and contexts. For example, NOVl nucleic acids and polypeptides can be used to identify proteins that are members of the neurotrophin family of proteins. The NOVl nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance NOVl activity or function. Specifically, the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., development and survival of certain sympathetic and sensory neurons in both the central and peripheral nervous systems. These molecules can be used to treat, e.g., proinflammatory disorder, immune disorder, and inflammatory disease.
In addition, the NOVl nucleic acid and polypeptide according to the invention are useful, inter alia, as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins. For example, the NOVl nucleic acid and polypeptide include structural motifs that are characteristic of proteins belonging to the family of nerve growth factors such as the neurotrophin proteins. Nerve growth factor (NGF) is the prototype for the neurotrophin family of polypeptides which are essential in the developments and survival of certain sympathetic and sensory neurons in both the central and peripheral nervous systems. NGF was discovered when mouse sarcoma tissue transplants in chicken embryos caused an increase in the size of spinal ganglia.
The NOVl nucleic acid and polypeptide, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in the mediation of the peripheral and central nervous system. As such the NOVl nucleic acid and polypeptide, antibodies and related compounds according to the invention may be used to treat immune and nervous system disorders, e.g., proinflammatory disorder, immune disorder, inflammatory disease, septic shock, arthritis, bone pain, or bone deformity.
The NOVl nucleic acid and polypeptide are useful for detecting specific cell types. For example, expression analysis has demonstrated that a NOVl nucleic acid is expressed in placenta and uterus.
Additional utilities for the NOVl nucleic acid and polypeptide according to the invention are disclosed herein. .
NOV2
The disclosed NOV2 nucleic acid (alternatively referred to herein as CG56275-01) encodes a novel Guanylate kinase-like protein and includes the 1336 nucleotide sequence (SEQ ID NO:3) shown in Table 2A. The novel NOV2 nucleic acid of the invention maps to chromosome 2. An open reading frame for the mature protein was identified beginning with an ATG initiation codon at nucleotides 3-5, and ending with a TGA stop codon at nucleotides 1326-1328. Putative untranslated regions, if any, are found upstream from the initiation codon and downstream from the termination codon. The start and stop codons are in bold letters.
Table 2A. NOV2 Nucleotide Sequence (SEQ ID NO:3)
CAATGAGGATTGTTTGTTTAGTGAAAAACCAACAGCCCCTGGGAGCCACCATCAAGCGCCACGAGATGACAGGGG ACATCTTGGTGGCCAGGATCATCCACGGTGGGCTGGCGGAGAGAAGTGGGTTGCTATATGCTGGAGACAAACTGG TAGAAGTGAATGGAGTTTCAGTTGAGGGACTGGACCCTGAACAAGTGATCCATATTCTGGCCATGTCTCGAGGCA CAATCATGTTCAAGGTGGTTCCAGTCTCTGACCCTCCTGTGAATAGCCAGCAGATGGTAAGAATTGTGTACGTCC GTGCCATGACTGAGTACTGGCCCCAGGAGGATCCCGACATCCCCTGCATGGACGCTGGATTGCCTTTCCAGAAGG GGGACATCCTCCAGATTGTGGACCAGAATGATGCCCTCTGGTGGCAGGCCCGAAAAATCTCAGACCCTGCTACCT GCGCTGGGCTTGTCCCTTCTAACCACCTTCTGAAGAGGAGGAAGCAACGGGAATTCTGGTGGTCTCAGCCGTACC AGCCTCACACCTGCCTCAAGTCAACCCTACAACTGAAGGAGGAGTTTGTTGGCTACGGTCAGAAGTTCTTTATAG GTAGGTCTCACCTCAGCCCGCTGCATGCCAGTGTGTGCTGCACCGGCAGCTGCTACAGTGCAGTGGGTGCCCCTT ACGAGGAGGTGGTGAGGTACCAGCGACGCCCTTCAGACAAGTACCGCCTCATAGTGCTCATGGGTATGTCCTTAG GACCCTCTGGTGTTGGAGTAAATGAGCTCAGAAGACAACTTATTGAATTTAATCCCAGCCATTTTCAAAGTGCTG TGCCAACTACTCGTACTAAAAAGAGTTACGAAATGAATGGGCGTGAGTATCACTATGTGTCCAAGGAAACATTTG AAAACCTCATATATAGTCACAGGAGGATGCTGGAGTATGGTGAGTACAAAGGCCACCTGTATGGCACTAGTGTGG ATGCTGTTCAAACAGTCCTTGTCGAAGGAAAGATCTGTGTCATGGACCTAGAGCCTCAGAATATGAGGTGTATGA AACAATCTCGGAAAAATGCCAAGGTTATTACTGACTACTATGTGGACATGAAGTTCAAGGTAAGAGCAAGTCAAA AACTAAAGGATGAAGACCTACAAGAGATGGAAAATTTAGCCCAAAGAATGGAAACTCAGTTTGGCCAATTTTTTG ATCATGTGATTGTGAATGACAGCTTGCACGATGCATGTGCCCAGTTGTTGTCTGCCATACAGAAGGCTCAGGAGG AGCCTCAGTGGGTACCAGCAACATGGATTTCCTCAGATACTGAGTCTCAATGAGACTTCTT
The NOV2 protein (SEQ ID NO:4) encoded by SEQ ID NO:3 is 441 amino acid residues in length and is presented using the one-letter amino acid code in Table 2B. The SignalP, Psort and/or Hydropathy results indicates that NOV2 has a signal peptide and is likely to be localized in the microbody (peroxisome) with a certainty of 0.3000. Alternatively, a NOV2 polypeptide is located to the nucleus with a certainty of 0.3000, the mitochondrial matrix space with a certainty of 0.1000, or the lysosome (lumen) with a certainty of 0.1000.
Table 2B. Encoded NOV2 Protein Sequence (SEQ ID NO:4)
MRIVCLVKNQQPLGATIKRHEMTGDILVARIIHGGLAERSGLLYAGDKLVEVNGVSVEGLDPEQVIHILAMSRGT IMFKVVPVSDPPVNSQQMVRIVYVRAMTEYWPQEDPDIPCMDAGLPFQKGDILQIVDQNDALWWQARKISDPATC AGLVPSNHLLKRRKQREFWWSQPYQPHTCLKSTLQLKEEFVGYGQKFFIGRSHLSPLHASVCCTGSCYSAVGAPY EEWRYQRRPSDKYRLIVLMGMSLGPSGVGVNELRRQLIEFNPSHFQSAVPTTRTKKSYEMNGREYHYVSKETFE NLIYSHRRMLEYGEYKGHLYGTSVDAVQTVLVEGKICVMDLEPQNMRCMKQSRKNAKVITDYYVDMKFKVRASQK LKDEDLQEMENLAQRMETQFGQFFDHVIVNDSLHDACAQLLSAIQKAQEEPQWVPATWISSDTESQ
Small nucleotide polymoφhisms (SNP) variants of NOV2 are disclosed in Example 2.
A search against the Patp database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 2C.
Table 2C. PatP Results for NOV2
Smallest
High Sum
Sequences Producing High-Scoring Segment Pairs: Score Prob P (N) patp:AAE11774 Human kinase (PKIN)-8 protein 2074 2.1e-214 patp:AAU07123 Human novel human protein, NHP #23 823 7.6e-82 patp:AAU07119 Human novel human protein, NHP #19 775 9.3e-77 patp:AAU07115 Human novel human protein, NHP #15 713 3.5e-70 patp:AAU07111 Human novel human protein, NHP #11 709 9.2e-70 hi a BLAST search of public sequence databases, it was found, for example, that the NOV2 nucleic acid sequence of this invention has 313 of 392 bases (79%) identical to a gb:GENBANK-rD:AB030499|acc:AB030499.1 mRNA from Rattus norvegicus (Rattus norvegicus mRNA for DLG6 alpha, complete eds). Further, the full amino acid sequence of the disclosed NOV2 protein of the invention has 346 of 441 amino acid residues (78%) identical to, and 380 of 441 amino acid residues (86%) similar to, the 441 amino acid residue ρtnr:SPTREMBL-ACC:Q9QYHl protein from Rattus norvegicus (Rat) (DLG6 ALPHA). The NOV2 protein of the invention also has homolgy to the proteins shown in the BLASTP data in Table 2D.
Figure imgf000026_0001
A multiple sequence alignment is given in Table 2E, with the NOV2 protein of the invention being shown in line 1 in a ClustalW analysis comparing NOV2 with related protein sequences of Table 2D.
Table 2E. ClustalW Analysis of NOV2
1. SEQ ID NO.: 4 NOV2 4. SEQ ID NO. 133 Q920P8
2. SEQ ID NO.: 131 Q96JB8 5. SEQ ID NO. 134 Q920P7
3. SEQ ID NO.: 132 Q96Q44 6. SEQ ID NO. 135 Q9QYH1
10 20 30 40 50 60
NOV2 Q96 B8 MIQSDKGADPPDKKDMKLSTATNPQNGLSQILRLVLQELSLFYSRDVNGVCLLYDLLHSP 60 Q96Q44 MIQSDKGADPPDKKDMKLSTATNPQNGLSQILRLVLQELSLFYGRDVNGVCLLYDLLHSP 60 Q920P8 1
Q920P7 1 Q9QYH1 1
70 80 90 100 110 120
N0V2
Q96JB8 WLQALLKIYDCLQEFKEKKLVPATPHAQVLSYEWELLRETPTSPEIQELRQMLQAPHFK 120
Q96Q44 LQALLKIYDCLQEFKEKKLVPATPHAQVLSYEWELLRETPTSPEIQELRQMLQAPHFK 120
Q920P8 1
Q920P7 1
Q9QYH1 1
Figure imgf000027_0001
Q920P7 SEELAE. Itj 259 Q9QYH1 S| KEEFVGffiGQSβFFIAGFR QQ|— CTfflSCYSAVGAPYΞE 221
Figure imgf000028_0001
The presence of identifiable domains in the disclosed NOV2 protein was determined by using Pfam and then determining the Interpro number. The results are listed in Table 2F with the statistics and domain description.
Figure imgf000028_0002
GK TRpVpRpgEvdGkdYhFVssrEemekdlaan . eFlEygef qgnyYGT
| | + + + | + | ++ | ++ ] + + | ++++ | ++ + | ++++ +++ | I I
NOV2 TRT-KKSYEMNGREYHYVS-KETFENLIYSHrRMLEYGEYKGHLYGT
GK sletvrqvakqgKiciLDvepQgvkrlrtaelsNPivvFIaPpSlqelek
++ ++++++ +1++++1+++1 ++ ++ + ++ + +++
NOV2 SVDAVQTVLVEGKICVMDLEPQNMRCMKQSRKN-AKVI TDYYVDMKF
GK rLegrnkesEes (SEQ ID NO: 136)
+ + ++ + NOV2 KVRASQKLKDED (SEQ ID NO:4)
Consistent with other known members of the guanylate kinase family of proteins, NOV2 contains guanylate kinase domains as illustrated in Table 2F.
The NOV2 nucleic acid, and the encoded polypeptide, according to the invention are useful in a variety of applications and contexts. For example, NOV2 nucleic acids and polypeptides can be used to identify proteins that are members of the guanylate kinase family of proteins. The NOV2 nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance NOV2 activity or function. Specifically, the nucleic acids and polypeptides according to the invention maybe used as targets for the identification of small molecules that modulate or inhibit, e.g., cell signaling pathways, cell junction organization, or transmembrane regulation.. These molecules can be used to treat, e.g., Von Hippel-Lindau (VHL) syndrome, diabetes, and tuberous sclerosis.
In addition, the NOV2 nucleic acid and polypeptide according to the invention are useful, ter alia, as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins. For example, the NOV2 nucleic acid and polypeptide include structural motifs that are characteristic of proteins belonging to the family of kinases such as the guanylate kinase proteins. Guanylate kinase is a critical enzyme for biosynthesis of GTP and dGTP, and its role in nucleotide metabolism makes it a target for cancer chemotherapy. The structure of mouse guanylate kinase (gmk) includes an N-terminal ATP binding motif and a neighboring guanylate kinase signature sequence (GKSS). The low molecular mass cytosolic forms of guanylate kinase, such as gmk and gukl, are implicated in the regulation of the supply of guanine nucleotides to cell signaling pathways, while the related families of high molecular mass and membrane-associated forms of guanylate kinase, such as MAGUK, CASK, SAP102, ZO-1, and MAGI-1, have roles in cell junction organization and transmembrane regulation. The NOV2 nucleic acid and polypeptide, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in the mediation of biosynthesis and nucleotide metabolism. As such the NOV2 nucleic acid and polypeptide, antibodies and related compounds according to the invention may be used to treat genetic conditions, e.g., Von Hippel-Lindau (VHL) syndrome, diabetes, or tuberous sclerosis.
The NOV2 nucleic acid and polypeptide are useful for detecting specific cell types. For example, expression analysis has demonstrated that a NOV2 nucleic acid is expressed in synovium/synovial membrane.
Additional utilities for the NOV2 nucleic acid and polypeptide according to the invention are disclosed herein.
NOV3
The disclosed NOV3 nucleic acid (alternatively referred to herein as CG53400-01) encodes a novel hypothetical 85.6 kDa-like protem and includes the 3089 nucleotide sequence (SEQ ID NO: 5) shown in Table 3 A. The novel NOV3 nucleic acid of the invention maps to chromosome 12.
An open reading frame for the mature protein was identified beginning with an ATG initiation codon at nucleotides 48-50, and ending with a TAA stop codon at nucleotides 3027- 3029. Putative untranslated regions, if any, are found upstream from the initiation codon and downstream from the termination codon. The start and stop codons are in bold letters.
Table 3A. NOV3 Nucleotide Sequence (SEQ ID NO:5)
GCTGTTCTGGGGAGAAATTGTTGAGTGTTTTCCACTTTAACCTTGCAATGGAATCAGCGGGGCCGCGCTCTCCCT GCAGCCGCCACCGCAGCCGCCGCCTGGGCCGCTCCGTGTCCCCGGTGGAGCCGCCGCCGCCGCCGCCGGGAGCTC GATGCGGACGGAGCCCGGGCCGAGCCATGGGGATCCTCAGCATCACGGACCAGCCGCCCCTGGTCCAGGCCATCT TTAGCCGAGATGTGGAGGAAGTGCGTTCCCTACTCTCGCAGAAGGAGAACATCAATGTGCTGGACCAAGAGAGGC GAACTCCATTGCATGCTGCTGCCTACGTAGGCGATGTCCCCATCCTCCAGTTGCTACTGATGTCAGGTGCTAATG TCAATGCTAAGGACACACTGTGGCTGACCCCTCTTCATCGTGCTGCTGCCTCCCGAAACGAGACGGTGAACCTGC TCCTCAACAAGGGAGCCAGCCTGAATGTCTGTGACAAAAAGGAGCGGCAGCCTCTGCATTGGGCAGCTTTTCTAG GGCACTTGGAGGTCCTAAAACTGCTGGTGGCACGGGGAGCAGACCTCGGCTGCAAGGACCGCAAGGGCTATGGGC TGCTCCATACAGCTGCTGCCAGTGGCCAGATTGAAGTGGTGAAGTACCTGCTTCGGATGGGAGCGGAGATCGATG AACCCAATGCTTTTGGAAACACAGCTTTGCACATCGCCTGCTACCTGGGCCAGGATGCTGTGGCTATTGAGCTGG TGAATGCCGGAGCCAATGTCAACCAGCCGAATGACAAGGGCTTCACGCCACTGCATGTGGCTGCAGTCTCGACCA ATGGCGCTCTCTGCTTGGAGCTACTGGTTAATAATGGGGCTGACGTCAACTACCAGAGCAAAGAAGGGAAAAGTC CTCTGCACATGGCTGCAATCCATGGCCGTTTCACACGCTCCCAGATCCTCATCCAGAATGGCAGCGAGATTGATT GTGCCGACAAATTTGGGAACACGCCACTGCATGTGGCTGCTCGATATGGACACGAGCTGCTCATCAGCACCCTCA TGACCAATGGCGCAGATACCGCCCGGCGTGGCATCCATGACATGTTCCCCCTGCACTTAGCTGTTCTCTTTGGAT TCTCTGACTGTTGTCGTAAGCTTCTTTCCTCAGGTCAGTTGTACAGCATTGTGTCTTCACTCAGCAATGAGCATG TGCTTTCAGCTGGGTTTGACATCAATACACCTGACAACCTTGGCCGTACCTGTCTTCATGCTGCTGCTTCCGGAG GGAATGTTGAATGTCTTAATTTGCTGTTGAGCAGTGGAGCTGACTTGAGGAGGAGGGACAAATTTGGCAGGACCC CACTGCACTATGCAGCTGCTAACGGTAGCTACCAGTGTGCAGTAACATTGGTGACTGCTGGGGCAGGTGTCAACG AGGCCGACTGTAAAGGCTGCTCTCCCCTCCACTACGCTGCCGCTTCTGACACTTACAGGAGAGCGGAACCCCATA CACCTTCCAGCCATGATGCCGAAGAGGACGAGCCACTGAAGGAGTCCCGCAGGAAGGAGGCCTTCTTCTGTCTGG AGTTCTTACTGGATAACGGTGCAGACCCCTCCCTGCGGGACAGGCAGGGCTACACAGCTGTGCACTATGCAGCCG CCTATGGCAACAGACAGAACCTCGAACTGCTCTTAGAAATGTCCTTTAACTGCCTGGAGGATGTGGAGAGCACCA TTCCAGTCAGCCCTTTGCACTTAGCTGCCTACAACGGTCACTGTGAAGCCTTGAAGACGCTGGCGGAGACGCTGG TGAATCTGGACGTAAGGGACCACAAGGGCCGGACCGCACTCTTCCTGGCCACGGAGCGCGGCTCTACTGAGTGTG TGGAGGTGCTTACAGCCCACGGCGCCTCTGCCCTCATCAAGGAGCGCAAGCGCAAGTGGACACCCCTGCACGCTG CTGCTGCCTCTGGCCACACTGACTCCCTGCACTTGCTGATCGACAGTGGGGAACGAGCTGACATCACAGATGTCA TGGATGCCTATGGACAGACCCCACTGATGCTGGCCATCATGAATGGCCATGTGGACTGTGTACATCTGCTGCTAG AGAAAGGATCCACAGCTGATGCTGCTGACCTCCGGGGCCGCACTGCCCTCCACCGCGGGGCAGTGACTGGCTGTG AGGACTGCCTGGCTGCCCTGCTGGACCACGACGCATTTGTGCTGTGCCGAGACTTTAAGGGCCGCACGCCCATTC ACCTGGCCTCAGCCTGTGGCCACACTGCAGTACTGCGGACCCTGCTGCAGGCTGCCCTTTCCACAGATCCCCTGG ATGCCGGGGTGGATTACAGCGGATACTCGCCCATGCACTGGGCCTCCTACACTGGACATGAAGATTGTCTGGAGT TGTTACTTGAACACAGCCCGTTTTCGTACCTGGAAGGAAACCCCTTCACTCCTTTGCACTGTGCAGTGATTAATA ACCAAGACAGCACCACAGAGATGCTACTGGGAGCTCTGGGTGCCAAGATTGTGAACAGCCGAGATGCCAAAGGAC GGACCCCCCTTCACGCCGCTGCCTTCGCGGACAATGTCTCTGGGCTCCGGATGCTGCTGCAGCATCAAGCTGAGG TGAACGCCACTGACCACATTGGCCGCACTGCGCTCATGACGGCGGCTGAGAACGGGCAGACCGCTGCTGTGGAAT TTCTGCTGTATCGAGGGAAGGCAGACCTTACTGTGTTGGATGAGAACAAGAACACGGCCCTCCACTTGGCTTGTA GCAAGGGCCATGAGAAATGTGCCCTCATGATCCTGGCAGAAACCCAAGACCTTGGCCTTATCAATGCTACCAACA GTGCGCTGCAGATGCCACTCCACATTGCTGCCCGGAATGGTCTAGCTTCTGTGGTACAGGCCCTGCTGAGTCATG GGGCCACAGTGCTGGCTGTGGATGAAGAAGGTGGGTGGGGTCTGGGGCCCCATGCCTCTCTTGGGTTTGGGGTCA GGGACATTCTTCAGGAGGTGACTTCTTAATCTTGCTATACATGGGATTTTCTTCCCAAGGGAACTCTTCAGAGCA GGGAGCCCACACCA
The NOV3 protein (SEQ ID NO:6) encoded by SEQ ID NO:5 is 993 amino acid residues in length and is presented using the one-letter amino acid code in Table 3B. The SignalP, Psort and/or Hydropathy results indicate that NOV3 has no known signal peptide and is likely to be localized in the mitochondrial matrix space with a certainty of 0.5083. Alternatively, a NOV3 polypeptide is located to the nucleus with a certainty of 0.3000, the mitochondrial inner membrane with a certainty of 0.2317, or the mitochondrial intermembrane space with a certainty of 0.2217.
Table 3B. Encoded NOV3 Protein Sequence (SEQ ID NO:6)
MESAGPRSPCSRHRSRRLGRSVSPVEPPPPPPGARCGRSPGRAMGILSITDQPPLVQAIFSRDVEEVRSLLSQKE NINVLDQERRTPLHAAAYVGDVPILQLLLMSGANVNAKDTL LTPLHRAAASRNETVNLLLNKGASLNVCDKKER QPLHAAFLGHLEVLKLLVARGADLGCKDRKGYGLLHTAAASGQIEWKYLLRMGAEIDEPNAFGNTALHIACYL GQDAVAIELVNAGANVNQPNDKGFTPLHVAAVSTNGALCLELLVNNGADVNYQSKEGKSPLHMAAIHGRFTRSQI LIQNGSEIDCADKFGNTPLHVAARYGHELLISTLMTNGADTARRGIHDMFPLHLAVLFGFSDCCRKLLSSGQLYS IVSSLSNEHVLSAGFDINTPDNLGRTCLHAAASGGNVECLNLLLSSGADLRRRDKFGRTPLHYAAANGSYQCAVT LVTAGAGVNEADCKGCSPLHYAAASDTYRRAEPHTPSSHDAΞEDEPLKESRRKΞAFFCLEFLLDNGADPSLRDRQ GYTAVHYAAAYGNRQNLELLLEMSFNCLEDVESTIPVSPLHLAAYNGHCEALKTLAETLVNLDVRDHKGRTALFL ATERGSTECVEVLTAHGASALIKERKRKTPLHAAAASGHTDSLHLLIDSGERADITDVMDAYGQTPLMLAIMNG HVDCVHLLLEKGSTADAADLRGRTALHRGAVTGCEDCLAALLDHDAFVLCRDFKGRTPIHLASACGHTAVLRTLL QAALSTDPLDAGVDYSGYSPMH ASYTGHEDCLELLLEHSPFSYLEGNPFTPLHCAVINNQDSTTEMLLGALGAK IVNSRDAKGRTPLHAAAFADNVSGLRMLLQHQAEVNATDHIGRTALMTAAENGQTAAVEFLLYRGKADLTVLDEN KNTALHLACSKGHEKCALMILAETQDLGLINATNSALQMPLHIAARNGLASWQALLSHGATVLAVDEEGGWGLG PHASLGFGVRDILQEVTS
Included in the invention are variants of the parent clone NOV3 as shown below in Table 3C. These novel variants were derived by laboratory cloning of cDNA fragments coding for a domain of the full length form of NOV3 (CG53400-01), between residues 596 and 968.
Figure imgf000032_0001
A search against the Patp database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 3D.
Table 3D. PatP Results for NOV3
Smallest
High Sum
Sequences Producing High-Scoring Segment Pairs: Score Prob P (N) patp:AAM39062 Human polypeptide 2704 3.6e-281 patp:AAU28174 Novel human secretory protein 1932 2.3e-199 ρatp:AAM40848 Human polypeptide 1621 2.1e-166 ρatp:AAU20496 Human secreted protein 1287 5.2e-131 patp:AAU25428 Human mddt protein from clone LG:893050.1:2000Febl8 1045 2.3e-105
In a BLAST search of public sequence databases, it was found, for example, that the NOV3 nucleic acid sequence of this invention has 1552 of 2369 bases (65%) identical to a gb:GENBANK-H):HSM801363|acc:AL133087.1 mRNA from Homo sapiens mRNA; cDNA DKFZρ434D2328 (from clone DKFZρ434D2328); partial eds. Further, the full amino acid sequence of the disclosed NOV3 protein of the invention has 498 of 791 amino acid residues (62%) identical to, and 600 of 791 amino acid residues (75%) similar to, the 791 amino acid residue ptnr:SPTREMBL-ACC:Q9UFA4 protein from Human (HYPOTHETICAL 85.6 KDA PROTEIN).
The NOV3 protein of the invention also has homolgy to the proteins shown in the BLASTP data in Table 3E.
Figure imgf000033_0001
A multiple sequence alignment is given in Table 3F, with the NOV3 protein of the invention being shown in line 1 in a ClustalW analysis comparing NOV3 with related protein sequences of Table 3E. Table 3F. ClustalW Analysis of NOV3
1. SEQ ID NO.: 6 NOV3 4. SEQ ID NO.: 139 Q9NCP8
2. SEQ ID NO.: 137 Q9UFA4 5. SEQ ID NO.: 140 T42714
3. SEQ ID NO.: 138 015084 6. SEQ ID NO.: 141 T42715
10 20 30 40 50 60
Figure imgf000034_0001
310 320 330 340 350 360
Figure imgf000035_0001
Figure imgf000036_0001
970 980 990 1000 1010 1020
N0V3 |EHSPFSYL GNP|nPLHCgVp NQ DSTTEMpJGiL@A 824
Q9UFA4 ( iEEHlICCFFRRKKFFiΪGGNNPPiΪBSpPLLHHcCSSlIϋ| lDDHH GGNNCCAAl|Lfflffl GGjglIDDSS 644
015084 EGAAEMfi igTLBA 819
Q9NCP8 lEl jFQKTiGNAΪlPLHC 'N-- jlSPHlTQvGSSPKgT|SGV-YIANGSGHDEPPHVGRKLS KSFL\ SSRBG 939
T42714 iii5liLVPslEQHLHFTREFDs|iSLRHYS AADTLDNVNLVSSPVHSGFL\ IΞRS G 996
T42715 l||||LVPS EQHLSFTREFDsiSLRHYS AADTLDNVNLVSSPVHSGFLv ύFj (H9RgG 975
1030 1040 1050 1060 1070 1080
Figure imgf000036_0002
T42715 ^RG|J3HHGMj3ll|PPRKCTAPTRITCRJ ^RHKLi! g|GLASRLV MgPAGgQ 1035
1090 1100 1110 1120 1130 1140
Figure imgf000037_0001
1150 1160 1170 1180 1190 1200
Figure imgf000037_0002
1210 1220 1230 1240 1250 1260
122
Figure imgf000037_0003
120
1270 1280 1290 1300 1310 1320
N0V3 993
Q9UFA4 791
015084 FNNIB GEQEYLYTDID 1049
Q9NCP8 B VAPEKLRKjS 1145
T42714 PSGEGVSNGYKGDATPNLRLLCSITS GTSPAQ EDΪTGTTPLTFIKDCVSFTTNVSARF 1285
T42715 PSGEGVSNGYKGDATPNLRLLCSITgGTSPAQWED|TGTTPLTFIKDCVSFTTNVSARF 1264
1330 1340 1350 1360 1370 1380
N0V3 993
Q9TJFA4 791
015084 --EL|DSDSETY 1059
Q9NCP8 VJJHVPKKK RFSLIW 1159
T42714 LADCiQVLETVGLASQLYRELICVPYMAKFWFAKTNDPVESSLRCFCMTDDRVDKTLEQ 1345
T42715 LADCIQVLETVGLASQLYRELICVPYMAKFWFAKTNDPVESSLRCFCMTDDRVDKTLEQ 1324
1390 1400 1410 1420 1430 1440
N0V3 993
Q9UFA4 791
015084 1059
Q9NCP8 1159
T42714 QENFEEVARSKDIEVLEGKPIYVDCYGNLAPLTKGGQQLVFNFYSFKENRLPFSIKIRDT 1405 T42715 QENFEEVARSKDIEVLEGKPIYVDCYGNLAPLTKGGQQLVFNFYSFKENRLPFSIKIRDT 1384
1450 1460 1470 1480 1490 1500 NOV3 993
Q9UFA4 791
015084 1059
Q9NCP8 -: 1159
T42714 SQEPCGRLSFLKΞPKTTKGLPQTAVCNLNITLPAHKKAEKADRRQSFASLALRKRYSYLT 1465
T42715 SQEPCGRLSFLKEPKTTKGLPQTAVCNLNITLPAHKKAEKADRRQSFASLALRKRYSYLT 1444
1510 1520 1530 1540 1550 1560
NOV3 993
Q9UFA4 791
015084 1059
Q9NCP8 1159
T42714 EPSMSPQSPCERTDIRMAIVADHLGLS TELARELNFSVDEINQIRVENPNSLISQSFML 1525
T42715 EPSMSPQSPCERTDIRMAIVADHLGLS TELARELNFSVDEINQIRVENPNSLISQSFML 1504
1570 1580 1590 1600 1610 1620
.... I .... I .... I .... I .... I .... I .... I .... I .... I .... I .... I .... I NOV3 993
Q9UFA4 791
015084 1059
Q9NCP8 1159
T42714 LKKWVTRDGKNATTDALTSVLTKINRIDIVTLLEGPIFDYGNISGTRSFADENNVFHDPV 1585 T42715 LKK VTRDGKNATTDALTSVLTKINRIDIVTLLEGPIFDYGNISGTRSFADENNVFHDPV 1564
1630 1640 1650 1660 1670 1680
NOV3 993
Q9UFA4 791
015084 1059
Q9NCP8 1159
T42714 D 1586
T42715 DGHPSFQVΞLETPMGLY TPPNPFQQDDHFSDISSIESPFRTPSRLSDGLVPSQGNIEHP 1624
1690 1700 1710 1720 1730 1740
N0V3 993
Q9TTFA4 791
015084 1059
Q9NCP8 1159
T42714 1586
T42715 TGGPPWTAEDTSLEDSKMDDSVTVTDPADPLDVDESQLKDLCQSECAQCWASVPGIPND 1684
1750 1760 1770 1780 1790 1800
N0V3 993
Q9UFA4 791
015084 1059
Q9NCP8 1159
T42714 1586
T42715 GRQAEPLRPQTRKVGMSSEQQEKGKSGPDEEVTEDKVKSLFEDIQLEEVEAEEMTEDQGQ 1744
1810 1820 1830 1840 1850 1860
N0 3 993
Q9UFA4 791 015084 1059
Q9NCP8 1159
T42714 G QNETPSGSLESPAQARRLTGGLLDRLDDSSDQARDSITSYL 1629
T42715 AMLNRVQRAELAMSSLAG QNETPSGSLESPAQARRLTGGLLDRLDDSSDQARDSITSYL 1804
1870 1880 1890 1900 1910 1920
NOV3 993
Q9UFA4 791
015084 1059
Q9NCP8 1159
T42714 TGEPGKIEANGNHTAEVIPEAKAKPYFPESQNDIGKQSIKENLKPKTHGCGRTEEPVSPL 1689
T42715 TGEPGKIEANGNHTAEVIPEAKAKPYFPESQNDIGKQSIKENLKPKTHGCGRTEEPVSPL 1864
1930 1940 1950 1960 1970 1980
NOV3 993
Q9UFA4 791
015084 1059
Q9NCP8 1159
T42714 TAYQKSLEETSKLVIEDAPKPCVPVGMKKMTRTTADGKARLNLQEEEGSTRSEPKQGEGY 17 9
T 2715 TAYQKSLEETSKLVIEDAPKPCVPVGMKKMTRTTADGKARLNLQΞEEGSTRSEPKQGEGY 1924
1990
NOV3 993
Q9UFA4 791
015084 1059
Q9NCP8 1159
T42714 KVKTKKEIRNVEKKTH 1765
T42715 KVKTKKEIRNVEKKTH 1940
The presence of identifiable domains in the disclosed NOV3 protein was determined by using Pfam and then determining the Interpro number. The results are listed in Table 3G with the statistics and domain description.
Figure imgf000039_0001
ANK dGrTPLHlAarnGhlewklLLeaGAdvnardk (SEQ ID NO: 143) |+|+ || |++ |+++++ +||+ ||++ +++
NOV3 LGRTCLHAAASGGNVECLNLLLSSGADLRRRDK (SEQ ID NO: 6)
Consistent with other known members of the 85.6kDa family of proteins, NOV3 contains ankyrin domains as illustrated in Table 3G.
The NOV3 nucleic acid, and the encoded polypeptide, according to the invention are useful in a variety of applications and contexts. For example, NOV3 nucleic acids and polypeptides can be used to identify proteins that are members of the ankyrin family of proteins. The NOV3 nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance NOV3 activity or function. Specifically, the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., red blood cell formation/organization, or signal transduction/cell activation. These molecules can be used to treat, e.g., spherocytosis.
In addition, the NOV3 nucleic acid and polypeptide according to the invention are useful, inter alia, as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins. For example, the NOV3 nucleic acid and polypeptide include structural motifs that are characteristic of proteins belonging to the family of transmembrane proteins/membrane skeleton proteins such as the ankyrin proteins. Ankyrin is a globular protein (200 kD) that links spectrin and an integral membrane protein (Band III) in the erythrocyte plasma membrane. Ankyrin belongs to a family of closely related polypeptides associated with the plasma membrane of cells in a variety of cell types (e.g. lymphocytes, platelets, fibroblasts and endothelial tissues). Ankyrin has been shown to underlie membrane proteins including CD44, the voltage-dependent sodium channel, NA+/K+ ATPase and the anion exchanger protein. Functional diversity between members of the ankyrin family is generated by the expression of multiple genes as well as alternative splicing of pre-mRna's. The formation of a direct connection between ankyrin and functionally important transmembrane proteins/membrane skeleton may be one of the earliest events to occur during signal transduction and cell activation.
The NOV3 nucleic acid and polypeptide, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in the mediation of signal transduction or cell activation. As such the NOV3 nucleic acid and polypeptide, antibodies and related compounds according to the invention may be used to treat genetic conditions, e.g., endometriosis, fertility, adrenoleukodystrophy, congenital adrenal hyperplasia, diabetes, Von Hippel-Lindau (vhl) syndrome , pancreatitis, obesity, hypeφarathyroidism, hypoparathyroidism, hyperthyroidism, hypothyroidism, SIDS, xerostomia, scleroderma, hypercalceimia, ulcers, cirrhosis, transplantation, inflammatory bowel disease, diverticular disease, hirschsprung's disease, crohn's disease, appendicitis, hemophilia, hypercoagulation, idiopathic thrombocytopenic puφura, autoimmume disease, allergies, immunodeficiencies, graft vesus host, anemia, ataxia- telangiectasia, lymphedema, tonsilitis, osteoporosis, hypercalceimia, arthritis, ankylosing spondylitis, scoliosis, tendinitis, muscular dystrophy, lesch-nyhan syndrome, myasthenia gravis, dental disease and infection, cardiomyopathy, atherosclerosis, hypertension, congenital heart defects, aortic stenosis, atrial septal defect (asd), atrioventricular (a-v) canal defect, ductus arteriosus, pulmonary stenosis, subaortic stenosis, ventricular septal defect (vsd), valve diseases, tuberous sclerosis, aneurysm, fibromuscular dysplasia, stroke, bleeding disorders, alzheimer's disease, parkinson's disease, huntington's disease, cerebral palsy, epilepsy, multiple sclerosis, ataxia-telangiectasia, leukodystrophies, behavioral disorders, addiction, anxiety, pain, neuroprotection, endocrine dysfunctions, growth and reproductive disorders, cystitis, incontinence, renal artery stenosis, interstitial nephritis, glomerulonephritis, polycystic kidney disease, systemic lupus erythematosus, renal tubular acidosis, iga nephropathy, or vesicoureteral reflux.
The NOV3 nucleic acid and polypeptide are useful for detecting specific cell types. For example, expression analysis has demonstrated that a NOV3 nucleic acid is expressed in adrenal gland/suprarenal gland, bone, brain, cartilage, cervix, coronary artery, platelets, kidney, kidney cortex, liver, mammary gland/breast, pancreas, placenta, salivary glands, spleen, synovium/synovial membrane, thymus, cerebral medulla/cerebral white matter, and left cerebellum. Additional utilities for the NOV3 nucleic acid and polypeptide according to the invention are disclosed herein.
NOV4
The NOV4 proteins descibed herein are novel myotonic dystrophy kinase-related CDC-42 binding kinase (MRCK)-like proteins. The NOV4 nucleic acids disclosed herein map to chromosome 1 lql3. Two alternative novel NOV4 nucleic acids and polypeptides are disclosed herein, namely NO V4a andNOV4b.
NOV4a
A NOV4 variant is NOV4a (alternatively referred to herein as CG56209-01), which encodes the 3835 nucleotide sequence (SEQ ID NO:7) shown in Table 4A. An open reading frame for the mature protein was identified beginning with an ATG codon at nucleotides 98-100 and ending with a TAG codon at nucleotides 3689-3691. Putative untranslated regions, if any, downstream from the termination codon and upstream from the initiation codon are underlined. The start and stop codons are in bold letters.
Table 4A. NOV4a Nucleotide Sequence (SEQ ID NO:7)
CGGACAGAGCCTCAGACGGTTGGGCGGACGGACGGCCCGACAGGCGGGCATGCGGGCGGCCAGACTGTAGCCGAG CAGCGAGGCTCCGGCCGCAGCCATGGAGCGGCGGCTGCGCGCGCTGGAGCAGCTGGCGCGGGGCGAGGCCGGCGG CTGCCCGGGGCTCGACGGCCTCCTAGATCTGCTGCTGGCGCTGCACCACGAGCTCAGCAGCGGCCCCCTACGGCG GGAGCGCAGCGTGGCGCAGTTCCTGAGCTGGGCCAGCCCCTTCGTATCAAAGGTGAAAGAACTGCGTCTGCAGAG AGATGACTTTGAGATCTTGAAGGTGATCGGCCGAGGAGCCTTTGGGGAGGTCACCGTGGTGAGGCAGAGGGACAC TGGGCAGATTTTTGCCATGAAAATGCTGCACAAGTGGGAGATGCTGAAGAGGGCTGAGACAGCCTGTTTCCGGGA GGAGCGGGATGTGCTCGTGAAAGGGGACAGCCGTTGGGTGACCACTCTGCACTATGCCTTCCAAGACGAGGAGTA CCTGTACCTTGTGATGGACTACTATGCTGGTGGGGACCTCCTGACGCTGCTGAGCCGCTTCGAGGACCGTCTCCC GCCCGAGCTGGCCCAGTTCTACCTGGCTGAGATGGTGCTGGCCATCCACTCGCTGCACCAGCTGGGTTATGTCCA CAGGGATGTCAAGCCAGACAACGTCCTGCTGGATGTGAACGGGCACATTCGCCTGGCTGACTTCGGCTCCTGCCT GCGTCTCAACACCAACGGCATGGTGGATTCATCAGTGGCAGTAGGGACGCCGGACTATATCTCCCCTGAGATCCT GCAGGCCATGGAGGAGGGCAAGGGCCACTACGGCCCACAGTGTGACTGGTGGTCGCTTGGAGTCTGCGCCTATGA GCTGCTCTTTGGGGAGACGCCCTTCTATGCTGAGTCCTTGGTGGAAACCTACGGCAAGATCATGAACCACGAGGA CCACCTGCAGTTCCCCCCGGACGTGCCTGACGTGCCAGCCAGCGCCCAAGACCTGATCCGCCAGCTGCTGTGTCG CCAGGAAGAGCGGCTAGGCCGTGGTGGGCTGGATGACTTCCGGAACCATCCTTTCTTCGAAGGCGTGGACTGGGA GCGGCTGGCGAGCAGCACGGCCCCCTATATTCCTGAGCTGCGGGGACCCATGGACACCTCCAACTTTGATGTGGA TGACGACACCCTCAACCATCCAGGGACCCTGCCACCGCCCTCCCACGGGGCCTTCTCCGGCCATCACCTGCCATT CGTGGGCTTCACCTACACCTCAGCTTGGGCTGCCCTGGAGCGGAAGCTCCAGTGTCTGGAGCAGGAGAAGCTCCC AGCTGGAGGAAGCCCGCAACTGAGGAAGGAGGTGGCCGCCCTGCGAGAGCAGCTGGAGCAGGCCCACAGCCACAG GCGTCTGCAGGAGGCCGAGAAGCAGAGCCAGGCCCTGCAACAGGAGCTCGCCATGCTGCGGGAGGAGCTGGAGCA GGAGAGCAAGCAGCGGCTGGAGGGTGAGCGGCGGGAGACGGAGAGCAACTGGGAGGCCCAGCTCGCCGACATCCT CAGCTGGGTGAATGATGAGAAGGTCTCAAGAGGCTACCTGCAGGCCCTGGCCACCAAGATGGCAGAGGAGCTGGA GTCCTTGAGGAACGTAGGCACCCAGGACCACCAGTGGAAGGCGCGGCGACTGCAGAAGATGGAGGCCTCGGCCAG GCTGGAGCTGCAGTCAGCGCTGGAGGCCGAGATCCGCGCCAAGCAGGGCCTGCAGGAGCGGCTGACACAGGTGCA GGAGGCCCAGCTGCAGGCTGAGGGCTGTCCCCCTCCCCAGCCCGGCTCACACACGCTGCGCCCCCGGAGCTTCCC ATCCCCGACCAAGTGTCTCCGCTGCACCTCGCTGATGCTGGGCCTGGGCCGCCAGGGCCTGGGTTGTGATTGCGG CTACTTTTGTCACACAACCTGTGCCCCACAGGCCCCACCCTGCCCCGTGCCCCCTGACCTCCTCCGCACAGCCCT GGGAGTACACCCCGAAACAGGCACAGGCACTGCCTATGAGGGCTTTCTGTCAGGTGTCCGGCGGGGCTGGCAGCG CGTGTTTGCTGCCCTGAGTGACTCACGCCTGCTGCTGTTTGACGCCCCTGACCTGAGGCTCAGCCCGCCCAGTGG GGCCCTCCTGCAGGTCCTAGATCTGAGGGACCCCCAGTTCTCGGCTACCCCTGTCCTGGCCTCTGATGTTATCCA TGCCCAATCCAGGGACCTGCCACGCATCTTTAGGGTGACAACCTCCCAGCTGGCAGTGCCGCCCACCACGTGCAC TGTGCTGCTGCTGGCAGAGAGCGAGGGGGAGCGGGAACGCTGGCTGCAGGTGCTGGGTGAGCTGCAGCGGCTGCT GCTGGACGCGCGGCCAAGACCCCGGCCCGTGTACACACTCAAGGAGGCTTACGACAACGGGCTGCCGCTGCTGCC TCACACGCTCTGCGCTGCCATCCTCGACCAGGATCGACTTGCGCTTGGCACCGAGGAGGGGCTCTTTGTCATCCA TCTGGACATCTTCCAGGTGGGGGAGTGCCGGCGCGTGCAGCAGCTGACCTTGAGCCCCAGTGCAGGCCTGCTGGT CGTGCTGTGTGGCCGCGGCCCCAGCGTGCGTCTCTTTGCCCTGGCGGAGCTGGAGAACATAGAGGTAGCAGGTGC CAAGATCCCCGAGTCTCGAGGCTGCCAGGTGCTGGCAGCTGGAAGCATCCTGCAGGCCCGCACCCCGGTGCTCTG TGTAGCCGTCAAGCGCCAGGTGCTCTGCTACCAGCTGGGCCCGGGCCCTGGGCCCTGGCAGCGCCGCATCCGTGA GCTGCAGGCACCTGCCACTGTGCAGAGCCTGGGGCTGCTGGGCGACCGGCTATGTGTGGGCGCCGCCGGTGGCTT TGCACTCTACCCGCTGCTCAACGAGGCTGCGCCGTTGGCGCTGGGGGCCGGTTTGGTGCCTGAGGAGCTGCCACC ATCCCGCGGGGGCCTGGGTGAGGCACTGGGTGCCGTGGAGCTTAGCCTCAGCGAGTTCCTGCTACTCTTCACCAC TGCTGGCATCTACGTGGATGGCGCAGGCCGCAAGTCTCTGTTCAGCGAGAACTCCATCGATGTGTTTGACGTGAG GAGGGCAGAATGGGTGCAGACCGTGCCGCTCAAGAAGGTGCGGCCCCTCAATCCAGAGGGCTCCCTGTTCCTCTA CGGCACCGAGAAGGACGAGTTCGACATCCCGGACCTCACCGACAACAGCCGGCGCCAGCTGTTCCGCACCAAGAG CAAGCGCCGCTTCTTTTTCCGCGTGTCGGAGGAGCAGCAGAAGCAGCAGCGCAGGGAGATGCTGAAGGACCCTTT TGTGCGCTCCAAGCTCATCTCGCCGCCTACCAACTTCAACCACCTAGTACACGTGGGCCCTGCCAACGGGCGGCC CGGCGCCAGGGACAAGTCCCCGGTTAGTCCTGCTCCAGAATTTGGAAATCCTAGTTTCCTCTCCTTCGTATCCCG AGTCTGGGACACAAAACTCCGCCCCCAGCCTATGAGCATCCTGAGCCCCGCCCTCTTCCTGACGAAACTGGCCCC GGATCAGAGCAGGACCTCCCTTACGCCACTGCACTCCAGCCTGGCCGACAGCAAGAGTCTGTCTCCCTCCTCCAC TCCCCATGAGCCCTAGGACGGGTCACTCATCCTCTCAGAGCCTCAGTTCCCAGCCCTGGAGGGAGATGAGGTTTC CCAGCCCCACAGGGCTGTTGTGAGGCTGACGTGCCCTCATGGCCAAGGGCTGTCTGTAGCCTGGCCCCCGTATCC TCTTGGGGTT
The NOV4a protein (SEQ ID NO:8) encoded by SEQ ID NO:7 is 1197 amino acid residues in length and is presented using the one-letter amino acid code in Table 4B. The SignalP, Psort and/or Hydropathy results indicate that NOV4a has no known signal peptide and is likely to be localized in the nucleus with a certainty of 0.7600. Alternatively, a NOV4a polypeptide is located to the microbody (peroxisome) with a certainty of 0.3114, the lysosome (lumen) with a certainty of 0.1772, or the mitochondrial matrix space with a certainty of 0.1000.
Table 4B. Encoded NOV4a Protein Sequence (SEQ ID NO: 8)
MERRLRALEQLARGEAGGCPGLDGLLDLLLALHHELSSGPLRRERSVAQFLSWASPFVSKVKELRLQRDDFEILK VIGRGAFGEVTWRQRDTGQIFAMKMLHKEMLKRAETACFREERDVLVKGDSRVTTLHYAFQDEEYLYLVMDY YAGGDLLTLLSRFEDRLPPELAQFYLAEMVLAIHSLHQLGYVHRDVKPDNVLLDVNGHIRLADFGSCLRLNTNGM VDSSVAVGTPDYISPEILQAMEEGKGHYGPQCD WSLGVCAYELLFGETPFYAESLVETYGKIMNHΞDHLQFPPD VPDVPASAQDLIRQLLCRQEERLGRGGLDDFRNHPFFEGVD ERLASSTAPYIPELRGPMDTSNFDVDDDTLNHP GTLPPPSHGAFSGHHLPFVGFTYTSAWAALERKLQCLEQEKLPAGGSPQLRKEVAALREQLEQAHSHRRLQEAΞK QSQALQQELAMLREELEQESKQRLEGERRETESNEAQLADILS VNDEKVSRGYLQALATKMAEELESLRNVGT QDHQ KARRLQKMEASARLELQSALEAEIRAKQGLQERLTQVQEAQLQAEGCPPPQPGSHTLRPRSFPSPTKCLR CTSLMLGLGRQGLGCDCGYFCHTTCAPQAPPCPVPPDLLRTALGVHPETGTGTAYEGFLSGVRRG QRVFAALSD SRLLLFDAPDLRLSPPSGALLQVLDLRDPQFSATPVLASDVIHAQSRDLPRIFRVTTSQLAVPPTTCTVLLLAΞS EGERER LQVLGELQRLLLDARPRPRPVYTLKEAYDNGLPLLPHTLCAAILDQDRLALGTEEGLFVIHLDIFQVG ECRRVQQLTLSPSAGLLWLCGRGPSVRLFALAELENIEVAGAKIPESRGCQVLAAGSILQARTPVLCVAVKRQV LCYQLGPGPGP QRRIRELQAPATVQSLGLLGDRLCVGAAGGFALYPLLNEAAPLALGAGLVPEELPPSRGGLGE ALGAVELSLSEFLLLFTTAGIYVDGAGRKSLFSΞNSIDVFDVRRAE VQTVPLKKVRPLNPEGSLFLYGTEKDΞF DIPDLTDNSRRQLFRTKSKRRFFFRVSEEQQKQQRREMLKDPFVRSKLISPPTNFNHLVHVGPANGRPGARDKSP VSPAPEFGNPSFLSFVSRVDTKLRPQPMSILSPALFLTKLAPDQSRTSLTPLHSSLADSKSLSPSSTPHEP
SNP variants of NOV4a are disclosed in Example 2. NOV4b
Alternatively, a NOV4 variant is NOV4b (alternatively referred to herein as CG56209-02), which includes the 3985 nucleotide sequence (SEQ ID NO:9) shown in Table 4C. An open reading frame for the mature protein was identified beginning with an ATG codon at nucleotides 98- 100 and ending with a TAG codon at nucleotides 3839-3841. Putative untranslated regions, if any, downstream from the termination codon and upstream from the initiation codon are underlined. The start and stop codons are in bold letters.
Table 4C. NOV4b Nucleotide Sequence (SEQ ID NO:9)
CGGACAGAGCCTCAGACGGTTGGGCGGACGGACGGCCCGACAGGCGGGCATGCGGGCGGCCAGACTGTAGCCGAG CAGCGAGGCTCCGGCCGCAGCCATGGAGCGGCGGCTGCGCGCGCTGGAGCAGCTGGCGCGGGGCGAGGCCGGCGG CTGCCCGGGGCTCGACGGCCTCCTAGATCTGCTGCTGGCGCTGCACCACGAGCTCAGCAGCGGCCCCCTACGGCG GGAGCGCAGCGTGGCGCAGTTCCTGAGCTGGGCCAGCCCCTTCGTATCAAAGGTGAAAGAACTGCGTCTGCAGAG AGATGACTTTGAGATCTTGAAGGTGATCGGCCGAGGAGCCTTTGGGGAGGTCACCGTGGTGAGGCAGAGGGACAC TGGGCAGATTTTTGCCATGAAAATGCTGCACAAGTGGGAGATGCTGAAGAGGGCTGAGACAGCCTGTTTCCGGGA GGAGCGGGATGTGCTCGTGAAAGGGGACAGCCGTTGGGTGACCACTCTGCACTATGCCTTCCAAGACGAGGAGTA CCTGTACCTTGTGATGGACTACTATGCTGGTGGGGACCTCCTGACGCTGCTGAGCCGCTTCGAGGACCGTCTCCC GCCCGAGCTGGCCCAGTTCTACCTGGCTGAGATGGTGCTGGCCATCCACTCGCTGCACCAGCTGGGTTATGTCCA CAGGGATGTCAAGCCAGACAACGTCCTGCTGGATGTGAACGGGCACATTCGCCTGGCTGACTTCGGCTCCTGCCT GCGTCTCAACACCAACGGCATGGTGGATTCATCAGTGGCAGTAGGGACGCCGGACTATATCTCCCCTGAGATCCT GCAGGCCATGGAGGAGGGCAAGGGCCACTACGGCCCACAGTGTGACTGGTGGTCGCTTGGAGTCTGCGCCTATGA GCTGCTCTTTGGGGAGACGCCCTTCTATGCTGAGTCCTTGGTGGAAACCTACGGCAAGATCATGAACCACGAGGA CCACCTGCAGTTCCCCCCGGACGTGCCTGACGTGCCAGCCAGCGCCCAAGACCTGATCCGCCAGCTGCTGTGTCG CCAGGAAGAGCGGCTAGGCCGTGGTGGGCTGGATGACTTCCGGAACCATCCTTTCTTCGAAGGCGTGGACTGGGA GCGGCTGGCGAGCAGCACGGCCCCCTATATTCCTGAGCTGCGGGGACCCATGGACACCTCCAACTTTGATGTGGA TGACGACACCCTCAACCATCCAGGGACCCTGCCACCGCCCTCCCACGGGGCCTTCTCCGGCCATCACCTGCCATT CGTGGGCTTCACCTACACCTCAGCTTGGGCTGCCCTGGAGCGGAAGCTCCAGTGTCTGGAGCAGGAGAAGCTCCC AGCTGGAGGAAGCCCGCAACTGAGGAAGGAGGTGGCCGCCCTGCGAGAGCAGCTGGAGCAGGCCCACAGCCACAG GCGTCTGCAGGAGGCCGAGAAGCAGAGCCAGGCCCTGCAACAGGAGCTCGCCATGCTGCGGGAGGAGCTGGAGCA GGAGAGCAAGCAGCGGCTGGAGGGTGAGCGGCGGGAGACGGAGAGCAACTGGGAGGCCCAGCTCGCCGACATCCT CAGCTGGGTGAATGATGAGAAGGTCTCAAGAGGCTACCTGCAGGCCCTGGCCACCAAGATGGCAGAGGAGCTGGA GTCCTTGAGGAACGTAGGCACCCAGGACCACCAGTGGAAGGCGCGGCGACTGCAGAAGATGGAGGCCTCGGCCAG GCTGGAGCTGCAGTCAGCGCTGGAGGCCGAGATCCGCGCCAAGCAGGGCCTGCAGGAGCGGCTGACACAGGTGCA GGAGGCCCAGCTGCAGGCTGAGGGCTGTCCCCCTCCCCAGCCCGGCTCACACACGCTGCGCCCCCGGAGCTTCCC ATCCCCGACCAAGTGTCTCCGCTGCACCTCGCTGATGCTGGGCCTGGGCCGCCAGGGCCTGGGTTGTGATTGCGG CTACTTTTGTCACACAACCTGTGCCCCACAGGCCCCACCCTGCCCCGTGCCCCCTGACCTCCTCCGCACAGCCCT GGGAGTACACCCCGAAACAGGCACAGGCACTGCCTATGAGGGCTTTCTGTCAGGTGTCCGGCGGGGCTGGCAGCG CGTGTTTGCTGCCCTGAGTGACTCACGCCTGCTGCTGTTTGACGCCCCTGACCTGAGGCTCAGCCCGCCCAGTGG GGCCCTCCTGCAGGTCCTAGATCTGAGGGACCCCCAGTTCTCGGCTACCCCTGTCCTGGCCTCTGATGTTATCCA TGCCCAATCCAGGGACCTGCCACGCATCTTTAGGGTGAGTGCCTGGTCCCAGCTGGCAGTGCCGCCCACCACGTG CACTGTGCTGCTGCTGGCAGAGAGCGAGGGGGAGCGGGAACGCTGGCTGCAGGTGCTGGGTGAGCTGCAGCGGCT GCTGCTGGACGCGCGGCCAAGACCCCGGCCCGTGTACACACTCAAGGAGGCTTACGACAACGGGCTGCCGCTGCT GCCTCACACGCTCTGCGCTGCCATCCTCGACCAGGATCGACTTGCGCTTGGCACCGAGGAGGGGCTCTTTGTCAT CCATCTGGACATCTTCCAGGTGGGGGAGTGCCGGCGCGTGCAGCAGCTGACCTTGAGCCCCAGTGCAGGCCTGCT GGTCGTGCTGTGTGGCCGCGGCCCCAGCGTGCGTCTCTTTGCCCTGGCGGAGCTGGAGAACATAGAGGTAGCAGG TGCCAAGATCCCCGAGTCTCGAGGCTGCCAGGTGCTGGCAGCTGGAAGCATCCTGCAGGCCCGCACCCCGGTGCT CTGTGTAGCCGTCAAGCGCCAGGTGCTCTGCTACCAGCTGGGCCCGGGCCCTGGGCCCTGGCAGCGCCGCATCCG TGAGCTGCAGGCACCTGCCACTGTGCAGAGCCTGGGGCTGCTGGGCGACCGGCTATGTGTGGGCGCCGCCGGTGG CTTTGCACTCTACCCGCTGCTCAACGAGGCTGCGCCGTTGGCGCTGGGGGCCGGTTTGGTGCCTGAGGAGCTGCC ACCATCCCGCGGGGGCCTGGGTGAGGCACTGGGTGCCGTGGAGCTTAGCCTCAGCGAGTTCCTGCTACTCTTCAC CACTGCTGGCATCTACGTGGATGGCGCAGGCCGCAAGTCTCGTGGCCACGAGCTGTTGTGGCCAGCAGCGCCCCC TGGCGTGCCCGCAGGGTATGCGGCCCCCTACCTGACAGTGTTCAGCGAGAACTCCATCGATGTGTTTGACGTGAG GAGGGCAGAATGGGTGCAGACCGTGCCGCTCAAGAAGGTGAGGGTCCGCCAGAGCCCTGGGCTGCCTCAGGTGCG GCCCCTCAATCCAGAGGGCTCCCTGTTCCTCTACGGCACCGAGAAGGTCCGCCTGACCTACCTCAGGAACCAGCT GGCAGGTGAGGGAGACGAGTTCGACATCCCGGACCTCACCGACAACAGCCGGCGCCAGCTGTTCCGCACCAAGAG CAAGCGCCGCTTCTTTTTCCGCGTGTCGGAGGAGCAGCAGAAGCAGCAGCGCAGGGAGATGCTGAAGGACCCTTT TGTGCGCTCCAAGCTCATCTCGCCGCCTACCAACTTCAACCACCTAGTACACGTGGGCCCTGCCAACGGGCGGCC CGGCGCCAGGGACAAGTCCCCGGTTAGTCCTGCTCCAGAATTTGGAAATCCTAGTTTCCTCTCCTTCGTATCCCG AGTCTGGGACACAAAACTCCGCCCCCAGCCTATGAGCATCCTGAGCCCCGCCCTCTTCCTGACGAAACTGGCCCC GGATCAGAGCAGGACCTCCCTTACGCCACTGCACTCCAGCCTGGCCGACAGCAAGAGTCTGTCTCCCTCCTCCAC TCCCCATGAGCCCTAGGACGGGTCACTCATCCTCTCAGAGCCTCAGTTCCCAGCCCTGGAGGGAGATGAGGTTTC CCAGCCCCACAGGGCTGTTGTGAGGCTGACGTGCCCTCATGGCCAAGGGCTGTCTGTAGCCTGGCCCCCGTATCC TCTTGGGGTT
The NOV4b protein (SEQ ID NO:10) encoded by SEQ ID NO:9 is 1247 amino acid residues in length and is presented using the one-letter amino acid code in Table 4D. The SignalP, Psort and/or Hydropathy results indicate that NOV4b has no known signal peptide and is likely to be localized in the nucleus with a certainty of 0.8800. Alternatively, a NOV4b polypeptide is located to the microbody (peroxisome) with a certainty of 0.3226, the lysosome (lumen) with a certainty of 0.1925, or the mitochondrial matrix space with a certainty of 0.1000.
Table 4D. Encoded NOV4b Protein Sequence (SEQ ID NO:10)
MERRLRALEQLARGEAGGCPGLDGLLDLLLALHHELSSGPLRRERSVAQFLS ASPFVSKVKELRLQRDDFEILK VIGRGAFGEVTWRQRDTGQIFAMKMLHKEMLKRAETACFREERDVLVKGDSR VTTLHYAFQDEEYLYLVMDY YAGGDLLTLLSRFEDRLPPELAQFYLAEMVLAIHSLHQLGYVHRDVKPDNVLLDVNGHIRLADFGSCLRLNTNGM VDSSVAVGTPDYISPEILQAMEEGKGHYGPQCDW SLGVCAYELLFGETPFYAESLVETYGKIMNHEDHLQFPPD VPDVPASAQDLIRQLLCRQEERLGRGGLDDFRNHPFFEGVD ERLASSTAPYIPELRGPMDTSNFDVDDDTLNHP GTLPPPSHGAFSGHHLPFVGFTYTSAWAALERKLQCLEQEKLPAGGSPQLRKEVAALREQLΞQAHSHRRLQEAEK QSQALQQELAMLREELEQESKQRLEGERRETESNWEAQLADILS VNDEKVSRGYLQALATKMAEELESLRNVGT QDHQ KARRLQKMEASARLELQSALEAEIRAKQGLQERLTQVQEAQLQAEGCPPPQPGSHTLRPRSFPSPTKCLR CTSLMLGLGRQGLGCDCGYFCHTTCAPQAPPCPVPPDLLRTALGVHPETGTGTAYEGFLSGVRRG QRVFAALSD SRLLLFDAPDLRLSPPSGALLQVLDLRDPQFSATPVLASDVIHAQSRDLPRIFRVSAWSQLAVPPTTCTVLLLAE SEGERER LQVLGELQRLLLDARPRPRPVYTLKEAYDNGLPLLPHTLCAAILDQDRLALGTEEGLFVIHLDIFQV GECRRVQQLTLSPSAGLLWLCGRGPSVRLFALAΞLENIEVAGAKIPESRGCQVLAAGSILQARTPVLCVAVKRQ VLCYQLGPGPGPWQRRIRELQAPATVQSLGLLGDRLCVGAAGGFALYPLLNEAAPLALGAGLVPEELPPSRGGLG EALGAVELSLSEFLLLFTTAGIYVDGAGRKSRGHELL PAAPPGVPAGYAAPYLTVFSENSIDVFDVRRAE VQT VPLKKVRVRQSPGLPQVRPLNPEGSLFLYGTEKVRLTYLRNQLAGEGDEFDIPDLTDNSRRQLFRTKSKRRFFFR VSEEQQKQQRREMLKDPFVRSKLISPPTNFNHLVHVGPANGRPGARDKSPVSPAPEFGNPSFLSFVSRVDTKLR PQPMSILSPALFLTKLAPDQSRTSLTPLHSSLADSKSLSPSSTPHEP
NO 4 Clones
Unless specifically addressed as NOV4a or NOV4b, any reference to NOV4 is assumed to encompass all variants. A search against the Patp database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 4E.
Table 4E. PatP Results for NOV4
Smallest
High Sum
Sequences Producing High-Scoring Segment Pairs: Score Prob P (N) patp:AAB42069 Human ORFX ORF1833 polypeptide sequence 1516 1.4e-189 patp:AAU17106 Novel signal transduction pathway protein - human 1471 1.6e-150 patp:AAR56979 Human myotonic dystrophy gene protein 1326 8.5e-138 patp:AAR38860 Myotonic dystrophy protein - Human 1314 2.6e-136 patp:AAR41000 Human brain cDNA clone C28 protein kinase 1215 2.2e-123
h a BLAST search of public sequence databases, it was found, for example, that the NOV4a nucleic acid sequence of this invention has 865 of 865 bases (100%) identical to a gb:GENBANK-ro:HSMDPKJN|acc:Y12337.1 mRNA from H.sapiens mRNA for myotonic dystrophy protein kinase like protein. Further, the full amino acid sequence of the disclosed NOV4a protein of the invention has 314 of 572 amino acid residues (54%) identical to, and 418 of 572 amino acid residues (73%) similar to, the 1732 amino acid residue ptnr:SPTREMBL- ACC:O54874 protein from Rattus norvegicus (Rat) (MYTONIC DYSTROPHY KINASE- RELATED CDC42-BINDING KINASE).
Additional BLAST results are shown in Table 4F.
Figure imgf000046_0001
Figure imgf000047_0002
A multiple sequence alignment is given in Table 4G, with the NOV4 proteins of the invention being shown in lines 1 and 2 in a ClustalW analysis comparing NOV4 with related protein sequences of Table 4F.
Table 4G. ClustalW Analysis of NOV4
1. SEQ ID NO.: 8 NOV4a 5. SEQ ID NO.: 146 001583
2. SEQ ID NO.: 10 NOV4b 6. SEQ ID NO.: 147 054874
3. SEQ ID NO.: 144 Q9W1B0 7. SEQ ID NO.: 148 054875
4. SEQ ID NO.: 145 044368
Figure imgf000047_0001
Figure imgf000048_0001
370 380 390 400 410 420
Figure imgf000048_0002
054874 SLTRKLQE(STQTVQALQYSTVDGPLTASKDLEIKSHKEJ| DEKJJRJKQVAEVNHLEQQ 512 054875 SLSRKLQE^TQTVQSLHGgTR-ALGNSNRDKE| -sERMRjSKMADSNRLERQ 510
Figure imgf000049_0001
670 680 690 700 710 720
NOV a 44
Figure imgf000049_0002
730 740 750 760 770 780
Figure imgf000049_0003
Figure imgf000050_0001
970 980 990 1000 1010 1020
NO 4a 576
N0V4b 576
Q9W1B0 PFQKKETELRDLQKGGLEYJS^SFLNKSTHHG-LSSAFFRDMSKNSEIIDSAESFGNESGD 926
044368 SFQKKETELRDFEKGGLEYBSSFLNKSTHHG-LSSAFFRDMSKNSEIIDSAESFGNESGD 902
001583 AMLKQQKNIENS- -SJJSAFSSTMGRGDLMISMNNDYEMSNSSLMRQEMISRQSTP 910
054874 IIEQLIKDTEE-LR- -gETtGVEHRDSQH-SFLAFLNTPTDALDQFERSPSCTP-AGK 978
054875 4QSLKKRMEEKFR- -^DTGLKLPDFQD-PIFEYFNTAPLAHDLTFRTSSASDQETQ 977
1030 1040 1050 1060 1070 1080
Figure imgf000050_0002
1090 1100 1110 1120 1130 1140
Figure imgf000050_0003
1210 1220 1230 1240 1250 1260
Figure imgf000051_0001
1270 1280 1290 1300 1310 1320
Figure imgf000051_0002
1330 1340 1350 1360 1370 1380
Figure imgf000051_0003
1390 1400 1410 1420 1430 1440
Figure imgf000051_0004
1450 1460 1470 1480 1490 1500
Figure imgf000051_0005
1510 1520 1530 1540 1550 1560
Figure imgf000051_0006
054875 |Dj| |P |PSLAJi|QQSF-3iC 5ii SE lajglBlc^SHi 1446
1570 1580 1590 1600 1610 1620
Figure imgf000052_0001
1630 1640 1650 1660 1670 1680
Figure imgf000052_0002
1690 1700 1710 1720 1730 1740
Figure imgf000052_0003
1750 1760 1770 1780 1790 1800
N0V4a ■-RPGARD- -KSPΪSPAPEFGNP SFLSFVSRVWDTK 1148
N0V4b --RPGARD- -KSPJSPAPEFGNP SFLSFVSRVWDTK 1198
Q9 1B0 -NQRJJJLDLPTTLET ADQACSPIIHS 1589
044368 -NQR|LDLPTTLET ADQACSPIIHS 1565
001583 _ 1558
054874 MNPRPQESRTVFSGSVSIPSITKSRPEPGRSMSASSGLSARSSAQNGSALKREFSGGSYN 1656
054875 LSVRPQPRR KSRILPQQASLGS- -LPSR NKPYVSWPSSGGSEP 1651
1810 1820 1830 1840 1850 1860
Figure imgf000052_0004
1870
N0V4a TPHEP 1197
N0V4b TPHEP 1247
Q9W1B0 GLSNND 1637 044368 GLSNND 1613
001583 VNND 1590
054874 KTKSLSLESTDRGSWDP 1732
054875 GASSL 1702
The presence of identifiable domains in the disclosed NOV4 protein was determined by using Pfam and then determining the Interpro number. The results are listed in Table 4H with the statistics and domain description.
Table 4H. Domain Analysis of NON4
PSSMs Producing Significant Alignments Score E (bits) Value p inase : domain 1 of 1 , from 71 to 337 231.5 1.3e-65
PKinase yelleklGeGsfGkVykakhk.tgkivAvKilkkesls 1
+++++++ I + |++|+| ++ +++++++ I+I+++++++ ++ ++ + Ν0V4 FEILKVIGRGAFGEVTWRQRdTGQIFAMKMLHKWEMLkraetacfR
PKinase rEiqilkrlsHpNIvrllgvfedtddhlylvmEymegGdLfdylrrng .p
I ++ + + + +++++++ +++++++++++ +|+| ++++++ ++
NOV4 EERDVLVKGDSRWVTTLHYAFQ-DEEYLYLVMDYYAGGDLLTLLSRFEdR
PKinase lsekeakkialQilrGleYLHsngivHRDLKpeNILldengtvKiaDFGL ++++ ++++ +++ ++ || + + I I I +| ++ I +| ++ +++++++ 1 1 1
NOV4 LPPELAQFYLAEMVLAIHSLHQLGYVHRDVKPDNVLLDVNGHIRLADFGS
PKinase Aril eklttfvGTpwYmmAPEvileg rgysskvDvWSlGv
+ +++++ + + +| I ++|+ + I |+ ++ ++++ ++++++ I + | I + | + NOV4 CLRLntngmVDSSVAVGTPDYI-SPEI-LQAmeegkGHYGPQCDWWSLGV
PKinase iLyElltggplfpgadlpaftggdevdqliifvlklPfsdelpktridpl
+ I ++++ ++ I ++ ++ NOV4 CAYELLFG ETPFYA ESLV
PKinase eelfrikkr..rlplpsncSee... lkdLlkkcLnkDPskRpGsatakei
+ + ++ ++++++ +++ +++ + ++++|++++|++ ++|+|++ ++
NOV4 ETYGKIMNHedHLQFPPDVPDVpasAQDLIRQLLCRQ-EERLGRGGLDDF
PKinase lnhpwf (SEQ ID NO: 149)
+++++ NOV4 RNHPFF (SEQ ID NO: 8)
Consistent with other known members of the myotonic dystrophy kinase-related Cdc42- binding kinases (MRCKs) family of proteins, NOV4 contains protein kinase domains as illustrated in Table 4H. NON4 nucleic acids, and the encoded polypeptides, according to the invention are useful in a variety of applications and contexts. For example, ΝON4 nucleic acids and polypeptides can be used to identify proteins that are members of the protein kinase family of proteins. The ΝON4 nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance ΝON4 activity or function. Specifically, the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., cytoskeletal reorganization or molecular switch mechanisms. These molecules can be used to treat, e.g., myotonic dystrophy, myotonic dystrophy type 2, proximal myotonic myopathy, or proximal myotonic dystrophy. In addition, various ΝOV4 nucleic acids and polypeptides according to the invention are useful, ter alia, as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins. For example, the NOV4 nucleic acids and their encoded polypeptides include structural motifs that are characteristic of proteins belonging to the family of protein kinases such as the MRCK protein. MRCK is a Ser/Thr kinase that is highly related to myotonic dystrophy kinase and ROKs. MRCK contains an N-terminal kinase domain, a coiled-coil region, a cysteine-rich domain (CR), a pleckstrin-like domain (PH), and a C-terminal p21 GTPase-binding domain (GBD). Two different MRCK genes are expressed in rat. MRCKa mRNA is enriched in brain and lung, while MRCKb mRNA is expressed in lung and kidney. MRCKa phosphorylates Ser/Thr residues in myelin basic protein, histone HI, and non-muscle myosin regulatory light chain. In HeLa cells, expression of kinase-dead MRCKa blocks Cdc42-dependent formation of focal complexes and peripheral microspikes, while in PC 12 cells MRCKa may act downstream of Cdc42 and Racl to promote neurite outgrowth.
The NOV4 nucleic acids and polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in the mediation of cell migration and differentiation. As such the NOV4 nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat muscle, or cell migration disorders, e.g., myotonic dystrophy, myotonic dystrophy type 2, proximal myotonic myopathy, proximal myotonic dystrophy, neuromuscular diseases associated with cardiomyopathy, multiple endocrine neoplasia type 1(MEN1), insulin dependent diabetes mellitus, familial paraganglioma type 2, spinocerebellar ataxia type 5, Bardet-Biedl syndrome, non-hodgkins lymphoma, cancers such as breast cancer, liver, lung, pancrease, and prostate cancers. The NON4 nucleic acids and polypeptides are useful for detecting specific cell types. For example, expression analysis has demonstrated that a ΝON4 nucleic acid is expressed in adrenal gland, bone marrow, brain - amygdala, brain - cerebellum, brain - hippocampus, brain - substantia nigra, brain - thalamus, brain -whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma - Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea, and uterus.
Additional utilities for ΝON4 nucleic acids and polypeptides according to the invention are disclosed herein.
ΝOV5
The disclosed NOV5 nucleic acid (alternatively referred to herein as CG56288-01) encodes a novel SI 00 calcium binding-like protein and includes the 332 nucleotide sequence (SEQ ID NO: 11) shown in Table 5 A. Although SignalP, Psort and/or hydropathy suggest that the SI 00 Calcium Binding Protein-like protein may be localized in the cytoplasm, the protein predicted here is similar to the SI 00 Calcium Binding Protein family, some members of which are secreted. Therefore it is likely that this novel SI 00 Calcium Binding Protein-like protein is available at the same sub-cellular localization and hence accessible to a diagnostic probe and for various therapeutic applications.
An open reading frame for the mature protein was identified beginning with an ATG initiation codon at nucleotides 8-10, and ending with a TGA stop codon at nucleotides 320-322. Putative untranslated regions, if any, are found upstream from the initiation codon and downstream from the termination codon. The start and stop codons are in bold letters.
Table 5A. NOV5 Nucleotide Sequence (SEQ ID NO:ll)
CTCCAACATGGCAAAAATCTCCAGCCCTACAGAGACTGTGCGGTGCATTCAGTCCCTGATTGCTGTTTTCCAGAA GTATGCTGGAAAGGATGGTTACAACCGCAATCTCTCCAAGACGGAGTTCCTAAGCTTCATGAATACAGAGCTGGC TGCCTTTACAAAGAACCAGAAGGACCCCGGTGTCCTTGACCGCATGAAGAAACTGGATGTCAGCAGCGATGGGCA GTTAGATTTCCCAAAATTTCTTAATCTGATTGGCGGCCTAGCTGTGGCTTGCCATGACTCCTTCCTCAAGGCTGT CCCTTCCCAGAAGTGGAACTGAGGACCCCATG
The NOV5 protein (SEQ ID NO: 12) encoded by SEQ ID NO: 11 is 104 amino acid residues in length and is presented using the one-letter amino acid code in Table 5B. The SignalP, Psort and/or Hydropathy results indicate that NON5 has no known signal peptide and is likely to be localized in the mitochondrial matrix space with a certainty of 0.5964. Alternatively, a ΝON5 polypeptide is located to the mitochondrial inner membrane with a certainty of 0.3037, the mitochondrial intermembrane space with a certainty of 0.3037, or the mitochondrial outer membrane with a certainty of 0.3037.
Table 5B. Encoded ΝOV5 Protein Sequence (SEQ ID NO:12)
MAKISSPTΞTVRCIQSLIAVFQKYAGKDGYNRNLSKTEFLSFMNTELAAFTKNQKDPGVLDRMKKLDVSSDGQLD FPKFLNLIGGLAVACHDSFLKAVPSQKWN
A search against the Patp database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 5C.
Table 5C. PatP Results for NO V5
Smallest
High Sum
Sequences Producing High-Scoring Segment Pairs: Score Prob P (N) patp:AAB58356 Lung cancer associated polypeptide sequence - human 467 4.0e-44 ρatp:AAB45541 Human S100A11 protein - Homo sapiens 467 4.0e-44 patp:AAU31484 Novel human secreted protein #1975 232 3.2e-19 patp:AAB45531 Human S 100A1 protein 167 2.5e-12 patp:AAM40258 Human polypeptide 167 2.5e-12
In a BLAST search of public sequence databases, it was found, for example, that the NOV5 nucleic acid sequence of this invention has 305 of 335 bases (91%) identical to a gb:GENBANK-ID:HUMS100CPl|acc:D49355.1 mRNA from Human mRNA for S100C protein, complete eds. Further, the full amino acid sequence of the disclosed NON5 protein of the invention has 102 of 103 amino acid residues (99%) identical to, and 102 of 103 amino acid residues (99%) similar to, the 104 amino acid residue ptnr:SPTREMBL-ACC:Q9UDP3 protein from Human (WUGSC:H_ΝH0456Ν16.1 PROTEIN). The NON5 protein of the invention also has homolgy to the proteins shown in the
BLASTP data in Table 5D.
Figure imgf000057_0001
A multiple sequence alignment is given in Table 5E, with the NON5 protein of the invention being shown in line 1 in a ClustalW analysis comparing ΝON5 with related protem sequences of Table 5D.
Table 5E. ClustalW Analysis of ΝON5
1. SEQ ID NO.: 12 NOV5 4. SEQ ID NO.: 152 P31949
2. SEQ ID NO. : 150 Q9UDP3 5. SEQ ID NO.: 153 P24480
3. SEQ ID NO.: 151 060417 6. SEQ ID NO.: 154 P50543
10 20 30 40 50 60
MAKISSPTETfflRCIWSLIAVFQKYAGKDGYNG fflLSKTEFLSFMNTELAAFTKNQKDPGVL 60
MAKISSPTΞTΒ|RCI[SLI VFQKYAGKDGYNB GT ISKTEFLSFMNTELAAFTKNQKDPGVL 60 MAKISSPTETERCIESLIAVFQKYAGKDGYNR GLSKTEFLSFMNTELAAFTKNQKDPGVL 60 MAKISSPTETERCIESLIAVFQKYAGKDGYNG "LSKTEFLSFMNTELAAFTKNQKDPGVL 60 SΞPTETERCIESLIAVFQKYAGKDG LSKTEFLSFMNTELAAFTKNQKDPGVL 57
PTETERCIESLIAVFQKY GKDGfiM MLSKTEFLSFMNTELAAFTKNQKDPGVL 55
Figure imgf000058_0001
The presence of identifiable domains in the disclosed NOV5 protein was determined by using Pfam and then determining the Interpro number. The results are listed in Table 5F with the statistics and domain description.
Figure imgf000058_0002
Consistent with other known members of the S100 family of proteins, NON5 contains S100 calcium binding domains as illustrated in Table 5F.
The ΝON5 nucleic acid, and the encoded polypeptide, according to the invention are useful in a variety of applications and contexts. For example, ΝON5 nucleic acids and polypeptides can be used to identify proteins that are members of the S100 family of proteins. The ΝOV5 nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance NOV5 activity or function. Specifically, the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., calcium regulation. These molecules can be used to treat, e.g., various cancers hke breast, lung, or colorectal.
In addition, the NOV5 nucleic acid and polypeptide according to the invention are useful, inter alia, as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins. For example, the NOV5 nucleic acid and polypeptide include structural motifs that are characteristic of proteins belonging to the family of S100 proteins. S100 proteins are expressed in a cell-type specific manner in higher organisms, including humans, and are involved in the calcium-regulated control of very diverse cellular processes. Proteins of the S100 family belong to the large group of EF-hand calcium-binding proteins. The NON5 nucleic acid and polypeptide, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in the mediation of calcium regulation. As such the ΝON5 nucleic acid and polypeptide, antibodies and related compounds according to the invention may be used to treat genetic conditions, e.g., various cancers like breast, lung, and colorectal, as well as heart disease such as myocardial ischemia. The ΝON5 nucleic acid and polypeptide are useful for detecting specific cell types. For example, expression analysis has demonstrated that a ΝON5 nucleic acid is expressed in elevated levels in colorectal cancers compared with that of normal colorectal mucosa, as well as in breast cancer-derived metastatic axillary lymph nodes, but not in normal lymph nodes or breast fibroadenomas. Accordingly, the ΝON5 nucleic acids and polypeptides, antibodies and related compounds accoring to the invention will have diagnostic and therapeutic applications in the detection of cancer, e.g., breast or colorectal cancer. A ΝON5 nucleic acid is also expressed in brain, lung, smooth muscle and keratinocyte tissue.
Additional utilities for the ΝON5 nucleic acid and polypeptide according to the invention are disclosed herein.
ΝOV6
The disclosed NOV6 nucleic acid (alternatively referred to herein as CG56048-01) encodes a novel olfactory receptor-like protein/G-protein coupled receptor (GPCR) protein and includes the 1121 nucleotide sequence (SEQ ID NO: 13) shown in Table 6A. An open reading frame for the mature protein was identified beginning with an ATG initiation codon at nucleotides 30-32, and ending with a TAG stop codon at nucleotides 1119- 1121. Putative untranslated regions, if any, are found upstream from the initiation codon and downstream from the termination codon. The start and stop codons are in bold letters. Table 6A. NOV6 Nucleotide Sequence (SEQ ID NO:13)
TTATTTCAAAAACTTTCGATACTGCTCCTATGGCTCCCCATGTCCGAATATGTATGCCCTTGACGGACGGCATTT CTTCATTTGAGGACCTCTTGGCTAACAATATCCTCAGAATATTTGTCTGGGTTATAGCTTTCATTACCTGCTTTG GAAATCTTTTTGTCATTGGCATGAGATCTTTCATTAAAGCTGAAAATACAACTCACGCTATGTCCATCAAAATCC TTTGTTGTGCTGATTGCCTGATGGGTGTTTACTTGTTCTTTGTTGGCATTTTCGATATAAAATACCGAGGGCAGT ATCAGAAGTATGCCTTGCTGTGGATGGAGAGCGTGCAGTGCCGCCTCATGGGGTTCCTGGCCATGCTGTCCACCG AAGTCTCTGTTCTGCTACTGACCTACTTGACTTTGGAGAAGTTCCTGGTCATTGTCTTCCCCTTCAGTAACATTC GACCTGGAAAACGGCAGACCTCAGTCATCCTCATTTGCATCTGGATGGCGGGATTTTTAATAGCTGTAATTCCAT TTTGGAATAAGGATTATTTTGGAAACTTTTATGGGAAAAATGGAGTATGTTTCCCACTTTATTATGACCAAACAG AAGATATTGGAAGCAAAGGGTATTCTCTTGGAATTTTCCTAGGTGTGAACTTGCTGGCTTTTCTCGTCATTGTGT TTTCCTATATTACTATGTTCTGTTCCATTCAAAAAACCGCCTTGCAGACCACAGAAGTAAGGAATTGTTTTGGAA GAGAGGTGGCTGTTGCAAATCGTTTCTTTTTTATAGTGTTCTCTGATGCCATCTGCTGGATTCCTGTATTTGTAG TTAAAATCCTTTCCCTCTTCCGGGTGGAAATTCCAGACACAATGACTTCCTGGATAGTGATTTTTTTCCTTCCAG TTAACAGTGCTTTGAATCCAATCCTCTATACTCTCACAACCAACTTTTTTAAGGACAAGTTGAAACAGCTGCTGC ACAAACATCAGAGGAAATCAATTTTCAAAATTAAAAAAAAAAGTTTATCTACATCCATTGTGTGGATAGAGGACT CCTCTTCCCTGAAACTTGGGGTTTTGAACAAAATAACACTTGGAGACAGTATAATGAAACCAGTTTCCTAG
The NOV6 protein (SEQ ID NO: 14) encoded by SEQ ID NO: 13 is 363 amino acid residues in length and is presented using the one-letter amino acid code in Table 6B. The SignalP, Psort and/or Hydropathy results indicate that NON6 has a signal peptide and is likely to be localized in the plasma membrane with a certainty of 0.6000. Alternatively, a ΝON6 polypeptide is located to the Golgi body with a certainty of 0.4000, the endoplasmic reticulum (membrane) with a certainty of 0.3000, or the mitochondrial inner membrane with a certainty of 0.0300. The SignalP indicates a likely cleavage site for a ΝON6 polypeptide is between positions 41 and 42, i.e., at the dash in the sequence CFG-ΝL.
Table 6B. Encoded ΝOV6 Protein Sequence (SEQ ID NO:14)
MAPHVRICMPLTDGISSFEDLLANNILRIFVWVIAFITCFGNLFVIGMRSFIKAENTTHAMSIKILCCADCLMGV YLFFVGIFDIKYRGQYQKYALLWMESVQCRLMGFLAMLSTEVSVLLLTYLTLEKFLVIVFPFSNIRPGKRQTSVI LICI MAGFLIAVIPFWNKDYFGNFYGKNGVCFPLYYDQTEDIGSKGYSLGIFLGVNLLAFLVIVFSYITMFCΞI QKTALQTTEVRNCFGREVAVANRFFFIVFSDAICWIPVFWKILSLFRVEIPDTMTS IVIFFLPVNSALNPILY TLTTNFFKDKLKQLLHKHQRKSIFKIKKKSLSTSIV IEDSSSLKLGVLNKITLGDSIMKPVS
SNP variants of NON6 are disclosed in Example 2.
A search against the Patp database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 6C.
Table 6C. PatP Results for ΝOV6 Smallest
High Sum
Sequences Producing High-Scoring Segment Pairs: Score Prob P (N) patp:AAU04370 Human G-protein coupled receptor, hRUPlό 1840 1.3e-189 ρatp:AAY42170 Human LGR7 long form protein sequence 1117 5.3e-113 patp:AAY42171 Human LGR7 short form protein sequence 1117 5.3e-113 patp:AAE02498 Human CON222 G protein-coupled receptor protein 1117 5.3e-113 patp:AAY57286 Human GPCR protein (HGPRP) sequence (clone ID 2488822) l l l l 2.3e-112
In a BLAST search of public sequence databases, it was found, for example, that the NOV6 nucleic acid sequence of this invention has 723 of 1057 bases (68%) identical to a gb:GENBANK-ID:AF190500|acc:AF190500.1 mRNA from Homo sapiens leucine-rich repeat- containing G protein-coupled receptor 7 (LGR7) mRNA, complete eds. Further, the full amino acid sequence of the disclosed NOV6 protein of the invention has 203 of 339 amino acid residues (59%) identical to, and 271 of 339 amino acid residues (79%) similar to, the 757 amino acid residue ptnr:TREMBLNEW-ACC:AAG17167 protein from Human (LEUCINE-RICH REPEAT-
CONTAΓNΓNG G PROTEΓN-COUPLED RECEPTOR 7).
The NOV6 protein of the invention also has homolgy to the proteins shown in the BLASTP data in Table 6D.
Figure imgf000061_0001
Figure imgf000062_0001
A multiple sequence alignment is given in Table 6E, with the NON6 protein of the invention being shown in line 1 in a ClustalW analysis comparing ΝON6 with related protein sequences of Table 6D.
Table 6E. ClustalW Analysis of ΝON6
1. SEQ ID NO. 14 NOV6 4. SEQ ID NO. 158 CAC38938
2. SEQ ID NO. 156 Q91ZZ5 5. SEQ ID NO. 159 Q9VBP0
3. SEQ ID NO. 157 Q9HBX9 6. SEQ ID NO. 160 P46023
10 20 30 40 50 60 NOV6 l
Q91ZZ5 1
Q9HBX9 1
CAC38938 1
Q9VBP0 1 P46023 MATMSGTTIVCLIYLTTMLGNSQGVNLKIESPSPPTLCSVEGTFHCDDGMLQCVLMGSKC 60
70 80 90 100 110 120
No 6 l Q91ZZ5 l
Q9HBX9 1
CAC38938 1
Q9VBP0 1
P46023 DGVSDCENGMDESVETCGCLQSEFQCNHTTCIDKILRCDRNDDCSNGLDERECDIYICPL 120
130 140 150 160 170 180
NOV6 l
Q91ZZ5 1 Q9HBX9 1
CAC38938 1
Q9VBP0 1
P46023 GTHVKWHNHFCVPRDKQCDFLDDCGDNSDEKICERRECVATEFKCNNSQCVAFGNLCDGL 180 190 200 210 220 230 240
NOV6 1
Q91ZZ5 1
Q9HBX9 1 CAC38938 1
Q9VBP0 1
P46023 VDCVDGSDEDQVACDSDKYFQCAEGSLIKKEFVCDGWVDCKLTFADELNCKLCDEDDFRC 240
250 260 270 280 290 300 Q91ZZ5 1
Q9HBX9 1
CAC38938 1
Q9VBP0 1
P46023 SDTRCIQKSNVCDGYCDCKTCDDEEVCANNTYGCPMDTKYMCRSIYGEPRCIDKDNVCNM 300
310 320 330 340 350 360
NOV6 1
Q91ZZ5 MWLLLHVILLTEVKDFALADS 21
Q9HBX9 MTSGSVFFYILIFGKYFSHG 20
CAC38938 1 Q9VBP0 1
P46023 INDCRDGNVGTDEYYCSNDSECKNFQAAMGFFYCPEERCLAKHLYCDLHPDCINGEDEQS 360
370 380 390 400 410 420 NOV6 1
Q91ZZ5 SMVAPLCPKGYFPCGNLTKCLPRAFHCDGVDDCGNGADEDNCGDTSGWTTIFGTVHGNVN 81
Q9HBX9 GGQDVKCSLGYFPCGNITKCLPQLLHCNGVDDCGNQADEDNCGDNNGWSMQFDKYFASYY 80
CAC38938 1
Q9VBP0 1 P46023 CLAPPKCSQDEFQCHHG-KCIPISKRCDSVHDCVDWSDEMNCENHQCAANMKSCLSGHCI 419
430 440 450 460 470 480
NOV6 1 Q91ZZ5 KVT LTQECFLS-QYPQHCYCRENELΞCVKADLKAVPKVSSN VTLLSLKKN 130
Q9HBX9 KMTSQYPFΞAETPECLVG-SVPVQCLCQGLELDCDETNLRAVPSVSSN VTAMSLQWN 136
CAC38938 1
Q9VBP0 1
P46023 EΞHKWCNFHRECPDGSDEKDCDPRPVCEANQFRCKNGQCIDPLQVCVKGDKYDGCADQSH 479
490 500 510 520 530 540
N0V6 1
Q91ZZ5 KIHRLPVKVFSRYTELRKIYLQHNCITHISRRAFLG LHNLQIL 173 Q9HBX9 LIRKLPPDCFKNYHDLQKLYLQNNKITSISIYAFRG LNSLTKL 179
CAC38938 1
Q9VBP0 1 P46023 LINCSQHICLEGQFRCRKSFCINQTKVCDGTVDCLQGMWDENNCRYWCPHGQAICQCEGV 539 550 560 570 580 590 600
NOV6 1
Q91ZZ5 YLSHNCITSLRPGIFKDLHQLAWLILDDNPITRISQKSFMGLNSLFFLPMVGNRLEALP- 232
Q9HBX9 YLSHNRITFLKPGVFEDLHRLEWLIIEDNHLSRISPPTFYGLNSLILLVLMNNVLTRLPD 239 CAC38938 1
Q9VBP0 1
P46023 TMDCTGQKLKEMPVQQMEEDLSKLMIGDNLLNLTSTTFSATYYDKVTYLDLSRNHLTEIP 599
610 620 630 640 650 660
NOV6 1
Q91ZZ5 ETLCAQMPQLNWVDLANNGIKYITNSTFLTCDSLTVLFLPRNQIGFVPEKTFSSLKNLGE 292 Q9HBX9 KPLCQHMPRLHWLDLEGNHIHNLRNLTFISCSNLTVLVMRKNKINHLNENTFAPLQKLDE 299
CAC38938 1
Q9VBP0 1
P46023 I SFQNMWKLTHLNLADNNITSLKNGSLLGLSNLKQLHINGNKIETIΞEDTFSSMIHLTV 659
670 680 690 700 710 720
NOV6
Q91ZZ5 LDLSSNMITKLPVHLFSDLHLLQKLNLSSNPLLYVHKNQFGSLKQLQSLDLERIEIPNIS 352
Q9HBX9 LDLGSNKIENLPPLIFKDLKELSQLNLSYNPIQKIQANQFDYLVKLKSLSLEGIEISNIQ 359
CAC38938 1
Q9VBP0 1
P46023 LDLSNQRLTHVYKNMFKGLKQITVLNISRNQINSIDNGAFNNLANVRLIDLSGNVIKDIG 719
Figure imgf000064_0001
970 980 990 1000 1010 1020
Figure imgf000064_0002
P 6023 SJggL wgggs VAK^RSAVRTAE^- - K PNA gRJgMTLEBMgjgFC^^IpGFy^AGAR 1013
1030 1040 1050 1060 1070 1080
Figure imgf000065_0001
1090 1100 1110 1120
NOV6 ιfflιτ| [G--DS'liK KPPVΛS 363
Q91ZZ5 ]y TE>ESS-LlI«G- IAUG-- Dffi 737
Q9HBX9 a SvbMWPLQEKJPPE[i MSPDHFTYPCEfflSLϋS [QSTRLNSYS-- 757
CAC38938 WPLQB SPPE| {g PPDDgffl:FTYPCEiSLlHQSTRLNSYS 396
Q9VBP0 TTGTATGS-sbHP DDFT£FA-K1A RCH 359
P46023 DDGTTHSYCEKKS-PYR^LEgKRLReJLNSSPPMYYNTELHSDS 1115
The presence of identifiable domains in the disclosed NOV6 protein was determined by using Pfam and then determining the Interpro number. The results are listed in Table 6F with the statistics and domain description.
Table 6F. Domain Analysis of NOV6
PSSMs Producing Significant Alignments Score E (bits) Value
7tm 1: domain 1 of 1, from 101 to 300 61.1 1.4e-18
7tm_l salCklvtaldwnmyaSillLtalSiDRYlAIvhPlryrrrrtspr ++ I ++ +++ +++ +|+++|+ ++++ +++I+ I ++ + +
NOV6 SVQCRLMGFLAMLSTEVSVLLLTYLTLEKFLVIVFPFSNIRPGK--R
7tm_l rAkwillvWvlalllslPpllf swvktveegngtlnvnvtvClidfpee
+ ++++++ I +++++++++ + ++++ +++ +|++ + +
NOV6 QTSVILICIWMAGFLIAVIP- -F-WNKDYFGNFY GKNGVCFPLYYDQ
7tm_l stasvstwlrsyvllstlvgFHPllvilvcYtrllrtlr
+ + + ++++ + +++++ ++ ++++++ I +++ ++++ ++++ +
NOV6 T EDIGSKGYSLGIFLGVNLLAFLVIVFSYITMFCSIQktalqttevr
7tm_l kaaktllvwwFvlCWlPyf ivllldtlc.lsiimsstCele
+ +++ ++ + +++ ++| I I +++ ++ + +
NOV6 ncf grevAVANRFFFIVFSDAICWIPVFWKILSLFRvEI
7tm_l rvlptallvtlwLay.vNsclNPilY (SEQ ID NO: 161)
+++++++++ + ++1+++11++1
NOV6 - -PDTMTSWIVIFFLpVNSALNPILY (SEQ ID NO: 14) Consistent with other known members of the olfactory receptor family of proteins, NON6 contains 7-transmembrane domains as illustrated in Table 6F.
The ΝON6 nucleic acid, and the encoded polypeptide, according to the invention are useful in a variety of applications and contexts. For example, ΝON6 nucleic acids and polypeptides can be used to identify proteins that are members of the olfactory receptor family of proteins. The ΝON6 nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance ΝON6 activity or function. Specifically, the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., cellular recognition, or G-protein-mediated transduction of odorant signals. These molecules can be used to treat, e.g., taste and scent detectability disorders, immune diseases, or signal transduction pathways.
In addition, the ΝON6 nucleic acid and polypeptide according to the invention are useful, ter alia, as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins. For example, the ΝON6 nucleic acid and polypeptide include structural motifs that are characteristic of proteins belonging to the family of olfactory receptor proteins. Olfactory receptors have great variety, exquisite specificity, high sensitivity and fast response. The human olfactory epithelium contains two to three thousand distinct olfactory receptors, a class of G-protein coupled receptors. The receptors consist of seven hydrophobic segments that span the cell membrane (trans-membrane domains I-NII), separated by hydrophilic segments that project into the intra- or extra-cellular space. Trans-membrane domains II-NII comprise a hypervariable segment that defines the ligand specificity of the receptor.
The ΝON6 nucleic acid and polypeptide, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in the mediation of signal transduction. As such the ΝON6 nucleic acid and polypeptide, antibodies and related compounds according to the invention may be used to treat, e.g., developmental diseases, MHC II and III diseases (immune diseases), taste and scent detectability disorders, Burkitt's lymphoma, corticoneurogenic disease, signal transduction pathway disorders, retinal diseases including those involving photoreception, cell growth rate disorders, cell shape disorders, feeding disorders, control of feeding, potential obesity due to over-eating, potential disorders due to starvation (lack of apetite), noninsulin-dependent diabetes mellitus (ΝTDDMl), bacterial, fungal, protozoal and viral infections (particularly infections caused by HIN-1 or HIN-2), pain, cancer (including but not limited to neoplasm, adenocarcinoma, lymphoma, prostate cancer, uterus cancer), anorexia, bulimia, asthma, parkinson's disease, acute heart failure, hypotension, hypertension, urinary retention, osteoporosis, crohn's disease, multiple sclerosis, and treatment of albright hereditary ostoeodystrophy, angina pectoris, myocardial infarction, ulcers, asthma, allergies, benign prostatic hypertrophy, and psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation, dentatorubro-pallidoluysian atrophy(DRPLA) hypophosphatemic rickets, autosomal dominant (2) acrocallosal syndrome and dyskinesias, such as huntington's disease or gilles de la tourette syndrome.
The NON6 nucleic acid and polypeptide are useful for detecting specific cell types. For example, expression analysis has demonstrated that a ΝON6 nucleic acid is predominantly expressed in olfactory epithelium and taste receptor cells of the tongue.
Additional utilities for the ΝON6 nucleic acid and polypeptide according to the invention are disclosed herein.
ΝON7
The ΝON7 proteins descibed herein are novel carbonate dehydratase-like proteins. The ΝON7 nucleic acids disclosed herein map to chromosome 15. Two alternative novel ΝON7 nucleic acids and polypeptides are disclosed herein, namely ΝON7a and ΝON7b. ΝOV7a A NON7 variant is ΝON7a (alternatively referred to herein as CG50365-01 ), which encodes the 828 nucleotide sequence (SEQ ID NO: 15) shown in Table 7A. An open reading frame for the mature protein was identified beginning with an ATG codon at nucleotides 16-18 and ending with a TAA codon at nucleotides 802-804. Putative untranslated regions, if any, downstream from the termination codon and upstream from the initiation codon are underlined. The start and stop codons are in bold letters.
Table 7A. NOV7a Nucleotide Sequence (SEQ ID NO:15)
CCACCCCGAGGGACCATGTCGAGGCTCAGCTGGGGATACCGCGAGCACAACGGTCCTATTCACTGGAAGGAATTT TTCCCTATTGCTGATGGTGATCAGCAATCTCCAATTGAGATTAAAACCAAAGAAGTGAAATATGACTCTTCCCTC CGACCACTTAGTATCAAGTATGACCCAAGCTCAGCTAAAATCATCAGCAACAGCGGCCATTCCTTCAATGTTGAC TTTGATGACACAGAGAACAAATCAGTTCTGCGTGGTGGTCCTCTCACTGGAAGCTACAGGTTACGGCAGGTTCAC CTTCACTGGGGGTCCGCTGATGACCACGGCTCCGAGCACATAGTAGATGGAGTGAGCTATGCTGCAGAGCTCCAT GTTGTTCACTGGAATTCAGACAAATACCCCAGCTTTGTTGAGGCAGCTCATGAACCAGATGGACTGGCTGTCTTG GGAGTGTTTTTACAGGTGGGTGAACCTAATTCCCAACTGCAAAAGATTACTGACACTTTGGATTCCATTAAAGAA AAGGGTAAACAAACTCGATTCACAAATTTTGACCTATTGTCTCTGCTTCCACCATCCTGGGACTACTGGACATAT CCTGGTTCTCTTACAGTTCCACCTCTTCTTGAGAGTGTCACATGGATTGTTTTAAAGCAACCTATAAACATCAGC TCTCAACAGCTGGCCAAATTTCGCAGTCTCCTGTGCACAGCGGAGGGTGAAGCAGCAGCTTTTCTGGTGAGCAAT CACCGCCCACCACAGCCTCTAAAGGGCCGCAAAGTGAGAGCCTCTTTCCATTAAAAATTGTCACCAATGAACTCC CCC
The NON7a protein (SEQ ID NO: 16) encoded by SEQ TD NO: 15 is 262 amino acid residues in length and is presented using the one-letter amino acid code in Table 7B. The SignalP, Psort and/or Hydropathy results indicate that NOV7a has no known signal peptide and is likely to be localized in the microbody (peroxisome) with a certainty of 0.7480. Alternatively, a NOV7a polypeptide is located to the mitochondrial matrix space with a certainty of 0.1000, the lysosome (lumen) with a certainty of 0.1000, or the endoplasmic reticulum (membrane) with a certainty of 0.1000.
Table 7B. Encoded NOV7a Protein Sequence (SEQ ID NO:16)
MSRLSWGYREHNGPIHWKEFFPIADGDQQSPIEIKTKEVKYDSSLRPLSIKYDPSSAKIISNSGHSFNVDFDDTE NKSVLRGGPLTGSYRLRQVHLHWGSADDHGSEHIVDGVSYAΆELHWHWNSDKYPSFVEAAHEPDGLAVLGVFLQ VGEPNSQLQKITDTLDSIKEKGKQTRFTNFDLLSLLPPSWDYWTYPGSLTVPPLLESVTWIVLKQPINISSQQLA KFRSLLCTAEGEAAAFLVSNHRPPQPLKGRKVRASFH
NOV7b
Alternatively, a NON7 variant is ΝON7b (alternatively referred to herein as CG50365-02), which includes the 833 nucleotide sequence (SEQ ID NO: 17) shown in Table 7C. An open reading frame for the mature protein was identified beginning with an ATG codon at nucleotides 21-23 and ending with a TAA codon at nucleotides 807-809. Putative untranslated regions, if any, downstream from the termination codon and upstream from the initiation codon are underlined. The start and stop codons are in bold letters.
Table 7C. NOV7b Nucleotide Sequence (SEQ ID NO.17)
ATGTCGAGGCTCAGCTGGGGATGTCGAGGCTCAGCTGGGGATACCGCGAGCACAACGGTCCTATTCACTGGAAGG AATTTTTCCCTATTGCTGATGGTGATCAGCAATCTCCAATTGAGATTAAAΆCCAAAGAAGTGAAATATGACTCTT CCCTCCGACCACTTAGTATCAAGTATGACCCAAGCTCAGCTAAAATCATCAGCAACAGCGGCCATTCCTTCAATG TTGACTTTGATGACACAGAGAACAAATCAGTTCTGCGTGGTGGTCCTCTCACTGGAAGCTACAGGTTACGGCAGG TTCACCTTCACTGGGGGTCCGCTGATGACCACGGCTCCGAGCACATAGTAGATGGAGTGAGCTATGCTGCAGAGC TCCATGTTGTTCACTGGAATTCAGACAAΆTACCCCAGCTTTGTTGAGGCAGCTCATGAACCAGATGGACTGGCTG TCTTGGGAGTGTTTTTACAGATTGGTGAACCTAΆTTCCCAΆCTGCAAAAGATTACTGACACTTTGGATTCCATTA AAGAAAAGGGTAAACAAACTCGATTCACAAATTTTGACCTATTGTCTCTGCTTCCACCATCCTGGGACTACTGGA CATATCCTGGTTCTCTTACAGTTCCACCTCTTCTTGAGAGTGTCACATGGATTGTTTTAAAGCAACCTATAAACA TCAGCTCTCAACAGCTGGCCAAATTTCGCAGTCTCCTGTGCACAGCGGAGGGTGAAGCAGCAGCTTTTCTGGTGA GCAATCACCGCCCACCACAGCCTCTAAAGGGCCGCAAAGTGAGAGCCTCTTTCCATTAAAAATTGTCACCAATGA ACTCCCCC
The NON7b protein (SEQ ID ΝO:18) encoded by SEQ ID NO:17 is 262 amino acid residues in length and is presented using the one-letter amino acid code in Table 7D. The SignalP, Psort and/or Hydropathy results indicate that NOV7b has no known signal peptide and is likely to be localized in the microbody (peroxisome) with a certainty of 0.7480. Alternatively, a NON7b polypeptide is located to the mitochondrial matrix space with a certainty of 0.1000, the lysosome (lumen) with a certainty of 0.1000, or the endoplasmic reticulum (membrane) with a certainty of 0.1000.
Table 7D. Encoded ΝOV7b Protein Sequence (SEQ ID NO:18)
MSRLSWGYREHNGPIHWKEFFPIADGDQQSPIEIKTKEVKYDSSLRPLSIKYDPSSAKIISNSGHSFNVDFDDTE KSVLRGGPLTGSYRLRQVHLHWGSADDHGSEHIVDGVSYAAELHWHWNSDKYPSFVEAAHEPDGLAVLGVFLQ IGEPNSQLQKITDTLDSIKEKGKQTRFTNFDLLSLLPPSWDYWTYPGSLTVPPLLESVTWIVLKQPINISSQQLA KFRSLLCTAEGEAAAFLVSNHRPPQPLKGRKVRASFH
NOV7 Clones
Unless specifically addressed as NOV7a or NON7b, any reference to ΝON7 is assumed to encompass all variants.
A search against the Patp database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table7E.
Table 7E. PatP Results for ΝOV7
Smallest
High Sum
Sequences Producing High-Scoring Segment Pairs: Score Prob P (N) ρatp:AAU19418 Human diagnostic and therapeutic polypeptide (DITHP) #4 1296 5.7e-132 patp:AAB63110 Human secreted protein sequence encoded by gene 27 964 8.7e-97 patp:AAG73863 Human colon cancer antigen protein 872 4.9e-87 ρatp:AAB59588 Human carbonic anhydrase isoform #1 870 8.0e-87 patp:AAW75702 Carbonic anhydrase II protein - Homo sapiens 858 1.5e-85 In a BLAST search of public sequence databases, it was found, for example, that the NON7a nucleic acid sequence of this invention has 551 of 793 bases (69%) identical to a gb:GEΝBAΝK-rD:lTUMCAIX|acc:M33987.1 mRNA from Human carbonic anhydrase I (CAT) mRNA, complete eds. Further, the full amino acid sequence of the disclosed NOV7a protein of the invention has 160 of 257 amino acid residues (62%) identical to, and 197 of 257 amino acid residues (76%) similar to, the 260 amino acid residue ptnr:SWISSPROT-ACC:Q92051 protein from Brachydanio rerio (Zebrafish) (Zebra danio) (CARBONIC ANHYDRASE (EC 4.2.1.1) (CARBONATE DEHYDRATASE)).
In a similar BLAST search of public sequence databases, it was found, for example, that the NOV7b nucleic acid sequence of this invention has 549 of 789 bases (69%) identical to a gb:GENBANK-ID:HSCAIR|acc:X05014.1 mRNA from Human cDNA for carbonic anhydrase I. Further, the full amino acid sequence of the disclosed NOV7b protein of the invention has 156 of 261 amino acid residues (59%) identical to, and 202 of 261 amino acid residues (77%) similar to, the 261 amino acid residue ptnr:pir-id:CRHUl protein from human (carbonate dehydratase (EC 4.2.1.1) I [validated]).
Additional BLAST results are shown in Table 7F.
Figure imgf000070_0001
Figure imgf000071_0002
A multiple sequence alignment is given in Table 7G, with the NON7 proteins of the invention being shown in lines 1 and 2 in a ClustalW analysis comparing ΝON7 with related protein sequences of Table 7F.
Table 7G. ClustalW Analysis of ΝON7
1. SEQ IDNO.: 16 NOV7a 5. SEQ ID NO.: 164 CRHU1
2. SEQ ID NO.: 18 NOV7b 6. SEQ ID NO.: 165 JN0836
3. SEQ ID NO.: 162 Q9D6N1 7. SEQ ID NO.: 166 P00917
4. SEQ ID NO.: 163 Q92051
Figure imgf000071_0001
190 200 210 220 230 240
Figure imgf000072_0001
250 260
Figure imgf000072_0002
The presence of identifiable domains in the disclosed NOV7 protein was determined by using Pfam and then determining the Interpro number. The results are listed in Table 7H with the statistics and domain description.
Table 7H. Domain Analysis of NOV7
PSSMs Producing Significant Alignments Score E (bits) Value carb anhydrase: domain 1 of 1, from 6 to 261 551.1 7.4e-162
Carb. Anhy. gYgehngpehsnnahvl hklyPiAnGGnCqGerQSPInlqtkeak l+l +++++ + i +++|+| |+ 1111+1++++++
NOV7 GYREHNGPIH KEFFPIAD GDQQSPIEIKTKEVK
Carb. Anhy. yDPsLkp slSYdaatakefeivNnGHsfqVeFdDsddksvlsGGPLpaG
+| +|++|++ I++ +++ + I ||+++|+|+|+ ++++++1111++
NOV7 YDSSLRPLSIKYDPSSAK- -IISNSGHSFNVDFDDTENKSV RGGPLTG-
Carb. Anhy. hpYRLkQfHFH GGAssddqGSEHTVDGkkYaaELHLVH NstKYgsyke
+III+I+I+III + +++llll 111++1++111+1111+ II ++ +
NOV7 -SYRLRQVHLHWG- -SADDHGSEHIVDGVSYAAELHWHWNSDKYPSFVE
Carb. Anhy. AvskpDGLAVlGvFlkvGdyqenpglqkwDaLssIktKGksatftnFDP
1+ ++11111+I+I+++I + + +++ +1 I++I+ II+++ +++II+
NOV7 AAHEPDGLAVLGVFLQVG- -EPNSQLQKITDTLDSIKEKGKQTRFTNFDL
Carb. Anhy. stLLPseklrDYWTYpGSLTTPP tEsVt iVlkepIsvSseQllkFRsL
+|||+ ++IIIII+IMI Ml I+I+I+I++ +1 +I++I+++M+I
NOV7 LSLLPP- -S DYWTYPGSLTVPPLLESVTWIV KQPINISSQQLAKFRSL
IfnaegeeevpGCdGimvdNyRPtQPLkgRvVrASF (SEQ ID NO: 167)
+ +++++ + ++ l+l I+| 11++|+|+| I I
Figure imgf000073_0001
Consistent with other known members of the carbonate dehydratase family of proteins, NOV7 contains carbonic anhydrase domains as illustrated in Table 7H.
NON7 nucleic acids, and the encoded polypeptides, according to the invention are useful in a variety of applications and contexts. For example, ΝON7 nucleic acids and polypeptides can be used to identify proteins that are members of the carbonate dehydratase family of proteins. The ΝON7 nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance ΝON7 activity or function. Specifically, the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., a variety of biological processes such as respiration, calcification, acid-base balance, bone resorption and the formation of aqueous humor, cerebrospinal fluid, saliva, and gastric acid. These molecules can be used to treat, e.g., hypertension, asthma, or emphysema.
In addition, various ΝON7 nucleic acids and polypeptides according to the invention are useful, ter alia, as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins. For example, the ΝON7 nucleic acids and their encoded polypeptides include structural motifs that are characteristic of proteins belonging to the lyase family such as the carbonic dehydratase protein. Carbonic dehydratase is an enzyme that catalyzes the equilibration of dissolved carbon dioxide and carbonic acid, speeding the movement of carbon dioxide from tissues to blood to alveolar air. It is a zinc metalloenzyme of great physiological importance.
The ΝON7 nucleic acids and polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in the mediation of CO2 transport. As such the ΝON7 nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat respiratory or CO2 transport disorders, e.g., lung cancer, hypertension, asthma, emphysema, or diabetes.
The ΝON7 nucleic acids and polypeptides are useful for detecting specific cell types. For example, expression analysis has demonstrated that a ΝON7 nucleic acid is expressed in lung.
Additional utilities for ΝON7 nucleic acids and polypeptides according to the invention are disclosed herein. NON8
The ΝON8 proteins descibed herein are novel carboxypeptidase-like proteins. The ΝON8 nucleic acids disclosed herein map to chromosome 2. Four alternative novel ΝOV8 nucleic acids and polypeptides are disclosed herein, namely NON8a, ΝON8b, ΝON8c and ΝON8d.
ΝOV8a
A NOV8 variant is NOV8a (alternatively referred to herein as CG55794-01), which encodes the 1196 nucleotide sequence (SEQ ID NO: 19) shown in Table 8 A. An open reading frame for the mature protein was identified beginning with an ATG codon at nucleotides 16-18 and ending with a TAA codon at nucleotides 1138-1140. Putative untranslated regions, if any, downstream from the termination codon and upstream from the initiation codon are underlined. The start and stop codons are in bold letters.
Table 8A. NOV8a Nucleotide Sequence (SEQ ID NO:19)
TTACTGTGTGGCAGAATGAAGCCTCTGCTTGAAACCCTTTATCTTTTGGGGATGCTGGTTCCTGGAGGGCTGGGA TATGATAGATCCTTAGCCCAACACAGACAAGAGATTGTGGACAAGTCAGTGAGTCCATGGAGCCTGGAGACGTAT TCCTATAACATATACCACCCCATGGGAGAGATCAATGAGTGGATGAGAGAGATCAGTGAGAAGTACAAGGAAGTG GTGACACAGCATTTCCTAGGAGTGACCTATGAGACCCACCCCATATATTATCTGAAGATCAGCCAACCATCTGGT AATCCCAAGAAAATCATTTGGATGGGCTGTGGAATTCACGCCAGAGAATGGATTGCTCCTGCTTTTTGCCAATGG TTCGTCAAAGAAATTCTACAAAACCATAAAGACAACTCAAGGATACGCAAGCTCCTTAGGAACCTGGACTTCTAT GTCCTTCCAGTTCTTAACATAGATGGTTATATCTACACTTGGACAACTGATCGTCTTTGGAGGAAATCCCGTTCA CCCCATAATAATGGCACATGTTTTGGGACGGATCTCAATCGAAATTTCAATGCATCTTGGTGTAGTATTGGTGCC TCTAGAAACTGCCAAGATCAAACATTCTGTGGGACAGGGCCAGTGTCTGAACCAGAGACTAAAGCTGTTGCCAGC TTCATAGAGAGCAAGAAGGATGATATTTTGTGCTTCCTGACCATGCACTCTTATGGGCAGTTAATTCTCACACCT TACGGCTACACCAAAAATAAATCAAGTAACCACCCAGAAATGATTCAAGTTGGACAGAAGGCAGCAAATGCATTG AAAGCAAAGTATGGAACCAATTATAGAGTTGGATCGAGTGCAGATATTTTATATGCCTCATCAGGGTCTTCAAGA GATTGGGCCCGAGACATTGGGATTCCCTTCTCATATACGTTTGAGCTGAGGGACAGTGGAACATATGGGTTTGTT CTGCCAGAAGCTCAGATCCAGCCCACCTGTGAGGAGACCATGGAGGCTGTGCTGTCAGTCCTGGATGATGTGTAT GCGAAACACTGGCACTCGGACAGTGCTGGAAGGGTGACATCTGCCACTATGCTGCTGGGCCTGCTGGTGTCCTGC ATGTCTCTTCTCTAAGTGCATCCTGCCCAGGCCTGCTCAACCCCAGTGGCATGAGTGTGGCTGGAGGAACG
The NOV8a protein (SEQ ID NO:20) encoded by SEQ ID NO: 19 is 374 amino acid residues in length and is presented using the one-letter amino acid code in Table 8B. The SignalP, Psort and/or Hydropathy results indicate that NOV8a has a signal peptide and is likely to be localized extracellularly at the plasma membrane with a certainty of 0.9190. Alternatively, a NON8a polypeptide is located to the lysosome (membrane) with a certainty of 0.2000, the microbody (peroxisome) with a certainty of 0.1292, or the endoplasmic reticulum (membrane) with a certainty of 0.1000. The SignalP indicates a likely cleavage site for a ΝON8a peptide is between positions 20 and 21, i.e., at the dash in the sequence GLG-YD. Table 8B. Encoded NOV8a Protein Sequence (SEQ ID NO:20)
M PLLETLYLLGMLVPGGLGYDRSLAQHRQEIVDKSVSPWSLETYSYNIYHPMGEINE MREISEKYKEWTQHF GVTYETHPIYYLKISQPSGNPK II MGCGIHAREWIAPAFCQWFVKEILQNH DNSRIRK LRNLDFYVLPV NIDGYIYTWTTDRLWRKSRSPHNNGTCFGTD NRNFNAS CSIGASRNCQDQTFCGTGPVSEPETKAVASFIESK KDDI CFLTMHSYGQLILTPYGYTKNKSSNHPE IQVGQKAANALKAKYGTNYRVGSSADILYASSGSSRD ARD IGIPFSYTFELRDSGTYGFVLPEAQIQPTCEET EAVLSVLDDVYAKHHSDSAGRVTSATMLLG LVSCMSLL
SNP variants of NON8a are disclosed in Example 2.
ΝON8b
Alternatively, a ΝON8 variant is ΝON8b (alternatively referred to herein as CG55794-03), which includes the 1222 nucleotide sequence (SEQ ID ΝO:21) shown in Table 8C. An open reading frame for the mature protein was identified beginning with an ATG codon at nucleotides 41-43 and ending with a TAA codon at nucleotides 1163-1165. Putative untranslated regions, if any, downstream from the termination codon and upstream from the initiation codon are underlined. The start and stop codons are in bold letters.
Table 8C. NOV8b Nucleotide Sequence (SEQ ID NO:21)
CCAGAGAGGCCCAGAATTTTCTAACTTACTGTGTGGCAGAATGAAGCCTCTGCTTGAAACCCTTTATCTTTTGGG GATGCTGGTTCCTGGAGGGCTGGGATATGATAGATCCTTAGCCCAACACAGACAAGAGATTGTGGACAAGTCAGT GAGTCCATGGAGCCTGGAGACGTATTCCTATAACATATACCACCCCATGGGAGAGATCTATGAGTGGATGAGAGA GATCAGTGAGAAGTACAAGGAAGTGGTGACACAGCATTTCCTAGGAGTGACCTATGAGACCCACCCCATATATTA TCTGAAGATCAGCCAACCATCTGGTAATCCCAAGAAAATCATTTGGATGGACTGTGGAATTCACGCCAGAGAATG GATTGCTCCTGCTTTTTGCCAATGGTTCGTCAAAGAAATTCTACAAAACCATAAAGACAACTCAAGGATACGCAA GCTCCTTAGGAACCTGGACTTCTATGTCCTTCCAGTTCTTAACATAGATGGTTATATCTACACTTGGACAACTGA TCGTCTTTGGAGGAAATCCCGTTCACCCCATAATAATGGCACATGTTTTGGGACGGATCTCAATCGAAATTTCAA TGCTTCTTGGTGTAGTATTGGTGCCTCTAGAAACTGCCAAGATCAAACATTCTGTGGGACAGGGCCAGTGTCTGA ACCAGAGACTAAAGCTGTTGCCAGCTTCATAGAGAGCAAGAAGGATGATATTTTGTGCTTCCTGACCATGCACTC TTATGGGCAGTTAATTCTCACACCTTACGGCTACACCAAAAATAAATCAAGTAACCACCCAGAAATGATTCAAGT TGGACAGAAGGCAGCAAATGCATTGAAAGCAAAGTATGGAACCAATTATAGAGTTGGATCGAGTGCAGATATTTT ATATGCCTCATCAGGGTCTTCAAGAGATTGGGCCCGAGACATTGGGATTCCCTTCTCATATACGTTTGAGCTGAG GGACAGTGGAACATATGGGTTTGTTCTGCCAGAAGCTCAGATCCAGCCCACCTGTGAGGAGACCATGGAGGCTGT GCTGTCAGTCCTGGATGATGTGTATGCGAAACACTGGCACTCGGACAGTGCTGGAAGGGTGACATCTGCCACTAT GCTGCTGGGCCTGCTGGTGTCCTGCATGTCTCTTCTCTAAGTGCATTCTGCCCAGGCCTGCTCAACCCCAGTGGC ATGAGTGTGGCTTGGAGGAACG
The NON8b protein (SEQ ID ΝO:22) encoded by SEQ ID NO:21 is 347 amino acid residues in length and is presented using the one-letter amino acid code in Table 8D. The
SignalP, Psort and/or Hydropathy results indicate that NON8b has a signal peptide and is likely to be localized extracellularly at the plasma membrane with a certainty of 0.9190. Alternatively, a NON8b polypeptide is located to the lysosome (membrane) with a certainty of 0.2000, the microbody (peroxisome) with a certainty of 0.1345, or the endoplasmic reticulum (membrane) with a certainty of 0.1000. The SignalP indicates a likely cleavage site for aΝON8b peptide is between positions 20 and 21, i.e., at the dash in the sequence GLG-YD.
Table 8D. Encoded ΝOV8b Protein Sequence (SEQ ID NO:22)
MKPLLETLYLLGMLVPGGLGYDRSLAQHRQEIVDKSVSPWSLETYSYNIYHPMGEIYEWMREISEKYKEWTQHF GVTYETHPIYY KISQPSGNPKKIIWMDCGIHAREWIAPAFCQ FVKEILQNHKDNSRIRKLRNDFYVLPV NIDGYIYT TTDRL RKSRSPHNNGTCFGTD NRNFNAS CSIGASRNCQDQTFCGTGPVSEPETKAVASFIESK KDDILCFLT HSYGQLILTPYGYTKNKSSNHPEMIQVGQKAANALKAKYGTNYRVGSSADILYASSGSSRDWARD IGIPFSYTFELRDSGTYGFVLPEAQIQPTCEET EAVLSVLDDVYAKHWHSDSAGRVTSATMLLGLLVSCMSLL
NOV8c
Alternatively, aNOV8 variant is NON8c (alternatively referred to herein as CG55794-06), which includes the 977 nucleotide sequence (SEQ ID ΝO:23) shown in Table 8E. An open reading frame for the mature protein was identified beginning with an ATG codon at nucleotides 41-43 and ending with a TAG codon at nucleotides 671-673. Putative untranslated regions, if any, downstream from the termination codon and upstream from the initiation codon are underlined. The start and stop codons are in bold letters.
Table 8E. NOV8c Nucleotide Sequence (SEQ ID NO:23)
CCAGAGAGGCCCAGAATTTTCTAACTTACTGTGTGGCAGAATGAAGCCTCTGCTTGAAACCCTTTATCTTTTGGG GATGCTGGTTCCTGGAGGGCTGGGATATGATAGATCCTTAGCCCAACACAGACAAGAGATTGTGGACAAGTCAGT GAGTCCATGGAGCCTGGAAACGTATTCCTATAACATATACCACCCCATGGGAGAGATCTATGAGTGGATGAGAGA GATCAGTGAGAAGTACAAGGAAGTGGTGACACAGCATTTCCTAGGAGTGACCTATGAGACCCACCCCATATATTA TCTGAAGATCAGCCAACCATCTGGTAATCCCAAGAAAATCATTTGGATGGACTGTGGAATTCACGCCAGAGAATG GATTGCTCCTGCTTTTTGCCAATGGTTCGTCAAAGAAATTCTACAAAACCATAAAGACAACTCAAGGATACGCAA GCTCCTTAGGAACCTGGACTTCTATGTCCTTCCAGTTCTTAACATAGATGGTTATATCTACACTTGGACAACTGA TCGTCTTTGGAGGAAATCCCGTTCACCCCATAATAATGGCACATGTTTTGGGACGGATCTCAATCGAAATTTCAA TGCTTCTTGGTGTAATTCAAGTTGGACAGAAGGCAGCAAATGCATTGAAAGCAAAGTATGGAACCAATTATAGAG TTGGATCGAGTGCAGATATTTTATATGCCTCATCAGGGTCTTCAAGAGATTGGGCCCGAGACATTGGGATTCCCT TCTCATATACGTTTGAGCTGAGGGACAGTGGAACATATGGGTTTGTTCTGCCAGAAGCTCAGATCCAGCCCACCT GTGAGGAGACCATGGAGGCTGTGCTGTCAGTCCTGGATGATGTGTATGCGAAACACTGGCACTCGGACAGTGCTG GAAGGGTGACATCTGCCACTATGCTGCTGGGCCTGCTGGTGTCCTGCATGTCTCTTCTCTAAGTGCATCCTGCCC AG
The NOV8c protein (SEQ ID NO:24) encoded by SEQ ID NO:23 is 210 amino acid residues in length and is presented using the one-letter amino acid code in Table 8F. The SignalP, Psort and/or Hydropathy results indicate that NONδc has a signal peptide and is likely to be localized extracellularly at the plasma membrane with a certainty of 0.3700. Alternatively, a ΝON8c polypeptide is located to the microbody (peroxisome) with a certainty of 0.2242, the lysosome (lumen) with a certainty of 0.1900, or the endoplasmic reticulum (membrane) with a certainty of 0.1000. The SignalP indicates a likely cleavage site for a ΝON8c peptide is between positions 20 and 21, i.e., at the dash in the sequence GLG-YD.
Table 8F. Encoded ΝON8c Protein Sequence (SEQ ID ΝO:24)
MKPLLETLY LGMLVPGGLGYDRSLAQHRQEIVDKSVSP SLETYSYNIYHP GEIYE MREISEKYKEWTQHF GVTYETHPIYY KISQPSGNPKKII MDCGIHARE IAPAFCQ FVKEILQNHKDNSRIRKLLRNLDFYV PVL NIDGYIYT TTDR WRKSRSPHNNGTCFGTDNRNFNASWCNSSWTEGSKCIESKVNQL
NOV8d Alternatively, a NON8 variant is ΝON8d (alternatively referred to herein as CG55794-07), which includes the 1378 nucleotide sequence (SEQ ID ΝO:25) shown in Table 8G. An open reading frame for the mature protein was identified beginning with an ATG codon at nucleotides 259-261 and ending with a TAA codon at nucleotides 1225-1227. Putative untranslated regions, if any, downstream from the termination codon and upstream from the initiation codon are underlined. The start and stop codons are in bold letters.
Table 8G. NOV8d Nucleotide Sequence (SEQ ID NO:25)
ACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAGGTGAAAATACATCAGCATGTGGGAAAGAGCAACGTTGATCG TCTTCACGGAAAGGCTGAGGACCCTGCGCCTACCACATGTTGGCCAGGGTGAGCAAGCAGTGAAAGAGAAAACAC TTTTTTCAAAAAGCCAACTGATCCTTAGCCCAACACAGACAAGAGATTGTGGACAAGTCAGTGAGTCCATGGAGC CTGGAGACGTATTCCTATAACATATACCACCCCATGGGAGAGATCTATGAGTGGATGAGAGAGATCAGTGAGAAG TACAAGGAAGTGGTGACACAGCATTTCCTAGGAGTGACCTATGAGACCCACCCCATATATTATCTGAAGATCAGC CAACCATCTGGTAATCCCAAGAAAATCATTTGGATGGACTGTGGAATTCACGCCAGAGAATGGATTGCTCCTGCT TTTTGCCAATGGTTCGTCAAAGAAATTCTACAAAACCATAAAGACAACTCAAGGATACGCAAGCTCCTTAGGAAC CTGGACTTCTATGTCCTTCCAGTTCTTAACATAGATGGTTATATCTACACTTGGACAACTGATCGTCTTTGGAGG AAATCCCGTTCACCCCATAATAATGGCACATGTTTTGGGACGGATCTCAATCGAAATTTCAATGCTTCTTGGTGT AGTATTGGTGCCTCTAGAAACTGCCAAGATCAAACATTCTGTGGGACAGGGCCAGTGTCTGAACCAGAGACTAAA GCTGTTGCCAGCTTCATAGAGAGCAAGAAGGATGATATTTTGTGCTTCCTGACCATGCACTCTTATGGGCAGTTA ATTCTCACACCTTACGGCTACACCAAAAATAAATCAAGTAACCACCCAGAAATGATTCAAGTTGGACAGAAGGCA GCAAATGCATTGAAAGCAAAGTATGGAACCAATTATAGAGTTGGATCGAGTGCAGATATTTTATATGCCTCATCA GGGTCTTCAAGAGATTGGGCCCGAGACATTGGGATTCCCTTCTCATATACGTTTGAGCTGAGGGACAGTGGAACA TATGGGTTTGTTCTGCCAGAAGCTCAGATCCAGCCCACCTGTGAGGAGACCATGGAGGCTGTGCTGTCAGTCCTG GATGATGTGTATGCGAAACACTGGCACTCGGACAGTGCTGGAAGGGTGACATCTGCCACTATGCTGCTGGGCCTG CTGGTGTCCTGCATGTCTCTTCTCTAAGTGCATTCTGCCCAGGCCTGCTCAACCCCAGTGGCATGAGTGTGGCTG GAGGAACGGTGTGTTATGGTTGTAAAGAAACCAAATAATTTAACTAAAAATACTTCCTATTTCAATAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAA The NON8d protein (SEQ TD ΝO:26) encoded by SEQ TD NO:25 is 322 amino acid residues in length and is presented using the one-letter amino acid code in Table 8H. The SignalP, Psort and/or Hydropathy results indicate that NOV8d has no known signal peptide and is likely to be localized in the cytoplasm at the endoplasmic reticulum (membrane) with a certainty of 0.8500. Alternatively, a NON8d polypeptide is located to the microbody (peroxisome) with a certainty of 0.4781, the plasma membrane with a certainty of 0.4400, or the mitochondrial inner membrane with a certainty of 0.1000.
Table 8H. Encoded ΝOV8d Protein Sequence (SEQ ID NO:26)
MGEIYEWMREISEKYKEWTQHF GVTYETHPIYYLKISQPSGNPKKIIWMDCGIHAREWIAPAFCQWFVKEILQ NHKDNSRIRKLRNLDFYVLPVLNIDGYIYT TTDRL RKSRSPHNNGTCFGTDLNRNFNAS CSIGASRNCQDQ TFCGTGPVSEPETKAVASFIESKKDDILCF TMHSYGQLI TPYGYTKNKSSNHPEMIQVGQKAANALKAKYGTN YRVGSSADILYASSGSSRD ARDIGIPFSYTFELRDSGTYGFVLPEAQIQPTCEETMEAVLSVLDDVYAHWHSD SAGRVTSATM LGLLVSCMS L
NOV8 Clones
Unless specifically addressed as NOV8a, NON8b, ΝON8c, or ΝON8d any reference to ΝON8 is assumed to encompass all variants.
A search against the Patp database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 81.
Table 81. PatP Results for ΝOV8
Smallest
High Sum
Sequences Producing High-Scoring Segment Pairs: Score Prob P (N) patp:AAG66547 Human secreted metallocarboxypeptidase-like polypeptide 2001 l.le-206 patp:AAG66565 Human secreted metallocarboxypeptidase-like variant polypeptide 1998 2.3e-206 patp:AAB74682 Human protease and protease inhibitor PPIM-15 1932 2.3e-199 patp:AAG66560 Human secreted metallocarboxypeptidase-like polypeptide 1899 7.3e-196 ρatp:AAG66566 Human secreted metallocarboxypeptidase-like polypeptide 1896 1.5e-195
In a BLAST search of public sequence databases, it was found, for example, that the NOV8a nucleic acid sequence of this invention has 584 of 914 bases (63%) identical to a gb:GENBANK-ID:AF190274|acc:AF190274.1 mRNA from Bothrops jararaca (Bothrops jararaca carboxypeptidase homolog mRNA, complete eds). Further, the full amino acid sequence of the disclosed NON8 a protein of the invention has 151 of 325 amino acid residues (46%) identical to, and 219 of 325 amino acid residues (67%) similar to, the 416 amino acid residue ρtnr:SPTREMBL-ACC:Q9PUF2 protein from Bothrops jararaca (Jararaca) (CARBOXYPEPTIDASE HOMOLOG).
In a similar BLAST search of public sequence databases, it was found, for example, that the ΝON8b nucleic acid sequence of this invention has 586 of 914 bases (64%) identical to a gb:GEΝBAΝK-ID:AF190274|acc:AF190274.1 mRNA from Bothrops jararaca (Bothrops jararaca carboxypeptidase homolog mRNA, complete eds). Further, the full amino acid sequence of the disclosed NOV8b protein of the invention has 152 of 325 amino acid residues (46%) identical to, and 219 of 325 amino acid residues (67%) similar to, the 416 amino acid residue ρtnr:SPTREMBL-ACC:Q9PUF2 protein from Bothrops jararaca (Jararaca) (CARBOXYPEPTIDASE HOMOLOG).
In a further BLAST search of public sequence databases, it was found, for example, that the NON8c nucleic acid sequence of this invention has 621 of 672 bases (92%) identical to a gb:GEΝBAΝK-ID:AX083139|acc:AX083139.1 mRNA from Homo sapiens (Sequence 42 from Patent WO0110903). Further, the full amino acid sequence of the disclosed NON8c protein of the invention has 83 of 176 amino acid residues (47%) identical to, and 122 of 176 amino acid residues (69%) similar to, the 422 amino acid residue ptnr:SPTREMBL-ACC:Q9EQN9 protein from Rattus norvegicus (Rat) (PRE-PROCARBOXYPEPTIDASE R).
In yet a further BLAST search of public sequence databases, it was found, for example, that the ΝON8d nucleic acid sequence of this invention has 1073 of 1077 bases (99%) identical to a gb:GEΝBAΝK-ID:AX083139|acc:AX083139.1 mRNA from Homo sapiens (Sequence 42 from Patent WO0110903). Further, the full amino acid sequence of the disclosed NOV8d protein of the invention has 142 of 283 amino acid residues (50%) identical to, and 199 of 283 amino acid residues (70%) similar to, the 416 amino acid residue ρtnr:SPTREMBL-ACC:Q9PUF2 protein from Bothrops jararaca (Jararaca) (CARBOXYPEPTIDASE HOMOLOG). Additional BLAST results are shown in Table 8 J.
Table 8J. NON8 BLASTP Results
Gene Index/ | Protein/Organism Length of | Identity (%) | Positives (%) | Expect Value
Figure imgf000080_0001
A multiple sequence alignment is given in Table 8K, with the NON8 proteins of the invention being shown in lines 1 through 4 in a ClustalW analysis comparing ΝON8 with related protein sequences of Table 8 J.
Table 8K. ClustalW Analysis of ΝON8
1. SEQ ID NO.: 20 NOV8a 6. SEQ ID NO.: 169 CAA03381
2. SEQ ED NO.: 22 NOV8b 7. SEQ ID NO.: 170 CAA03380
3. SEQ ID NO.: 24 NOV8c 8. SEQ ID NO.: 171 CAA03377
4. SEQ ID NO.: 26 NOV8d 9. SEQ ID NO.: 172 Q9JHH6
5. SEQ ID NO.: 168 Q9PUF2
10 20 30 40 50 60
NOV8a 1
NOV8b 1
NOV8c 1 NOV8d 1
Q9PUF2 M PLLFLIGATSAFAETTVHRFDGEKVYRVTPRNEDEVYFLNYLANIVQVDF RPDSVEL 60
CAA03381 1
CAA03380 1
CAA03377 1 Q9JHH6 MKLHGLGILVAIILYEQHGFAFQSGQVLSALPRTSRQVQLLQNLTTTYEWLWQPVTAEF 60
70 80 90 100 110 120
NOV8a MKPLLET-LY jϊJ GJ^PGG gYDRSJ^QHR@Ei;v K:SVS WS LET(Ygg 47
Figure imgf000081_0001
Figure imgf000082_0001
The presence of identifiable domains in the disclosed NOV8 protein was determined by using Pfam and then determining the Interpro number. The results are listed in Table 8L with the statistics and domain description.
Table 8L. Domain Analysis of NOV8
PSSMs Producing Significant Alignments Score E (bits) Value
Zn_carbOpept : domain 1 of 1 , from 50 to 332 384.8 8.7e-112
Zn_CarbOpept YhnleeiyawlDllvsnfPdLvskvsiGksyeGRdlkvLKisdnpat |+ +++ +++ ++ ++ + +++ +| +++ +++ I I ++
NOV8 YHPMGEINE MREISEKY EWTQHFLGVTYΞTHPIYYLKISQP--S Zn_CarbOpept genePevfavag iHAREwvtsAtllwllkelvanYgsDktitklldgld +++++++++ ++ + [ I I I ++++ I ++++++++++++ ++++++++++++
NOV8 GNPKKII MGCG-IHARE IAPAFCQWFVKEILQNHKDNSRIR LLRNLD
Zn__CarbOpept lfyilpvfNpDGyaYsittdSyRmWRKtRspnagsfcvGtDpNRN yaqw
++++++ 1 +1 1 ++1 +++++ l+l I I I ++ + ++ I+I + III+++ +
NOV8 -FYVLPVLNIDGYIYT TTD- -RLWR SRSPHNNGTCFGTDLNRNFNAS Zn__CarbOpept ggmgassysPcSetYeGtapfSepEtkavedfirswlgGGkgnlkayltf +++++++ + ++++|+ + I ++ I +++++ ++ + ++ I ++++
NOV8 CSIGASR-NCQDQTFCGTGPVSEPETKAVASFIESKKD DILCFLTM Zn_CarbOpept HsYSqlllyPYgydynlnpdandldelsdlkiaadalsarhgtyYtlglp I+I+++++ ||+++ ++ ++ + +++ +++++++++++++ I +++ + NOV8 HSYGQLILTPYGYTKNKSSNHPEMIQVG--QKAANALKAKYGTNYRVG-S Zn_CarbOpept gsstlYpasAGGsdDwaydvgiikyaftfElrpdtgsyGnPCFllPeeql +++++ I +++ I ++ I ++ + + +++++++I ++ +++ +| |++|+ +|
NOV8 SADILYASS-GSSRDWARDIG-IPFSYTFELR-DSGTYG FVLPEAQI Zn_CarbOpept iptgsee (SEQ ID NO: 173)
+++++ + NOV8 QPTCE-E (SEQ ID NO: 20)
Consistent with other known members of the carboxypeptidase family of proteins, NOV8 contains a zinc carboxypeptidase domain as illustrated in Table 8L.
NON8 nucleic acids, and the encoded polypeptides, according to the invention are useful in a variety of applications and contexts. For example, ΝON8 nucleic acids and polypeptides can be used to identify proteins that are members of the carboxypeptidase family of proteins. The ΝON8 nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance ΝON8 activity or function. Specifically, the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., digestion or hydrolysis of polypeptide chains. These molecules can be used to treat, , e.g., pancreatitis, ulcers, inflammatory bowel disease, diverticular disease, Crohn's disease, appendicitis, or obesity.
In addition, various ΝON8 nucleic acids and polypeptides according to the invention are useful, ter alia, as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins. For example, the ΝON8 nucleic acids and their encoded polypeptides include structural motifs that are characteristic of proteins belonging to the carboxypeptidase family. Carboxypeptidase B, (CPB) like carboxypeptidase A, is a pancreatic exopeptidase. Unlike carboxypeptidase A, however, carboxypeptidase B catalyzes the hydrolysis of the peptide bonds involving basic amino acids lysine, arginine and ornithine. This hydrolysis occurs at the C-terminal bond in these polypeptides.
The ΝON8 nucleic acids and polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in the mediation of hydrolysis. As such the ΝON8 nucleic acids and polypeptides, antibodies and related compounds according to the invention maybe used to treat digestive disorders, e.g., xerostomia, hypercalceimia, ulcers, Non Hippel-Lindau (NHL) syndrome, cirrhosis, transplantation, inflammatory bowel disease, diverticular disease, hirschsprung's disease, crohn's disease, appendicitis, stroke, tuberous sclerosis, anxiety, pain, endocrine dysfunctions, nueroprotection, diabetes, obesity, growth and reproductive disorders, myasthenia gravis.
The NON8 nucleic acids and polypeptides are useful for detecting specific cell types. For example, expression analysis has demonstrated that a ΝON8 nucleic acid is expressed in pooled mammalian tissue, small intestine, and spinal cord.
Additional utilities for ΝON8 nucleic acids and polypeptides according to the invention are disclosed herein.
ΝON9 The disclosed ΝON9 nucleic acid (alternatively referred to herein as CG56463-01) encodes a novel neurotransmitter receptor-like protein and includes the 1142 nucleotide sequence (SEQ ID ΝO:27) shown in Table 9A. The NON9 nucleic acid disclosed herein maps to chromosome 6p23.
An open reading frame for the mature protein was identified beginning with an GCT codon at nucleotides 3-5, and ending with a TAA stop codon at nucleotides 1020-1022. Putative untranslated regions, if any, are found upstream from the initiation codon and downstream from the termination codon. The start and stop codons are in bold letters.
Table 9A. ΝOV9 Nucleotide Sequence (SEQ ID NO:27)
AGGCTGTGGAGCTGTGTTACAAGAACGTGAACGAATCCTGCATTAAAACTCCTTACTCGCCAGGTCCTCGATCTA TCCTCTACGCCGTCCTTGGTTTTGGGGCTGTGCTGGCAGCGTTTGGAAACTTACTGGTCATGATTGCTATCCTTC ACTTCAAACAACTGCACACACCTACAAACTTTCTGATTGCGTCGCTGGCCTGTGCTGACTTCTTGGTGGGAGTCA CTGTGATGCCCTTCAGCACAGTGAGGTCTGTGGAGAGCTGTTGGTACTTTGGGGACAGTTACTGTAAATTCCATA CATGTTTTGACACATCCTTCTGTTTTGCTTCTTTATTTCATTTATGCTGTATCTCTGTTGATAGATACATTGCTG TTACTGATCCTCTGACCTATCCAACCAAGTTTACTGTGTCAGTTTCAGGGATATGCATTGTTCTTTCCTGGTTCT TTTCTGTCACATACAGCTTTTCGATCTTTTACACGGGAGCCAACGAAGAAGGAATTGAGGAATTAGTAGTTGCTC TAACCTGTGTAGGAGGCTGCCAGGCTCCACTGAATCAAAACTGGGTCCTACTTTGTTTTCTTCTATTCTTTATAC CCAATGTCGCCATGGTGTTTATATACAGTAAGATATTTTTGGTGGCCAAGCATCAGGCTAGGAAGATAGAAAGTA CAGCCAGCCAAGCTCAGTCCTCCTCAGAGAGTTACAAGGAAAGAGTAGCAAAAAGAGAGAGAAAGGCTGCCAAAA CTTTGGGAATTGCTATGGCAGCATTTCTTGTCTCTTGGCTACCATACCTCGTTGATGCAGTGATTGATGCTTATA TGAATTTTATAACTCCTCCTTATGTTTATGAGATTTTAGTTTGGTGTGTTTATTATAATTCAGCTATGAACCCCT TGATTTATGCTTTCTTTTACCAATGGTTTGGGAAGGCAATAAAACTTATTGTAAGCGGCAAGGTCTTAAGGACTG ATTCGTCAACAACTAATTTATTTTCTGAAGAAGTAGAGACAGATTAAAAACATTACTGTAGAGACCTCAAAACTA ACTTGAAATTAAGGTCAAGTGCAAAAATAAACACTTGGACATAGAGAGGCAAGCATGATCATATGCCAAGTTGTA GGACAATACATTCAATC The NON9 protein (SEQ ID ΝO:28) encoded by SEQ ID NO:27 is 339 amino acid residues in length and is presented using the one-letter amino acid code in Table 9B. The SignalP, Psort and/or Hydropathy results indicate that NON9 has a signal peptide and is likely to be localized in the plasma membrane with a certainty of 0.6000. Alternatively, aΝON9 polypeptide is located to the Golgi body with a certainty of 0.4000, the endoplasmic reticulum (membrane) with a certainty of 0.3000, or the mitochondrial inner membrane with a certainty of 0.0300. The SignalP indicates a likely cleavage site for a ΝON9 polypeptide is between positions 47 and 48, i.e., at the dash in the sequence MIA-IL.
Table 9B. Encoded ΝOV9 Protein Sequence (SEQ ID NO:28)
AVELCYKNVNESCIKTPYSPGPRSILYAVLGFGAVLAAFGNLLVMIAILHFKQLHTPTNFLIASLACADFLVGVT VMPFSTVRSVESC YFGDSYCKFHTCFDTSFCFASLFHLCCISVDRYIAVTDPLTYPT FTVSVSGICIVLS FF SVTYSFSIFYTGANEEGIEELWALTCVGGCQAPLNQN VLLCFLLFFIPNVAMVFIYSKIFLVAKHQARKIEST ASQAQSSSESY ERVAKRERKAAKTLGIAMAAFLVS LPYLVDAVIDAYMNFITPPYVYEILVWCVYYNSAMNPL IYAFFYQWFGKAIKLIVSGKVLRTDSSTTNLFSEEVETD
SNP variants of NON9 are disclosed in Example 2.
A search against the Patp database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 9C.
Table 9C. PatP Results for ΝON9
Smallest
High Sum
Sequences Producing High-Scoring Segment Pairs: Score Prob P (Ν) patp:AAB18764 Amino acid sequence of the human SΝORF1 receptor 1782 1.8e-183 patp:AAB 18765 Amino acid sequence of the rat SΝORF1 receptor 1592 2.5e-163 patp:AAG80970 Human nGPCR40 #2 1307 3.9e-133 patp:AAG72611 Human OR-like polypeptide query sequence 1257 7.8e-128 patp:AAU25611 Human G Protein-Coupled Receptor (GPCR) polypeptide #58 1251 3.4e-127
In a BLAST search of public sequence databases, it was found, for example, that the ΝON9 nucleic acid sequence of this invention has 601 of 1000 bases (60%) identical to a gb:GEΝBAΝK-ID:AR035954|acc:AR035954.1 mRNA from Unknown. (Sequence 1 from patent US 5871967). Further, the full amino acid sequence of the disclosed NON9 protein of the invention has 152 of 330 amino acid residues (46%) identical to, and 216 of 330 amino acid residues (65%) similar to, the 337 amino acid residue ptnr:SPTREMBL-ACC:O14804 protein from Human (PUTATIVE NEUROTRANSMITTER RECEPTOR).
The NOV9 protein of the invention also has homolgy to the proteins shown in the BLASTP data in Table 9D.
Figure imgf000086_0002
A multiple sequence alignment is given in Table 9E, with the NON9 protein of the invention being shown in line 1 in a ClustalW analysis comparing ΝON9 with related protein sequences of Table 9D.
Table 9E. ClustalW Analysis of ΝOV9
1. SEQ ID NO.: 28 NOV9 4. SEQ ID NO.: 176 Q923Y2
2. SEQ ID NO.: 174 Q96RI9 5. SEQ ID NO.: 177 Q923X5
3. SEQ ID NO.: 175 Q923Y6 6. SEQ ID NO.: 178 Q923X8
Q923X8 - θl-PHg i^τ?l^κ!-1fflE-gSϊ3fl.tiΛA{Fiar-^^ 35
Figure imgf000087_0001
N0V9 ETD 339
Q96RI9 ETD 348
Q923Y6 GAG 338
Q923Y2 358
Q923X5 358
Q923X8 333 The presence of identifiable domains in the disclosed NON9 protein was determined by using Pfam and then determining the Interpro number. The results are listed in Table 9F with the statistics and domain description.
Table 9F. Domain Analysis of ΝOV9
PSSMs Producing Significant Alignments Score E (bits) Value
7tm 1: domain 1 of 2, from 40 to 156 144.4 8.9e-45
7tm_l GNlLVi1 il t klrtptnifilNLAvADLLf111lppwalyy1 g II+II+++++ +++++++++++ +|| I I+I+++++++++++ ++
NOV9 GNLLVMIAILHFKQLHTPTNFLIASLACADFLVGVTVMPFSTVRSVE
7tm_l gsed pfGsalCklvtaldwnmyaSillLtalSiDRYlAIv Plryrrr
+ I + | + I ++ +++++ ++++ I ++ I ++ I I + I I I + I +++ I + ++++
NOV9 - - SC YFGDS CKFHTCFDTSFCFASLFHLCCIS VDRYIAVTDPLTYPTK
7tm__l rtsprrAkwillv vlalllsl (SEQ ID NO: 179)
+ + + + +++ I ++++ ++
NOV9 FT-VSVSGICIVLS FFSVTYSF (SEQ ID NO:28)
7tm 1: domain 2 of 2, from 190 to 302 82.0 4.1e-25
7tm__l llstlvgFllPllvilvcYtrllrtlr
++++++ I ++ I + +++ I + I + +++++ ++ +++ ++ ++++++
NOV9 VLLCFLLFFIPNVAMVFIYSKIFLVAKhqarkiestasqaqsssesy
7tm__l kaaktllvwwFvlC lPyfivllldtlc. IsiimsstCe
+++ ++++++++++++ + +|+++|+|+ + +++++++++
NOV9 kervakrerKAAKTLGIAMAAFLVSWLPYLVDAVIDAYMnFI
7tm__l lervlptallvtlwLayvNsclNPilY (SEQ ID NO : 180 ) ++++++ + +++ + I+++M +II
NOV9 TPPYVYEILV CVYYNSAMNPLIY (SEQ ID NO : 28 )
Consistent with other known members of the neurotransmitter recpetor family of proteins, NOV9 contains 7-transmembrane domains as illustrated in Table 9F.
The NOV9 nucleic acid, and the encoded polypeptide, according to the invention are useful in a variety of applications and contexts. For example, NOV9 nucleic acids and polypeptides can be used to identify proteins that are members of the neurotransmitter receptor family of proteins. The NOV9 nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance NOV9 activity or function. Specifically, the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., cellular recognition, or G-protein-mediated transduction of neuromuscular/synaptic signals. These molecules can be used to treat, e.g., neurological disorders, immune diseases, or signal transduction pathways. h addition, the NON9 nucleic acid and polypeptide according to the invention are useful, inter alia, as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins. For example, the ΝON9 nucleic acid and polypeptide include structural motifs that are characteristic of proteins belonging to the family of neurotransmitter receptor proteins. Nerve cells are highly specialized for cell-to-cell communication. A number of small molecules called neurofransmitters act as the actual signals, released from one nerve cell only to dock on another cell. There are numerous subtypes of receptor for any given neurotransmitter. Docking molecules, or receptors, act as gates, triggered by the neurotransmitter. When a neurotransmitter molecule fits into a receptor, it typically opens the gate, allowing ions to travel through the cell's membrane. The ions, in turn, excite the cell. If the receiving cell is a nerve cell, this excitation can lead to release of its own neurofransmitters. The vast majority of neurotransmitter receptors belong to a class of proteins known as the seφentine receptors. This class exhibits a characteristic transmembrane structure: spaning the cell membrane, seven times. The link between neurofransmitters and intracellular signaling is carried out by association either with G-proteins (small GTP -binding and hydrolyzing proteins) or with protein kinases, or by the receptor itself in the form of a ligand-gated ion channel (for example, the acetylcholine receptor). An additional characteristic of neurotransmitter receptors is that they are subject to ligand-induced desensitization: such that they can become unresponsive upon prolonged exposure to their neurotransmitter.
The NOV9 nucleic acid and polypeptide, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in the mediation of signal transduction. As such the NON9 nucleic acid and polypeptide, antibodies and related compounds according to the invention maybe used to treat, e.g., leukemia, acute nonlymphocytic, spinocerebellar ataxia-1, or neurological disorders.
The ΝON9 nucleic acid and polypeptide are useful for detecting specific cell types. For example, expression analysis has demonstrated that a ΝON9 nucleic acid is predominantly expressed in skeletal muscle and selected areas of the brain. Additional utilities for the NON9 nucleic acid and polypeptide according to the invention are disclosed herein.
ΝON10
The disclosed ΝON10 nucleic acid (alternatively referred to herein as CG56321-01) encodes a novel proto-oncogene MAF-like protein and includes the 1189 nucleotide sequence (SEQ ID ΝO:29) shown in Table 10A. The NON10 nucleic acid disclosed herein maps to chromosome 8.
An open reading frame for the mature protein was identified beginning with an ATG initiation codon at nucleotides 85-87, and ending with a TAG stop codon at nucleotides 1144- 1146. Putative untranslated regions, if any, are found upstream from the initiation codon and downstream from the termination codon. The start and stop codons are in bold letters.
Table 10A. ΝOV10 Nucleotide Sequence (SEQ ID NO:29)
GGGAGCAGGGGGGGGAGAGGCCTGCAGCTCCCCCACCACTCCCACGCCGCCCGTCGGGGCGCGGCCGGGCGCGGG
CCCCGGGCGATGGCCGCGGAGCTGGCGATGGGCGCCGAGCTGCCCAGCAGCCCGCTGGCCATCGAGTACGTCAAC
GACTTCGACCTGATGAAGTTCGAGGTGAAGAAGGAGCCTCCCGAGGCCGAGCGCTTCTGCCACCGCCTGCCGCCA
GGCTCGCTGTCCTCGACGCCGCTCAGCACGCCCTGCTCCTCCGTGCCCTCCTCGCCCAGCTTCTGCGCGCCCAGC
CCGGGCACCGGCGGCGGCGGCGGCGCGGGGGGCGGCGGCGGCTCGTCTCAGGCCGGGGGCGCCCCCGGGCCGCCG
AGCGGGGGCCCCGGCGCCGTCGGGGGCACCTCGGGGAAGCCGGCGCTGGAGGATCTGTACTGGATGAGCGGCTAC
CAGCATCACCTCAACCCCGAGGCGCTCAACCTGACGCCCGAGGACGCGGTGGAGGCGCTCATCGGCAGCGGCCAC
CACGGCGCGCACCACGGCGCGCACCACCCGGCGGCCGCCGCAGCCTACGAGGCTTTCCGCGGCCCGGGCTTCGCG
GGCGGCGGCGGAGCGGACGACATGGGCGCCGGCCACCACCACGGCGCGCACCACGCCGCCCACCATCACCACGCC
GCCCACCACCACCACCACCACCACCACCACCATGGCGGCGCGGGACACGGCGGTGGCGCGGGCCACCACGTGCGC
CTGGAGGAGCGCTTCTCCGACGACCAGCTGGTGTCCATGTCGGTGCGCGAGCTGAACCGGCAGCTCCGCGGCTTC
AGCAAGGAGGAGGTCATCCGGCTCAAGCAGAAGCGGCGCACGCTCAAGAACCGCGGCTACGCGCAGTCCTGCCGC
TTCAAGCGGGTGCAGCAGCGGCACATTCTGGAGAGCGAGAAGTGCCAACTCCAGAGCCAGGTGGAGCAGCTGAAG-
CTGGAGGTGGGGCGCCTGGCCAAAGAGCGGGACCTGTACAAGGAGAAATACGAGAAGCTGGCGGGCCGGGGCGGC
CCCGGGAGCGCGGGCGGGGCCGGTTTCCCGCGGGAGCCTTCGCCGCCGCAGGCCGGTCCCGGCGGGGCCAAGGGC
ACGGCCGACTTCTTCCTGTAGGCGCCGGACCCCGAGCCCGCGCCGCCGTCGCCGGGGACAAGTT
The NOV10 protein (SEQ ID NO:30) encoded by SEQ ID NO:29 is 353 amino acid residues in length and is presented using the one-letter amino acid code in Table 10B. The SignalP, Psort and/or Hydropathy results indicate that NOV10 has no known signal peptide and is likely to be localized in the nucleus with a certainty of 0.7600. Alternatively, a NONIO polypeptide is located to the microbody (peroxisome) with a certainty of 0.5418, the lysosome (lumen) with a certainty of 0.1882, or the mitochondrial matrix space with a certainty of 0.1000. Table 10B. Encoded NOV10 Protein Sequence (SEQ ID NO:30)
MAAELAMGAΞLPΞSPLAIEYVNDFDLMKFEVKKEPPΞAERFCHRLPPGSLSSTPLSTPCSSVPSSPSFCAPSPGT GGGGGAGGGGGSSQAGGAPGPPSGGPGAVGGTSGKPALEDLY MSGYQHHLNPEALNLTPEDAVEALIGSGHHGA HHGAHHPAAAAAYEAFRGPGFAGGGGADDMGAGHHHGAHHAAHHHHAAHHHHHHHHHHGGAGHGGGAGHHVRLEE RFSDDQLVSMSVRELNRQLRGFSKEEVIRLKQKRRTLKNRGYAQSCRFKRVQQRHILESEKCQLQSQVEQLKLEV GRLAKERDLYKEKYEKLAGRGGPGSAGGAGFPREPSPPQAGPGGAKGTADFFL
A search against the Patp database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table IOC.
Table IOC. PatP Results for NONIO
Smallest
High Sum
Sequences Producing High-Scoring Segment Pairs: Score Prob P (Ν) patp:AAB94964 Human protein sequence 670 1.2e-65 patp : AAY31232 Human c-Maf protein 452 1.6e-42 parp:AAB43201 Human ORFX ORF2965 polypeptide sequence 260 1.6e-28 patp:AAE03948 Human gene 1 encoded secreted protein fragment 253 1.9e-21 patp:AAB93529 Human protein sequence 238 7.5e-20
h a BLAST search of public sequence databases, it was found, for example, that the ΝON10 nucleic acid sequence of this invention has 258 of 322 bases (80%) identical to a gb:GEΝBAΝK-ID:AF034693|acc:AF034693.1 mRNA from Coturnix japonica (Coturnix coturnix japonica bZip transcription factor MafA (mafA) gene, complete eds). Further, the full amino acid sequence of the disclosed NONIO protein of the invention has 134 of 249 amino acid residues (53%) identical to, and 151 of 249 amino acid residues (60%) similar to, the 311 amino acid residue ptnr:SPTREMBL-ACC:Q90370 protein from Coturnix coturnix japonica (Japanese quail) (MAFB PROTEIN). The NOVl 0 protein of the invention also has homolgy to the proteins shown in the
BLASTP data in Table 10D.
Figure imgf000091_0001
Figure imgf000092_0003
A multiple sequence alignment is given in Table 10E, with the NONIO protein of the invention being shown in line 1 in a ClustalW analysis comparing ΝON10 with related protein sequences of Table 10D.
Table 10E. ClustalW Analysis of ΝON10
1. SEQ ID NO.: 30 NOV10 4. SEQ ID NO.: 183 Q98UK4
2. SEQ ID NO.: 181 Q90370 5. SEQ ID NO.: 184 Q98UK5
3. SEQ ID NO.: 182 Q90888 6. SEQ ID NO.: 185 073679
Figure imgf000092_0001
129
Figure imgf000092_0002
129
Figure imgf000093_0001
The presence of identifiable domains in the disclosed NONIO protein was determined by using Pfam and then determining the Interpro number. The results are listed in Table 10F with the statistics and domain description.
Figure imgf000093_0002
MAF askeskaqsaqlsptSppgaghvapaSagggggaGSgytimsgvGGG
+++ ++++ ++ ++ +++++ I NOVl0 HHGAHHAAHHHHAA HHHHHHHHHHGGAG
MAF sCvitRAALqPCRPLnqqlmsssgsvspsagsGapSplsledrfSDeqLV
++ ] + + +++++ ] ] ++| I NOV10 HGGGAGHHVRLEERFSDDQLV
MAF smSVRELNRtLkLRGlskeEwRLKQKRRTLKNRGYAqsCRaKRvqQkhe
++ 1 I I I I I I +||| ++++1 ++1 I 111 I I I I 111 I I I ++ 1 I +1 I ++ 1 ++
NOVl0 SMSVRELNR--QLRGFSKEEVIRLKQKRRTLKNRGYAQSCRFKRVQQRHI
MAF LEkeKaqLaqQleqLkeEvsrlarErDaykakyerLlsfivpvARsPvsg
I I++ I++ I + I+++ I+ I+++++ I+|++++++++ I+ + +
NOV10 LESEKCQLQSQVEQLKLEVGRLAKERDLYKEKYEKLAGRG GP
MAF ageagsssdsp (SEQ ID NO: 186)
++ ++++ + + NOV10 GSAGGAGFPRE (SEQ ID NO: 30)
Consistent with other known members of the basic leucine zipper (bZip) transcription factor family of proteins, NONIO contains MAF domains as illustrated in Table 10F.
The ΝON10 nucleic acid, and the encoded polypeptide, according to the invention are useful in a variety of applications and contexts. For example, ΝON10 nucleic acids and polypeptides can be used to identify proteins that are members of the bZip transcription factor family of proteins. The ΝON10 nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance ΝON10 activity or function. Specifically, the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., gene regulation/expression. These molecules can be used to treat, e.g., autoimmune disorders or antioxidant induction of molecules such as ΝQO1, GST,
Ya, or other detoxification enzymes. hi addition, the ΝON10 nucleic acid and polypeptide according to the invention are useful, ter alia, as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins. For example, the ΝON10 nucleic acid and polypeptide include structural motifs that are characteristic of proteins belonging to the family of bZip transcription factor proteins. Transcription factors are proteins that bind to the enhancer or promoter regions and interact such that transcription occurs from only a small group of promoters in any cell. Most transcription factors can bind to specific DΝA sequences, and these trans- regulatory proteins can be grouped together in families based on similarities in structure. Within such a family, proteins share a common framework structure in their respective DNA-binding sites, and slight differences in the amino acids at the binding site can alter the sequence of the DNA to which it binds. In addition to having this sequence-specific DNA-binding domain, transcription factors contain a domain involved in activating the transcription of the gene whose promoter or enhancer it has bound. Usually, this trans-activating domain enables that transcription factor to interact with proteins involved in binding RNA polymerase. This interaction often enhances the efficiency with which the basal transcriptional complex can be built and bind RNA polymerase II. There are several families of transcription factors.
The bZip transcription factor family of proteins are dimers, each of whose subunits contains a basic DNA-binding domain at the carboxyl end, followed closely by an a helix containing several leucine residues. These leucines are placed in the helix such that they interact with similarly spaced leucine residues on other bZip proteins to form a "leucine zipper" between them, causing dimers to form. This domain is followed by a regulatory domain that can interact with the promoter to stimulate or repress transcription. The NONIO nucleic acid and polypeptide, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in the mediation of gene transcription. As such the ΝON10 nucleic acid and polypeptide, antibodies and related compounds according to the invention may be used to treat, e.g., anemia, ataxia-telangiectasia, autoimmume disease, cancer, immunodeficiencies, hemophilia, hypercoagulation, idiopathic thrombocytopenic purpura, allergies, transplantation, graft versus host disease (GNHD), lymphaedema, systemic lupus erythematosus, asthma, emphysema, scleroderma, ARDS, diabetes, renal artery stenosis, interstitial nephritis, glomerulonephritis, polycystic kidney disease, systemic lupus erythematosus, renal tubular acidosis, IgA nephropathy, hypercalceimia, or Lesch-Νyhan syndrome. The ΝON10 nucleic acid and polypeptide are useful for detecting specific cell types. For example, expression analysis has demonstrated that a ΝON10 nucleic acid is expressed in blood, lymphocyte, whole embryo, lung, pancreas, kidney and eye.
Additional utilities for the ΝON10 nucleic acid and polypeptide according to the invention are disclosed herein. NOV11
The NOVl 1 proteins descibed herein are novel lysyl oxidase-like proteins. The NOVl 1 nucleic acids disclosed herein map to chromosome 10. Two alternative novel NOVl 1 nucleic acids and polypeptides are disclosed herein, namely NON1 la and ΝON1 lb.
ΝOVlla
A ΝON11 variant is ΝON1 la (alternatively referred to herein as CG56381-01), which encodes the 2599 nucleotide sequence (SEQ ID ΝO:31) shown in Table 1 IA. An open reading frame for the mature protein was identified beginning with an ATG codon at nucleotides 78-80 and ending with a TGA codon at nucleotides 2568-2570. Putative untranslated regions, if any, downstream from the termination codon and upstream from the imtiation codon are underlined. The start and stop codons are in bold letters.
Table 11A. NOVlla Nucleotide Sequence (SEQ ID NO:31)
CCGCCGCGGCGCCCGCCCAGCCCCGGACTGTCCGCGCTCCATCTGGTATCTTGGCCTCAGCTGTCCTTGAAGTCA CCATGGCGTGGTCCCCACCAGCCACCCTCTTTCTGTTCCTGCTGCTGCTAGGCCAGCCCCCTCCCAGCAGGCCAC AGTCACTGGGCACCACTAAGCTCCGGCTGGTGGGCCCAGAGAGCAAGCCAGAGGAGGGCCGCCTGGAGGTGCTGC ACCAGGGCCAGTGGGGCACCGTGTGTGATGACAACTTTGCTATCCAGGAGGCCACAGTGGCTTGCCGCCAGCTGG GCTTCGAAGCTGCCTTGACCTGGGCCCACAGTGCCAAGTACGGCCAAGGGGAGGGACCCATCTGGCTGGACAATG TGCGCTGTGTGGGCACAGAGAGCTCCTTGGACCAGTGCGGGTCTAATGGCTGGGGAGTCAGTGACTGCAGTCACT CAGAAGACGTAGGGGTGATATGCCACCCCCGGCGCCATCGTGGCTACCTTTCTGAAACTGTCTCCAATGCCCTTG GGCCCCAGGTGAGGAGGCTGGGCCGGCGGCTGGAGGAGGTGCGGCTCAAGCCCATCCTTGCCAGTGCCAAGCAGC ATAGCCCAGTGACCGAGGGAGCCGTGGAGGTGAAGTATGAGGGCCACTGGCGGCAGGTGTGTGACCAGGGCTGGA CCATGAACAACAGCAGGGTGGTGTGCGGGATGCTGGGCTTCCCCAGCGAGGTGCCTGTCGACAGCCACTACTACA GGCTGAAGAGCCTGACGAATAAGAACTCCTTCTGGATCCACCAGGTCACCTGCCTGGGGACAGAGCCCCACATGG CCAACTGCCAGGTGCAGGTGGCTCCAGCCCGGGGCAAGCTGCGGCCAGCCTGCCCAGGTGGCATGCACGCTGTGG TCAGCTGTGTGGCAGGGCCTCACTTCCGCCCACCGAAGACAAAGCCACAACGCAAAGGGTCCTGGGCAGAGGAGC CGAGGGTGCGCCTGCGCTCCGGGGCCCAGGTGGGCGAGGGCCGGGTGGAAGTGCTCATGAACCGCCAGTGGGGCA CGGTCTGTGACCACAGGTGGAACCTCATCTCTGCCAGTGTCGTGTGTCGTCAGCTGGGCTTTGGCTCTGCTCGGG AGGCCCTCTTTGGGGCCCGGCTGGGCCAAGGGCTAGGGCCCATCCACCTGAGTGAGGTGCGCTGCAGGGGATATG AGCGGACCCTCAGCGACTGCCCTGCCCTGGAAGGGTCCCAGAATGGTTGCCAACATGAGAATGATGCTGCTGTCA GGTGCAATGTCCCTAACATGGGCTTTCAGAATCAGGTGCGCTTGGCTGGTGGGCGTATCCCTGAGGAGGGGCTAT TGGAGGTGCAGGTGGAGGTGAACGGGGTCCCACGCTGGGGGAGCGTGTGCAGTGAAAACTGGGGGCTCACCGAAG CCATGGTGGCCTGCCGACAGCTCGGCCTGGGTTTTGCCATCCATGCCTACAAGGAAACCTGGTTCTGGTCGGGGA CGCCAAGGGCCCAGGAGGTGGTGATGAGTGGGGTGCGCTGCTCAGGCACAGAGCTGGCCCTGCAGCAGTGCCAGA GGCACGGGCCGGTGCACTGCTCCCACGGTGGCGGGCGCTTCCTGGCTGGAGTCTCCTGCATGGACAGTGCACCAG ACCTGGTGATGAACGCCCAGCTAGTGCAGGAGACGGCCTACTTGGAGGACCGCCCGCTCAGCCAGCTGTATTGTG CCCACGAGGAGAACTGCCTCTCCAAGTCTGCGGATCACATGGACTGGCCCTACGGATACCGCCGCCTATTGCGCT TCTCCACACAGATCTACAATCTGGGCCGGACTGACTTTCGTCCAAAGACTGGACGCGATAGCTGGGTTTGGCACC AGTGCCACAGGCATTACCACAGCATTGAGGTCTTCACCCACTACGACCTCCTCACTCTCAATGGCTCCAAGGTGG CTGAGGGGCACAAGGCCAGCTTCTGTCTGGAGGACACAAACTGCCCCACAGGACTGCAGCGGCGCTACGCATGTG CCAACTTTGGAGAACAGGGAGTGACTGTAGGCTGCTGGGACACCTACCGGCATGACATTGATTGCCAGTGGGTGG ATATCACAGATGTGGGCCCCGGGAATTATATCTTCCAGGTGATTGTGAACCCCCACTATGAAGTGGCAGAGTCAG ATTTCTCCAACAATATGCTGCAGTGCCGCTGCAAGTATGATGGGCACCGGGTCTGGCTGCACAACTGCCACACAG GTGAATTCATACCCAGCCAATGCAGAACTCTCCCTGGAGCAGGAACAGCGTCTCAGGAACAACCTCATCTGAAGC TGTCACTGCACACTCCTAGCTGCTGCCGATACACCAGATACCTCAGCTTATTGGAGCCATGCCCTTCACAGAGTC CCAACTCAGAGGAAAAGGGCCAGTGCCAAGGGGCACCAAGAACCTGCTCAGGAAGCCTTTTGATGGCAAGATCAC
Figure imgf000097_0001
The NONl la protein (SEQ ID ΝO:32) encoded by SEQ ID NO:31 is 830 amino acid residues in length and is presented using the one-letter amino acid code in Table 1 IB. The SignalP, Psort and/or Hydropathy results indicate that NOVl la has a signal peptide and is likely to be localized in the lysosome (lumen) with a certainty of 0.4247. Alternatively, a NOVl la polypeptide is located extracellularly with a certainty of 0.3700, the microbody (peroxisome) with a certainty of 0.1250, or the endoplasmic reticulum (membrane) with a certainty of 0.1000. The SignalP indicates a likely cleavage site for a NOVl la peptide between positions 24 and 25, i.e., at the dash in the sequence SRP-QS.
Table 11B. Encoded NOVlla Protein Sequence (SEQ ID NO:32)
MAWSPPATLFLFLLLLGQPPPSRPQSLGTTKLRLVGPESKPEEGRLEVLHQGQ GTVCDDNFAIQEATVACRQLG FEAALTWAHSAKYGQGEGPI LDNVRCVGTESSLDQCGSNG GVSDCSHSEDVGVICHPRRHRGYLSETVSNALG PQVRRLGRRLΞEVRLKPILASAKQHSPVTEGAVEVKYEGH RQVCDQG TMNNSRWCGMLGFPSEVPVDSHYYR LKSLTNKNSFWIHQVTCLGTEPHMANCQVQVAPARGKLRPACPGGMHAWSCVAGPHFRPPKTKPQRKGSWAEΞP RVRLRSGAQVGEGRVEVLMNRQ GTVCDHRNLISASWCRQLGFGSAREALFGARLGQGLGPIHLSEVRCRGYE RTLSDCPALEGSQNGCQHENDAAVRCNVPNMGFQNQVRLAGGRIPEEGLLEVQVEVNGVPR GSVCSENWGLTEA MVACRQLGLGFAIHAYKETWFWSGTPRAQEWMSGVRCSGTELALQQCQRHGPVHCSHGGGRFLAGVSCMDSAPD LVMNAQLVQETAYLEDRPLSQLYCAHEENCLSKSADHMD PYGYRRLLRFSTQIYNLGRTDFRPKTGRDSWVHQ CHRHYHSIEVFTHYDLLTLNGSKVAEGHKASFCLEDTNCPTGLQRRYACANFGEQGVTVGCWDTYRHDIDCQ VD ITDVGPGNYIFQVIVNPHYEVAESDFSNNMLQCRCKYDGHRVWLHNCHTGEFIPSQCRTLPGAGTASQEQPHLKL SLHTPSCCRYTRYLSLLEPCPSQSPNSΞEKGQCQGAPRTCSGSLLMARSPIQMVLLPQDGSGPAPKGLWPMEYVL QALLS
SNP variants of NONl la are disclosed in Example 2.
ΝONllb
Alternatively, a ΝON11 variant is ΝON1 lb (alternatively referred to herein as CG56381- 02), which includes the 2592 nucleotide sequence (SEQ ID ΝO:33) shown in Table 1 IC. An open reading frame for the mature protein was identified beginning with an ATG codon at nucleotides 46-48 and ending with a TGA codon at nucleotides 2314-2316. Putative untranslated regions, if any, downstream from the termination codon and upstream from the initiation codon are underlined. The start and stop codons are in bold letters.
Table 11C. NOVllb Nucleotide Sequence (SEQ ID NO:33)
CGCGCTCCATCTGGTATCTTGGCCTCAGCTGTCCTTGAAGTCACCATGGCGTGGTCCCCACCAGCCACCCTCTTT CTGTTCCTGCTGCTGCTAGGCCAGCCCCCTCCCAGCAGGCCACAGTCACTGGGCACCACTAAGCTCCGGCTGGTG GGCCCAGAGAGCAAGCCAGAGGAGGGCCGCCTGGAGGTGCTGCACCAGGGCCAGTGGGGCACCGTGTGTGATGAC AACTTTGCTATCCAGGAGGCCACAGTGGCTTGCCGCCAGCTGGGCTTCGAAGCTGCCTTGACCTGGGCCCACAGT GCCAAGTACGGCCAAGGGGAGGGACCCATCTGGCTGGACAATGTGCGCTGTGTGGGCACAGAGAGCTCCTTGGAC CAGTGCGGGTCTAATGGCTGGGGAGTCAGTGACTGCAGTCACTCAGAAGACGTAGGGGTGATATGCCACCCCCGG CGCCATCGTGGCTACCTTTCTGAAACTGTCTCCAATGCCCTTGGGCCCCAGGGCCAGCGGCTGGAGGAGGTGCGG CTCAAGCCCATCCTTGCCAGTGCCAAGCAGCATAGCCCAGTGACCGAGGGAGCCGTGGAGGTGAAGTATGAGGGC CACTGGCGGCAGGTGTGTGACCAGGGCTGGACCATGAACAACAGCAGGGTGGTGTGCGGGATGCTGGGCTTCCCC AGCGAGGTGCCTGCCGACAGCCACTACTACAGGAAAGTCTGGGATCTGAAGATGAGGGACCCTAAGTCTAGGCTG AAGAGCCTGACGAATAAGAACTCCTTCTGGATCCACCAGGTCACCTGCCTGGGGACAGAGCCCCACATGGCCAAC TGCCAGGTGCAGGTGGCTCCAGCCCGGGGCAAGCTGCGGCCAGCCTGCCCAGGTGGCATGCACGCTGTGGTCAGC TGTGTGGCAGGGCCTCACTTCCGCCCACCGAAGACAAAGCCACAACGCAAAGGGTCCTGGGCAGAGGAGCCGAGG GTGCGCCTGCGCTCCGGGGCCCAGGTGGGCGAGGGCCGGGTGGAAGTGCTCATGAACCGCCAGTGGGGCACGGTC TGTGACCACAGGTGGAACCTCATCTCTGCCAGTGTCGTGTGTCGTCAGCTGGGCTTTGGCTCTGCTCGGGAGGCC CTCTTTGGGGCCCGGCTGGGCCAAGGGCTAGGGCCCATCCACCTGAGTGAGGTGCGCTGCAGGGGATATGAGCGG ACCCTCAGCGACTGCCCTGCCCTGGAAGGGTCCCAGAATGGTTGCCAACATGAGAATGATGCTGCTGTCAGGTGC AATGTCCCTAACATGGGCTTTCAGAATCAGGTGCGCTTGGCTGGTGGGCGTATCCCTGAGGAGGGGCTATTGGAG GTGCAGGTGGAGGTGAACGGGGTCCCACGCTGGGGGAGCGTGTGCAGTGAAAACTGGGGGCTCACCGAAGCCATG GTGGCCTGCCGACAGCTCGGCCTGGGTTTTGCCATCCATGCCTACAAGGAAACCTGGTTCTGGTCGGGGACGCCA AGGGCCCAGGAGGTGGTGATGAGTGGGGTGCGCTGCTCAGGCACAGAGCTGGCCCTGCAGCAGTGCCAGAGGCAC GGGCCGGTGCACTGCTCCCACGGTGGCGGGCGCTTCCTGGCTGGAGTCTCCTGCATGGACAGTGCACCAGACCTG GTGATGAACGCCCAGCTAGTGCAGGAGACGGCCTACTTGGAGGACCGCCCGCTCAGCCAGCTGTATTGTGCCCAC GAGGAGAACTGCCTCTCCAAGTCTGCGGATCACATGGACTGGCCCTACGGATACCGCCGCCTATTGCGCTTCTCC ACACAGATCTACAATCTGGGCCGGACTGACTTTCGTCCAAAGACTGGACGCGATAGCTGGGTTTGGCACCAGTGT CACAGGCATTACCACAGCATTGAGGTCTTCACCCACTACGACCTCCTCACTCTCAATGGCTCCAAGGTGGCTGAG GGGCACAAGGCCAGCTTCTGTCTGGAGGACACAAACTGCCCCACAGGACTGCAGCGGCGCTACGCATGTGCCAAC TTTGGAGAACAGGGAGTGACTGTAGGCTGCTGGGACACCTACCGGCATGACATTGATTGCCAGTGGGTGGATATC ACAGATGTGGGCCCCGGGAATTATATCTTCCAGGTGATTGTGAATCCCCACTATGAAGTGGCAGAGTCAGATTTC TCCAACAATATGCTGCAGTGCCGCTGCAAGTATGATGGGCACCGGGTCTGGCTGCACAACTGCCACACAGGGAAT TCATACCCAGCCAATGCAGAACTCTCCCTGGAGCAGGAACAGCGTCTCAGGAACAACCTCATCTGAAGCTGTCAC TGCACACTCCTAGCTGCTGCCGATACACCAGATACCTCAGCTTATTGGAGCCATGCCCTTCACAGAGTCCCAACT CAGAGGAAAAGGGCCAGTGCCAAGGGGCACCAAGAACCTGCTCAGGAAGCCTTTTGATGGCAAGATCACCAATCC AGATGGTATTGCTCCCTCAGGATGGCTCTGGGCCTGCCCCTAAGGGCCTGTGGCCTATGGAATATGTCCTCCAGG CTTTGCTTAGCTGAGCTCCCCTTCTGTAAGGAAACCCAGTCA
The NONl lb protein (SEQ ID ΝO:34) encoded by SEQ ID NO:33 is 756 amino acid residues in length and is presented using the one-letter amino acid code in Table 1 ID. The SignalP, Psort and/or Hydropathy results indicate that NOVl lb has a signal peptide and is likely to be localized in the lysosome (lumen) with a certainty of 0.4302. Alternatively, a NOVl lb polypeptide is located extracellularly with a certainty of 0.3700, the microbody (peroxisome) with a certainty of 0.1403, or the endoplasmic reticulum (membrane) with a certainty of 0.1000. The SignalP indicates a likely cleavgae site for a NONl lb peptide between positions 24 and 25, i.e., at the dash in the sequence SRP-QS.
Table 11D. Encoded ΝONllb Protein Sequence (SEQ ID ΝO:34)
MA SPPATLFLFLLLLGQPPPSRPQSLGTTKLRLVGPESKPEEGRLEVLHQGQWGTVCDDNFAIQEATVACRQLG FEAALTAHSAKYGQGEGPI LDNVRCVGTESSLDQCGSNG GVSDCSHSEDVGVICHPRRHRGYLSETVSNALG PQGQRLEEVRLKPILASAKQHSPVTEGAVEVKYEGH RQVCDQG TMNNSRWCGMLGFPSEVPADSHYYRKV D LKMRDPKSRLKSLTNKNSF IHQVTCLGTEPHMANCQVQVAPARGKLRPACPGGMHAWSCVAGPHFRPPKTKPQ RKGS AEEPRVRLRSGAQVGEGRVEVLMNRQ GTVCDHR NLISASWCRQLGFGSAREALFGARLGQGLGPIHL SEVRCRGYERTLSDCPALEGSQNGCQHENDAAVRCNVPNMGFQNQVRLAGGRIPEEGLLEVQVΞVNGVPR GSVC SENWGLTEAMVACRQLGLGFAIHAYKET FWSGTPRAQEWMSGVRCSGTELALQQCQRHGPVHCSHGGGRFLAG VSCMDSAPDLVMNAQLVQETAYLEDRPLSQLYCAHEENCLSKSADHMD PYGYRRLLRFSTQIYNLGRTDFRPKT GRDSWVWHQCHRHYHSIEVFTHYDLLTLNGSKVAEGHKASFCLEDTNCPTGLQRRYACANFGEQGVTVGC DTYR HDIDCQ VDITDVGPGNYIFQVIVNPHYΞVAESDFSNNMLQCRCKYDGHRV LHNCHTGNSYPANAELSLEQEQR LRNNLI
NOV11 Clones
Unless specifically addressed as NONl la or ΝON1 lb, any reference to ΝOV11 is assumed to encompass all variants. A search against the Patp database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table HE.
Table HE. PatP Results for NO VII
Smallest
High Sum
Sequences Producing High-Scoring Segment Pairs: Score Prob P (N) patp : AAB 19127 Polypeptide isolated from lymph node stromal cells of fsn -/- mice 3450 0.0 patp:AAB49534 Clone HOHEC84 #1 - Homo sapiens 2947 6.4e-307 patp:AAB00077 Human lysyl oxidase related protein (Lor) 2281 2.4e-236 ρatp:AAB00073 Human lysyl oxidase related protein (Lor)-2 2279 3.9e-236 patpAAEl 1935 Human CG153 (or C593) receptor protein variant #2 2265 1.2e-234
In a BLAST search of public sequence databases, it was found, for example, that the
NOVl la nucleic acid sequence of this invention has 1075 of 1078 bases (99%) identical to a gb:GENBANK-ID:AK025542|acc:AK025542.1 mRNA from Homo sapiens cDNA: FLJ21889 fis, clone HEP03178. Further, the full amino acid sequence of the disclosed NONl la protein of the invention has 404 of 705 amino acid residues (57%) identical to, and 523 of 705 amino acid residues (74%) similar to, the 774 amino acid residue ρtnr:SPTREMBL-ACC:Q9Y4K0 protein from Human (LYSYL OXIDASE-RELATED PROTEIN).
In a similar BLAST search of public sequence databases, it was found, for example, that the NOVl lb nucleic acid sequence of this invention has 1070 of 1076 bases (99%) identical to a gb:GENBANK-ID:AK025542|acc:AK025542.1 mRNA from Homo sapiens cDNA: FLJ21889 fis, clone HEP03178. Further, the full amino acid sequence of the disclosed NONl lb protein of the invention has 360 of 616 amino acid residues (58%) identical to, and 456 of 616 amino acid residues (74%) similar to, the 895 amino acid residue ptnr:SPTREMBL-ACC:Q9W6Nl protein from Perca flavescens (Yellow perch) (LYSYL OXIDASE RELATED PROTEIN HOMOLOG). Additional BLAST results are shown in Table 1 IF.
Figure imgf000100_0001
A multiple sequence alignment is given in Table 11G, with the NONl 1 proteins of the invention being shown in lines 1 and 2 in a ClustalW analysis comparing ΝON11 with related protein sequences of Table 1 IF.
Table 11 G. ClustalW Analysis of ΝON11
1. SEQ ID NO. 32 NOVl la 5. SEQ ID NO. 189 Q96DY1
2. SEQ ID NO. 34 NOVl lb 6. SEQ ID NO. 190 Q924C6
3. SEQ ID NO. 187 Q96JB6 7. SEQ ID NO. 191 Q9Y4K0
4. SEQ FD NO. 188 Q96PC0
Figure imgf000101_0001
250 260 270 280 290 300
..■■[..■■I.... i .... i ....|....ι....|....ι....ι....[....ι....|
NOVlla ja jtøilg ggg ^jg^^^g^l^fc^^^g^J^^afcg^j^^^gj^^^^^g^j^ 261 NOVl lb ^^^^^S^npπgRgg^^^^^^^^^ffl^^^^^^^^R^^g^g 270
Q96JB6 ^^fe^^Mg^^D^Λ^^^^^^^^^^^gϊ^^^^^^^^HjSj^^^S 270
Q96PC0 ^^B^^ffl55gBjD^ ^^^^^^^^^^^^^^^^^^!^^W^^g^B 270
Q96DY1 ^^S^^KB^jDgfflR^^^^^^ffl^^^^g^ro^^HJ^^^^^g^W 270
Q924C6 ^TS^^^^^^2c3^^K^^^^^^^ ^§3^^^^BF^^^ gK^^^^^^ 271 Q9Y4K0 G RTYNΪK BK- - MF @^-EQRY^PFS I)ST^EA1ISSBKLG Q ^ D 284
Figure imgf000101_0002
Figure imgf000102_0001
430 440 450 460 470 480
NOVlla CPALEGSQNGCQHΞNDAAVRCNVPNMGFQNQVRLAGGRIPEEGLLEVQVEVNGVPRWGS 440 NOVllb CPALEGSQNGCQHENDAAVRCNVPNMGFQNQVRLAGGRIPEEGLLEVQVEVNGVPR GSV 449 Q96JB6 CPALEGSQNGCQHENDAAVRCNVPNMGFQNQVRLAGGRIPEEGLLEVQVEVNGVPR GSV 449 Q96PC0 CPALEGSQNGCQHENDAAVRCNVPNMGFQNQVRLAGGRIPEEGLLEVQVEVNGVPRWGSV 449 Q96DY1 CPALEGSQNGCQHENGAAVRCNVPNMGFQNQVRLAGGRIPEEGLLEVQVEVNGVPRWGSVI 449 Q924C6 450 Q9Y4K0 BKFNAEB 463
490 500 510 520 530 540
NOVlla 2SENWGLTEAMVACRQLGLGFAIHAYKETWFWSGTPRAQEWMSGVRCSGTELALQQCQR 500 NOVllb SENWGLTEAMVACRQLGLGFAIHAYKETWFWSGTPRAQEWMSGVRCSGTELALQQCQR 509 Q96JB6 SENWGLTEAMVACRQLGLGFAIHAYKETWFWSGTPRAQEWMSGVRCSGTELALQQCQR 509 Q96PC0 SENWGLTEAMVACRQLGLGFAIHAYKETWFWSGTPRAQΞWMgGVRCSGTELALQQCQR 509 Q96DY1 SENWGLTEAMVACRQLGLGFAIHAYKETWFWSGTPRAQEWMSGVRCSGTELALQQCQR 509 Q924C6 510 Q9Y4K0 !RH 523
550 560 570 580 590 600
NOVlla HGHPVHCSHGGGRFLAGVSCMDSAPDLVMNAQLVQETAYLEDRPLSQLYCAHEENCLSK; 559 NOVllb HGIPVHCSHGGGRFLAGVSCMDSAPDLVMNAQLVQETAYLEDRPLSQLYCAHEENCLSKS 568 Q96JB6 HGIPVHCSHGGGRFLAGVSCMDSAPDLVMNAQLVQETAYLEDRPLSQLYCAHEENCLSK: 568 Q96PC0 HGIPVHCSHGGGRFLAGVSCMDSAPDLVMNØQL^QETAYLEDRPLSQLYCAHEENCLSK! 568 Q96DY1 HGIPVHCSHGGGRFLAGVSCMDSAPDLVMNAQLVQETAYLEDRPLSQLYCAHEΞNCLSK; 568 Q924C6 569 Q9Y4K0 583
Figure imgf000102_0002
NOVlla MGSKVAEGHKASFCLEDTNCPTGLQRRYACANFGEQGVTVGCWDTYRHDIDCQ VDITDV 679 NOVllb NGSKVAEGHKASFCLEDTNCPTGLQRRYACANFGEQGVTVGC DTYRHDIDCQ VDITDV 688 Q96 B6 NGSKVAEGHKASFCLEDTNCPTGLQRRYACANFGEQGVTVGC DTYRHDIDCQ VDITDV 688 Q96PC0 MGSKVAΞGHKASFCLEDTNCPTGLQRRYACANFGEQGVTVGC DTYRHDIDCQWVDITDV 688 Q96DY1 WGSKVAEGHKASFCLΞDTNCPTGLQRRYACANFGEQGVTVGCWDTYRHDIDCQWVDITDV 688 Q924C6 SjgvHajjaMMwajl OIMSHMMM iH 689 Q9Y4 0 EGDffS 3EI JDUSYRHDIDCQWVDITDV jjj 703
Figure imgf000103_0001
790 800 810 820 830 840
NOVlla TSSQEQPHfflKELSlHTPSCCRYTRYLSLLEPCPSQSPNSEEKGQCQGAPRTCSGSLLMARS 799
NOVllb JSLEQEQRLRNNLI 756 Q96JB6 JSLEQEQRLRNNLI 756 Q96PC0 SLEQEQRLRNNLI 756 Q96DY1 iSLEQEQRLRNNLI 756
Q924C6 JSLEQEQRLRNNLI 757
Q9Y4K0 KKFiaJHFS GULWOLS PO - 774
850 860 870
NOVlla PIQMVLLPQDGSGPAPKGL PMEYVLQALLS 830
NOVllb 756 Q96JB6 756 Q96PC0 756 Q96DY1 756 Q924C6 757 Q9Y4K0 774
The presence of identifiable domains in the disclosed NONl 1 protein was determined by using Pfam and then determining the Interpro number. The results are listed in Table 1 IH with the statistics and domain description.
Figure imgf000103_0002
LOX RrLLRFssrvkNlGrADFrPkapRhs e HsCHqHYHSmdeFtHYDLLda
I+IIII+ ++ 1+1+ 11+1+ +1 +I+II+II+IIII+++I+IIIII++
NOVl1 RRLLRFSTQIYNLGRTDFRPKTGRDS VHQCHRHYHSIEVFTHYDLLTL LOX ngtkKVAEGHKASFCLEDteCdegvlkRYaCtnhGtQGlsvGCyDtYraD
++ II 11.1111111111 + I + + +11+1 +++1+11 +++11+1 +1 ++1
NOVl1 NGS-KVAEGHKASFCLΞDTNCPTGLQRRYACANFGEQGVTVGCWDTYRHD LOX IDCQ iDITDvkPGnYILkVevNPkyevaESDFtNNwrCnikYdGhrvy
1 1 1 1 1 + 1 1 11 + 11 +1 I++1 +11 ++++1 I I I +1 I ++1 +++1 +1 ++++
NOV11 IDCQ VDITDVGPGNYIFQVIVNPHYEVAESDFSNNMLQCRCKYDGHRV
LOX asnChigda (SEQ ID NO: 192)
++|+ ++ NOV11 LHNCHTGEF (SEQ ID NO: 32)
Consistent with other known members of the copper-dependent amine oxidase family of proteins, NONl 1 contains a lysyl oxidase domain as illustrated in Table 1 IH.
ΝON11 nucleic acids, and the encoded polypeptides, according to the invention are useful in a variety of applications and contexts. For example, ΝON11 nucleic acids and polypeptides can be used to identify proteins that are members of the copper-dependent amine oxidase family of proteins. The ΝON11 nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance ΝON11 activity or function. Specifically, the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., crosslinking of extracellular matrix proteins. These molecules can be used to treat, e.g., autoimmune disease, allergies, immunodeficiencies, asthma, psoriasis, acne, or pigmentation disorders.
In addition, various ΝON11 nucleic acids and polypeptides according to the invention are useful, ter alia, as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins. For example, the ΝON11 nucleic acids and their encoded polypeptides include structural motifs that are characteristic of proteins belonging to the lysyl oxidase family. Lysyl oxidase (LOX) is a secreted enzyme that cross-links collagen and elastin, and thus is critical for the integrity of the extracellular matrix, the breakdown of which contributes to cancer invasion and metastasis. LOX is also important to the health of connective tissues and arteries. Lysyl oxidase requires a copper co-factor and therefore its activity can be lowered by a dietary deficiency. The NONl 1 nucleic acids and polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in the mediation of crosslinking and biogenesis of connective tissue matrices. As such the ΝON11 nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat digestive disorders, e.g., diabetes, Non Hippel-Lindau (NHL) syndrome, pancreatitis, obesity, endometriosis, fertility, hemophilia, hypercoagulation, idiopathic thrombocytopenic purpura, autoimmume disease, allergies, immunodeficiencies, transplantation, graft versus host disease (GNHD), lymphaedema, osteoporosis, hypercalceimia, arthritis, ankylosing spondylitis, scoliosis, systemic lupus erythematosus, asthma, emphysema, scleroderma, allergy, ARDS, renal artery stenosis, interstitial nephritis, glomerulonephritis, polycystic kidney disease, systemic lupus erythematosus, renal tubular acidosis, IgA nephropathy, Lesch-Νyhan syndrome, psoriasis, actinic keratosis, tuberous sclerosis, acne, hair growth/loss, allopecia, pigmentation disorders, and endocrine disorders.
The ΝON11 nucleic acids and polypeptides are useful for detecting specific cell types. For example, expression analysis has demonstrated that a ΝON11 nucleic acid is expressed in kidney, lung, lymphoid tissue, mammary gland/breast, ovary, pancreas, testis, uterus, and bone.
Additional utilities for ΝON11 nucleic acids and polypeptides according to the invention are disclosed herein.
ΝON12
The ΝON12 proteins descibed herein are novel phosphatase-like proteins. The ΝON12 nucleic acids disclosed herein map to chromosome 17. Two alternative novel ΝON12 nucleic acids and polypeptides are disclosed herein, namely ΝON12a and ΝON12b. ΝON12a A ΝON12 variant is ΝON12a (alternatively referred to herein as CG56436-01), which encodes the 1002 nucleotide sequence (SEQ ID ΝO:35) shown in Table 12A. An open reading frame for the mature protein was identified beginning with an ATG codon at nucleotides 101-103 and ending with a TGA codon at nucleotides 902-904. Putative untranslated regions, if any, downstream from the termination codon and upstream from the initiation codon are underlined. The start and stop codons are in bold letters. Table 12A. NOV12a Nucleotide Sequence (SEQ ID NO:35)
GCCGGGACAGACCTCTGCTGCCGCCGCCCCCACGAACGTGTGACGACGGCTGGAGGCCAACAGAGTCCCTACAGG TGGTGCTCACGGTAATGCACCGACAATGAGTGGCTGTTTTCCAGTTTCTGGCCTCCGCTGCCTATCTAGGGACGG CAGGATGGCCGCGCAGGGCGCGCCGCGCTTCCTCCTGACCTTCGACTTCGACGAGACTATCGTGGACGAAAACAG CGACGATTCGATCGTGCGCGCCGCGCCGGGCCAGCGGCTCCCGGAGAGCCTGCGAGCCACCTACCGCGAGGGCTT CTACAACGAGTACATGCAGCGCGTCTTCAAGTACCTGGGCGAGCAGGGCGTGCGGCCGCGGGACCTGAGCGCCAT CTACGAAGCCATCCCTTTGTCGCCAGGCATGAGCGACCTGCTGCAGTTTGTGGCAAAACAGGGCGCCTGCTTCGA GGTGATTCTCATCTCCGATGCCAACACCTTTGGCGTGGAGAGCTCGCTGCGCGCCGCCGGCCACCACAGCCTGTT CCGCCGCATCCTCAGCAACCCGTCGGGGCCGGATGCGCGGGGACTGCTGGCTCTGCGGCCGTTCCACACACACAG CTGCGCGCGCTGCCCCGCCAACATGTGCAAGCACAAGGTGCTCAGCGACTACCTGCGCGAGCGGGCCCACGACGG CGTGCACTTCGAGCGCCTCTTCTACGTGGGCGACGGCGCCAACGACTTCTGCCCCATGGGGCTGCTGGCGGGCGG CGACGTGGCCTTCCCGCGCCGCGGCTACCCCATGCACCGCCTCATTCAGGAGGCCCAGAAGGCCGAGCCCAGCTC GTTCCGCGCCAGCGTGGTGCCCTGGGAAACGGCTGCAGATGTGCGCCTCCACCTGCAACAGGTGCTGAAGTCGTG CTGAGTCTGGCCGCCTGCAGGGGGGTACCCGGGCCAACGGCGGAGGGGGCGGGGAAGGGAGATTCGGCAAAGACA GCTTTACTACTCCCTTTTCCCTTTGGC
The NOV12a protein (SEQ TD NO:36) encoded by SEQ ID NO:35 is 267 amino acid residues in length and is presented using the one-letter amino acid code in Table 12B. The SignalP, Psort and/or Hydropathy results indicate that NON 12a has no known signal peptide and is likely to be localized in the mitochondrial matrix space with a certainty of 0.4728. Alternatively, a ΝON12a polypeptide is located in the microbody (peroxisome) with a certainty of 0.2224, the lysosome (lumen) with a certainty of 0.1905, or the mitochondrial inner membrane with a certainty of 0.1762.
Table 12B. Encoded ΝOV12a Protein Sequence (SEQ ID NO:36)
MSGCFPVSGLRCLSRDGRMAAQGAPRFLLTFDFDETIVDENSDDSIVRAAPGQRLPESLRATYREGFYNEYMQRV FKYLGEQGVRPRDLSAIYEAIPLSPGMSDLLQFVAKQGACFEVILISDANTFGVESSLRAAGHHSLFRRILSNPS GPDARGLLALRPFHTHSCARCPANMCKHKVLSDYLRERAHDGVHFERLFYVGDGANDFCPMGLLAGGDVAFPRRG YPMHRLIQEAQKAEPSSFRASWP ETAADVRLHLQQVLKSC
SNP variants of NON12a are disclosed in Example 2.
ΝOV12b
Alternatively, a NOV12 variant is NOV12b (alternatively referred to herein as CG56436- 02), which includes the 903 nucleotide sequence (SEQ ID NO:37) shown in Table 12C. An open reading frame for the mature protein was identified beginning with an ATG codon at nucleotides 4-6 and ending with a TGA codon at nucleotides 805-807. Putative untranslated regions, if any, downstream from the termination codon and upstream from the initiation codon are underlined. The start and stop codons are in bold letters. Table 12C. NON12b Nucleotide Sequence (SEQ ID NO:37)
ACAATGAGTGGCTGTTTTCCAGTTTCTGGCCTCCGCTGCCTATCTAGGGACGGCAGGATGGCCGCGCAGGGCGCG CCGCGCTTCCTCCTGACCTTCGACTTCGACGAGACTATCGTGGACGAAAACAGCGACGATTCGATCGTGCGCGCC GCGCCGGGCCAGCGGCTCCCGGAGAGCCTGCGAGCCACCTACCGCGAGGGCTTCTACAACGAGTACATGCAGCGC GTCTTCAAGTACCTGGGCGAGCAGGGCGTGCGGCCGCGGGACCTGAGCGCCATCTACGAAGCCATCCCTTTGTCG CCAGGCATGAGCGACCTGCTGCAGTTTGTGGCAAAACAGGGCGCCTGCTTCGAGGTGATTCTCATCTCCGATGCC AACACCTTTGGCGTGGAGAGCTCGCTGCGCGCCGCCGGCCACCACAGCCTGTTCCGCCGCATCCTCAGCAACCCG TCGGGGCCGGATGCGCGGGGACTGCTGGCTCTGCGGCCGTTCCACACACACAGCTGCGCGCGCTGCCCCGCCAAC ATGTGCAAGCACAAGGTGCTCAGCGACTACCTGCGCGAGCGGGCCCACGACGGCGTGCACTTCGAGCGCCTCTTC TACGTGGGCGACGGCGCCAACGACTTCTGCCCCATGGGGCTGCTGGCGGGCGGCGACGTGGCCTTCCCGCGCCGC GGCTACCCCATGCACCGCCTCATTCAGGAGGCCCAGAAGGCCGAGCCCAGCTCGTTCCGCGCCAGCGTGGTGCCC TGGGAAACGGCTGCAGATGTGCGCCTCCACCTGCAACAGGTGCTGAAGTCGTGCTGAGTCTGGCCGCCTGCAGGG GGGTACCCGGGCCAACGGCGGAGGGGGCGGGGAAGGGAGATTCGGCAAAGACAGCTTTACTACTCCCTTTTCCCT TTG
The NOV12b protem (SEQ ID NO:38) encoded by SEQ ID NO:37 is 267 amino acid residues in length and is presented using the one-letter amino acid code in Table 12D. The SignalP, Psort and/or Hydropathy results predict that NOV12b has no known signal peptide and is likely to be localized in the mitochondrial matrix space with a certainty of 0.4728. Alternatively, a NON 12b polypeptide is located in the microbody (peroxisome) with a certainty of 0.2224, the lysosome (lumen) with a certainty of 0.1905, or the mitochondrial inner membrane with a certainty of 0.1762.
Table 12D. Encoded ΝOV12b Protein Sequence (SEQ ID NO:38)
MSGCFPVSGLRCLSRDGRMAAQGAPRFLLTFDFDETIVDENSDDSIVRAAPGQRLPESLRATYREGFYNEYMQRV FKYLGEQGVRPRDLSAIYEAIPLSPGMSDLLQFVAKQGACFEVILISDANTFGVESSLRAAGHHSLFRRILSNPS GPDARGLLALRPFHTHSCARCPANMCKHKVLSDYLRERAHDGVHFERLFYVGDGANDFCPMGLLAGGDVAFPRRG YPMHR IQEAQKAEPSSFRASWP ETAAD RLHLQQVLKSC
NOV12 Clones
Unless specifically addressed as NOV12a or NOV12b, any reference to NOV12 is assumed to encompass all variants. A search against the Patp database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 12E.
Table 12E. PatP Results for NOV12 Smallest
High Sum
Sequences Producing High-Scoring Segment Pairs: Score Prob P (N) patp:AAB42487 Human ORFX ORF2251 polypeptide sequence 1409 6.1e-144 ρatp:AAB52146 Human secreted protein encoded by cDNA #44 738 7.7e-73 patp:AAB52178 Human secreted protein BLAST search protein 706 1.9e-69 patp:AAB52177 Human secreted protein BLAST search protein 475 5.7e-45 patp:AAM93066 Human digestive system antigen 347 2.1e-31
In a BLAST search of public sequence databases, it was found, for example, that the NOVl 2a nucleic acid sequence of this invention has 609 of 916 bases (66%) identical to a gb:GENBANK-ID:GGA6529|acc:AJ006529.1 mRNA from Gallus gallus (Gallus gallus mRNA for putative phosphatase). Further, the full amino acid sequence of the disclosed NON12a protein of the invention has 159 of 265 amino acid residues (60%) identical to, and 199 of 265 amino acid residues (75%) similar to, the 268 amino acid residue ρtnr:SPTREMBL-ACC:O73884 protein from Gallus gallus (Chicken) (PUTATIVE PHOSPHATASE). hi a similar BLAST search of public sequence databases, it was found, for example, that the ΝON12b nucleic acid sequence of this invention has 579 of 865 bases (66%) identical to a gb:GEΝBAΝK-ID:GGA6529|acc:AJ006529.1 mRNA from Gallus gallus (Gallus gallus mRNA for putative phosphatase). Further, the full amino acid sequence of the disclosed NON12b protein of the invention has 159 of 265 amino acid residues (60%) identical to, and 199 of 265 amino acid residues (75%) similar to, the 268 amino acid residue ptnr:SPTREMBL-ACC:O73884 protein from Gallus gallus (Chicken) (PUTATIVE PHOSPHATASE).
Additional BLAST results are shown in Table 12F.
Figure imgf000108_0001
Figure imgf000109_0003
A multiple sequence alignment is given in Table 12G, with the NON 12 proteins of the invention being shown in lines 1 and 2 in a ClustalW analysis comparing ΝON12 with related protein sequences of Table 12F.
Table 12G. ClustalW Analysis of ΝON12
1. SEQ ID NO.: 36 NOV12a 5. SEQ ID NO.: 195 Q9VWF0
2. SEQ ID NO.: 38 NOV12b 6. SEQ ID NO.: 196 Q9SU92
3. SEQ ID NO.: 193 073884 7. SEQ ID NO.: 197 Q9FZ62
4. SEQ ID NO.: 194 Q9D9M5
Figure imgf000109_0001
130 140 150 160 170 180
Figure imgf000109_0002
Figure imgf000110_0001
310
NOVl2a 267 NOV12b 267 073884 268 Q9D9M5 241 Q9V F0 RASAVAGPTKSPN 306 Q9SU92 245 Q9FZ62 EPIQVPLNLVK- - 279
The presence of identifiable domains in the disclosed NOV12 protein was determined by using Pfam and then determining the Interpro number. The results are listed in Table 12H with the statistics and domain description.
Figure imgf000110_0002
++ +++ + ++ + + ++ ++ |++ ++
NOV12 EVIL IsDANTFGVESSLRaAGHHSLFRRILS NPSGPDAR
Hyd. lalerlgvkpeevg p .kvlmvGDgi
++ + + +++ + + +++ ++ +++ +++ +++ ++| |+ NOVl2 GLL LRPFHTHSCArcpanmckhkvlsdylrerahdgvhFeRLFYVGDGA
Hyd. nD. apalaaAGvgvamgngg (SEQ ID NO: 198)
+| +++ I I +++ +++ NOV12 NDfCPMGLLAGGDVAFPRRG (SEQ ID NO: 36)
Consistent with other known members of the protein phosphatase family of proteins, NOV12 contains a hydrolase domain as illustrated in Table 12H.
NON12 nucleic acids, and the encoded polypeptides, according to the invention are useful in a variety of applications and contexts. For example, ΝON12 nucleic acids and polypeptides can be used to identify proteins that are members of the protein phosphatase family of proteins. The ΝON12 nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance ΝON12 activity or function. Specifically, the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., biological processes that control cell growth and homeostasis. These molecules can be used to treat, e.g., hyper/hypothyroidism, endometriosis, fertility, transplantation, hypogonadism, Alzheimer's disease, Parkinson's disease, neurodegeneration, or grwoth disorders. hi addition, various ΝOV12 nucleic acids and polypeptides according to the invention are useful, ter alia, as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins. For example, the NON 12 nucleic acids and their encoded polypeptides include structural motifs that are characteristic of proteins belonging to the protein phosphatase family. The major protein phosphatases in all cells are highly conserved and widely distributed. They are integrally associated with the regulation of many neuronal functions and have been implicated in the etiology of several neurological disorders. Their involvement in the specific control of individual neuronal functions requires the specific regulation of distinct pools of protein phosphatase inside the cell. This is believed to be mediated by specific proteins which both target the enzyme to specific subcellular locations and modulate its activity towards colocalised substrates. The NON12 nucleic acids and polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in the mediation of cell signaling/signal transduction. As such the ΝON12 nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat, e.g., hyperthyroidism, hypothyroidism, endometriosis, fertility, Non Hippel-Lindau (NHL) syndrome, cirrhosis, transplantation, hypogonadism, Alzheimer's disease, stroke, tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, cerebral palsy, epilepsy, Lesch-Νyhan syndrome, multiple sclerosis, ataxia-telangiectasia, leukodystrophies, behavioral disorders, addiction, anxiety, pain, neurodegeneration, endocrine dysfunctions, diabetes, obesity, growth and reproductive disorders, systemic lupus erythematosus, autoimmune disease, asthma, emphysema, scleroderma, allergy, ARDS, renal artery stenosis, interstitial nephritis, glomerulonephritis, polycystic kidney disease, renal tubular acidosis, IgA nephropathy, hemophilia, hypercoagulation, idiopathic thrombocytopenic purpura, immunodeficiencies, and graft versus host disease.
The ΝON12 nucleic acids and polypeptides are useful for detecting specific cell types. For example, expression analysis has demonstrated that a ΝON12 nucleic acid is expressed in bone marrow, brain, kidney, liver, lung, lung pleura, pituitary gland, placenta, and thyroid.
Additional utilities for ΝON12 nucleic acids and polypeptides according to the invention are disclosed herein.
ΝON13
The disclosed ΝON13 nucleic acid (alternatively referred to herein as CG56441-01) encodes a novel chloride channel protein CLC-KA-like protein and includes the 1991 nucleotide sequence (SEQ ID ΝO:39) shown in Table 13A.
An open reading frame for the mature protein was identified beginning with an ATG initiation codon at nucleotides 8-10, and ending with a TAG stop codon at nucleotides 1973-1975. Putative untranslated regions, if any, are found upstream from the initiation codon and downstream from the termination codon. The start and stop codons are in bold letters.
Table 13A. NOV13 Nucleotide Sequence (SEQ ID NO:39)
GGGCCTGATGGAGGAGTTTGTGGGGCTGCGTGAGGGCTTCTCAGGGGACCCTGTGACTCTGCAGGAGCTGTGGGG CCCCTGTCCCCACATCCGCCGAGCCATCCAAGGTGGCCTGGAGTGGCTAAAGCAGAAGGTGTTCCGCCTGGGAGA AGACTGGTACTTCCTGATGACCCTCGGGGTGCTCATGGCCCTGGTCAGCTATGCCATGAACTTTGCCATCGGGTG TGTGGTCCGAGGCTTCTCCCAGAGCATCACGCCCTCCTCTGGAGGTTCTGGAATCCCGGAGCTGAAGACCATGTT GGCGGGTGTGATCTTGGAGGACTACCTGGATATCAAGAACTTTGGGGCCAAGGTGGTGGGCCTCTCCTGCACCCT GGCCACCGGCAGCACCCTGTTCCTGGGCAAAGTGGGCCCTTTCGTGCACTTGTCTGTAATGATCGCTGCCTACCT GGGCCGTGTGCGCACCACGACCATCGGGGAGCCTGAGAACAAGAGCAAGCAAAACGAAATGCTGGTGGCAGCGGC GGCAGTGGGCGTGGCCACAGTCTTTGCAGCTCCCTTCAGCGGCGTCCTGTTCAGCATCGAGGTCATGTCTTCCCA CTTCTCTGTCCGGGATTACTGGAGGGGCTTCTTTGCGGCCACCTGCGGGGCCTTCATATTCCGGCTCCTGGCAGT CTTCAACAGCGAGCAGGAGACCATCACCTCCCTCTACAAGACCAGTTTCCGGGTGGACGTTCCCTTCGACCTGCC TGAGATCTTCTTTTTTGTGGCGCTGGGTGGCATCTGCGGCGTCCTGAGCTGTGCTTACCTCTTCTGTCAGCGAAC CTTCCTCAGCTTCATCAAGACCAATCGGTACAGCTCCAAACTGCTGGCTACTAGCAAGCCTGTGTACTCCGCTCT GGCCACCTTGCTTCTCGCCTCCATCACCTACCCGCCTGGTGTGGGCCACTTCCTAGCTTCTCGGCTGTCCATGAA GCAGCATCTGGACTCGCTGTTCGACAACCACTCCTGGGCGCTGATGACCCAGAACTCCAGCCCACCCTGGCCCGA GGAGCTCGACCCCCAGCACCTTTGGTGGGAATGGTACCACCCGCGGTTCACCATCTTTGGGACCCTTGCCTTCTT CCTGGTTATGAAGTTCTGGATGCTGATTCTGGCCACCACCATCCCCATGCCTGCCGGGTACTTCATGCCCATCTT TATCCTTGGAGCTGCCATCGGGCGCCTCTTGGGAGAGGCTCTTGCCGTCGCCTTCCCTGAGGGCATTGTGACTGG AGGGGTTACCAATCCCATCATGCCCGGGGGGTATGCTCTGGCAGGGGCTGCAGCCTTCTCAGGGGCTGTGACCCA CACCATCTCCACGGCGCTGCTGGCCTTTGAGCTGACCGGCCAGATAGTGCATGCACTGCCCGTGCTGATGGCGGT GCTGGCAGCCAACGCCATTGCACAGAGCTGCCAGCCCTCCTTCTATGATGGCACCATCATTGTCAAGAAGCTGCC ATACCTGCCACGGATTCTGGGCCGCAACATCGGCTCCCACCATGTGAGGGTGGAGCACTTCATGAACCACAGCAT CACCACACTGGCCAAGGACACGCCGCTGGAGGAGGTGGTCAAGGTTGTGACCTCCACAGACGTGACCGAGTATCC CCTGGTGGAGAGCACAGAGTCCCAGATCCTGGTAGGCATCGTGCAGAGGGCCCAGCTGGTGCAGGCCCTCCAGGC TGAGCCTCCTTCCAGGGCTCCAGGACACCAGCAGCGTCTCCAGGACATCTTGGCCAGGGGCTGCCCCACGGAACC AGTGACCCTGACGCTATTCTCAGAGACCACCTTGCACCAGGCACAAAACCTCTTTAAGCTGTTGAACCTTCAGTC CCTCTTCGTGACATCGCGGGGCAGAGCTGTGGGCTGCGTGTCCTGGGTGGAGATGAAGAAAGCAATTTCCAACCT GACAAATCCGCCAGCTCCAAAGTGAGCCGGCCCAGCAAGAT
The NON13 protein (SEQ ID ΝO:40) encoded by SEQ ID NO:39 is 655 amino acid residues in length and is presented using the one-letter amino acid code in Table 13B. The SignalP, Psort and/or Hydropathy results indicate that NOVl 3 has a signal peptide and is likely to be localized at the plasma membrane with a certainty of 0.6000. Alternatively, a NON13 polypeptide is located to the Golgi body with a certainty of 0.4000, the endoplasmic reticulum (membrane) with a certainty of 0.1882, or the microbody (peroxisome) with a certainty of 0.3000. The SignalP indicates a likely cleavage site for a ΝON13 peptide between positions 66 and 67, i.e., at the dash in the sequence SYA-MΝ.
Table 13B. Encoded ΝOV13 Protein Sequence (SEQ ID NO:40)
MEEFVGLREGFSGDPVTLQELWGPCPHIRRAIQGGLEWLKQKVFRLGED YFLMTLGVLMALVSYAMNFAIGCW RGFSQSITPSSGGSGIPELKTMLAGVILEDYLDIKNFGAKWGLSCTLATGSTLFLGKVGPFVHLSVMIAAYLGR VRTTTIGEPENKSKQNEMLVAAAAVGVATVFAAPFSGVLFSIEVMSSHFSVRDY RGFFAATCGAFIFRLLAVFN SEQETITSLYKTSFRVDVPFDLPEIFFFVALGGICGVLSCAYLFCQRTFLSFIKTNRYSSKLLATSKPVYSALAT LLLASITYPPGVGHFLASRLSMKQHLDSLFDNHS ALMTQNSSPPWPEELDPQHLWWEWYHPRFTIFGTLAFFLV MKFWMLILATTIPMPAGYFMPIFILGAAIGRLLGEALAVAFPEGIVTGGVTNPIMPGGYALAGAAAFSGAVTHTI STALLAFELTGQIVHALPVLMAVLAANAIAQSCQPSFYDGTIIVKKLPYLPRILGRNIGSHHVRVEHFMNHSITT LAKDTPLEEWKWTSTDVTEYPLVESTESQILVGIVQRAQLVQALQAEPPSRAPGHQQRLQDILARGCPTEPVT LTLFSETTLHQAQNLFKLLNLQSLFVTSRGRAVGCVS VEMKKAISNLTNPPAPK SNP variants of NOVl 3 are disclosed in Example 2.
A search against the Patp database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 13C.
Table 13C. PatP Results for NOV13
Smallest
High Sum
Sequences Producing High-Scoring Segment Pairs: Score Prob P (N) patp:AAY13937 Human CLCNKB protein 2754 0.0 patp:AAR60336 ClC-Kl protein - Rattus rattus - Sprague-Da ley 2331 2.3e-273 patp:AAY69633 Human gastric chloride channel C1C-2G 1216 1.6e-132 patp:AAY69631 Rabbit gastric chloride channel C1C-2G 1211 5.5e-132 patp:AAY69632 Rat brain chloride channel C1C-2 1207 1.5e-131
h a BLAST search of public sequence databases, it was found, for example, that the NON 13 nucleic acid sequence of this invention has 1768 of 1779 bases (99%) identical to a gb:GEΝBAΝK-TD:HSCLCHPRA|acc:Z30643.1 mRNA from H.sapiens mRNA for chloride channel (putative) 2139bp. Further, the full amino acid sequence of the disclosed NON13 protein of the invention has 578 of 579 amino acid residues (99%) identical to, and 578 of 579 amino acid residues (99%) similar to, the 687 amino acid residue ρtnr:SWISSPROT-ACC:P51800 protein from Human (CHLORIDE CHANNEL PROTEIN CLC-KA (CLC-K1)).
The NONl 3 protein of the invention also has homolgy to the proteins shown in the BLASTP data in Table 13D.
Figure imgf000114_0001
Figure imgf000115_0002
A multiple sequence alignment is given in Table 13E, with the NONl 3 protein of the invention being shown in line 1 in a ClustalW analysis comparing ΝON13 with related protein sequences of Table 13D.
Table 13E. ClustalW Analysis of ΝON13
1. SEQ ID NO.: 40 NOV13 4. SEQ ID NO.: 201 P51803
2. SEQ ID NO.: 199 P51800 5. SEQ ID NO.: 202 P51804
3. SEQ ID NO.: 200 P51801 6. SEQ ID NO.: 203 A57713
Figure imgf000115_0001
190 200 210 220 230 240 NOVl3 GRVRTnTIGEgENKSKQNEMLVAAAAVGVATVFAAPFSGVLFSIEVMSSHFSVraDY RGI 208
P51800 GRVRTnTIGE|ENKSKQNEMLVAAAAVGVATVFAAPFSGVLFSIEVMSSHFSVgDY RGI 240
P51801 GRVRTHTIGEHENKSKQNEMLVAAAAVGVATVF^PFSGVLFSIEVMSSHFSVWDYWRGI 240
P51803 ^VGVATVFAAPFSGVLFHIEVMSSHFSVWDYWRG 240
P51804 MaMHMK _ s iis AAVGVATVFAAPFSGVLFglEVMSSHFSV DY RG 240
A57713 ' WGVATVFAAPFSGVLFS IEVMS SHFS V Y RGE 240
Figure imgf000116_0001
370 380 390 400 410 420
NOVl3 LFDNHSWALMTWNSSPPWPEEBDPQHLWWEWYHPRFTIFGTLAFFLVMKFWMLILATTI: 388
P51800 LFDNHSWALMTGNSSPPWP EFFLDPQHL EWYHPRFTIFGTLAFFLVMKFWMLILAT I] 420
P51801 LFDNHSWALMTINSSPP P EBDPQHL E YHPRFTIFGTLAFFLVMKF MLILATTI: 420
P51803 ■.nRigtaaas-MaA^piBgwawMgiawjaaaaiwa^ rnibviBins 420
P51804 PQHL WEWYHPRFTIFGTLAFFLVMKFWMLILATTI: 420
A57713 SEEBSSSbϋSplBawCT-BiLT-BCT 420
Figure imgf000116_0002
490 500 510 520 530 540
NOVl3 riSTALLAFELTGQIVHALPVLMAVLAANAIAQSCQPSFYDGTIMVKKLPYLPRIjlGRj 508
P51800 riSTALLAFELTGQIVHALPVLMAVLAANAIAQSCQPSFYDGTinVKKLPYLPRIfflGR! 540
P51801 QI HALPVLMAVLAANAI QSCQP 540
P51803 OIVHALPVLMAVLAANAIAOSCOP; Pβ 540
P51804 ISTALLAFELTGQIVHALPVLMAVLAANAIAQSCQPSFYDGTIlVKKLPYLPrøiraGRBI 540
A57713 Wi i igMMwiaaa iieMai Aiaaffa'jiiMrjiiRMgoil 540
Figure imgf000116_0003
Figure imgf000117_0001
The presence of identifiable domains in the disclosed NON 13 protein was determined by using Pfam and then determimng the Interpro number. The results are listed in Table 13F with the statistics and domain description.
Figure imgf000117_0002
NOVl3 PVYSALATLLLASITYPPGVGHFLASRLSMKQ--HLDSLFDNHS A1MTQ CLC dlasllcdtpedavlslfdhwngpgegdfsaftlLlllliakfiltiltf +++++++++ +++++++ ++++ ++++++++I++++++++++ ++++ NOVl3 NSSPP PEELDPQHL --EWYH PRFTIFGTLAFFLVMKFWMLILAT CLC GigvPgGlFvPslviGAavGrlvGiaverlimavlsHdwfpiglfcegfp ++++I+I I+|++++| I++|+++| + +++ +++++++ + + NOVl3 TIPMPAGYFMPIFILGAAIGRLLG EALAVA FPEGIVTGGVT CLC dcilePGlYAwGAAAflgGwrmTVslaVIvfElTGnlsvilPlMvAvl
++++ II +II++IIM+ + ++++1 ++++++ +|+| i++ + +]+++!++ NOVl3 NPIM-PGGYALAGAAAFSG-AVTHTISTALLAFELTGQIVHALPVLMAVL CLC iakavadslg (SEQ ID NO: 204)
+++++++++ NOVl3 AANAIAQSCQ (SEQ ID NO: 40)
Consistent with other known members of the voltage-gated chloride channel family of proteins, NON 13 contains CLC domains as illustrated in Table 13F.
The ΝON13 nucleic acid, and the encoded polypeptide, according to the invention are useful in a variety of applications and contexts. For example, ΝON13 nucleic acids and polypeptides can be used to identify proteins that are members of the voltage-gated chloride chaimel family of proteins. The ΝON13 nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance ΝON13 activity or function. Specifically, the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., physiological functions such as cell volume regulation, membrane potential stabilization, signal transduction, or transepithelial transport. These molecules can be used to treat, e.g., diseases associated with the kidney such as renal artery stenosis, diabetes, or renal tubular acidosis.
In addition, the ΝON13 nucleic acid and polypeptide according to the invention are useful, inter alia, as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins. For example, the ΝON13 nucleic acid and polypeptide include structural motifs that are characteristic of proteins belonging to the family of voltage-gated chloride channel proteins. All functionally characterized members of the CLC family transport chloride, some in a voltage-regulated process. These channels serve a variety of physiological functions such as cell volume regulation, membrane potential stabilization, signal transduction, and transepithelial transport. The NONl 3 nucleic acid and polypeptide, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in the mediation of kidney diseases. As such the ΝON13 nucleic acid and polypeptide, antibodies and related compounds according to the invention may be used to treat, e.g., diabetes, autoimmune disease, renal artery stenosis, interstitial nephritis, glomerulonephritis, polycystic kidney disease, systemic lupus erythematosus, renal tubular acidosis, IgA nephropathy, hypercalceimia, or Lesch-Νyhan syndrome.
The ΝON13 nucleic acid and polypeptide are useful for detecting specific cell types. For example, expression analysis has demonstrated that a ΝON13 nucleic acid is expressed in the kidney.
Additional utilities for the ΝON13 nucleic acid and polypeptide according to the invention are disclosed herein.
ΝOV14 The disclosed NOV14 nucleic acid (alternatively referred to herein as CG56443-01) encodes a novel mast cell function-associated antigen (MAFA)-like protein and includes the 645 nucleotide sequence (SEQ ID NO:41) shown in Table 14A. The NON14 nucleic acid disclosed herein maps to chromosome 19.
An open reading frame for the mature protein was identified beginning with an ATG initiation codon at nucleotides 31-33, and ending with a TAA stop codon at nucleotides 604-606. Putative untranslated regions, if any, are found upstream from the initiation codon and downstream from the termination codon. The start and stop codons are in bold letters.
Table 14A. ΝON14 Nucleotide Sequence (SEQ ID NO:41)
ACTGGAGTGTTGCTACAAAGATACCCCAAAATGTGGAAGCAACTGTGGAACTGGGTAACAGGCCTTCCAGAAAGC CCCCAATTTGAGTCCCATCAAAGGTTAGTTCTTCTGCCTATTCTTGAAATTCATGTAAACTCAAAATCTTACAGA ATGTATTCATTCTGTTTGGGTTTCTTAACTCTTGTGAGACAGAGTCTTGCTCTGTCACCCAGGCTGGAATGCAGT GGCGCCATCTCGGCTCACTGCAAGATCTGTGAGCCGTGCCCTACGTCGTGGCTGCCCTTCGGGGGCTCCTGCTAC TATTTCTCTGTGCCGAAGACCACGTGGGCAGAGGCGCAGGGCCACTGCGCCGATGCCAGCGCACATCTGGCTGCC TTCCCAGAAGATAGGAAAGTCGCCTTTTATTCTGTACTTTTGGGTAGGTGCCTCTTCGGAATAGGCCTGGCCAGA GTGGGTGGGTGGAGGTGGCAGGTGGCACCGGGGACCCAGATAGATGCACCCGCAGTAGGACAAGGGGCCTGCTTC TGTCAGGAAAGCATTTCTGGTCTTCCTGCCTCGGAACTCAGGCTGGAAAAGTGGTGGCACTGCTCAAAAACACTG CAATAACAAACCACAGATGTCTGTTCCAAAGATTACAATCAAAAC The NON14 protein (SEQ ID ΝO:42) encoded by SEQ ID NO:41 is 191 amino acid residues in length and is presented using the one-letter amino acid code in Table 14B. The SignalP, Psort and/or Hydropathy results indicate that NOV14 has a signal peptide and is likely to be localized at the plasma membrane with a certainty of 0.7900. Alternatively, a NOV14 polypeptide is located to the microbody (peroxisome) with a certainty of 0.5804, the Golgi body with a certainty of 0.3000, or the endoplasmic reticulum (membrane) with a certainty of 0.2000. The SignalP indicates a likely cleavage site for a NON14 peptide between positions 57 and 58, i.e:, at the dash in the sequence SLA-LS.
Table 14B. Encoded ΝOV14 Protein Sequence (SEQ ID NO:42)
M KQL NWVTGLPESPQFESHQRLVLLPILEIHVNSKSYRMYSFCLGFLTLVRQSLALSPRLECSGAISAHCKIC EPCPTS LPFGGSCYYFSVPKTTWAEAQGHCADASAHLAAFPEDRKVAFYSVLLGRCLFGIGLARVGG R QVAP GTQIDAPAVGQGACFCQESISGLPASELRLEKW HCSKTLQ
A search against the Patp database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 14C.
Table 14C. PatP Results for NOV14
Smallest
High Sum
Sequences Producing High-Scoring Segment Pairs: Score Prob P (N) patp:AAEl 1760 Mouse mast cell function associated antigen (MAFA) protein 266 8.1e-23 patp:AAR77033 Mammalian mast cell function-associated antigen (MAFA) 252 2.5e-21 patp:AAW88277 Rat mast cell function-associated antigen (MAFA) 252 2.5e-21 patp : AAE 11761 Rat mast cell function associated antigen (MAFA) protein 252 2.5e-21 patp:AAM25760 Human protein sequence 239 5.9e-20
In a BLAST search of public sequence databases, it was found, for example, that the
NON 14 nucleic acid sequence of this invention has 109 of 151 bases (72%) identical to a gb:GEΝBAΝK-ID:HSA007973|acc:AJ007973.1 mRNA from Homo sapiens LGMD2B gene.
Further,the full amino acid sequence of the disclosed NON 14 protein of the invention has 62 of 179 amino acid residues (34%) identical to, and 87 of 179 amino acid residues (48%) similar to, the 188 amino acid residue ptnr:SPTREMBL-ACC:O88713 protein from Mouse (MAST CELL
FUNCTION-ASSOCIATED ANTIGEN 2F1 (MAFA)). The NON 14 protein of the invention also has homolgy to the proteins shown in the BLASTP data in Table 14D.
Figure imgf000121_0002
A multiple sequence alignment is given in Table 14E, with the NON 14 protein of the invention being shown in line 1 in a ClustalW analysis comparing ΝON14 with related protein sequences of Table 14D.
Table 14E. ClustalW Analysis of ΝON14
1. SEQ ID NO.: 42 NOV14 4. SEQ ID NO.: 207 075613
2. SEQ ID NO.: 205 088713 5. SEQ ID NO.: 208 Q96E93
3. SEQ ID NO.: 206 Q64335 6. SEQ ID NO.: 209 043198
Figure imgf000121_0001
Figure imgf000122_0001
The presence of identifiable domains in the disclosed NON14 protein was determined by using Pfam and then determining the Interpro number. The results are listed in Table 14F with the statistics and domain description.
Figure imgf000122_0002
Consistent with other known members of the MAFA family of proteins, NOV14 contains an extracellular link (Xlink) domains as illustrated in Table 14F. The NON14 nucleic acid, and the encoded polypeptide, according to the invention are useful in a variety of applications and contexts. For example, ΝON14 nucleic acids and polypeptides can be used to identify proteins that are members of the MAFA family of proteins. The ΝON14 nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance ΝON14 activity or function. Specifically, the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., cellular activation/cascade, allergic response, or the release of mediators such as histamine. These molecules can be used to treat, e.g., atopic disorders such as asthma, allergies, cancers such as lymphoma, or immunological disorders. In addition, the ΝON14 nucleic acid and polypeptide according to the invention are useful, ter alia, as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins. For example, the ΝOV14 nucleic acid and polypeptide include structural motifs that are characteristic of proteins belonging to the family of MAFA proteins. Mast cells are part of the immune system. They carry Fcepsilon type receptors on their surface to which IgE antibodies bind specifically. Crosslinking by multivalent antigens initiates a biochemical cascade which causes the secretion of neuroxransmitters and thus allergic reaction of the immediate type. It was recently discovered that this membrane protein carries an Immune Receptor Tyrosine based Inhibition Motif (ITIM).
The NON14 nucleic acid and polypeptide, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in the mediation of immune response, cancer, or atopy. As such the ΝON14 nucleic acid and polypeptide, antibodies and related compounds according to the invention may be used to treat, e.g., cancer, autoimmune disease, allergies, immunodeficiencies, transplantation, graft versus host disease (GNHD), or lymphaedema. The ΝON14 nucleic acid and polypeptide are useful for detecting specific cell types. For example, expression analysis has demonstrated that a ΝON14 nucleic acid is expressed in lymph, testis, liver, breast, melanocyte, heart, uterus, brain, and spleen.
Additional utilities for the ΝOV14 nucleic acid and polypeptide according to the invention are disclosed herein. NON15
The ΝON15 proteins descibed herein are novel murine epidermal growth factor-6 (MEGF6). The ΝON15 nucleic acids disclosed herein map to chromosome 1. Six alternative novel ΝON15 nucleic acids and polypeptides are disclosed herein, namely ΝON15a, ΝON15b, ΝON15c, ΝON15d, ΝON15e and ΝOV15f.
NOV15a
A NON 15 variant is ΝON15a (alternatively referred to herein as CG56449-01), which encodes the 7337 nucleotide sequence (SEQ ID ΝO:43) shown in Table 15A. An open reading frame for the mature protein was identified beginning with an ATG codon at nucleotides 1-3 and ending with a TGA codon at nucleotides 4213-4215. Putative untranslated regions, if any, downstream from the termination codon and upstream from the imtiation codon are underlined. The start and stop codons are in bold letters.
Table 15A. NOV15a Nucleotide Sequence (SEQ ID NO:43)
ATGCCCATGGGACATTCTGACAGGTGGTCTTGGCGTCTCCTGAGGCTGGCACTGCCACTCCCAGTCTGGTTGCCG GCTGGGGGTGGCCGAGGCGCTGACTCTCCATGTCTCTGTTCCAGGCCCCACGTGTGTGCTGAGCAGGAGCTGACC CTGGTGGGCCGCCGCCAGCCGTGCGTGCAGGCCTTAAGCCACACGGTGCCGGTGTGGAAGGCCGGCTGTGGGTGG CAGGCGTGGTGCGTGGGTCATGAGCGGAGGACCGTCTACTACATGGGCTACAGGCAGGTGTATACCACGGAGGCC CGGACCGTGCTCAGGTGCTGCCGAGGGTGGATGCAGCAGCCCGACGAGGAGGGCTGCCTCTCGGATGTGGGTGAG TGTGCCAACGCCAACGGGGGCTGTGCGGGTCGGTGCCGGGACACCGTGGGGGGCTTCTACTGCCGCTGGCCCCCC CCCAGCCACCAGCTGCAGGGTGATGGCGAGACTTGCCAAGATGTGGACGAATGCCGAACCCACAACGGTGGCTGC CAGCACCGGTGCGTGAACACCCCAGGCTCCTACCTCTGTGAGTGCAAGCCCGGCTTCCGGCTCCACACTGACAGC AGGACCTGCGCCATTAACTCCTGCGCCCTGGGCAATGGCGGCTGCCAGCACCACTGTGTCCAGCTCACAATCACT CGGCATCGCTGCCAGTGCCGGCCCGGGTTCCAGCTCCAGGAGGACGGCAGGCATTGTGTCCGTAGAAGCCCGTGT GCCAACAGGAACGGCAGCTGCATGCACAGGTGCCAGGTGGTCCGGGGCCTCGCCCGCTGTGAGTGCCACGTGGGC TATCAGCTAGCAGCGGACGGCAAGGCCTGTGAAGATGTGGACGAATGTGCCGCAGGGCTGGCCCAGTGTGCCCAT GGCTGCCTCAACACCCAGGGGTCCTTCAAGTGCGTGTGTCACGCGGGCTATGAGCTGGGCGCCGATGGCCGGCAG TGCTACCGTATTGAGATGGAAATCGTGAACAGCTGTGAGGCCAACAACGGCGGCTGCTCCCATGGCTGCAGCCAC ACCAGTGCTGGGCCCCTGTGCACCTGTCCCCGCGGCTACGAGCTGGACACAGATCAGAGGACCTGCATCAGATGT CGACGACTGTGCAGACAGCCCGTGCTGCAGCAGGTGTGCACCAACAACCCTGGCGGGTACGAGTGCGGCTGCTAC GCCGGCTACCGGCTCAGTGCCGATGGCTGCGGCTGCGAGGATGTGGATGAGTGCGCCTCCAGCCGTGGCGGCTGC GAGCACCACTGCACCAACCTGGCCGGCTCCTTCCAGTGCTCCTGCGAGGCCGGCTACCGGCTGCACGAGGACCGT AGGGGCTGCAGCGCCCTGGAGGAGCCGATGGTGGACCTGGACGGCGAGCTGCCTTTCGTGCGGCCCCTGCCCCAC ATTGCCGTGCTCCAGGACGAGCTGCCGCAACTCTTCCAGGATGACGACGTCGGGGCCGATGAGGAAGAGGCAGAG TTGCGGGGCGAACACACGCTCACAGAGAAGTTTGTCTGCCTGGATGACTCCTTTGGCCATGACTGCAGCTTGACC TGTGATGACTGCAGGAACGGAGGGACCTGCCTCCTGGGCCTGGATGGCTGTGATTGCCCCGAGGGCTGGACTGGG CTCATCTGCAATGAGAGTTGTCCTCCGGACACCTTTGGGAAGAACTGCAGCTTCTCCTGCAGCTGTCAGAATGGT GGGACCTGCGACTCTGTCACGGGGGCCTGCCGCTGCCCCCCGGGTGTCAGTGGAACTAACTGTGAGGATGGCTGC CCCAAGGGCTACTATGGCAAGCACTGTCGCAAGAAATGCAACTGTGCCAACCGGGGCCGGTGCCACCGCCTCTAC GGGGCCTGCCTCTGCGACCCAGGGCTCTACGGCCGCTTCTGCCACCTCGCCTGCCCGCCGTGGGCCTTTGGGCCG GGCTGCTCGGAGGAGTGCCAGTGTGTGCAGCCCCACACGCAGTCCTGTGACAAGAGGGATGGCAGCTGCTCCTGC AAGGCTGGCTTCCGGGGCGAGCGCTGTCAGGCAGAGTGTGAGCCGGGCTACTTTGGGCCGGGGTGCTGGCAGGCA TGCACCTGCCCAGTGGGCGTGGCCTGTGACTCCGTGAGCGGCGAGTGTGGGAAGCGGTGTCCTGCTGGCTTCCAG GGAGAGGACTGTGGCCAAGAGTGCCCGGTGGGGACCTTTGGCGTGAACTGCTCGAGCTCCTGCTCCTGTGGGGGG GCCCCCTGCCACGGGGTCACGGGGCAGTGCCGGTGTCCGCCGGGGAGGACTGGGGAAGACTGTGAGGCAGGTGAG TGTGAGGGCCTCTGGGGGCTGGGCTGCCAGGAGATCTGCCCAGCATGCCATAACGCTGCTCGCTGCGACCCTGAG ACCGGAGCCTGCCTGTGCCTCCCTGGCTTTGTCGGCAGCCGCTGCCAGGACTGTGAGGCAGGCTGGTATGGTCCC AGCTGCCAGACAATGTGCTCTTGTGCCAATGATGGGCACTGCCACCAAGACACGGGACACTGCAGCTGTGCCCCC GGGTGGACCGGCTTTAGCTGCCAGAGAGCCTGTGATACTGGGCACTGGGGACCTGACTGCAGCCACCCCTGCAAC TGCAGCGCTGGCCACGGGAGCTGTGATGCCATCAGCGGCCTGTGTCTGTGTGAGGCTGGCTACGTGGGCCCGCGG TGCGAGCAGTCAGAGTGTCCCCAGGGCCACTTTGGGCCCGGCTGTGAGCAGCGGTGCCAGTGTCAGCATGGAGCA GCCTGTGACCACGTCAGCGGGGCCTGCACCTGCCCGGCCGGCTGGAGGGGCACCTTCTGCGAGCATGCCTGCCCG GCCGGCTTCTTTGGATTGGACTGTCGCAGTGCCTGCAACTGCACCGCCGGAGCTGCCTGTGATGCCGTGAATGGC TCCTGCCTCTGCCCCGCTGGCCGCCGGGGCCCCCGCTGTGCCGAGAGTGCCTGCCCAGCCCACACCTACGGGCAC AATTGCAGCCAGGCCTGTGCCTGCTTTAACGGGGCCTCCTGTGACCCTGTCCACGGGCAGTGCCACTGTGCCCCT GGCTGGATGGGGCCCTCCTGCCTGCAGGCCTGCCCTGCCGGCCTGTACGGCGACAACTGTCGGCATTCCTGCCTC TGCCAGAACGGAGGGACCTGTGACCCTGTCTCAGGCCACTGTGCGTGCCCAGAGGGCTGGGCCGGCCTGGCCTGT GAGGTAGAGTGCCTCCCCCGGGACGTCAGAGCTGGCTGCCGGCACAGCGGCGGTTGCCTCAACGGGGGCCTGTGT GACCCGCACACGGGCCGCTGCCTCTGCCCAGCCGGCTGGACTGGGGACAAGTGTCAGAGCCCTGCAGCCTGTGCC AAGGGCACATTCGGGCCTCACTGTGAGGGGCGCTGTGCCTGCCGGTGGGGAGGCCCCTGCCACCTTGCCACCGGG GCCTGCCTCTGCCCTCCGGGGTGGCGGGGGCCTCATCTTTCTGCAGCCTGCCTGCGGGGCTGGTTTGGAGAGGCC TGTGCCCAGCGCTGCAGCTGCCCGCCTGGCGCTGCCTGCCACCACGTCACTGGGGCCTGCCGCTGTCCCCCTGGC TTCACTGGCTCCGGCTGCGAGCAGGCCTGCCCACCCGGCAGCTTTGGGGAGGACTGTGCGCAGATGTGCCAGTGT CCCGGTGAGAACCCGGCCTGCCACCCTGCCACCGGGACCTGCTCATGTGCTGCTGGCTACCACGGCCCCAGCTGC CAGCAACGATGTCCGCCCGGGCGGTATGGGCCAGGCTGTGAACAGCTGTGTGGGTGTCTCAACGGGGGCTCCTGT GATGCGGCCACGGGGGCCTGCCGCTGCCCCACTGGGTTCCTCGGGACGGACTGCAACCTCACCTGTCCGCAGGGC CGCTTCGGCCCCAACTGCACCCACGTGTGTGGGTGTGGGCAGGGGGCGGCCTGCGACCCTGTGACCGGCACCTGC CTCTGCCCCCCGGGGAGAGCCGGCGTCCGCTGTGAGCGAGGCTGCCCCCAGAACCGGTTTGGCGTGGGCTGCGAG CACACCTGCTCCTGCAGAAATGGGGGCCTGTGCCACGCCAGCAAGCGGCAGCTGCTCCTGTGGCCTGGGCTGGAC GGGGCGGCACTGCGAGCTGGCCTGTCCCCCTGGGCGCTACGGAGCCGCCTGCCATCTGGAGTGCTCCTGCCACAA CAACAGCACGTGTGAGCCTGCCACGGGCACCTGCCGCTGCGGCCCCGGCTTCTATGGCCAGGCCTGCGAGCACCC CTGTCCCCCTGGCTTCCACGGGGCTGGCTGCCAGGGGTTGTGCTGGTGTCAACATGGAGCCCCCTGCGACCCCAT CAGTGGCCGATGCCTCTGCCCTGCCGGCTTCCACGGCCACTTCTGTGAGAGGGGGTGTGAGCCAGGTTCATTTGG AGAGGGCTGCCACCAGCGCTGTGACTGTGACGGGGGGGCACCCTGTGACCCTGTCACCGGTCTCTGCCTTTGCCC ACCAGGGCGCTCAGGAGCCACCTGTAACCTGGATTGCAGAAGGGGCCAGTTTGGGCCCAGCTGCACCCTGCACTG TGACTGCGGGGGTGGGGCTGACTGCGACCCTGTCAGTGGGCAGTGTCACTGTGTGGATGGCTACATGGGGCCCAC GTGCCGGGAAGGTGGGCCCCTCCGGCTCCCCGAGAACCCGTCCTTAGCCCAGGGCTCAGCGGGCACACTGCCCGC CTCCAGCAGACCCACATCCCGGAGCGGTGGACCAGCGAGGCACTAGTAGAGGCAGTCCCGTGGAGCCCGCCTCTC CAGTCCCAGCCAGAGGGGACCCTGGCCTTTGGTGACCACTGAGAAGGACACTTCACGGGCCCAGAGCTCCTGGTA CTGCCCTTCCTTTGAGGGCCGTGGAGGGCTGTGGACAGCCCAGCAACCTGTCGCTCTTGGAGGCTGGTGTGGCCT TGAGGAGGGAAGCCTCGCATGGCCGCTGGAAGAGAGGCGCCTCCTGGCCTGGCTCTGCAGAACCCAGGGGCACGC TCTGGGCCTGGGCTGAGGAAGTCCCGCTCTCCCCGCGGCTCTGAGTTGGACTGAGGACAGGTGTGGGCGCCAGTG TGGGTGCAGGCGCAGGTGCAGGCACAGGGCCACTGTCCTCCAGGCAGGCTTTTTGGTGCTAGGCCCTGGGACTGG AAGTCGCCCAGCCCGTATTTATGTAAAGGTATTTATGGGCCACTGCACATGCCCGCTGCAGCCCTGGGATCAGCT GGAAGCTGCCTGTCATCTCCTGCCCAATCCCCAGAAACCCTGATTCAGGTCTGCAGGCTCCTGCGGGCTCACCAG GCTGCTGGCTCCGGTACCATGTAAACCTAGGAAGGTAAAGGAGCAGGCAACCTCCTCGTGGCCTGTGTGTTTGCT GTGTTACGTGGACTCTGTGTGGGCTCCTCCCTGGGGCCCGGCCAGCATAACGGTGCACCCAGGGACCTCCCAGTG CACCCGGGGCCCTTTGCAGGGGTGGGGGTGCCACACAAGTGAAGAAGTTGGGACTCATCTCAGTTCCCAGTGCTA TTGAGGAGAACGCTGGGGCTGCATTCATTACCGCTGAGACCCAGAGACTGGCTGTTCCCAGAGAATGGCCCAGGG GGAGGAGGGCTGGTGTGGAGGGGCAACCTGGACTGAGGCCGAACTCCCTTGGGCTCACCCCACCCACCCCTACCT GAGCATCAGCAGTGGGGGGAGGGCAGCATCGCAGGGGCAGGGACTCCCTGGGTGAGGACAGACCAGCCCTCCCGA GCACCTGGCACTCATGGGCTGAGGCTGACTTCTCCTGGAAGAAGGGCCCAGAGTGGAAGGAAGAGGCAGAGGGTA GAGGTGGTGGCTGGGGGCTCCTCTGCAGAGTGGGGTGGCCAATGGAGAGGGCTGCACTCACACCGCAACATAGGA CTCTCTCTCCCTTAAGAAGGCCCCCTTAGGGTCTGGGCTGCCGCCCCCATCACCCTAAAACCAGCCAAGGTAGCT GAGGCCCCAGGGCAGACAATTTCACCAGCAGGANGAGGAGGAGTCCAGTGAGCTTGGTTGCTCACAGACAGCAAG GGAGCTGTCACAGAGGAAGCTGATGAATGGACCGCTGTGGGGAGACTTTAAAGTAGAACAGTGATAAGGGAGGGC AGGATGGTGGGGATGCAGAAGCAGCAGCCAGAGAGAGACGGACTGGGGTGCAGACGGAGTGTGGAAAACGCATAC CTTGAAATGAAGCATCCAGCAGATGGGGTGAGTGGATACAGCTCAGGAGATTCTCCCAGGAATAGCAGGGAGGCG TAAAGAGAGACAACGTACAGAGATAGATGAΆTGGAAΆTGGGTAAGGGAGGTGTTCATTCACATCCATCTAACTGC AAAATACAAAΆGTAAGAAGTCATTGACATGAAGCAACGACGACCAAGACGTTCTCAGATCTAAAGGTGAATGATC TCAGTCAGCCTGGAAATGCACAAGGTGGAAΆAATAACATAAAAΆAGCCATAAGACCTTGAAGAACATCAATGTCA AAGATAAATTCTAAAGTCCCAGAGAΆAAAAGAATGGGAATCAAATTGACCTCAGACTATACGTGAGAAACACGGA GAGCCAGAAAΆCTGTGATGTTCCATCCTCAGAGTTTGAAGGAAATATTTGAAGGCTGAΆTTTTACATCCAGCTAΆ ACTATCAAAGGCATGCAAAGTCCATGTTATTCTTAGGCCTTCAAGGCCTCGGCCATTTTTCTACAGAΆAAGCCTG ATTTTAAAATGCTCTTAGAGACGTTCTCCAGCCAGAAGAGAAAGAAGCCAGGAGGGTGCTCTGAGATATTCAGTC ACCACAGTTCCCAAATGGCCTAGGAATTCAGAGAGTCAGAATATCACCATTACTCCCCAATGGGAACCCCCGACA GTCTCAGCATGGTGTGAGGGTGTGGACGGGGGGCCTGGCAGGTACCAΆTCACTCATCCCGCTCAGTGAAGACACA GTGTTCAGCTACGGAAGCCATAAGGCAGGCCGAGCTTCTGCCCATCCGGAGGAAATCTCAGCTATCCAACGGCGG TCAGGAGCAGAGGAΆΆATAAAGCAGAΆTAΆCTAGAAAΆCACGCTCACAGATCCTAATGTTAACGGTTACAAATGA CGACGGAAAAACAAACTCCTGACCATATATTATATAGTTTCAAGCAGCAAGAAGGAGGATATTGAACATTCTCAA CACACATAATAAACGCTTGAGATGATGATATGCTCATTACCCTGATTTGATCACTAGACATNCCATGTATCAAAA CATCACTGTGTATCCGATGAATATCTACAATTATTGTCAATTAAAAACATCATTAAAAACAA
The NOV15aprotein (SEQ ID NO:44) encoded by SEQ ID NO:43 is 1404 amino acid residues in length and is presented using the one-letter amino acid code in Table 15B. Although the SignalP, Psort and/or Hydropathy results indicate that NOVl5a has a signal peptide and is likely to be localized in the mitochondrial matrix space with a certainty of0.4753, the NOV15a protein disclosed here is similar to the EGF family, some members ofwhich are released extracellularly. Alternatively, a NONl5apolypeptide is located to the microbody (peroxisome) with a certainty of0.3000, the mitochondrial innermembrane with a certainty of0.1802, or the mitochondrial hitermembrane space with a certainty of0.1802. The SignalP indicates a likely cleavage site for a ΝON15a peptide is betweenpositions 31 and 32, i.e., at the dash in the sequence GRG-AD.
Table 15B. Encoded ΝOV15a Protein Sequence (SEQ ID NO:44)
MPMGHSDRWS RLLRLALPLPVWLPAGGGRGADSPCLCSRPHVCAEQELTLVGRRQPCVQALSHTVPV KAGCG QA CVGHERRTVYYMGYRQVYTTEARTVLRCCRGWMQQPDEEGCLSDVGECANANGGCAGRCRDTVGGFYCR PP PSHQLQGDGETCQDVDECRTHNGGCQHRCVNTPGSYLCECKPGFRLHTDSRTCAINSCALGNGGCQHHCVQLTIT RHRCQCRPGFQLQEDGRHCVRRSPCANRNGSCMHRCQWRGLARCECHVGYQLAADGKACEDVDECAAGLAQCAH GCLNTQGSFKCVCHAGYΞLGADGRQCYRIEMΞIVNSCEANNGGCSHGCSHTSAGPLCTCPRGYELDTDQRTCIRC RRLCRQPVLQQVCTNNPGGYECGCYAGYRLSADGCGCEDVDECASSRGGCEHHCTNLAGSFQCSCEAGYRLHEDR RGCSALEEPMVDLDGELPFVRPLPHIAVLQDELPQLFQDDDVGADEEEAELRGEHTLTEKFVCLDDSFGHDCSLT CDDCRNGGTCLLGLDGCDCPEG TGLICNESCPPDTFGKNCSFSCSCQNGGTCDSVTGACRCPPGVSGTNCEDGC PKGYYGKHCRKKCNCANRGRCHRLYGACLCDPGLYGRFCHLACPP AFGPGCSEECQCVQPHTQSCDKRDGSCSC KAGFRGERCQAECEPGYFGPGC QACTCPVGVACDSVSGECGKRCPAGFQGEDCGQECPVGTFGVNCSSSCSCGG APCHGVTGQCRCPPGRTGEDCEAGECEGL GLGCQEICPACHNAARCDPETGACLCLPGFVGSRCQDCEAG YGP SCQTMCSCANDGHCHQDTGHCSCAPGWTGFSCQRACDTGHWGPDCSHPCNCSAGHGSCDAISGLCLCEAGYVGPR CEQSECPQGHFGPGCEQRCQCQHGAACDHVSGACTCPAGRGTFCΞHACPAGFFGLDCRSACNCTAGAACDAVNG SCLCPAGRRGPRCAESACPAHTYGHNCSQACACFNGASCDPVHGQCHCAPGWMGPSCLQACPAGLYGDNCRHSCL CQNGGTCDPVSGHCACPEGWAGLACEVECLPRDVRAGCRHSGGCLNGGLCDPHTGRCLCPAG TGDKCQSPAACA KGTFGPHCEGRCACR GGPCHLATGACLCPPG RGPHLSAACLRGWFGEACAQRCSCPPGAACHHVTGACRCPPG FTGSGCEQACPPGSFGEDCAQMCQCPGENPACHPATGTCSCAAGYHGPSCQQRCPPGRYGPGCEQLCGCLNGGSC DAATGACRCPTGFLGTDCNLTCPQGRFGPNCTHVCGCGQGAACDPVTGTCLCPPGRAGVRCERGCPQNRFGVGCE HTCSCRNGGLCHASKRQLLL PGLDGAALRAGLSPALRSRLPSGVLLPQQQHV SNP variants of NOVl 5a are disclosed in Example 2.
NOV15b
Alternatively, a NOVl 5 variant is NOVl 5b (alternatively referred to herein as CG56449- 02), which includes the 7319 nucleotide sequence (SEQ ID NO:45) shown in Table 15C. An open reading frame for the mature protein was identified beginning with an ATG codon at nucleotides 1-3 and ending with a TGA codon at nucleotides 4195-4197. Putative untranslated regions, if any, downstream from the termination codon and upstream from the initiation codon are underlined. The start and stop codons are in bold letters.
Table 15C. NON15b Nucleotide Sequence (SEQ ID NO:45)
ATGCCCATGGGACATTCTGACAGGTGGTCTTGGCGTCTCCTGAGGCTGGCACTGCCACTCCCAGTCTGGTTGCCG GCTGGGGGTGGCCGAGGCGCTGACTCTCCATGTCTCTGTTCCAGGCCCCACGTGTGTGCTGAGCAGGAGCTGACC CTGGTGGGCCGCCGCCAGCCGTGCGTGCAGGCCTTAAGCCACACGGTGCCGGTGTGGAAGGCCGGCTGTGGGTGG CAGGCGTGGTGCGTGGGTCATGAGCGGAGAACCGTCTACTACATGGGCTACAGGCAGGTGTATACCACGGAGGCC CGGACCGTGCTCAGGTGCTGCCGAGGGTGGACGCAGCAGCCCGACGAGGAGGGCTGCCTCTCGGCTGAATGCAGC GCCAGCCTCTGTTTTCACGGTGGCCGTTGTGTGCCAGGCTCAGCCCAGCCGTGTCACTGTCCCCCCGGCTTCCAG GGACCCCGCTGTCAGTATGATGTGGACGAATGCCGAACCCACAACGGTGGCTGCCAGCACCGGTGCGTGAACACC CCAGGCTCCTACCTCTGTGAGTGCAAGCCCGGCTTCCGGCTCCACACTGACAGCAGGACCTGCCTGGCCATTAAC TCCTGCGCCCTGGGCAATGGCGGCTGCCAGCACCACTGTGTCCAGCTCACAATCACTCGGCATCGCTGCCAGTGC CGGCCCGGGTTCCAGCTCCAGGAGGACGGCAGGCATTGTGTCCGTAGAAGCCCGTGTGCCAACAGGAACGGCAGC TGCATGCACAGGTGCCAGGTGGTCCGGGGCCTCGCCCGCTGTGAGTGCCACGTGGGCTATCAGCTAGCAGCGGAC GGCAAGGCCTGTGAAGATGTGGACGAATGTGCCGCAGGGCTGGCCCAGTGTGCCCATGGCTGCCTCAACACCCAG GGGTCCTTCAAGTGCGTGTGTCACGCGGGCTATGAGCTGGGCGCCGATGGCCGGCAGTGCTACCGTATTGAGATG GAAATCGTGAACAGCTGTGAGGCCAACAACGGCGGCTGCTCCCATGGCTGCAGCCACACCAGTGCTGGGCCCCTG TGCACCTGTCCCCGCGGCTACGAGCTGGACACAGATCAGAGGACCTGCATCAGATGTCGACGACTGTGCAGACAG CCCGTGCTGCAGCAGGTGTGCACCAACAACCCTGGCGGGTACGAGTGCGGCTGCTACGCCGGCTACCGGCTCAGT GCCGATGGCTGCGGCTGCGAGGATGTGGATGAGTGCGCCTCCAGCCGTGGCGGCTGCGAGCACCACTGCACCAAC CTGGCCGGCTCCTTCCAGTGCTCCTGCGAGGCCGGCTACCGGCTGCACGAGGACCGTAGGGGCTGCAGCGCCCTG GAGGAGCCGATGGTGGACCTGGACGGCGAGCTGCCTTTCGTGCGGCCCCTGCCCCACATTGCCGTGCTCCAGGAC GAGCTGCCGCAACTCTTCCAGGATGACGACGTCGGGGCCGATGAGGAAGAGGCAGAGTTGCGGGGCGAACACACG CTCACAGAGAAGTTTGTCTGCCTGGATGACTCCTTTGGCCATGACTGCAGCTTGACCTGTGATGACTGCAGGAAC GGAGGGACCTGCCTCCTGGGCCTGGATGGCTGTGATTGCCCCGAGGGCTGGACTGGGCTCATCTGCAATGAGAGT TGTCCTCCGGACACCTTTGGGAAGAACTGCAGCTTCTCCTGCAGCTGTCAGAATGGTGGGACCTGCGACTCTGTC ACGGGGGCCTGCCGCTGCCCCCCGGGTGTCAGTGGAACTAACTGTGAGGATGGCTGCCCCAAGGGCTACTATGGC AAGCACTGTCGCAAGAAATGCAACTGTGCCAACCGGGGCCGGTGCCACCGCCTCTACGGGGCCTGCCTCTGCGAC CCAGGGCTCTACGGCCGCTTCTGCCACCTCGCCTGCCCGCCGTGGGCCTTTGGGCCGGGCTGCTCGGAGGAGTGC CAGTGTGTGCAGCCCCACACGCAGTCCTGTGACAAGAGGGATGGCAGCTGCTCCTGCAAGGCTGGCTTCCGGGGC GAGCGCTGTCAGGCAGAGTGTGAGCCGGGCTACTTTGGGCCGGGGTGCTGGCAGGCATGCACCTGCCCAGTGGGC GTGGCCTGTGACTCCGTGAGCGGCGAGTGTGGGAAGCGGTGTCCTGCTGGCTTCCAGGGAGAGGACTGTGGCCAA GAGTGCCCGGTGGGGACCTTTGGCGTGAACTGCTCGAGCTCCTGCTCCTGTGGGGGGGCCCCCTGCCACGGGGTC ACGGGGCAGTGCCGGTGTCCGCCGGGGAGGACTGGGGAAGACTGTGAGGCAGGTGAGTGTGAGGGCCTCTGGGGG CTGGGCTGCCAGGAGATCTGCCCAGCATGCCATAACGCTGCTCGCTGCGACCCTGAGACCGGAGCCTGCCTGTGC CTCCCTGGCTTTGTCGGCAGCCGCTGCCAGGACTGTGAGGCAGGCTGGTATGGTCCCAGCTGCCAGACAATGTGC TCTTGTGCCAATGATGGGCACTGCCACCAAGACACGGGACACTGCAGCTGTGCCCCCGGGTGGACCGGCTTTAGC TGCCAGAGAGCCTGTGATACTGGGCACTGGGGACCTGACTGCAGCCACCCCTGCAACTGCAGCGCTGGCCACGGG AGCTGTGATGCCATCAGCGGCCTGTGTCTGTGTGAGGCTGGCTACGTGGGCCCGCGGTGCGAGCAGTCAGAGTGT CCCCAGGGCCACTTTGGGCCCGGCTGTGAGCAGCGGTGCCAGTGTCAGCATGGAGCAGCCTGTGACCACGTCAGC GGGGCCTGCACCTGCCCGGCCGGCTGGAGGGGCACCTTCTGCGAGCATGCCTGCCCGGCCGGCTTCTTTGGATTG GACTGTCGCAGTGCCTGCAACTGCACCGCCGGAGCTGCCTGTGATGCCGTGAATGGCTCCTGCCTCTGCCCCGCT GGCCGCCGGGGCCCCCGCTGTGCCGAGAGTGCCTGCCCAGCCCACACCTACGGGCACAATTGCAGCCAGGCCTGT GCCTGCTTTAACGGGGCCTCCTGTGACCCTGTCCACGGGCAGTGCCACTGTGCCCCTGGCTGGATGGGGCCCTCC TGCCTGCAGGCCTGCCCTGCCGGCCTGTACGGCGACAACTGTCGGCATTCCTGCCTCTGCCAGAACGGAGGGACC TGTGACCCTGTCTCAGGCCACTGTGCGTGCCCAGAGGGCTGGGCCGGCCTGGCCTGTGAGGTAGAGTGCCTCCCC CGGGACGTCAGAGCTGGCTGCCGGCACAGCGGCGGTTGCCTCAACGGGGGCCTGTGTGACCCGCACACGGGCCGC TGCCTCTGCCCAGCCGGCTGGACTGGGGACAAGTGTCAGAGCCCTGCAGCCTGTGCCAAGGGCACATTCGGGCCT CACTGTGAGGGGCGCTGTGCCTGCCGGTGGGGAGGCCCCTGCCACCTTGCCACCGGGGCCTGCCTCTGCCCTCCG GGGTGGCGGGGGCCTCATCTTTCTGCAGCCTGCCTGCGGGGCTGGTTTGGAGAGGCCTGTGCCCAGCGCTGCAGC TGCCCGCCTGGCGCTGCCTGCCACCACGTCACTGGGGCCTGCCGCTGTCCCCCTGGCTTCACTGGCTCCGGCTGC GAGCAGGCCTGCCCACCCGGCAGCTTTGGGGAGGACTGTGCGCAGATGTGCCAGTGTCCCGGTGAGAACCCGGCC TGCCACCCTGCCACCGGGACCTGCTCATGTGCTGCTGGCTACCACGGCCCCAGCTGCCAGCAACGATGTCCGCCC GGGCGGTATGGGCCAGGCTGTGAACAGCTGTGTGGGTGTCTCAACGGGGGCTCCTGTGATGCGGCCACGGGGGCC TGCCGCTGCCCCACTGGGTTCCTCGGGACGGACTGCAACCTCACCTGTCCGCAGGGCCGCTTCGGCCCCAACTGC ACCCACGTGTGTGGGTGTGGGCAGGGGGCGGCCTGCGACCCTGTGACCGGCACCTGCCTCTGCCCCCCGGGGAGA GCCGGCGTCCGCTGTGAGCGAGGCTGCCCCCAGAACCGGTTTGGCGTGGGCTGCGAGCACACCTGCTCCTGCAGA AATGGGGGCCTGTGCCACGCCAGCAAGCGGCAGCTGCTCCTGTGGCCTGGGCTGGACGGGGCGGCACTGCGAGCT GGCCTGTCCCCCTGGGCGCTACGGAGCCGCCTGCCATCTGGAGTGCTCCTGCCACAACAACAGCACGTGTGAGCC TGCCACGGGCACCTGCCGCTGCGGCCCCGGCTTCTATGGCCAGGCCTGCGAGCACCCCTGTCCCCCTGGCTTCCA CGGGGCTGGCTGCCAGGGGTTGTGCTGGTGTCAACATGGAGCCCCCTGCGACCCCATCAGTGGCCGATGCCTCTG CCCTGCCGGCTTCCACGGCCACTTCTGTGAGAGGGGGTGTGAGCCAGGTTCATTTGGAGAGGGCTGCCACCAGCG CTGTGACTGTGACGGGGGGGCACCCTGTGACCCTGTCACCGGTCTCTGCCTTTGCCCACCAGGGCGCTCAGGAGC CACCTGTAACCTGGATTGCAGAAGGGGCCAGTTTGGGCCCAGCTGCACCCTGCACTGTGACTGCGGGGGTGGGGC TGACTGCGACCCTGTCAGTGGGCAGTGTCACTGTGTGGATGGCTACATGGGGCCCACGTGCCGGGAAGGTGGGCC CCTCCGGCTCCCCGAGAACCCGTCCTTAGCCCAGGGCTCAGCGGGCACACTGCCCGCCTCCAGCAGACCCACATC CCGGAGCGGTGGACCAGCGAGGCACTAGTAGAGGCAGTCCCGTGGAGCCCGCCTCTCCAGTCCCAGCCAGAGGGG ACCCTGGCCTTTGGTGACCACTGAGAAGGACACTTCACGGGCCCAGAGCTCCTGGTACTGCCCTTCCTTTGAGGG CCGTGGAGGGCTGTGGACAGCCCAGCAACCTGTCGCTCTTGGAGGCTGGTGTGGCCTTGAGGAGGGAAGCCTCGC ATGGCCGCTGGAAGAGAGGCGCCTCCTGGCCTGGCTCTGCAGAACCCAGGGGCACGCTCTGGGCCTGGGCTGAGG AAGTCCCGCTCTCCCCGCGGCTCTGAGTTGGACTGAGGACAGGTGTGGGCGCCAGTGTGGGTGCAGGCGCAGGTG CAGGCACAGGGCCACTGTCCTCCAGGCAGGCTTTTTGGTGCTAGGCCCTGGGACTGGAAGTCGCCCAGCCCGTAT TTATGTAAAGGTATTTATGGGCCACTGCACATGCCCGCTGCAGCCCTGGGATCAGCTGGAAGCTGCCTGTCATCT CCTGCCCAATCCCCAGAAACCCTGATTCAGGTCTGCAGGCTCCTGCGGGCTCACCAGGCTGCTGGCTCCGGTACC ATGTAAACCTAGGAAGGTAAAGGAGCAGGCAACCTCCTCGTGGCCTGTGTGTTTGCTGTGTTACGTGGACTCTGT GTGGGCTCCTCCCTGGGGCCCGGCCAGCATAACGGTGCACCCAGGGACCTCCCAGTGCACCCGGGGCCCTTTGCA GGGGTGGGGGTGCCACACAAGTGAAGAAGTTGGGACTCATCTCAGTTCCCAGTGCTATTGAGGAGAACGCTGGGG CTGCATTCATTACCGCTGAGACCCAGAGACTGGCTGTTCCCAGAGAATGGCCCAGGGGGAGGAGGGCTGGTGTGG AGGGGCAACCTGGACTGAGGCCGAACTCCCTTGGGCTCACCCCACCCACCCCTACCTGAGCATCAGCAGTGGGGG GAGGGCAGCATCGCAGGGGCAGGGACTCCCTGGGTGAGGACAGACCAGCCCTCCCGAGCACCTGGCACTCATGGG CTGAGGCTGACTTCTCCTGGAAGAAGGGCCCAGAGTGGAAGGAAGAGGCAGAGGGTAGAGGTGGTGGCTGGGGGC TCCTCTGCAGAGTGGGGTGGCCAATGGAGAGGGCTGCACTCACACCGCAACATAGGACTCTCTCTCCCTTAAGAA GGCCCCCTTAGGGTCTGGGCTGCCGCCCCCATCACCCTAAAACCAGCCAAGGTAGCTGAGGCCCCAGGGCAGACA ATTTCACCAGCAGGANGAGGAGGAGTCCAGTGAGCTTGGTTGCTCACAGACAGCAAGGGAGCTGTCACAGAGGAA GCTGATGAATGGACCGCTGTGGGGAGACTTTAAAGTAGAACAGTGATAAGGGAGGGCAGGATGGTGGGGATGCAG AAGCAGCAGCCAGAGAGAGACGGACTGGGGTGCAGACGGAGTGTGGAAAACGCATACCTTGAAATGAAGCATCCA GCAGATGGGGTGAGTGGATACAGCTCAGGAGATTCTCCCAGGAATAGCAGGGAGGCGTAAAGAGAGACAACGTAC AGAGATAGATGAATGGAAATGGGTAAGGGAGGTGTTCATTCACATCCATCTAACTGCAAAATACAAAAGTAAGAA GTCATTGACATGAAGCAACGACGACCAAGACGTTCTCAGATCTAAAGGTGAATGATCTCAGTCAGCCTGGAAATG CACAAGGTGGAAAAATAACATAAAAAAGCCATAAGACCTTGAAGAACATCAATGTCAAAGATAAATTCTAAAGTC CCAGAGAAAAAAGAATGGGAATCAAATTGACCTCAGACTATACGTGAGAAACACGGAGAGCCAGAAAACTGTGAT GTTCCATCCTCAGAGTTTGAAGGAAATATTTGAAGGCTGAATTTTACATCCAGCTAAACTATCAAAGGCATGCAA AGTCCATGTTATTCTTAGGCCTTCAAGGCCTCGGCCATTTTTCTACAGAAAAGCCTGATTTTAAAATGCTCTTAG AGACGTTCTCCAGCCAGAAGAGAAAGAAGCCAGGAGGGTGCTCTGAGATATTCAGTCACCACAGTTCCCAAATGG CCTAGGAATTCAGAGAGTCAGAATATCACCATTACTCCCCAATGGGAACCCCCGACAGTCTCAGCATGGTGTGAG GGTGTGGACGGGGGGCCTGGCAGGTACCAATCACTCATCCCGCTCAGTGAAGACACAGTGTTCAGCTACGGAAGC CATAAGGCAGGCCGAGCTTCTGCCCATCCGGAGGAAATCTCAGCTATCCAACGGCGGTCAGGAGCAGAGGAAAAT AAAGCAGAATAACTAGAAAACACGCTCACAGATCCTAATGTTAACGGTTACAAATGACGACGGAAAAACAAACTC CTGACCATATATTATATAGTTTCAAGCAGCAAGAAGGAGGATATTGAACATTCTCAACACACATAATAAACGCTT GAGATGATGATATGCTCATTACCCTGATTTGATCACTAGACATNCCATGTATCAAAACATCACTGTGTATCCGAT GAATATCTACAATTATTGTCAATTAAAAACATCATTAAAAACAA
The NOVl 5b protein (SEQ ID NO:46) encoded by SEQ ID NO:45 is 1398 amino acid residues in length and is presented using the one-letter amino acid code in Table 15D. Although the SignalP, Psort and/or Hydropathy results indicate that NON 15b has a signal peptide and is likely to be localized in the mitochondrial matrix space with a certainty of 0.4753, the ΝON15b protein disclosed here is similar to the EGF family, some members of which are released extracellularly. Alternatively, a ΝON15b polypeptide is located to the microbody (peroxisome) with a certainty of 0.3000, the mitochondrial inner membrane with a certainty of 0.1802, or the mitochondrial intermembrane space with a certainty of 0.1802. The SignalP indicates a likely cleavage site for a ΝON15b peptide is between positions 31 and 32, i. e. , at the dash in the sequence GRG-AD.
Table 15D. Encoded ΝOV15b Protein Sequence (SEQ ID NO:46)
MPMGHSDRWS RLLRLALPLPVWLPAGGGRGADSPCLCSRPHVCAEQELTLVGRRQPCVQALSHTVPV KAGCG QACVGHERRTVYYMGYRQVYTTEARTVLRCCRG TQQPDEΞGCLSAECSASLCFHGGRCVPGSAQPCHCPPGFQ GPRCQYDVDECRTHNGGCQHRCVNTPGSYLCECKPGFRLHTDSRTCLAINSCALGNGGCQHHCVQLTITRHRCQC RPGFQLQEDGRHCVRRSPCANRNGSCMHRCQWRGLARCECHVGYQLAADGKACEDVDECAAGLAQCAHGCLNTQ GSFKCVCHAGYELGADGRQCYRIEMEIVNSCEANNGGCSHGCSHTSAGPLCTCPRGYELDTDQRTCIRCRRLCRQ PVLQQVCTNNPGGYECGCYAGYRLSADGCGCEDVDECASSRGGCEHHCTNLAGSFQCSCEAGYRLHEDRRGCSAL EEPMVDLDGELPFVRPLPHIAVLQDELPQLFQDDDVGADEEEAELRGEHTLTEKFVCLDDSFGHDCSLTCDDCRN GGTCLLGLDGCDCPEG TGLICNESCPPDTFGKNCSFSCSCQNGGTCDSVTGACRCPPGVSGTNCEDGCPKGYYG KHCRKKCNCANRGRCHRLYGACLCDPGLYGRFCHLACPPWAFGPGCSEECQCVQPHTQSCDKRDGSCSCKAGFRG ΞRCQAECEPGYFGPGC QACTCPVGVACDSVSGECGKRCPAGFQGEDCGQECPVGTFGVNCSSSCSCGGAPCHGV TGQCRCPPGRTGEDCEAGECEGL GLGCQEICPACHNAARCDPETGACLCLPGFVGSRCQDCEAG YGPSCQTMC SCANDGHCHQDTGHCSCAPGWTGFSCQRACDTGH GPDCSHPCNCSAGHGSCDAISGLCLCEAGYVGPRCEQSEC PQGHFGPGCEQRCQCQHGAACDHVSGACTCPAG RGTFCEHACPAGFFGLDCRSACNCTAGAACDAVNGSCLCPA GRRGPRCAΞSACPAHTYGHNCSQACACFNGASCDPVHGQCHCAPG MGPSCLQACPAGLYGDNCRHSCLCQNGGT CDPVSGHCACPEGWAGLACEVECLPRDVRAGCRHSGGCLNGGLCDPHTGRCLCPAG TGDKCQSPAACAKGTFGP HCEGRCACRWGGPCHLATGACLCPPGRGPHLSAACLRG FGEACAQRCSCPPGAACHHVTGACRCPPGFTGSGC EQACPPGSFGEDCAQMCQCPGENPACHPATGTCSCAAGYHGPSCQQRCPPGRYGPGCEQLCGCLNGGSCDAATGA CRCPTGFLGTDCNLTCPQGRFGPNCTHVCGCGQGAACDPVTGTCLCPPGRAGVRCERGCPQNRFGVGCEHTCSCR NGGLCHASKRQLLLWPGLDGAALRAGLSP ALRSRLPSGVLLPQQQHV NON15c
Alternatively, a ΝON15 variant is ΝON15c (alternatively referred to herein as CG56449- 03), which includes the 4733 nucleotide sequence (SEQ ID ΝO:47) shown in Table 15E. An open reading frame for the mature protein was identified beginning with an ATG codon at nucleotides 1-3 and ending with a TAG codon at nucleotides 4351-4353. Putative untranslated regions, if any, downstream from the termination codon and upstream from the initiation codon are underlined. The start and stop codons are in bold letters.
Table 15E. NOV15c Nucleotide Sequence (SEQ ID NO:47)
ATGCCCATGGGACATTCTGACAGGTGGTCTTGGCGTCTCCTGAGGCTGGCACTGCCACTCCCAGTCTGGTTGCCG GCTGGGGGTGGCCGAGGCGCTGACTCTCCATGTCTCTGTTCCAGGCCCCACGTGTGTGCTGAGCAGGAGCTGACC CTGGTGGGCCGCCGCCAGCCGTGCGTGCAGGCCTTAAGCCACACGGTGCCGGTGTGGAAGGCCGGCTGTGGGTGG CAGGCGTGGTGCGTGGGTCATGAGCGGAGAACCGTCTACTACATGGGCTACAGGCAGGTGTATACCACGGAGGCC CGGACCGTGCTCAGGTGCTGCCGAGGGTGGACGCAGCAGCCCGACGAGGAGGGCTGCCTCTCGGCTGAATGCAGC GCCAGCCTCTGTTTTCACGGTGGCCGTTGTGTGCCAGGCTCAGCCCAGCCGTGTCACTGTCCCCCCGGCTTCCAG GGACCCCGCTGTCAGTATGATGTGGACGAATGCCGAACCCACAACGGTGGCTGCCAGCACCGGTGCGTGAACACC CCAGGCTCCTACCTCTGTGAGTGCAAGCCCGGCTTCCGGCTCCACACTGACAGCAGGACCTGCCTGGCCATTAAC TCCTGCGCCCTGGGCAATGGCGGCTGCCAGCACCACTGTGTCCAGCTCACAATCACTCGGCATCGCTGCCAGTGC CGGCCCGGGTTCCAGCTCCAGGAGGACGGCAGGCATTGTGTCCGTAGAAGCCCGTGTGCCAACAGGAACGGCAGC TGCATGCACAGGTGCCAGGTGGTCCGGGGCCTCGCCCGCTGTGAGTGCCACGTGGGCTATCAGCTAGCAGCGGAC GGCAAGGCCTGTGAAGATGTGGACGAATGTGCCGCAGGGCTGGCCCAGTGTGCCCATGGCTGCCTCAACACCCAG GGGTCCTTCAAGTGCGTGTGTCACGCGGGCTATGAGCTGGGCGCCGATGGCCGGCAGTGCTACCGTATTGAGATG GAAATCGTGAACAGCTGTGAGGCCAACAACGGCGGCTGCTCCCATGGCTGCAGCCACACCAGTGCTGGGCCCCTG TGCACCTGTCCCCGCGGCTACGAGCTGGACACAGATCAGAGGACCTGCATCAGATGTCGACGACTGTGCAGACAG CCCGTGCTGCAGCAGGTGTGCACCAACAACCCTGGCGGGTACGAGTGCGGCTGCTACGCCGGCTACCGGCTCAGT GCCGATGGCTGCGGCTGCGAGGATGTGGATGAGTGCGCCTCCAGCCGTGGCGGCTGCGAGCACCACTGCACCAAC CTGGCCGGCTCCTTCCAGTGCTCCTGCGAGGCCGGCTACCGGCTGCACGAGGACCGTAGGGGCTGCAGCGCCCTG GAGGAGCCGATGGTGGACCTGGACGGCGAGCTGCCTTTCGTGCGGCCCCTGCCCCACATTGCCGTGCTCCAGGAC GAGCTGCCGCAACTCTTCCAGGATGACGACGTCGGGGCCGATGAGGAAGAGGCAGAGTTGCGGGGCGAACACACG CTCACAGAGAAGTTTGTCTGCCTGGATGACTCCTTTGGCCATGACTGCAGCTTGACCTGTGATGACTGCAGGAAC GGAGGGACCTGCCTCCTGGGCCTGGATGGCTGTGATTGCCCCGAGGGCTGGACTGGGCTCATCTGCAATGAGAGT TGTCCTCCGGACACCTTTGGGAAGAACTGCAGCTTCTCCTGCAGCTGTCAGAATGGTGGGACCTGCGACTCTGTC ACGGGGGCCTGCCGCTGCCCCCCGGGTGTCAGTGGAACTAACTGTGAGGATGGCTGCCCCAAGGGCTACTATGGC AAGCACTGTCGCAAGAAATGCAACTGTGCCAACCGGGGCCGGTGCCACCGCCTCTACGGGGCCTGCCTCTGCGAC CCAGGGCTCTACGGCCGCTTCTGCCACCTCGCCTGCCCGCCGTGGGCCTTTGGGCCGGGCTGCTCGGAGGAGTGC CAGTGTGTGCAGCCCCACACGCAGTCCTGTGACAAGAGGGATGGCAGCTGCTCCTGCAAGGCTGGCTTCCGGGGC GAGCGCTGTCAGGCAGAGTGTGAGCTGGGCTACTTTGGGCCGGGGTGCTGGCAGGCATGCACCTGCCCAGTGGGC GTGGCCTGTGACTCCGTGAGCGGCGAGTGTGGGAAGCGGTGTCCTGCTGGCTTCCAGGGAGAGGACTGTGGCCAA GAGTGCCCGGTGGGGACGTTTGGCGTGAACTGCTCGAGCTCCTGCTCCTGTGGGGGGGCCCCCTGCCACGGGGTC ACGGGGCAGTGCCGGTGTCCACCGGGGAGGACTGGGGAAGACTGTGAGGCAGATTGTCCCGAGGGCCGCTGGGGG CTGGGCTGCCAGGAGATCTGCCCAGCATGCCAGCACGCTGCCCGCTGCGACCCTGAGACCGGAGCCTGCCTGTGC CTCCCTGGCTTCGTCGGCAGCCGCTGCCAGGACGTGTGCCCAGCAGGCTGGTATGGTCCCAGCTGCCAGACAAGG TGCTCTTGTGCCAATGATGGGCACTGCCACCCAGCCACCGGACACTGCAGCTGTGCCCCCGGGTGGACCGGCTTT AGCTGCCAGAGAGCCTGTGATACTGGGCACTGGGGACCTGACTGCAGCCACCCCTGCAACTGCAGCGCTGGCCAC GGGAGCTGTGATGCCATCAGCGGCCTGTGTCTGTGTGAGGCTGGCTACGTGGGCCCGCGGTGCGAGCAGCAGTGT CCCCAGGGCCACTTTGGGCCCGGCTGTGAGCAGCTGTGCCAGTGTCAGCATGGAGCAGCCTGTGACCACGTCAGC GGGGCCTGCACCTGCCCGGCCGGCTGGAGGGGCACCTTCTGCGAGCATGCCTGCCCGGCCGGCTTCTTTGGATTG GACTGTCGTAGTGCCTGCAACTGCACCGCCGGAGCTGCCTGTGATGCCGTGAATGGCTCCTGCCTCTGCCCCGCT GGCCGCCGGGGCCCCCGCTGTGCCGAGACCTGCCCTGCCGGCCTGTACGGCGACAACTGTCGGCATTCCTGCCTC TGCCAGAACGGAGGGACCTGTGACCCTGTCTCAGGCCACTGTGCGTGCCCAGAGGGCTGGGCCGGCCTGGCCTGT GAGAAGGAGTGCCCCCCCCGGGACGTCAGAGCTGGCTGCCGGCACAGCGGTGGTTGCCTCAACGGGGGCCTGTGT GACCCGCACACGGGCCGCTGCCTCTGCCCAGCCGGCTGGGCTGGGGACAAGTGTCAGAGCCCCTGCCTGCGGGGC TGGTTTGGAGAGGCCTGTGCCCAGCACTGCAGCTGCCCGCCTGGCGCTGCCTGCCACCACGTCACTGGGGCCTGC CGCTGTCCCCCTGGCTTCACTGGCTCCGGCTGCGAGCAGGGATGTCCGCCCGGGCGGTATGGGCCAGGCTGTGAA CAGCTGTGTGGGTGTCTCAACGGGGGCTCCTGTGATGCGGCCACGGGGGCCTGCCGCTGCCCCACTGGGTTCCTC GGGACGGACTGCAACCTCACCTGTCCGCAGGGCCGCTTCGGCCCCAACTGCACCCACGTGTGTGGGTGTGGGCAG GGGGCGGCCTGCGACCCTGTGACCGGCACCTGCCTCTGCCCCCCGGGGAGAGCCGGCGTCCGCTGTGAGCGAGGC TGCCCCCAGAACCGGTTTGGCGTGGGCTGCGAGCACACCTGCTCCTGCAGAAATGGGGGCCTGTGCCACGCCAGC AACGGCAGCTGCTCCTGTGGCCTGGGCTGGACGGGGCGGCACTGCGAGCTGGCCTGTCCCCCTGGGCGCTACGGA GCCGCCTGCCATCTGGAGTGCTCCTGCCACAACAACAGCACGGGTGAGCCTGCCACGGGCACCTGCCGCTGCGGC CCCGGCTTCTATGGCCAGGCCTGCGAGCACCCCTGTCCCCCTGGCTTCCACGGGGCTGGCTGCCAGGGGTTGTGC TGGTGTCAACATGGAGCCCCCTGCGACCCCATCAGTGGCCGATGCCTCTGCCCTGCCGGCTTCCACGGCCACTTC TGTGAGAGGGGGTGTGAGCCAGGTTCATTTGGAGAGGGCTGCCACCAGCGCTGTGACTGTGACGGGGGGGCACCC TGTGACCCTGTCACCGGTCTCTGCCTTTGCCCACCAGGGCGCTCAGGAGCCACCTGTAACCTGGATTGCAGAAGG GGCCAGTTTGGGCCCAGCTGCACCCTGCACTGTGACTGCGGGGGTGGGGCTGACTGCGACCCTGTCAGTGGGCAG TGTCACTGTGTGGATGGCTACATGGGGCCCACGTGCCGGGAAGGTGGGCCCCTCCGGCTCCCCGAGAACCCGTCC TTAGCCCAGGGCTCAGCGGGCACACTGCCCGCCTCCAGCAGACCCACATCCCGGAGCGGTGGACCAGCGAGGCAC TAGTAGAGGCAGTCCCGTGGAGCCCGCCTCTCCAGTCCCAGCCAGAGGGGACCCTGGCCTTTGGTGACCACTGAG AAGGACACTTCACGGGCCCAGAGCTCCTGGTACTGCCCTTCCTTTGAGGGCCGTGGAGGGCTGTGGACAGCCCAG CAACCTGTCGCTCTTGGAGGCTGGTGTGGCCTTGAGGAGGGAAGCCTCGCATGGCCGCTGGAAGAGAGGCGCCTC CTGGCCTGGCTCTGCAGAACCCAGGGGCACGCTCTGGGCCTGGGCTGAGGAAGTCCCGCTCTCCCCGCGGCTCTG AGTTGGACTGAGGACAGGTGTGGGCGCCAGTGTGGGTGCAGGCGCAGGTGCAGGCACAGGGCCACTGTCCTCCAG GCAGGCTT
The NONl 5c protein (SEQ ID ΝO:48) encoded by SEQ ID NO:47 is 1450 amino acid residues in length and is presented using the one-letter amino acid code in Table 15F. Although the SignalP, Psort and/or Hydropathy results indicate that NOV15c has a signal peptide and is likely to be localized in the mitochondrial matrix space with a certainty of 0.4753, the NOVl 5c protein disclosed here is similar to the EGF family, some members of which are released extracellularly. Alternatively, a NOV15c polypeptide is located to the cytoplasm with a certainty of 0.4500, the mitochondrial inner membrane with a certainty of 0.1802, or the mitochondrial intermembrane space with a certainty of 0.1802. The SignalP indicates a likely cleavage site for a NON15c peptide is between positions 31 and 32, i.e., at the dash in the sequence GRG-AD.
Table 15F. Encoded ΝON15c Protein Sequence (SEQ ID ΝO:48)
MPMGHSDR SWRLLRLALPLPVWLPAGGGRGADSPCLCSRPHVCAEQΞLTLVGRRQPCVQALSHTVPV KAGCG QA CVGHΞRRTVYYMGYRQVYTTEARTVLRCCRGWTQQPDEEGCLSAECSASLCFHGGRCVPGSAQPCHCPPGFQ GPRCQYDVDECRTHNGGCQHRCVNTPGSYLCECKPGFRLHTDSRTCLAINSCALGNGGCQHHCVQLTITRHRCQC RPGFQLQEDGRHCVRRSPCANRNGSCMHRCQWRGLARCECHVGYQLAADGKACEDVDECAAGLAQCAHGCLNTQ GSFKCVCHAGYELGADGRQCYRIEMEIVNSCEANNGGCSHGCSHTSAGPLCTCPRGYELDTDQRTCIRCRRLCRQ PVLQQVCTNNPGGYECGCYAGYRLSADGCGCEDVDECASSRGGCEHHCTNLAGSFQCSCEAGYRLHEDRRGCSAL EEPMVDLDGELPFVRPLPHIAVLQDELPQLFQDDDVGADEEΞAELRGEHTLTEKFVCLDDSFGHDCSLTCDDCRN GGTCLLGLDGCDCPEG TGLICNESCPPDTFGKNCSFSCSCQNGGTCDSVTGACRCPPGVSGTNCEDGCPKGYYG KHCRKKCNCANRGRCHRLYGACLCDPGLYGRFCHLACPP AFGPGCSEECQCVQPHTQSCDKRDGSCSCKAGFRG ERCQAECELGYFGPGC QACTCPVGVACDSVSGECGKRCPAGFQGEDCGQECPVGTFGVNCSSSCSCGGAPCHGV TGQCRCPPGRTGEDCEADCPEGRWGLGCQEICPACQHAARCDPETGACLCLPGFVGSRCQDVCPAGWYGPSCQTR CSCANDGHCHPATGHCSCAPG TGFSCQRACDTGH GPDCSHPCNCSAGHGSCDAISGLCLCEAGYVGPRCEQQC PQGHFGPGCEQLCQCQHGAACDHVSGACTCPAG RGTFCEHACPAGFFGLDCRSACNCTAGAACDAVNGSCLCPA GRRGPRCAETCPAGLYGDNCRHSCLCQNGGTCDPVSGHCACPEGWAGLACEKECPPRDVRAGCRHSGGCLNGGLC DPHTGRCLCPAGWAGDKCQSPCLRG FGEACAQHCSCPPGAACHHVTGACRCPPGFTGSGCEQGCPPGRYGPGCE QLCGCLNGGSCDAATGACRCPTGFLGTDCNLTCPQGRFGPNCTHVCGCGQGAACDPVTGTCLCPPGRAGVRCERG CPQNRFGVGCEHTCSCRNGGLCHASNGSCSCGLG TGRHCELACPPGRYGAACHLECSCHNNSTGEPATGTCRCG PGFYGQACEHPCPPGFHGAGCQGLCWCQHGAPCDPISGRCLCPAGFHGHFCERGCEPGSFGEGCHQRCDCDGGAP CDPVTGLCLCPPGRSGATCNLDCRRGQFGPSCTLHCDCGGGADCDPVSGQCHCVDGYMGPTCREGGPLRLPENPS LAQGSAGTLPASSRPTSRSGGPARH
NON15d
Alternatively, a ΝON15 variant is ΝON15d (alternatively referred to herein as CG56449- 04), which includes the 877 nucleotide sequence (SEQ ID ΝO:49) shown in Table 15G. An open reading frame for the mature protein was identified beginning with an ATG codon at nucleotides 25-27 and ending with a TAG codon at nucleotides 535-537. Putative untranslated regions, if any, downstream from the termination codon and upstream from the initiation codon are underlined. The start and stop codons are in bold letters.
Table 15G. NOV15d Nucleotide Sequence (SEQ ID NO:49)
CCGGAGCTGCCTGTGATGCCGTGAATGGCTCCTGCCTCTGCCCCGCTGGCCGCCGGGGCCCCCGCTGTGCCGAGA CCTGCCCTGCCGGCCTGTACGGCGACAACTGTCGGCATTCCTGCCTCTGCCAGAACGGAGGGACCTGTGACCCTG TCTCAGGCCTGCGAGCACCCCTGTCCCCCTGGCTTCCACGGGGCTGGCCGCCAGGGGTTGTGCTGGTGTCAACAT GGAGCCCCCTGCGACCCCATCAGTGGCCGATGCCTCTGCCCTGCCGGCTTCCACGGCCACTTCTGTGAGAGGGAT TGCAGAAGGGGCCAGTTTGGGCCCAGCTGCACCCTGCACTGTGACTGCGGGGGTGGGGCTGACTGCGACCCTGTC AGTGGGCAGTGTCACTGTGTGGATGGCTACATGGGGCCCACGTGCCGGGAAGGTGGGCCCCTCCGGCTCCCCGAG AACCCGTCCTTAGCCCAGGGCTCAGCGGGCACACTGCCCGCCTCCAGCAGACCCACATCCCGGAGCGGTGGACCA GCGAGGCACTAGTAGAGGCAGTCCCGTGGAGCCCGCCTCTCCAGTCCCAGCCAGAGGGGACCCTGGCCTTTGGTG ACCACTGAGAAGGACACTTCACGGGCCCAGAGCTCCTGGTACTGCCCTTCCTTTGAGGGCCGTGGAGGGCTGTGG ACAGCCCAGCAACCTGTCGCTCTTGGAGGCTGGTGTGGCCTTGAGGAGGGAAGCCTCGCATGGCCGCTGGAAGAG AGGCGTCTCCTGGCCTGGCTCTGCAGAACCCAGGGGCACGCTCTGGGCCTGGGCTGAGGAAGTCCCGCTCTCCCG CGGCTCTGAGTTGGACTGAGGACAGGTGTGGGCGCCAGTGTGGGTGCAGGCG
The NOV15d protein (SEQ ID NO:50) encoded by SEQ ID NO:49 is 170 amino acid residues in length and is presented using the one-letter amino acid code in Table 15H. The SignalP, Psort and/or Hydropathy results indicate that NON15d has no known signal peptide and is likely to be localized in the cytoplasm with a certainty of 0.6500. Alternatively, a ΝON15d polypeptide is located to the lysosome (lumen) with a certainty of 0.1853, or the mitochondrial matrix space with a certainty of 0.1000. Table 15H. Encoded NON15d Protein Sequence (SEQ ID ΝO:50)
MAPASAPLAAGAPAVPRPALPACTATTVGIPASARTEGPVTLSQACEHPCPPGFHGAGRQGLC CQHGAPCDPIS GRCLCPAGFHGHFCERDCRRGQFGPSCTLHCDCGGGADCDPVSGQCHCVDGYMGPTCREGGPLRLPENPSLAQGS AGTLPASSRPTSRSGGPARH
NOV15e
Alternatively, a NON 15 variant is ΝON15e (alternatively referred to herein as CG56449- 06), which includes the 7334 nucleotide sequence (SEQ ID ΝO:51) shown in Table 151. An open reading frame for the mature protem was identified beginning with an ATG codon at nucleotides 1-3 and ending with a TGA codon at nucleotides 4210-4212. Putative untranslated regions, if any, downstream from the termination codon and upstream from the initiation codon are underlined. The start and stop codons are in bold letters.
Table 151. NOV15e Nucleotide Sequence (SEQ ID NO:51)
ATGCCCATGGGACATTCTGACAGGTGGTCTTGGCGTCTCCTGAGGCTGGCACTGCCACTCCCAGTCTGGTTGCCG GCTGGGGGTGGCCGAGGCGCTGACTCTCCATGTCTCTGTTCCAGGCCCCACGTGTGTGCTGAGCAGGAGCTGACC CTGGTGGGCCGCCGCCAGCCGTGCGTGCAGGCCTTAAGCCACACGGTGCCGGTGTGGAAGGCCGGCTGTGGGTGG CAGGCGTGGTGCGTGGGTCATGAGCGGAGGACCGTCTACTACATGGGCTACAGGCAGGTGTATACCACGGAGGCC CGGACCGTGCTCAGGTGCTGCCGAGGGTGGATGCAGCAGCCCGACGAGGAGGGCTGCCTCTCGGATGTGGGTGAG TGTGCCAACGCCAACGGGGGCTGTGCGGGTCGGTGCCGGGACACCGTGGGGGGCTTCTACTGCCGCTGGCCCCCC CCCAGCCACCAGCTGCAGGGTGATGGCGAGACTTGCCAAGATGTGGACGAATGCCGAACCCACAACGGTGGCTGC CAGCACCGGTGCGTGAACACCCCAGGCTCCTACCTCTGTGAGTGCAAGCCCGGCTTCCGGCTCCACACTGACAGC AGGACCTGCGCCATTAACTCCTGCGCCCTGGGCAATGGCGGCTGCCAGCACCACTGTGTCCAGCTCACAATCACT CGGCATCGCTGCCAGTGCCGGCCCGGGTTCCAGCTCCAGGAGGACGGCAGGCATTGTGTCCGTAGAAGCCCGTGT GCCAACAGGAACGGCAGCTGCATGCACAGGTGCCAGGTGGTCCGGGGCCTCGCCCGCTGTGAGTGCCACGTGGGC TATCAGCTAGCAGCGGACGGCAAGGCCTGTGAAGATGTGGACGAATGTGCCGCAGGGCTGGCCCAGTGTGCCCAT GGCTGCCTCAACACCCAGGGGTCCTTCAAGTGCGTGTGTCACGCGGGCTATGAGCTGGGCGCCGATGGCCGGCAG TGCTACCGTATTGAGATGGAAATCGTGAACAGCTGTGAGGCCAACAACGGCGGCTGCTCCCATGGCTGCAGCCAC ACCAGTGCTGGGCCCCTGTGCACCTGTCCCCGCGGCTACGAGCTGGACACAGATCAGAGGACCTGCATCAGATGT CGACGACTGTGCAGACAGCCCGTGCTGCAGCAGGTGTGCACCAACAACCCTGGCGGGTACGAGTGCGGCTGCTAC GCCGGCTACCGGCTCAGTGCCGATGGCTGCGGCTGCGAGGATGTGGATGAGTGCGCCTCCAGCCGTGGCGGCTGC GAGCACCACTGCACCAACCTGGCCGGCTCCTTCCAGTGCTCCTGCGAGGCCGGCTACCGGCTGCACGAGGACCGT AGGGGCTGCAGCGCCCTGGAGGAGCCGATGGTGGACCTGGACGGCGAGCTGCCTTTCGTGCGGCCCCTGCCCCAC ATTGCCGTGCTCCAGGACGAGCTGCCGCAACTCTTCCAGGATGACGACGTCGGGGCCGATGAGGAAGAGGCAGAG TTGCGGGGCGAACACACGCTCACAGAGAAGTTTGTCTGCCTGGATGACTCCTTTGGCCATGACTGCAGCTTGACC TGTGATGACTGCAGGAACGGAGGGACCTGCCTCCTGGGCCTGGATGGCTGTGATTGCCCCGAGGGCTGGACTGGG CTCATCTGCAATGAGAGTTGTCCTCCGGACACCTTTGGGAAGAACTGCAGCTTCTCCTGCAGCTGTCAGAATGGT GGGACCTGCGACTCTGTCACGGGGGCCTGCCGCTGCCCCCCGGGTGTCAGTGGAACTAACTGTGAGGATGGCTGC CCCAAGGGCTACTATGGCAAGCACTGTCGCAAGAAATGCAACTGTGCCAACCGGGGCCGGTGCCACCGCCTCTAC GGGGCCTGCCTCTGCGACCCAGGGCTCTACGGCCGCTTCTGCCACCTCGCCTGCCCGCCGTGGGCCTTTGGGCCG GGCTGCTCGGAGGAGTGCCAGTGTGTGCAGCCCCACACGCAGTCCTGTGACAAGAGGGATGGCAGCTGCTCCTGC AAGGCTGGCTTCCGGGGCGAGCGCTGTCAGGCAGAGTGTGAGCCGGGCTACTTTGGGCCGGGGTGCTGGCAGGCA TGCACCTGCCCAGTGGGCGTGGCCTGTGACTCCGTGAGCGGCGAGTGTGGGAAGCGGTGTCCTGCTGGCTTCCAG GGAGAGGACTGTGGCCAAGAGTGCCCGGTGGGGACCTTTGGCGTGAACTGCTCGAGCTCCTGCTCCTGTGGGGGG GCCCCCTGCCACGGGGTCACGGGGCAGTGCCGGTGTCCGCCGGGGAGGACTGGGGAAGACTGTGAGGCAGGTGAG TGTGAGGGCCTCTGGGGGCTGGGCTGCCAGGAGATCTGCCCAGCATGCCATAACGCTGCTCGCTGCGACCCTGAG ACCGGAGCCTGCCTGTGCCTCCCTGGCTTTGTCGGCAGCCGCTGCCAGGACTGTGAGGCAGGCTGGTATGGTCCC AGCTGCCAGACAATGTGCTCTTGTGCCAATGATGGGCACTGCCACCAAGACACGGGACACTGCAGCTGTGCCCCC GGGTGGACCGGCTTTAGCTGCCAGAGAGCCTGTGATACTGGGCACTGGGGACCTGACTGCAGCCACCCCTGCAAC TGCAGCGCTGGCCACGGGAGCTGTGATGCCATCAGCGGCCTGTGTCTGTGTGAGGCTGGCTACGTGGGCCCGCGG TGCGAGCAGTCAGAGTGTCCCCAGGGCCACTTTGGGCCCGGCTGTGAGCAGCGGTGCCAGTGTCAGCATGGAGCA GCCTGTGACCACGTCAGCGGGGCCTGCACCTGCCCGGCCGGCTGGAGGGGCACCTTCTGCGAGCATGCCTGCCCG GCCGGCTTCTTTGGATTGGACTGTCGCAGTGCCTGCAACTGCACCGCCGGAGCTGCCTGTGATGCCGTGAATGGC TCCTGCCTCTGCCCCGCTGGCCGCCGGGGCCCCCGCTGTGCCGAGACCTGCCCAGCCCACACCTACGGGCACAAT TGCAGCCAGGCCTGTGCCTGCTTTAACGGGGCCTCCTGTGACCCTGTCCACGGGCAGTGCCACTGTGCCCCTGGC TGGATGGGGCCCTCCTGCCTGCAGGCCTGCCCTGCCGGCCTGTACGGCGACAACTGTCGGCATTCCTGCCTCTGC CAGAACGGAGGGACCTGTGACCCTGTCTCAGGCCACTGTGCGTGCCCAGAGGGCTGGGCCGGCCTGGCCTGTGAG GTAGAGTGCCTCCCCCGGGACGTCAGAGCTGGCTGCCGGCACAGCGGCGGTTGCCTCAACGGGGGCCTGTGTGAC CCGCACACGGGCCGCTGCCTCTGCCCAGCCGGCTGGACTGGGGACAAGTGTCAGAGCCCTGCAGCCTGTGCCAAG GGCACATTCGGGCCTCACTGTGAGGGGCGCTGTGCCTGCCGGTGGGGAGGCCCCTGCCACCTTGCCACCGGGGCC TGCCTCTGCCCTCCGGGGTGGCGGGGGCCTCATCTTTCTGCAGCCTGCCTGCGGGGCTGGTTTGGAGAGGCCTGT GCCCAGCGCTGCAGCTGCCCGCCTGGCGCTGCCTGCCACCACGTCACTGGGGCCTGCCGCTGTCCCCCTGGCTTC ACTGGCTCCGGCTGCGAGCAGGCCTGCCCACCCGGCAGCTTTGGGGAGGACTGTGCGCAGATGTGCCAGTGTCCC GGTGAGAACCCGGCCTGCCACCCTGCCACCGGGACCTGCTCATGTGCTGCTGGCTACCACGGCCCCAGCTGCCAG CAACGATGTCCGCCCGGGCGGTATGGGCCAGGCTGTGAACAGCTGTGTGGGTGTCTCAACGGGGGCTCCTGTGAT GCGGCCACGGGGGCCTGCCGCTGCCCCACTGGGTTCCTCGGGACGGACTGCAACCTCACCTGTCCGCAGGGCCGC TTCGGCCCCAACTGCACCCACGTGTGTGGGTGTGGGCAGGGGGCGGCCTGCGACCCTGTGACCGGCACCTGCCTC TGCCCCCCGGGGAGAGCCGGCGTCCGCTGTGAGCGAGGCTGCCCCCAGAACCGGTTTGGCGTGGGCTGCGAGCAC ACCTGCTCCTGCAGAAATGGGGGCCTGTGCCACGCCAGCAAGCGGCAGCTGCTCCTGTGGCCTGGGCTGGACGGG GCGGCACTGCGAGCTGGCCTGTCCCCCTGGGCGCTACGGAGCCGCCTGCCATCTGGAGTGCTCCTGCCACAACAA CAGCACGTGTGAGCCTGCCACGGGCACCTGCCGCTGCGGCCCCGGCTTCTATGGCCAGGCCTGCGAGCACCCCTG TCCCCCTGGCTTCCACGGGGCTGGCTGCCAGGGGTTGTGCTGGTGTCAACATGGAGCCCCCTGCGACCCCATCAG TGGCCGATGCCTCTGCCCTGCCGGCTTCCACGGCCACTTCTGTGAGAGGGGGTGTGAGCCAGGTTCATTTGGAGA GGGCTGCCACCAGCGCTGTGACTGTGACGGGGGGGCACCCTGTGACCCTGTCACCGGTCTCTGCCTTTGCCCACC AGGGCGCTCAGGAGCCACCTGTAACCTGGATTGCAGAAGGGGCCAGTTTGGGCCCAGCTGCACCCTGCACTGTGA CTGCGGGGGTGGGGCTGACTGCGACCCTGTCAGTGGGCAGTGTCACTGTGTGGATGGCTACATGGGGCCCACGTG CCGGGAAGGTGGGCCCCTCCGGCTCCCCGAGAACCCGTCCTTAGCCCAGGGCTCAGCGGGCACACTGCCCGCCTC CAGCAGACCCACATCCCGGAGCGGTGGACCAGCGAGGCACTAGTAGAGGCAGTCCCGTGGAGCCCGCCTCTCCAG TCCCAGCCAGAGGGGACCCTGGCCTTTGGTGACCACTGAGAAGGACACTTCACGGGCCCAGAGCTCCTGGTACTG CCCTTCCTTTGAGGGCCGTGGAGGGCTGTGGACAGCCCAGCAACCTGTCGCTCTTGGAGGCTGGTGTGGCCTTGA GGAGGGAAGCCTCGCATGGCCGCTGGAAGAGAGGCGCCTCCTGGCCTGGCTCTGCAGAACCCAGGGGCACGCTCT GGGCCTGGGCTGAGGAAGTCCCGCTCTCCCCGCGGCTCTGAGTTGGACTGAGGACAGGTGTGGGCGCCAGTGTGG GTGCAGGCGCAGGTGCAGGCACAGGGCCACTGTCCTCCAGGCAGGCTTTTTGGTGCTAGGCCCTGGGACTGGAAG TCGCCCAGCCCGTATTTATGTAAAGGTATTTATGGGCCACTGCACATGCCCGCTGCAGCCCTGGGATCAGCTGGA AGCTGCCTGTCATCTCCTGCCCAATCCCCAGAAACCCTGATTCAGGTCTGCAGGCTCCTGCGGGCTCACCAGGCT GCTGGCTCCGGTACCATGTAAACCTAGGAAGGTAAAGGAGCAGGCAACCTCCTCGTGGCCTGTGTGTTTGCTGTG TTACGTGGACTCTGTGTGGGCTCCTCCCTGGGGCCCGGCCAGCATAACGGTGCACCCAGGGACCTCCCAGTGCAC CCGGGGCCCTTTGCAGGGGTGGGGGTGCCACACAAGTGAAGAAGTTGGGACTCATCTCAGTTCCCAGTGCTATTG AGGAGAACGCTGGGGCTGCATTCATTACCGCTGAGACCCAGAGACTGGCTGTTCCCAGAGAATGGCCCAGGGGGA GGAGGGCTGGTGTGGAGGGGCAACCTGGACTGAGGCCGAACTCCCTTGGGCTCACCCCACCCACCCCTACCTGAG CATCAGCAGTGGGGGGAGGGCAGCATCGCAGGGGCAGGGACTCCCTGGGTGAGGACAGACCAGCCCTCCCGAGCA CCTGGCACTCATGGGCTGAGGCTGACTTCTCCTGGAAGAAGGGCCCAGAGTGGAAGGAAGAGGCAGAGGGTAGAG GTGGTGGCTGGGGGCTCCTCTGCAGAGTGGGGTGGCCAATGGAGAGGGCTGCACTCACACCGCAACATAGGACTC TCTCTCCCTTAAGAAGGCCCCCTTAGGGTCTGGGCTGCCGCCCCCATCACCCTAAAACCAGCCAAGGTAGCTGAG GCCCCAGGGCAGACAATTTCACCAGCAGGANGAGGAGGAGTCCAGTGAGCTTGGTTGCTCACAGACAGCAAGGGA GCTGTCACAGAGGAAGCTGATGAATGGACCGCTGTGGGGAGACTTTAAAGTAGAACAGTGATAAGGGAGGGCAGG ATGGTGGGGATGCAGAAGCAGCAGCCAGAGAGAGACGGACTGGGGTGCAGACGGAGTGTGGAAAACGCATACCTT GAAATGAAGCATCCAGCAGATGGGGTGAGTGGATACAGCTCAGGAGATTCTCCCAGGAATAGCAGGGAGGCGTAA AGAGAGACAACGTACAGAGATAGATGAATGGAAATGGGTAAGGGAGGTGTTCATTCACATCCATCTAACTGCAAA ATACAAAAGTAAGAAGTCATTGACATGAAGCAACGACGACCAAGACGTTCTCAGATCTAAAGGTGAATGATCTCA GTCAGCCTGGAAATGCACAAGGTGGAAAAATAACATAAAAAAGCCATAAGACCTTGAAGAACATCAATGTCAAAG ATAAATTCTAAAGTCCCAGAGAAAAAAGAATGGGAATCAAATTGACCTCAGACTATACGTGAGAAACACGGAGAG CCAGAAAACTGTGATGTTCCATCCTCAGAGTTTGAAGGAAATATTTGAAGGCTGAATTTTACATCCAGCTAAACT ATCAAAGGCATGCAAAGTCCATGTTATTCTTAGGCCTTCAAGGCCTCGGCCATTTTTCTACAGAAAAGCCTGATT TTAAAATGCTCTTAGAGACGTTCTCCAGCCAGAAGAGAAAGAAGCCAGGAGGGTGCTCTGAGATATTCAGTCACC ACAGTTCCCAAATGGCCTAGGAATTCAGAGAGTCAGAATATCACCATTACTCCCCAATGGGAACCCCCGACAGTC TCAGCATGGTGTGAGGGTGTGGACGGGGGGCCTGGCAGGTACCAATCACTCATCCCGCTCAGTGAAGACACAGTG TTCAGCTACGGAAGCCATAAGGCAGGCCGAGCTTCTGCCCATCCGGAGGAAATCTCAGCTATCCAACGGCGGTCA GGAGCAGAGGAAAATAAAGCAGAATAACTAGAAAACACGCTCACAGATCCTAATGTTAACGGTTACAAATGACGA CGGAAAAACAAACTCCTGACCATATATTATATAGTTTCAAGCAGCAAGAAGGAGGATATTGAACATTCTCAACAC ACATAATAAACGCTTGAGATGATGATATGCTCATTACCCTGATTTGATCACTAGACATNCCATGTATCAAAACAT CACTGTGTATCCGATGAATATCTACAATTATTGTCAATTAAAAACATCATTAAAAACAA
The NON15e protein (SEQ ID ΝO:52) encoded by SEQ ID NO:51 is 1403 amino acid residues in length and is presented using the one-letter amino acid code in Table 15J. Although the SignalP, Psort and/or Hydropathy results indicate that NOV15e has a signal peptide and is likely to be localized in the mitochondrial matrix space with a certainty of 0.4753, the NON15e protein disclosed here is similar to the EGF family, some members of which are released extracellularly. Alternatively, a ΝON15e polypeptide is located to the microbody (peroxisome) with a certainty of 0.3000, the mitochondrial inner membrane with a certainty of 0.1802, or the mitochondrial intermembrane space with a certainty of 0.1802. The SignalP indicates a hkely cleavage site for a ΝON15e peptide is between positions 31 and 32, i. e. , at the dash in the sequence GRG-AD.
Table 15J. Encoded ΝOV15e Protein Sequence (SEQ ID NO:52)
MPMGHSDRWS RLLRLALPLPV LPAGGGRGADSPCLCSRPHVCAEQELTLVGRRQPCVQALSHTVPV KAGCGW QA CVGHERRTVYYMGYRQVYTTEARTVLRCCRG MQQPDEEGCLSDVGECANANGGCAGRCRDTVGGFYCR PP PSHQLQGDGETCQDVDECRTHNGGCQHRCVNTPGSYLCECKPGFRLHTDSRTCAINSCALGNGGCQHHCVQLTIT RHRCQCRPGFQLQEDGRHCVRRSPCANRNGSCMHRCQWRGLARCECHVGYQLAADGKACEDVDECAAGLAQCAH GCLNTQGSFKCVCHAGYELGADGRQCYRIEMEIVNSCEANNGGCSHGCSHTSAGPLCTCPRGYELDTDQRTCIRC RRLCRQPVLQQVCTNNPGGYECGCYAGYRLSADGCGCEDVDECASSRGGCEHHCTNLAGSFQCSCEAGYRLHEDR RGCSALEEPMVDLDGELPFVRPLPHIAVLQDELPQLFQDDDVGADEEEAELRGEHTLTEKFVCLDDSFGHDCSLT CDDCRNGGTCLLGLDGCDCPEG TGLICNESCPPDTFGKNCSFSCSCQNGGTCDSVTGACRCPPGVSGTNCEDGC PKGYYGKHCRKKCNCANRGRCHRLYGACLCDPGLYGRFCHLACPPWAFGPGCSEECQCVQPHTQSCDKRDGSCSC KAGFRGERCQAECEPGYFGPGC QACTCPVGVACDSVSGECGKRCPAGFQGEDCGQECPVGTFGVNCSSSCSCGG APCHGVTGQCRCPPGRTGEDCEAGECEGL GLGCQEICPACHNAARCDPETGACLCLPGFVGSRCQDCEAG YGP SCQTMCSCANDGHCHQDTGHCSCAPG TGFSCQRACDTGHWGPDCSHPCNCSAGHGSCDAISGLCLCEAGYVGPR CEQSECPQGHFGPGCEQRCQCQHGAACDHVSGACTCPAG RGTFCEHACPAGFFGLDCRSACNCTAGAACDAVNG SCLCPAGRRGPRCAETCPAHTYGHNCSQACACFNGASCDPVHGQCHCAPGWMGPSCLQACPAGLYGDNCRHSCLC QNGGTCDPVSGHCACPEGWAGLACEVECLPRDVRAGCRHSGGCLNGGLCDPHTGRCLCPAGWTGDKCQSPAACAK GTFGPHCEGRCACRWGGPCHLATGACLCPPGWRGPHLSAACLRGWFGEACAQRCSCPPGAACHHVTGACRCPPGF TGSGCEQACPPGSFGEDCAQMCQCPGENPACHPATGTCSCAAGYHGPSCQQRCPPGRYGPGCEQLCGCLNGGSCD AATGACRCPTGFLGTDCNLTCPQGRFGPNCTHVCGCGQGAACDPVTGTCLCPPGRAGVRCERGCPQNRFGVGCEH TCSCRNGGLCHASKRQLLL PGLDGAALRAGLSPWALRSRLPSGVLLPQQQHV NON15f
Alternatively, aΝON15 variant is ΝON15f (alternatively referred to herein as CG56449- 08), which includes the 4835 nucleotide sequence (SEQ ID ΝO:53) shown in Table 15K. An open reading frame for the mature protein was identified beginning with an ATG codon at nucleotides 1-3 and ending with a TAG codon at nucleotides 4732-4734. Putative untranslated regions, if any, downstream from the termination codon and upstream from the initiation codon are underlined. The start and stop codons are in bold letters.
Table 15K. NOVl 5f Nucleotide Sequence (SEQ ID NO:53)
ATGTCGTTCCTTGAAGAGGCGAGGGCAGCGGGGCGCGCGGTGGTCCTGGCGTTGGTGCTGCTGCTGCTCCCCGCC GTGCCCGTGGGCGCCAGCGTTCCGCCGCGGCCCCTGCTCCCGCTGCAGCCCGGCATGCCCCACGTGTGTGCTGAG CAGGAGCTGACCCTGGTGGGCCGCCGCCAGCCGTGCGTGCAGGCCTTAAGCCACACGGTGCCGGTGTGGAAGGCC GGCTGTGGGTGGCAGGCGTGGTGCGTGGGTCATGAGCGGAGGACCGTCTACTACATGGGCTACAGGCAGGTGTAT ACCACGGAGGCCCGGACCGTGCTCAGGTGCTGCCGAGGGTGGATGCAGCAGCCCGACGAGGAGGGCTGCCTCTCG GATGTGGGTGAGTGTGCCAACGCCAACGGGGGCTGTGCGGGTCGGTGCCGGGACACCGTGGGGGGCTTCTACTGC CGCTGGCCCCCCCCCAGCCACCAGCTGCAGGGTGATGGCGAGACTTGCCAAGATGTGGACGAATGCCGAACCCAC AACGGTGGCTGCCAGCACCGGTGCGTGAACACCCCAGGCTCCTACCTCTGTGAGTGCAAGCCCGGCTTCCGGCTC CACACTGACAGCAGGACCTGCGCCATTAACTCCTGCGCCCTGGGCAATGGCGGCTGCCAGCACCACTGTGTCCAG CTCACAATCACTCGGCATCGCTGCCAGTGCCGGCCCGGGTTCCAGCTCCAGGAGGACGGCAGGCATTGTGTCCGT AGAAGCCCGTGTGCCAACAGGAACGGCAGCTGCATGCACAGGTGCCAGGTGGTCCGGGGCCTCGCCCGCTGTGAG TGCCACGTGGGCTATCAGCTAGCAGCGGACGGCAAGGCCTGTGAAGATGTGGACGAATGTGCCGCAGGGCTGGCC CAGTGTGCCCATGGCTGCCTCAACACCCAGGGGTCCTTCAAGTGCGTGTGTCACGCGGGCTATGAGCTGGGCGCC GATGGCCGGCAGTGCTACCGTATTGAGATGGAAATCGTGAACAGCTGTGAGGCCAACAACGGCGGCTGCTCCCAT GGCTGCAGCCACACCAGTGCTGGGCCCCTGTGCACCTGTCCCCGCGGCTACGAGCTGGACACAGATCAGAGGACC TGCATCAGATGTCGACGACTGTGCAGACAGCCCGTGCTGCAGCAGGTGTGCACCAACAACCCTGGCGGGTACGAG TGCGGCTGCTACGCCGGCTACCGGCTCAGTGCCGATGGCTGCGGCTGCGAGGATGTGGATGAGTGCGCCTCCAGC CGTGGCGGCTGCGAGCACCACTGCACCAACCTGGCCGGCTCCTTCCAGTGCTCCTGCGAGGCCGGCTACCGGCTG CACGAGGACCGTAGGGGCTGCAGCGCCCTGGAGGAGCCGATGGTGGACCTGGACGGCGAGCTGCCTTTCGTGCGG CCCCTGCCCCACATTGCCGTGCTCCAGGACGAGCTGCCGCAACTCTTCCAGGATGACGACGTCGGGGCCGATGAG GAAGAGGCAGAGTTGCGGGGCGAACACACGCTCACAGAGAAGTTTGTCTGCCTGGATGACTCCTTTGGCCATGAC TGCAGCTTGACCTGTGATGACTGCAGGAACGGAGGGACCTGCCTCCTGGGCCTGGATGGCTGTGATTGCCCCGAG GGCTGGACTGGGCTCATCTGCAATGAGAGTTGTCCTCCGGACACCTTTGGGAAGAACTGCAGCTTCTCCTGCAGC TGTCAGAATGGTGGGACCTGCGACTCTGTCACGGGGGCCTGCCGCTGCCCCCCGGGTGTCAGTGGAACTAACTGT GAGGATGGCTGCCCCAAGGGCTACTATGGCAAGCACTGTCGCAAGAAATGCAACTGTGCCAACCGGGGCCGGTGC CACCGCCTCTACGGGGCCTGCCTCTGCGACCCAGGGCTCTACGGCCGCTTCTGCCACCTCGCCTGCCCGCCGTGG GCCTTTGGGCCGGGCTGCTCGGAGGAGTGCCAGTGTGTGCAGCCCCACACGCAGTCCTGTGACAAGAGGGATGGC AGCTGCTCCTGCAAGGCTGGCTTCCGGGGCGAGCGCTGTCAGGCAGAGTGTGAGCCGGGCTACTTTGGGCCGGGG TGCTGGCAGGCATGCACCTGCCCAGTGGGCGTGGCCTGTGACTCCGTGAGCGGCGAGTGTGGGAAGCGGTGTCCT GCTGGCTTCCAGGGAGAGGACTGTGGCCAAGAGTGCCCGGTGGGGACCTTTGGCGTGAACTGCTCGAGCTCCTGC TCCTGTGGGGGGGCCCCCTGCCACGGGGTCACGGGGCAGTGCCGGTGTCCGCCGGGGAGGACTGGGGAAGACTGT GAGGCAGGTGAGTGTGAGGGCCTCTGGGGGCTGGGCTGCCAGGAGATCTGCCCAGCATGCCATAACGCTGCTCGC TGCGACCCTGAGACCGGAGCCTGCCTGTGCCTCCCTGGCTTTGTCGGCAGCCGCTGCCAGGACTGTGAGGCAGGC TGGTATGGTCCCAGCTGCCAGACAATGTGCTCTTGTGCCAATGATGGGCACTGCCACCAAGACACGGGACACTGC AGCTGTGCCCCCGGGTGGACCGGCTTTAGCTGCCAGAGAGCCTGTGATACTGGGCACTGGGGACCTGACTGCAGC CACCCCTGCAACTGCAGCGCTGGCCACGGGAGCTGTGATGCCATCAGCGGCCTGTGTCTGTGTGAGGCTGGCTAC GTGGGCCCGCGGTGCGAGCAGTCAGAGTGTCCCCAGGGCCACTTTGGGCCCGGCTGTGAGCAGCGGTGCCAGTGT CAGCATGGAGCAGCCTGTGACCACGTCAGCGGGGCCTGCACCTGCCCGGCCGGCTGGAGGGGCACCTTCTGCGAG CATGCCTGCCCGGCCGGCTTCTTTGGATTGGACTGTCGCAGTGCCTGCAACTGCACCGCCGGAGCTGCCTGTGAT GCCGTGAATGGCTCCTGCCTCTGCCCCGCTGGCCGCCGGGGCCCCCGCTGTGCCGAGAGTGCCTGCCCAGCCCAC ACCTACGGGCACAATTGCAGCCAGGCCTGTGCCTGCTTTAACGGGGCCTCCTGTGACCCTGTCCACGGGCAGTGC CACTGTGCCCCTGGCTGGATGGGGCCCTCCTGCCTGCAGGCCTGCCCTGCCGGCCTGTACGGCGACAACTGTCGG CATTCCTGCCTCTGCCAGAACGGAGGGACCTGTGACCCTGTCTCAGGCCACTGTGCGTGCCCAGAGGGCTGGGCC GGCCTGGCCTGTGAGGTAGAGTGCCTCCCCCGGGACGTCAGAGCTGGCTGCCGGCACAGCGGCGGTTGCCTCAAC GGGGGCCTGTGTGACCCGCACACGGGCCGCTGCCTCTGCCCAGCCGGCTGGACTGGGGACAAGTGTCAGAGCCCT GCAGCCTGTGCCAAGGGCACATTCGGGCCTCACTGTGAGGGGCGCTGTGCCTGCCGGTGGGGAGGCCCCTGCCAC CTTGCCACCGGGGCCTGCCTCTGCCCTCCGGGGTGGCGGGGGCCTCATCTTTCTGCAGCCTGCCTGCGGGGCTGG TTTGGAGAGGCCTGTGCCCAGCGCTGCAGCTGCCCGCCTGGCGCTGCCTGCCACCACGTCACTGGGGCCTGCCGC TGTCCCCCTGGCTTCACTGGCTCCGGCTGCGAGCAGGCCTGCCCACCCGGCAGCTTTGGGGAGGACTGTGCGCAG ATGTGCCAGTGTCCCGGTGAGAACCCGGCCTGCCACCCTGCCACCGGGACCTGCTCATGTGCTGCTGGCTACCAC GGCCCCAGCTGCCAGCAACGATGTCCGCCCGGGCGGTATGGGCCAGGCTGTGAACAGCTGTGTGGGTGTCTCAAC GGGGGCTCCTGTGATGCGGCCACGGGGGCCTGCCGCTGCCCCACTGGGTTCCTCGGGACGGACTGCAACCTCACC TGTCCGCAGGGCCGCTTCGGCCCCAACTGCACCCACGTGTGTGGGTGTGGGCAGGGGGCGGCCTGCGACCCTGTG ACCGGCACCTGCCTCTGCCCCCCGGGGAGAGCCGGCGTCCGCTGTGAGCGAGGCTGCCCCCAGAACCGGTTTGGC GTGGGCTGCGAGCACACCTGCTCCTGCAGAAATGGGGGCCTGTGCCACGCCAGCAACGGCAGCTGCTCCTGTGGC CTGGGCTGGACGGGGCGGCACTGCGAGCTGGCCTGTCCCCCTGGGCGCTACGGAGCCGCCTGCCATCTGGAGTGC TCCTGCCACAACAACAGCACGTGTGAGCCTGCCACGGGCACCTGCCGCTGCGGCCCCGGCTTCTATGGCCAGGCC TGCGAGCACCCCTGTCCCCCTGGCTTCCACGGGGCTGGCTGCCAGGGGTTGTGCTGGTGTCAACATGGAGCCCCC TGCGACCCCATCAGTGGCCGATGCCTCTGCCCTGCCGGCTTCCACGGCCACTTCTGTGAGAGGGGGTGTGAGCCA GGTTCATTTGGAGAGGGCTGCCACCAGCGCTGTGACTGTGACGGGGGGGCACCCTGTGACCCTGTCACCGGTCTC TGCCTTTGCCCACCAGGGCGCTCAGGAGCCACCTGTAACCTGGATTGCAGAAGGGGCCAGTTTGGGCCCAGCTGC ACCCTGCACTGTGACTGCGGGGGTGGGGCTGACTGCGACCCTGTCAGTGGGCAGTGTCACTGTGTGGATGGCTAC ATGGGGCCCACGTGCCGGGAAGCGGGCACACTGCCCGCCTCCAGCAGACCCACATCCCGGAGCGGTGGACCAGCG AGGCACTAGTAGAGGCAGTCCCGTGGAGCCCGCCTCTCCAGTCCCAGCCAGAGGGGACCCTGGCCTTTGGTGACC ACTGAGAAGGACACTTCACGGGCCCAGAGCTCCTG
The NONl 5f protem (SEQ ID ΝO:54) encoded by SEQ ID NO:53 is 1577 amino acid residues in length and is presented using the one-letter amino acid code in Table 15L. The SignalP, Psort and/or Hydropathy results indicate that NOVl 5f has a signal peptide and is likely to be localized extracellularly with a certainty of 0.8200. Alternatively, a NOVl 5f polypeptide is located to the lysosome (lumen) with a certainty of 0.1900, the endoplasmic reticulum (membrane) with a certainty of 0.1000, or the endoplasmic reticulum (lumen) with a certainty of 0.1000. The SignalP indicates a likely cleavage site for a NON15f peptide is between positions 30 and 31, i.e., at the dash in the sequence NGA-SN.
Table 15L. Encoded NO V15f Protein Sequence (SEQ ID NO:54)
MSFLEEARAAGRAWLALVLLLLPAVPVGASVPPRPLLPLQPGMPHVCAEQELTLVGRRQPCVQALSHTVPVWKA GCG QA CVGHERRTVYYMGYRQVYTTEARTVLRCCRG MQQPDEEGCLSDVGECANANGGCAGRCRDTVGGFYC R PPPSHQLQGDGETCQDVDECRTHNGGCQHRCVNTPGSYLCECKPGFRLHTDSRTCAINSCALGNGGCQHHCVQ LTITRHRCQCRPGFQLQEDGRHCVRRSPCANRNGSCMHRCQWRGLARCECHVGYQLAADGKACEDVDECAAGLA QCAHGCLNTQGSFKCVCHAGYELGADGRQCYRIEMEIVNSCEANNGGCSHGCSHTSAGPLCTCPRGYELDTDQRT CIRCRRLCRQPVLQQVCTNNPGGYECGCYAGYRLSADGCGCEDVDECASSRGGCEHHCTNLAGSFQCSCEAGYRL HEDRRGCSALEEPMVDLDGELPFVRPLPHIAVLQDELPQLFQDDDVGADEEEAELRGEHTLTEKFVCLDDSFGHD CSLTCDDCRNGGTCLLGLDGCDCPEGWTGLICNESCPPDTFGKNCSFSCSCQNGGTCDSVTGACRCPPGVSGTNC EDGCPKGYYGKHCRKKCNCANRGRCHRLYGACLCDPGLYGRFCHLACPP AFGPGCSEECQCVQPHTQSCDKRDG SCSCKAGFRGERCQAECEPGYFGPGCWQACTCPVGVACDSVSGECGKRCPAGFQGEDCGQECPVGTFGVNCSSSC SCGGAPCHGVTGQCRCPPGRTGEDCEAGECEGL GLGCQEICPACHNAARCDPETGACLCLPGFVGSRCQDCEAG WYGPSCQTMCSCANDGHCHQDTGHCSCAPGWTGFSCQRACDTGHWGPDCSHPCNCSAGHGSCDAISGLCLCEAGY VGPRCEQSECPQGHFGPGCEQRCQCQHGAACDHVSGACTCPAG RGTFCEHACPAGFFGLDCRSACNCTAGAACD AVNGSCLCPAGRRGPRCAESACPAHTYGHNCSQACACFNGASCDPVHGQCHCAPG MGPSCLQACPAGLYGDNCR HSCLCQNGGTCDPVSGHCACPEG AGLACEVECLPRDVRAGCRHSGGCLNGGLCDPHTGRCLCPAGWTGDKCQSP AACAKGTFGPHCEGRCACRWGGPCHLATGACLCPPG RGPHLSAACLRGWFGEACAQRCSCPPGAACHHVTGACR CPPGFTGSGCEQACPPGSFGEDCAQMCQCPGENPACHPATGTCSCAAGYHGPSCQQRCPPGRYGPGCEQLCGCLN GGSCDAATGACRCPTGFLGTDCNLTCPQGRFGPNCTHVCGCGQGAACDPVTGTCLCPPGRAGVRCERGCPQNRFG VGCEHTCSCRNGGLCHASNGSCSCGLG TGRHCELACPPGRYGAACHLECSCHNNSTCEPATGTCRCGPGFYGQA CEHPCPPGFHGAGCQGLC CQHGAPCDPISGRCLCPAGFHGHFCERGCEPGSFGEGCHQRCDCDGGAPCDPVTGL CLCPPGRSGATCNLDCRRGQFGPSCTLHCDCGGGADCDPVSGQCHCVDGYMGPTCREAGTLPASSRPTSRSGGPA RH
NOV15 Clones
Unless specifically addressed as NOV15a, NOV15b, NOV15c, NOV15d, NOV15e, or NOV15f any reference to NON15 is assumed to encompass all variants. A search against the Patp database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 15M.
Table 15M. PatP Results for ΝOV15
Smallest
High Sum
Sequences Producing High-Scoring Segment Pairs: Score Prob P (N) patp:AAY72091 Human serine protease #2 encoded by clone HMGBM65 2570 5.8e-267 patp:AAB66267 Human TANGO 272 1416 l.le-144 patp: AAY72715 HFICU08 clone human attractin-like protein 1396 1.5e-142 patp:AAB66269 Rat TANGO 272 1200 8.6e-122 patp:AAG75479 Human colon cancer antigen protein 945 3.4e-94
In a BLAST search of public sequence databases, it was found, for example, that the
NOV15a nucleic acid sequence of this invention has 2717 of 3360 bases (80%) identical to a gb:GENBANK-rD:AB011532|acc:AB011532.1 mRNA from Rattus norvegicus mRNA for MEGF6, complete eds. Further, the full amino acid sequence of the disclosed NON15a protein of the invention has 1060 of 1364 amino acid residues (77%) identical to, and 1147 of 1364 amino acid residues (84%) similar to, the 1574 amino acid residue ptnr:SPTREMBL-ACC:O88281 protein from Rat (MEGF6).
In a similar BLAST search of public sequence databases, it was found, for example, that the ΝON15b nucleic acid sequence of this invention has 2624 of 3343 bases (78%) identical to a gb:GEΝBAΝK-ID:AB011532|acc:AB011532.1 mRNA from Rattus norvegicus mRNA for MEGF6, complete eds. Further, the full amino acid sequence of the disclosed NONl 5b protein of the invention has 1045 of 1363 amino acid residues (76%) identical to, and 1131 of 1363 amino acid residues (82%) similar to, the 1574 amino acid residue ptnr:SPTREMBL-ACC:O88281 protein from Rat (MEGF6). In a similar BLAST search of public sequence databases, it was found, for example, that the ΝON15c nucleic acid sequence of this invention has 3219 of 4514 bases (71%) identical to a gb:GEΝBAΝK-ID:AB011532|acc:AB011532.1 mRNA from Rattus norvegicus mRNA for MEGF6, complete eds. Further, the full amino acid sequence of the disclosed NONl 5c protein of the invention has 966 of 1426 amino acid residues (67%) identical to, and 1062 of 1426 amino acid residues (74%) similar to, the 1574 amino acid residue ptnr:SPTREMBL-ACC:O88281 protein from Rat (MEGF6).
In a similar BLAST search of public sequence databases, it was found, for example, that the ΝON15d nucleic acid sequence of this invention has 650 of 687 bases (94%) identical to a gb:GEΝBAΝK-ID:AB011539|acc:AB011539.1 mRNA from Homo sapiens mRNA for MEGF6, partial eds. Further, the full amino acid sequence of the disclosed NOV15d protein of the invention has 106 of 141 amino acid residues (75%) identical to, and 108 of 141 amino acid residues (76%) similar to, the 153 amino acid residue ptnr:SPTREMBL-ACC:O75095 protein from Human (MEGF6).
In a further BLAST search of public sequence databases, it was found, for example, that the NOV15e nucleic acid sequence of this invention has 1072 of 1072 bases (100%) identical to a gb:GENBANK-lD:AB011539|acc:AB011539.1 mRNA from Homo sapiens mRNA for MEGF6, partial eds. Further, the full amino acid sequence of the disclosed NOV15e protein of the invention has 1059 of 1363 amino acid residues (77%) identical to, and 1147 of 1363 amino acid residues (84%) similar to, the 1574 amino acid residue ptnr:SPTREMBL-ACC:O88281 protein
Figure imgf000139_0001
yet a further BLAST search of public sequence databases, it was found, for example, that the NON15f nucleic acid sequence of this invention has 2755 of 3390 bases (81%) identical to a gb:GEΝBAΝK-ID:AB011532|acc:AB011532.1 mRNA from Rattus norvegicus mRNA for MEGF6, complete eds. Further, the full amino acid sequence of the disclosed NOV15f protein of the invention has 1222 of 1562 amino acid residues (78%) identical to, and 1322 of 1562 amino acid residues (84%) similar to, the 1574 amino acid residue ρtnr:SPTREMBL-ACC:O88281 protein from Rat (MEGF6).
Additional BLAST results are shown in Table 15N.
Figure imgf000140_0002
A multiple sequence alignment is given in Table 15O, with the NON15 proteins of the invention being shown in lines 1 through 6 in a ClustalW analysis comparing ΝON15 with related protein sequences of Table 15Ν.
Table 15O. ClustalW Analysis of NOV15
1. SEQ ID NO.: 44 NOV15a 6. SEQ ID NO.: 54 NOV15f
2. SEQ ID NO.: 46 NOV15b 7. SEQ ID NO.: 211 088281
3. SEQ ID NO.: 48 NOV15c 8. SEQ ID NO.: 212 Q9TVQ2
4. SEQ ID NO.: 50 NOV15d 9. SEQ ID NO.: 213 T27283
5. SEQ ID NO.: 52 NOV15e 10. SEQ ID NO.: 214 Q96KG6
11. SEQ ID NO.: 215 Q96KG7
6 6 6
Figure imgf000140_0001
Figure imgf000141_0001
Q96KG7
Figure imgf000141_0002
Figure imgf000142_0001
Figure imgf000143_0001
NOVl5e JLDgELPFVjRP LPHIA LQI LPQSFQD ■ VGADgEEAlLRgEHpj 506 NOVl5f MVDLDHELPFVRP LPHIAVLQS SJLPQIJFQD I- VGADrøEEAEI-R@EHti3 510 088281 VTO DHRLPF R LPHI VLRB R JFQD ■ YG- - -AEEEAAAAELR@EHjE 508 Q9TVQ2 MNTGfflcAQLCKlsrRKGSRRCQCFAG iyAHr gKSCpA S •SADIFSNDIR ■YS3K?P@LDSSΪ 523 T27283 fclNNGfflcAQLc NRKGSRRCQCFAGYI " " 2KSCKAAS •SADIFSNDIR lYSEVPlJLDsS, 571 Q96KG6 -- 158 Q96KG7 -- 239
Figure imgf000143_0002
Q96 G7 239
Figure imgf000143_0003
730 740 750 760 770 780
Figure imgf000143_0004
Figure imgf000144_0003
Figure imgf000144_0001
970 980 990 1000 1010 1020
Figure imgf000144_0002
Figure imgf000145_0001
1030 1040 1050 1060 1070 1080
Figure imgf000145_0002
1150 1160 1170 1180 1190 1200
Figure imgf000145_0003
1210 1220 1230 1240 1250 1260
Figure imgf000145_0004
Figure imgf000146_0001
1270 1280 1290 1300 1310 1320
Figure imgf000146_0002
1330 1340 1350 1360 1370 1380
Figure imgf000146_0003
1390 1400 1410 1420 1430 1440
Figure imgf000146_0004
1450 1460 1470 1480 1490 1500
Figure imgf000146_0005
Q96KG7 LKNVNPSJKREJPVGDSJTGTLPADWK^GYLNE3GAFG| ^SYMGKSLKDLGKNS-EYNS^NBJ 1021
1510 1520 1530 1540 1550 1560
Figure imgf000147_0001
1570 1580 1590 1600 1610 1620
Figure imgf000147_0002
1630 1640 1650 1660 1670 1680
Figure imgf000147_0003
1690 1700 1710 1720 1730 1740
NOV15a 1404
NOV15b 1398
NOVl5c FGEGCHQRCDCDGG- -APCDPVTGLCLCPPGRSGATCNLDCRRGQFGPSC 1382
NOV15d 170
NOV15e 1403
NOVl5f FGEGCHQRCDCDGG APCDPVTGLCLCPPGRSGATCNLDCRRGQFGPSC 1525 088281 FGDGCLQQCNCHTG VPCDPISGLCLCPPGRTGAACDLDCRRGRFGPGC 1520 Q9TVQ2 LGDKCEQKCSEGSFGPACSQQCNCGKYKCDATDGKCICPVGRHGPLCEEECRPGRYGQSC 1608 T27283 LVQNIEFF 1620 Q96KG6 969 Q96KG7 1140 1750 1760 1770 1780 1790 1800
NOV15a 1404
NOV15b 1398
NOV15C TLHCDCGGGADCDPVSGQCHCVDGYMGPTCREGGPLRLPENPSLAQGSAGTLPASSRPTS 1442
NOV15d 170
NOV15e 1403
NOV15f TLHCDCGGGADCDPVSGQCHCVDGYMGPTCREAG -TLPASSRPTS 1569
088281 ALRCDCGGGADCDPISGQCHCVDSYMGPTCREVP -TQISSSRPAP 1564
Q9TVQ2 QNKCQCFNGATCDARTGQCSCSPGWLGPTCQIEMMDP- -NNVANRGDLP 1655
T27283 1620
Q96KG6 969
Q96KG7 1140
1810
NOVl5a 1404
NOVl5b 1398
NOVl5c RSGGPARH- - 1450
NOVl5d 170
NOVl5e 1403 NOVl5f RSGGPARH-- 1577
088281 QHPSSRAMKH 1574
Q9TVQ2 EDWEWRKKR- 1664
T27283 1620 Q96KG6 969
Q96KG7 1140
The presence of identifiable domains in the disclosed NOVl 5 protein was determined by using Pfam and then determining the Inteφro number. The results are listed in Table 15P with the statistics and domain description.
Table 15P. Domain Analysis of NOVl 5
PSSMs Producing Significant Alignments Score E (bits) Value
EGF : domain 2 of 27 , from 168 to 203 38.8 1.2e-07
EGF Capnn . pCsngGtCvntpggssdnfggytCeCppGdyylsytG rC (SEQ ID NO: 16) I ++++I++ + I +++++ ++ I + I ++ I ++++ + ++ I
NOVl5 CRTHNgGCQH- -RCVNTPG SYLCECKPG-FRLHTDSRTC (SEQ ID NO: 44)
EGF: domain 3 of 27, from 208 to 244 34.2 3e-06
EGF Capnn. CsngGtCvntpggssdnfggytCeCppGdyylsytGkrC (SEQ ID NO: 217) )++++++I ++ |+ + + ++I+I +| ++++ +|++| NOVl5 CALGNgGCQH--HCVQLTI TRHRCQCRPG-FQLQEDGRHC (SEQ ID NO: 44)
EGF: domain 4 of 27, from 250 to 285 33.9 3.7Θ-06 EGF Capnn. pCsngGtCvntpggssdnfggytCeCppGdyylsytGkrC (SEQ ID NO .-218]
|+ ++ |++ +|+ +++ +I+I++I ++++ +|+ I N0V15 CANRNgSCMH--RCQWRG LARCECHVG-YQLAADGKAC (SEQ ID NO: 44)
EGF: domain 5 of 27, from 291 to 326 29.5 7.9e-05
EGF Capnn.pCsngGtCvntpggssdnfggytCeCppGdyylsytGkrC (SEQ ID NO: 219) |+ + I + |+++ + +++1+1+ I ++++ +|++|
N0V15 CAAGLaQCAH--GCLNTQG SFKCVCHAG-YELGADGRQC (SEQ ID NO:44)
Consistent with other known members of the MEGF6 family of proteins, NON 15 contains an epithelial growth factor (EGF) domain as illustrated in Table 15P.
ΝON15 nucleic acids, and the encoded polypeptides, according to the invention are useful in a variety of applications and contexts. For example, ΝON15 nucleic acids and polypeptides can be used to identify proteins that are members of the EGF family of proteins. The ΝON15 nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance ΝON15 activity or function. Specifically, the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., cell adhesion or receptor-ligand interactions. These molecules can be used to treat, e.g., neurodegenerative disorders such as Alzheimers or Parkinson's disease, or connective tissue disorders such as Marfan syndrome, . hi addition, various ΝON15 nucleic acids and polypeptides according to the invention are useful, inter alia, as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins. For example, the ΝON15 nucleic acids and their encoded polypeptides include structural motifs that are characteristic of proteins belonging to the MEGF family. Proteins belonging to the MEGF/Fibrillin family of proteins share a common feature of having epidermal growth factor (EGF)-like motifs. Examples of proteins containing EGF-like motifs include the MEGF proteins, which are expressed in the brain and are involved in neural development and function, the fibrillins, which are involved in extracellular matrix structure and maintenance, and the notch proteins (MEGF6), which are thought to be involved in mediating cell-fate decisions during hematopoiesis and neural development. Thus, such proteins play a critical role in a number of extracellular events, including cell adhesion and receptor-ligand interactions. Defects in these proteins can have profound effects on cellular and extracellular physiology and structure. For example, a mutation in fibrillin 1 causes Marfan syndrome, a disease that involves connective tissue, bone and lung manifestations.
The NON 15 nucleic acids and polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in the mediation of cellular and extracellular physiology. As such the ΝON15 nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat, e.g., cancer, trauma, bacterial and viral infections, regeneration (in vitro and in vivo), fertility, endometriosis, cardiomyopathy, atherosclerosis, hypertension, congenital heart defects, aortic stenosis, atrial septal defect (ASD), atrioventricular (A-N) canal defect, ductus arteriosus, pulmonary stenosis, subaortic stenosis, ventricular septal defect (NSD), valve diseases, tuberous sclerosis, scleroderma, obesity, transplantation, anemia, bleeding disorders, transplantation, diabetes, autoimmune disease, renal artery stenosis, interstitial nephritis, glomerulonephritis, polycystic kidney disease, systemic lupus erythematosus, renal tubular acidosis, IgA nephropathy, hypercalceimia, Lesch-Νyhan syndrome, systemic lupus erythematosus, autoimmune disease, asthma, emphysema, allergy, ARDS, von Hippel-Lindau (NHL) syndrome, Alzheimer's disease, stroke, hypercalceimia, Parkinson's disease, Huntington's disease, cerebral palsy, epilepsy, Lesch- Νyhan syndrome, multiple sclerosis, ataxia-telangiectasia, leukodystrophies, behavioral disorders, addiction, anxiety, pain, neurodegeneration, Hirschsprung's disease , Crohn's Disease, and appendicitis. The ΝON15 nucleic acids and polypeptides are useful for detecting specific cell types.
For example, expression analysis has demonstrated that a ΝON15 nucleic acid is expressed in: brain, colon, frontal lobe, heart, kidney, lung, mammary gland/breast, ovary, prostate, and vein.
Additional utilities for ΝON15 nucleic acids and polypeptides according to the invention are disclosed herein.
ΝOV16
The disclosed NOV16 nucleic acid (alternatively referred to herein as AL359846_A_dal) encodes a novel G-protein coupled receptor (GPCR)-like protein and includes the 990 nucleotide sequence (SEQ ID NO: 55) shown in Table 16 A. The NON 16 nucleic acid disclosed herein maps to chromosome 4. An open reading frame for the mature protein was identified beginning with an ATG initiation codon at nucleotides 3-5, and ending with a TGA stop codon at nucleotides 945-947. Putative untranslated regions, if any, are found upstream from the initiation codon and downstream from the termination codon. The start and stop codons are in bold letters.
Table 16A. NON16 Nucleotide Sequence (SEQ ID NO:55)
ACATGGAGACAAGAAATTACTCTGCCATGACTGAATTCTTTCTGGTGGGGCTTTCCCAATATCCAGAGCTCCAGC TTTTTCTGTTCCTGCTCTGCCTCATCATGTACATGATAATCCTCCTGGGAAATAGCCTCCTCATTATCATCACCA TCTTGGATTCTCGCCTCCATACTCCCATGTATTTCTTTCTTGGAAACCTCTCATTCTTGGACATCTGTTACACAT CCTCATCCATTCCTCCAATGCTTATTATATTTATGTCTGAGAGAAAATCCATCTCCTTCATTGGCTGTGCTCTGC AGATGGTTATGTCCCTTGGCTTGGGCTCCACTGAGTGTGTCCTCCTGGCTGTGATGGCCTATGACCACTATGTGG CCATCTGCAACCCACTGAGGTACTCCATCATCATGAACGGAGTGCTGTATGTGCAAATGGCTGCATGGTCCTGGA TCATAGGCTGTCTGACCTCCCTATTGCACACAGTTCTGACAATGATGTTGCCTTTCTGTGGGAATAATGTCATTG ATCATATTACCTGTGAAATTTTGGCCCTTCTAAAACTTGTTTGTTCAGATATCACCATCAATGTGCTTATCATGA CAGTGACAAATATTGTTTCACTGGTGATTCTTCTACTGTTAATTTTCATCTCCTATGTGTTTATTCTCTCTTCCA TCCTGAGAATTAATTGTGCTGAGGGAAGAAAGAAAGCCTTCTCTACCTGTTCAGCGCACTCGATTGTGGTCATCT TATTCTACGGTTCAGCCCTTTTTATGTACATGAAACCCAAGTCAAAGAACACTAATACATCTGATGAGATTATTG GGCTGTCTTATGGAGTGGTAAGCCCAATGTTAAATCCCATCATCTATAGCCTCAGGAATAAAGAGGTCAAAGAGG CTGTAAAGAAAGTCCTGAGCAGACATCTGCATTTATTGAAAATGTGAAAAACCTTGGGCATGCGATATCCTCAAT GGGGCAAGAGAGCTT
The NOV16 protein (SEQ ID NO:56) encoded by SEQ ID NO:55 is 314 amino acid residues in length and is presented using the one-letter amino acid code in Table 16B. The SignalP, Psort and/or Hydropathy results indicate that NON 16 has a signal peptide and is likely to be localized at the plasma membrane with a certainty of 0.6000. Alternatively, a ΝON16 polypeptide is located to the Golgi body with a certainty of 0.4000, the endoplasmic reticulum (membrane) with a certainty of 0.3000, or the microbody (peroxisome) with a certainty of 0.3000. The SignalP indicates a likely cleavage site for a ΝON16 peptide between positions 43 and 44, i.e., at the dash in the sequence GΝS-LL.
Table 16B. Encoded ΝOV16 Protein Sequence (SEQ ID NO:56)
METRNYSAMTEFFLVGLSQYPELQLFLFLLCLIMYMIILLGNSLLIIITILDSRLHTPMYFFLGNLSFLDICYTS SSIPPMLIIFMSERKSISFIGCALQMVMSLGLGSTECVLLAVMAYDHYVAICNPLRYSIIMNGVLYVQMAAWSWI IGCLTSLLHTVLTMMLPFCGNNVIDHITCEILALLKLVCSDITINVLIMTVTNIVSLVILLLLIFISYVFILSSI LRINCAEGRKKAFSTCSAHSIWILFYGSALFMYMKPKSKNTNTSDEIIGLSYGWSPMLNPIIYSLRNKEVKEA VKKVLSRHLHLLKM A search against the Patp database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 16C.
Table 16C. PatP Results for NON16
Smallest
High Sum
Sequences Producing High-Scoring Segment Pairs: Score Prob P (Ν) patp:AAU24629 Human olfactory receptor AOLFR123 1575 1.6e-161 patp:AAG71424 Human olfactory receptor polypeptide 1569 6.8e-161 patp:AAG72315 Human olfactory receptor polypeptide 1377 1.5e-140 ρatp:AAG71954 Human olfactory receptor polypeptide 1028 1.4e-103 patp:AAG72652 Murine OR-like polypeptide query sequence 991 1.2e-99
h a BLAST search of public sequence databases, it was found, for example, that the ΝON16 nucleic acid sequence of this invention has 555 of 804 bases (69%) identical to a gb:GEΝBAΝK-ID:MMU133424|acc:AJ133424.1 mRNA from Mus musculus or37a gene. Further, the full amino acid sequence of the disclosed NON 16 protein of the invention has 189 of 313 amino acid residues (60%) identical to, and 246 of 313 amino acid residues (78%) similar to, the 318 amino acid residue ptnr:SPTREMBL-ACC:Q9QZ21 protein from Mouse (OLFACTORY RECEPTOR).
The ΝON16 protein of the invention also has homolgy to the proteins shown in the BLASTP data in Table 16D.
Figure imgf000152_0001
Figure imgf000153_0002
A multiple sequence alignment is given in Table 16E, with the NON 16 protein of the invention being shown in line 1 in a ClustalW analysis comparing ΝON16 with related protein sequences of Table 16D.
Table 16E. ClustalW Analysis of ΝON16
1. SEQ ID NO.: 56 NOV16 4. SEQ ID NO.: 222 Q9QZ20
2. SEQ ID NO.: 220 Q9QZ21 5. SEQ ID NO.: 223 Q9QZ19
3. SEQ ID NO. : 221 Q9QZ22 6. SEQ ID NO.: 224 Q9NQN1
10 20 30 40 50 60
Figure imgf000153_0001
250 260 270 280 290 300
Figure imgf000154_0001
310 320
Figure imgf000154_0002
The presence of identifiable domains in the disclosed NONl 6 protein was determined by using Pfam and then determining the Interpro number. The results are listed in Table 16F with the statistics and domain description.
Figure imgf000154_0003
Consistent with other known members of the GPCR family of proteins, NON16 contains a 7-transmembrane (7tm_l) domain as illustrated in Table 16F.
The ΝON16 nucleic acid, and the encoded polypeptide, according to the invention are useful in a variety of applications and contexts. For example, ΝON16 nucleic acids and polypeptides can be used to identify proteins that are members of the GPCR family of proteins. The ΝON16 nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance ΝON16 activity or function. Specifically, the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., cell recognition or signal transduction. These molecules can be used to treat, e.g., taste and scent detectability disorders, weight disorders, immune diseases, or signal transduction pathways.
In addition, the ΝON16 nucleic acid and polypeptide according to the invention are useful, inter alia, as novel members of the protem families according to the presence of domains and sequence relatedness to previously described proteins. For example, the ΝON16 nucleic acid and polypeptide include structural motifs that are characteristic of proteins belonging to the family of GPCR proteins. The human GPCR genes are generally intron-less and belong to four gene subfamilies, displaying great sequence variability. These genes are dominantly expressed in olfactory epithelium. Olfactory receptors (ORs) have been identified as extremely large family of GPCRs in a number of species. As members of the GPCR family, these receptors share a seven transmembrane domain structure with many neurotransmitter and hormone receptors, and are likely to underlie the recognition and G-protein-mediated transduction of odorant signals. Like GPCRs, the ORs they can be expressed in a variety of tissues where they are thought to be involved in recognition and transmission of a variety of signals.
The ΝON16 nucleic acid and polypeptide, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in the mediation of signal transduction. As such the ΝON16 nucleic acid and polypeptide, antibodies and related compounds according to the invention maybe used to treat, e.g., developmental diseases, MHCII and III diseases (immune diseases), taste and scent detectability disorders, Burkitt's lymphoma, corticoneurogenic disease, signal transduction pathway disorders, retinal diseases including those involving photoreception, cell growth rate disorders, cell shape disorders, feeding disorders, control of feeding, potential obesity due to over-eating, potential disorders due to starvation (lack of apetite), noninsulin-dependent diabetes mellitus (NTDDM1), bacterial, fungal, protozoal and viral infections (particularly infections caused by H1N-1 or HIN-2), pain, cancer (including but not limited to Neoplasm; adenocarcinoma; lymphoma; prostate cancer; uterus cancer), anorexia, bulimia, asthma, Parkinson's disease, acute heart failure, hypotension, hypertension, urinary retention, osteoporosis, Crohn's disease, multiple sclerosis, angina pectoris, myocardial infarction, ulcers, asthma, allergies, benign prostatic hypertrophy, or psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, and severe mental retardation.
The NONl 6 nucleic acid and polypeptide are useful for detecting specific cell types. For example, expression analysis has demonstrated that a ΝON16 nucleic acid is expressed in olfactory neuroepithelium and the heart.
Additional utilities for the ΝON16 nucleic acid and polypeptide according to the invention are disclosed herein.
ΝON17
The ΝON17 proteins descibed herein are novel transporter-like proteins. Two alternative novel ΝON17 nucleic acids and polypeptides are disclosed herein, namely ΝON17a and ΝON17b.
ΝON17a is directed to a transporter protein having a hydrophilic amino terminus containing sequences enriched in proline (P), glutamate (E), serine (S), and threonine (T), i.e.,
PEST-containing transporter. The ΝON17a nucleic acid disclosed herein maps to chromosome 6. ΝON17b is directed to a Νa+ independent aromatic amino acid transporter. The NON17b nucleic acid maps to chromosome 5.
ΝON17a
A ΝON17 variant is ΝON17a (alternatively referred to herein as CG56459-01), which encodes the 1875 nucleotide sequence (SEQ ID ΝO:57) shown in Table 17A. An open reading frame for the mature protein was identified beginning with an ATG codon at nucleotides 5-7 and ending with a TAA codon at nucleotides 1823-1825. Putative untranslated regions, if any, downstream from the termination codon and upstream from the imtiation codon are underlined. The start and stop codons are in bold letters. Table 17A. NONl 7a Nucleotide Sequence (SEQ ID NO:57)
GCTCATGCTTATGGGCGGGGTGACCCACACATCTGTGCCCCTCTCTGAGCAGGAGGAGGCCCCGTCGCAGACGCG CGCGCAGACAGCGTCTGCCGCGGGCACCTGGGGCCGCGCGCCGCGGGGCGCCCCGCCTCCGCTCTCCGAGGCCCA ATCATCTGGAGGCTGTGGGGGCACGTCCCGCTCCCGGCCACGCCCCCAGCCGGCGGGGCGGGGGCTCGCGTCCCT CGCGCTTCTCCGGCGCCTGAGGGGCCCGCCTCGGGCCATGGTGCTCTCCCAGGAGGAGCCGGACTCCGCGCGGGG CACGAGCGAGGCGCAGCCGCTCGGCCCCGCGCCCACGGGGGCCGCTCCGCCGCCCGGCCCGGGACCCTCGGACAG CCCCGAGGCGGCTGTCGAGAAGGTGGAGGTGGAGCTGGCGGGGCCGGCGACCGCGGAGCCCCATGAGCCCCCCGA ACCCCCCGAGGGCGGCTGGGGCTGGCTGGTGATGCTGGCGGCCATGTGGTGCAACGGGTCGGTGTTCGGCATCCA GAACGCTTGCGGGGTGCTCTTCGTGTCCATGCTGGAAACCTTCGGCTCCAAAGACGATGACAAGATGGTCTTTAA GACAGCATGGGTAGGTTCTCTCTCCATGGGGATGATTTTCTTTTGCTGCCCAATAGTCAGCGTCTTCACAGACCT ATTTGGTTGTCGGAAAACAGCTGTCGTGGGTGCTGCTGTTGGATTTGTTGGGCTCATGTCCAGTTCTTTTGTAAG TTCCATCGAGCCTCTGTACCTTACCTATGGAATCATATTTGCCTGCGGCTGCTCCTTTGCATACCAGCCTTCATT GGTCATTTTGGGACACTATTTCAAGAAGCGCCTTGGACTGGTGAATGGCATTGTCACTGCTGGCAGCAGTGTCTT CACAATCCTGCTGCCTTTGCTCTTAAGGGTTCTGATTGACAGCGTGGGCCTCTTTTACACATTGAGGGTGCTCTG CATCTTCATGTTTGTTCTCTTTCTGGCTGGCTTTACTTACCGACCTCTTGCTACCAGTACCAAAGATAAAGAGAG TGGAGGTAGCGGATCCTCCCTCTTTTCCAGGAAAAAGTTCAGTCCTCCAAAAAAAATTTTCAATTTTGCCATCTT CAAGGTGACAGCTTATGCAGTGTGGGCAGTTGGAATACCACTTGCACTTTTTGGATACTTTGTGCCTTATGTTCA CTTGGTGAGTATGCTCCTTCACAAACATGTAAATGAAAGATTTCAAGATGAAAAAAATAAAGAGGTTGTTCTCAT GTGCATTGGCGTCACTTCAGGAGTTGGACGACTGCTCTTTGGCCGGATTGCAGATTATGTGCCTGGTGTGAAGAA GGTTTATCTACAGGTACTTTCCTTTTTCTTCATTGGTCTGATGTCCATGATGATTCCTCTGTGTAGCATCTTTGG GGCCCTCATTGCTGTGTGCCTCATCATGGGTCTCTTCGATGGATGCTTCATTTCCATTATGGCTCCCATAGCCTT TGAGTTAGTTGGTGCCCAGGATGTCTCCCAAGCAATTGGATTTCTGCTCGGATTCATGTCTATACCCATGACTGT TGGCCCACCCATTGCAGGTTTACTTCGTGACAAACTGGGCTCCTATGATGTGGCATTCTACCTCGCTGGAGTCCC TCCCCTTATTGGAGGTGCTGTGCTTTGTTTTATCCCGTGGATCCATAGTAAGAAGCAAAGAGAGATCAGTAAAAC CACTGGAAAAGAAAAGATGGAGAAAATGTTGGAAAACCAGAACTCTCTGCTGTCAAGTTCATCTGGAATGTTCAA GAAAGAATCTGACTCTATTATTTAATATCTTACATACCTCCACCAGACTGGACTTGCTTTTTGAATTTTAAGCAA
The NOV17a protein (SEQ ID NO:58) encoded by SEQ ID NO:57 is 606 amino acid residues in length and is presented using the one-letter amino acid code in Table 17B. The SignalP, Psort and/or Hydropathy results indicate that NOV17a has no known signal peptide and is likely to be localized at the plasma membrane with a certainty of 0.8000. Alternatively, a NON17a polypeptide is located in the Golgi body with a certainty of 0.4000, the endoplasmic reticulum (membrane) with a certainty of 0.3000, or the mitochondrial inner membrane with a certainty of 0.0300.
Table 17B. Encoded ΝOV17a Protein Sequence (SEQ ID NO:58)
MLMGGVTHTSVPLSΞQEEAPSQTRAQTASAAGT GRAPRGAPPPLSEAQSSGGCGGTSRSRPRPQPAGRGLASLA LLRRLRGPPRAMVLSQEEPDSARGTSEAQPLGPAPTGAAPPPGPGPSDSPEAAVEKVEVELAGPATAΞPHEPPΞP PΞGG GWLVMLAAM CNGSVFGIQNACGVLFVSMLETFGSKDDDKMVFKTAWVGSLSMGMIFFCCPIVSVFTDLF GCRKTAWGAAVGFVGLMSSSFVSSIEPLYLTYGII ACGCSFAYQPSLVILGHYFKKRLGLVNGIVTAGSSVFT ILLPLLLRVLIDSVGLFYTLRVLCIFMFVLFLAGFTYRPLATSTKDKESGGSGSSLFSRKKFSPPKKIFNFAIFK VTAYAVAVGIPLALFGYFVPYVHLVSMLLHKHVNERFQDEKNKEVVLMCIGVTSGVGRLLFGRIADYVPGVKKV YLQVLSFFFIGLMSMMIPLCSIFGALIAVCLIMGLFDGCFISIMAPIAFELVGAQDVSQAIGFLLGFMSIPMTVG PPIAGLLRDKLGSYDVAFYLAGVPPLIGGAVLCFIP IHSKKQREISKTTGKEKMEKMLENQNSLLSSSSGMFKK ESDSII
SNP variants of NON17a are disclosed in Example 2.
NO VI 7b
Alternatively, a NON 17 variant is ΝON17b (alternatively referred to herein as CG56459- 02), which includes the 1605 nucleotide sequence (SEQ ID ΝO:59) shown in Table 17C. An open reading frame for the mature protein was identified beginning with an ATG codon at nucleotides 31-33 and ending with a TAA codon at nucleotides 1576- 1578. Putative untranslated regions, if any, downstream from the termination codon and upstream from the initiation codon are underlined. The start and stop codons are in bold letters.
Table 17C. NOV17b Nucleotide Sequence (SEQ ID NO:59)
CTCCGGCGCCTGAGGGGCCCGCCTCGGGCCATGGTGCTCTCCCAGGAGGAGCCGGACTCCGCGCGGGGCACGAGC GAGGCGCAGCCGCTCGGCCCCGCGCCCACGGGGGCCGCTCCGCCGCCCGGCCCGGGACCCTCGGACAGCCCCGAG GCGGCTGTCGAGAAGGTGGAGGTGGAGCTGGCGGGGCCGGCGACCGCGGAGCCCCATGAGCCCCCCGAACCCCCC GAGGGCGGCTGGGGCTGGCTGGTGATGCTGGCGGCCATGTGGTGCAACGGGTCGGTGTTCGGCATCCAGAACGCT TGCGGGGTGCTCTTCGTGTCCATGCTGGAAACCTTCGGCTCCAAAGACGATGACAAGATGGTCTTTAAGACAGCA TGGGTAGGTTCTCTCTCCATGGGGATGATTTTCTTTTGCTGCCCAATAGTCAGTGTCTTCACAGACCTATTTGGT TGTCGGAAAACAGCTGTCGTGGGTGCTGCTGTTGGATTTGTTGGGCTCATGTCCAGTTCTTTTGTAAGTTCCATC GAGCCTCTGTACCTTACCTATGGAATCATATTTGCCTGCGGCTGCTCCTTTGCATACCAGCCTTCATTGGTCATT TTGGGACACTATTTCAAGAAGCGCCTTGGACTGGTGAATGGCATTGTCACTGCTGGCAGCAGTGTCTTCACAATC CTGCTGCCTTTGCTCTTAAGGGTTCTGATTGACAGCGTGGGCCTCTTTTACACATTGAGGGTGCTCTGCATCTTC ATGTTTGTTCTCTTTCTGGCTGGCTTTACTTACCGACCTCTTGCTACCAGTACCAAAGATAAAGAGAGTGGAGGT AGCGGATCCTCCCTCTTTTCCAGGAAAAAGTTCAGTCCTCCAAAAAAAATTTTCAATTTTGCCATCTTCAAGGTG ACAGCTTATGCAGTGTGGGCAGTTGGAATACCACTTGCACTTTTTGGATACTTTGTGCCTTATGTTCACTTGATG AAACATGTAAATGAAAGATTTCAAGATGAAAAAAATAAAGAGGTTGTTCTCATGTGCATTGGCGTCACTTCAGGA GTTGGACGACTGCTCTTTGGCCGGATTGCAGATTATGTGCCTGGTGTGAAGAAGGTTTATCTACAGGTACTCTCC TTTTTCTTCATTGGTCTGATGTCCATGATGATTCCTCTGTGTAGCATCTTTGGGGCCCTCATTGCTGTGTGCCTC ATCATGGGTCTCTTCGATGGATGCTTCATTTCCATTATGGCTCCCATAGCCTTTGAGTTAGTTGGTGCCCAGGAT GTCTCCCAAGCAATTGGATTTCTGCTCGGATTCATGTCTATACCCATGACTGTTGGCCCACCCATTGCAGGTTTA CTTCGTGACAAACTGGGCTCCTATGATGTGGCATTCTACCTCGCTGGAGTCCCTCCCCTTATTGGAGGTGCTGTG CTTTGTTTTATCCCGTGGATCCATAGTAAGAAGCAAAGAGAGATCAGTAAAACCACTGGAAAAGAAAAGATGGAG AAAATGTTGGAAAACCAGAACTCTCTGCTGTCAAGTTCATCTGGAATGTTCAAGAAAGAATCTGACTCTATTATT TAATATCTTACATACCTCCACCAGACTGGA
The NON17b protein (SEQ ID ΝO:60) encoded by SEQ ID NO:59 is 515 amino acid residues in length and is presented using the one-letter amino acid code in Table 17D. The SignalP, Psort and or Hydropathy results indicate that NOVl 7b has no known signal peptide and is likely to be localized at the plasma membrane with a certainty of 0.8000. Alternatively, a NOVl 7b polypeptide is located in the Golgi body with a certainty of 0.4000, the endoplasmic reticulum (membrane) with a certainty of 0.3000, or the mitochondrial inner membrane with a certainty of 0.0300.
Table 17D. Encoded NOVl 7b Protein Sequence (SEQ ID NO:60)
MVLSQEEPDSARGTSEAQPLGPAPTGAAPPPGPGPSDSPEAAVEKVEVELAGPATAEPHEPPEPPEGG G LVML AAM CNGSVFGIQNACGVLFVSMLETFGSKDDDKMVFKTA VGSLSMGMIFFCCPIVSVFTDLFGCRKTAWGAA VGFVGLMSSSFVSSIEPLYLTYGIIFACGCSFAYQPSLVILGHYFKKRLGLVNGIVTAGSSVFTILLPLLLRVLI DSVGLFYTLRVLCIFMFVLFLAGFTYRPLATSTKDKΞSGGSGSSLFSRKKFSPPKKIFNFAIFKVTAYAVWAVGI PLALFGYFVPYVHLMKHVNERFQDEKNKEVVLMCIGVTSGVGRLLFGRIADYVPGVKKVYLQVLSFFFIGLMSMM IPLCSIFGALIAVCLIMGLFDGCFISIMAPIAFELVGAQDVSQAIGFLLGFMSIPMTVGPPIAGLLRDKLGSYDV AFYLAGVPPLIGGAVLCFIP IHSKKQREISKTTGKEKMEKMLENQNSLLSSSSGMFKKESDSII
NOV17 Clones
Unless specifically addressed as NOVl 7a or NOV17b, any reference to NOV17 is assumed to encompass all variants.
A search against the Patp database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 17E.
Table 17E. PatP Results for NOV17
Smallest
High Sum
Sequences Producing High-Scoring Segment Pairs: Score Prob P (N) ρatp:AAE07068 Human gene 18 encoded secreted protein HKZC 47 712 4.4e-70 patp:AAM93737 Human polypeptide 308 1.5e-41 patp:AAY31642 Human transport-associated ρrotein-4 (TRANP-4) 442 1.8e-41 patp:AAB88570 Human hydrophobic domain containing protein clone HP03612 #34 297 2.5e-25 patp:AAE06594 Human protein having hydrophobic domain, HP03949 162 6.3e-20
h a BLAST search of public sequence databases, it was found, for example, that the NOV17a nucleic acid sequence of this invention has 687 of 711 bases (96%) identical to a gb:GENBANK-ID:AFl 16652|acc:AFl 16652.1 mRNA from Homo sapiens PRO0813 mRNA, complete eds. Further, the full amino acid sequence of the disclosed NONl 7a protein of the invention was found to have 283 of 564 amino acid residues (50%) identical to, and 363 of 564 amino acid residues (64%) similar to, the 613 amino acid residue ptnr:SWTSSPROT-ACC:P36021 protein from Human (X-LIΝKED PEST-COΝTAIΝIΝG TRANSPORTER). h a similar BLAST search of public sequence databases, it was found, for example, that the NON17b nucleic acid sequence of this invention has 1363 of 1605 bases (84%) identical to a gb:GEΝBAΝK-ID:AB047324|acc:AB047324.1 mRNA from Rattus norvegicus TATl mRNA, complete eds. Further the full amino acid sequence of the disclosed NOVl 7b protein of the invention was found to have 435 of 515 amino acid residues (84%) identical to, and 463 of 515 amino acid residues (89%) similar to, the 514 amino acid residue ptnr:TREMBLNEW- ACC:BAB55595 protein from Rat (TATl PROTEIN).
Additional BLAST results are shown in Table 17F.
Figure imgf000160_0001
A multiple sequence alignment is given in Table 17G, with the NONl 7 proteins of the invention being shown in lines 1 and 2 in a ClustalW analysis comparing ΝON17 with related protein sequences of Table 17F.
Table 17G. ClustalW Analysis of ΝON17
1. SEQ ID NO.: 58 NOVl 7a 5. SEQ ID NO.: 228 070324
2. SEQ ID NO.: 60 NOV17b 6. SEQ ID NO.: 229 Q9P1I2 3. SEQ ID NO.: 226 Q91Y77 7. SEQ ID NO.: 230 AAH17968
4. SEQ ID NO.: 227 P36021
10 20 30 40 50 60
NOV17a -MLMGGVTHTSVPLSEQEEAPSQTRAQTASAAGT GRAPRGAPPPLSEAQSSGGC 54 NOV17b Q91Y77 P36021 MGRGGGGLDVGGGGEGSRDRLSRDGLAS GAEPGGGGSGSGSSSPPSSSSCSSRNKYQPQ 60 070324 MALP SPASEEAEGPCQEANQEYQEP 25 Q9P1I2 1 AAH17968
Figure imgf000161_0001
AAH17968
Figure imgf000161_0002
AAH17968
Figure imgf000161_0003
Figure imgf000162_0001
AAH17968 1
Figure imgf000162_0002
AAH17968 1
Figure imgf000162_0003
550 560 570 580 590 600
NOVl7a :PMTVGPPIAGLLRDKLGSYDVAFYLAGVPPFFLIGGAVLCFIPWIHSKKQREISK ITGKΞI 579 NOV17b :PMTVGPPIAGLLRDKLGSYDVAFYLAGVPPSIGGAVLCFIP IHSKKQREISKHTGKEI 488 Q91Y77 487 P36021 3PPIAGLLR 590 070324 3PPIAGLLR iQRMFKSEQi 542 Q9P1I2 CPMTVGPPIAGLLRDKLGSYDVAFYLAGVPPFFLLGGAVLCFIP IHSKKQREISKIJTGKE] 174 AAH17968 :PMTVGPPIAGLLRDKLGSYDVAFYLAGVPP!IGGAVLCFIP IHSKKQREIS STGKE: 174
Figure imgf000162_0004
The presence of identifiable domains in the disclosed NON 17 protein was determined by using Pfam and then determining the Interpro number. The results are listed in Table 17H with the statistics and domain description.
Figure imgf000163_0001
Figure imgf000164_0001
Consistent with other known members of the proton-linked monocarboxylate transporter (MCTs) family of proteins, NON17 contains a transporter domain as illustrated in Table 17H.
ΝON17 nucleic acids, and the encoded polypeptides, according to the invention are useful in a variety of applications and contexts. For example, ΝON17 nucleic acids and polypeptides can be used to identify proteins that are members of the MCTs family of proteins. The ΝON17 nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance ΝON17 activity or function. Specifically, the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., cellular metabolism, or transport of monocarboxylates such as lactate and pyruvate. These molecules can be used to treat, e.g., infantile sialic storage disease.
In addition, various ΝON17 nucleic acids and polypeptides according to the invention are useful, inter alia, as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins. For example, the ΝON17 nucleic acids and their encoded polypeptides include structural motifs that are characteristic of proteins belonging to the MCTs family. Monocarboxylates such as lactate and pyruvate play a pivotal role in cellular physiology of most mammalian cells. Lactic acid, in particular, is produced in huge amounts as an end-product of glycolysis. Some tissues, such as white skeletal muscle, red blood cells and tumor cells, rely on this pathway to produce majority of their ATP under normal physiological conditions, while all tissues become dependent on this pathway during hypoxia or ischaemia. Two molecules of lactic acid are generated for every glucose molecule during glycolysis. Lactic acid must be transported out of the cell if high rates of glycolysis are to be maintained. Accumulation of lactic acid leads to a decrease in intracellular pH and cessation of glycolysis. Lactic acid transport is carried out by a recently identified family of proton-linked monocarboxylate transporters (MCTs) located at the plasma membrane. At least 9 MCTs
(MCTl-9)-related genes have so far been identified in mammals, each having a different tissue distribution. MCTs also mediate the transport of many other metabolically important monocarboxylates such as pyruvate, the branched-chain oxo acids derived from leucine, valine and isoleucine, and the ketone bodies acetoacetate, -hydroxybutyrate and acetate. The NONl 7 nucleic acids and polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in the mediation of cellular metabolism and transport. As such the ΝON17 nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat, e.g., Salla disease, infantile sialic acid storage disease, cystinosis, or streptozotocin-induced diabetes.
The ΝON17 nucleic acids and polypeptides are useful for detecting specific cell types. For example, expression analysis has demonstrated that a ΝON17a nucleic acid is expressed in adrenal gland, bone marrow, brain - amygdala, brain - cerebellum, brain - hippocampus, brain - substantia nigra, brain - thalamus, brain -whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma - Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea, and uterus. Conversely, a ΝON17b nucleic acid is expressed in parathyroid gland, liver, colon, muscle, brain, placenta, vulva, testis, lung, kidney, skin, and colon adenocarcinoma.
Additional utilities for ΝON17 nucleic acids and polypeptides according to the invention are disclosed herein.
ΝON18
The ΝON18 proteins descibed herein are novel olfactory receptor/G-protein coupled receptor (GPCR)-like proteins. Two alternative novel ΝON18 nucleic acids and polypeptides are disclosed herein, namely ΝON18a and ΝON18b.
ΝON18a
AΝON18 variant is ΝON18a (alternatively referred to herein as CG56510-01), which encodes the 1001 nucleotide sequence (SEQ ID ΝO:61) shown in Table 18A. An open reading frame for the mature protein was identified beginning with an ATG codon at nucleotides 37-39 and ending with a TGA codon at nucleotides 958-960. Putative untranslated regions, if any, downstream from the termination codon and upstream from the imtiation codon are underlined. The start and stop codons are in bold letters. Table 18A. NON18a Nucleotide Sequence (SEQ ID NO:61)
GTGTTCCATAGATTATTTTGTCTTTTGTCTGAAGTGATGCTGAATACAACCTCAGTCACCGAATTTCTCCTCTTG GGAGTGACAGACATTCAAGAACTGCAGCCTTTTCTCTTCGTGGTTTTCCTCACCATCTACTTCATCAGTGTGACT GGGAATGGAGCCGTTCTGATGATTGTCATCTCCGATCCTAGACTCCATTCCCTTATGTATTTCTTCCTGGGAAAC CTGTCCTACCTGGATATCTGTTACTCTACGGTGACACTGCCAAAAATGCTGCAGAACTTTCTCTCTACACACAAA GCAATTTCTTTCTTGGGATGCATAAGCCAGCTTCATTTCTTCCACTTCCTGGGCAGCACGGAGTCCATGTTGTTC GCCGTGATGGCATTTGACCTCTCTGTGGCTATCTGCAAGCCACTTCGCTACACTGTCATCATGAACCCTCAGCTC TGTACCCAGATGGCCATCACAATCTGGGTCATTGGTTTTTTCCATGCCCTGCTGCACTCCGTAATGACTTCTCGC TTGAACTTCTGTGGTTCCAACCGTATCCATCATTTTCTCTGTGATATTAAGCCATTGCTAAAGCTGGCCTGTGGG AACACTGAGCTTAATCAGTGGCTACTCAGTACTGTCACGGGGACAATTGCCATGGGCCCCTTCTTTCTGACACTT CTCTCCTATTTCTACATTATCACTTATCTCTTCTTCAAGACCCGTTCTTGTAGCATGCTCTGTAAAGCACTGTCC ACTTGTGCCTCCCACTTCATGGTAGTTATTCTTTTCTATGCACCTGTTCTTTTCACCTATATCCATCCTGCGTTA GAGAGCTTCATGGACCAGGACCGGATTGTTGCCATCATGTACACTGTGGTCACTCCTGTACTAAACCCACTGATC TATACTTTGAGGAACAAGGAAGTGAAGGGGGCCTTGGGTAGAGTGATCAGAAGGCTTTGATTTGAATAAACCAGA GAACTCTACTGAGGCATAAATAACCA
The NOV18a protein (SEQ ID NO:62) encoded by SEQ ID NO:61 is 307 amino acid residues in length and is presented using the one-letter amino acid code in Table 18B. The SignalP, Psort and/or Hydropathy results indicate that NONl 8a has a signal peptide and is likely to be localized at the plasma membrane with a certainty of 0.6000. Alternatively, a ΝON18a polypeptide is located in the Golgi body with a certainty of 0.4000, the endoplasmic reticulum (membrane) with a certainty of 0.3000, or the microbody (peroxisome) with a certainty of 0.3000. The SignalP indicates a likely cleavage site for a ΝON18a peptide between positions 39 and 40, i.e., at the dash in the sequence NTG-ΝG.
Table 18B. Encoded ΝOV18a Protein Sequence (SEQ ID NO:62)
MLNTTSVTEFLLLGVTDIQΞLQPFLFWFLTIYFISVTGNGAVLMIVISDPRLHSLMYFFLGNLSYLDICYSTVT LPKMLQNFLSTHKAISFLGCISQLHFFHFLGSTESMLFAVMAFDLSVAICKPLRYTVIMNPQLCTQMAITI VIG FFHALLHSVMTSRLNFCGSNRIHHFLCDIKPLL LACGNTELNQ LLSTVTGTIAMGPFFLTLLSYFYIITYLFF KTRSCSMLCKALSTCASHFMWILFYAPVLFTYIHPALESFMDQDRIVAIMYTWTPVLNPLIYTLRNKEVKGAL GRVIRRL
SNP variants of NOVl 8a are disclosed in Example 2.
NONlδb Alternatively, a ΝON18 variant is ΝON18b (alternatively referred to herein as CG56510-
02), which includes the 1101 nucleotide sequence (SEQ ID ΝO:63) shown in Table 18C. An open reading frame for the mature protein was identified beginning with an ATG codon at nucleotides 148-150 and ending with a TGA codon at nucleotides 1069-1071. Putative untranslated regions, if any, downstream from the termination codon and upstream from the initiation codon are underlined. The start and stop codons are in bold letters.
Table 18C. NOV18b Nucleotide Sequence (SEQ ID NO:63)
TAAGCTTCTATACAACTTCTGAGGTTTGGAAGAAGTACAACAGTACTCTCCTTCCAAGTATCTTTGGCTTGGTGA GAAAATTCTGAGCCGGAAGGATTCTGATTGCGATTAGTGTTCCATAGATTATTTTGTCTTTTGTCTGAAGTGATG CTGAATACAACCTCAGTCACCGAATTTCTCCTCTTGGGAGTGACAGACATTCAAGAACTGCAGCCTTTTCTCTTC GTGGTTTTCCTCACCATCTACTTCATCAGTGTGACTGGGAATGGAGCCGTTCTGATGATTGTCATCTCCGATCCT AGACTCCATTCCCTTATGTATTTCTTCCTGGGAAACCTGTCCTACCTGGATATCTGTTACTCTACGGTGACACTG CCAAAAATGCTGCAGAACTTTCTCTCTACACACAAAGCAATTTCTTTCTTGGGATGCATAAGCCAGCTTCATTTC TTCCACTTCCTGGGCAGCACGGAGTCCATGTTGTTCGCCGTGATGGCATTTGACCTCTCTGTGGCTATCTGCAAG CCACTTCGCTACACTGTCATCATGAACCCTCAGCTCTGTACCCAGATGGCCATCACAATCTGGGTCATTGGTTTT TTCCATGCCCTGCTGCACTCCGTAATGACTTCTCGCTTGAACTTCTGTGGTTCCAACCGTATCCATCATTTTCTC TGTGATATTAAGCCATTGCTAAAGCTGGCCTGTGGGAACACTGAGCTTAATCAGTGGCTACTCAGTACTGTCACG GGGACAATTGCCATGGGCCCCTTCTTTCTGACACTTCTCTCCTATTTCTACATTATCACTTATCTCTTCTTCAAG ACCCGTTCTTGTAGCATGCTCTGTAAAGCACTGTCCACTTGTGCCTCCCACTTCATGGTAGTTATTCTTTTCTAT GCACCTGTTCTTTTCACCTATATCCATCCTGCGTTAGAGAGCTTCATGGACCAGGACCGGATTGTTGCCATCATG TACACTGTGGTCACTCCTGTACTAAACCCACTGATCTATACTTTGAGGAACAAGGAAGTGAAGGGGGCCTTGGGT AGAGTGATCAGAAGGCTTTGATTTGAATAAACCAGAGAACTCTACTGAGGC
The NOVl 8b nucleic acid (SEQ ID NO:63) encodes the NOVl 8a protein SEQ ID NO:62.
NOV18 Clones
Unless specifically addressed as NONl 8a or ΝON18b, any reference to ΝON18 is assumed to encompass all variants.
A search against the Patp database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 18D.
Table 18D. PatP Results for ΝOV18
Smallest
High Sum
Sequences Producing High-Scoring Segment Pairs: Score Prob P (N) patρ:AAG72203 Human olfactory receptor polypeptide 1590 4.0e-163 patp:AAG72870 Human olfactory receptor data exploratorium sequence 1590 4.0e-163 patp :AAG71661 Human olfactory receptor polypeptide 1344 4.7e-137 patp:AAG72212 Human olfactory receptor polypeptide 1344 4.7e-137 patp:AAG72633 Murine OR-like polypeptide query sequence 1296 5.7e-132 h a BLAST search of public sequence databases, it was found, for example, that the NONl 8 nucleic acid sequences of this invention have 590 of 911 bases (64%) identical to a gb:GEΝBAΝK-ID:AF101730|acc:AF101730.1 mRNA from Pan troglodytes isolate PTOR1E1 olfactory receptor gene, complete eds. Further, the full amino acid sequence of the disclosed NON 18 protein of the invention has 205 of 306 amino acid residues (66%) identical to, and 251 of 306 amino acid residues (82%) similar to, the 316 amino acid residue ptnπSPTREMBL- ACC:Q9UGF7 protein from Human (BA150A6.1 (NOVEL 7 TRANSMEMBRANE RECEPTOR (RHODOPSIN FAMILY) (OLFACTORY RECEPTOR LIKE) PROTErN (HS6M1-27))). While it has not been annotated as an olfactory receptor, genomic clone Genbank TD AL049739.2 shows 100% homology to NOVl 8.
Additional BLAST results are shown in Table 18E.
Figure imgf000168_0001
Figure imgf000169_0003
A multiple sequence alignment is given in Table 18F, with the NON 18 protein of the invention being shown in line 1 in a ClustalW analysis comparing ΝON18 with related protein sequences of Table 18E.
Table 18F. ClustalW Analysis of ΝON18
1. SEQ ID NO.: 62 NOVl 8 4. SEQ ID NO.: 234 Q920Y8
2. SEQ ID NO.: 232 P58182 5. SEQ ID NO.: 235 Q920Z0
3. SEQ ID NO.: 233 Q920Y9 6. SEQ ID NO.: 236 CAC44547
Figure imgf000169_0001
130 140 150 160 170 180
Figure imgf000169_0002
190 200 210 220 230 240
Figure imgf000170_0001
The presence of identifiable domains in the disclosed NON 18 protein was determined by using Pfam and then determimng the Inteφro number. The results are listed in Table 18G with the statistics and domain description.
Table 18G. Domain Analysis of NO VI 8
PSSMs Producing Significant Alignments Score E (bits) Value
7tm 1 : domain 1 of 1 , from 39 to 289 68.8 5.6e-21
7tm GNlLVilvilrtkklrtptnifilNLAvADLLflltlppwalyylvg ||+ I +++++ ++++ ++++++ 1 1 + I ++++++ ++ ++ +++ NOVl8 GNGAVLMIVISDPRLHSLMYFFLGNLSYLDICYSTVTLPKMLQNFLS 7tm gsedWpfGsalCklvtaldwnmyaSillLtalSiDRYlAIvhPlryrrr
++ ++ ++ I + ++ + + + ++ +++++ | + | | ++ | +++ ++ NOVl8 - -THKAISFLGCISQLHFFHFLGSTESMLFAVMAFDLSVAICKPLRYTVI 7tm rtsprrAkwillv vlalllslPpllfswvktveegngtlnvnvtvCli ++ ++++ + + +I++++ ++++ ++ ++++++++++ + ++ I ++ NOVl8 MN-PQLCTQMAITIWVIGFFHALLHSVM-TSRLNFCGSNR- -IHHFLCDI 7tm dfpeestasvst lrsyvllstlvgFllPllvilvcYtrllrtlr
+++ + ++ + + | +++ + I + I + ++ ++++ NOVl8 KPLLKLACGNTELNQ LLSTVTGTIAMGPFFLTLLSYFYIITYLFfktrs 7tm .kaaktll wwFylCWlPyfiyllldtlc . lsii sstCelerv + +++ + + +++++ + | + + ++ ++
N0V18 CSmlcKALSTCASHFMWILFYAPVLFTYIHPALEsFM- - -
7tm lptallvtl LayvNsclNPilY (SEQ ID NO : 237 )
+ +++++++++ +++ ii+ii
N0V18 -DQDRIVAIMYTWTPVLNPLIY (SEQ ID NO : 62 )
Consistent with other known members of the olfactory receptor family of proteins, NONl 8 contains 7-transmembrane domains as illustrated in Table 18G.
The ΝON18 nucleic acids, and the encoded polypeptide, according to the invention are useful in a variety of applications and contexts. For example, ΝON18 nucleic acids and polypeptides can be used to identify proteins that are members of the olfactory receptor family of proteins. The ΝON18 nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance ΝON18 activity or function. Specifically, the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., cellular recognition, or G-protein-mediated transduction of odorant signals. These molecules can be used to treat, e.g., taste and scent detectability disorders, immune diseases, or signal transduction pathways.
In addition, the ΝON18 nucleic acids and polypeptide according to the invention are useful, wzter alia, as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins. For example, the ΝON18 nucleic acids and polypeptide include structural motifs that are characteristic of proteins belonging to the family of olfactory receptor proteins. Olfactory receptors have great variety, exquisite specificity, high sensitivity and fast response. The human olfactory epithelium contains two to three thousand distinct olfactory receptors, a class of G-protein coupled receptors. The receptors consist of seven hydrophobic segments that span the cell membrane (trans-membrane domains I-NII), separated by hydrophilic segments that project into the intra- or extra-cellular space. Trans-membrane domains II-NII comprise a hypervariable segment that defines the ligand specificity of the receptor.
The ΝON18 nucleic acids and polypeptide, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in the mediation of signal transduction. As such the ΝON18 nucleic acids and polypeptide, antibodies and related compounds according to the invention may be used to treat, e.g., developmental diseases, MHC II and III diseases (immune diseases), taste and scent detectability disorders, Burkitt's lymphoma, corticoneurogenic disease, signal transduction pathway disorders, retinal diseases including those involving photoreception, cell growth rate disorders, cell shape disorders, feeding disorders, control of feeding, potential obesity due to over-eating, potential disorders due to starvation (lack of apetite), noninsulin-dependent diabetes mellitus (NTDDM1), bacterial, fungal, protozoal and viral infections (particularly infections caused by HIV-1 or HIN-2), pain, cancer (including but not limited to neoplasm, adenocarcinoma, lymphoma, prostate cancer, uterus cancer), anorexia, bulimia, asthma, parkinson's disease, acute heart failure, hypotension, hypertension, urinary retention, osteoporosis, crohn's disease, multiple sclerosis, and treatment of albright hereditary ostoeodystrophy, angina pectoris, myocardial infarction, ulcers, asthma, allergies, benign prostatic hypertrophy, and psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation, dentatorubro-pallidoluysian atrophy(DRPLA) hypophosphatemic rickets, autosomal dominant (2) acrocallosal syndrome and dyskinesias, such as huntington's disease or gilles de la tourette syndrome.
The ΝON18 nucleic acids and polypeptide are useful for detecting specific cell types. For example, expression analysis has demonstrated that a ΝON18 nucleic acid is predominantly expressed in olfactory epithelium and taste receptor cells of the tongue. However, it is also expressed in apical microvilli of the retinal pigment epithelium, arterial (aortic), basal forebrain, brain, Burkitt lymphoma cell lines, coφus callosum, cardiac (atria and ventricle), caudate nucleus, CΝS and peripheral tissue, cerebellum, cerebral cortex, colon, cortical neurogenic cells, endothelial (coronary artery and umbilical vein) cells, palate epithelia, eye, neonatal eye, frontal cortex, fetal hematopoietic cells, heart, hippocampus, hypothalamus, leukocytes, liver, fetal liver, lung, lung lymphoma cell lines, fetal lymphoid tissue, adult lymphoid tissue, those that express MHC II and III nervous, medulla, subthalamic nucleus, ovary, pancreas, pituitary, placenta, pons, prostate, putamen, serum, skeletal muscle, small intestine, smooth muscle (coronary artery in aortic) spinal cord, spleen, stomach, testis, thalamus, and thymus tissue.
Additional utilities for the ΝON18 nucleic acid and polypeptide according to the invention are disclosed herein.
ΝOV19 A NOV19 polypeptide has been identified as a Major Duchenne Muscular Dystrophy
(DP71)-like protein. The novel NOV19 nucleic acid sequences maps to the chromosome Xp21.2. Two alternative novel NOV19, NOV19a and NOV19b, nucleic acids and encoded polypeptides are provided.
NOV19a
A NOV19 variant is the novel NOV19a (alternatively referred to herein as CG56574-01), which includes the 2463 nucleotide sequence (SEQ ID NO: 64) shown in Table 19 A. A NOVl 9a ORF begins with a TGG initiation codon at nucleotides 3-5 and ends with a TAA codon at nucleotides 2106-2108. Putative untranslated regions upstream from the initiation codon and downstream from the termination codon are underlined in Table 19A, and the start and stop codons are in bold letters.
Table 19A. NOV19a Nucleotide Sequence (SEQ ID NO:64)
GGTGGGCGAGCCGACACACGCCCGCCCCGTCTGGGGGCAGCGCCCCCTCCCCGGCCCGCCCGCGCCG GCTCCTCCGCAGTGCTTTCAGCTGTGAGCTTGGGCGGCGGCGGCGGCGGCGCTCCACTTTCGGGGAG CCCGGCGGCTCTGGGAAGCTCACTCCTCCACTCGTACCCACACTCGACCGCGGAGCCCTTGCAGCCA TGAGGGAACAGCTCAAAGGCCACGAGACTCAAACAACTTGCTGGGACCATCCCAAAATGACAGAGCT CTACCAGTCTTTAGCTGACCTGAATAATGTCAGATTCTCAGCTTATAGGACTGCCATGAAACTCCGA AGACTGCAGAAGGCCCTTTGCTGGGATCTCTTGAGCCTGTCAGCTGCATGTGATGCCTTGGACCAGC ACAACCTCAAGCAAAATGACCAGCCCATGGATATCCTGCAGATTATTAATTGTTTGACCACTATTTA TGACCGCCTGGAGCAAGAGCACAACAATTTGGTCAACGTCCCTCTCTGCGTGGATATGTGTCTGAAC TGGCTGCTGAATGTTTATGATACGGGACGAACAGGGAGGATCCGTGTCCTGTCTTTTAAAACTGGCA TCATTTCCCTGTGTAAAGCACATTTGGAAGACAAGTACAGAAACCTTTTCAAGCAAGTGGCAAGTTC AACAGGATTTTGTGACCAGCGCAGGCTGGGCCTCCTTCTGCATGATTCTATCCAAATTCCAAGACAG TTGGGTGAAGTTGCATCCTTTGGGGGCAGTAACATTGAGCCAAGTGTCCGGAGCTGCTTCCAATTTG CTAATAATAAGCCAGAGATCGAAGCGGCCCTCTTCCTAGACTGGATGAGACTGGAACCCCAGTCCAT GGTGTGGCTGCCCGTCCTGCACAGAGTGGCTGCTGCAGAAACTGCCAAGCATCAGGCCAAATGTAAC ATCTGCAAAGAGTGTCCAATCATTGGATTCAGGTACAGGAGTCTAAAGCACTTTAATTATGACATCT GCCAAAGCTGCTTTTTTTCTGGTCGAGTTGCAAAAGGCCATAAAATGCACTATCCCATGGTGGAATA TTGCACTCCGACTACATCAGGAGAAGATGTTCGAGACTTTGCCAAGGTACTAAAAAACAAATTTCGA ACCAAAAGGTATTTTGCGAAGCATCCCCGAATGGGCTACCTGCCAGTGCAGACTGTCTTAGAGGGGG ACAACATGGAAACTCCCGTTACTCTGATCAACTTCTGGCCAGTAGATTCTGCGCCTGCCTCGTCCCC TCAGCTTTCACACGATGATACTCATTCACGCATTGAACATTATGCTAGCAGGCTAGCAGAAATGGAA AACAGCAATGGATCTTATCTAAATGATAGCATCTCTCCTAATGAGAGCATAGATGATGAACATTTGT TAATCCAGCATTACTGCCAAAGTTTGAACCAGGACTCCCCCCTGAGCCAGCCTCGTAGTCCTGCCCA GATCTTGATTTCCTTAGAGAGTGAGGAAAGAGGGGAGCTAGAGAGAATCCTAGCAGATCTTGAGGAA GAAAACAGGAATCTGCAAGCAGAATATGACCGTCTAAAGCAGCAGCACGAACATAAAGGCCTGTCCC CACTGCCGTCCCCTCCTGAAATGATGCCCACCTCTCCCCAGAGTCCCCGGGATGCTGAGCTCATTGC TGAGGCCAAGCTACTGCGTCAACACAAAGGCCGCCTGGAAGCCAGGATGCAAATCCTGGAAGACCAC AATAAACAGCTGGAGTCACAGTTACACAGGCTAAGGCAGCTGCTGGAGCAACCCCAGGCAGAGGCCA AAGTGAATGGCACAACGGTGTCCTCTCCTTCTACCTCTCTACAGAGGTCCGACAGCAGTCAGCCTAT GCTGCTCCGAGTGGTTGGCAGTCAAACTTCGGACTCCATGGGTGAGGAAGATCTTCTCAGTCCTCCC CAGGACACAAGCACAGGGTTAGAGGAGGTGATGGAGCAACTCAACAACTCCTTCCCTAGTTCAAGAG GACACAATGTAGGAAGTCTTTTCCACATGGCAGATGATTTGGGCAGAGCGATGGAGTCCTTAGTATC AGTCATGACAGATGAAGAAGGAGCAGAATAAATGTTTTACAACTCCTGATTCCCGCATGGTTTTTAT AATATTCATACAACAAAGAGGATTAGACAGTAAGAGTTTACAAGAAATAAATCTATATTTTTGTGAA GGGTAGTGGTATTATACTGTAGATTTCAGTAGTTTCTAAGTCTGTTATTGTTTTGTTAACAATGGCA GGTTTTACACGTCTATGCAATTGTACAAAAAAGTTATAAGAAAACTACATGTAAAATCTTGATAGCT AAATAACTTGCCATTTCTTTATATGGAACGCATTTTGGGTTGTTTAAAAATTTATAACAGTTATAAA GAAAGATTGTAAACTAAAGTGTGCTTTATAAAAAAAGTTGTTTATAAAAAC The NOV19a polypeptide (SEQ ID NO:65) encoded by SEQ ID NO:64 is 701 amino acid residues in length and is presented using the one-letter amino acid code in Table 19B. The Psort profile for the NOVl 9a predicts that this peptide is likely to be localized at the nucleus with a certainty of 0.9700.
Table 19B. NOV19a protein sequence (SEQ ID NO:65) ASRHTPAPSGGSAPSPARPRRLLRSAFSCELGRRRRRRSTFGΞPGGSGKLTPPLVPTLDRGALAAM REQLKGHETQTTCWDHPKMTELYQSLADLNNVRFSAYRTAMKLRRLQKALCWDLLSLSAACDALDQH NLKQNDQPMDILQIINCLTTIYDRLEQEHNNLVNVPLCVDMCLN LLNVYDTGRTGRIRVLSFKTGI ISLCKAHLEDKYRNLFKQVASSTGFCDQRRLGLLLHDSIQIPRQLGEVASFGGSNIEPSVRSCFQFA NNKPEIEAALFLD MRLEPQSMVWLPVLHRVAAAETAKHQAKCNICKECPIIGFRYRSLKHFNYDIC QSCFFSGRVAKGHKMHYPMVEYCTPTTSGEDVRDFAKVLKNKFRTKRYFAKHPRMGYLPVQTVLEGD NMETPVTLINFWPVDSAPASSPQLSHDDTHSRIEHYASRLAEMΞNSNGSYLNDSISPNESIDDEHLL IQHYCQSLNQDSPLSQPRSPAQILISLESEERGELERILADLEEENRNLQAEYDRLKQQHEHKGLSP LPSPPEMMPTSPQSPRDAELIAEAKLLRQHKGRLEARMQILEDHNKQLESQLHRLRQLLEQPQAEAK VNGTTVSSPSTSLQRSDSSQPMLLRWGSQTSDSMGEEDLLSPPQDTSTGLEEVMEQLNNSFPSSRG HNVGSLFHMADDLGRAMESLVSVMTDEEGAE
NOV19b
Alternatively, a NOVl 9 variant is the novel NOVl 9b (alternatively referred to herein as CG56574-02), which includes the 2005 nucleotide sequence (SEQ ID NO:66) shown in Table
19C. NOV19b was created by polymerase chain reaction (PCR) using the primers: 5'
GAAGCTCACTCCTCCACTCGTACC 3' (SEQ ID NO:237) and 5'
ATGAATATTATAAAAACCATGCGGGAA 3' (SEQ ID NO:238). Primers were designed based on in silico predictions of the full length or some portion (one or more exons) of the cDNA/protein sequence of the invention. The PCR product derived by exon linking, covering the entire open reading frame, was cloned into the pCR2.1 vector from Invitrogen to provide clone
127720: :M18533r3_0_1.698587.P22.
The NOVl 9b ORF begins with a Kozak consensus ATG initiation codon at nucleotides
53-55 and ends with a TAA codon at nucleotides 1958-1960. Putative untranslated regions upstream from the initiation codon and downstream from the termination codon are underlined in
Table 19C, and the start and stop codons are in bold letters.
Table 19C. NOV19b Nucleotide Sequence (SEQ ID NO:66)
GAAGCTCACTCCTCCACTCGTACCCACACTCGACCGCGGAGCCCTTGCAGCCATGAGGGAACAGCTC AAAGGCCACGAGACTCAAACAACTTGCTGGGACCATCCCAAAATGACAGAGCTCTACCAGTCTTTAG CTGACCTGAATAATGTCAGATTCTCAGCTTATAGGACTGCCATGAAACTCCGAAGACTGCAGAAGGC CCTTTGCTTGGATCTCTTGAGCCTGTCAGCTGCATGTGATGCCTTGGACCAGCACAACCTCAAGCAA AATGACCAGCCCATGGATATCCTGCAGATTATTAATTGTTTGACCACTATTTATGACCGCCTGGAGC AAGAGCACAACAATTTGGTCAACGTCCCTCTCTGCGTGGATATGTGTCTGAACTGGCTGCTGAATGT TTATGATACGGGACGAACAGGGAGGATCCGTGTCCTGTCTTTTAAAACTGGCATCATTTCCCTGTGT AAAGCACATTTGGAAGACAAGTACAGATACCTTTTCAAGCAAGTGGCAAGTTCAACAGGATTTTGTG ACCAGCGCAGGCTGGGCCTCCTTCTGCATGATTCTATCCAAATTCCAAGACAGTTGGGTGAAGTTGC ATCCTTTGGGGGCGGTAACATTGAGCCAAGTGTCCGGAGCTGCTTCCAATTTGCTAATAATAAGCCA GAGATCGAAGCGGCCCTCTTCCTAGACTGGATGAGACTGGAACCCCAGTCCATGGTGTGGCTGCCCG TCCTGCACAGAGTGGCTGCTGCAGAAACTGCCAAGCATCAGGCCAAATGTAACATCTGCAAAGAGTG TCCAATCATTGGATTCAGGTACAGGAGTCTAAAGCACTTTAATTATGACATCTGCCAAAGCTGCTTT TTTTCTGGTCGAGTTGCAAAAGGCCATAAAATGCACTATCCCATGGTGGAATATTGCACTCCGACTA CATCAGGAGAAGATGTTCGAGACTTTGCCAAGGTACTAAAAAACAAATTTCGAACCAAAAGGTATTT TGCGAAGCATCCCCGAATGGGCTACCTGCCAGTGCAGACTGTCTTAGAGGGGGACAACATGGAAACT CCCGTTACTCTGATCAACTTCTGGCCAGTAGATTCTGCGCCTGCCTCGTCCCCTCAGCTTTCACACG ATGATACTCATTCACGCATTGAACATTATGCTAGCAGGCTAGCAGAAATGGAAAACAGCAATGGATC TTATCTAAATGATAGCATCTCTCCTAATGAGAGCATAGATGATGAACATTTGTTAATCCAGCATTAC TGCCAAAGTTTGAACCAGGACTCCCCCCTGAGCCAGCCTCGTAGTCCTGCCCAGATCTTGATTTCCT TAGAGAGTGAGGAAAGAGGGGAGCTAGAGAGAATCCTAGCAGATCTTGAGGAAGAAAACAGGAATCT GCAAGCAGAATATGACCGTCTAAAGCAGCAGCACGAACATAAAGGCCTGTCCCCACTGCCGTCCCCT CCTGAAATGATGCCCACCTCTCCCCAGAGTCCCCGGGATGCTGAGCTCATTGCTGAGGCCAAGCTAC TGCGTCAACACAAAGGCCGCCTGGAAGCCAGGATGCAAATCCTGGAAGACCACAATAAACAGCTGGA GTCACAGTTACACAGGCTAAGGCAGCTGCTGGAGCAACCCCAGGCAGAGGCCAAAGTGAATGGCGCA ACGGTGTCCTCTCCTTCTACCTCTCTACAGAGGTCCGACAGCAGTCAGCCTATGCTGCTCCGAGTGG TTGGCAGTCAAACTTCGGACTCCATGGGTGAGGAAGATCTTCTCAGTCCTCCCCAGGACACAAGCAC AGGGTTAGAGGAGGTGATGGAGCAACTCAACAACTCCTTCCCTAGTTCAAGAGGACACAATGTAGGA AGTCTTTTCCACATGGCAGATGATTTGGGCAGAGCGATGGAGTCCTTAGTATCAGTCATGACAGATG AAGAAGGAGCAGAATAAATGTTTTACAACTCCTGATTCCCGCATGGTTTTTATAATATTCAT
Variant sequences of NOVl 9b are included in Example 2. A variant sequence can include a single nucleotide polymoφhism (SNP). A SNP can, in some instances, be referred to as a "cSNP" to denote that the nucleotide sequence containing the SNP originates as a cDNA.
The NOV19b protein (SEQ ID NO:67) encoded by SEQ ID NO:66 is 635 amino acid residues in length and is presented using the one-letter code in Table 19D. The Psort profile for NOVl 9b predicts that this sequence is likely to be localized at the cytoplasm with a certainty of 0.4500. The Signal P predicts a likely cleavage site for a NOVl 9b peptide is between positions 64 and 65, i.e., at the dash in the sequence CDA-LD.
Table 19D. NOV19b protein sequence (SEQ ID NO:67)
MREQLKGHETQTTC DHPKMTELYQSLADLNNVRFSAYRTAMKLRRLQKALCLDLLSLSAACDALDQ HNLKQNDQPMDILQIINCLTTIYDRLEQEHNNLVNVPLCVDMCLNWLLNVYDTGRTGRIRVLSFKTG IISLCKAHLEDKYRYLFKQVASSTGFCDQRRLGLLLHDSIQIPRQLGEVASFGGGNIEPSVRSCFQF ANNKPEIEAALFLDWMRLEPQSMV LPVLHRVAAAETAKHQAKCNICKECPIIGFRYRSLKHFNYDI CQSCFFSGRVAKGHKMHYPMVEYCTPTTSGEDVRDFAKVLKNKFRTKRYFAKHPRMGYLPVQTVLEG DNMETPVTLINFWPVDSAPASSPQLSHDDTHSRIEHYASRLAEMENSNGSYLNDSISPNESIDDEHL LIQHYCQSLNQDSPLSQPRSPAQILISLESEERGELΞRILADLEEENRNLQAEYDRLKQQHEHKGLS PLPSPPEMMPTSPQSPRDAELIAEAKLLRQHKGRLEARMQILEDHNKQLESQLHRLRQLLEQPQAEA KVNGATVSSPSTSLQRSDSSQPMLLRWGSQTSDSMGEEDLLSPPQDTSTGLEEVMEQLNNSFPSSR GHNVGSLFHMADDLGRAMESLVSVMTDEEGAE NON19 Clones
Unless specifically addressed as ΝON19a or ΝON19b, any reference to ΝON19 is assumed to encompass all variants. ΝON19a polypeptide is longer than the ΝON19b polypeptide, having an additional 66 amino acids on the Ν-terminus. ΝON19a also differs from ΝON19b at four amino acid residues [aa 119 (W>L); aa 215 (Ν>Y); aa 255 (S>G); aa 607 (T>A)] as shown Table 19G.
A search against the Patp database, a proprietary database that contains sequences published in patents and patent publications, yielded several homologous proteins shown in Table 19E.
Table 19E. Patp results for NON19
Smallest
Sum
Reading High Prob
Sequences producing High-scoring Segment Pairs: Frame Score P(Ν)
>patp:AAY59237 A rod shortened dystrophin (deltaDysAx2) +1 3127 0.0
>patp:AAY59238 A rod shortened dystrophin (deltaDysAxll) +1 3127 0.0
>patp:AAY59239 A rod shortened dystrophin (deltaDysAH3) +1 3127 0.0
>patp:AAY59240 A rod shortened dystrophin (deltaDysM3) +1 3127 0.0
>patp:AAY59242 A rod shortened dystrophin (deltaDysH4) +1 3127 0.0
>patp:AAP90290 Human Duchenne muscular dystrophy gene +1 3120 0.0
In a BLAST search of public sequence databases, it was found, for example, that the NOVl 9a nucleic acid sequence of this invention has 1793 of 1798 bases (99%) identical to a gb:GENBANK-ID:HSDMDR|acc:X14298.1 mRNA from Homo sapiens (Human mRNA for MAJOR DUCHENNE MUSCULAR DYSTROPHY PROTEIN (DP71)). The NOVl 9a polypeptide sequence of the invention was found to have 620 of 635 amino acid residues (97%) identical to, and 620 of 635 amino acid residues (97%) similar to, the 622 amino acid residue ptnr:SPTREMBL-ACC:Q02295 protein from Homo sapiens (MAJOR DUCHENNE MUSCULAR DYSTROPHY PROTEIN (DP71)).
Similarly, it was found, for example, that the NOV 19b nucleic acid sequence of this invention has 1793 of 1798 bases (99%) identical to a gb:GENBANK-ID:E30218|acc:E30218.1 mRNA from unidentified (Shortened dystrophin). The NOVl 9b polypeptide sequence of the invention also was found to have 620 of 635 amino acid residues (97%) identical to, and 620 of 635 amino acid residues (97%) similar to, the 622 amino acid residue ptnπSPTREMBL- ACC:Q02295 protein from Homo sapiens (MAJOR DUCHENNE MUSCULAR DYSTROPHY PROTEIN (DP71)). Additional BLAST results are shown in Table 19F.
Figure imgf000177_0002
A multiple sequence alignment is given in Table 19G, with the NOVl 9 protein of the invention being shown on line 1, in a ClustalW analysis comparing NOVl 9 with related protein sequences disclosed in Table 19F.
Table 19G. Information for the ClustalW proteins:
1. >NOV19a; SEQ ID NO:65
2. >NOV19b; SEQ ID NO:67
3. >Q02295/DMD PROTEIN [Homo sapiens]; SEQ ID NO:239
4. >P11532/ Dystrophin [Homo sapiens]; SEQ ID NO:240
5. >Q14205/ Dystrophin [Homo sapiens]; SEQ DD NO:241
2810 2820 2830 2840 2850
NOV19a -tøASRJJJT- - - - pggsggS gs PAR- NOVl9b Q02295 P11532 EASSDQ KRLgLSLQELLV LQLKDDELSRQ^I^DFgAVQKQNDVHRA Q14205 EASSDQHκRLfflLS QELLV LQLKDDELSRQ^lfflDFBAVQKQNDVHRA
2860 2870 2880 2890 2900
NOVl9a NOVl9b Q02295 P11532 FKRELKTKEPVIMSTLETVRIFLTEQPLEGLEKLYQE^ELPPEERAQNV Q14205 FKRELKTKEPVIMSTLETVRIFLTEQPLEGLEKLYQEfflELPPEERAQNV
Figure imgf000177_0001
Q02295 P11532 Tg SKQAEEWTEWEKLNLHSADWQRKIDETLEGLQELQGATDELDLKL Q14205 Tg SKQAEEVNTEWEKLNLHSADWQRKIDETLEBLQELQHATDELDLKL
29G0 2970 2980 2990 3000
NOVl 9 a -@KLTJjJP JJVPTJJD- -jjgJAL] MREQLK NOVl 9b MREQLK
Figure imgf000178_0001
3010 3020 3030 3040 3050
NOVl9 NOVl9b Q02295 P11532 QLTTLGIQLSPYNLSTLEDLNTRWKLLQVAVEDRVRQLHEAHRDFGPASQ Q14205 QLTTLGIQLSPYNLSTLEDLNTRWKLLQVAVEDRVRQLHEAHRDFGPASQ
3060 3070 3080 3090 3100
NOVl9a HETQTTCWDHPKMTELYQSLADLNN NOVl9b HETQTTCWDHPKMTELYQSLADLNN Q02295 HETQTTCWDHPKMTELYQSLADLNN P11532 HFLSTSVQGPWERAISPNKVPYYI HETQTTCWDHPKMTELYQSLADLNN Q14205 HFLSTSVQGPWERAISPNVPYYI HETQTTCWDHPKMTELYQSLADLNN
3110 3120 3130 3140 3150
NOV19a VRFSAYRTAMKLRRLQKALC|Ϊ|DLLSLSAACDALDQHNLKQNDQPMD I Q I NOV19b VRFSAYRTAMKLRRLQKALCLDLLSLSAACDALDQHNLKQNDQPMDILQI Q02295 \7RFSAYRTAMKLRRLQKALCLDLLSLSAACDALDQHNLKQNDQPMDILQI P11532 VRFSAYRTAMKLRRLQKALCLDLLSLSAACDALDQHNLKQNDQPMDILQI Q14205 VRFSAYRTAMKLRRLQKALCLDLLSLSAACDALDQHNLKQNDQPMDILQI
3160 3170 3180 3190 3200
:NCLTTIYDRLEQEHNNLVNVPLCVDMCLNWLLNVYDTGRTGRIRVLSFI INCLTTIYDRLEQEHNNLVNVPLCVDMCLNLLNVYDTGRTGRIRVLSFI NCLTTIYDRLEQEHNNLVNVPLCVDMCLNWLLNVYDTGRTGRIRVLSFI INCLTTIYDRLEQEHNNLVNVPLCVDMCLNWLLNVYDTGRTGRIRVLSFK :NCLTTIYDRLEQEHNNLVNVPLCVDMCLNWLLNVYDTGRTGRIRVLSFI
3210 3220 3230 3240 3250 ..I
NOVl9a ΓGIISLCKAHLEDKYRJSJLFKQVASSTGFCDQRRLGLLLHDSIQIPRQLGE NOVl9b ΓGIISLCKAHLEDKYRYLFKQVASSTGFCDQRRLGLLLHDSIQIPRQLGE Q02295 ΓGIISLCKAHLEDKYRYLFKQVASSTGFCDQRRLGLLLHDSIQIPRQLGE P11532 ΓGIISLCKAHLEDKYRYLFKQVASSTGFCDQRRLGLLLHDSIQIPRQLGE Q14205 ΓGIISLCKAHLEDKYRYLFKQVASSTGFCDQRRLGLLLHDSIQIPRQLGE
3260 3270 3280 3290 3300 ..I....I ....I...
NOVl9a VASFGGSNIEPSVRSCFQFANNKPEIEAALFLDWMRLEPQSMVWLPVLHR NOVl9b VASFGGSNIEPSVRSCFQFANNKPEIEAALFLDWMRLEPQSMVWLPVLHR Q02295 VASFGGSNIEPSVRSCFQFANNKPEIEAALFLDWMRLEPQSMVWLPVLHR P11532 VASFGGSNIEPSVRSCFQFANNKPEIEAALFLDWMRLEPQSMVWLPVLHR Q14205 VASFGGSNIEPSVRSCFQFANNKPEIEAALFLDWMRLEPQSMVWLPVLHR
3310 3320 3330 3340 3350
NOVl9a VAAAETAKHQAKCNICKECPIIGFRYRSLKHFNYDICQSCFFSGRVAKGH NOVl9b AAAETAKHQAKCNICKECPIIGFRYRSLKHFNYDICQSCFFSGRVAKGH Q02295 VAAAETAKHQAKCNICKECPIIGFRYRSLKHFNYDICQSCFFSGRVAKGH P11532 VAAAETAKHQAKCNICKECPIIGFRYRSLKHFNYDICQSCFFSGRVAKGH Q14205 VAAAETAKHQAKCNICKECPIIGFRYRSLKHFNYDICQSCFFSGRVAKGH
3360 3370 3380 3390 3400
..I
NOVl9a KMHYPMVEYCTPTTSGEDVRDFAKVLKNKFRTKRYFAKHPRMGYLPVQTV NOVl 9b liiBli Q02295 ^IHYPMVEYCTPTTSGEDVRDFAKVLKNKFRTKRYFAKHPRMGYLPVQT^ P11532 YIHYPMVEYCTPTTSGEDVRDFAKVLKNKFRTKRYFAKHPRMGYLPVQT-' Q14205 MHYPMVEYCTPTTSGEDVRDFAKVLKNKFRTKRYFAKHPRMGYLPVOT'*
3410 3420 3430 3440 3450
N0V19a JEGDNMETPVTLINFWPVDSAPASSPQLSHDDTHSRIEHYASRLAEMEN! NOVl9b JEGDNMETPVTLINFWPVDSAPASSPQLSHDDTHSRIEHYASRLAEMENS Q02295 iEGDNMEτBHBB--B-βPASSpQLSHDDTHSRIEHYASRLAEMENS P11532 ILEGDNMETPVTLINFWPVDSAPASSPQLSHDDTHSRIEHYASRLAEMENS| Q14205 ILEGDNMETPVTLINFWPVDSAPASSPQLSHDDTHSRIEHYASRLAEMENSI
3460 3470 3480 3490 3500
NOVl9a NOVl9b IGSYLNDSISPNESIDDEHLLIQHYCQSLNQDSPLSQPRSPAQILISLE! Q02295 ΓGSYLNDSISPNESIDDEHLLIQHYCQSLNQDSPLSQPRSPAQILISLE! P11532 ΓGSYLNDSISPNESIDDEHLLIQHYCQSLNQDSPLSQPRSPAQILISLE; Q14205 SYLNDSISPNESIDDEHLLIOHYCOSLNODSPLSOPRSPAOILISLE!
3510 3520 3530 3540 3550
NOVl9a EERGELERILADLEEENRNLQAEYDRLKQQHEHKGLSPLPSPPEMMPTSP NOVl9b EERGELERILADLEEENRNLQAEYDRLKQQHEHKGLSPLPSPPEMMPTSP Q02295 EERGELERILADLEEENRNLQAEYDRLKQQHEHKGLSPLPSPPEMMPTSP P11532 EERGELERILADLEEENRNLQAEYDRLKQQHEHKGLSPLPSPPEMMPTSP Q14205 EERGELERILADLEEENRNLQAEYDRLKQQHEHKGLSPLPSPPEMMPTSP
3560 3570 3580 3590 3600
I ..I...
NOVl9a QSPRDAELIAEAKLLRQHKGRLEARMQILEDHNKQLESQLHRLRQLLEQI NOVl9b QSPRDAELIAEAKLLRQHKGRLEARMQILEDHNKQLESQLHRLRQLLEQI Q02295 SPRDAELIAEAKLLRQHKGRLEARMQILEDHNKQLESQLHRLRQLLEQI P11532 QSPRDAELIAEAKLLRQHKGRLEARMQILEDHNKQLESQLHRLRQLLEQI Q14205 QSPRDAELIAEAKLLRQHKGRLEARMQILEDHNKQLESQLHRLRQLLEQI
3610 3620 3630 3640 3650
..I....I ..1...
NOV19a AEAKV GTTVSSPSTSLQRSDSSQPMLLRVVGSQTSDSMGEEDLLSPPς NOV19b 2AEAKVNG^TVSSPSTSLQRSDSSQPMLLRWGSQTSDSMGEEDLLSPPς Q02295 )AEAKVNGTTVSSPSTSLQRSDSSQPMLLRWGSQTSDSMGEEDLLSPPC P11532 JAEAKVNGTTVSSPSTSLQRSDSSQPMLLRWGSQTSDSMGEEDLLSPPς Q14205 AEAKVNGTTVSSPSTSLQRSDSSQPMLLRWGSQTSDSMGEEDLLSPPC
3660 3670 3680 3690 3700 . . } . . . .1 ..I... 1
NOVl9a DTSTGLEEVMEQLNNSFPSSRGHNVGSLFHMADDLGRAMESLVSVMTDEE NOVl9b DTSTGLEEVMEQLNNSFPSSRGHNVGSLFHMADDLGRAMESLVSVMTDEE Q02295 DTSTGLEEVMEQLNNSFPSSRGHNVGSLFHMADDLGRAMESLVSVMTDEEI P11532 DTSTGLEEVMEQLNNSFPSSRGEN "" --iKPlR--EDT Q14205 DTSTGLEEVMEQLNNSFPSSRGSN --HKPHR--EDTB
Figure imgf000179_0001
Q14205 The NOVl 9 Clustal W alignment shown in Table 19G was modified to begin at amino residue 2801. The data in Table 19G includes all of the regions overlapping with the NOV19 protein sequences.
The presence of identifiable domains in the protein disclosed herein was determined by searches using algorithms such as PROSITE, Blocks, Pfam, ProDomain, Prints and then determining the Inteφro number by crossing the domain match (or numbers) using the Interpro website (http:www.ebi.ac.uk/interpro/). Table 19H lists the domain description from DOMAIN analysis results against NOVl 9.
Figure imgf000180_0001
Consistent with other known members of the DP71 family of proteins, NOVl 9 contains a zinc finger ZZ domain as illustrated in Table 19H. NOVl 9 nucleic acids, and the encoded polypeptides, according to the invention are useful in a variety of applications and contexts. For example, NOVl 9 nucleic acids and polypeptides can be used to identify proteins that are members of the DP71 family of proteins. The NOV19 nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance NOVl 9 activity or function. Specifically, the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., cellular activation, cellular metabolism and signal transduction. These molecules can be used to treat, e.g., deafness 4, congenital sensorineural, Duchenne muscular dystrophy, Becker muscular dystroph, cardiomyopathy, dilated, X-linked, McLeod phenotype, Lesch-Nyhan syndrome, myasthenia gravis, Adrenal hypoplasia, congenital, with hypogonadotropic hypogonadism, Dosage-sensitive sex reversal, Glycerol kinase deficiency; Gonadal dysgenesis, XY female type, Hyperglycerolemia, diabetes, obesity, and Retinitis pigmentosa-6.
In addition, various NOVl 9 nucleic acids and polypeptides according to the invention are useful, ter alia, as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins. For example, the NOVl 9 nucleic acids and their encoded polypeptides include structural motifs that are characteristic of proteins belonging to the family of DP71 such as the Major Duchenne Muscular Dystrophy proteins involved in skeletal muscle and nerve physiology.
Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder which manifest as a progressive degeneration of muscles and results in death. A less severe disorder, Becker's muscular dystrophy (BMD), is allelic to DMD. Some 30% of DMD patients also suffer also from mental retardation. The DMD gene is the largest known gene, consisting of almost 0.1% of the human genome (2,500 Kbp). The product of the DMD gene in the muscle, dystrophin, is a 427 kDa protein translated from a 14 kb mRNA. Dystrophin is a rod-shaped protein consisting of an actin binding N-terminal domain, a large domain of spectrin-like repeats, a cystein-rich domain with potential Ca2+ binding sites, and a C-terminal domain. A very similar isoform of dystrophin, encoded by the same gene, is found in the brain. The expression of the two isoforms is regulated by two promoters. One is active in muscle cells and glia cells. The other is active mainly in neurons. A 70.8 kDa protein, called Dp71, is the product of a promoter located between exons 62 and 63 of the DMD gene.
Dp71 is of special interest as it consists of the cysteine-rich and C-terminal domains of dystrophin, but lacks the actin binding domain and the spectrin-like repeats. Dp71 is by far the major product of the DMD gene in brain and many other nonmuscle tissues. Analysis of the expression of the DMD gene products during development has shown that Dp71 is already expressed in the embryonic stem cells. The known dystrophins and their mRNAs are detected only after differentiation of specialized cell types.
The NOV19 nucleic acids and polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in the mediation of muscle and nerve physiology. As such, the NOVl 9 nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat muscle and nervous system disorders, e.g., Duchenne muscular dystrophy, Becker muscular dystroph, cardiomyopathy, dilated, X-linked, McLeod phenotype, Lesch-Nyhan syndrome, myasthenia gravis.
The NOV19 nucleic acids and polypeptides are useful for detecting specific cell types. For example, expression analysis has demonstrated that a NOVl 9 nucleic acid is expressed in
Adipose, Aorta, Vein, Umbilical Vein, Adrenal Gland/Suprarenal gland, Pancreas, Thyroid, Parotid Salivary glands, Stomach, Liver, Colon, Bone Marrow, Peripheral Blood, Spleen, Lymph node, Tonsils, Bone, Cartilage, Muscle, Skeletal Muscle, Brain, Cerebellum, Left cerebellum, Thalamus, Pituitary Gland, Temporal Lobe, Amygdala, Substantia Nigra, Hippocampus, Spinal Chord, Cervix, Mammary gland/Breast, Ovary, Placenta, Uterus, Oviduct/Uterine Tube/Fallopian tube, Prostate, Testis, Lung, Kidney, Retina, Cochlea, and Foreskin.
Additional utilities for NOVl 9 nucleic acids and polypeptides according to the invention are disclosed herein.
NOV20 A NOV20 polypeptide has been identified as a G Protein-Coupled Receptor RTA
(GPCR)-like protein. The novel NO V20 nucleic acid sequences maps to the chromosomell. Two alternative novel NOV20, NOV20a and NOV20b, nucleic acids and encoded polypeptides are provided.
NOV20a A NOV20 variant is the novel NOV20a (alternatively referred to herein as CG56517-01), which includes the 1219 nucleotide sequence (SEQ ID NO: 68) shown in Table 20 A. A NOV20a ORF begins with a Kozak onsensus sequence ATG initiation codon at nucleotides 31-33 and ends with a TGA codon at nucleotides 1051-1053. Putative untranslated regions upstream from the initiation codon and downstream from the termination codon are underlined in Table 20A, and the start and stop codons are in bold letters.
Table 20A. NOV20a Nucleotide Sequence (SEQ ID NO:68)
AGCAGGGGCCCAGACGCGCCAGGCCTGGAGATGGCTGGAAACTGCTCCTGGGAGGCCCATCCCGGCA ACAGGAACAAGATGTGCCCTGGCCTGAGCGAGGCCCCGGAACTCTACAGCCGGGGCTTCCTGACCAT CGAGCAGATCGCGATGCTGCCGCCTCCGGCCGTCATGAACTACATCTTCCTGCTCCTCTGCCTGTGT GGCCTGGTGGGCAACGGGCTGGTCCTCTGGTTTTTCGGCTTCTCCATCAAGAGGAACCCCTTCTCCA TCTACTTCCTGCACCTGGCCAGCGCCGATGTGGGCTACCTCTTCAGCAAGGCGGTGTTCTCCATCCT GAACACGGGGGGCTTCCTGGGCACGTTTGCCGACTACATCCGCAGCGTGTGCCGGGTCCTGGGGCTC TGCATGTTCCTTACCGGCGTGAGCCTCCTGCCGGCCGTCAGCGCGTCAGCGTGCGCCTCGGTCATCT TCCCCGCCTGGTACTGGCGCCGGCGGCCCAAGCGCCTGTCGGCCGTGGTGTGCGCCCTGCTGTGGGT CCTGTCCCTCCTGGTCACCTGCCTGCACAACTACTTCTGCGTGTTCCTGGGCCGCGGGGCCCCGGGC GCGTGCTGCAGGCACATGGACATCTTCCTGGGCATCCTCCTGTTCCTGCTCTGCTGCCCGCTCATGG TGCTGCCCTGCCTGGCCCTCATCCTGCACGTGGAGTGCGGGCCCGACGGGCCACGCTCTGCCAAGCT CAAGCACGTCATCCTGGCCATGGTCTCCGTCTTCCTGGTGTCCTCCATCTACTTAGGGATCGACTGG TTCCTCTTCTGGGTCTTCCAGATCCCGGCCCCCTTCCCCGAGTACGTCACTGACCTGTGCATCTGCA TCAACAGCAGCGCCAAGCCCATCGTCTACTTCCTGGCCGGGAGGACAAGTCGCAGCGGCTGTTGGAG CCTTAGGGTGGTCTTCAGTGGGGCCTGCGGGACGGCGCTGACTGGGGGATGTCGGGGCAGCACGCTC AACACAGTCACCATGGAGATGCAGTGTCCCCCGGGGAACGCCTCCTGAGACTGCAGCGCCTGGAGGA GGCAGTGGCAGGAATCGTGCTCCAAGACTCTTCTGCTGTGGACAGGAATGGGCACTAGTTCTGAGTC CATACAGGAGAGGAAAGATCTGTATGCTCTCCTCGGGCCTTCTTCTCCCTGGGACTGTGGAACTCAG GTAGTGTCTGGGC
The NOV20a polypeptide (SEQ ID NO:69) encoded by SEQ ID NO:68 is 340 amino acid residues in length and is presented using the one-letter amino acid code in Table 20B. The Psort profile for the NOV20 a and NOV20b proteins predicts that this peptides are likely to be localized at the plasma membrane with a certainty of 0.6000. The Signal P predicts a likely cleavage site for a NOV20 peptide is between positions 67 and 68, i.e., at the dash in the sequenceVLW-FF.
Table 20B. NOV20a protein sequence (SEQ ID NO:69)
MAGNCSWEAHPGNRNKMCPGLSEAPELYSRGFLTIEQIAMLPPPAVMNYIFLLLCLCGLVGNGLVLW FFGFSIKRNPFSIYFLHLASADVGYLFSKAVFSILNTGGFLGTFADYIRSVCRVLGLCMFLTGVSLL PAVSASACASVIFPA YWRRRPKRLSAWCALLWVLSLLVTCLHNYFCVFLGRGAPGACCRHMDIFL GILLFLLCCPLMVLPCLALILHVECGPDGPRSAKLKHVILAMVSVFLVSSIYLGIDWFLFWVFQIPA PFPEYVTDLCICINSSAKPIVYFLAGRTSRSGCWSLRWFSGACGTALTGGCRGSTLNTVTMEMQCP
PGNAS NON20b
Alternatively, a ΝON20 variant is the novel ΝON20b (alternatively referred to herein as CG56517-02), which includes the 1113 nucleotide sequence (SEQ ID ΝO:70) shown in Table 20C. NON20b was created by polymerase chain reaction (PCR) using the primers: 5'- ATCAGGACAGCTGCAGGTGGGT-3' (SEQ ID ΝO:242) and 5'- TCTCCTGTATGGACTCAGAAGAAGGTG-3' (SEQ ID NO:243). Primers were designed based on in silico predictions of the full length or some portion (one or more exons) of the cDNA/protein sequence of the invention. The PCR product derived by exon linking, covering the entire open reading frame, was cloned into the pCR2.1 vector from Invitrogen to provide clone CG56517-01.698754. Al 3. The NOV20b ORF begins with a Kozak consensus ATG initiation codon at nucleotides
73-75 and ends with a TGA codon at nucleotides 1102-1104. Putative untranslated regions upstream from the initiation codon and downstream from the termination codon are underlined in Table 20C, and the start and stop codons are in bold letters. Table 20C. NOV20b Nucleotide Sequence (SEQ ID NO:70)
ATCAGGACAGCTGCAGGTGGGTGTGCAGACTGGTGAGCTGCCAGCAGGGGCCCAGACGCGCCAGGCC TGGAGATGGCTGGAAACTGCTCCTGGGAGGCCCATCCCGGCAACAGGAACAAGATGTGCCCTGGCCT GAGCGAGGCCCCGGAACTCTACAGCCGGGGCTTCCTGACCATCGAGCAGATCGCGATGCTGCCGCCT CCGGCCGTCATGAACTACATCTTCCTGCTCCTCTGCCTGTGTGGCCTGGTGGGCAACGGGCTGGTCC TCTGGTTTTTCGGCTTCTCCATCAAGAGGAACCCCTTCTCCATCTACTTCCTGCACCTGGCCAGCGC CGATGTGGGCTACCTCTTCAGCAAGGCGGTGTTCTCCATCCTGAACACGGGGGGCTTCCTGGGCACG TTTGCCGACTACATCCGCAGCGTGTGCCGGGTCCTGGGGCTCTGCATGTTCCTTACCGGCGTGAGCC TCCTGCCGGCCGTCAGCGCCGAGCGCTGCGCCTCGGTCATCTTCCCCGCCTGGTACTGGCGCCGGCG GCCCAAGCGCCTGTCGGCCGTGGTGTGCGCCCTGCTGTGGGTCCTGTCCCTCCTGGTCACCTGCCTG CACAACTACTTCTGCGTGTTCCCGGGCCGCGGGGCCCCCGGCGCGGCCTGCAGGCACATGGACATCT TCCTGGGCATCCTCCTGTTCCTGCTCTGCTGCCCGCTCATGGTGCTGCCCTGCCTGGCCCTCATCCT GCACGTGGAGTGCCGGGCCCGACGGCGCCAGCGCTCTGCCAAGCTCAACCACGTCATCCTGGCCATG GTCTCCGTCTTCCTGGTGTCCTCCATCTACTTAGGGATCGACTGGTTCCTCTTCTGGGTCTTCCAGA TCCCGGCCCCCTTCCCCGAGTACGTCACTGACCTGTGCATCTGCATCAACAGCAGCGCCAAGCCCAT CGTCTACTTCCTGGCCGGGAGGGACAAGTCGCAGCGGCTGTGGGAGCCGCTCAGGGTGGTCTTCCAG CGGGCCCTGCGGGACGGCGCTGAGCTGGGGGAGGCCGGGGGCAGCACGCCCAACACAGTCACCATGG AGATGCAGTGTCCCCCGGGGAACGCCTCCTGAGACTCCAGC
The NOV20b protein (SEQ ID NO:71) encoded by SEQ ID NO:70 is 343 amino acid residues in length and is presented using the one-letter code in Table 20D.
Table 20D. NOV20b protein sequence (SEQ ID NO:71)
MAGNCSWEAHPGNRNKMCPGLSEAPELYSRGFLTIEQIAMLPPPAVMNYIFL LCLCGLVG NGLVL FFGFSIKRNPFSIYFLHLASADVGY FSKAVFSILNTGGFLGTFADYIRSVCRVL GLCMFLTGVSLLPAVSAERCASVIFPA Y RRRPKRLSAWCALLWVLSLLVTCLHNYFCV FPGRGAPGAACRHMDIFLGI LFLLCCPLMVLPCLALILHVECRARRRQRSAKLNHVILAM VSVFLVSSIYLGID FLF VFQIPAPFPEYVTDLCICINSSAKPIVYFLAGRDKSQRL EP LRWFQRALRDGAELGEAGGSTPNTVT E QCPPGNAS .
NOV20 Clones
Unless specifically addressed as NON20a or ΝON20b, any reference to ΝON20 is assumed to encompass all variants. ΝON20b has four frame-shifts at position 762, 959, 986, and 1042 bp, respectively, when compared with ΝON20a. These frame-shifts result in numerous amino acid differences between ΝON20a and ΝON20b.
A search against the Patp database, a proprietary database that contains sequences published in patents and patent publications, yielded several homologous proteins shown in Table 20E and Table 20F. Table 20E. Patp results for NON20a
Smallest Sum
Reading High Prob
Sequences producing High-scoring Segment Pairs: Frame Score P(Ν)
>patp:AAB88477 Human membrane clone PSEC0142 +1 1625 7.9e-167
>patp:AAR97222 Human G-protein coupled receptor +1 1591 3.2e-163
>patp:AAR96145 G protein coupled receptor protein +1 1404 2.1Θ-143
Table 20F. Patp results for ΝON20b
Smallest Sum
Reading High Prob
Sequences producing High-scoring Segment Pairs: Frame Score P(N)
>patp:AAB88477 Human membrane clone PSEC0142 +1 1826 3.9e-188
>patp:AAR97222 Human G-protein coupled receptor +1 1792 1.6e-184
>patp:AAR961 5 G protein coupled receptor protein +1 1589 5.1e-163
h a BLAST search of public sequence databases, it was found, for example, that the NON20a nucleic acid sequence of this invention has 840 of 1032 bases (81%) identical to a gb:GEΝBAΝK-ID:RATRTA|acc:M35297.1 mRNA from Rattus norvegicus probableG protein- coupled receptor (RTA) mRNA, complete eds. NON20a protein of the invention was found to have 265 of 343 amino acid residues (77%) identical to, and 280 of 343 amino acid residues (81%) similar to, the 343 amino acid residue ptnr:SWISSPROT-ACC:P23749 protein from probable Rattus norvegicus G protein-coupled receptor RTA.
Similarly, it was found, for example, that the ΝON 20b nucleic acid sequence of this invention has 903 of 1086 bases (83%) identical to a gb:GEΝBAΝK-ID:RATRTA|acc:M35297.1 mRNA from Rattus norvegicus G protein- coupled receptor (RTA) mRNA, complete eds. NON20b protein of the invention was found to have 291 of 343 amino acid residues (84%) identical to, and 307 of 343 amino acid residues (89%) similar to, the 343 amino acid residue ptnr:SWISSPROT-ACC:P23749 protein from Rattus norvegicus probable G protein-coupled receptor RTA.
Additional BLAST results are shown in Table 20G.
Figure imgf000186_0001
A multiple sequence alignment is given in Table 201, with the NOV20 protein of the invention being shown on line 1, in a ClustalW analysis comparing NOV20 with related protein sequences disclosed in Table 20G and Table 20H.
Table 201. Information for the ClustalW proteins:
1. >NOV20a; SEQ ID NO:69
2. >NOV20b; SEQ ID NO:71
3. >Q96AM1/ Hypothetical Protein 38.2 [Homo sapiens]; SEQ ID NO-.244
4. >CAC39840/ Sequence 321 from Patient EP1067182 [Homo sapiens]; SEQ ID NO:245
5. >P23749/ Probable G protein-coupled receptor RTA [Homo sapiens]; SEQ D3 NO:246
6. >Q91ZB6/ G protein-coupled receptor [Mus musculus]; SEQ DD NO:247
10 20 30 40 50
I 1 . 1 . . . 1 . . 1 . 1 . . ] . 1
NOV20a MAGNCSWEAHPGNRNKMCPGLSEAPELYSRGFLTIEQIAMLPPPAVMNYI NOV20b MAGNCSWEAHPGNRNKMCPGLSEAPELYSRGFLTIEQIAMLPPPAVMNYI
Figure imgf000187_0001
60 70 80 90 100 .1.. .1..
NOV20a FLLLCLCGLVGNGLVLWFFGFSIKRNPFSIYFLHLASADVGYLFSKAVF!
NOV20b FLLLCLCGLVGNGLVLWFFGFSIKRNPFSIYFLHLASADVGYLFSKAVF;
Q96AM1 FLLLCLCGLVGNGLVLWFFGFSIKRNPFSIYFLHLASADVGYLFSKAVF!
CAC39840 FLLLCLCGLVGNGLVLWFFGFSIKRNPFSIYFLHLASADVGYLFSKAVFS
P23749 JCGLVC 4k«ιvtj»i'?i3a«as««:«rjτιaciϊ*»'<a-ι:ι-»i«^»JGiM«>aB»Wij
Q91ZB6 PFSIYFLHLASAD
110 120 130 140 150
NOV20a :LNTGGFLGTFADYIRSVCRV GLCMFLTGVSLLPAVSA|22CASVIFPA^
NOV20b :LNTGGFLGTFADYIRSVCRVLGLCMFLTGVSLLPAVSAERCASVIFPAW
Q96AM1 :LNTGGFLGTFADYIRSVCRVLGLCMFLTGVSLLPAVSAERCASVIFPAW
CAC39840 :LNTGGFLGTFADYIRSVCRVLGLCMFLTGVSLLPAVSAERCASVIFPAW
P23749 m
Q91ZB6
160 170 180 190 200
NOV20a YWRRRPKRLSAWCALL VLSLLVTCLHNYFCVFLGRGAPGAgjCRHMDII
NOV20b RRRPKRLSAWCALL VLSLLVTCLHNYFCVFgGRGAPGAACRHMDIF
Q96AM1 Y RRRPKRLSAWCALL VLSLLVTCLHNYFCVFLGRGAPGAACRHMDIF
CAC39840 Y RRRPKRLSAWCALL VLSLLVTCLHNYFCVFLGRGAPGAACRHMDII
P23749 IHEBSB ΪLNl
Q91ZB6 lESaB
Figure imgf000187_0002
260 270 280 290 300
NOV20a NOV20b iSIYLGID FLFWVFQIPAPFPEYVTDLCICINSSAKPIVYFLAGRDKSQ Q96AM1 SSIYLGID FLF VFQIPAPFPEYVTDLCICINSSAKPIVYFLAGRDKSQ CAC39840 iSIYLGID FLF VFQIPAPFPEYVTDLCICINSSAKPIVYFLAGRDKSQ P23749 iSIYLGIDWFLFWVFQIPAPFPEYVTDLCICINSSAKPIVYFLAGRDKSQ Q91ZB6 !SIYLGID FLF VFQIPAPFPEYVTDLCICINSSAKPIVYFLAGRDKSQ
Figure imgf000187_0003
The presence of identifiable domains in the protein disclosed herein was determined by searches using algorithms such as PROSITE, Blocks, Pfam, ProDomain, Prints and then determining the Interpro number by crossing the domain match (or numbers) using the Interpro website (http:www.ebi.ac.uk/interpro/). The DOMAIN analysis results indicate that the NOV20 protein contains the following protein domain (as defined by Interpro): domain name 7tm_l 7 transmembrane receptor (rhodopsin family). DOMAIN results for NOV20 were collected from the Conserved Domain Database (CDD) with Reverse Position Specific BLAST. This BLAST samples domains found in the Smart and Pfam collections.
As discussed below, the NOV20 protein of the invention contained significant homology to the 7tm_l domain. This indicates that the NON20 sequence has properties similar to those of other proteins known to contain this 7tm_l domain and similar to the properties of these domains. The 254 amino acid domain termed 7tm_l (SEQ ID ΝO:248; Pfam Ace. No. 00001) a seven transmembrane receptor (rhodopsin family), is shown in Table 20J.
Table 20J. 7tm_l, 7 transmembrane receptor domain (SEQ ID NO:248)
GNLLVILVILRTKKLRTPTNIFLLNLAVADLLFLLTLPP ALYYLVGGDWVFGDALCKLVGALFWNGYASILLLTAISIDRYL AIVHPLRYRRIRTPRRAKVLILLVWVLALLLSLPPLLFS LRTVEEGNTTVCLIDFPEESVKRSYVLLSTLVGFVLPLLVILVC YTRI RTLRKRARSQRSLKRRSSSERKAAKMLLWWVFVLC LPYHIVLLLDSLCLLSIWRVLPTALLITL LAYVNSCLNPI IY
The DOMAIN results are listed in Table 20K and Table 20L with the statistics and domain description. An alignment of NON20a residues 61-290 (SEQ ID ΝO:68) with the full 7tm_l domain, residues 1-254 (SEQ ID NO:248), are shown in Table 20K. A similar alignment of NOV20b residues 61-290 (SEQ ID NO:70), are shown in Table 20L. This indicates that the NON20 sequences have properties similar to those of other proteins known to contain this domain as well as to the 254 amino acid 7tm domain (SEQ ID ΝO:248). For Table 20K and Table 20L, fully conserved single residues are indicated by the vertical line and "strong" semi-conserved residues are indicated by the "plus sign." The "strong" group of conserved amino acid residues may be any one of the following groups of amino acids: STA, NEQK, NHQK, NDEQ, QHRK,
MΓLV, MΓLF, HY, FYW.
Table 20K Domain Analysis of NOV20a PSSMs producing significant alignments: Score E (bits) value gnl|Pfam|pfam00001 7tm_l, 7 transmembrane receptor (rhodopsin family) 72.2 4.9e-22
*->GNlLVilvilrtkklrtptnifilNLAvADLLflltlppwalyylvg
II+II++ +++ I+I++I++1+II II +l++ ++++++ I
NOV20a 61 GNGLVL FFGF-SIKRNPFSIYFLHLASADVGYLFSKAVFSILNTGG 106 gsedWpfGsalCklvtaldwn yaSillLtalSiDRYlAIvhPlryrrr + I ++ + I +++ + +++ I I I I + +++ I + 1 + 1 I
NOV20a 107 - -FLGTFADYIRSVCRVLGLCMFLTGVSLLPAVSASACASVIFPAWY RR 154 rtsprrAkwillvWvlalllslPpllf swvktveegngtlnvnvtvCli I + | + + | | ++ | I I I + | I ++ ++ + | + + | +
NOV20a 155 RP-KRLSAWCALL VLSLLVTCLHNYFCVFLGRGAP GACCRH 196 dfpeestasvstwlrsyvllstlvgFllPllvilvcYtrllrtlr
+ ++| +|++ I ++ |+ I I +M++ ++++++
NOV20a 197 M DIFLGILLFLLCCPLMVLPCLALILHVECgpdgp 231
... kaaktllvwwFvlC lPyfivllldtlc . IsiimsstCelervlp
++ |+ +++|+ I+II+++ + I ++ I +++++++ I
NOV20a 232 rsaKLKHVILAMVSVFLVSSIYLGID FLF VFqIP AP 269 tallvtlwLayvNsclNPiIY<-* (SEQ ID NO: 248)
++ +| I ++ ++| I I ]+ i
NOV20a 270 FPEYVTDLCICINSSAKPIVY 290 (SEQ ID NO: 69)
Table 20L Domain Analysis of NOV20b
PSSMs producing significant alignments: Score E (bits) value gnl|Pfam|pfam00001 7tm_l, 7 transmembrane receptor (rhodopsin family) 102.2 1.7e-31
*->GNlLVilvilrtkklrtptnifilNLAvADLLflltlppwalyylvg I I + 1 I ++ +++ I + 1 ++ 1 ++ 1 + 1 I || + I ++ ++++++ I
NOV20b 61 GNGLVL FFGF-SIKRNPFSIYFLHLASADVGYLFSKAVFSILNTGG 106 gsed pfGsalCklvtaldwnmyaSillLtalSiDRYlAIvhPlryrrr
' + I ++ + I +++ + +++ | | I I + 1 + +++ I + 1 + 1 1
NOV2 Ob 107 - -FLGTFADYIRSVCRVLGLCMFLTGVSLLPAVSAERCASVIFPAWYWRR 154 rtsprrAkwillvWvlalllslPpllf swvktveegngtlnvnvtvCli I + | + + I I ++ ] I I I + 1 I ++ ++ + 1 + ++ I + + | +
NOV20b 155 RP-KRLSAWCALLWVLSLLVTCLHNYF- -CVFPGRGAP GAACRH 196 dfpeestasvstwlrsyvllstlvgFllPllvilvcYtrllrtlr
+ ++ I +|++ I ++ |+ I I +||++ ++ +++
NOV20b 197 M DIFLGILLFLLCCPLMVLPCLALILHVΞCrarrr 231
. kaaktllvwwFvlCWlPyfivllldtlc . IsiimsstCelervl +++ |+ +++ i + 1+11+++ + i ++ i +++++++
NOV20b 232 qrsaKLNHVILAMVSVFLVSSIYLGIDWFLFWVFqIP A 269 ptallvtlwLayvNsclNPilY<-* (SEQ ID NO: 248)
I++ +1 I ++ ++1 I I I+|
NOV20b 270 PFPEYVTDLCICINSSAKPIVY 291 (SEQ ID NO: 71)
Consistent with other known members of the GPCR family of proteins, NOV20 contains 7tm_l 7 transmembrane receptor (rhodopsin family) domain as illustrated in Table 20K and Table 20L, as well as homology and cellular localization, i.e. plasma membrane.
NOV20 nucleic acids, and the encoded polypeptides, according to the invention are useful in a variety of applications and contexts. For example, NOV20 nucleic acids and polypeptides can be used to identify proteins that are members of the GPCR family of proteins. The NOV20 nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance NON20 activity or function. Specifically, the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., cellular signal transduction. These molecules can be used to treat, e.g., cancer, immune disorders, and endocrine disorders. hi addition, various ΝON20 nucleic acids and polypeptides according to the invention are useful, inter alia, as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins. For example, the ΝON20 nucleic acids and their encoded polypeptides include 7tm_l 7 transmembrane receptor (rhodopsin family) domain and sequence homology that are characteristic of proteins belonging to the family of GPCR such as the G protein-coupled receptor (RTA). The GPCRl protein of the invention has a high homology to the 7tm_l domain (PFam Ace. No. pfamOOOOl). The 7tm_l domain is from the 7 transmembrane receptor family, which includes a number of different proteins, including, for example, serotonin receptors, dopamine receptors, histamine receptors, andrenergic receptors, cannabinoid receptors, angiotensin II receptors, chemokine receptors, opioid receptors, G-protein coupled receptor (GPCR) proteins, olfactory receptors (OR), and the like.
G-Protein Coupled Receptor proteins ("GPCRs") have been identified as a large family of G protein-coupled receptors in a number of species. These receptors share a seven transmembrane domain structure with many neurotransmitter and hormone receptors, and are likely to underlie the recognition and G-protein-mediated transduction of various signals. Human GPCR generally do not contain introns and belong to four different gene subfamilies, displaying great sequence variability. These genes are dominantly expressed in olfactory epithelium. See, e.g., Ben-Arie et al., Hum. Mol. Genet. 3:229-235 (1994); and, Online Mendelian Inheritance in Man ("OMIM") entry # 164342 (http://www.ncbi.nlm.nih.gov/ entrez/ dispomim.cgi?).
The NON20 nucleic acids and polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in the mediation of cellular signal transduction. As such the ΝON20 nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat a wide range of disorders such as cancer, immune disorders, endocrine disorders and other diseases, e.g., developmental diseases; MHCπ and III diseases (immune diseases); taste and scent detectability disorders; Burkitt's lymphoma; corticoneurogenic disease; signal transduction pathway disorders; metabolic pathway disorders; retinal diseases including those involving photoreception; cell growth rate disorders; cell shape disorders; metabolic disorders; feeding disorders; control of feeding; the metabolic syndrome X; wasting disorders associated with chronic diseases; obesity; potential obesity due to over-eating or metabolic disturbances; potential disorders due to starvation (lack of appetite); diabetes; noninsulin-dependent diabetes mellitus (ΝIDDM); infectious disease; bacterial, fungal, protozoal and viral infections (particularly infections caused by HIN-1 or HIN-2); pain; cancer (including but not limited to neoplasm; adenocarcinoma; lymphoma; prostate cancer; uterus cancer); cancer-associated cachexia; anorexia; bulimia; asthma; Parkinson's disease; acute heart failure; hypotension; hypertension; urinary retention; osteoporosis; Crohn's disease; multiple sclerosis; Albright Hereditary Ostoeodystrophy; angina pectoris; myocardial infarction; ulcers; allergies; benign prostatic hypertrophy; and psychotic and neurological disorders; including anxiety; schizophrenia; manic depression; delirium; dementia; neurodegenerative disorders; Alzheimer's disease; severe mental retardation; Dentatorubro-pallidoluysian atrophy (DRPLA); Hypophosphatemic rickets; autosomal dominant (2) Acrocallosal syndrome and dyskinesias, such as Huntington's disease or Gilles de la Tourette syndrome; immune disorders;
Adrenoleukodystrophy; Congenital Adrenal Hypeφlasia; Hemophilia; Hypercoagulation; Idiopathic thrombocytopenic purpura; autoimmume disease; immunodeficiencies; transplantation; Non Hippel-Lindau (NHL) syndrome; Stroke; Tuberous sclerosis; hypercalceimia; Cerebral palsy; Epilepsy; Lesch-Νyhan syndrome; Ataxia-telangiectasia; Leukodystrophies; Behavioral disorders; Addiction; Νeuroprotection; Cirrhosis; Transplantation; Systemic lupus erythematosus; Emphysema; Scleroderma; ARDS; Renal artery stenosis; Interstitial nephritis; Glomerulonephritis; Polycystic kidney disease; Systemic lupus erythematosus; Renal tubular acidosis; IgA nephropathy; Cardiomyopathy; Atherosclerosis; Congenital heart defects; Aortic stenosis ; Atrial septal defect (ASD); Atrioventricular (A-N) canal defect; Ductus arteriosus; Pulmonary stenosis ; Subaortic stenosis; Ventricular septal defect (NSD); valve diseases; Scleroderma; fertility; Pancreatitis; Endocrine dysfunctions; Growth and reproductive disorders; Inflammatory bowel disease; Diverticular disease; Leukodystrophies; Graft vesus host; Hyperthyroidism; Endometriosis; and hematopoietic disorders.
The ΝON20 nucleic acids and polypeptides are useful for detecting specific cell types. For example, expression analysis has demonstrated that a ΝON20 nucleic acid is expressed in Brain, Synovium/Synovial membrane.
Additional utilities for ΝOV20 nucleic acids and polypeptides according to the invention are disclosed herein.
NOV21 A NOV21 polypeptide has been identified as a TFIIIC box B-binding subunit-like protein
(also referred to as CG56500-01). The disclosed novel NOV21 nucleic acid (SEQ ΪD NO:72) of 6921 nucleotides is shown in Table 21A. The cDNA coding for the NON21 was cloned by polymerase chain reaction (PCR) using the following primers: 5'-CCATGGGCCGACCGGCTC- 3' (SEQ ID ΝO:249) and 5'-TGGCGGGCTTCCTCGTCATC-3' (SEQ ID NO:250) on the following pools of human cDNAs: Pool 1 - adrenal gland, bone marrow, brain - amygdala, brain - cerebellum, brain - hippocampus, brain - substantia nigra, brain - thalamus, brain -whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma - Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea, uterus. The novel NON21 nucleic acid sequences maps to the chromosomel6pl2.
An ORF begins with an ATG initiation codon at nucleotides 61-63 and ends with a TAG codon at nucleotides 6313-6315. Aputative untranslated region and/or downstream from the termination codon is underlined in Table 21 A, and the start and stop codons are in bold letters.
Table 21 A. ΝOV21 Nucleotide Sequence (SEQ ID NO:72)
ATGGACCAAGGCCCTTGGCGGTGCGTTGCGCACCCCCGGGGCGCCGCGACTGAAGTAGCAATGGACG CGCTGGAGTCGTTGTTGGACGAAGTCGCTCTGGAGGGGCTCGATGGCCTGTGTCTGCCAGCGCTGTG GAGCCGGCTGGAGACGCGAGTGCCGCCCTTCCCGCTGCCTTTGGAACCCTGCACGCAGGAGTTTCTC TGGCGGGCCCTCGCCACGCACCCGGGCATCAGCTTCTATGAGGAGCCTCGGGAGCGACCCGACCTAC AGCTCCAGGACCGGTATGAAGAAATTGATTTGGAAACTGGAATTTTGGAGTCTAGGAGGGACCCGGT GGCTTTGGAGGATGTCTACCCCATTCATATGATCTTAGAGAATAAGGATGGCATCCAGGGCTCATGC CGCTACTTTAAGGAGAGGAAAAACATTACCAATGACATCAGAACCAAGTCCTTGCAGCCTCGCTGTA CAATGGTGGAACCCTTTGACAGGTGGGGGAAGAAACTGATCATCGGTTCCCTCCCAGCCCATGCGGT ACAGGCCCTTGATAGCCCAGGAGGGGGATCCCGACCTGAAGCTGCCCGACTTCTCCTACTGCATCCT GGAACGGCTAGGCCGGTCCAGGTGCAAGGGGAGCTCCAGCGAGACCTTCACACCACTGCTTTCAAGG TTGATGCTGGGAAGCTGCACTATCACAGAAAAATTTTGAACAAAAACGGGCTGATTACAATGCAGTC CCATGTGATCCGATTACCCACTGGAGCCCAGCAACACTCAATCCTCCTCCTACTGAACCGGTTTCAT GTGGACAGGAGGAGCAAATACGACATCCTCATGGAGAAGCTTTCGGTCATGCTGAGCACACGGACTA ACCACATAGAGACGCTGGGAAAGCTGAGGGAAGAGCTGGGGCTGTGCGAAAGGACGTTTAAGCGTCT GTACCAGTATATGCTGAACGCCGGGCTAGCCAAGGTGGTGTCTCTTCGCTTGCAAGAGATCCACCCT GAATGTGGACCTTGTAAGACAAAGAAAGGGACCGACGTCATGGTTCGGTGCCTCAAGCTGCTGAAGG AATTTAAACGGAATGACCATGATGATGACGAGGACGAGGAGGTCATCTCCAAGACAGTGCCTCCAGT GGACATTGTGTTCGAGCGGGATATGCTCACACAGACCTACGACCTCATTGAGCGCAGAGGCACGAAA GGAATTTCCCAAGCTGAAATCCGAGTGGCTATGAATGTGGGAAAACTAGAAGCAAGAATGCTGTGCC GACTTCTTCAAAGATTCAAAGTTGTCAAGGGATTCATGGAAGACGAAGGTCGGCAGCGAACCACCAA GTACATTTCCTGCGTGTTTGCAGAGGAGAGCGACCTAAGCCGGCAGTACCAAAGAGAGAAGGCCCGC AGCGAGCTCTTGACCACCGTGAGCCTGGCGTCTATGCAGGAGGAGTCGCTTCTGCCTGAAGGCGAGG ACACCTTCCTCTCTGAGTCGGACAGTGAGGAGGAGAGGAGCAGCAGCAAGCGGAGAGGCAGAGGGTC CCAGAAAGACACAAGAGCCTCTGCAAACCTCCGGCCCAAGACCCAGCCTCATCACTCCACCCCAACC AAGGGTGGGTGGAAAGTTGTAAACCTACACCCATTGAAAAAGCAGCCGCCCTCCTTCCCAGGAGCTG CTGAAGAGAGAGCCTGCCAGAGCCTTGCCAGCAGGGACAGCCTCTTAGATACCAGCAGCGTCTCAGA ACCCAACGTGTCCTTTGTCTCCCACTGTGCGGACAGCAACAGTGGTGACATAGCTGTGATCGAGGAG GTCCGGATGGAAAACCCAAAGGAGAGTAGCAGTTCCCTGAAGACTGGGAGGCACAGCTCAGGCCAAG ACAAACCACACGAAACTTACCGACTGCTGAAACGCAGGAATCTGATCATAGAAGCTGTCACCAATCT TCGCTTAATCGAGAGTTTATTCACGATTCAGAAGATGATCATGGATCAGGAGAAGCAGGAAGGCGTG TCCACCAAGTGCTGCAAGAAGTCCATTGTCCGCTTGGTGCGGAACCTGTCTGAGGAAGGTCTCTTGC GATTGTATCGGACCACTGTCATTCAAGATGGCATCAAGAAGAAGGTGGATCTGGTGGTGCACCCGTC CATGGACCAGAACGACCCTCTAGTGAGAAGTGCCATCGAGCAGGTCCGCTTCCGGATCTCCAATTCA AGCACAGCCAACAGGGTTAAAACTTCCCAGCCTCCAGTGCCCCAAGGGGAGGCAGAAGAAGACAGTC AAGGAAAAGAGGGCCCAAGTGGATCAGGGGACTCTCAGCTGAGTGCTTCCTCTAGATCAGAAAGTGG ACGGATGAAAAAAAGTGATAATAAAATGGGCATAACCCCGCTTAGAAATTATCACCCCATTGTAGTT CCCGGACTGGGGCGTTCTCTAGGATTTCTGCCCAAAATGCCTCGCCTGCGGGTGGTCCACATGTTTC TGTGGTACCTCATCTACGGGCACCCTGCCAGCAACACCGTGGAGAAGCCAAGCTTCATCAGTGAACG GAGAACGATAAAGCAGGAGTCAGGCAGGGCAGGCGTCCGGCCGTCCTCCTCTGGAAGTGCCTGGGAG GCCTGCTCTGAAGCCCCATCTAAAGGCAGCCAAGATGGTGTCACCTGGGAGGCTGAAGTGGAGCTTG CCACGGAGACAGTGTATGTCGACGATGCCTCGTGGATGCGCTACATCCCCCCAATCCCAGTCCACAG GGACTTCGGCTTTGGCTGGGCTCTCGTCAGCGACATCCTCCTCTGCCTTCCCCTCTCCATCTTCATC CAGATTGTGCAAGTCAGCTACAAGGTGGACAACCTGGAGGAATTTCTGAACGACCCGCTGAAGAAGC ACACGCTGATCCGCTTTCTCCCCAGGCCCATTCGGCAGCAGCTTCTGTACAAGAGGCGTTACATTTT TTCGGTGGTGGAGAACCTTCAGAGGCTGTGCTACATGGGGGTGCTACAGTTTGGTCCCACGGAAAAG TTTCAGGATAAAGATCAGGTCTTTATCTTCTTGAAGAAGAATGCAGTCATTGTTGACACTACCATCT GCGACCCACATTACAACCTGGGCCGCAGGAGGCGGCCCTTCGAGAGGCGCCTCTATGTCCTGAACTC AATGCAGGATGTGGAAAACTACTGGTTTGACCTGCAGTGCGTCTGCCTCAACACCCCACTAGGCGTG GTGCGCTGCCCGCGCGTCAGGAAGAACAGCAGCACAGACCAGGGCAGCGACGAGGAGGGCAGCCTGC AGAAGGAGCAGGAGAGCGCCATGGACAAGCACAACCTGGAGCGCAAGTGCGCCATGCTGGAGTACAC CACTGGAAGCCGTGAGGTGGTGGATGAAGGCTTGATCCCTGGAGATGGGCTGGGTGCCGCAGGGCTC GATTCCAGCTTCTACGGACACCTCAAGCGCAACTGGATCTGGACCAGCTACATCATCAACCAGGCCA AAAAGGAGAACACTGCCGCAGAGAATGGACTCACAGTGAGGCTCCAGACATTTCTGTCCAAGCGCCC AATGCCCCTCAGTGCCAGAGGCAACAGCAGGTTGAATATTTGGGGGGAAGCAAGAGTAGGCTCCGAG CTCTGTGCTGGCTGGGAAGAGCAGTTTGAGGTGGACCGAGAGCCCTCGCTGGACCGAAACCGGAGAG TGAGGGGTGGGAAAAGCCAGAAGCGGAAGCGGCTGAAGAAGGACCCTGGGAAGAAGATCAAGAGAAA GAAGAAAGGAGAGTTCCCAGGAGAAAAAAGCAAAAGGCTGCGCTACCATGATGAAGCCGACCAGAGT GCCCTGCATCGGATGACGCGGCTTCGTGTCACCTGGTCTATGCAGGAGGATGGGCTGCTTGTGCTGT GCCGCATTGCCAGCAATGTCCTCAACACCAAGGTGAAGGGTCCATTTGTCACCTGGCAGGTGGTACG GGACATTTTGCATGCCACGTTTGAAGAGTCTTTGGATAAAACATCTCATTCCCTTGGACGAAGAGCT CGCTACATAGTCAAAAACCCACAGGCCTATCTCAACTATAAAGTGTGCCTGGCCGAGGTGTACCAGG ATAAAGCACTTGTTGGAGATTTCATGAATCGAAGAGGTGACTATGATGACCCAAAGGTTTGTGCCAA CGAGTTTAAAGAATTTGTGGAGAAGCTTAAAGAAAAGTTCAGTTCAGCCCTAAGGAATTCTAACCTT GAAATCCCAGACACACTCCAGGAGCTGTTCGCCAGGTACCGAGTTTTGGCAATTGGGGATGAAAAAG ATCAAACCAGGAAAGAGGATGAACTTAACAGCGTGGATGACATCCACTTTCTGGTGCTTCAGAACCT GATCCAGAGCACGCTGGCCCTCTCAGACAGTCAGATGAAGTCCTACCAGTCATTCCAGACTTTCCGC CTCTATCGGGAGTACAAGGACCACGTTCTTGTGAAGGCCTTCATGGAGTGCCAGAAGAGGAGCTTGG TCAACCGGCGCCGGGTCAACCACACGCTGGGCCCCAAGAAGAACCGGGCCCTCCCCTTCGTGCCAAT GTCCTACCAGCTATCCCAGACCTACTACAGGATTTTTACGTGGCGATTTCCAAGCACCATCTGCACG GAGTCATTCCAGTTTTTGGACAGAATGCGGGCTGCCGGCAAGTTGGACCAGCCTGATCGTTTCTCTT TCAAAGACCAGGATAATAACGAGCCCACAAACGACATGGTGGCCTTTTCACTGGACGGCCCTGGAGG AAATTGTGTGGCCGTCCTGACCCTCTTCTCTCTGGGCCTCATTTCTGTGGATGTCAGGATCCCGGAG CAGATCATCGTGGTAGACAGCTCAATGGTGGAGAATGAGGTCATCAAAAGCTTGGGGAAGGACGGCA GCCTGGAGGATGACGAGGATGAAGAGGATGACTTGGACGAAGGTGTAGGGGGCAAGCGCCGGAGCAT GGAGGTGAAACCTGCGCAAGCCTCCCACACCAACTACCTGCTGATGAGGGGCTACTACTCCCCCGGC ATCGTCAGCACCCGCAACCTCAACCCCAACGACAGCATTGTGGTCAACTCCTGCCAGATGAAGTTCC AGCTCCGCTGCACCCCTGTGCCCGCCCGGCTCAGGCCCGCTGCCGCTCCTCTGGAAGAGCTAACAAT GGGAACCTCCTGCCTCCCTGATACGTTCACCAAGCTGATAAACCCCCAGGAAAACACCTGCAGCTTG GAGGAGTTTGTCCTCCAGCTGGAGCTGTCTGGGTATAGTCCCGAAGACCTGACTGCTGCCTTGGAGA TCTTGGAAGCCATTATAGCCACGGGTTGTTTTGGGATTGACAAGGAGGAGCTGCGCAGACGGTTCTC GGCCTTGGAGAAGGCAGGTGGTGGGCGCACCAGGACATTCGCAGATTGCATCCAGGCCCTCCTGGAG CAGCATCAGGTGCTGGAGGTCGGTGGCAACACTGCGCGCCTGGTAGCCATGGGCTCTGCCTGGCCTT GGCTCCTGCACTCCGTGCGGCTGAAAGACAGAGAAGACGCCGACATCCAGAGAGAAGACCCCCAGGC CAGACCCCTGGAGGGGTCTTCCAGTGAGGACAGCCCCCCCGAGGGGCAGGCACCTCCTTCTCACAGC CCCCGGGGCACCAAGAGGCGCGCCAGCTGGGCCAGTGAGAATGGGGAGACCGACGCCGAGGGCACCC AGATGACCCCTGCCAAGAGGCCAGCGCTCCAGGACTCAAATTTGGCCCCCAGCCTTGGGCCCGGAGC TGAAGATGGGGCAGAAGCCCAGGCCCCATCTCCACCCCCAGCTCTTGAAGACACCGCTGCAGCGGGA GCAGCACAGGAAGACCAAGAGGGTGTCGGGTTCACAGAGAGTTTCGGAGCTGCCAACATCTCCCAGG CAGCACGGGAAAGGGACTGTGAGAGTGTCTGCTTCATCGGCCGGCCGTGGCGTGTCGTGGATGGCCA CCTGAACCTTCCTGTATGCAAGGGTATGATGGAGGCCATGCTGTACCACATCATGACCAGGCCTGGC ATCCCCGAGAGCTCCCTGCTGCGCCACTACCAGGGGGTCCTGCAGCCCGTCGCCGTGCTGGAGTTGC TCCAGGGCCTGGAGTCCCTCGGCTGCATCCGGAAGCGCTGGCTGAGAAAGCCAAGGCCTGTCTCGCT CTTCTCTACACCCGTGGTGGAAGAGGTGGAAGTGCCCTCCAGCCTGGACGAGAGCCCCATGGCTTTC TATGAGCCCACCTTGGACTGTACCCTCCGGCTGGGCCGTGTGTTCCCCCACGAGGTCAACTGGAACA AGTGGATCCACCTCTAGGACCCCTGTGGGCGTCCCCTCCCTCCCAGCCACCGCCTGCCACACCACTC CTGCCTGGTGCTCGGCAGACCCCACTGTGCCCTGGCCTTGGGTCTGCCGAGCCTCCTGCAGCAGGGG ACGGGTGCTTTGGCCAGAGTCACAGACTGACACGTTTCCCACTGTACTGGAACTCTGGAAAGAGGGG CTCCCCGACCTGCCCATCCCCCAGGCTCTTCTGGGCCTTCCCCTTGGGAACTGGCCTCATCACACTG GGAGTTGGTGCTTCTTGTCTCTGGGTCTCCAGAGTTTGCCCCGCCTGTGCACACCTCACATTCCAGA CTCTAGCCATCTCGGCAGGATCTCCTGGCTCCTTGAGTGCCCAGGTGCCACCAAGAGGAAGGGCCTT GTGGGATACACCTTGCAGAATAGGGATCGGTGTGCCCCGCTGCGAGGGGCCCCCCATGGGGGCTGTG GCCCCTCCGCAGGCAGGACATCCCAACCCCTGGCTGGGACTGAACCACCCAGAGCGGAGCGGCTCCC TTTTCAGCCTTGTGAGTCACCTGGCAGGCCCCAGCTGGGCTGGCTGTCCGTGTCCCTCAGCCTGGCT GGTGATTCCTTGCAGGAGGG
The NON21 protein (SEQ ID ΝO:73) encoded by SEQ ID NO:72 is 2084 amino acid residues in length and is presented using the one-letter amino acid code in Table 21B. NOV21 has two SNP variants, whose variant positions for its nucleotide and amino acid sequences is numbered according to SEQ ID NOS:72 and 73, respectively. A variant sequence can include a single nucleotide polymorphism (SNP). A SNP can, in some instances, be referred to as a "cSNP" to denote that the nucleotide sequence containing the SNP originates as a cDNA.
NOV21 has at least two variants. Variant 13376755 is a G to A SNP at 129 bp of the nucleotide sequence that results in no change in the protein sequence (silent), and variant clOO.2613 is an insertion of nucleotide C before 5780 bp of the nucleotide sequence that results in a frameshift with all amino acids after 1907 being discordant with the original protein sequence.
Psort analysis predicts the NOV21 protein of the invention to be localized in the nucleus with a certainty of 0.8000, as expected by a transcription factor subunit. As expected for a member of the TFIIIC box B-binding subunit protein family, no identifiable domains with significant score were identified in the NON21 polypeptide by searches using algorithms such as PROSITE, Blocks, Pfam, ProDomain, Prints.
Table 21B. Encoded ΝON21 protein sequence (SEQ ID ΝO:73)
MDALESLLDEVALEGLDGLCLPAL SRLETRVPPFPLPLEPCTQEFL RALATHPGISFYEEPRERPDLQLQD RYEEIDLETGILESRRDPVALEDVYPIHMILENKDGIQGSCRYFKERKNITNDIRTKSLQPRCTMVEPFDR G KKLIIGSLPAHAVQALDSPGGGSRPEAARLLLLHPGTARPVQVQGELQRDLHTTAFKVDAGKLHYHRKILNKN GLITMQSHVIRLPTGAQQHSILLLLNRFHVDRRSKYDILMEKLSVMLSTRTNHIETLGKLREELGLCERTFKR LYQYMLNAGLAKWSLRLQEIHPECGPCKTKKGTDVMVRCLKLLKEFKRNDHDDDEDEEVISKTVPPVDIVFE RDMLTQTYDLIERRGTKGISQAEIRVAMNVGKLEARMLCRLLQRFKWKGFMEDEGRQRTTKYISCVFAEESD LSRQYQREKARSELLTTVSLASMQEESLLPEGEDTFLSESDSEEERSSSKRRGRGSQKDTRASANLRPKTQPH HSTPTKGG KWNLHPLKQPPSFPGAAEERACQSLASRDSLLDTSSVSEPNVSFVSHCADSNSGDIAVIEEV RMENPKESSSSLKTGRHSSGQDKPHETYRLLKRRNLIIEAVTNLRLIESLFTIQKMIMDQEKQEGVSTKCCKK SIVRLVRNLSEEGLLRLYRTTVIQDGIKKKVDLWHPSMDQNDPLVRSAIEQVRFRISNSSTANRVKTSQPPV PQGEAEEDSQGKEGPSGSGDSQLSASSRSESGRMKKSDNKMGITPLRNYHPIWPGLGRSLGFLPKMPRLRW HMFLWYLIYGHPASNTVEKPSFISERRTIKQESGRAGVRPSSSGSA EACSEAPSKGSQDGVT EAEVELATE TVYVDDASWMRYIPPIPVHRDFGFGWALVSDILLCLPLSIFIQIVQVSYKVDNLEEFLNDPLKKHTLIRFLPR PIRQQLLYKRRYIFSWENLQRLCYMGVLQFGPTEKFQDKDQVFIFLKKNAVIVDTTICDPHYNLGRRRRPFE RRLYVLNSMQDVENY FDLQCVCLNTPLGWRCPRVRKNSSTDQGSDEEGSLQKEQESAMDKHNLERKCAMLE YTTGSREWDEGLIPGDGLGAAGLDSSFYGHLKRN IWTSYIINQAKKENTAAENGLTVRLQTFLSKRPMPLS ARGNSRLNIWGEARVGSELCAG EEQFEVDREPSLDRNRRVRGGKSQKRKRLKKDPGKKIKRKKKGEFPGEKS KRLRYHDEADQSALHRMTRLRVT SMQEDGLLVLCRIASNVLNTKVKGPFVTWQWRDILHATFEESLDKTSH SLGRRARYIVKNPQAYLNYKVCLAEVYQDKALVGDFMNRRGDYDDPKVCANEFKEFVEKLKEKFSSALRNSNL EIPDTLQELFARYRVLAIGDEKDQTRKEDELNSVDDIHFLVLQNLIQSTLALSDSQMKSYQSFQTFRLYREYK DHVLVKAFMECQKRSLVNRRRVNHTLGPKKNRALPFVPMSYQLSQTYYRIFT RFPSTICTESFQFLDRMRAA GKLDQPDRFSFKDQDNNEPTNDMVAFSLDGPGGNCVAVLTLFSLGLISVDVRIPEQIIWDSSMVENEVIKSL GKDGSLEDDEDEEDDLDEGVGGKRRSMEVKPAQASHTNYLLMRGYYSPGIVSTRNLNPNDSIWNSCQMKFQL RCTPVPARLRPAAAPLEELTMGTSCLPDTFTKLINPQENTCSLEEFVLQLELSGYSPEDLTAALEILEAIIAT GCFGIDKEELRRRFSALEKAGGGRTRTFADCIQALLEQHQVLEVGGNTARLVAMGSAWP LLHSVRLKDREDA DIQREDPQARPLEGSSSEDSPPEGQAPPSHSPRGTKRRASWASENGETDAEGTQMTPAKRPALQDSNLAPSLG PGAEDGAEAQAPSPPPALEDTAAAGAAQEDQEGVGFTESFGAANISQAARERDCESVCFIGRPWRWDGHLNL PVCKGMMEAMLYHIMTRPGIPESSLLRHYQGVLQPVAVLELLQGLESLGCIRKRWLRKPRPVSLFSTPWEEV EVPSSLDESPMAFYEPTLDCTLRLGRVFPHEVN NKWIHL
A search against the Patp database, a proprietary database that contains sequences published in patents and patent publications, yielded several homologous proteins shown in Table 21C. Table 21C. Patp results for NON21
Smallest Sum
Reading High Prob
Sequences producing High-scoring Segment Pairs: Frame Score P(Ν)
>patp:AAM32653 Peptide #6690 encoded by probe +1 519 5.2e-48
>patp:AAM59814 Human brain expressed single exon probe +1 519 5.2e-48
>patp:AAM72401 Human bone marrow expressed probe +1 519 5.2e-48
>patp:AAM34175 Peptide #8212 encoded by probe +1 491 4.9e-48
>patp:AAM74000 Human bone marrow expressed probe +1 491 4.9e-48
>patp:AAM60401 Human brain expressed single exon probe +1 343 2.7e-29
>patp:AAM73037 Human bone marrow expressed probe +1 343 2.7e-29
>patp:AAM33554 Peptide #7591 encoded by probe +1 324 2.8e-27
>patp:AAM60680 Human brain expressed single exon probe +1 324 2.8e-27
>patp:AAM73352 Human bone marrow expressed probe +1 324 2.8e-27
hi a BLAST search of public sequence databases, it was found, for example, that the nucleic acid sequence of this invention has 6006 of 6134 bases (97%) identical to a gb:GENBANK-ID:HSU02619|acc:U02619.1 mRNA from Homo sapiens (Human TFIIIC Box B- binding subunit mRNA, complete eds). The NON21 polypeptide was found to have 1937 of 1948 amino acid residues (99%) identical to, and 1939 of 1948 amino acid residues (99%) similar to, the 2109 amino acid residue ptnr:SPTREMBL-ACC:Q12789 protein from Homo sapiens (TFIIIC BOX B-BΓΝDIΝG SUBUNIT (TRANSCRIPTION FACTOR (TFIIIC) ALPHA CHAIN) (3' PARTIAL)). The NON21 polypeptide lacks 25 internal amino acids, when compared to ptnr:SPTREMBL-ACC:Q12789 protein from Homo sapiens (Human) (TFIIIC BOX B-BΓΝDIΝG SUBUNIT (TRANSCRIPTION FACTOR (TFIIIC) ALPHA CHAIN) (3' PARTIAL)).
NON21 also has homology to the proteins shown in the BLASTP data in Table 21D.
Figure imgf000196_0001
Figure imgf000197_0001
A multiple sequence alignment is given in Table 2 IE, with the NOV21 protein being shown on line 1 in Table 2 IE in a ClustalW analysis, and comparing the NOV21 protein with the related protein sequences shown in Table 21D. This BLASTP data is displayed graphically in the ClustalW in Table 21E.
Table 21E. ClustalW Analysis of NON21
1) > ΝOV21; SEQ D3 NO:73
2) >Q12789/ TFHIC BOX B-BhNDING SUBUNIT (TRANSCRIPTION FACTOR (TFIIIC) ALPHA CHAIN) (3' PARTIAL) [Homo sapiens]; SEQ DD NO:251
3) >Q9Y4W9/ TRANSCRIPTION FACTOR (TFIIIC) ALPHA CHAIN, PARTIAL [Homo sapiens]; SEQ TD NO:252
4) >Q63505/ TRANSCRIPTION FACTOR IIIC ALPHA-SUBUNIT [Rattus norvegicus]; SEQ DO NO:253
5) >Q123838 / TFIIIC ALPHA SUBUNIT [Homo sapiens]; SEQ K> NO:254
6) >013129/ Transcription factor IIIC alpha chain [Homo sapiens]; SEQ TD NO:255
10 20 30 40 50
....|....|....|....|....|....|....|....|....|....]
NOV21 MDALESLLDEVALEGLDGLCLPAL SRLETRVPPFPLPLEPCTQEFL RA
Q12789 MDALESLLDEVALEGLDGLCLPAL SRLETRVPPFPLPLEPCTQEFL RA
Q9Y4 9
Q63505 MDALESLLDEVALEGLDGLCLPALWSRLESRSPAFPLPLEPYTQEFLWRA
Q12838
B56011
60 70 80 90 100
....|....|....|....|_L^1....|....|....|....| ....|
NOV21 LATHPGISFYEEPRERPDLC LQDRYEEIDLETGILESRRDPVALEDVYPI
Q12789 LATHPGISFYEEPRERPDLC LQDRYEEIDLETGILESRRDPVALEDVYPI
Q9Y4 9
Q63505 ]WTHPGISFYEEPRERPDLQlW»kW^WA^^i*^»1J»l^T^»uW»W
Q12838 NS* AQDRYEEIDLETGILESRRDPVALEDVYP:
B56011 .QDRYEEIDLETGILESRRDPVALEDVYP:
110 120 130 140 150 NOV21 IMI ENKDGIQGSCRYFKERKNITNDIRTKSLQPRCTMVEISFDRWGKKLJ
Q12789 3MI ENKDGIQGSCRYFKERKNITNDIRTKSLQPRCTMVEraFDRWGKKL]
Q9Y4W9
Q63 05 l liiιi!fl Λfta-^^W«MM«OIieMMwW;ryVi^la^;iiiDDrofBy'sSs sπmgsiScιW5iJ»l)iJ[5ijgΑBsld'!'^{4!4-ii.
Q12838 IMILENKDGIQGSCRYFKERKNITNDIRTKSLQPRCTMVEΘFDRWGKKL
B56011 MILENKDGIQGSCRYFKERKNITNDIRTKSLOPRCTMVESFDRWGKKL 160 170 180 190 200
NOV21 GSLPGHAVQOTDSPGGΣSRPEAARLLLLHPGTARPVQ 2GELQRDLHT1
Q12789 GSLPBHAVQSDSPGSGSRPEAARLLLLHPGTARPVQ DGELQRDLHTT
Q9Y4W9
Q63505 ASQDMRYRailGLESDPDLKLPDFSYCILERLGRSRW >MM,M.W.uύ.
Q12838 ifASQg IlGQES DPDLKLPDFSYCILERLGRSRWBgjst-roWEsE.tM..llll.
B56011 ASQg IIGQES PDLKLPDFSYCILERLGRSRWS QGELQRDLHTT
210 220 230 240 250
N0V21 FKVDAGKLHYHRKILNKNGLITMQSHVIRLPTGAQQHSILLLLNRFHV
Q12789 ^FKVDAGKLHYHRKILNKNGLITMQSHVIRLPTGAQQHSILLLLNRFHVE
Q9Y4 9
Q63505 iFKVDAGKLHYHRKILNKNGLITMQSHVIRLPTGAQQHSILLLLNRFHVI
Q12838 i.FKVDAGKLHYHRKILNKNGLITMQSHVIRLPTGAQQHSILLLLNRFHV
B56011 LFKVDAGKLHYHRKILNKNGLITMQSHVIRLPTGAQQHSILLLLNRFHV
260 270 280 290 300
RRSKYDILMEKLSVMLSTRTNHIETLGKLREELGLCERTFKRLYQYMLNi RRSKYDILMEKLSVMLSTRTNHIETLGKLREELGLCERTFKRLYQYMLN] HsKYDILMEKLSVMLSTRTNHIETLGKLREELGLCERTFKRLYQYMLNi RRSKYDILMEKLSJJJMLSTRgN@IETLGKLREELGLCERTFKRLYQYMLNi RRSKYDILMEKLSVMLSTRTNHIETLGKLREELGLCERTFKRLYQYMLNi RRSKYDILMEKLSVMLSTRTNHIETLGKLREELGLCERTFKRLYQYMLNi
310 320 330 340 350
N0V21 ΪLAKWSLRLQEIHPECGPCKTKKGTDVMVRCLKLLKEFKR^BNDHDDL
Q12789 3LAKWSLRLQEIHPECGPCKTKKGTDVMVRCLKLLKEFKR^HNDHDDI
Q9Y4W9 !LAKWSLRLQEIHPECGPCKTKKGTDVMVRCLKLLKEFKR^HNDHDD
Q63505 nwawAtta-gi-msMa >awa«eιιιHMi.mMwιιaιmaaigRiϊlκMEDBBiBBB
Q12838 LAKWSLRLQEIHPECGPCKTKKGTDVMVRCLKLLKEFKR
B56011 LAKWSLRLQEIHPECGPCKTKKGTDVMVRCLKLLKEFKR
360 370 380 390 400
NOV21 EDEEVISKTVPPVDIVFERDMLTQTYDLIERRGTKGISQAEIRVAMNVGK
Q12789 EDEEVISKTVPPVDIVFERDMLTQTYDLIERRGTKGISQAEIRVAMNVGK
Q9Y4W9 EDEEVISKTVPPVDIVFERDMLTQTYDLIERRGTKGISQAEIRVAMNVGK
Q63505 SDEESISK^VPPVDIVFERDMLTQTY^LIERRGTKGISQAEIRVAMNVGK
Q12838 EDEEVISKTVPPVDIVFERDMLTQTYDLIERRGTKGISQAEIRVAMNVGK
B56011 EDEEVISKTVPPVDIVFERDMLTQTYDLIERRGTKGISQAEIRVAMNVGK
410 420 430 440 450
I ....!....I ....I ..
N0V21 EARMLCRLLQRFKWKGFMEDEGRQRTTKYISCVFAEESDLSRQYQREK
Q12789 EARMLCRLLQRFKWKGFMEDEGRQRTTKYISCVFAEESDLSRQYQREK
Q9Y4 9 LEARMLCRLLQRFKWKGFMEDEGRQRTTKYISCVFAEESDLSRQYQREK
Q63505 LEARMLCRLLQRFKWKGFMEDEGRQRTTKYISCVFAEESDLSRQYGREK
Q12838 LEARMLCRLLQRFKWKGFMEDEGRQRTTKYISCVFAEESDLSRQYQREK
B56011 LEARMLCRLLQRFKWKGFMEDEGRQRTTKYISCVFAEESDLSRQYQREK
460 470 480 490 500 I....I.... I.... I..
NOV21 RSELLTTVSLASMQEESLLPEGEDTFLSESDSEEERSSSBKRRGRGSQK
Q12789 ARSELLTTVSLASMQEESLLPEGEDTFLSESDSEEERSSSBKRRGRGSQK
Q9Y4W9 ARSELLTTVSLASMQEESLLPEGEDTFLSESDSEEERSSSIKRRGRGSQK
Q63505 ARgELLTTVSLAsEQJ3ESLi5JPEGE[^FLSJ3s[ SEEE sgsEκR GRGS[^
Q12838 ARSELLTTVSLASMQEESLLPEGEDTFLSESDSEEERSSSIKRRGRGSQK
B56011 ARSELLTTVSLASMQEESLLPEGEDTFLSESDSEEERSSSBKRRGRGSQK 510 520 530 540 550
NOV21 3TRASANLRPKTQPHHSTPTKGGWKVVNLHPLKKQPPSFPGAAEERACQ!
Q12789 ITRASANLRPKTQPHHSTPTKGG KWNLHPLKKQPPSFPGAAEERACQJ
Q9Y4W9 TRASANLRPKTQPHHSTPTKGGWKWNLHPLKKQPPSFPGAAEERACQ;
Q63505 ASg^P^gPHHSTPgκGG KV§NLHPLK gj^JH3 2ERg3g!
Q12838 )TRASANLRPKTQPHHSTPTKGGWKWNLHPLKKQPPSFPGAAEERACQ!
B56011 DTRASANLRPKTQPHHSTPTKGG KWNLHPLKKQPPSFPGAAEERACQ;
560 570 580 590 600
NOV21 ASRDSLLDTSSVSEPNVSFVSHCADSNSGDIAVIEEVRMENPKESSSSI
Q12789 LJASRDSLLDTSSVSEPNVSFVSHCADSNSGDIAVIEEVRMENPKESSSSL
Q9Y4 9 LASRDSLLDTSSVSEPNVSFVSHCADSNSGDIAVIEEVRMENPKESSSSI
Q63505 ΑHRDCT DTSSBSEi-faBgFiH3Hl5i!'lDSi5sGDIAVIEEVRi-BlMPKEE!gas
Q12838 JASRDSLLDTSSVSEPNVSFVSHCADSNSGDIAVIEEVRMENPKESSSSI
B56011 JASRDSLLDTSSVSEPNVSFVSHCADSNSGDIAVIEEVRMENPKESSSSL
610 620 630 640 650
NOV21 KTGRHSSGQDKPHETYRLLKRRNLIIEAVTNLRLIESLFTIQKMIMDQE1
Q12789 KTGRHSSGQDKPHETYRLLKRRNLIIEAVTNLRLIESLFTIQKMIMDQEI
Q9Y4 9 KTGRHSSGQDKPHETYRLLKRRNLIIEAVTNLRLIESLFTIQKMIMDQE1.
Q63505
Q12838 KTGRHSSGQDKPHETYRLLKRRNLIIEAVTNLRLIESLFTIQKMIMDQEI
B56011 TGRHSSGQDKPHETYRLLKRRNLIIEAVTNLRLIESLFTIQKMIMDQE
660 670 680 690 700
)EGVSTKCCKKSIVRLVRNLSEEGLLRLYRTTVIQDGIKKKVDLWHPSI» JEGVSTKCCKKSIVRLVRNLSEEGLLRLYRTTVIQDGIKKKVDLWHPSIV )EGVSTKCCKKSIVRLVRNLSEEGLLRLYRTTVIQDGIKKKVDLWHPSI*
JEGVSTKCCKKSIØRLVRNLSEEGLLRLYRTTVIQDGIKKKVDLWHPSIV )EGVSTKCCKKSIVRLVRNLSEEGLLRLYRTTVIQDGIKKKVDLWHPS]Y 3EGVSTKCCKKSIVRLVRNLSEEGLLRLYRTTVIQDGIKKKVDLWHPSN
Figure imgf000199_0001
B56011
Figure imgf000200_0001
B56011
Figure imgf000200_0002
B56011
Figure imgf000200_0003
B56011
Figure imgf000200_0004
B56011
Figure imgf000200_0005
B56011
Figure imgf000200_0006
B56011
Figure imgf000200_0007
B56011
Figure imgf000201_0001
B56011
Figure imgf000201_0002
B56011
1310 1320 1330 1340 1350 ..I...
NOV21 rRDILHATFEESLDKTSHStøGRRARYIVKNE γιM________ ___
Q12789 ύJΛΛMitMMHsMim
Q9Y4W9 rRDILHATFEESLDKTSHSfflGRRARYIVKNPQ£ L -,-'«M«--».l!Ht»,rtl»l'#ιll
Q63505 rRDILHATFEESLDKTSHSfflGRRARYIVKNPQAS ,MέiύlMAAMifi- B
Q12838
B56011
Figure imgf000201_0003
B56011
Figure imgf000201_0004
B56011
Figure imgf000201_0005
B56011
Figure imgf000201_0006
B56011
Figure imgf000202_0001
B56011
Figure imgf000202_0002
B56011
Figure imgf000202_0003
B56011
Figure imgf000202_0004
B56011
Figure imgf000202_0005
B56011
Figure imgf000202_0006
B56011
Figure imgf000202_0007
B56011
Figure imgf000203_0001
B56011
Figure imgf000203_0002
B56011
Figure imgf000203_0003
B56011
Figure imgf000203_0004
B56011
2160
NOV21
Q12789
Q9Y4W9
Q63505 SHSCYQSSAQPSTGVATSR
Q12838
B56011
Consistent with other known members of the TFHTC box B-binding subunit family of proteins, NOV21 has, for example, homology to other members of the TFIIIC box B-binding subunit family and nuclear localization. NOV21 nucleic acids, and the encoded polypeptides, according to the invention are useful in a variety of applications and contexts. For example, NOV21 nucleic acids and polypeptides can be used to identify proteins that are members of the TFIIIC box B-binding subunit family of proteins. The NOV21 nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance NOV21 activity or function. Specifically, the nucleic acids and polypeptides according to the invention maybe used as targets for the identification of small molecules that modulate or inhibit, e.g., gene transcription. These molecules can be used to treat, e.g., cancer and viral disease. i addition, various NOV21 nucleic acids and polypeptides according to the invention are useful, ter alia, as novel members of the protein families according to the sequence relatedness to previously described proteins. For example, the NOV20 nucleic acids and their encoded polypeptides show homology to proteins belonging to the family of TFIIIC box B-binding subunit proteins such as the α-chain of the Human TFIIIC box B-binding subunit.
Transcription factor IIIC (TFIIIC) is a multisubunit basic TF for RNA polymerase III. It initiates transcription complex assembly on tRNA and related genes by binding to the internal box B promoter element and is also required for transcription of 5S rRNA and other stable nuclear and cytoplasmic RNAs transcribed by polymerase III. In mammalian cells, regulation of TFIIIC activity controls overall polymerase III transcription in response to growth factors and viral infection. A full-length cDNA (and genomic DNA from the transcription initiation region) encoding the box B binding subunit of human TFIIIC, the 243-kDa alpha subunit has been reported and shown to encode a component of TFIIIC. (L'Etoile et al., Proc Natl Acad Sci USA, 91: 1652-6 (1994)).
The NOV21 nucleic acids and polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in gene regulation. As such the NOV21 nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat cancer and viral infections, e.g., TFIIIC box B-binding subunit protein is cleaved and inactivated by the poliovirus-encoded 3C protease during poliovirus infection (Shen et al., Mol. Cell. Biol, 16: 4163-71 (1996)).
The NON21 nucleic acids and polypeptides are useful for detecting specific cell types. For example, expression analysis has demonstrated that a ΝON21 nucleic acid is expressed in adrenal gland, bone marrow, brain - amygdala, brain - cerebellum, brain - hippocampus, brain - substantia nigra, brain - thalamus, brain -whole, fetal brain, fetal'kidney, fetal liver, fetal lung, heart, kidney, lymphoma - Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea, uterus, Adipose, Aorta, Bone, Bronchus, Cartilage, Cervix, Chorionic Nillus, Colon, Coronary Artery, Dermis, Epidermis, Hypothalamus, Liver, Lung, Lymph node, Lymphoid tissue, Myometrium, Ovary, Peripheral Blood, Respiratory Bronchiole, Retina, Right Cerebellum, Svnovium/Synovial membrane, Temporal Lobe, Thymus, Tonsils, Umbilical Vein, Vein, Vulva, and Whole Organism.
Additional utilities forΝOV21 nucleic acids and polypeptides according to the invention are disclosed herein.
NOV22
A NOV22 polypeptide has been identified as a Nucleoside Diphosphate Kinase B-like protein (also referred to as CG56475-01). The disclosed novel NOV22 nucleic acid (SEQ ID NO: 74) of 473 nucleotides is shown in Table 22 A. The novel NOV22 nucleic acid sequences maps to the chromosome 2.
An ORF begins with an ATG initiation codon at nucleotides 12-14 and ends with a TAA codon at nucleotides 464-466. A putative untranslated region and/or downstream from the termination codon is underlined in Table 22 A, and the start and stop codons are in bold letters.
Table 22A. NOV22 Nucleotide Sequence (SEQ ID NO:74)
ATCCTCAGGCCATGGCCAACACTGAGAGCATCATTATCAATCCGAGTGCTGTTCAGCACAGCCTGGT GGGTGAAATCATCAAATACTCTGAGCAGAAGGGATTCTACCTGGTGACCATGAAGTTCCTTCGGGCC TCTGAGAAACCCCTGAAGGAGCACTACACTAACCTGAAAGACCACCCATTCTTCCCGGACCTTGTGA AGTACATGAACTCAGGGCAGGTTGTGGCCATGGTCCTGGAGGGGCTGAATGTGGCAAAGACAGGGCT AAGGATGCTTGGGGAGACCAATTCATTGGGCTCTATGCTAGAGACTATTATTCGCAGGGACTTCTGC GCTAAAATAGGCGGGAACGTCATTGGTGGCAGTGATTCATTACAAAGTGCTGGCAAAGAAATGGCTA AATGGCTTAAAGAAGAAGAACTGGTTGACTACAAATCTCGTGCCTATGACAAGATCTATGATAAAAA GGAG
The NOV22 protein (SEQ ID NO:75) encoded by SEQ ID NO:74 is 181 amino acid residues in length and is presented using the one-letter amino acid code in Table 22B. Psort analysis predicts the NOV22 protein of the invention to be localized in the cytoplasm with a certainty of 0.6500. Table 22B. Encoded NON22 protein sequence (SEQ ID ΝO:75)
MANTESIIINPSAVQHSLVGEIIKYSEQKGFYLVTMKFLRASEKPLKEHYTNLKDHPFFPDLVKY NSGQWA ^WLEGLNVAKTGLRMLGETNS GSMLETIIRRDFCA IGG VIGGSDS QSAGKEM K LKEEELVDYKSRAY DKI YDKKEVKAAVCLDAVPGS DTALHP IDLE Al G
A search against the Patp database, a proprietary database that contains sequences published in patents and patent publications, yielded several homologous proteins shown in Table 22C.
Table 22C. Patp results for NON22
Smallest Sum
Reading High Prob
Sequences producing High- scoring Segment Pairs : Frame Score P (Ν)
>patp :AAY07000 mm23 -H2 protein sequence +1 468 3 . 2e-44
>patp : AAB14812 Human nm23 protein nm23 -H2S +1 468 3 . 2e-44
In a BLAST search of public sequence databases, it was found, for example, that the nucleic acid sequence of this invention has 376 of 475 bases (79%) identical to a gb:GENBANK- ID:HUMPUF|acc:Ll 6785.1 mRNA from Homo sapiens (c-myc transcription factor (puf) mRNA, complete eds). The NOV22 polypeptide of the invention was found to have 100 of 152 amino acid residues (65%) identical to, and 113 of 152 amino acid residues (74%) similar to, the 152 amino acid residue ptnr:SWISSPROT-ACC:P22392 protein from Homo sapiens (NUCLEOSIDE DIPHOSPHATE KINASE B (EC 2.7.4.6) (NDK B) (NDP KTNASE B) (NM23-H2) (C-MYC PURΓNE-BΓNDING TRANSCRIPTION FACTOR PUF)).
NON22 also has homology to the proteins shown in the BLASTP data in Table 22D.
Figure imgf000206_0001
Figure imgf000207_0003
A multiple sequence alignment is given in Table 22E, with the NOV22 protein being shown on line 1 in Table 22E in a ClustalW analysis, and comparing the NOV22 protein with the related protein sequences shown in Table 22D. This BLASTP data is displayed graphically in the ClustalW in Table 22E.
Table 22E. ClustalW Analysis of NON22
1) > ΝOV22; SEQ TD NO:75
2) > P22392/ Nucleoside diphosphate kinase B (EC 2.7.4.6) [Homo sapiens]; SEQ ID NO:256
3) > PI 9804/ Nucleoside diphosphate kinase B (EC 2.7.4.6) [Rattus norvegicus]; SEQ TD NO:257
4) > Q01768/ Nucleoside diphosphate kinase B (EC 2.7.4.6) [Mus musculus]; SEQ ID NO:258
5) > 057535/ Nucleoside diphosphate kinase [Gallus gallus]; SEQ ID NO:259
6) > P15532/ Nucleoside diphosphate kinase A (EC 2.7.4.6) [Mus musculus]; SEQ TD NO:260
Figure imgf000207_0001
60 10 80 90 100
.... I .... I ....|....I.... I .... I .... I .... |....|.... I
NOV22
P22392 QHYIDLKDRPFFPGLVKYMNSGPWAMVBEGLNWKTGRVMLGETNPADS
P19804 QHYIDLKDRPFFPGLVKYMNSGPWAMVIEGLNWKTGRVMLGETNPADS
Q01768 QHYIDLKDRPFFPGLVKYI^SGPVVAMVIEGLNVVKTGRVMLGETNPADS
057535 QHYIDLKDRPFHPGLVKYMNSGPWAMMEGLNWKTGRVMLGETNPADS
P15532 Ei;MTiww^tjaaaa [e)-vτf;y<aiHWM.-*AVjavτi-iMe>wι!i.wi^wrvjθitιe)-ij.ι?i!jι
Figure imgf000207_0002
Figure imgf000208_0001
160 170 180
NOV 2 KIHDKKEVKAAVCLDAVPGSLDTALHPIDLEAIG
P22392
P19804
Q01768
057535
P15532
The presence of identifiable domains in the protem disclosed herein was determined by searches using algorithms such as PROSITE, Blocks, Pfam, ProDomain, Prints and then determining the Interpro number by crossing the domain match (or numbers) using the Interpro website (http:www.ebi.ac.uk/interpro/). Table 22F lists the domain description from DOMAIN analysis results against NOV22.
Figure imgf000208_0002
Consistent with other known members of the subunit family of proteins, NOV22 has, for example, an Nucleoside Diphosphate Kinase (NDK) signature sequence and homology to other members of the Nucleoside Diphosphate Kinase B family.
NOV22 nucleic acids, and the encoded polypeptides, according to the invention are useful in a variety of applications and contexts. For example, NOV22 nucleic acids and polypeptides can be used to identify proteins that are members of the Nucleoside Diphosphate Kinase B family of proteins. The NOV22 nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance NOV22 activity or function. Specifically, the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., nucleic acid synthesis, CTP for lipid synthesis, UTP for polysaccharide synthesis and GTP for protein elongation, signal transduction and microtubule polymerization. These molecules can be used to treat disorders of metabolism, cellular growth and differentiation, e.g., cancer. hi addition, various NOV22 nucleic acids and polypeptides according to the invention are useful, inter alia, as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins. For example, the NOV20 nucleic acids and their encoded polypeptides show homology to proteins belonging to the family of Nucleoside Diphosphate Kinase B such as human nicleoside diphosphate kinase B (EC 2.7.4.6). Nucleoside diphosphate kinases (EC 2.7.4.6) (NDK) are enzymes required for the synthesis of nucleoside triphosphates (NTP) other than ATP. They provide NTPs for nucleic acid synthesis, CTP for lipid synthesis, UTP for polysaccharide synthesis and GTP for protein elongation, signal transduction and microtubule polymerization (Parks, R. and Agarwal R., hi: The enzymes - Group transfer. Boyer P.D. (Ed.) Academic Press, New York, 1973, pp.307-334). hi eukaryotes, there is a small family of NDK isozymes each of which acts in a different subcellular compartment and or has a distinct biological function. Eukaryotic NDK isozymes are hexamers of two highly related chains (A and B) (Gilles, et al., J. Biol. Chem. 266: 8784-8789 (1991)). By random association (A6, A5B...AB5, B6), these two kinds of chain form isoenzymes differing in their isoelectric point.
NDK are proteins of 17 Kd that act via a ping-pong mechanism in which a histidine residue is phosphorylated, by transfer of the terminal phosphate group from ATP. hi the presence of magnesium, the phosphoenzyme can transfer its phosphate group to any NDP, to produce an NTP.
NDK isozymes have been sequenced from prokaryotic and eukaryotic sources. It has also been shown that the Drosophila awd (abnormal wing discs) protem, is a microtubule-associated NDK (Biggs et al., Cell 63: 933-940 (1990)). Mammalian NDK is also known as metastasis inhibition factor nm23. The sequence of NDK has been highly conserved through evolution. There is a single histidine residue conserved in all known NDK isozymes within the NDK signature, which is involved in the catalytic mechanism (Gilles, et al., J. Biol. Chem. 266: 8784- 8789 (1991)). The NOV22 nucleic acids and polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in cellular growth and metabolism. As such the NOV22 nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat cancer, e.g., atherosclerosis, aneurysm, hypertension, fibromuscular dysplasia, stroke, scleroderma, obesity, transplantation, myocardial infarction, embolism, cardiovascular disorders, bypass surgery, fertility disorders, myasthenia gravis, leukodystrophies, pain, neuroprotection, systemic lupus erythematosus, autoimmune disease, asthma, emphysema, scleroderma, allergy, ARDS and other diseases, disorders and conditions of the like.
The NOV22 nucleic acids and polypeptides are useful for detecting specific cell types. For example, expression analysis has demonstrated that a NOV22 nucleic acid is expressed in lymphocyte, placental, liver, cardiovascular, nervous, respiratory, and immune systems, and this protein is found in reduced amount in tumor cells of high metastasic potential. Accordingly, the NOV22 nucleic acids, polypeptides, antibodies and related compounds according to the invention will have diagnostic and therapeutic applications in the detection of cancer.
Additional utilities for NOV22 nucleic acids and polypeptides according to the invention are disclosed herein.
NON23
A ΝON23 polypeptide has been identified as a T-cell-like protein (also referred to as CG56352-02). The disclosed novel ΝON23 nucleic acid (SEQ ID ΝO:76) of 1326 nucleotides is shown in Table 23 A. An ORF begins with an ATG initiation codon at nucleotides 19-21 and ends with a TAG codon at nucleotides 1324- 1326. A putative untranslated region and/or downstream from the termination codon is underlined in Table 23 A, and the start and stop codons are in bold letters. The NON23 nucleotide sequence maps to chromosome 5.
Table 23A. ΝON23 Nucleotide Sequence (SEQ ID NO:76)
TGGGGGCGGCTACTGCTCATGTGATTGTGGAGTAGACAGTTGGAAGAAGTACCCAGTCCATTTGGAG AGTTAAAACTGTGCCTAACAGAGGTGTCCTCTGACTTTTCTTCTGCAAGCTCCATGTTTTCACATCT TCCCTTTGACTGTGTCCTGCTGCTGCTGCTACTACTTACAACCCTGTTCTCCCGTGTTCACAGAATT GGGCCACAATTCTCTCCTAGGGCAGTGTTTCTGAAAGTGAGCAGACAAAATGGGGTAGGGAAACAAT CAGATAACGCATTTGTGTCTGGCCAGGGTGACCGCACCGGTAAAGATGAGCTATCAATCTGCATTGC ACAGCTGACCAGGAATTTCCTTGTGGGCAAAATATGGGGGAGTAGCTTCCTCTTTATTCTGTTAGAC ATGGCTTGCAGTTTTCCTGAAATGGAGTTACCTCACTCACCGCTTGAGTCTTGGCTCTCCTTCTCTC TCTATGCAGGGTCCTCAGAAGTGGAATACAGAGCGGAGGTCGGTCAGAATGCCTATCTGCCCTGCTT CTACACCCCAGCCGCCCCAGGGAACCTCGTGCCCGTCTGCTGGGGCAAAGGAGCCTGTCCTGTGTTT GAATGTGGCAACGTGGTGCTCAGGACTGATGAAAGGGATGTGAATTATTGGACATCCAGATACTGGC TAAATGGGGATTTCCGCAAAGGAGATGTGTCCCTGACCATAGAGAATGTGACTCTAGCAGACAGTGG GATCTACTGCTGCCGGATCCAAATCCCAGGCATAATGAATGATGAAAAATTTAACCTGAAGTTGGTC ATCAAACCAGCCAAGGTCACCCCTGCACCGACTCGGCAGAGAGACTTCACTGCAGCCTTTCCAAGGA TGCTTACCACCAGGGGACATGGCCCACATGATGGTGGTTCTTGTCTTTCACTTCCAGATGTAAGACT CACCCAAATATCCACATTGGCCAATGAGTTACGGGACTCTAGATTGGCCAATGACTTACGGGACTCT GGAGCAACCATCAGAATAGGCATCTACATCGGAGCAGGGATCTGTGCTGGGCTGGCTCTGGCTCTTA TCTTCGGCGCTTTAATTTTCAAATGTTATTCTCATAGCAAAGAGAAGATACAGAATTTAAGCCTCAT CTCTTTGGCCAACCTCCCTCCCTCAGGATTGGCAAATGCAGTAGCAGAGGGAATTCGCTCAGAAGAA AACATCTATACCATTGAAGAGAACGTATATGAAGTGGAGGAGCCCAATGAGTATTATTGCTATGTCA GCAGCAGGCAGCAACCCTCACAACCTTTGGGTTGTCGCTTTGCAATGCCATAG The NON23 protein (SEQ ID ΝO:77) encoded by SEQ ID NO:76 is 401 amino acid residues in length and is presented using the one-letter amino acid code in Table 23B. NON23 has one SΝP variants, whose variant positions for its nucleotide and amino acid sequences is numbered according to SEQ ID ΝOS:76 and 77, respectively. A variant sequence can include a single nucleotide polymorphism (SNP). A SNP can, in some instances, be referred to as a "cSNP" to denote that the nucleotide sequence containing the SNP originates as a cDNA. Variant 13376756 is a G to T SNP at 839 bp of the nucleotide sequence that results in an Arg to Leu change at amino acid 240 of protein sequence.
Psort analysis predicts the NOV23 protein of the invention to be localized in the endoplasmic reticulum (membrane) with a certainty of 0.6850. The Signal P predicts a likely cleavage site for a NOV23 peptide is between positions 26 and 27, i.e., at the dash in the sequence VHR-IG.
Table 23B. Encoded NOV23 protein sequence (SEQ ID NO:77) FSHLPFDCVLLLLLLLTTLFSRVHRIGPQFSPRAVFLKVSRQNGVGKQSDNAFVSGQGDRTGKD ELSICIAQLTRNFLVGKIWGSSFLFILLDMACSFPEMELPHSPLESWLSFSLYAGSSEVEYRAEV GQNAYLPCFYTPAAPGNLVPVC GKGACPVFECGNWLRTDERDVNYWTSRYWLNGDFRKGDVSL TIENVTLADSGIYCCRIQIPGIMNDEKFNLKLVIKPAKVTPAPTRQRDFTAAFPR LTTRGHGPH DGGSCLSLPDVRLTQISTLANELRDSRLANDLRDSGATIRIGIYIGAGICAGLALALIFGALIFK CYSHSKEKIQNLSLISLANLPPSGLANAVAEGIRSEENIYTIEENVYEVEEPNEYYCYVSSRQQP SQPLGCRFAMP
A search against the Patp database, a proprietary database that contains sequences published in patents and patent publications, yielded several homologous proteins shown in Table 23C.
Table 23C. Patp results for NON23
Smallest Sum
Reading High Prob
Sequences producing High- scoring Segment Pairs: Frame Score P(Ν)
>patp:AA 01049 Product 200 gene expressed T helper cells +1 1420 6.6e-153
>patp:AAY97058 Human T helper cell gene 200 product +1 1420 6.6e-153
>patp:AAB51104 Human 200 gene product +1 1420 6.6e-153
>patp:AAB59169 Human 200 gene protein +1 1420 6.6e-153
>patp:AAB81518 Human TH1 specific 200 gene product +1 1420 6.6e-153
In a BLAST search of public sequence databases, it was found, for example, that the NOV23 polypeptide of the invention was found to have 274 of 298 amino acid residues (91%) identical to, and 278 of 298 amino acid residues (93%) similar, to the 301 amino acid residue ptr:SPTREMBL-ACC:Q96K94 cDNA FLJ14428 FIS, clone HEMBA1006293 (Homo sapiens). NOV23 also has homology to the proteins shown in the BLASTP data in Table 23D.
Figure imgf000212_0002
A multiple sequence alignment is given in Table 23E, with the NOV23 protein being shown on line 1 in Table 23E in a ClustalW analysis, and comparing the NOV23 protein with the related protein sequences shown in Table 23D. This BLASTP data is displayed graphically in the ClustalW in Table 23E.
Table 23E. ClustalW Analysis of NOV23
1) > NOV23; SEQ TD NO:77
2) > Q96K94/cDNA FLJ14428 FIS [Homo sapiens]; SEQ TD NO:261
3) > AAL35776/ TIM3 [Mus musculus]; SEQ TD NO:262
4) > 054947/ Kidney Injury Molecule-1 Precursor [Rattus norvegicus]; SEQ TD NO-.263
5) > AAL35774/ TJM1 [Mus musculus]; SEQ ID NO:264
6) > 043656/ Hepatitis A Virus Cellular Receptor 1; SEQ TD NO:265
Figure imgf000212_0001
60 70 80 90 100
NOV23 DNAFVSGQGDRTGKDELSICIAQLTRNFLVGKTWGSSFLFILLDMACSFP
Q96K94 LTR
AAL35776 LAR
054947
AAL35774
043656
Figure imgf000213_0001
310 320 330 340 350
NOV23 LSLgDVR
Q96K94 GSLgDIN
AAL35776 VTLHNNN
054947 THKHEI TFYA HETTAEVTETP
AAL35774 THK SEPT-- -TFCP HETTAEVTGIP
043656 TSVSVTTTVSTFVPPMPLPRQNHEPVATSPSSPQPAETHPTTLQGAIRRE
Figure imgf000213_0002
Figure imgf000214_0001
The presence of identifiable domains in the protem disclosed herein was determined by searches using algorithms such as PROSITE, Blocks, Pfam, ProDomain, Prints and then determimng the Interpro number by crossing the domain match (or numbers) using the Interpro website (http:www.ebi.ac.uk/inte ro/). Table 23F lists the domain description from DOMAIN analysis results against NOV23.
Figure imgf000214_0002
Consistent with other known members of the immunoglobulin superfamily class of proteins, e.g., T-cell proteins, NOV23 has, for example, an immunoglobulin signature sequence and homology to the 'human gene 200' protein (WO 1049), may represent a previously unknown splice variant. NOV23 nucleic acids, and the encoded polypeptides, according to the invention are useful in a variety of applications and contexts. For example, NOV23 nucleic acids and polypeptides can be used to identify proteins that are members of the T-cell family of proteins. The NOV23 nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance NOV23 activity or function. Specifically, the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit immune function. These molecules can be used to treat inflammation, allergies, and other immune disorders.
In addition, various NOV23 nucleic acids and polypeptides according to the invention are useful, inter alia, as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins. For example, the NOV23 nucleic acids and their encoded polypeptides show homology to proteins belonging to the immunoglobulin superfamily such as kidney injury molecule-1 (KIM-1). KIM-1 seems to play a role in cell adhesion.
The basic structure of immunoglobulin (lg) molecules is a tetramer of two light chains and two heavy chains linked by disulfide bonds (Gough, Trends Biochem. Sci. 6: 203-205 (1981)). There are two types of light chains: kappa and lambda, each composed of a constant domain (CL) and a variable domain (VL). There are five types of heavy chains: alpha, delta, epsilon, gamma and mu, all consisting of a variable domain (VH) and three (in alpha, delta and gamma) or four (in epsilon and mu) constant domains (CHI to CH4). The major histocompatibility complex (MHC) molecules are made of two chains, h class I the alpha chain is composed of three extracellular domains, a transmembrane region and a cytoplasmic tail (Klein and Figueroa, Immunol. Today 7: 41-44 (1986)). The beta chain (beta-2- microglobulin) is composed of a single extracellular domain. In class II (Figueroa and Klein, J. Immunol. Today 7: 78-81 (1986)), both the alpha and the beta chains are composed of two extracellular domains, a transmembrane region and a cytoplasmic tail. It is known that the lg constant chain domains and a single extracellular domain in each type of MHC chains are related (Orr et al., Nature 282: 266-270 (1979); Cushley and Owen, Immunol. Today 4: 88-92 (1983)).
These homologous domains are approximately one hundred amino acids long and include a conserved intradomain disulfide bond. Members of the immunoglobulin superfamily are found in hundreds of proteins of different functions. Examples include antibodies, the giant muscle kinase titin and receptor tyrosine kinases. Immunoglobulin-like domains may be involved in protein-protein and protein-ligand interactions.
The NOV23 nucleic acids and polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in cellular growth and metabolism. As such the NOV23 nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to immune disorders, e.g., inflammation, allergies, autoimmune disease, and asthma.
The NOV23 nucleic acids and polypeptides are useful for detecting specific cell types. For example, expression analysis has demonstrated that a NOV23 nucleic acid is differentially expressed in mononuclear cells, B and CD4+ lymphocytes, as well as secondary Thl, -2, and Trl cells. Accordingly, the NOV23 nucleic acids, polypeptides, antibodies and related compounds according to the invention will have diagnostic and therapeutic applications in the detection of immune disorders and mflammation.
Additional utilities for NOV23 nucleic acids and polypeptides according to the invention are disclosed herein.
NOV24
A NON24 polypeptide has been identified as a Organin Anion Transporter (OAT)-like protein. The novel ΝON24 nucleic acid sequences maps to the chromosomell. Two alternative novel ΝON24, ΝON24a and ΝON24b, nucleic acids and encoded polypeptides are provided.
ΝOV24a
A NOV24 variant is the novel NOV24a (alternatively referred to herein as CG56062-01), which includes the 1741 nucleotide sequence (SEQ ID NO:78) shown in Table 24A. A NON24a ORF begins with a Kozak onsensus sequence ATG initiation codon at nucleotides 5-7 and ends with a TAA codon at nucleotides 1724-1726. Putative untranslated regions upstream from the initiation codon and downstream from the termination codon are underlined in Table 24A, and the start and stop codons are in bold letters.
Table 24A. ΝOV24a Nucleotide Sequence (SEQ ID NO:78)
GCCTATGGCCATGGCCTTCACAGACCTGCTGGATGCTCTGGGCAGCATQGGCCGCTTCCAGCTCAAG CACACAGCCCTGCTGCTGCTGCCCTGCGGCCTGCTGGCCTGCCACAACTTCCTGCAGAACTTCACCG CCGCTGTCCCCCCCCACCACTGCCGGGGCCCTGCCAACCACACTGAGGCCTCCACCAACGACTCGGG GGCCTGGCTGAGGGCCACCATACCCCTGGACCAGCTTGGGGCCCCTGAGCCCTGCCGGCGCTTCACC AAGCCTCAGTGGGCCCTGCTGAGCCCCAACTCCTCCATCCCGGGCGCGGCCACGGAGGGCTGCAAGG ACGGCTGGGTCTATAACCGCAGTGTTTTCCCGTCCACCATCGTGATGGAGGTCAGAAGGGGCTGGGT GTGTGGGGGGGCTGCTGCCGAGGCCCAGTCTGAAGCGCCCATGTCTTCCCTGCAGTGGGATCTGGTG TGTGAGGCCCGCACTCTCCGAGACCTGGCGCAGTCCGTCTACATTGCCGGGGTGCTGGTGGGGGCTG CCGTGTTTGGCAGCTTGGCAGACAGGCTGGGCTGCAAGGGCCCCCTGGTCTGGTCCTACCTGCAGCT GGCAGCTTCGGGGGCCGCCACAGCGTATTTCAGCTCCTTCAGTGCCTATTGCGTCTTCCGGTTCCTG ATGGGCATGACCTTCTCTGGCATCGTGGAGTGGATGCCCACACGGGGCCGGACTGTGGCGGGTATTT TGCTGGGGTATTCCTTCACCCTGGGCCAGCTCATCCTGGCTGGGGTAGCCTACCTGATTCGCCCCTG GCGGTGCCTGCAGTTTGCCATCTCTGCTCCTTTCCTGATCTTTTTCCTCTATTCTTGGTGGCTTCCA GAGTCATCCCGCTGGCTCCTCCTGCATGGCAAGTCCCAGTTAGCTGTACAGAATCTGCAGAAGGTGG CTGCAATGAACGGGAGGAAGCAGGAAGGGGAAAGGCTGACCAAGGAGGTGATGAGCTCCTACATCCA AAGCGAGTTTGCAAGTGTCTGCACCTCCAACTCAATCTTGGACCTCTTCCGAACCCCGGCCATCCGC AAGGTCACATGCTGTCTCATGGTGATTTGGTTCTCCAACTCTGTGGCTTACTATGGCCTGGCCATGG ACCTGCAGAAGTTTGGGCTCAGCCTATACCTGGTGCAGGCCCTGTTTGGAATCATCAACATCCCGGC CATGCTGGTGGCCACCGCCACCATGATTTACGTGGGCCGCCGTGCCACGGTGGCCTCCTTCCTCATC CTGGCCGGGCTCATGGTGATCGCCAACATGTTTGTGCCAGAAGGTACGCAGATCCTGTGCACAGCCC AGGCAGCGCTGGGCAAAGGCTGCCTGGCCAGCTCCTTCATCTGTGTGTACCTGTTTACCGGCGAGCT GTACCCCACGGAGATCAGGCAGATGGGGATGGGCTTTGCCTCTGTCCACGCCCGCCTCGGGGGCCTG ACGGCGCCCCTGGTTACCACACTTGGGGAATACAGCACCATCCTGCCACCCGTGAGCTTTGGGGCCA CCGCAATCCTGGCTGGGCTGGCCGTCTGCTTCCTGACTGAGACCCGCAACATGCCCCTGGTGGAGAC CATCGCAGCCATGGAGAGGAGGGTCAAAGAAGGCTCTTCCAAGAAACATGTAGAAGAGAAGAGTGAA GAAATTTCTCTTCAGCAGCTGAGAGCATCTCCCCTCAAAGAGACCATCTAAGCTGCCTGGAACCTG
The NON24a polypeptide (SEQ ID ΝO:79) encoded by SEQ ID NO:78 is 573 amino acid residues in length is presented using the one-letter amino acid code in Table 24B. NON24a has one SΝP variant, whose variant position for its nucleotide and amino acid sequences is numbered according to SEQ ID ΝOS:78 and 79, respectively. A variant sequence can include a single nucleotide polymorphism (SNP). A SNP can, in some instances, be referred to as a "cSNP" to denote that the nucleotide sequence containing the SNP originates as a cDNA. NON24b Variant 13374434 is a C to T SΝP at 190 bp of the nucleotide sequence that result does not result in a change in the protein sequence (silent).
The Psort profile for the ΝOV24a and NOV24b proteins predicts that this peptides are likely to be localized at the plasma membrane with a certainty of 0.6000. The Signal P predicts a likely cleavage site for a NON24a peptide is between positions 34 and 35, i.e., at the dash in the sequence LLA-CH. A similar cleavage sit is predicted between positions 41 and 42 in ΝON24b.
Table 24B. ΝON24a protein sequence (SEQ ID ΝO:79)
MAMAFTDLLDALGSMGRFQLNHTALLLLPCGLLACHNFLQNFTAAVPPHHCRGPANHTEAS TNDSGA LRATIPLDQLGAPEPCRRFTKPQWALLSPNSSIPGAATEGCKDG VYNRSVFPS TIVMEVRRG VCGGAAAEAQSEAPMSSLQ DLVCEARTLRDLAQSVYIAGVLVGAAVFGSL ADRLGCKGPLVWSYLQLAASGAATAYFSSFSAYCVFRFLMGMTFSGIVE MPTRGRTVAGI LLGYSFTLGQLILAGVAYLIRP RCLQFAISAPFLIFFLYS LPESSRWLLLHGKSQLAV QNLQKVAAMNGRKQEGERLTKEVMSSYIQSEFASVCTSNSILDLFRTPAIRKVTCCL VIW FSNSVAYYGLAMDLQKFGLSLYLVQALFGIINIPAMLVATATMIYVGRRATVASFLILAGL MVIANMFVPEGTQILCTAQAALGKGCLASSFICVYLFTGELYPTEIRQ G GFASVHARLG GLTAPLVTTLGEYSTI PPVSFGATAILAGLAVCFLTETRN PLVETIAAMERRVKEGSSK KΗVEEKSEEISLQQLRASPLKETI
NOV24b
Alternatively, a NOV24 variant is the novel NOV24b (alternatively referred to herein as CG56062-02), which includes the 1690 nucleotide sequence (SEQ ID NO:80) shown in Table
24C. NON24b sequence was cloned by the polymerase chain reaction (PCR) using the primers: 5' CATGGCCTTCACAGACCTGCT 3' (SEQ ID ΝO:266) and 5' CAGGTTCCAGGCAGCTTAGATG 3' (SEQ ID NO:267). Primers were designed based on in silico predictions of the full length or some portion (one or more exons) of the cDNA/protein sequence of the invention. These primers were used to amplify a cDNA from a pool containing expressed human sequences derived from the following tissues: adrenal gland, bone marrow, brain - amygdala, brain - cerebellum, brain - hippocampus, brain - substantia nigra, brain - thalamus, brain -whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma - Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea and uterus.
The NOV24b ORF begins with a Kozak consensus ATG initiation codon at nucleotides 5- 7 and ends with a TAA codon at nucleotides 1673-1675. Putative untranslated regions upstream from the initiation codon and downstream from the termination codon are underlined in Table 24C, and the start and stop codons are in bold letters.
Table 24C. NOV24b Nucleotide Sequence (SEQ ID NO:80)
GCCTATGGCCATGGCCTTCACAGACCTGCTCATGGCCTTCACAGACCTGCTGGATGCTCTGGGCAGC ATGGGCCGCTTCCAGCTCAACCACACAGCCCTGCTGCTGCTGCCCTGCGGCCTGCTGGCCTGCCACA ACTTCCTGCAGAACTTCACGGCCGCTGTCCCCCCCCACCACTGCCGGGGCCCTGCCAACCACACTGA GGACTCCACCAACGACTCGGGGGCCTGGCTGAGGGCCACCATACCCCTGGACCAGCTTGGGGCCCCT AAGCCCTGCCGGCGCTTCACCAAGCCTCAGTGGGCCCTGCTGAGCCCCAACTCCTCCATCCCGGGCG CGGCCACGGAGGGCTGCAAGGACGGCTGGGTCTATAACCGCAGTGTTTTCCCGTCCACCATCGTGAT GGAGTGGGATCTGGTGTGTGAGGCCCGCACTCTCCGAGACCTGGTGCAGTCCGTCTACATGGCCGGG GTGCTGGTGGGGGCTGCCGTGTTTGGCAGCTTGGCAGACAGGCTGGGCTGCAAGGGCCCCCTGGTCT GGTCCTACCTGCAGCTGGCAGCTTCGGGGGCCGCCACAGCGTATTTCAGCTCCTTCAGTGCCTATTG CGTCTTCCGGTTCCTGATGGGCATGACCTTCTCTGGCATCGTGGAGTGGATGCCCACACGGGGCCGG ACTGTGGGGGGTATTTTGCTGGGGTATTCCTTCACCCTGGGCCAGCTCATCCTGGCTGGGGTAGCCT ACCTGATTCGCCCCTGGCGGTGCCTGCAGTTTGCCATCTCTGCTCCTTTCCTGATCTTTTTCCTCTA TTCTTGGTGGCTTCCAGAGTCATCCCGCTGGCTCCTCCTGCATGGCAAGTCCCAGTTAGCTGTACAG AATCTGCAGAAGGTGGCTGCAATGAACGGGAGGAAGCAGGAAGGGGAAAGGCTGACCAAGGAGGTGA TGAGCTCCTACATCCAAAGCGAGTTTGCAAGTGTCTGCACCTCCAACTCAATCTTGGACCTCTTCCG AACCCCGGCCATCCGCAAGGTCACATGCTGTCTCATGGTGATTTGGTTCTCCAACTCTGTGGCTTAC TATGGCCTGGCCATGGACCTGCAGAAGTTTGGGCTCAGCCTATACCTGGTGCAGGCCCTGTTTGGAA TCATCAACATCCCGGCCATGCTGGTGGCCACCGCCACCATGATTTACGTGGGCCGCCGTGCCACGGT GGCCTCCTTCCTCATCCTGGCCGGGCTCATGGTGATCGCCAACATGTTTGTGCCAGAAGGTACGCAG ATCCTGTGCACAGCCCAGGCAGCGCTGGGCAAAGGCTGCCTGGCCAGCTCCTTCATCTGTGTGTACC TGTTTACCGGCGAGCTGTACCCCACGGAGATCAGGCAGATGGGGATGGGCTTTGCCTCTGTCCACGC CCGCCTCGGGGGCCTGACGGCGCCCCTGGTTACCACACTTGGGGAATACAGCACCATCCTGCCACCC GTGAGCTTTGGGGCCACCGCAATCCTGGCTGGGCTGGCCGTCTGCTTCCTGACTGAGACCCGCAACA TGCCCCTGGTGGAGACCATCGCAGCCATGGAGAGGAGGGTCAAAGAAGGCTCTTCCAAGAAACATGT AGAAGAGAAGAGTGAAGAAATTTCTCTTCAGCAGCTGAGAGCATCTCCCCTCAAAGAGACCATCTAA GCTGCCTGGAACCTG The NON24b protein (SEQ ID ΝO:81) encoded by SEQ ID NO:80 is 556 amino acid residues in length and is presented using the one-letter code in Table 24D.
Table 24D. NOV24b protein sequence (SEQ ID NO:81)
LYLAJ LAFTDLLMAFTDLLDALGSMGRFQLNHTALLLLPCGLLACHNFLQNFTAAVPPHHCRGPANHTED STNDSGA LRATIPLDQLGAPKPCRRFTKPQWALLSPNSSIPGAATEGCKDGWVYNRSVFPSTIVME DLVCEARTLRDLVQSVYMAGVLVGAAVFGSLADRLGCKGPLV SYLQLAASGAATAYFSSFSAYCV FRFLMGMTFSGIVEWMPTRGRTVAGILLGYSFTLGQLILAGVAYLIRP RCLQFAISAPFLIFFLYS WWLPESSR LLLHGKSQLAVQNLQKVAAMNGRKQEGERLTKEV SSYIQSEFASVCTSNSILDLFRT PAIRKVTCCLMVI FSNSVAYYGLAMDLQ FGLSLYLVQALFGIINIPA LVATATMIYVGRRATVA SFLILAGLMVIANMFVPEGTQILCTAQAALGKGCLASSFICVYLFTGELYPTEIRQMGMGFASVHAR LGGLTAPLVTTLGEYSTILPPVSFGATAILAGLAVCFLTETRNMPLVETIAAMERRVKEGSSKKHVE EKSEEISLQQLRASPLKETI
NOV24 Clones
Unless specifically addressed as NON24a or ΝON24b, any reference to ΝON24 is assumed to encompass all variants. ΝON24b polypeptide sequence is 17 amino acids shorter than ΝON24a polypeptide and also has 13 different amono acids shown in Table 24E.
Table 24E. Information for the ClustalW proteins:
10 20 30 40 50
ΝOV24a MAFTDLLDALGSMGRFQLNHTALLLLPCGLLACHNFLQNF' NOV24b lAMAFTDLL AFTDLLDALGSMGRFQLNHTALLLLPCGLLACHNFLQNF1:
Figure imgf000219_0001
160 170 180 190 200
NOV 4a EAPMSSLO D t^ JB35^!S^3roi^^-W AA*iJe^lb JiRJLlGrfC«tKMGdPjLV NOV24b ARTLRDLVOSVYMAGBLVGAAVFGSLADI'" KGPLV
210 220 230 240 250
NOV24a WSYLQLAASGAATAYFSSFSAYCVFRFLMGMTFSGIVEWflPTRGRTVAGI NOV24b WSYLQLAASGAATAYFSSFSAYCVFRFLMGMTFSGIVEWMPTRGRTVAGI
260 270 280 290 300
NOV24a LLGYSFΠLGQLILAGVAYLIRPWRCLQFAISAPFLIFFLYSWWLPESSRW NOV24b LGYSFBLGQLILAGVAYLIRPWRCLQFAISAPFLIFFLYS WLPESSRWI
Figure imgf000220_0001
360 370 380 390 400
NOV24a ftHftWII KMlfi N0V24b ilLDLFRTPAIRKVTCCLMVIWFSNSVAYYGLAMDLOKFGLSLYLVOAL
410 420 430 440 450
NOV24a ;iINIPAMLVATraTMIYVGRRATVASFLILAGLMVIANMFVPEGTQILC NOV24b ΪIINIPAMLVATBTMIYVGRRATVASFLILAGLMVIANMFVPEGTQILC^
460 470 480 490 500
NOV24a IVQAALGKGCLAS S F I CVYLFTGEJYPTE IRQMGMGFASVHΘRLGGLTAPL NOV 4b ^QAALGKGCLASSFICVYLFTGELYPTEIRQMGMGFASVHIRLGGLTAPL
510 520 530 540 550
NOV24a fTTLGEYSTILPPVSFGATAILAGLAVCFLTETRNMPLVETIAAMERRVI NOV24b fTTLGEYSTILPPVSFGATAILAGLAVCFLTETRNMPLVETIAAMERRVI
560 570
NOV24a nsinaam aatanaiw Qfflffi N0V 4b
A search against the Patp database, a proprietary database that contains sequences published in patents and patent publications, yielded several homologous proteins shown in Table 24F and Table 24G.
Table 24F. Patp results for NOV24a
Smallest Sum
Reading High Prob
Sequences producing High-scoring Segment Pairs: Frame Score P(N)
>patp:AAB36553 Mouse organic anion transporter 5 (OATP5) +1 978 l.le-126
>patp:AAY92903 Rat cerebral OAT3 +1 1009 6.6e-125
>patp:AAB47274 hOAT3 +1 992 3.2e-123
>patp:AAY92902 Human cerebral OAT3 +1 991 4.1e-123
>patp:AAW44195 Mouse osteoclast transporter protein +1 990 9.7e-122
Table 24G. Patp results for NOV24b
Smallest Sum
Reading High Prob
Sequences producing High- scoring Segment Pairs: Frame Score P(N)
>patp:AAY44278 Human organic anion transporter +1 1256 1.0e-127
>patp:AAB47271 hOATl +1 1256 1.0e-127
>patp.-AA 88 88 Rat organic anion transporter OAT-1 +1 1254 1.6e-127
>patp:AA 88489 Human organic anion transporter OAT-1 +1 1249 5.5e-127
>patp:AAY92903 Rat cerebral 0AT3 +1 1239 6.3e-126 In a BLAST search of public sequence databases, it was found, for example, that the NOV24a nucleic acid sequence of this invention has 680 of 1082 bases (62%) identical to a gb:GENBANK-ID:OCU242871|acc:AJ242871.1 mRNA from Oryctolagus cuniculus (Oryctolagus cuniculus mRNA for renal organic anion transporter 1 (rbOATl)) (Fig. 3A). NON24a polypeptide was found to have 196 of 424 amino acid residues (46%) identical to, and 277 of 424 amino acid residues (65%) similar to, the 536 amino acid residue ptar.SPTREMBL- ACC:Q9R1U7 protein from Rattus norvegicus (ORGANIC ANION TRANSPORTER 3).
Similarly, it was found, for example, that the NON 24b nucleic acid sequence of this invention has 713 of 1132 bases (62%) identical to a gb:GEΝBAΝK- ID:AF097491|acc:AF097491.1 mRNA fromHomo sapiens (Homo sapiens organic anion transporter 3 (OAT3) mRNA, complete eds). NOV24b was found to have 246 of 522 amino acid residues (47%) identical to, and 328 of 522 amino acid residues (62%) similar to, the 563 amino acid residue ρtnr:SPTREMBL-ACC:O95742 protein from Homo sapiens (RENAL ORGANIC ANION TRANSPORT PROTEIN 1).
Additional BLAST results are shown in Table 24Η and Table I.
Table 24H. BLAST results for NOV24a
Gene Index/ Protein/ Organism Length Identity Positives Expect Identifier (aa) (%) ____. gi 117472512 I ref |XP_ similar to 705 338/400 341/400 0.0 061724.1 I (XM 061724 putative renal (84%) (84%) organic anion transporter 1 [Homo sapiens] gi|3831566|gb|AAC70 putative renal 550 236/550 314/550 le-119 004. ll (AF057039) organic anion (42%) (56%) transporter 1 [Homo sapiens] gi I 4759042 I ref |NP_0 solute carrier 550 236/550 314/550 le-118 04781. ll (NM 004790) family 22 (42%) (56%) (organic anion transporter) , member 6 ; renal organic anion transporter 1 [Homo sapiens] gi I 4579723 | dbj |BAA7 hOATl-1 [Homo 563 236/550 314/550 le-ll£ 5072.1) (AB009697) sapiens] (42%) (56%) gi|2687858|emb|CAB0 renal organic 562 237/557 323/557 le-llf 9724. ll (Z97028) anion transporter (42%) (57%) [Pseudopleuronect es americanus]
Figure imgf000222_0001
A multiple sequence alignment is given in Table 24J, with the NOV24a protein of the invention being shown on line 1, in a ClustalW analysis comparing NOV24a with related protein sequences disclosed in Table 24H.
Table 24J. Information for the ClustalW proteins (NON24a):
1. >ΝOV24a; SEQ TD NO:79
2. >gi|17472512|/ similar to putative renal organic anion transporter [Homo sapiens]; SEQ DD NO:268
3. >gi|3831566|/ putative renal organic anion transporter 1 [Homo sapiens]; SEQ TD NO:269
4. >gi|4759042|/ solute carrier family 22 [Homo sapiens]; SEQ TD NO:270
5. >gi|4579723|/ hOATl-1 [Homo sapiens]; SEQ D NO:271
6. >gi|2687858|/ renal organic anion transporter [Pseudopleuronectes americanus]; SEQ DD NO:272
Figure imgf000223_0001
Figure imgf000224_0001
460 470 480 490 500
NOV24a gi|l747251 TRPAQSCPGNRRFGSRTPGLANRTR IGAMSKCFASLPAGSRAGLAPGIN gi|3831566 gi|4759042 gi|4579723 gi|2687858
A similar multiple sequence alignment is given in Table 24K, with the NOV24b protein of the invention being shown on line 1, in a ClustalW analysis comparing NON24b with related protein sequences disclosed in Table 241.
Table 24K. Information for the ClustalW proteins (ΝON24b): 1. >ΝOV24b; SEQ DD NO:'81
2. >gi|17472512|/ similar to putative renal organic anion transporter 1 [Homo sapiens]; SEQ DD NO:273 3. >gi|4579723|/ hOATl-l [Homo sapiens]; SEQ DD NO:274
4. >gi|3831566|/ putative renal organic anion transporter 1 [Homo sapiens]; SEQ DD NO:275
5. >gi|4759042|/ solute carrier family 22 [Homo sapiens]; SEQ DD NO:276
6. >gi|8393886|/ organic cationic transporter-like 1 [Rattus norvegicus]; SEQ DD NO:277
10 20 30 40 50
NOV24b MAM FTDLLl!l^τBHtiDALiaSMB!3Mτ.ιNH ffll.LB3cGiiLSci!iSFiBBΪ gi I 4579723 ΦiFNDLLQQVGGVGRFQQIQVTLWLPLLLMASHNTLQNF' gi]3831566 AFNDLLQQVGGVGRFQQIQVTLWLPLLLMASHNTLQNFI gi|4759042 AFNDLLQQVGGVGRFQQ I QVTL WLPLLLMASHNTLQNFI gi I 8393886 ιaraa ιwnιK[»gι«w. )aat]τ.fwwi.iMaϊB i3iιιιιιa aa:i> M-iW>ι
Figure imgf000225_0001
210 220 230 240 250
NOV2 b laviaa iSMmo -iMalBIBiaTRGiaTVAmiWILiMMSFTWca gi|4579723 AFRLLSGMALAGISLNCMTLNVEWMPIHTRACVGTLIGYVYSLGQFL gi|3831566 'CAFRLLSGMALAGISLNCMTLNVEWMPIHTRACVGTLIGYVYSLGQFL gi j 4759042 rCAFRLLSGMALAGISLNCMTLNVEWMPIHTRACVGTLIGYVYSLGQF gi|8393886 K/GTLIGYVYSLG
NOV24b AGVAYIHimaWRWILOlajaHSAPF gi|4579723 AGVAYAVPHWRHLQLLVSAPFFAFFIYSWFFIESARWHSSSGRLDLTLRA gi |3831566 AGVAYAVPHWRHLQLLVSAPFFAFFIYSWFFIESARWHSSSGRLDLTLR gi|4759042 AGVAYAVPHWRHLQLLVSAPFFAFFIYSWFFIESARWHSSSGRLDLTLR gi|8393886 l^ιι»»ΛttagiMa.ιι»i-ft7-Bavιs^
310 320 330 340 350
NOV24b !raκEMβRKQi^E 3τκi^SSYlHs FASVCTS ilLD|iFJ3τ|3Aliaκ gi|4579723 LQRVARINGKREEGAKLSMEVLRASLQKELTMGKGQASAMELLRCPTLRH gi|3831566 LQRVARINGKREEGAKLSMEVLRASLQKELTMGKGQASAMELLRCPTLRH gi|4759042 LQRVARINGI REEGAKLSMEVLRASLQKELTMGKGQASAMELLRCPTLRH gi I 8393886 WfWj;>«>w:θ)aawΛ:««wτiBit aMτ,i-««r>)i«aw>τ,s)i t{*h JfaWiBiι<>i>r ι>dιrt*ifii
Figure imgf000226_0001
410 420 430 440 450
.■•■ l ....| ....l ....) ....l ....l ....| ....| ....l ....l
NOV24b TMΠ ATVASFffllH^LM IASlMFvΘEGTQH CiΪAQSABB^SEsE gi I 4579723 NSLGRRPAQMAALLLAGICILLNGVIPQDQSIVRTSLAVLGKGCLAASi gi|3831566 4/INSLGRRPAQMAALLLAGI CILLNGVI PQDQS IVRTSLAVLGKGCLAAS gi|4759042 tfTNSLGRRPAQMAALLLAGICILLNGVIPQDQSIVRTSLAVLGKGCLAAS gi I 8393886 M«^>-Mwa;ijiWιawsii)aftτe.<rt*-wι«eiι>«aκsHTMii:aiMWi JKrt;<««iffj,qκ
460 470 480 490 500
NOV24b 3ι^γilFBB3iBSE!is βSFivHSaLHGLra ΓTLGIYSTILSP gi I 4579723 FNCIFLYTGELYPTMIRQTGMGMGSTMARVGSIVSPLVSMTAELYPSMPL gi j 3831566 FNCIFLYTGELYPTMIRQTGMGMGSTMARVGSIVSPLVSMTAELYPSMPL gi|4759042 FNCIFLYTGELYPTMIRQTGMGMGSTMARVGSIVSPLVSMTAELYPSMPL gi j 8393886 ιιι\Ha»ιικciL,mαιcιai-araaιcB«--τ/aa-iaaa»ifaajH-«aaαιa--
510 520 530 540 550
NOV24b VSF^TAILΞGLAVCFBτ[^RN {S3vEgiAA Rig- - ■-VK---EGSS gi|4579723 FIYGAVPVAASAVTVLLPETLGQPLPDTVQDLES PTQKEAGIYPR gi(3831566 FIYGAVPVAASAVTVLLPETLGQPLPDTVQDLES gi j 4759042 FIYGAVPVAASAVTVLLPETLGQPLPDTVQDLES gi j 8393886 ASAVTΘLLPETLGQPLPDTVQDL
560 570
NOV24b gKHVEEKSEEISLQQ iJgR; gEP KE I - gi|4579723 gi|3831566 CQTRQQQEHQKYMVPLQASAQEKNG gi j 759042 CQTRQQQEHQKYMVPLQASAQEKNGL gi j 8393886
The presence of identifiable domains in the NOV24 proteins disclosed herein was determined by searches using algorithms such as PROSITE, Blocks, Pfam, ProDomain, Prints and then determining the Interpro number by crossing the domain match (or numbers) using the Interpro website (http:www.ebi.ac.uk/interpro/). The DOMAIN results are listed in Table 24L and Table 24M with the statistics and domain description. This indicates that the NOV24 sequences have properties similar to those of other proteins known to contain these domains.
Figure imgf000226_0002
I protein
Figure imgf000227_0001
Consistent with other known members of the OAT family of proteins, NON24 has, for example, sugar transporter domain and homology to other members of the OAT Protein family. ΝON24 nucleic acids, and the encoded polypeptides, according to the invention are useful in a variety of applications and contexts. For example, ΝON24 nucleic acids and polypeptides can be used to identify proteins that are members of the OAT family of proteins. The ΝON24 nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance ΝON24 activity or function. Specifically, the nucleic acids and polypeptides according to the invention maybe used as targets for the identification of small molecules that modulate or inhibit, e.g., molecular transport. These molecules can be used to treat, e.g., cancer, immune disorders, and kidney disorders.
In addition, various ΝON24 nucleic acids and polypeptides according to the invention are useful, inter alia, as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins. It is known that many members of the organic anion transporter (OAT), organic cation transporter (OCT), and organic anion- transporting polypeptide (oatp) gene families mediate the transport of diverse organic anions and cations. It has also been suggested that ATP-dependent primary active transporters such as MDRl/P-glycoprotein and the multidrug resistance-associated protein (MRP) gene family function as efflux pumps of renal tubular cells for more hydrophobic molecules and anionic conjugates.
A number transporters, such as the p-aminohippurate/dicarboxylate exchanger OAT1, the anion/sulfate exchanger SAT1, the peptide transporters PEPT1 and PEPT2, and the nucleoside transporters CΝT1 and CΝT2, are key proteins in organic anion handling that possess the same characteristics as has been predicted from previous physiological studies. The role of other cloned transporters, such as MRP1, MRP2, OATP1, OAT-K1, and OAT-K2, is still poorly characterized, whereas the only information that is available on the homologs OAT2, OAT3, OATP3, and MRP3-6 is that they are expressed in the kidney, but their localization, not to mention their function, remains to be elucidated. The organic anion transporter 3 belongs to sugar transporter family. The sugar transporters belong to a family of membrane proteins responsible for the transport of various sugars in a wide range of prokaryotic and eukaryotic organisms. These integral membrane proteins are predicted to comprise twelve membrane spanning domains. It is likely that the transporters have evolved from an ancient protein present in living organisms before the divergence into prokaryotes and eukaryotes. In mammals, these proteins are expressed in a number of organs.
The NON24 nucleic acids and polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in the mediation of molecular transport. As such the ΝON24 nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat a wide range of disorders such as cancer, kidney disorders, immune disorders and other diseases, e.g., Non Hippel-Lindau (NHL) syndrome, Cirrhosis,Transplantation, Osteoporosis, Hypercalceimia, Arthritis, Ankylosing spondylitis, Scoliosis, Diabetes, Autoimmune disease, Renal artery stenosis, Interstitial nephritis, Glomerulonephritis, Polycystic kidney disease, Systemic lupus erythematosus, Renal tubular acidosis, IgA nephropathy, Lesch-Νyhan syndrome renal malfunction, nephrotoxicity, disease associated with cytotoxic drug, osteoporosis, osteopetrosis resistance, liver diseases, and heart diseases.
The ΝON24 nucleic acids and polypeptides are useful for detecting specific cell types. For example, expression analysis has demonstrated that a ΝON24 nucleic acid is expressed in Bone Marrow, Kidney, Intestine, Liver membrane, adrenal gland, bone marrow, brain - amygdala, brain - cerebellum, brain - hippocampus, brain - substantia nigra, brain - thalamus, brain -whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma - Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea and uterus.
Additional utilities for ΝON24 nucleic acids and polypeptides according to the invention are disclosed herein. NON25
A ΝON25 polypeptide has been identified as a Ficolin-like protein. Eight alternative novel ΝON25, ΝON25a, ΝON25b, ΝON25c, ΝON25d, ΝON25e, ΝON25f, ΝON25g, andΝON25h, nucleic acids and encoded polypeptides are provided. The novel ΝON25 nucleic acid sequences maps to the chromosome 9q34. ΝOV25a
A NOV25 variant is the novel NOV25a (alternatively referred to herein as 152736829), which includes the 1082 nucleotide sequence (SEQ ID NO: 82) shown in Table 25 A. NOV25a sequence was cloned by polymerase chain reaction (PCR) using the following primers: GCTCGCTGTCCTGCTAGTCTTGTT (SEQ ID NO:278) and
AGAAACATAATTCTCCCTCTGGTGAGG (SEQ ID NO:279) on the following pool of human cDNAs: Pool 1 - Adrenal gland, bone marrow, brain - amygdala, brain - cerebellum, brain - hippocampus, brain - substantia nigra, brain - thalamus, brain -whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma - Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea, uterus. A NON25a ORF begins with a ATG initiation codon at nucleotides 16-18 and ends with a TAG codon at nucleotides 928-930. Putative untranslated regions upstream from the initiation codon and downstream from the termination codon are underlined in Table 25 A, and the start and stop codons are in bold letters.
Table 25A. ΝON25a Nucleotide Sequence (SEQ ID NO:82)
CTGAGTGGAGCCACCATGGCCCGGGGGCTCGCTGTCCTGCTAGTCTTGTTCCTGCATATCAAGAACC TGCCTGCCCAGGCTGCGGACACATGTCCAGAGGTGAAGGTGGTGGGCCTGGAGGGCTCTGACAAGCT CACCATTCTCCGAGGCTGCCCGGGGCTGCCCGGGGCCCCAGGGCCAAAGGGAGAGGCAGGTGTCATT GGAGAGAGAGGAGAACGCGGTCTCCCTGGAGCCCCTGGAAAGGCAGGACCAGTGGGGCCCAAAGGAG ACCGAGGAGAGAAGGGGATGCGTGGAGAGAAAGGAGACGCTGGGCAGTCTCAGTCGTGTGCGACAGG CCCACGCAACTGCAAGGACCTGCTAGACCGGGGGTATTTCCTGAGCGGCTGGCACACCATCTACCTG CCCGACTGCCGGCCCCTGACTGTGCTCTGTGACATGGACACGGACGGAGGGGGCTGGACCGTTTTCC AGCGGAGGATGGATGGCTCTGTGGACTTCTATCGGGACTGGGCCGCATACAAGCAGGGCTTCGGCAG TCAGCTGGGGGAGTTCTGGCTGGGGAACGACAACATCCACGCCCTGACTGCCCAGGGAAGCAGCGAG CTCCGTGTAGACCTGGTGGACTTTGAGGGCAACCACCAGTTTGGTAAGTACAAATCATTCAAGGTGG CTGACGAGGCAGAGAAGTACAAGCTGGTACTGGGAGCCTTTGTCGGGGGCAGTGCGGACCAAGACAA TGATGTGAGTTCTTCGAATTGTGCTGAGAAGTTCCAGGGAGCCTGGTGGTACGCCGACTGTCATGCT TCAAACCTCAATGGTCTCTACCTCATGGGACCCCATGAGAGCCATGCCAATGGTATCAACTGGAGTG CGGCGAAGGGGTACAAATATAGCTACAAGGTGTCAGAGATGAAGGTGCGGCCCGCCTAGACGGGCCA GGACCCCTCCACATGCACCTGCTAGTGGGGAGGCCACACCCACAAGCGCTGCGTCGTGGAAGTCACC CCATTTCCCCAGCCAGACACACTCCCATGACGCCCACAGCTGCCCCTTTGCCCCCAGCTCAGTCAAG CCGCCACATG The NON25a polypeptide (SEQ ID ΝO:83) encoded by SEQ ID NO:82 is 304 amino acid residues in length and is presented using the one-letter amino acid code in Table 25B. NON25a has one SΝP variant, whose variant position for its nucleotide and amino acid sequences is numbered according to SEQ ID ΝOS:82 and 83, respectively. A variant sequence can include a single nucleotide polymorphism (SNP). A SNP can, in some instances, be referred to as a "cSNP" to denote that the nucleotide sequence containing the SNP originates as a cDNA. NOV25a Variant 13374708 is a G to A SNP at 774 bp of the nucleotide sequence that results in no change in the protein sequence (silent). The Psort profile for the NOV25a predicts that this peptide is likely to be localized extracellularly with a certainty of 0.0.4944. The Signal P predicts a likely cleavage site for a NOV25a peptide is between positions 22 and 23, i.e., at the dash in the sequence AQA-AD.
Table 25B. NOV25a protein sequence (SEQ ID NO:83)
MARGLAVLLVLFLHIKNLPAQAADTCPΞVKWGLEGSDKLTILRGCPGLPGAPGPKGEAGVIGERGE RGLPGAPGKAGPVGPKGDRGEKGMRGEKGDAGQSQSCATGPRNCKDLLDRGYFLSGWHTIYLPDCRP LTVLCDMDTDGGG TVFQRRMDGSVDFYRDAAYKQGFGSQLGEF LGNDNIHALTAQGSSΞLRVDL VDFEGNHQFAKYKSFKVADEAEKYKLVLGAFVGGSADQDNDVSSSNCAEKFQGA YADCHASNLNG LYLMGPHESHANGINWSAAKGYKYSYKVSEMKVRPA
NOV25b
Alternatively, a NOV25 variant is the novel NON25b (alternatively referred to herein as CG56653-02), which includes the 1332 nucleotide sequence (SEQ ID ΝO:84) shown in Table 25C. NOV25b was cloned by polymerase chain reaction (PCR) using the following primers: GCTCGCTGTCCTGCTAGTCTTGTT (SEQ ID NO:280) and
AGAAACATAATTCTCCCTCTGGTGAGG (SEQ ID NO:281) on the following pool of human cDNAs: Pool 1 - Adrenal gland, bone marrow, brain - amygdala, brain - cerebellum, brain - hippocampus, brain - substantia nigra, brain - thalamus, brain -whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma - Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea, uterus. The NON25b ORF begins with a Kozak consensus ATG initiation codon at nucleotides 183-185 and ends with a TAG codon at nucleotides 1107-1109. Putative untranslated regions upstream from the initiation codon and downstream from the termination codon are underlined in Table 25C, and the start and stop codons are in bold letters.
Table 25C. NOV25b Nucleotide Sequence (SEQ ID NO:84)
TTTTAGGTCTGTTTGTCGTAGGCAGATGGAGCTTGTTATAATTATGCCTCATAGGGATAGTACAAGG AAGGGGTAGGCTATGTGTTTTGTCAGGGAGTTGAGAAACTGTGGCACAAGGCGAGAGCTGGTTTCCT CTGCCCTGTTAGAGCTGGGGGACTCTTCAGAGTCAAAGGCCAGAGAGCATGGAGCTGAGTGGAGCCA CCATGGCCCGGGGGCTCGCTGTCCTGCTAGTCTTGTTCCTGCATATCAAGAACCTGCCTGCCCAGGC TGCGGACACATGTCCAGAGGTGAAGGTGGTGGGCCTGGAGGGCTCTGGCAAGCTCACCATTCTCCGA GGCTGCCCGGGGCTGCCCGGGGCCCCAGGGCCAAAGGGAGAGGCAGGTGTCATTGGAGAGAGAGGAG ACCGAGGAGAGAAGGGGATGCGTGGAGAGAAAGGAGACGCTGGGCAGTCTCAGTCGTGTGCGACAGG CCCACGCAACTGCAAGGACCTGCTAGACCGGGGGTATTTCCTGAGCGGCTGGCACACCATCTACCTG CCCGACTGCCGGCCCCTGACTGTGCTCTGTGACATGGACACGGACGGAGGGGGCTGGACCGTTTTCC AGCGGAGGATGGATGGCTCTGTGGACTTCTATCGGGACTGGGCCGCATACAAGCAGGGCTTCGGCAG TCAGCTGGGGGAGTTCTGGCTGGGGAATGACAACATCCACGCCCTGACTGCCCAGGGAAGCAGCGAG CTCCGTGTAGACCTGGTGGACTTTGAGGGCAACCACCAGTTTGCTAAGTACAAATCATTCAAGGTGG CTGACGAGGCAGAGAAGTACAAGCTGGTACTGGGAGCCTTTGTCGGGGGCAGTGCGGGTAATTCTCT AACGGGCCACAACAACAACTTCTTCTCCACCAAAGACCAAGACAATGATGTGAGTTCTTCGAATTGT GCTGAGAAGTTCCAAGGAGCCTGGTGGTACGCCGACTGTCATGCTTCAAACCTCAATGGTCTCTACC TCATGGGACCCCATGAGAGCTATGCCAATGGTATCAACTGGAGTGCGGCGAAGGGGTACAAATATAG CTACAAGGTGTCAGAGATGAAGGTGCGGCCCGCCTAGACGGGCCAGGACCCCTCCACATGCACCTGC TAGTGGGGAGGCCACACCCACAAGCGCTGCGTCGTGGAAGTCACCCCATTTCCCCAGCCAGACACAC TCCCATGACGCCCACAGCTGCCCCTTTGCCCCCAGCTCAGTCAAGCCGCCACATGCCCACAACCTCA CCAGAGGGAGAATTATGTTTCTAAATATGTTTACTTTTGGGGACAGAAAAAAAAAAAAA
The NON25b protein (SEQ ID ΝO:85) encoded by SEQ ID NO:84 is 308 amino acid residues in length is presented using the one-letter code in Table 25D. The Psort profile for NOV25b predicts that this sequence is likely to be localized extracellularly with a certainty of 0.4500. The Signal P predicts a likely cleavage site for a NOV25b peptide is between positions 29 and 30, i.e., at the dash in the sequence AQA-AD.
Table 25D. NOV25b protein sequence (SEQ ID NO:85)
MELSGATMARGLAVLLVLFLHIKNLPAQAADTCPEVKWGLΞGSGKLTILRGCPGLPGAPGPKGEAG VIGERGDRGEKGMRGEKGDAGQSQSCATGPRNCKDLLDRGYFLSGHTIYLPDCRPLTVLCDMDTDG GGWTVFQRRMDGSVDFYRDWAAYKQGFGSQLGEF LGNDNIHALTAQGSSELRVDLVDFEGNHQFAK Y SFKVADEAEKYKLVLGAFVGGSAGNSLTGHNNNFFST DQDNDVSSSNCAEKFQGAWYADCHAS NLNGLYLMGPHESYANGINWSAAKGYKYSYKVSEMKVRPA
NOV25c Alternatively, a NOV25 variant is the novel NOV25c (alternatively referred to herein as
CG56653-03), which includes the 728 nucleotide sequence (SEQ ID NO:86) shown in Table 25E. NOV25c was cloned by the polymerase chain reaction (PCR) using the primers: 5' GCTCGCTGTCCTGCTAGTCTTGTT 3' (SEQ ID NO:282) and 5*
AGAAACATAATTCTCCCTCTGGTGAGG 3' (SEQ ID NO:283). Primers were designed based on in silico predictions of the full length or some portion (one or more exons) of the cDNA/protein sequence of the invention. These primers were used to amplify a cDNA from a pool containing expressed human sequences derived from the following tissues: adrenal gland, bone marrow, brain - amygdala, brain - cerebellum, brain - hippocampus, brain - substantia nigra, brain - thalamus, brain -whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma - Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea and uterus. The NON25c ORF begins with an ORF identified at nucleotides 1-2 and ends with a TAG codon at nucleotides 574-576. Putative untranslated regions upstream from the initiation codon and downstream from the termination codon are underlined in Table 25E, and the start and stop codons are in bold letters.
Table 25E. ΝON25c Nucleotide Sequence (SEQ ID NO:86)
CTGCATATCAAGAACCTGCCTGCCCAGGCTGCGGACACATGTCCAGAGGTGAAGGTGGTGGGCCTGG AGGGCTCTGACAAGCTCACCATTCTCCGAGGCTGCCCGGGGCTGCCCGGGGCCCCAGGGCCAAAGGG AGAGGCAGGTGTCATTGGAGAGAGAGGAGAACGCGGTCTCCCTGGAGCCCCTGGAAAGGCAGGACCA GTGGGGCCCAAAGGAGACCGAGGAGAGAAGGGGATGCGTGGAGAGAAAGGAGACGCTGGGCAGTCTC AGTCGTGTGCGACAGGCCCACGCAACTGCAAGGACCTGCTAGACCGGGGGTATTTCCTGAGCGGCTG GCACACCATCTACCTGCCCGACTGCCGGCCCCTGACTGTGCTCTGTGACATGGACACGGACGGAGGG GGCTGGACCGTTTTCCAGGGAGCCTGGTGGTACGCCGACTGTCATGCTTCAAACCTCAATGGTCTCT ACCTCATGGGACCCCATGAGAGCTATGCCAATGGTATCAACTGGAGTGCGGCGAAGGGGTACAAATA TAGCTACAAGGTGTCAGAGATGAAGGTGCGGCCCGCCTAGACGGGCCAGGACCCCTCCACATGCACC TGCTAGTGGGGAGGCCACACCCACAAGCGCTGCGTCGTGGAAGTCACCCCATTTCCCCAGCCAGACA CACTCCCATGACGCCCACAGCTGCCCCTTTGCCCCCAGCTCAGTCAAGCCGCCACATG
The NOV25c protein (SEQ ID NO:87) encoded by SEQ ID NO:86 is 191 amino acid residues in length is presented using the one-letter code in Table 25F. The Psort profile for NOV25c predicts that this sequence is likely to be localized in the cytoplasm with a certainty of 0.4500.
Table 25F. NOV25c protein sequence (SEQ ID NO:87)
LHIKNLPAQAADTCPEVKWGLEGSDKLTILRGCPGLPGAPGPKGEAGVIGERGERGLPGAPGKAGP VGPKGDRGEKGMRGEKGDAGQSQSCATGPRNCKDLLDRGYFLSG HTIYLPDCRPLTVLCDMDTDGG G TVFQGA YADCHASNLNGLYLMGPHESYANGIN SAAKGYKYSYKVSEMKVRPA NOV25d Alternatively, a NOV25 variant is the novel NOV25d (alternatively referred to herein as
CG56653-04), which includes the 1104 nucleotide sequence (SEQ ID NO:88) shown in Table 25G. NOV25d was cloned by the polymerase chain reaction (PCR). Primers were designed based on in silico predictions of the full length or some portion (one or more exons) of the cDNA/protein sequence of the invention. These primers were used to amplify a cDNA from a pool containing expressed human sequences derived from the following tissues: adrenal gland, bone marrow, brain - amygdala, brain - cerebellum, brain - hippocampus, brain - substantia nigra, brain - thalamus, brain -whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma - Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea and uterus. The NOV25d ORF begins with a Kozak consensus ATG initiation codon at nucleotides 16-18 and ends with a TAG codon at nucleotides 883-885. Putative untranslated regions upstream from the imtiation codon and downstream from the termination codon are underlined in Table 25G, and the start and stop codons are in bold letters.
Table 25G. NOV25d Nucleotide Sequence (SEQ ID NO:88)
CTGAGTGGAGCCACCATGGCCCGGGGGCTCGCTGTCCTGCTAGTCTTGTTCCTGCATATCACGAACC TGCCTGCCCAGGCTGCGGACACATGTCCAGAGGTGAAGGTGGTGGGCCTGGAGGGCTCTGACAAGCT CACCATTCTCCGAGGCTGCCCGGGGCTGCCCGGGGCCCCAGGACCAAAGGGAGAGGCAGGTGTCATT GGAGAGAAAGGAGACGCTGGGCAGTCTCAGTCGTGTGCGACAGGCCCACGCAACTGCAAGGACCTGC TAGACCGAGGGTATTTCCTGAGCGGCTGGCACACCATCTACCTGCCCGACTGCCGGCCCCTTACTGT GCTCTGTGACATGGATACGGACGGAGGGGGCTGGACCGTTTTCCAGCGGAGGATGGATGGCTCTGTG GACTTCTATCGGGACTGGGCCGCATACAAGCAGGGCTTCGGCAGTCAGCTGGGGGAGTTCTGGCTGG GGAATGACAACATCCACGCCCTGACTGCCCAGGGAAGCAGCGAGCTCCGTGTAGACCTGGTGGACTT TGAGGGCAACCACCAGTTTGCTAAGTACAAATCATTCAAGGTGGCTGACGAGGCAGAGAAGTACAAG CTGGTACTGGGAGCCTTTGTCGGGGGCAGTGCGGGTAATTCTCTAACGGGCCACAACAACAACTTCT TCTCCACCAAAGACCAAGACAATGATGTGAGTTCTTCGAATTGTGCTGAGAAGTTCCAGGGAGCCTG GTGGTACGCCGACTGTCATGCTTCAAACCTCAATGGTCTCTACCTCATGGGACCCCATGAGAGCTAT GCCAATGGTATCAACTGGAGTGCGGCGAAGGGGTACAAATATAGCTACAAGGTGTCAGAGATGAAGG TGCGGCCCGCCTAGACGGGCCAGGACCCCTCCACATGCACCTGCTAGTGGGGAGGCCACACCCACAA GCGCTGCGTCGTGGAAGTCACCCCATTTCCCCAGCCAGACACACTCCCATGACGCCCACAGCTGC.ee CTTTGCCCCCAGCTCAGTCAAGCCGCCACATGCCCACAACCTCACCAGAGGGAGAATTATGTTTCTA AATATGTTTACTTTGGGACAGAAAAAAAAAAA The NOV25d protein (SEQ ID NO:89) encoded by SEQ ID NO:88 is 289 amino acid residues in length is presented using the one-letter code in Table 25H. The Psort profile for NOV25d predicts that this sequence is likely to be localized extracellularly with a certainty of 0.6472. The Signal P predicts a likely cleavage site for a NOV25d peptide is between positions 22 and 23, i.e., at the dash in the sequence AQA-AD.
Table 25H. NOV25d protein sequence (SEQ ID NO:89)
MARGLAVLLVLFLHITNLPAQAADTCPEVKWGLEGSDKLTILRGCPGLPGAPGPKGEAGVIGEKGD AGQSQSCATGPRNCKDLLDRGYFLSGWHTIYLPDCRPLTVLCDMDTDGGG TVFQRRMDGSVDFYRD WAAYKQGFGSQLGEFWLGNDNIHALTAQGSSELRVDLVDFEGNHQFAKYKSFKVADEAEKYLVLGA FVGGSAGNSLTGHNNNFFSTKDQDNDVSSSNCAEKFQGAW YADCHASNLNGLYLMGPHESYANGIN SAAKGYKYSYKVSE KVRPA
NON25e
Alternatively, a ΝON25 variant is the novel ΝON25e (alternatively referred to herein as CG56653-06), which includes the 988 nucleotide sequence (SEQ ID ΝO:90) shown in Table 251. NON25e was cloned by the polymerase chain reaction (PCR) using the primers: 5'GTTTTGTCAGGGAGTTGAGAAACTGTG 3' (SEQ ID ΝO:284) and 5' GAAATGGGGTGACTTCCACGAC 3' (SEQ ID NO:285). Primers were designed based on in silico predictions of the full length or some portion (one or more exons) of the cDNA/protein sequence of the invention. These primers were used to amplify a cDNA from a pool containing expressed human sequences derived from the following tissues: adrenal gland, bone marrow, brain - amygdala, brain - cerebellum, brain - hippocampus, brain - substantia nigra, brain - thalamus, brain -whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma - Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea and uterus. The NON25e ORF begins with a Kozak consensus ATG initiation codon at nucleotides 56-58 and ends with a TAG codon at nucleotides 905-907. Putative untranslated regions upstream from the initiation codon and downstream from the termination codon are underlined in Table 251, and the start and stop codons are in bold letters.
Table 251. ΝOV25e Nucleotide Sequence (SEQ ID NO:90)
GTTTCCTCTGCCCTGTTAGAGCTGGGGGACTCTTCAGAGTCAAAGGCCAGAGAGCATGGAGCTGAGT GGAGCCACCATGGCCCGGGGGCTCGCTGTCCTGCTAGTCTTGTTCCTGCATATCAAGAACCTGCCTG CCCAGGCTGCGGACACATGTCCAGAGGTGAAGGTGGTGGGCCTGGAGGGCTCTGACAAGCTCACCAT TCTCCGAGGCTGCCCGGGGCTGCCCGGGGCCCCAGGGCCAAAGGGAGAGGCAGGTGTCATTGGAGAG AGAGGAGAACGCGGTCTCCCTGGAGCCCCTGGAAAGGCAGGACCAGTGGGGCCCAAAGGAGACCGAG GAGAGAAGGGGATGCGTGGAGAGAAAGGAGACGCTGGGCAGTCTCAGTCGTGTGCGACAGGTCCACG CAACTGCAAGGACCTGCTAGACCGGGGGTATTTCCTGAGCGGCTGGCACACCATCTACCTGCCCGAC TGCCGGCCCCTGACTGTGCTCTGTGACATGGACACGGACGGAGGGGGCTGGACCGTTTTCCAGCGGA GGATGGATGGCTCTGTGGACTTTGAGGGCAACCACCAGTTTGCTAAGTACAAATCATTCAAGGTGGC TGACGAGGCAGAGAAGTACAAGCTGGTACTGGGAGCCTTTGTCGGGGGCAGTGCGGGTAATTCTCTA ACGGGCCACAACAACAACTTCTTCTCCACCAAAGACCAAGACAATGATGTGAGTTCTTCGAATTGTG CTGAGAAGTTCCAAGGAGCCTGGTGGTACGCCGACTGTCATGCTTCAAACCTCAATGGTCTCTACCT CATGGGACCCCATGAGAGCTATGCCAATGGTATCAACTGCAGTGCGGCGAAGGGGTACAAATATAGC TACAAGGTGTCAGAGATGAAGGTGCGGCCCGCCTAGACGGGCCAGGACCCCTCCACATGCACCTGCT AGTGGGGAGGCCACACCCACAAGCGCTGCGTCGTGGAAGTCACCCATTTC
The NON25e protein (SEQ ID ΝO:91) encoded by SEQ ID NO:90 is 283 amino acid residues in length is presented using the one-letter code in Table 25 J. A variant sequence can include a single nucleotide polymorphism (SNP). A SNP can, in some instances, be referred to as a "cSNP" to denote that the nucleotide sequence containing the SNP originates as a cDNA. A NON24b Variant is a G to T SΝP at 89 bp of the nucleotide sequence.
The Psort profile for ΝOV25e predicts that this sequence is likely to be localized extracellularly with a certainty of 0.6711. The Signal P predicts a likely cleavage site for a NOV25e peptide is between positions 29 and 30, i.e., at the dash in the sequence AQA-AD.
Table 25J. NOV25e protein sequence (SEQ ID NO:91)
MELSGATMARGLAVLLVLFLHIKNLPAQAADTCPEVKWGLEGSDKLTILRGCPGLPGAPGPKGEAG VIGERGERGLPGAPGKAGPVGPKGDRGEKGMRGEKGDAGQSQSCATGPRNCKDLLDRGYFLSG HTI YLPDCRPLTVLCDMDTDGGG TVFQRRMDGSVDFEGNHQFAKYKSFKVADEAΞKYKLVLGAFVGGSA GNSLTGHNNNFFSTKDQDNDVSSSNCAEKFQGA YADCHASNLNGLYLMGPHESYANGINCSAAKG YKYSYKVSEMKVRPA
NOV25f
Alternatively, a NOV25 variant is the novel NON25f (alternatively referred to herein as CG56653-01), which includes the 1194 nucleotide sequence (SEQ ID ΝO:92) shown in Table 25K. The NOV25f ORF begins with a Kozak consensus ATG initiation codon at nucleotides 15- 18 and ends with a TAG codon at nucleotides 973-975. Putative untranslated regions upstream from the imtiation codon and downstream from the termination codon are underlined in Table 25K, and the start and stop codons are in bold letters. Table 25K. NO V25f Nucleotide Sequence (SEQ ID NO:92)
CTGAGTGGAGCCACCATGGCCCGGGGGCTCGCTGTCCTGCTAGTCTTGTTCCTGCATATCAAGAACC TGCCTGCCCAGGCTGCGGACACATGTCCAGAGGTGAAGGTGGTGGGCCTGGAGGGCTCTGACAAGCT CACCATTCTCCGAGGCTGCCCGGGGCTGCCCGGGGCCCCAGGGCCAAAGGGAGAGGCAGGTGTCATT GGAGAGAGAGGAGAACGCGGTCTCCCTGGAGCCCCTGGAAAGGCAGGACCAGTGGGGCCCAAAGGAG ACCGAGGAGAGAAGGGGATGCGTGGAGAGAAAGGAGACGCTGGGCAGTCTCAGTCGTGTGCGACAGG CCCACGCAACTGCAAGGACCTGCTAGACCGGGGGTATTTCCTGAGCGGCTGGCACACCATCTACCTG CCCGACTGCCGGCCCCTGACTGTGCTCTGTGACATGGACACGGACGGAGGGGGCTGGACCGTTTTCC AGCGGAGGATGGATGGCTCTGTGGACTTCTATCGGGACTGGGCCGCATACAAGCAGGGCTTCGGCAG TCAGCTGGGGGAGTTCTGGCTGGGGAACGACAACATCCACGCCCTGACTGCCCAGGGAAGCAGCGAG CTCCGTGTAGACCTGGTGGACTTTGAGGGCAACCACCAGTTTGCTAAGTACAAATCATTCAAGGTGG CTGACGAGGCAGAGAAGTACAAGCTGGTACTGGGAGCCTTTGTCGGGGGCAGTGCGGGTAATTCTCT AACGGGCCACAACAACAACTTCTTCTCCACCAAAGACCAAGACAATGATGTGAGTTCTTCGAATTGT GCTGAGAAGTTCCAGGGAGCCTGGTGGTACGCCGACTGTCATGCTTCAAACCTCAATGGTCTCTACC TCATGGGACCCCATGAGAGCTATGCCAATGGTATCAACTGGAGTGCGGCGAAGGGGTACAAATATAG CTACAAGGTGTCAGAGATGAAGGTGCGGCCCGCCTAGACGGGCCAGGACCCCTCCACATGCACCTGC TAGTGGGGAGGCCACACCCACAAGCGCTGCGTCGTGGAAGTCACCCCATTTCCCCAGCCAGACACAC TCCCATGACGCCCACAGCTGCCCCTTTGCCCCCAGCTCAGTCAAGCCGCCACATGCCCACAACCTCA CCAGAGGGAGAATTATGTTTCTAAATATGTTTACTTTGGGACAGAAAAAAAAAAA
The NOV25f protein (SEQ ID NO:93) encoded by SEQ ID NO:92 is 319 amino acid residues in length is presented using the one-letter code in Table 25L. The Psort profile for NON25f predicts that this sequence is likely to be localized extracellularly with a certainty of 0.4944.
Table 25L. ΝON25f protein sequence (SEQ ID ΝO:93)
MARGLAVLLVLFLHI NLPAQAADTCPEVKWGLEGSDKLTILRGCPGLPGAPGPKGEAGVIGERGE RGLPGAPGKAGPVGPKGDRGEKGMRGEKGDAGQSQSCATGPRNCKDLLDRGYFLSGWHTIYLPDCRP LTVLCDMDTDGGG TVFQRRMDGSVDFYRDWAAYKQGFGSQLGEF LGNDNIHALTAQGSSELRVDL VDFEGNHQFAYKSFIWADEAEKYKiVLGAFVGGSAGNSLTGHNNNFFSTKDQDNDVSSSNCAEKFQ GAWYADCHASNLNGLYL GPHESYANGIN SAAKGYKYSYKVSEMKVRPA
NOV25g Alternatively, a NON25 variant is the novel ΝON25g (alternatively referred to herein as
CG56653-09), which includes the 1144 nucleotide sequence (SEQ ID ΝO:94) shown in Table 25M. NON25g was derived by laboratory cloning of cDΝA fragments, by in silico prediction of the sequence. cDΝA fragments covering either the full length of the DΝA sequence, or part of the sequence, or both, were cloned, hi silico prediction was based on sequences available in Curagen's proprietary sequence databases or in the public human sequence databases, and provided either the full length DΝA sequence, or some portion thereof. The ΝON25g ORF begins with a Kozak consensus ATG initiation codon at nucleotides 183-185 and ends with a TAG codon at nucleotides 981-983. Putative untranslated regions upstream from the initiation codon and downstream from the termination codon are underlined in Table 25M, and the start and stop codons are in bold letters.
Table 25M. NOV25g Nucleotide Sequence (SEQ ID NO:94)
TTTTAGGTCTGTTTGTCGTAGGCAGATGGAGCTTGTTATAATTATGCCTCATAGGGATAGTACAAGG AAGGGGTAGGCTATGTGTTTTGTCAGGGAGTTGAGAAACTGTGGCACAAGGCGAGAGCTGGTTTCCT CTGCCCTGTTAGAGCTGGGGGACTCTTCAGAGTCAAAGGCCAGAGAGCATGGAGCTGAGTGGAGCCA CCATGGCCCGGGGGCTCGCTGTCCTGCTAGTCTTGTTCCTGCATATCAAGAACCTGCCTGCCCAGGC TGCGGACACATGTCCAGAGGTGAAGGTGGTGGGCCTGGAGGGCTCTGACAAGCTCACCATTCTCCGA GGCTGCCCGGGGCTGCCCGGGGCCCCAGGGCCAAAGGGAGAGGCAGGTGTCATTGGAGAGAGAGGAG AACGCGGTCTCCCTGGAGCCCCTGGAAAGGCAGGACCAGTGGGGCCCAAAGGAGACCGAGGAGAGAA GGGGATGCGTGGAGAGAAAGGAGACGCTGGGCAGTCTCAGTCGTGTGCGACAGGCCCACGCAACTGC AAGGACCTGCTAGACCGGGGGTATTTCCTGAGCGGCTGGCACACCATCTACCTGCCCGACTGCCGGC CCCTGACTGTGCTCTGTGACATGGACACGGACGGAGGGGGCTGGACCGTTTTCCAGCGGAGGATGGA TGGCTCTGTGGACTTCTATCGGGACTGGGCCGCATACAAGCAGGGCTTCGGCAGTCAGCTGGGGGGT AATTCTCTAACGGGCCACAACAACAACTTCTTCTCCACCAAAGACCAAGACAATGATGTGAGTTCTT CGAATTGTGCTGAGAAGTTCCAAGGAGCCTGGTGGTACGCCGACTGTCATGCTTCAAACCTCAATGG TCTCTACCTCATGGGACCCCATGAGAGCTATGCCAATGGTATCAACTGGAGTGCGGCGAAGGGGTAC AAATATAGCTACAAGGTGTCAGAGATGAAGGTGCGGCCCGCCTAGACGGGCCAGGACCCCTCCACAT GCACCTGCTAGTGGGGAGGCCACACCCACAAGCGCTGCGTCGTGGAAGTCACCCCATTTCCCCAGCC AGACACACTCCCATGACGCCCACAGCTGCCCCTTTGCCCCCAGCTCAGTCAAGCCGCCACATGCCCA CAACC
The NOV25g protein (SEQ ID NO:95) encoded by SEQ ID NO:94 is 266 amino acid residues in length is presented using the one-letter code in Table 25N. The Psort profile for NOV25g predicts that this sequence is likely to be localized extracellularly with a certainty of 0.6711. The Signal P predicts a likely cleavage site for a NOV25g peptide is between positions 29 and 30, i.e., at the dash in the sequence AQA-AD.
Table 25N. NOV25g protein sequence (SEQ ID NO:95)
MELSGATMARGLAVLLVLFLHIKNLPAQAADTCPEVKWGLEGSDKLTILRGCPGLPGAPGPKGEAG VIGERGERGLPGAPGKAGPVGPKGDRGEKGMRGΞKGDAGQSQSCATGPRNCKDLLDRGYFLSG HTI YLPDCRPLTVLCDMDTDGGGWTVFQRRMDGSVDFYRD AAYKQGFGSQLGGNSLTGHNNNFFSTKDQ DNDVSSSNCAEKFQGA YADCHASNLNGLYLMGPHESYANGINWSAAKGYKYSYKVSEMKVRPA
NON25h Alternatively, a ΝON25 variant is the novel ΝON25h (alternatively referred to herein as
CG56653-01 & CG56653-02 assembly 169319361), which includes the 900 nucleotide sequence
(SEQ ID ΝO:96) shown in Table 250. The NOV25h ORF begins at nucleotides 1-2 and ends with a TAG codon at nucleotides 898. Table 25O. NON25h Nucleotide Sequence (SEQ ID NO:96)
GGATCCGCGGACACATGTCCAGAGGTGAAGGTGGTGGGCCTGGAGGGCTCTGACAAGCTCACCATTC TCCGAGGCTGCCCGGGGCTGCCCGGGGCCCCAGGGCCAAAGGGAGAGGCAGGTGTCATTGGAGAGAG AGGAGAACGCGGTCTCCCTGGAGCCCCTGGAAAGGCAGGACCAGTGGGGCCCAAAGGAGACCGAGGA GAGAAGGGGATGCGTGGAGAGAAAGGAGACGCTGGGCAGTCTCAGTCGTGTGCGACAGGCCCACGCA ACTGCAAGGACCTGCTAGACCGGGGGTATTTCCTGAGCGGCTGGCACACCATCTACCTGCCCGACTG CCGGCCCCTGACTGTGCTCTGTGACATGGACACGGACGGAGGGGGCTGGACCGTTTTCCAGCGGAGG ATGGATGGCTCTGTGGACTTCTATCGGGACTGGGCCGCATACAAGCAGGGCTTCGGCAGTCAGCTGG GGGAGTTCTGGCTGGGGAACGACAACATCCACGCCCTGACTGCCCAGGGAAGCAGCGAGCTCCGTGT AGACCTGGTGGACTTTGAGGGCAACCACCAGTTTGCTAAGTACAAATCATTCAAGGTGGCTGACGAG GCAGAGAAGTACAAGCTGGTACTGGGAGCCTTTGTCGGGGGCAGTGCGGGTAATTCTCTAACGGGCC ACAACAACAACTTCTTCTCCACCAAAGACCAAGACAATGATGTGAGTTCTTCGAATTGTGCTGAGAA GTTCCAGGGAGCCTGGTGGTACGCCGACTGTCATGCTTCAAACCTCAATGGTCTCTACCTCATGGGA CCCCATGAGAGCTATGCCAATGGTATCAACTGGAGTGCGGCGAAGGGGTACAAATATAGCTACAAGG TGTCAGAGATGAAGGGGCCCGCCCTCGAG
The NOV25h protein (SEQ ID NO:97) encoded by SEQ ID NO:96 is 300 amino acid residues in length is presented using the one-letter code in Table 25P. The Psort profile for NON25h predicts that this sequence is likely to be localized extracellularly with a certainty of 0.4500.
Table 25P. ΝOV25h protein sequence (SEQ ID NO:97)
GSADTCPEVKWGLEGSDKLTILRGCPGLPGAPGPKGEAGVIGERGERGLPGAPGKAGPVGPKGDRG EKGMRGEKGDAGQSQSCATGPRNCKDLLDRGYFLSG HTIYLPDCRPLTVLCDMDTDGGG TVFQRR MDGSVDFYRDWAAYKQGFGSQLGEF LGNDNIHALTAQGSSELRVDLVDFEGNHQFAKYKSFKVADE AEKYKLVLGAFVGGSAGNSLTGHNNNFFSTKDQDNDVSSSNCAEKFQGA WYADCHASNLNGLYLMG PHESYANGIN SAAKGYKYSYKVSEMKGPALE
NOV25 Clones
Unless specifically addressed as NOV25a, NOV25b, NOV25c, NOV25d, NON25e, ΝON25f, ΝON25g, or ΝON25h , any reference to ΝON25 is assumed to encompass all variants. Further, Patp, BLAST, and DOMAIN analyses are presented for NOV25b, the longest NOV25 polypetide sequence.
A search against the Patp database, a proprietary database that contains sequences published in patents and patent publications, yielded several homologous proteins shown in Table 25Q. Table 25Q. Patp results for NON25
Smallest Sum
Reading High Prob Sequences producing High- scoring Segment Pairs: Frame Score P(Ν)
>patp:AAR94183 Human 35 kDa opsonin protein P35 +1 1272 2.0e-129
>patp:AAR94179 Human 35 kDa opsonin protein P35 fragment +1 1225 1.9e-124
>patp:AAR30971 TGF-beta-1 binding protein +1 1200 8.6e-122
>patp:AAR94178 Human 35 kDa opsonin protein P35 fragment +1 1022 6.2e-103
>patp:AAB29658 Human membrane-associated protein HUMAP-15 +1 746 l.le-73
NOV25 polypeptides are ficolin-like proteins with sequence homology to the Fibrinogen protein family. In a BLAST search of public sequence databases, it was found, for example, that the NOV25b nucleic sequence of this invention has 956 of 982 bases (97%) identical to a gb:GENBANK-ID:S80990|acc:S80990.1 mRNA from Homo sapiens [ficolin (human, uterus, mRNA, 1736 nt)]. The full NOV25b polypeptide sequence was found to have 252 of 276 amino acid residues (91%) identical to, and 258 of 276 amino acid residues (93%) similar to, the 319 amino acid residue ptnr:SPTREMBL-ACC:Q92596 protein from Homo sapiens.
Additional BLAST results are shown in Table 25R.
Figure imgf000239_0001
A multiple sequence aligmnent is given in Table 25S, with the NOV25 protein of the invention being shown on line 1, in a ClustalW analysis comparing NOV25 with related protein sequences disclosed in Table 25R.
Table 25S. Information for the ClustalW proteins: 1. >NOV25a; SEQ DD NO:83
2. >NOV25b; SEQ TD NO:85
3. >NOV25c; SEQ TD NO:87
4. >NOV25d; SEQ DD NO:89
5. >NOV25e; SEQ TD NO:91 6. >NOV25f; SEQ TD NO:93
7. >NOV25g; SEQ BD NO:95
8. >NOV25h; SEQ BD NO:97
9. >GI|8051584/ FICOLIN 1 PRECURSOR [Homo sapiens]; SEQ BD NO:286
10.>GI|1312416/ FCN1_HUMAN FICOLIN 1 PRECURSOR [Homo sapiens]; SEQ BD NO:287 11 GI|2135117/ FICOLIN- 1 PRECURSOR Homo sapiens]; SEQ D) NO:288 12 GI|423207|/ FICOLIN-BETA [Sus scrofa]; SEQ TD NO:289 13 GI|1675844/ FICOLIN B [Rattus norvegicus]; SEQ ID NO:290
10 20 30 40 50
NOV25a lARGLAVLLVLFLHIKNLPAQAADTCPEVKWGLEGSDKLTl;
NOV25b MELSGAT lARGLAVLLVLFLHIKNLPAQAADTCPEVKVVGLEGSgJKLTIl
NOV25C JHIKNLPAQAADTCPEVKWGLEGSDKLTII
NOV25d ARGLAVLLVLFLHI0NLPAQAADTCPEVKWGLEGSDKLTII
NOV25e MELSGATIMARGLAVLLVLFLHIKNLPAQAADTCPEVKWGLEGSDKLTIL
NOV25f MARGLAVLLVLFLHIKNLPAQAADTCPEV WGLEGSDKLTII
NOV25g MELSGA1: MARGLAVLLVLFLHIKNLPAQAADTCPEVKWGLEGSDKLTII
NOV25h ΓCPEVKWGLEGSDKLTII
GI 8051584 MELSGAT U3TCPEVKWGLEGSDKLTII
GI 1312416 MELSGAT J ARGLAΛ^LVLFLHIKNLPAQAADTCPEVKWGLEGSDKLTII
GI 2135117 MARGLAVLLVLFLHIKNLPAQAADTCPEVKWGLEGSDKLTII
GI 423207| MELSRVAVΘLgPTGQ|lL| JSFQTΠ JØAQAADTCPEVKVVGLEGSDKLHII GI 1675844 UJv sSAlFl HSfflCVTEWTLHkWrillM8>8_tJi*tiT,Dπ
60 70 80 90 100
■ I . . . 1 . .
NOV25a 3LPGAPGPKGEAGVIGERGERGLPGAPGKAGPVGPKGDRGEKGMRC: NOV25b 3LPGAPGPKGEAGVIGERGgH^^^^^^^^HDRGEKGMRG NOV25C 3LPGAPGPKGEAGVIGERGERGLPGAPGKAGPVGPKGDRGEKGMRC NOV25d 3LPGAPGPKGEAGVIG N0V25e 3LPGAPGPKGEAGVIGERGERGLPGAPGKAGPVGPKGDRGEKGMRC NOV25f 3LPGAPGPKGEAGVIGERGERGLPGAPGKAGPVGPKGDRGEKGMR NOV25g 3LPGAPGPKGEAGVIGERGERGLPGAPGKAGPVGPKGDRGEKGMRG NOV25h 3LPGAPGPKGEAGVIGERGERGLPGAPGKAGPVGPKGDRGEKGMRG
GI 8051584 3LPGAPGPKGEAGVIGERGERGLPGAPGKAGPVGPKGDRGEKGMRG GI 1312416 3LPGAPGPKGEAGVIGERGERGLPGAPGKAGPVGPKGDRGEKGMRG GI 2135117 3LPGAPGPKGEAGVIGERGERGLPGAPGKAGPVGPKGDRGEKGMRG GI 423207| ιrrø«»ytfny«im[«y: «- "røA KPK[ei:>lrJe!sπ GI 1675844 ϊwLmgSti-WBAK
110 120 130 140 150
NOV25a WMMMW W'VUMur M:<ιιnιt>)r{ei,«»tl-iWi»ki»,««l9«*);,9-ytfJ-l«ιJύιn N0V25b EKGDAGQSQSCATGPRNCKDLLDRGYFLSGWHTIYLPDCRPLTVLCDMDT NOV25C EKGDAGQSQSCATGPRNCKDLLDRGYFLSG HTIYLPDCRPLTVLCDMDT NOV 5d JKGDAGQSQSCATGPRNCKDLLDRGYFLSG HTIYLPDCRPLTVLCDMDT NOV25e EKGDAGQSQSCATGPRNCKDLLDRGYFLSG HTIYLPDCRPLTVLCDMDT NOV 5f EKGDAGQSQSCATGPRNCKDLLDRGYFLSG HTIYLPDCRPLTVLCDMDT NOV25g EKGDAGQSQSCATGPRNCKDLLDRGYFLSGWHTIYLPDCRPLTVLCDMDT NOV25h EKGDAGQSQSCATGPRNCKDLLDRGYFLSG HTIYLPDCRPLTVLCDMDT
GI 8051584 EKGDAGQSQSCATGPRNCKDLLDRGYFLSG HTIYLPDCRPLTVLCDMDT GI 1312416 EKGDAGQSQSCATGPRNCKDLLDRGYFLSGWHJJJIYLPDCRPLTVLCDMDT GI 2135117 EKGDAGQSQSCATGPRNCKDLLDRGYFLSGWHTIYLPDCRPLTVLCDMDT GI 423207| GI 1675844
160 170 180 190 200
I I ..I.. ..I.. ..I
NOV25a DGGG TVFQRRMDGSVDFYRD AAYKQGFGSQLGEFWLGNDNIHALTAQG
NOV25b DGGGWTVFQRRMDGSVDFYRD AAYKQGFGSQLGEFWLGNDNIHALTAQG
NOV25C DGGGWTVFQ
NOV 5d DGGG TVFQRRMDGSVDFYRD AAYKQGFGSQLGEF LGNDNIHALTAQC
NOV25e DGGG TVFQRRMDGSVDFJ3
NOV25f DGGG TVFQRRMDGSVDFYRDWAAYKQGFGSQLGEFWLGNDNIHALTAQC
NOV25g DGGGWTVFQRRMDGSVDFY
NOV25h DGGG TVFQRRMDGSVDFYRDWAAYKQGFGSQLGEF LGNDNIHALTAQG
GI 8051584 DGGG TVFQRRMDGSVDFYRD AAYKQGFGSQLGEFWLGNDNIHALTAQG
GI 1312416 DGGG TVFQRRMDGSVDFYRD AAYKQGFGSQLGEFWLGNDNIHALTAQG
GI 2135117 DGGG TVFQRRMDGSVDFYRD AAYKQGFGSQLGEF LGNDNIHALTAQG
GI 423207| DGGG TVFQRR§DGSVDFYRD AAYKSGFGSQLGEF LGNDgjIHALTAQG GI 1675844 FtsumTsaimuBttRiβimBimi tim
210 220 230 240 250
Figure imgf000241_0001
260 270 280 290 300
NOV25a DQDNDVSSSNCAEKFQGAWWYADCHASNLNGLYLMGPH
NOV25b LTGHNNNFFSTKDQDNDVSSSNCAEKFQGAW YADCHASNLNGLYLMGPH
NOV25C AW YADCHASNLNGLYLMGPH
NOV25d LTGHNNFFSTKDQDNDVSSSNCAEKFQGAWWYADCHASNLNGLYLMGPH
NOV25e LTGHNNFFSTKDQDNDVSSSNCAEKFQGAW YADCHASNLNGLYLMGPH
NOV25f LTGHNNFFSTKDQDNDVSSSNCAEKFQGAW YADCHASNLNGLYLMGPH
NOV25g LTGHNNNFFSTKDQDNDVSSSNCAEKFQGAWWYADCHASNLNGLYLMGPH
NOV25h LTGHNNNFFSTKDQDNDVSSSNCAEKFQGAWWYADCHASNLNGLYLMGPH
GI 8051584 LTGHNNNFFSTKDQDNDVSSSNCAEKFQGAWWYADCHASNLNGLYLMGPH
GI 1312416 LTGHNNNFFSTKDQDNDVSSSNCAEKFQGAW YADCHAS LNGLYLMGPH
GI 2135117 LTGHNMNFFSTKDQDNDVSSSNCAEKFQGAWYADCHASNLNGLYLMGPH
GI 423207| GI 1675844
310 320 330 NOV25a SSjgANGINWSAAKGYKYSYKVSEMKVRPA NOV25b iSYANGIN SAAKGYKYSYKVSEMKVRPA NOV25C SSYANGINWSAAKGYKYSYKVSEMKVRPA NOV25d ESYANGINWSAAKGYKYSYKVSEMKVRPA N0V25e ΪSYANGINgSAAKGYKYSYKVSEMKVRPA NOV25f iSYANGINWSAAKGYKYSYKVSEMKVRPA NOV25g SYANGINWSAAKGYKYSYKVSEMKVRPA N0V25h ΞYANGIN SAAKGYKYSYKVSEMK3 PA Gl| 8051584 JSYANGINWSAAKGYKYSYKVSEMKVRPA GI j 1312416 :SYANGINWSAAKGYKYSYKVSEMKVRPA GI J2135117 ;SYANGINWSAAKGYKYSYKVSEMKVRPA GI j 423207 j GI 1675844
The presence of identifiable domains in the protein disclosed herein was determined by searches using algorithms such as PROSITE, Blocks, Pfam, ProDomain, Prints and then determining the Inteφro number by crossing the domain match (or numbers) using the Interpro website (http:www.ebi.ac.uk/interpro/). Table 25T lists the domain description from DOMAIN analysis results against NON25.
Figure imgf000242_0001
Consistent with other known members of the Fibrinogen family of proteins, e.g., ficolins, NOV25 contains fibrinogen and collagen domains as illustrated in Table 25T (Ohashi and Erickson, J. Biol. Chem., 272: 14220-6 (1997)). NOV25 nucleic acids, and the encoded polypeptides, according to the invention are useful in a variety of applications and contexts. For example, NOV25 nucleic acids and polypeptides can be used to identify proteins that are members of the Fibrinogen family of proteins. The NOV25 nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance NOV25 activity or function. Specifically, the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., cellular activation, cellular metabolism, host defense and signal transduction. These molecules can be used to treat, e.g., arthritis, autoimmune disease, immunodeficiencies, anemia, ataxia-telangiectasia, hemophilia, hypercoagulation, idiopathic thrombocytopenic purpura, graft versus host disease, endometriosis, fertility, systemic lupus erythematosus, asthma, emphysema, scleroderma, allergies, ARDS, hypercoagulation, as well as other diseases, disorders and conditions.
In addition, various NON25 nucleic acids and polypeptides according to the invention are useful, ter alia, as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins. For example, the ΝON25 nucleic acids and their encoded polypeptides include structural motifs that are characteristic of proteins belonging to the ficolin family of proteins involved in cytokine and steroid physiology (Ohashi and Erickson, J. Biol. Chem., 272: 14220-6 (1997)).
Ficolin was originally isolated as a protein from pig uterus membrane extracts that bound transforming growth factor- (Ichijo et al, J. Biol. Chem., 266: 22459-64 91991)). Ficolins have also been identified from human blood as a corticosteroid binding protein, termed hucolin (Edgar,
FEBS Lett., 375: 159-61 (1995)), an elastin binding protem, termed EBP-37 (Harumiya et al, J.
Biochem., 117: 1029-35 (1995)), and a GlcΝAc binding lectin, termed P35 (Matsushita et al, J.
Biol. Chem. 271: 2448-54 (1996)). Ficolin cDΝAs, are termed human ficolin (Lu et al, Biochem. J., 313: 473-8 (1996)), ficolin-1 (Harumiya et al, J. Biochem., 120: 745-51 (1996)), and P35- related gene (Endo et al, Genomics, 36: 515-21, (1996)) have been cloned.
The amino acid sequence of ficolins consist of a short Ν-terminal domain, a middle collagen-like domain, and aC-terminal fibrinogen-like (fbg)l domain (Ohashi and Erickson, J.
Biol. Chem., 272: 14220-6 (1997)). The collagen domains assemble these proteins into trimers, and electron microscopy shows that four or six trimers are connected together by the Ν-terminal domain, leaving the C-terminal lectin domains to project in a multimeric array (13-17). Like Clq and collectins play roles in immune defense, ficolins have been implicated in a similar role, in that human plasma ficolin (P35) is a lectin that binds to the carbohydrate of bacterial surface (Lu et al, Immunology, 89: 289-94 (1996)) and enhances opsonic activity of white blood cells, e.g., polymorphonuclear neutrophils (Matsushita et al, J. Biol. Chem., 271 : 2448-54 (1996)). Ficolin may play a role in alleviating inflammation in joints and other sites of inflammation.
Indeed, experiments comparing the gene expression levels of matched white blood cell fraction, e.g., peripheral blood lymphocytes (PBLs), and synovial fluid from three Rheumatoid
Arthritis (RA) patients showed significantly lower level of ΝON25f of the present invention in the RA patients. Specifically, the ΝON25f gene was found to be upregulated 26-fold The presence of this ficolin in the PBLs was, on average, 26-fold over the level found in the synoviocytes of these RA patients.
Based on the surprising result from GeneCalling that Ficolin is upregulated to such a high extent in PBLs vs synoviocytes in RA patients, and coupling this knowledge with the TaqMan profiles presented below, ficolin may be useful as a protein therapeutic in RA patients and other patients with autoimmune diseases. Ficolin, or pharmaceutically active portions thereof, may be administered to RA patients directly into the joint space of the knee or other joint space to alleviate inflammation and promote healing.
Protein therapeutics designed with the protein encoded for by NON25 could function as an opsinin to target and eliminate bacteria by complement -mediated destruction. These proteins could be important for the treatment of bacterial septicemia. Ficolins may also have the ability to bind to elastins. Elastins are functionally important for lung alveolar development and inactivation of these proteins can lead to emphysema-like disease. Antibodies against ΝON25 may prevent tissue destruction mediated by ficolin activity during emphysema, asthma and arthritis
The ΝON25 nucleic acids and polypeptides are useful for detecting specific cell types. For example, expression analysis has demonstrated that a ΝON25 nucleic acid is expressed in white blood cells, Aorta, Colon, Bone Marrow, Joints, Peripheral Blood, Spleen, Pituitary Gland, Mammary gland/Breast, Uterus, Prostate, Lung, and Kidney. . Additional utilities for ΝON25 nucleic acids and polypeptides according to the invention are disclosed herein.
ΝON26
A ΝON26 polypeptide has been identified as a ficolin-like protein (also referred to as 152736833). The disclosed novel ΝON26 nucleic acid (SEQ ID ΝO:98) of 779 nucleotides is shown in Table 26A. The cDNA coding for the NON26 was cloned by polymerase chain reaction (PCR) using the following primers: GCTCGCTGTCCTGCTAGTCTTGTT (SEQ ID ΝO:291) and AGAAACATAATTCTCCCTCTGGTGAGG (SEQ ID NO:292) on the following pool of human cDNAs: Pool 1 - Adrenal gland, bone marrow, brain - amygdala, brain - cerebellum, brain - hippocampus, brain - substantia nigra, brain - thalamus, brain -whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma - Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea, uterus. The novel NON26 nucleic acid sequences maps to the chromosome 9.
An ORF begins with an Kozak consensus ATG initiation codon at nucleotides 16-18 and ends with a TAG codon at nucleotides 625-627. A putative untranslated region and/or downstream from the termination codon is underlined in Table 26A, and the start and stop codons are in bold letters.
Table 26A. ΝON26 Nucleotide Sequence (SEQ ID NO:98)
CTQAGTGGAGCCACCATGGCCCGGGGGCTCGCTGTCCTGCTAGTCTTGTTCCTGCATATCAAGAACC TGCCTGCCCAGGCTGCGGACACATGTCCAGAGGTGAAGGTGGTGGGCCTGGAGGGCTCTGACAAGCT CACCATTCTCCGAGGCTGCCCGGGGCTGCCCGGGGCCCCAGGGCCAAAGGGAGAGGCAGGTGTCATT GGAGAGAGAGGAGAACGCGGTCTCCCTGGAGCCCCTGGAAAGGCAGGACCAGTGGGGCCCAAAGGAG ACCGAGGAGAGAAGGGGATGCGTGGAGAGAAAGGAGACGCTGGGCAGTCTCAGTCGTGTGCGACAGG CCCACGCAACTGCAAGGACCTGCTAGACCGGGGGTATTTCCTGAGCGGCTGGCACACCATCTACCTG CCCGACTGCCGGCCCCTGACTGTGCTCTGTGACATGGACACGGACGGAGGGGGCTGGACCGTTTTCC AGGGAGCCTGGTGGTACGCCGACTGTCATGCTTCAAACCTCAATGGTCTCTACCTCATGGGACCCCA TGAGAGCTATGCCAATGGTATCAACTGGAGTGCGGCGAAGGGGTACAAATATAGCTACAAGGTGTCA GAGATGAAGGTGCGGCCCGCCTAGACGGGCCAGGACCCCTCCACATGCACCTGCTAGTGGGGAGGCC ACACCCACAAGCGCTGCGTCGTGGAAGTCACCCCATTTCCCCAGCCAGACACACTCCCATGACGCCC ACAGCTGCCCCTTTGCCCCCAGCTCAGTCAAGCCGCCACATG
The NOV26 protein (SEQ ID NO:99) encoded by SEQ ID NO:98 is 203 amino acid residues in length and is presented using the one-letter amino acid code in Table 26B. Psort analysis predicts the NON26 protein of the invention to be localized extracellularly with a certainty of 0.4944. The Signal P predicts a likely cleavage site for a ΝON26 peptide is between positions 22 and 23, i.e., at the dash in the sequence AQA-AD.
Table 26B. Encoded ΝOV26 protein sequence (SEQ ID NO:99)
MARGLAVLLVLFLHIKNLPAQAADTCPEVKVVGLEGSDKLTILRGCPGLPGAPGP GEAGVIGER GERGLPGAPGKAGPVGPKGDRGEKGMRGEKGDAGQSQSCATGPRNCKDLLDRGYFLSG HTIYLP DCRPLTVLCDMDTDGGG TVFQGA YADCHASNLNGLYLMGPHESYANGIN SAAKGYKYSYKV SEMKVRPA A search against the Patp database, a proprietary database that contains sequences published in patents and patent publications, yielded several homologous proteins shown in Table 26C.
Table 26C. Patp results for NON26
Smallest Sum
Reading High Prob Sequences producing High-scoring Segment Pairs: Frame Score P(Ν)
>patp:AAR94183 Human 35 kDa opsonin protein P35 +1 560 2.9e-78
>patp:AAR30971 TGF-beta-1 binding protein +1 582 7.7e-78
>patp:AAR94179 Human 35 kDa opsonin P35 fragment (III) +1 539 5.4e-75
>patp:AAB19732 Human SECX Clone 4437909.0.4 +1 200 9.7e-27
>patp:AAB19733 Human SECX Clone 4437909.0.55 +1 200 9.7e-27
In a BLAST search of public sequence databases, it was found, for example, that the nucleic acid sequence of this invention has 487 of 496 bases (98%) identical to a gb:GENBANK- ID:D83920|acc:D83920.1 mRNA from Homo sapiens (Human uterus mRNA for human ficolin-1, complete eds). The full amino acid sequence of the protein of the invention was found to have 152 of 152 amino acid residues (100%) identical to, and 152 of 152 amino acid residues (100%) similar to, the 319 amino acid residue ptnr:SPTREMBL-ACC:Q92596 protein from Homo sapiens (FICOLIN).
NOV26 also has homology to the proteins shown in the BLASTP data in Table 26D.
Figure imgf000246_0001
A multiple sequence alignment is given in Table 26E, with the NOV26 protein being shown on line 1 in Table 26E in a ClustalW analysis, and comparing the NOV26 protem with the related protein sequences shown in Table 26D. This BLASTP data is displayed graphically in the ClustalW in Table 26E.
Table 26E. ClustalW Analysis of NOV26
1) > NOV26; SEQ TD NO:99
2) > gi|8051584/ ficolin 1 precursor [Homo sapiens]; SEQ TD NO:293
3) > gi|2135117/ ficolin 1 precurson [Homo sapiens]; SEQ BD NO.-294
4) > gi|1312416/ FCNl_Human Ficolin 1 precursor; SEQ ID NO:295
5) > gi|1669354/ P35-related protein [Homo sapiens] ; SEQ ID NO:296
6) > gi|423207|/ ficolin-beta [Pig]; SEQ TD NO:297
Figure imgf000247_0001
110 120 130 140 150
NOV26 EKGDAGQSQSCATGPRNCKDLLDRGYFLSGWHTIYLPDCRPLTVLCDMDT gi I 8051584 EKGDAGQSQSCATGPRNCKDLLDRGYFLSG HTIYLPDCRPLTVLCDMDT gi|2135117 EKGDAGQSQSCATGPRNCKDLLDRGYFLSG HTIYLPDCRPLTVLCDMDT gij 1312416 EKGDAGQSQSCATGPRNCKDLLDRGYFLSG HJJJLYLPDCRPLTVLCDMDT gijl669354 SKGDAGQSQSCATGPRNCKDLLDRGYFLSG HTIYLPDCRPLTVLCDMDT gi|423207| J-^E !^ι iEH^ϊa33τ!a5FflB|τigS}Hf5
160 170 180 190 200
I
NOV26 DGGGWTVFQ_ gi|8051584 DGGGWTVFQRRjgDGSVDFYRDWAAYKgGFGSQLGEFWLGNDgjIHALTAQG gi|2135117 DGGG TVFQRRBDGSVDFYRDWAAYKBGFGSQLGEFWLGNDSIHALTAQG gij 1312416 DGGG TVFQRR"DGSVDFYRD AAYKMGFGSQLGEF LGND"IHALTAQG gijl669354 DGGG TVFQR gi|423207| !lsιtιe>ϊiτ<Ma'«atwfc A'<aRl35ewM-iM!aa';ιaB »iHπϊrjt.>j»ιt] 0
Figure imgf000247_0002
gi|423207|
Figure imgf000248_0001
The presence of identifiable domains in the protein disclosed herein was determined by searches using algorithms such as PROSITE, Blocks, Pfam, ProDomain, Prints and then determining the Interpro number by crossing the domain match (or numbers) using the Interpro website (http:www.ebi.ac.uk/interpro/). Table 26F lists the domain description from DOMAIN analysis results against NOV26.
Figure imgf000248_0002
Consistent with other known members of the Fibrinogen family of proteins, e.g., ficolin, NON26 contains fibrinogen and collagen domains as illustrated in Table 25T (Ohashi and Erickson, J. Biol. Chem., 272: 14220-6 (1997)). ΝON26 nucleic acids, and the encoded polypeptides, according to the invention are useful in a variety of applications and contexts. For example, ΝON26 nucleic acids and polypeptides can be used to identify proteins that are members of the Fibrinogen family of proteins. The ΝON26 nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance ΝON26 activity or function. Specifically, the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., cellular activation, cellular metabolism, host defense and signal transduction. These molecules can be used to treat, e.g., arthritis, autoimmune disease, immunodeficiencies, anemia, ataxia-telangiectasia, hemophilia, emphysema, hypercoagulation, idiopathic thrombocytopenic purpura, graft versus host disease, endometriosis, fertility, systemic lupus erythematosus, asthma, emphysema, scleroderma, allergies, ARDS, hypercoagulation, as well as other diseases, disorders and conditions. hi addition, various NON26 nucleic acids and polypeptides according to the invention are useful, inter alia, as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins. For example, the ΝOV26 nucleic acids and their encoded polypeptides include structural motifs that are characteristic of proteins belonging to the ficolin family of proteins involved in cytokine and steroid physiology (Ohashi and Erickson, J. Biol. Chem., 272: 14220-6 (1997)).
Ficolin was originally isolated as a protein from pig uterus membrane extracts that bound transforming growth factor- (Ichijo et al, J. Biol. Chem., 266: 22459-64 91991)). Ficolins have also been identified from human blood as a corticosteroid binding protein, termed hucolin (Edgar, FEBS Lett., 375: 159-61 (1995)), an elastin binding protein, termed EBP-37 (Harumiya et al, J. Biochem., 117: 1029-35 (1995)), and a GlcNAc binding lectin, termed P35 (Matsushita et al, J. Biol. Chem. 271 : 2448-54 (1996)). Ficolin cDNAs, are termed human ficolin (Lu et al, Biochem. J., 313: 473-8 (1996)), ficolin-1 (Harumiya et al, J. Biochem., 120: 745-51 (1996)), and P35- related gene (Endo et al, Genomics, 36: 515-21, (1996)) have been cloned.
The amino acid sequence of ficolins consist of a short N-terminal domain, a middle collagen-like domain, and a C-terminal fibrino gen-like (fbg)l domain (Ohashi and Erickson, J. Biol. Chem., 272: 14220-6 (1997)). The collagen domains assemble these proteins into trimers, and electron microscopy shows that four or six trimers are connected together by the N-terminal domain, leaving the C-terminal lectin domains to project in a multimeric array (13-17). Like Clq and collectins play roles in immune defense, ficolins have been implicated in a similar role, in that human plasma ficolin (P35) is a lectin that binds to the carbohydrate of bacterial surface (Lu et al, Immunology, 89: 289-94 (1996)) and enhances opsonic activity of white blood cells, e.g., polymoφhonuclear neutrophils (Matsushita et al, J. Biol. Chem., 271: 2448-54 (1996)). Ficolin may play a role in alleviating inflammation in joints and other sites of inflammation. Protein therapeutics designed with the protein encoded for by NOV26 could function as an opsinin to target and eliminate bacteria by complement -mediated destruction. These proteins could be important for the treatment of bacterial septicemia. Ficolins may also have the ability to bind to elastins. Elastins are functionally important for lung alveolar development and inactivation of these proteins can lead to emphysema-like disease. Antibodies against NOV26 may prevent tissue destruction mediated by ficolin activity during emphysema, asthma and arthritis
The NOV26 nucleic acids and polypeptides are useful for detecting specific cell types. For example, expression analysis has demonstrated that a NOV25 nucleic acid is expressed in peripheral blood leukocytes, uterus, spleen, lung, and thymus.
Additional utilities for NOV26 nucleic acids and polypeptides according to the invention are disclosed herein.
NON27
A ΝON27 polypeptide has been identified as a peroxisomal Ca-dependent solute carrierlike protein (also referred to as CG56262-01). The disclosed novel ΝON27 nucleic acid (SEQ ID ΝO:100) of 1551 nucleotides is shown in Table 27A. An ORF begins with a Kozak consensus ATG initiation codon at nucleotides 108-110 and ends with a TGA codon at nucleotides 1512- 1514. A putative untranslated region and/or downstream from the termination codon is underlined in Table 27 A, and the start and stop codons are in bold letters. The novel NON27 nucleic acid sequences maps to the chromosome 19.
Table 27A. ΝON27 Nucleotide Sequence (SEQ ID NO:100)
GCGGCCGCGGGAGCTGACCCTGCGGGGTCCCGGGGGGGGAGGGGGAGCCGCGAAGCCCCCACTGAGG CCGCCGCTGCCGGGCCTCCCCTCCCCCCCGGGCGGGCGCCATGCGGGGGAGCCCGGGCGACGCGGAG CGGCGGCAGCGCTGGGGTCGCCTGTTCGAGGAGCTGGACAGTAACAAGGATGGCCGCGTGGACGTGC ACGAGTTGCGCCAGGGGCTGGCCAGGCTGGGCGGGGGCAACCCAGACCCCGGCGCCCAACAGGGTAT CTCCTCTGAGGGTGATGCTGACCCAGATGGCGGGCTCGACCTGGAGGAATTTTCCCGCTATCTGCAG GAGCGGGAACAGCGTCTGCTGCTCATGTTTCACAGTCTTGACCGGAACCAGGATGGTCACATTGATG TCTCTGAGATCCAACAGAGTTTCCGAGCTCTGGGCATTTCCATCTCGCTGGAGCAGGCTGAGAAAAT TTTGCACAGCATGGACCGAGACGGCACAATGACCATTGACTGGCAAGAATGGCGCGACCACTTCCTG TTGCATTCGCTGGAAAATGTGGAGGACGTGCTGTATTTCTGGAAGCATTCCACGGTCCTGGACATTG GCGAGTGCCTGACAGTGCCGGACGAGTTCTCAAAGCAAGAGAAGCTGACGGGCATGTGGTGGAAACA GCTGGTGGCCGGCGCAGTGGCAGGTGCCGTGTCACGGACAGGCACGGCCCCTCTGGACCGCCTCAAG GTCTTCATTCAGGTCCATGCCTCAAAGACCAACCGGCTGAACATCCTTGGGGGGCTTCGAAGCATGG TCCTTGAGGGAGGCATCCGCTGCCTGTGGCGCGGCAATGGTATTAATGTACTCAAGATTGCCCCCGA GTCAGCTATCAAGTTCATGGCCTATGAACAGGTGAGGAGGGCCATCCTGGGGCAGCAGGAGACACTG CATGTGCAGGAGCGCTTCGTGGCTGGCTCCCTGGCTGGTGCCACAGCCCAAACCATCATTTACCCTA TGGAGGTGCTGAAGACGCGGCTGACCTTGCGCCGGACGGGCCAGTATAAGGGGCTGCTGGACTGCGC CAGGCGTATCCTGGAGAGGGAGGGGCCCCGTGCCTTCTACCGCGGCTACCTCCCCAACGTGCTGGGC ATCATCCCCTATGCGGGCATCGACCTGGCCGTCTACGAGGTCCTGAAGAACTGGTGGCTTCAGCAGT ACAGCCACGACTCGGCAGACCCAGGCATCCTCGTGCTCCTGGCCTGCGGTACCATATCCAGCACCTG CGGCCAGATAGCCAGTTACCCGCTGGCCCTGGTCCGGACCCGCATGCAGGCACAAGCCTCCATCGAG GGTGGCCCCCAGCTGTCCATGCTGGGTCTGCTACGTCACATCCTGTCCCAGGAGGGCATGCGGGGCC TCTACCGGGGGATCGCCCCCAACTTCATGAAGGTTATTCCAGCTGTGAGCATCTCCTATGTGGTCTA CGAGAACATGAAGCAGGCCTTGGGGGTCACGTCCAGGTGAGGGACCCGGAGCCCGTCCCCCCAATCC CTCACCCCCC
The NOV27 protein (SEQ ID NO:101) encoded by SEQ ID NO:100 is 468 amino acid residues in length and is presented using the one-letter amino acid code in Table 27B. Psort analysis predicts the NOV27 protein of the invention to be localized in the cytoplasm with a certainty of 0.4500. NOV27 has a SNP variant, whose variant position for its nucleotide and amino acid sequence is numbered according to SEQ ID NOS:100 and 101, respectively. A variant sequence can include a single nucleotide polymorphism (SNP). A SNP can, in some instances, be referred to as a "cSNP" to denote that the nucleotide sequence containing the SNP originates as a cDNA.
NOV27 has at least one variant. NOV27 variant 13376757 is a G to A SNP at 1529 bp of the nucleotide sequence that results in no change in the protein sequence since the SNP is not in the amino acid coding region.
Table 27B. Encoded NOV27 protein sequence (SEQ ID NO:101)
MRGSPGDAERRQR GRLFEELDSNKDGRVDVHELRQGLARLGGGNPDPGAQQGISSEGDADPDGG LDLEEFSRYLQEREQRLLLMFHSLDRNQDGHIDVSEIQQSFRALGISISLEQAEKILHSMDRDGT MTID QEWRDHFLLHSLENVEDVLYF HSTVLDIGECLTVPDEFSKQEKLTGM WKQLVAGAVA GAVSRTGTAPLDRL VFIQVHAS TNRLNILGGLRSMVLEGGIRCLWRGNGINVLKIAPESAI F MAYEQVRRAILGQQETLHVQERFVAGSLAGATAQTIIYPMΞVLKTRLTLRRTGQYKGLLDCARRI LEREGPRAFYRGYLPNVLGIIPYAGIDLAVYEVLKN WLQQYSHDSADPGILVLLACGTISSTCG QIASYPLALVRTRMQAQASIEGGPQLSMLGLLRHILSQEGMRGLYRGIAPNFMKVIPAVSISYW YENMKQALGVTSR
A search against the Patp database, a proprietary database that contains sequences published in patents and patent publications, yielded several homologous proteins shown in Table
27C.
Table 27C. Patp results for NOV27
Smallest Sum
Reading High Prob Sequences producing High-scoring Segment Pairs: Frame Score P(N)
>patp:AAU27697 Human full-length polypeptide sequence #22 +1 2403 2.8e-249
• >patp:AAU27869 Human contig polypeptide sequence #22 +1 2403 2.8e-249
>patp:AAM79077 Human protein +1 1543 3.8e-158
>patp:AAY66718 Membrane-bound protein PRO1106 +1 1536 2.1e-157
>patp:AAB65241 Human PRO1106 (UNQ549) +1 1536 2.1e-157 hi a BLAST search of public sequence databases, it was found, for example, that the nucleic acid sequence of this invention has 886 of 1379 bases (64%) identical to a gb:GENBANK-ID:AF123303|acc:AF123303.1 mRNA from Homo sapiens (calcium-binding transporter mRNA, partial eds). The full amino acid sequence of the protein of the invention was found to have 280 of 461 amino acid residues (60%) identical to, and 365 of 461 amino acid residues (79%) similar to, the 475 amino acid residue ptnr:SPTREMBL-ACC:O18757 protein from Oryctolagus cuniculus (PEROXISOMAL CA-DEPENDENT SOLUTE CARRIER).
NON27 also has homology to the proteins shown in the BLASTP data in Table 27D.
Figure imgf000253_0002
A multiple sequence alignment is given in Table 27E, with the NOV27 protein being shown on line 1 in Table 27E in a ClustalW analysis, and comparing the NOV27 protein with the related protein sequences shown in Table 27D. This BLASTP data is displayed graphically in the ClustalW in Table 27E.
Table 27E. ClustalW Analysis of NOV27
1) > NOV27; SEQ ID NO:101
2) > gi|1343086/ hypothetical protein MGC2615 [Homo sapiens]; SEQ TD NO.-298
3) > gi| 1654952/ unnamed protem product [Homo sapiens] ; SEQ TD NO:299
4) > gi| 1562085/ KIAA1896 protem [Homo sapiens] ; SEQ ID NO:300
5) > gijl 136034/ peroxisomal Ca-dependent solute carrier [Oryctolagus cuniculus] ; SEQ TD NO:301
6) > gi|1804356/ Unknown (protein for MGC:28954) [Mus musculus] ; SEQ DD NO:302
10 20 30 40 50
SELLLRPGTTAVLAHLKKQETAPACRASSLRTPGQSSQQLCSQRALVL S
Figure imgf000253_0001
60 70 80 90 100 ....|....j....|....|....|....|....|....|....|....|
NOV27 MRGSPGDAERRQg gi 11343086 MRGSPGDAERRQg gi I 1654952 gi j 1562085 HPSKVNSRHRRRLEETVMLQMLWHFLASFFPRAGCHGSREGDDREVgGTP gi 11136034 MLRWLRGFVLPTAACQGAEPPTg gij 1804356
Figure imgf000254_0001
Figure imgf000255_0001
The presence of identifiable domains in the protein disclosed herein was determined by searches using algorithms such as PROSITE, Blocks, Pfam, ProDomain, Prints and then determining the Interpro number by crossing the domain match (or numbers) using the Interpro website (http:www.ebi.ac.uk/interpro/). Table 27F lists the domain description from DOMAIN analysis results against NOV27.
Figure imgf000255_0002
Consistent with other known members of the mitochondrial carrier proteins, e.g., peroxisomal Ca-dependent solute carrier family of proteins, NOV27 contains EF hand calcium binding domains and mitochondrial carrier transport signature domains as illustrated in Table 27F. NOV27 nucleic acids, and the encoded polypeptides, according to the invention are useful in a variety of applications and contexts. For example, NOV27 nucleic acids and polypeptides can be used to identify proteins that are members of the peroxisomal Ca-dependent solute carrier family of proteins. The NOV27 nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance NOV27 activity or function. Specifically, the NOV27 nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., transport facilitation. These molecules can be used to treat, e.g., cancer, trauma, regeneration (in vitro and in vivo), viral/bacterial/parasitic infections, atherosclerosis, aneurysm, hypertension, fibromuscular dysplasia, stroke, scleroderma, obesity, Von Hippel-Lindau (VHL) syndrome, Alzheimer's disease, stroke, tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, cerebral palsy, epilepsy, Lesch-Nyhan syndrome, multiple sclerosis, ataxia-telangiectasia, leukodystrophies, behavioral disorders, addiction, anxiety, pain, diabetes, autoimmune disease, renal artery stenosis, interstitial nephritis, glomerulonephritis, polycystic kidney disease, systemic lupus erythematosus, renal tubular acidosis, IgA nephropathy, hypercalceimia, muscular dystrophy, Lesch-Nyhan syndrome, myasthenia gravis and other diseases, disorders and conditions of the like. hi addition, various NOV27 nucleic acids and polypeptides according to the invention are useful, inter alia, as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins. For example, the NOV27 nucleic acids and their encoded polypeptides include structural motifs and homology that are characteristic of proteins belonging to the family of mitochondrial carrier proteins such as the peroxisomal Ca-dependent solute carrier proteins.
Many calcium-binding proteins belong to the same evolutionary family and share a type of calcium-binding domain known as the EF-hand. This type of domain consists of a twelve residue loop flanked on both side by a twelve residue alpha-helical domain. Different types of substrate carrier proteins involved in energy transfer are found in the inner mitochondrial membrane such as the ADP, ATP carrier protein (AAC) (ADP/ATP translocase), the 2-oxoglutarate/malate carrier protein (OGCP), the phosphate carrier protein, which transports phosphate groups from the cytosol into the mitochondrial matrix all share a common carrier protein motif. NOV27 also resembles the peroxisomal Ca-dependent solute carrier from rabbit. Although the Psort suggests that this is a cytosolic protein rather than mitochondrial, it is hypothesized that it might function in the uncoupling of ATP translocation and play a role in metabolic disease.
The NOV27 nucleic acids and polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications for the treatment of metabolic disorders. As such the NOV27 nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat metabolic disorders, e.g., cancer, trauma, regeneration (in vitro and in vivo), viral/bacterial/parasitic infections, atherosclerosis, aneurysm, hypertension, fibromuscular dysplasia, stroke, scleroderma, obesity, Von Hippel- Lindau (VHL) syndrome, Alzheimer's disease, stroke, tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, cerebral palsy, epilepsy, Lesch-Nyhan syndrome, multiple sclerosis, ataxia-telangiectasia, leukodystrophies, behavioral disorders, addiction, anxiety, pain, diabetes, autoimmune disease, renal artery stenosis, interstitial nephritis, glomerulonephritis, polycystic kidney disease, systemic lupus erythematosus, renal tubular acidosis, IgA nephropathy, hypercalceimia, muscular dystrophy, Lesch-Nyhan syndrome, and myasthenia gravis.
The NOV27 nucleic acids and polypeptides are useful for detecting specific cell types. For example, expression analysis has demonstrated that a NOV27 nucleic acid is expressed in Adrenal Gland/Suprarenal gland, Amygdala, Aorta, Brain, Bronchus, Cerebral Medulla/Cerebral white matter, Cervix, Coronary Artery, Frontal Lobe, Heart, Kidney, Liver, Lung, Mammary gland/Breast, Ovary, Oviduct/Uterine Tube/Fallopian tube, Parietal Lobe, Peripheral Blood, Pituitary Gland, Prostate, Retina, Skeletal Muscle, Spinal Chord, Spleen, Substantia Nigra, Temporal Lobe, Testis, Thalamus, Thymus, Thyroid, and Vein .
Additional utilities for NOV27 nucleic acids and polypeptides according to the invention are disclosed herein. NOV28
A NOV28 polypeptide has been identified as a Sodium-glucose cotransporter (SGLT)-like protein (also referred to as CG56559-01). The disclosed novel NOV28 nucleic acid (SEQ ID NO:102) of 1900 nucleotides is shown in Table 28A. The novel NOV28 nucleic acid sequences maps to the chromosome 17.
An ORF begins with a Kozak consensus ATG initiation codon at nucleotides 51-53 and ends with a TGA codon at nucleotides 1851-1853. Aputative untranslated region and/or downstream from the termination codon is underlined in Table 28A, and the start and stop codons are in bold letters.
Table 28A. NOV28 Nucleotide Sequence (SEQ ID NO:102)
TTGGCCCTCAGTCCCTCGGGCTCATACCTAGTGCCTGCGGCAGGACAGCCATGGCCGCCAACTCCAC CAGCGACCTCCACACTCCCGGGACGCAGCTGAGCGTGGCTGACATCATCGTCATCACTGTGTATTTT GCTCTGAACGTGGCCGTGGGCATATGGTCCTCTTGTCGGGCCAGTAGGAACACGGTGAATGGCTACT TCCTGGCAGGCCGGGACATGACGTGGTGGCCGATTGGAGCCTCCCTCTTCGCCAGCAGCGAGGGCTC TGGCCTCTTCATTGGACTGGCGGGCTCAGGCGCGGCAGGAGGTCTGGCCGTGGCAGGCTTCGAGTGG AATGCCACGTACGTGCTGCTGGCACTGGCATGGGTGTTCGTGCCCATCTACATCTCCTCAGAGATCG TCACCTTACCTGAGTACATTCAGAAGCGCTACGGGGGCCAGCGGATCCGCATGTACCTGTCTGTCCT GTCCCTGCTACTGTCTGTCTTCACCAAGATATCGGCCCTGGACCTGTACGCGGGGGCTCTGTTTGTG CACATCTGCCTGGGCTGGAACTTCTACCTCTCCACCATCCTCACGCTCGGCATCACAGCCCTGTACA CCATCGCAGGTACTGGCGGCCTGGCTGCTGTAATCTACACGGACGCCCTGCAGACGCTCATCATGGT GGTGGGGGCTGTCATCCTGACAATCAAAGCTTTTGACCAGATCGGTGGTTACGGGCAGCTGGAGGCA GCCTACGCCCAGGCCATTCCCTCCAGGACCATTGCCAACACCACCTGCCACCTGCCACGTACAGACG CCATGCACATGTTTCGAGACCCCCACACAGGGGACCTGCCGTGGACCGGGATGACCTTTGGCCTGAC CATCATGGCCACCTGGTACTGGTGCACCGACCAGGTGATCGTGCAGCGATCACTGTCAGCCCGGGAC CTGAACCATGCCAAGGCGGGCTCCATCCTGGCCAGCTACCTCAAGATGCTCCCCATGGGCCTGATCA TCATGCCGGGCATGATCAGCCGCGCATTGTTCCCAGATGATGTGGGCTGCGTGGTGCCGTCCGAGTG CCTGCGGGCCTGCGGGGCCGAGGTCGGCTGCTCCAACATCGCCTACCCCAAGCTGGTCATGGAACTG ATGCCCATCGGTCTGCGGGGGCTGATGATCGCAGTGATGCTGGCGGCGCTCATGTCGTCGCTGACCT CCATCTTCAACAGCAGCAGCACCCTCTTCACTATGGACATCTGGAGGCGGCTGCGTCCCCGCTCCGG CGAGCGGGAGCTCCTGCTGGTGGGACGGTTGGTCATAGTGGCACTCATCGGCGTGAGTGTGGCCTGG ATCCCCGTCCTGCAGGACTCCAACAGCGGGCAACTCTTCATCTACATGCAGTCAGTGACCAGCTCCC TGGCCCCACCAGTGACTGCAGTCTTTGTCCTGGGCGTCTTCTGGCGACGTGCCAACGAGCAGCAGGG GGCCTTCTGGGGCCTGATAGCAGGGCTGGTGGTGGGGGCCACGAGGCTGGTCCTGGAATTCCTGAAC CCAGCCCCACCGTGCGGAGAGCCAGACACGCGGCCAGCCGTCCTGGGGAGCATCCACTACCTGCACT TCGCTGTCGCCCTCTTTGCACTCAGTGGTGCTGTTGTGGTGGCTGGAAGCCTGCTGACCCCACCCCC ACAGAGTGTCCAGATTGAGAACCTTACCTGGTGGACCCTGGCTCAGGATGTGCCCTTGGGAACTAAA GCAGGTGATGGCCAAACACCCCAGAAACACGCCTTCTGGGCCCGTGTCTGTGGCTTCAATGCCATCC TCCTCATGTGTGTCAACATATTCTTTTATGCCTACTTCGCCTGACACTGCCATCCTGGACAGAAAGG CAGGAGCTCTGAGTCCTCAGGTCC
The NOV28 protein (SEQ ID NO:103) encoded by SEQ ID NO:102 is 600 amino acid residues in length and is presented using the one-letter amino acid code in Table 28B. NOV28 has at least five SNP variants, whose variant positions for its nucleotides and amino acids sequences is numbered according to SEQ ID NOS:102 and 103, respectively. A variant sequence can include a single nucleotide polymorphism (SNP). A SNP can, in some instances, be referred to as a "cSNP" to denote that the nucleotide sequence containing the SNP originates as a cDNA.
NOV28 variant 13376762 is an A to T SNP at 158 bp of the nucleotide sequence that results in no change in the protein sequence (silent), variant 13376761 is a C to T SNP at 491 bp of the nucleotide sequence that results in no change in the protein sequence (silent), variant 13376760 is a T to C SNP at 565 bp of the nucleotide sequence that results in a Leu to Pro change at amino acid 172 of protein sequence, variant 13376759 is a C to T SNP at 867 bp of the nucleotide sequence that results in no change in the protein sequence (silent), and variant 13376758 is a C to T SNP at 1762 bp of the nucleotide sequence that results in a Pro to Leu change at amino acid 571 of protein sequence.
Psort analysis predicts the NOV28 protein of the invention to be localized to the plasma membrane with a certainty of 0.8200. The Signal P predicts a likely cleavage site for a NOV28 peptide is between positions 42 and 43, i.e., at the dash in the sequence CRA-SR.
Table 28B. Encoded NOV28 protein sequence (SEQ ID NO:103)
MAANSTSDLHTPGTQLSVADIIVITVYFALNVAVGIWSSCRASRNTVNGYFLAGRDMTWWPIGASLFASSEGS GLFIGLAGSGAAGGLAVAGFEWNATYVLLALAWVFVPIYISSEIVTLPEYIQKRYGGQRIRMYLSVLSLLLSV FTKISALDLYAGALFVHICLGWNFYLSTILTLGITALYTIAGTGGLAAVIYTDALQTLIMWGAVILTIKAFD QIGGYGQLEAAYAQAIPSRTIANTTCHLPRTDAMHMFRDPHTGDLPWTGMTFGLTIMATWYWCTDQVIVQRSL SARDLNHAKAGSILASYLKMLPMGLIIMPGMISRALFPDDVGCVVPSECLRACGAEVGCSNIAYPKLVMELMP IGLRGLMIAVMLAALMSSLTSIFNSSSTLFTMDIWRRLRPRSGERELLLVGRLVIVALIGVSVAWIPVLQDSN SGQLFIYMQSVTSSLAPPVTAVFVLGVFWRRANEQQGAFWGLIAGLWGATRLVLEFLNPAPPCGEPDTRPAV LGSIHYLHFAVALFALSGAVWAGSLLTPPPQSVQIENLTWWTLAQDVPLGTKAGDGQTPQKHAFWARVCGFN
Al LLMCVNI FFYAYFA
A search against the Patp database, a proprietary database that contains sequences published in patents and patent publications, yielded several homologous proteins shown in Table 28C.
Table 28C. Patp results for NOV28
Smallest Sum
Reading High Prob
Sequences producing High-scoring Segment Pairs: Frame Score P(N)
>patp:AAE06614 Human protein +1 3051 6.1e-318
>patp:AAE08088 Human transporter-related protein #35 +1 3051 6.1e-318
>patp:AAR73595 Cotransporter protein SGLT1 +1 1669 5.2e-177
>patp:AAR73593 Cotransporter protein SNST1 +1 1655 4.2e-175
>patp:AAB60093 Human transport protein TPPT-13 +1 1622 3.1e-168 hi a BLAST search of public sequence databases, it was found, for example, that the nucleic acid sequence of this invention has 1451 of 1839 bases (78%) identical to a gb:GENBANK-ID.OCU08813|acc:U08813.1 mRNA from Oryctolagus cuniculus (Na+/glucose cotransporter-related protein mRNA, complete eds). NON28 polypeptide of the invention was found to have 532 of 600 amino acid residues (88%) identical to, and 560 of 600 amino acid residues (93%) similar to, the 597 amino acid residue ptnr:SPTREMBL-ACC:Q28610 protein from Oryctolagus cuniculus (ΝA+/GLUCOSE COTRANSPORTER-RELATED PROTEIN).
NOV28 also has homology to the proteins shown in the BLASTP data in Table 28D.
Figure imgf000260_0001
A multiple sequence alignment is given in Table 28E, with the NOV28 protein being shown on line 1 in Table 28E in a ClustalW analysis, and comparing the NOV28 protein with the related protein sequences shown in Table 28D. This BLASTP data is displayed graphically in the ClustalW in Table 28E. Table 28E. ClustalW Analysis of NOV28
1) > NOV28; SEQ TD NO:103
2) > gi|520469|/ 597 aa protein related to Na/glucose cotransporters [Oryctolagus cuniculus]; SEQ BD NO:303
3) > gi|1655393/unnamed protein product [Homo sapiens]; SEQ TD NO:304
4) > gi|9588428/ dJ1024N4.1 (novel Sodium:solute symporter family member similar to SLC5A1 (SGLTl) [Homo sapiens]; SEQ ED NO:305
5) > gi|631592|/ glucose transport protein [sheep]; SEQ TD NO:306
6) > gij 1709219/ SL51_sheep sodium/glucose cotransporter 1 Na+/glucose cotransporter 1) (high affinity sodium-glucose cotransporter); SEQ TD NO:307
Figure imgf000261_0001
Figure imgf000262_0001
610 620 630 640 650
NOV28 TP- røGMTP-
EBGBTP-
HIHIlAREDALEIDTEASEEKKGCLRQAYDMFCGLDQQKGPKMTKEEEAAM
Figure imgf000263_0001
lAREDALEIDTEASEEKKGCLRQAYDMFCGLDQQKGPKMTKEEEAAM
Figure imgf000263_0002
The presence of identifiable domains in the protein disclosed herein was determined by searches using algorithms such as PROSITE, Blocks, Pfam, ProDomain, Prints and then determining the Interpro number by crossing the domain match (or numbers) using the Interpro website (http:www.ebi.ac.uk/interpro/). Table 28F lists the domain description from DOMAIN analysis results against NON28.
Figure imgf000263_0003
Consistent with other known members of the sodium:solute symporter family (SSF), the
NON28 Νa+/glucose transporter-like protein contains the sodium:solute symporter family domain and an integral membrane domain as illustrated in Table 28F (Ohashi and Erickson, J. Biol. Chem., 272: 14220-6 (1997)). NOV28 nucleic acids, and the encoded polypeptides, according to the invention are useful in a variety of applications and contexts. For example, NON28 nucleic acids and polypeptides can be used to identify proteins that are members of the sodium: solute symporter family of proteins. The ΝON28 nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance ΝON28 activity or function. Specifically, the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., cellular activation and cellular metabolism. These molecules can be used to treat, e.g., for metabolic diseases such cancer, trauma, regeneration (in vitro and in vivo), viral/bacterial/parasitic infections, diabetes, autoimmune disease, renal artery stenosis, interstitial nephritis, glomerulonephritis, polycystic kidney disease, systemic lupus erythematosus, renal tubular acidosis, IgA nephropathy, hypercalceimia, Lesch-Nyhan syndrome, cardiomyopathy, atherosclerosis, hypertension, congenital heart defects, aortic stenosis, atrial septal defect (ASD), atrioventricular (A-N) canal defect, ductus arteriosus, pulmonary stenosis, subaortic stenosis, ventricular septal defect (VSD), valve diseases, tuberous sclerosis, scleroderma, obesity, transplantation and other diseases, disorders and conditions of the like. hi addition, various ΝON28 nucleic acids and polypeptides according to the invention are useful, inter alia, as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins. For example, the ΝON28 nucleic acids and their encoded polypeptides include structural motifs that are characteristic of proteins belonging to the SSF family such as the Νa+/glucose transporter proteins involved in renal transport and metabolism. (Ohashi and Erickson, J. Biol. Chem., 272: 14220-6 (1997)).
Integral membrane proteins that mediate the intake of a wide variety of molecules with the concomitant uptake of sodium ions are grouped into a number of distinct families. One of these families, known as the SSF, consists of integral membrane proteins that are predicted to comprise at least ten membrane spanning domains. Members of the SSF catalyze solute:Na+ symport
(Reizer et al,. Biochem. Biophys. Acta, 1197: 133-166 (1994)) can transport sugars, amino acids, nucleosides, inositols, vitamins, urea or anions, depending on the system. Members of the SSF family have been identified in bacteria, archaea and animals, and all functionally well characterized members catalyze solute uptake via Na+ symport. The NON28 nucleic acids and polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in the mediation of metabolism and immune function and renal physiology. As such, the ΝON28 nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat metabolic, immune and renal disorders, e.g., metabolic diseases such as diabetes and hypertension, or cancer, trauma, regeneration (in vitro and in vivo), viral/bacterial/parasitic infections, diabetes, autoimmune disease, renal artery stenosis, interstitial nephritis, glomerulonephritis, polycystic kidney disease, systemic lupus erythematosus, renal tubular acidosis, IgA nephropathy, hypercalceimia, Lesch-Nyhan syndrome, cardiomyopathy, atherosclerosis, hypertension, congenital heart defects, aortic stenosis, atrial septal defect (ASD), atrioventricular (A-V) canal defect, ductus arteriosus, pulmonary stenosis, subaortic stenosis, ventricular septal defect (VSD), valve diseases, tuberous sclerosis, scleroderma, obesity, transplantation and other diseases, disorders and conditions of the like. The NOV28 nucleic acids and polypeptides are useful for detecting specific cell types. For example, expression analysis has demonstrated that a NON28 nucleic acid is expressed in Kidney and Heart .
Additional utilities for ΝON28 nucleic acids and polypeptides according to the invention are disclosed herein.
ΝON29
A ΝON29 polypeptide has been identified as a Νa+/glucose transporter-like protein. Six alternative novel NOV29, NOV29a, NOV29b, NOV29c, NOV29d, NOV29e, and NOV29f, nucleic acids and encoded polypeptides are provided. The novel NON29 nucleic acid sequences maps to the chromosome 1.
ΝON29a
A ΝON29 variant is the novel ΝON29a (alternativelyreferred to herein as CG56557-01), which includes the 2147 nucleotide sequence (SEQ ID ΝO:104) shown in Table 29A. A NOV29a ORF begins with a Kozak consensus ATG initiation codon at nucleotides 66-68 and ends with a TGA codon at nucleotides 2118-2120. Putative untranslated regions upstream from the initiation codon and downstream from the termination codon are underlined in Table 29A, and the start and stop codons are in bold letters.
Table 29A. NON29a Nucleotide Sequence (SEQ ID NO:104)
TTAAAGGAAΆGGAATGAAGCCAGGAGCGCCTCAΆAGTCCAGCCTGCTGTTGACCAACACTAΆCAGAT GAGCAAGGAGCTGGCAGCAATGGGGCCTGGAGCTTCAGGGGACGGGGTCAGGACTGAGACAGCTCCA CACATAGCACTGGACTCCAGAGTTGGTCTGCACGCCTACGACATCAGCGTGGTGGTCATCTACTTTG TCTTCGTCATTGCTGTGGGGATCTGGTCGTCCATCCGTGCAAGTCGAGGGACCATTGGCGGCTATTT CCTGGCCGGGAGGTCCATGAGCTGGTGGCCAGTGGGAGCATCTCTGATGTCCAGCAATGTGGGCAGT GGCTTGTTCATCGGCCTGGCTGGGACAGGGGCTGCCGGAGGCCTTGCCGTAGGTGGCTTCGAGTGGA ACGTAAGGAAGCTGGCCTGGTTTCTCGTCTTCGTCCCTGTGTACATCGCAGCAGGTGTGGTCACAAT GCCGCAGTATCTGAAGAAGCGATTTGGGGGCCAGAGGATCCAGGTGTACATGTCTGTCCTGTCTCTC ATCCTCTACATCTTCACCAAGATCTCGGTAGACATCTTCTCTGGAGCCCTCTTCATCCAGATGGCAT TGGGCTGGAACCTGTACCTCTCCACAGGGATCCTGCTGGTGGTGACTGCCGTCTACACCATTGCAGG TGGCCTCATGGCCGTGATCTACACAGATGCTCTGCAGACGGTGATCATGGTAGGGGGAGCCCTGGTC CTCATGTTTCAGGACGTGGGCTGGTACCCAGGCCTGGAGCAGCGGTACAGGCAGGCCATCCCTAATG TCACAGTCCCCAACACCACCTGTCACCTCCCACGGCCCGATGCTTTCCACATTCTTCGGGACCCTGT GAGCGGGGACATCCCTTGGCCAGGTCTCATTTTCGGGCTCACAGTGCTGGCCACCTGGTGTTGGTGC ACAGACCAGGTCATTGTGCAGCGGTCTCTCTCGGCCAAGAGTCTGTCTCATGCCAAGGGAGGCTCCG TGCTGGGGGGCTACCTGAAGATCCTCCCCATGTTCTTCATCGTCATGCCTGGCATGATCAGCCGGGC CCTGTTCCCAGACGAGGTGGGCTGCGTGGACCCTGATGTCTGCCAAAGAATCTGTGGGGCCCGAGTG GGATGTTCCAACATTGCCTACCCTAAGTTGGTCATGGCCCTCATGCCTGTTGGTCGGGGGCTGATGA TTGCCGTGATCATGGCCGCTCTCATGAGCTCACTCACCTCCATCTTCAACAGCAGCAGCACCCTGTT CACCATTGATGTGTGGCAGCGCTTCCGCAGGAAGTCAACAGAGCAGGAGCTGATGGTGGTGGGCAGG GTGTTTGTGGTGTTCCTGGTTGTCATCAGCATCCTCTGGATCCCCATCATCCAAAGCTCCAACAGTG GGCAGCTCTTCGACTACATCCAGGCTGTCACCAGTTACCTGGCCCCACCCATCACCGCTCTCTTCCT GCTGGCCATCTTCTGCAAGAGGGTCACAGAGCAGGGAGCTTTCTGGGGCCTCGTGTTTGGCCTGGGA GTGGGGCTTCTGCGTATGATCCTGGAGTTCTCATACCCAGCGCCAGCCTGTGGGGAGGTGGACCGGA GGCCAGCAGTGCTGAAGGACTTCCACTACCTGTACTTTGCAATCCTCCTCTGCGGGCTCACTGCCAT CGTCATTGTCATTGTCAGCCTCTGTACAACTCCCATCCCTGAGGAACAGGCAAGTCGCCTCACATGG TGGACTCGGAACTGCCCCCTCTCTGAGCTGGAGAAGGAGGCCCCCCCATACTTTCCATCAGTATCTC ACCATCTCTCTCCCTCCCCTACTCTCCTATCACTTTCCTTTCTCCAATCTTCTTTGCCTCTCCCCTC CTGCTCTCCTTTGTCTTCTGGCTTTGTCCCTCCAGCCCCAAGCAGGTCCTGGGGAAAGTTGCTCTGG AGCTGGTTCTGTGGGCTCTCTGGAACACCGGAGCAGGCCCTGAGCCCAGCAGAGAAGGCTGCGCTAG AACAGAAGCTGACAAGCATTGAGGAGGAGCCACTCTGGAGACATGTCTGCAACATCAATGCTGTCCT TTTGCTGGCCATCAACATCTTCCTCTGGGGCTATTTTGCGTGATTCCACAGACCTGGCTTCAGTGTA GAC
The NON29a polypeptide (SEQ ID NO: 105) encoded by SEQ ID NO: 104 is 684 amino acid residues in length and is presented using the one-letter amino acid code in Table 29B. NOV29a has two SNP variants, whose variant positions for their nucleotide and amino acid sequences is numbered according to SEQ ID NOS: 104 and 105, respectively. A variant sequence can include a single nucleotide polymorphism (SNP). A SNP can, in some instances, be referred to as a "cSNP" to denote that the nucleotide sequence containing the SNP originates as a cDNA. NOV29a Variant 13374708 is a G to A SNP at 774 bp of the nucleotide sequence that results in no change in the protein sequence (silent). NOV29a variant 13375611 is a T to C SNP at 1572 bp of the nucleotide sequence that results in a Ser to Pro change at amino acid 503 of protein sequence, and NON29a variant 13375610 is a T to C SΝP at 1684 bp of the nucleotide sequence that results in a Nal to Ala change at amino acid 540 of protein sequence.
The Psort profile for the ΝON29a predicts that this peptide is likely to be localized at the plasma membrane with a certainty of 0.8000. The Signal P predicts a likely cleavage site for a ΝON29a peptide is between positions 59 and 60, i.e., at the dash in the sequence IRA-SR.
Table 29B. ΝOV29a protein sequence (SEQ ID NO: 105) SKELAAMGPGASGDGVRTETAPHIALDSRVGLHAYDISVWIYFVFVIAVGI SSIRASRGTIGGY FLAGRSMS PVGASLMSSNVGSGLFIGLΆGTGAAGGLAVGGFE NVRKLA FLVFVPVΎIAAGWT MPQYLKKRFGGQRIQVYMSVLSLILYIFTKISVDIFSGALFIQMALGNLYLSTGILLWTAVYTIA GGLMAVIYTDALQTVIMVGGALVLMFQDVG YPGLEQRYRQAIPNVTVPNTTCHLPRPDAFHILRDP VSGDIPWPGLIFGLTVLATWCWCTDQVIVQRSLSAKSLSHAKGGSVLGGYLKILPMFFIVMPGMISR ALFPDEVGCVDPDVCQRICGARVGCSNIAYPKLVMALMPVGRGLMIAVIMAALMSSLTSIFNSSSTL FTIDVWQRFRRKSTEQELMWGRVFWFLWISIL IPIIQSSNSGQLFDYIQAVTSYLAPPITALF LLAIFCKRVTEQGAFWGLVFGLGVGLLRMILEFSYPAPACGEVDRRPAVLKDFHYLYFAILLCGLTA IVIVIVSLCTTPIPEEQASRLT WTRNCPLSELE EAPPYFPSVSHHLSPSPTLLSLSFLQSSLPLP SCSPLSSGFVPPAPSRSWGKLL S FCGLSGTPEQALSPAEKAALEQLTSIEEEPLWRHVCNINAV LLLAINIFLWGYFA
NON29b
Alternatively, a ΝON29 variant is the novel ΝON29b (alternatively referred to herein as CG56557-02), which includes the 797 nucleotide sequence (SEQ ID ΝO:106) shown in Table 29C. NON29b was cloned by polymerase chain reaction (PCR) using the primers: 5' GTCAGGACTGAGACAGCTCCACAC 3' (SEQ ID ΝO.-308) and 5'
CTGAAGCCAGGTCTGTGGAATCAC 3' (SEQ ID NO:309). Primers were designed based on in silico predictions of the full length or some portion (one or more exons) of the cDN A/protein sequence of the invention. These primers were used to amplify a cDNA from a pool containing expressed human sequences derived from the following tissues: adrenal gland, bone marrow, brain - amygdala, brain - cerebellum, brain - hippocampus, brain - substantia nigra, brain - thalamus, brain -whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma - Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea and uterus. The PCR product derived by exon linking, covering the entire open reading frame, was cloned into the pCR2.1 vector from Invitrogen to provide clone 57808::ba252a4.698037.N15.
The NON29b ORF begins with a Kozak consensus ATG initiation codon at nucleotides 19-21 and ends with a TGA codon at nucleotides 775-777. Putative untranslated regions upstream from the initiation codon and downstream from the termination codon are underlined in Table 29C, and the start and stop codons are in bold letters.
Table 29C. ΝON29b Nucleotide Sequence (SEQ ID NO:106)
AGCAΆGGAGCTGGCAGCAATGGGGCCTGGAGCTTCAGGGGACGGGGTCAGGACTGAGACAGCTCCAC ACATAGCACTGGACTCCAGAGTTGGTCTGCACGCCTACGACATCAGCGTGGTGGTCATCTACTTTGT CTTCGTCATTGCTGTGGGGATCTGGTCGTCCATCTTCTGCAAGAGGGTCACAGAGCCCGGAGCTTTC TGGGGCCTCGTGTTTGGCCTGGGAGTGGGGCTTCTGCGTATGATCCTGGAGTTCTCATACCCAGCGC CAGCCTGTGGGGAGGTGGACCGGAGGCCAGCAGTGCTGAAGGACTTCCACTACCTGTACTTTGCAAT CCTCCTCTGCGGGCTCACTGCCATCGTCATTGTCATTGTCAGCCTCTGTACAACTCCCATCCCTGAG GAACAGCTCACACGCCTCACATGGTGGACTCGGAACTGCCCCCTCTCTGAGCTGGAGAAGGAGGCCC CGAGAGCACACCGGAGATATCCGAGAGGCCAGCCGGGGAGTGCCCTGCAGGAGGTGGAGCGGCAGA GAACTCGAGCCTGGGCCAGGAGCAGCCTGAAGCCCCAAGCAGGTCCTGGGGAAAGTTGCTCTGGAGC TGGTTCTGTGGGCTCTCTGGAACACCGGAGCAGGCCCTGAGCCCAGCAGAGAAGGCTGCGCTAGAAC AGAAGCTGACAAGCATTGAGGAGGAGCCACTCTGGAGACATGTCTGCAACATCAATGCTGTCCTTTT GCTGGCCATCAACATCTTCCTCTGGGGCTATTTTGCGTGATTCCACAGACCTGGCTTCAG
The NON29b protein (SEQ ID ΝO:107) encoded by SEQ ID NO:106 is 252 amino acid residues in length is presented using the one-letter code in Table 29D. The Psort profile for NON29b predicts that this sequence is likely to be localized at the plasma membrane with a certainty of 0.6000. The Signal P predicts a likely cleavage site for a ΝON29b peptide is between positions 52 and 53, i.e., at the dash in the sequence IFC-KR.
Table 29D. ΝON29b protein sequence (SEQ ID ΝO:107)
MGPGASGDGVRTETAPHIALDSRVGLHAYDISVWIYFVFVIAVGI SSIFCKRVTEPGAF GLVFG LGVGLLRMILEFSYPAPACGEVDRRPAVLKDFHYLYFAILLCGLTAIVIVIVSLCTTPIPEEQLTRL TWWTRNCPLSELEKEAHESTPEISERPAGECPAGGGAAENSSLGQEQPEAPSRSWGKLLWSWFCGLS GTPEQALSPAEKAALEQKLTSIEΞEPLWRHVCNINAVLLLAINIFLWGYFA
NOV29c
Alternatively, a NOV29 variant is the novel NOV29c (alternatively referred to herein as CG56557-03), which includes the 2278 nucleotide sequence (SEQ ID NO:108) shown in Table 29E. The NOV29c ORF begins with a Kozak consensus sequence ATG identified at nucleotides 20-22 and ends with a TGA codon at nucleotides 2248-2250. Putative untranslated regions upstream from the initiation codon and downstream from the termination codon are underlined in Table 29E, and the start and stop codons are in bold letters.
Table 29E. NOV29c Nucleotide Sequence (SEQ ID NO:108)
GAGCAAGGAGCTGGCAGCAATGGGGCCTGGAGCTTCAGGGGACGGGGTCAGGACTGAGACAGCTCCA CACATAGCACTGGACTCCGGAGTTGGTCTGCACGCCTACGACATCAGCGTGGTGGTCATCTACTTTG TCTTCGTCATTGCTGTGGGGATCTGGTCGTCCATCCGTGCAAGTCGAGGGACCATTGGCGGCTATTT CCTGGCCGGGAGGTCCATGAGCTGGTGGCCAATTGGAGCATCTCTGATGTCCAGCAATGTGGGCAGT GGCTTGTTCATCGGCCTGGCTGGGACAGGGGCTGCCGGAGGCCTTGCCGTAGGTGGCTTCGAGTGGA ACATGAGGAAATCAAGGTCTGGAGGAGACAGAGGGATCCATCCAAGGTCACACGGGAGGACTGGGGT CAGGTCCCAGGTCTCTTATTTCTCTGTTCGGGGGCCTCCCACAGCACAGCACTGCCTCTGGGTGGGA AGCCGCCCCTCTGTCTACATCCAGGACCTGGATACCTTCTTCTTCTCCCCACTCTCCCAGGCAACCT GGCTGCTCCTGGCCCTTGGCTGGGTCTTCGTCCCTGTGTACATCGCAGCAGGTGTGGTCACAATGCC GCAGTATCTGAAGAAGCGATTTGGGGGCCAGAGGATCCAGGTGTACATGTCTGTCCTGTCTCTCATC CTCTACATCTTCACCAAGATCTCGACTGACATCTTCTCTGGAGCCCCCTTCATCCAGATGGCATTGG GCTGGAACCTGTACCTCTCCACAGGGATCCTGCTGGTGGTGACTGCCGTCTACACCATTGCAGGTGG CCTCATGGCCGTGATCTACACAGATGCTCTGCAGACGGTGATCATGGTAGGGGGAGCCCTGGTCCTC ATGTTTCAGGACGTGGGCTGGTACCCAGGCCTGGAGCAGCGGTACAGGCAGGCCATCCCTAATGTCA CAGTCCCCAACACCACCTGTCACCTCCCACGGCCCGATGCTTTCCACATTCTTCGGGACCCTGTGAG CGGGGACATCCCTTGGCCAGGTCTCATTTTCGGGCTCACAGTGCTGGCCACCTGGTGTTGGTGCACA GACCAGGTCATTGTGCAGCGGTCTCTCTCGGCCAAGAGTCTGTCTCATGCCAAGGGAGGCTCCGTGC TGGGGGGCTACCTGAAGATCCTCCCCATGTTCTTCATCGTCATGCCTGGCATGATCAGCCGGGCCCT GTTCCCAGACGAGGTGGGCTGCGTGGACCCTGATGTCTGCCAAAGAATCTGTGGGGCCCGAGTGGGA TGTTCCAACATTGCCTACCCTAAGTTGGTCATGGCCCTCATGCCTGTTGGTCGGGGGCTGATGATTG CCGTGATCATGGCCGCTCTCATGAGCTCACTCACCTCCATCTTCAACAGCAGCAGCACCCTGTTCAC CATTGATGTGTGGCAGCGCTTCCGCAGGAAGTCAACAGAGCAGGAGCTGATGGTGGTGGGCAGGGTG TTTGTGGTGTTCCTGGTTGTCATCAGCATCCTCTGGATCCCCATCATCCAAAGCTCCAACAGTGGGC AGCTCTTCGACTACATCCAGGCTGTCACCAGTTACCTGGCCCCACCCATCACCGCTCTCTTCCTGCT GGCCATCTTCTGCAAGAGGGTCACAGAGCAGGGAGCTTTCTGGGGCCTCGTGTTTGGCCTGGGAGTG GGGCTTCTGCGTATGATCCTGGAGTTCTCATACCCAGCGCCAGCCTGTGGGGAGGTGGACCGGAGGC CAGCAGTGCTGAAGGACTTCCACTACCTGTACTTTGCAATCCTCCTCTGCGGGCTCACTGCCATCGT CATTGTCATTGTCAGCCTCTGTACAACTCCCATCCCTGAGGAACAGGCAAGTCGCCTCACATGGTGG ACTCGGAACTGCCCCCTCTCTGAGCTGGAGAAGGAGGCCCCCCCATACTTTCCATCAGTATCTCACC ATCTCTCTCCCTCCCCTACTCTCCTATCACTTTCCTTTCTCCAATCTTCTTTGCCTCTCCCCTCCTG CTCTCCTTTGTCTTCTGGCTTTGTCCCTCCAGCCCCAAGCAGGTCCTGGGGAAAGTTGCTCTGGAGC TGGTTCTGTGGGCTCTCTGGAACACCGGAGCAGGCCCTGAGCCCAGCAGAGAAGGCTGCGCTAGAAC AGAAGCTGACAAGCATTGAGGAGGAGCCACTCTGGAGACATGTCTGCAACATCAATGCTGTCCTTTT GCTGGCCATCAACATCTTCCTCTGGGGCTATTTTGCGTGATTCCACAGACCTGGCTTCAGTGTAGAC
The NON29c protein (SEQ ID NO: 109) encoded by SEQ ID NO: 108 is 743 amino acid residues in length is presented using the one-letter code in Table 29F. The Psort profile for NOV29c predicts that this sequence is likely to be localized in the cytoplasm with a certainty of 0.8000. The Signal P predicts a likely cleavage site for a NOV29c peptide is between positions 52 and 53, i.e., at the dash in the sequence IRA-SR.
Table 29F. NON29c protein sequence (SEQ ID ΝO:109)
MGPGASGDGVRTETAPHIALDSGVGLHAYDISVWIYFVFVIAVGIWSSIRASRGTIGGYFLAGRSM SW PIGASLMSSNVGSGLFIGLAGTGAAGGLAVGGFE NMRKSRSGGDRGIHPRSHGRTGVRSQVSY FSVRGPPTAQHCL VGSRPSVYIQDLDTFFFSPLSQATWLLLALG VFVPVYIAAGWTMPQYLKKR FGGQRIQVYMSVLSLILYIFTKISTDIFSGAPFIQMALGWNLYLSTGILLWTAVYTIAGGLMAVIY TDALQTVIMVGGALVLMFQDVG YPGLEQRYRQAIPNVTVPNTTCHLPRPDAFHILRDPVSGDIPWP GLIFGLTVLATWC CTDQVIVQRSLSAKSLSHAKGGSVLGGYLKILPMFFIVMPGMISRALFPDEVG CVDPDVCQRICGARVGCSNIAYPIOiVMALMPVGRGLMIAVIMAALMSSLTSIFNSSSTLFTIDV QR FRR STEQELMWGRVFWFLWISIL IPIIQSSNSGQLFDYIQAVTSYLAPPITALFLLAIFCKR VTEQGAFWGLVFGLGVGLLRMILEFSYPAPACGEVDRRPAVLKDFHYLYFAILLCGLTAIVIVIVSL CTTPIPEEQASRLT TRNCPLSELE EAPPYFPSVSHHLSPSPTLLSLSFLQSSLPLPSCSPLSSG FVPPAPSRSWGKLLWSWFCGLSGTPEQALSPAEKAALEQKLTSIEEEPLWRHVCNINAVLLLAINIF LWGYFA NON29d
Alternatively, a ΝON29 variant is the novel ΝON29d (alternatively referred to herein as CG56653-04), which includes the 1969 nucleotide sequence (SEQ ID ΝO:110) shown in Table 29G. The NOV29d ORF begins with a Kozak consensus ATG initiation codon at nucleotides 18- 20 and ends with a TGA codon at nucleotides 847-849. Putative untranslated regions upstream from the initiation codon and downstream from the termination codon are underlined in Table 29G, and the start and stop codons are in bold letters.
Table 29G. NOV29d Nucleotide Sequence (SEQ ID NO.-110)
AGCAAGGAGCTGGCAGCAATGGGGCCTGGAGCTTCAGGGGACGGGGTCAGGACTGAGACAGCTCCAC ACATAGCACTGGACTCCAGAGTTGGTCTGCACGCCTACGACATCAGCGTGGTGGTCATCTACTTTGT CTTCGTCATTGTTGTGGGGATCTGGTCGTCCATCCGTGCAAGTCGAGGGACCATTGGCGGCTATTTC CTGGCCCCACCCATCACCGCTCTCTTCCTGCTGGCCATCTTCTGCAAGAGGGTCACAGAGCCCGGAG CTTTCTGGGGCCTCGTGTTTGGCCTGGGAGTGGGGCTTCTGCGTATGATCCTGGAGTTCTCATACCC AGCGCCAGCCTGTGGGGAGGTGGACCGGAGGCCAGCAGTGCTGAAGGACTTCCACTACCTGTACTTT GCAATCCTCCTCTGCGGGCTCACTGCCATCGTCATTGTCATTGTCAGCCTCTGTACAACTCCCATCC CTGAGGAACAGCTCACACGCCTCACATGGTGGACTCGGAACTGCCCCCTCTCTGAGCTGGAGAAGGA GGCCCACGAGAGCACACCGGAGATATCCGAGAGGCCAGCCGGGGAGTGCCCTGCAGGAGGTGGAGCG GCAGAGAACTCGAGCCTGGGCCAGGAGCAGCCTGAAGCCCCAAGCAGGTCCTGGGGAAAGTTGpTCT GGAGCTGGTTCTGTGGGCTCTCTGGAACACCGGAGCAGGCCCTGAGCCCAGCAGAGAAGGCTGCGCT AGAACAGAAGCTGACAAGCATTGAGGAGGAGCCACTCTGGAGACATGTCTGCAACATCAATGCTGTC CTTTTGCTGGCCATCAACATCTTCCTCTGGGGCTATTTTGCGTGATTCCACAGACCTGGCTTCAGTG TAGACAGATTAAACAAAGCCCAAGCCTGTCAGCCACAGAAACAGGCTCTCCTCTTACTTTGCTGTCT AAACTGGAGATCACAGAAGTCAAGACTGCAAGCTCCCCTGAAGAGAATCCAACTCAACCTGCACACT TGACAAGTGGAGAAACAGAAGCTCAGAGAGAGCACTGGGTTTGTTCAGGACCACCCAGAAGGTGTCA CACGGGGTTTCCCCACTCTTTCTGATATATTGCCTTACAGACCTACCTCAAACACACTGTTTCCACC CTCTTCTTGAATGTATTCAGTAGCCTTTACTGAATGTGTGTCTTGAGAGTAGAAAAATGGAGGATAC AAGAAAAGGAGCAGGAAGAAATTTGCAAAAATCCAAGAGCACCTTTGCTCCCCCTTATCCTCCTTCC TCTTCCCCTTTCTAGTTCCCCTACCTCTCTATCTTTCTATTCTCACCAATAATCTCTTTGTTGCATG AATTTACCCAGGAGAGTCCTATATTTCCATTGGTGGCTCCACAGTGGTGGCTGTCAGACCCGAAGGG GTGGGGAGCCAAGGGTGGACTTTAAGCATGGTGACAGATGGTATTTTGGGCAGAAAGCTCTTAGACA ATGGACTATCCAAAGCACTATTTAAATTCTGCCTCTTCCTACTCTCTAACCCAAATATGCACAAACT CTCTATGGCCTTGAGAAGCAGTTGGAGAGACATGACTTGTTAAAACCTCAAGGAATCAAGACATGTT ACTCTGTATTTAAGGGTAAGCCCCACAGCGGGCAGCACAAACAGCCTGGGAGCCACTGTGCCTGTGC TTCTCTGTCCTTCTCCCTTTGCTTGCCATGAATCCGCATACCTTGGAATACACTGTGACCCCAGTTA AGTGTCCCTTCGCCAGGAAGCTGCCGCAACGTCCAGACCTGGGTCAAGTTCCCACTCCTGCTCCCAT AGCCTTGACCTGCTTCTGTCACAGCACTGATCACACTGAGATGGAAGACTCCAGGGGGCAAGGACCA AGGGCCATATCCCAAGTGACTTTGTACCCAGAAAATAACAGCTGTTCAATAAATGTGTATTGAGTTA ATTAGTTAAAAAAAAAAAAAAAAAAA
The NOV29d protein (SEQ ID NO:lll) encoded by SEQ ID NO:110 is 276 amino acid residues in length is presented using the one-letter code in Table 29H. The Psort profile for NON29d predicts that this sequence is likely to be localized at the plasma membrane with a certainty of 0.6000. The Signal P predicts a likely cleavage site for a ΝON29d peptide is between positions 52 and 53, i.e., at the dash in the sequence IRA-SR.
Table 29H. ΝON29d protein sequence (SEQ ID ΝO:lll)
MGPGASGDGVRTETAPHIALDSRVGLHAYDISVWIYFVFVIWGI SSIRASRGTIGGYFLAPPIT ALFLLAIFC RVTEPGAF GLVFGLGVGLLRMILEFSYPAPACGEVDRRPAVLKDFHYLYFAILLCG LTAIVIVIVSLCTTPIPEEQLTRLTW TRNCPLSELEKEAHESTPEISERPAGΞCPAGGGAAENSSL GQEQPEAPSRS GKLL SWFCGLSGTPEQALSPAEKAALEQKLTSIEEΞPL RHVCNINAVLLLAIN IFL GYFA
NOV29e
Alternatively, a NON29 variant is the novel ΝON29e (alternatively referred to herein as CG56557-05), wliich includes the 2162 nucleotide sequence (SEQ ID ΝO:112) shown in Table 291. The NOV29e ORF begins with a Kozak consensus ATG initiation codon at nucleotides 21- 23 and ends with a TAG codon at nucleotides 2133-2135. Putative untranslated regions upstream from the initiation codon and downstream from the termination codon are underlined in Table 291, and the start and stop codons are in bold letters.
Table 291. NOV29e Nucleotide Sequence (SEQ ID NO:112)
TGAGCAAGGAGCTGGCAGCAATGGGGCCTGGAGCTTCAGGGGACGGGGTCAGGACTGAGACAGCTCC ACACATAGCACTGGACTCCAGAGTTGGTCTGCACGCCTACGACATCAGCGTGGTGGTCATCTACTTT GTCTTCGTCATTGCTGTGGGGATCTGGTCGTCCATCCGTGCAAGTCGAGGGACCATTGGCGGCTATT TCCTGGCCGGGAGGTCCATGAGCTGGCGGCCAATTGGAGCATCTCTGATGTCCAGCAATGTGGGCAG TGGCTTGTTCATCGGCCTGGCTGGGACAGGGGCTGCCGGAGGCCTTGCCGTAGGTGGCTTCGAGTGG AACATGAGGAAATCAAGGTCTGGAGGAGACAGAGGGATCCATCCAAGGTCACACGGGAGGACTGGGG TCAGGTCCCAGGCAACCTGGCTGCTCCTGGCCCTTGGCTGGGTCTTCGTCCCTGTGTACATCGCAGC AGGTGTGGTCACAATGCCGCAGTATCTGAAGAAGCGATTTGGGGGCCAGAGGATCCAGGTGTACATG TCTGTCCTGTCTCTCATCCTCTACATCTTCACCAAGATCTCGACTGACATCTTCTCTGGAGCCCTCT TCATCCAGATGGCATTGGGCTGGAACCTGTACCTCTCCACAGGGATCCTGCTGGTGGTGACTGCCGT CTACACCATTGCAGGTGGCCTCATGGCCGTGATCTACACAGATGCTCTGCAGACGGTGATCATGGTA GGGGGAGCCCTGGTCCTCATGTTTCAGGACGTGGGCTGGTACCCAGGCCTGGAGCAGCGGTACAGGC AGGCCATCCCTAATGTCACAGTCCCCAACACCACCTGTCACCTCCCACGGCCCGATGCTTTCCACAT TCTTCGGGACCCTGTGAGCGGGGACATCCCTTGGCCAGGTCTCATTTTCGGGCTCACAGTGCTGGCC ACCTGGTGTTGGTGCACAGACCAGGTCATTGTGCAGCGGTCTCTCTCGGCCAAGAGTCTGTCTCATG CCAAGGGAGGCTCCGTGCTGGGGGGCTACCTGAAGATCCTCCCCATGTTCTTCATCGTCATGCCTGG CATGATCAGCCGGGCCCTGTTCCCAGACGAGGTGGGCTGCGTGGACCCTGATGTCTGCCAAAGAATC TGTGGGGCCCGAGTGGGATGTTCCAACATTGCCTACCCTAAGTTGGTCATGGCCCTCATGCCTGTTG GTCGGGGGCTGATGATTGCCGTGATCATGGCCGCTCTCATGAGCTCACTCACCTCCATCTTCAACAG CAGCAGCACCCTGTTCACCATTGATGTGTGGCAGCGCTTCCGCAGGAAGTCAACAGAGCAGGAGCTG ATGGTGGTGGGCAGGGTGTTTGTGGTGTTCCTGGTTGTCATCAGCATCCTCTGGATCCCCATCATCC AAAGCTCCAACAGTGGGCAGCTCTTCGACTACATCCAGGCTGTCACCAGTTACCTGGCCCCACCCAT CACCGCTCTCTTCCTGCTGGCCATCTTCTGCAAGAGGGTCACAGAGCAGGGAGCTTTCTGGGGCCTC GTGTTTGGCCTGGGAGTGGGGCTTCTGCGTATGATCCTGGAGTTCTCATACCCAGCGCCAGCCTGTG GGGAGGTGGACCGGAGGCCAGCAGTGCTGAAGGACTTCCACTACCTGTACTTTGCAATCCTCCTCTG CGGGCTCACTGCCATCGTCATTGTCATTGTCAGCCTCTGTACAACTCCCATCCCTGAGGAACAGGCA AGTCGCCTCACATGGTGGACTCGGAACTGCCCCCTCTCTGAGCTGGAGAAGGAGGCCCCCCCATACT TTCCATCAGTATCTCACCATCTCTCTCCCTCCCCTACTCTCCTATCACTTTCCTTTCTCCAATCTTC TTTGCCTCTCCCCTCCTGCTCTCCTTTGTCTTCTGGCTTTGTCCCTCCAGCCCCAAGCAGGTCCTGG GGAAAGTTGCTCTGGAGCTGGTTCTGTGGGCTCTCTGGAACACCGGAGCAGGCCCTGAGCCCAGCAG AGAAGGCTGCGCTAGAACAGAAGCTGACAAGCATTGAGGAGGAGCCACTCTGGAGACATGTCTGCAA CATCAATGCTGTCCTTTTGCTGGCCATCAACATCTTCCTCTGGGGCTATTTTGCGTGATTCCACAGA CCTGGCTTCAGTGTAGAC
The NON29e protein (SEQ ID ΝO:113) encoded by SEQ ID NO:112 is 704 amino acid residues in length is presented using the one-letter code in Table 29J. The Psort profile for NON29e predicts that this sequence is likely to be localized at the plasma membrane with a certainty of 0.8000. The Signal P predicts a likely cleavage site for aΝON29e peptide is between positions 52 and 53, i.e., at the dash in the sequence IRA-SR.
Table 29J. ΝON29e protein sequence (SEQ ID ΝO:113) GPGASGDGVRTETAPHIALDSRVGLHAYDISWVIYFVFVIAVGI SSIRASRGTIGGYFLAGRSM S RPIGASLMSSNVGSGLFIGLAGTGAAGGLAVGGFE NMRKSRSGGDRGIHPRΞHGRTGVRSQAT LLLALG VFVPVYIAAGWTMPQYLKKRFGGQRIQVYMSVLSLILYIFTKISTDIFSGALFIQMALG NLYLSTGILLWTAVYTIAGGLMAVIYTDALQTVIMVGGALVLMFQDVG YPGLEQRYRQAIPNVT VPNTTCHLPRPDAFHILRDPVSGDIP PGLIFGLTVLAT CWCTDQVIVQRSLSAKSLSHAKGGSVL GGYLKILPMFFIVMPGMISRALFPDEVGCVDPDVCQRICGARVGCSNIAYPKLVMALMPVGRGLMIA VIMAALMSSLTSIFNSSSTLFTIDV QRFRRKSTEQELMWGRVFWFLWISIL IPIIQSSNSGQ LFDYIQAVTSYLAPPITALFLLAIFCKRVTEQGAFWGLVFGLGVGLLRMILEFSYPAPACGEVDRRP AVLKDFHYLYFAILLCGLTAIVIVIVSLCTTPIPEEQASRLTW TRNCPLSELEKEAPPYFPSVSHH LSPSPTLLSLSFLQSSLPLPSCSPLSSGFVPPAPSRSWGKLL S FCGLSGTPEQALSPAEKAALEQ KLTSIEEEPLWRHVCNINAVLLLAINIFL GYFA
NOV29f
Alternatively, a NON29 variant is the novel ΝON29f (alternatively referred to herein as CG56557-06), which includes the 875 nucleotide sequence (SEQ ID ΝO:114) shown in Table 29K. NOV25e was cloned by the polymerase chain reaction (PCR) using the primers: 5' GTCAGGACTGAGACAGCTCCACAC 3' (SEQ ID NO:310) and 5' CTGAAGCCAGGTCTGTGGAATCAC 3' (SEQ ID NO:311). Primers were designed based on in silico predictions of the full length or some portion (one or more exons) of the cDNA/protein sequence of the invention. These primers were used to amplify a cDNA from a pool containing expressed human sequences derived from the following tissues: adrenal gland, bone marrow, brain - amygdala, brain - cerebellum, brain - hippocampus, brain - substantia nigra, brain - thalamus, brain -whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma - Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea and uterus. The PCR product derived by exon linking, covering the entire open reading frame, was cloned into the ρCR2.1 vector from Invitrogen to provide clone 57808::ba252a4.698037.Nl.
The NOV29f ORF begins with a Kozak consensus ATG imtiation codon at nucleotides 19-21 and ends with a TGA codon at nucleotides 847-849. Putative untranslated regions upstream from the imtiation codon and downstream from the termination codon are underlined in Table 29K, and the start and stop codons are in bold letters.
Table 29K. NON29f Nucleotide Sequence (SEQ ID NO:114)
AGCAAGGAGCTGGCAGCAATGGGGCCTGGAGCTTCAGGGGACGGGGTCAGGACTGAGACAGCTCCAC ACATAGCACTGGACTCCAGAGTTGGTCTGCACGCCTACGACATCAGCGTGGTGGTCATCTACTTTGT CTTCGTCATTGTTGTGGGGATCTGGTCGTCCATCCGTGCAAGTCGAGGGACCATTGGCGGCTATTTC CTGGCCCCACCCATCACCGCTCTCTTCCTGCTGGCCATCTTCTGCAAGAGGGTCACAGAGCCCGGAG CTTTCTGGGGCCTCGTGTTTGGCCTGGGAGTGGGGCTTCTGCGTATGATCCTGGAGTTCTCATACCC AGCGCCAGCCTGTGGGGAGGTGGACCGGAGGCCAGCAGTGCTGAAGGACTTCCACTACCTGTACTTT GCAATCCTCCTCTGCGGGCTCACTGCCATCGTCATTGTCATTGTCAGCCTCTGTACAACTCCCATCC CTGAGGAACAGCTCACACGCCTCACATGGTGGACTCGGAACTGCCCCCTCTCTGAGCTGGAGAAGGA GGCCCACGAGAGCACACCGGAGATATCCGAGAGGCCAGCCGGGGAGTGCCCTGCAGGAGGTGGAGCG GCAGAGAACTCGAGCCTGGGCCAGGAGCAGCCTGAAGCCCCAAGCAGGTCCTGGGGAAAGTTGCTCT GGAGCTGGTTCTGTGGGCTCTCTGGAACACCGGAGCAGGTCCTGAGCCCAGCAGAGAAGGCTGCGCT AGAACAGAAGCTGACAAGCATTGAGGAGGAGCCACTCTGGAGACATGTCTGCAACATCAATGCTGTC CTTTTGCTGGCCATCAACATCTTCCTCTGGGGCTATTTTGCGTGATTCCACAGACCTGGCTTCAGTG TAGA
Variant sequences of NON29f are included in Example 2. A variant sequence can include a single nucleotide polymorphism (SΝP).
The ΝON29f protein (SEQ ID ΝO:115) encoded by SEQ ID NO:114 is 319 amino acid residues in length is presented using the one-letter code in Table 29L. The Psort profile for NO V29f predicts that this sequence is likely to be localized at the plasma membrane with a certainty of 0.6000. The Signal P predicts a likely cleavage site for a NO V29f peptide is between positions 52 and 53, i.e., at the dash in the sequence IRA-SR.
Table 29L. NOV29f protein sequence (SEQ ID NO-.115)
MGPGASGDGVRTETAPHIALDSRVGLHAYDISWVIYFVFVIWGIWSSIRASRGTIGGYFLAPPIT ALFLLAIFCKRVTEPGAF GLVFGLGVGLLRMILEFSYPAPACGEVDRRPAVLKDFHYLYFAILLCG LTAIVIVIVSLCTTPIPEEQLTRLT WTRNCPLSELEKEAHESTPEISERPAGECPAGGGAAENSSL GQEQPEAPSRS GKLLWS FCGLSGTPEQVLSPAE AALEQKLTSIEEEPL RHVCNINAVLLLAIN IFL GYFA
NOV29 Clones
Unless specifically addressed as NOV29a, NON29b, ΝON29c, ΝON29d, ΝON29e, or ΝON29f, any reference to ΝON29 is assumed to encompass all variants. Further, Patp, BLAST, and DOMAIN analyses are presented for NON29c, the longest ΝON29 polypeptide sequence.
A search against the Patp database, a proprietary database that contains sequences published in patents and patent publications, yielded several homologous proteins shown in Table29M.
Table 29M. Patp results for ΝON29
Smallest Sum
Reading High Prob
Sequences producing High-scoring Segment Pairs: Frame Score P(Ν)
>patp:AAE06614 Human protein +1 1567 1.4e-199
>patp:AAE08088 Human transporter-related protein #35 +1 1567 1.4e-199
>patp:AAR73595 Cotransporter protein SGLTl +1 1474 4.1e-196
>patp:AAR73593 Cotransporter protein SΝST1 +1 1508 l.le-185
>patp:AAB60093 Human transport protein TPPT-13 +1 1531 2.7e-184
NON29 polypeptides are ficolin-like proteins with sequence homology to the Fibrinogen protein family. In a BLAST search of public sequence databases, it was found, for example, that the ΝON29c nucleic sequence of this invention has 660 of 938 bases (70%) identical to a gb:GEΝBAΝK-ID:RΝU03120|acc:U03120.1 Rattus norvegicus (Sprague-Dawley sodium- glucose cotransporter 1 mRNA, complete eds - Rattus norvegicus, 2627 bp). The full NOV29c polypeptide sequence was found to have 401 of 446 amino acid residues (89%) identical to, and 401 of 446 amino acid residues (91%) similar to, the 552 amino acid residue gi|9588428|emb|CAC00574.1] (AL109659) dJ1024N4.1 (novel Sodium:solute symporter family member similar to SLC5 Al (SGLTl)) from Homo sapiens.
Additional BLAST results are shown in Table 25N.
Figure imgf000275_0001
A multiple sequence alignment is given in Table 290, with the NON29 protein of the invention being shown on line 1, in a ClustalW analysis comparing ΝON29 with related protein sequences disclosed in Table 25Ν.
Table 29O. Information for the ClustalW proteins:
1. >NOV29a; SEQ ID NO:105
2. >NOV29b; SEQ ID NO:107
3. >NOV29c; SEQ TD NO:109
4. >NOV29d; SEQ ID NOrlll
5. >NOV29e; SEQ ID NO-.113
6. >NOV29f; SEQ TD NO:115
7. >GI|9588428/ dJ1022N4.1 (sodiumrsolute symporter family member) [Homo sapiens]; SEQ ID NO:312
8. >GI|631592|/ glucose transport protein [sheep]; SEQ DD NO:313
9. >GI| 1709219/ SL51_Sheep sodium/glucose cotransporter 1 [sheep]; SEQ TD NO:314
10. >GI|631593|/glucose transport protein homologue [sheep]; SEQ ID NO:315
11. >GI|6563312/SGLT1 protein [Mus musculus]; SEQ DO NO:316
10 20 30 40 50
NOV29a MSKEL2 MGPGASGDGVRTETAPHIALDSgVGLHAYDISVWIYFVFVIj NOV29b MGPGASGDGVRTETAPHIALDSraVGLHAYDISVWIYFVFVIJ NOV29C MGPGASGDGVRTETAPHIALDsSvGLHAYDISVWIYFVFVlJ NOV29d MGPGASGDGVRTETAPHIALDS SVGLHAYDISVWIYFVFVI|
NOV29e MGPGASGDGVRTETAPHIALDS SVGLHAYDISVWIYFVFVIi
NOV29f MGPGASGDGVRTETAPHIALDS SVGLHAYDISVWIYFVFVIj gi I 9588428 MGPGASGDGVRTETAPHIALDSSVGLHAYDISVWIYFVFVIi gi|631592| DSSTLSPPATDTAER SAIBSIBIB gij 1709219 3SST SPPATATAEJ3LQBYERIR- gij 631593 I DSSTWSPPATATAE LQ YERIR- giJ6563312 jSSTLSPAj TATDggIPSYERIR-
Figure imgf000276_0001
GVRSOVSYFSVRGPPTA GVR-
Figure imgf000276_0002
Figure imgf000276_0003
gi j 6563312 IIΪ^ECT.R] 3^κia7aiπ ^i^l ^ti^li^fealΛ^^elJli3^Lga
260 270 280 290 300
NOV29a YLSTG- -ILLWTAVYTIAGGLMAVIYTDALQTVI NOV29b NOV29C YLSTG- -ILLWTAVYTIAGGLMAVIYTDALQTVI NOV29d NOV29e YLSTG- -ILLWTAVYTIAGGLMAVIYTDALQTVI NOV29f
91 9588428
631592 I gi 1709219 gi 631593 I gi 6563312
310 320 330 340 350
NOV29 MVGGALVLMFQDVGWYPGLEQRYRQAIPNVTVPNTTCHLPRPDAFHILRD NOV29b NOV29C MVGGALVLMFQDVGWYPGLEQRYRQAIPNVTVPNTTCHLPRPDAFHILRD NOV29d NOV29e MVGGALVLMFQDVGWYPGLEQRYRQAIPNVTVPNTTCHLPRPDAFHILRD NOV29f gi 9588428 gi 631592 I gi 1709219 g 631593 I gi 6563312
360 370 380 390 400
NOV29a PVSGDIPWPGLIFGLTVLATWCWCTDQVIVQRSLSAKSLSHAKGGSVLGG
NOV29b
NOV29C PVSGDIPWPGLIFGLTVLATWCWCTDQVIVQRSLSAKSLSHAKGGSVLGG
NOV29d
NOV29e PVSGDIPWPGLIFGLTVLATWCWCTDQVIVQRSLSAKSLSHAKGGSVLGG
Figure imgf000277_0001
410 420 430 440 450
NOV29a YLKILPMFFIVMPGMISRALFPDEVGCVDPDVCQRICGARVGCSNIAYPK
NOV29b
NOV29C YLKILPMFFIVMPGMISRALFPDEVGCVDPDVCQRICGARVGCSNIAYPK
NOV29d
NOV29e YLKILPMFFIVMPGMISRALFPDEVGCVDPDVCQRICGARVGCSNIAYPK
Figure imgf000277_0002
460 470 180 490 500
NOV29a LVMALMPVGRGLMIAVIMAALMSSLTSIFNSSSTLFTIDVWQRFRRKSTE NOV29b NOV29C LVMALMPVGRGLMIAVIMAALMSSLTSIFNSSSTLFTIDVWQRFRRKSTE N0V29d N0V29e LVMALMPVGRGLMIAVIMAALMSSLTSIFNSSSTLFTIDVWQRFRRKSTE
Figure imgf000278_0001
gi I 6563312 FGttAIIiA^YWCπnnBRORCltSAKNMSHVKAGCTLCGYI.KLffPMFT.MVM
Figure imgf000279_0001
The NON29 Clustal W alignment shown in Table 290 was modified to end at amino residue 900. The data in Table 290 includes all of the regions overlapping with the NON29 protein sequences.
The presence of identifiable domains in the protein disclosed herein was determined by searches using algorithms such as PROSITE, Blocks, Pfam, ProDomain, Prints and then determining the Interpro number by crossing the domain match (or numbers) using the Interpro website (http:www.ebi.ac.uk/interpro/). Table 29P lists the domain description from DOMAIN analysis results against NON29c.
Figure imgf000280_0001
Consistent with other known members of the sodium: solute symporter family (SSF), the
ΝON29 Νa+/glucose transporter-like protein contains the sodium: solute symporter family domain and an integral membrane domain as illustrated in Table 25T (Ohashi and Erickson, J. Biol. Chem., 272: 14220-6 (1997)). NON29 nucleic acids, and the encoded polypeptides, according to the invention are useful in a variety of applications and contexts. For example, ΝON29 nucleic acids and polypeptides can be used to identify proteins that are members of the sodium: solute symporter family of proteins. The ΝON29 nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance ΝON29 activity or function. Specifically, the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., cellular activation and cellular metabolism. These molecules can be used to treat, e.g., for metabolic diseases such as diabetes and hypertension, or cancer, trauma, regeneration (in vitro and in vivo), viral/bacterial/parasitic infections, diabetes, autoimmune disease, renal artery stenosis, interstitial nephritis, glomerulonephritis, polycystic kidney disease, systemic lupus erythematosus, renal tubular acidosis, IgA nephropathy, hypercalceimia, Lesch-Νyhan syndrome, cirrhosis, transplantation, infertility and other diseases, disorders and conditions of the like. In addition, various NON29 nucleic acids and polypeptides according to the invention are useful, ter alia, as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins. For example, the ΝON29 nucleic acids and their encoded polypeptides include structural motifs that are characteristic of proteins belonging to the SSF family such as the Νa+/glucose transporter proteins involved in renal transport and metabolism. (Ohashi and Erickson, J. Biol. Chem., 272: 14220-6 (1997)).
Integral membrane proteins that mediate the intake of a wide variety of molecules with the concomitant uptake of sodium ions are grouped into a number of distinct families. One of these families, known as the SSF, consists of integral membrane proteins that are predicted to comprise at least ten membrane spanning domains. Members of the SSF catalyze solute:Na+ symport
(Reizer et al.,. Biochem. Biophys. Acta, 1197: 133-166 (1994)) can transport sugars, amino acids, nucleosides, inositols, vitamins, urea or anions, depending on the system. Members of the SSF family have been identified in bacteria, archaea and animals, and all functionally well characterized members catalyze solute uptake via Na+ symport. The NON29 nucleic acids and polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in the mediation of metabolism and immune function and renal physiology . As such, the ΝON29 nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat metabolic, immune and renal disorders, e.g., metabolic diseases such as diabetes and hypertension, or cancer, trauma, regeneration (in vitro and in vivo), viral/bacterial/parasitic infections, diabetes, autoimmune disease, renal artery stenosis, interstitial nephritis, glomerulonephritis, polycystic kidney disease, systemic lupus erythematosus, renal tubular acidosis, IgA nephropathy, hypercalceimia, Lesch-Νyhan syndrome, cirrhosis, transplantation, or infertility. The ΝON29 nucleic acids and polypeptides are useful for detecting specific cell types. For example, expression analysis has demonstrated that a ΝON29 nucleic acid is expressed in Kidney, Liver, Testis, Whole Organism. .
Additional utilities for ΝON29 nucleic acids and polypeptides according to the invention are disclosed herein.
ΝON30 A NON30 polypeptide has been identified as a Sodium-glucose cotransporter (SGLT)-like protein (also referred to as CG56398-01). The disclosed novel ΝON30 nucleic acid (SEQ ID ΝO:116) of 2105 nucleotides is shown in Table 30A. The novel NOV30 nucleic acid sequences maps to the chromosome 16.
An ORF begins with a Kozak consensus ATG initiation codon at nucleotides 31-33 and ends with a TAA codon at nucleotides 2056-2058. A putative untranslated region and/or downstream from the termination codon is underlined in Table 30A, and the start and stop codons are in bold letters.
Table 30A. NON30 Nucleotide Sequence (SEQ ID NO:116)
CCTCAGGATCCAGAGGTCTCGTTCAGGACCATGGAGAGCGGCACCAGCAGCCCTCAGCCTCCACAGT TAGATCCCCTGGATGCGTTTCCCCAGAAGGGCTTGGAGCCTGGGGACATCGCGGTGCTAGTTCTGTA CTTCCTCTTTGTCCTGGCTGTTGGACTATGGTCCACAGTGAAGACCAAAAGAGACACAGTGAAAGGC TACTTCCTGGCTGGAGGGGACATGGTGTGGTGGCCAGTGGGTGCATCCTTGTTTGCCAGCAATGTTG GAAGTGGACATTTCATTGGCCTGGCAGGGTCAGGTGCTGCTACGGGCATTTCTGTATCAGCTTATGA ACTTAATGGCTTGTTTTCTGTGCTGATGTTGGCCTGGATCTTCCTACCCATCTACATTGCTGGTCAG GTGACCACGATGCCAGAATACCTACGGAAGCGCTTCGGTGGCATCAGAATCCCCATCATCCTGGCTG TACTCTACCTATTTATCTACATCTTCACCAAGATCTCGGTAGACATGTATGCAGGTGCCATCTTCAT CCAGCAGTCTTTGCACCTGGATCTGTACCTGGCCATAGTTGGGCTACTGGCCATCACTGCTGTATAC ACGGTTGCTGGTGGCCTGGCTGCTGTGATCTACACGGATGCCCTGCAGACGCTGATCATGCTTATAG GAGCGCTCACCTTGATGGGCTACAGTTTCGCCGCGGTTGGTGGGATGGAAGGACTGAAGGAGAAGTA CTTCTTGGCCCTGGCTAGCAACCGGAGTGAGAACAGCAGCTGCGGGCTGCCCCGGGAAGATGCCTTC CATATTTTCCGAGATCCGCTGACATCTGATCTCCCGTGGCCGGGGGTCCTATTTGGAATGTCCATCC CATCCCTCTGGTACTGGTGCACGGATCAGGTAATTGTCCAGCGGACTCTGGCTGCCAAGAACCTGTC CCATGCCAAAGGAGGTGCTCTGATGGCTGCATACCTGAAGGTGCTGCCCCTCTTCATAATGGTGTTC CCTGGGATGGTCAGCCGCATCCTCTTCCCAGATCAAGTGGCCTGTGCAGATCCAGAGATCTGCCAGA AGATCTGCAGCAACCCCTCAGGCTGTTCGGACATCGCGTATCCCAAACTCGTGCTGGAACTCCTGCC CACAGGTCTCCGTGGGCTGATGATGGCTGTGATGGTGGCGGCTCTCATGTCCTCCCTCACCTCCATC TTTAACAGTGCCAGCACCATCTTCACCATGGACCTCTGGAATCACCTCCGGCCTCGGGCATCTGAGA AGGAGCTCATGATTGTGGGCAGGGTGTTTGTGCTGCTGCTGGTCCTGGTCTCCATCCTCTGGATCCC TGTGGTCCAGGCCAGCCAGGGCGGCCAGCTCTTCATCTATATCCAGTCCATCAGCTCCTACCTGCAG CCGCCTGTGGCGGTGGTCTTCATCATGGGATGTTTCTGGAAGAGGACCAATGAAAAGGGTGCCTTCT GGGGCCTGATCTCGGGCCTGCTCCTGGGCTTGGTTAGGCTGGTCCTGGACTTTATTTACGTGCAGCC TCGATGCGACCAGCCAGATGAGCGCCCGGTCCTGGTGAAGAGCATTCACTACCTCTACTTCTCCATG ATCCTGTCCACGGTCACCCTCATCACTGTCTCCACCGTGAGCTGGTTCACAGAGCCACCCTCCAAGG AGATGGTCAGCCACCTGACCTGGTTTACTCGTCACGACCCCGTGGTCCAGAAGGAACAAGCACCACC AGCAGCTCCCTTGTCTCTTACCCTCTCTCAGAACGGGATGCCAGAGGCCAGCAGCAGCAGCAGCGTC CAGTTCGAGATGGTTCAAGAAAACACGTCTAAAACCCACAGCGGTGACATGACCCCAAAGCAGTCCA AAGTGGTGAAGGCCATCCTGTGGCTCTGTGGAATACAGGAGAAGGGCAAGGAAGAGCTCCCGGCCAG AGCAGAAGCCATCATAGTTTCCCTGGAAGAAAACCCCTTGGTGAAGACCCTCCTGGACGTCAACCTC ATTTTCTGCGTGAGCTGCGCCATCTTTATCTGGGGCTATTTTGCTTAGTGTGGGGTGAACCCAGGGG TCCAAACTCTGTTTCTCTTCAGTGCTCC
The NOV30 protein (SEQ ID NO:117) encoded by SEQ ID NO:116 is 675 amino acid residues in length and is presented using the one-letter amino acid code in Table 30B. Psort analysis predicts the NON30 protein of the invention to be localized to the plasma membrane with a certainty of 0.8000. The Signal P predicts a likely cleavage site for aNON30 peptide is between positions 50 and 51, i.e., at the dash in the sequenceNKT-KR.
Table 30B. Encoded ΝOV30 protein sequence (SEQ ID NO:117)
MESGTSSPQPPQLDPLDAFPQKGLEPGDIAVLVLYFLFVLAVGLWSTVKTKRDTVKGYFLAGGDM WWPVGASLFASNVGSGHFIGLAGSGAATGISVSAYELNGLFSVLMLAWIFLPIYIAGQVTTMPE YLRKRFGGIRIPIILAVLYLFIYIFTKISVDMYAGAIFIQQSLHLDLYLAIVGLLAITAVYTVAG GLAAVIYTDALQTLI LIGALTLMGYSFAAVGGMEGLKEKYFLALASNRSΞNSSCGLPREDAFHI FRDPLTSDLP PGVLFGMSIPSLWY CTDQVIVQRTLAAKNLSHAKGGALMAAYLKVLPLFIMVF PGMVSRILFPDQVACADPEICQKICSNPSGCSDIAYPKLVLΞLLPTGLRGLM AVMVAAL SSLT SIFNSASTIFTMDL NHLRPRASEKELMIVGRVFVLLLVLVSIL IPWQASQGGQLFIYIQSIS SYLQPPVAWFI GCFWKRTNEKGAF GLISGLLLGLVRLVLDFIYVQPRCDQPDERPVLVKSIH YLYFSMILSTVTLITVSTVSWFTEPPS EMVSHLT FTRHDPWQKEQAPPAAPLSLTLSQNGMP EASSSSSVQFEMVQENTSKTHSGDMTPKQSKWKAILWLCGIQEKGKEELPARAEAIIVSLEENP LVKTLLDVNLIFCVSCAIFIWGYFA
A search against the Patp database, a proprietary database that contains sequences published in patents and patent publications, yielded several homologous proteins shown in Table 30C.
Table 30C. Patp results for NOV30
Smallest Sum
Reading High Prob
Sequences producing High-scoring Segment Pairs: Frame Score P(N)
>patp:AAB60093 Human transport protein TPPT-13 +1 3461 0.0
>patp:AAB85102 Novel human transporter protein (NHP) +1 3455 0.0
>patp:AAR73595 Cotransporter protein SGLTl +1 1729 2.1e-184
>patp:AAR73593 Cotransporter protein SNST1 +1 1726 1.6e-177
>patp:AAY31221 Human SAAT1 protein +1 1629 2.9e-174
In a BLAST search of public sequence databases, it was found, for example, that the NOV30 nucleic acid sequence of this invention has 1764 of 2068 bases (85%) identical to a gb:GENBANK-ID:RABSGCTP|acc:D16226.1 mRNA from Oryctolagus cuniculus (Rabbit mRNA for sodium-glucose cotransporter, complete eds). NON30 polypeptide was found to have 568 of 675 amino acid residues (84%) identical to, and 624 of 675 amino acid residues (92%) similar to, the 674 amino acid residue ptnr:SPTREMBL-ACC:Q28728 protein from Oryctolagus cuniculus (ONE OF THE MEMBERS OF SODIUM-GLUCOSE COTRANSPORTER FAMILY).
NON30 also has homology to the proteins shown in the BLASTP data in Table 30D. Table 30D. BLAST results for NOV30
Gene Index/ Protein/ Organism Length Identity Positives Expect Identifier (aa) (%) (%) gi 117941285 I ref |NP_ putative sodium- 675 597/675 597/675 0.0 443176.2| coupled (88%) (88%) (NM 052944) cotransporter RKST1 [Homo sapiens] gi 115419543 I gb I AAK9 putative sodium- 675 596/675 596/675 0.0
7053.l|AF292385_l coupled (88%) (88%)
(AF292385) cotransporter RKST1 [Homo sapiens] gi|473969|dbj |BAA03 one of the 674 503/675 550/675 0.0 753. ll (D16226) members of (74%) (80%) sodium-glucose cotransporter f mily
[Oryctolagus cuniculus] gi 116165175 I ref |XP_ putative sodium- 548 483/548 483/548 0.0 056259. l| coupled (88%) (88%) (XM 056259) cotransporter RKST1 [Homo sapiens] gi I 2564063 | d j |BAA2 Na+-glucose 673 791 bits 409/679 0.0 2950. ll (AB008225) cotransporter (2042) (60%) type 1 (SGLT-1) - like protein [Xenopus laevis]
A multiple sequence aligmnent is given in Table 30E, with the NON30 protein being shown on line 1 in Table 30E in a ClustalW analysis, and comparing the ΝON30 protein with the related protein sequences shown in Table 30D. This BLASTP data is displayed graphically in the ClustalW in Table 30E.
Table 30E. ClustalW Analysis of ΝOV30
1) > NOV30; SEQ BD NO:117
2) > gi|1794128/ putative sodium-coupled cotransporter RKSTf [Homo sapiens]; SEQ TD NO:317
3) > gi|1541954/ putative sodium-coupled cotransporter RSTK1 [Homo sapiens]; SEQ TD NO:318
4) > gi|473969|/ member of sodium-glucose cotransporter family [Oryctolagus cuniculus]; SEQ DO NO:319
5) > gi|1616517/ putative sodium-coupled cotransporter RKST1 [Homo sapiens]; SEQ TD NO:320
6) > gi|2564063/ Na+-glucose cotransporter type 1 (SGLT-l)-like protein [Xenopus laevis]; SEQ ID NO:321
10 20 30 40 50
....j ....| .. ■■i ....| ....| ....i ....| ....| ....| ....i
NOV30 ffi^GnS^J» JOLiro*tkyj»K<GI-\-nri-> A^
Figure imgf000285_0001
gi|2564063 ffflπfiBfflep<affaqκfii^Mi^τ«a tna^^
110 120 130 140 150
Figure imgf000285_0002
160 170 180 190 200
Figure imgf000285_0003
NOV30
Figure imgf000285_0005
Figure imgf000285_0004
260 270 280 290 300
NOV30 GLPREDAFHIFRDPLTSDLP PGVLFGMSIPSL YWCTDQVIVQRTLAAK gi 1794128 GLPREDAFHIFRDPLTSDLP PGVLFGMSIPSL YWCTDQVIVQRTLAAK gi 1541954 GLPREDAFHIFRDPLTSDLP PGVLFGMSIPSL Y CTDQVIVQRTLAAK gi 473969| GLPREDAFHJJFRDPLTSDLPWPGØLFGMSIPSLWY CTDQVIVQRLAAK gi 1616517 GLPREDAFHIFRDPLTSDLPWPGVLFGMSIPSLWYWCTDQVIVQRTLAAK gi 2564063 5iιaaMw^a:«ιaaMaviιw»>->a?ιatiSivM&τ><5B«tι?r«'JWHtι»tfiifctιwasiisCTa 310 320 330 340 350
NOV30 NLSHAKGGALMAAYLKVLPLFIMVFPGMVSRILFPDQVACADPEICQKIC gi 1794128 NLSHAKGGALMAAYLKVLPLFIMVFPGMVSRILFPDQVACADPEICQKIC gi 1541954 NLSHAKGGALMAAYLKVLPLFIMVFPGMVSRILFPDQVACADPEICQKIC gi 473969| NLSHAKGGILMAAYLKVLPLFIMVFPGMVSRILFPDQVACADPEØCQSEC gi 1616517 NLSHAKGGALMAAYLKVLPLFIMVFPGMVSRILFPDQVACADPEICQKIC gi 2564063 iSiiHJ^gABSilL^ISiWWJBansM^ fi.^IIStϊBπW
360 370 380 390 400 I....I....I....I.. ..I.... I
NOV30 SNPSGCSDIAYPKLVLELLPTGLRGLMMAVMVAALMSSLTSIFNSASTIF gi 11794128 SNPSGCSDI AYPKLVLELLPTGLRGLMMAVMVAALMSSLTS I FNSASTI F gij 1541954 SNPSGCSDIAYPKLVLELLPTGLRGLMMAVMVAALMSSLTSIFNSASTIF gij 473969 I JJNPSGCSDIAYPKLVLELLPTGLRGLMMAVMVAALMSSLTSIFNSASTIF gijl616517 SNPSGCSDI AYPKLVLELLPTGLRGLMMAVMVAALMSSLTS I FNSASTI F gi|2564063 Qιaa««^ι«»iV«at<MΛiιawna^«ι<r<«-iιaιaiSwaιiraj-ιoB^-wκ^»!.CTWi>i-
410 420 430 440 450
Figure imgf000286_0001
510 520 530 540 550
NOV30 YVQPRCDQPDERPVLVKSIHYLYFSMILSTVTLITVSTVS FTEPPSKEM gi 1794128 VQPRCDQPDERPVLVKSIHYLYFSMILSTVTLITVSTVS FTEPPSKEM gi 1541954 YVQPRCDQPDERPVLVKSIHYLYFSMILSTVTLITVSTVS FTEPPSKEM gi 473969| YVJ-^P^CJ|θPDERP[-^IθTRHYLYFSMILSE!vτBiTVpro!lsi-fflTEPPSKEM i 1616517 YVQPRCDQPDERPVLVKSIHYLYFSMILSTVTLITVSTVS FTEPPSKEM gi 2564063 gTJaiGV^Y Jgig gSUJG LgjLVgVA^I
560 570 580 590 600
..I ..
NOV30 VSHLT FTRHDPWQKEQAPPAAPLSLTLSQNGMPEASSSSSVQFEMVQE gi 11794128 VSHLT FTRHDPWQKEQAPPAAPLSLTLSQNGMPEASSSSSVQFEMVQE gi 11541954 VSHLTWFTRHDP QKEQAPPAAPLSLTLSQNGMPEASSSSSVQFEMVQE gij 473969 I HsHLTWF RgDP oκgoigpBl HpLB^LsgiNGlf^E iasgsπoBEπ«lθE gij 1616517 VSHLT FTRHDPWQKEQAPPAAPLSLTLSQNGMPEASSSSSVQFEMVQE giJ2564063 ltøRI-Uil.WaFlHAB{«EDPVETNHRJ3 AEl«3lSWEDl|giEEPHTTSTD
Figure imgf000286_0002
660 670 ..I.. I I
NOV30 EENPLVKTLLDVNL I FCVS CAI FI WGYFA gi 11794128 EENPLVKTLLDVNLIFCVSCAIFI GYFA gi 11541954 |EENPLVKTLLDVNLIFCVSCAIFIWGYFA giJ473969| |EENPS KTL D S53CEBCAE3F3WGYFA gij 1616517 EENPLVKTLLDVNLIFCVSCAIFIWGYFA gij 2564063 -^R^LjgQVJJ TA gLgMg G g gj gG The presence of identifiable domains in the protein disclosed herein was determined by searches using algorithms such as PROSITE, Blocks, Pfam, ProDomain, Prints and then determimng the Interpro number by crossing the domain match (or numbers) using the Interpro website (http:www.ebi.ac.uk/interpro/). Table 30F lists the domain description from DOMAIN analysis results against NOV30.
Figure imgf000287_0001
Consistent with other known members of the sodium:solute symporter family (SSF), the NOV30 Na+/glucose transporter-like protein contains the sodium:solute symporter family domain and an integral membrane domain as illustrated in Table 30F (Ohashi and Erickson, J. Biol.
Chem., 272: 14220-6 (1997)). NOV30 nucleic acids, and the encoded polypeptides, according to the invention are useful in a variety of applications and contexts. For example, NON30 nucleic acids and polypeptides can be used to identify proteins that are members of the sodium: solute symporter family of proteins. The ΝON30 nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance ΝON30 activity or function. Specifically, the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., cellular activation and cellular metabolism. These molecules can be used to treat, e.g., for metabolic diseases such as cancer, trauma, regeneration (in vitro and in vivo), viral/bacterial/parasitic infections, hyperparathyroidism, hypoparathyroidism ,inflammatory bowel disease, diverticular disease, Non Hippel-Lindau (VHL) syndrome, Alzheimer's disease, stroke, tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, cerebral palsy, epilepsy, Lesch-Νyhan syndrome, multiple sclerosis, ataxia-telangiectasia, leukodystrophies, behavioral disorders, addiction, anxiety, pain, neurodegeneration, systemic lupus erythematosus, autoimmune disease, asthma, emphysema, scleroderma, allergy, ARDS, diabetes, autoimmune disease, renal artery stenosis, interstitial nephritis, glomerulonephritis, polycystic kidney disease, systemic lupus erythematosus, renal tubular acidosis, IgA nephropathy, hypercalceimia, inflammatory bowel disease, diverticular disease and other diseases, disorders and conditions of the like.
In addition, various NON30 nucleic acids and polypeptides according to the invention are useful, inter alia, as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins. For example, the ΝON30 nucleic acids and their encoded polypeptides include structural motifs that are characteristic of proteins belonging to the SSF family such as the Νa+/glucose transporter proteins involved in renal transport and metabolism. (Ohashi and Erickson, J. Biol. Chem., 272: 14220-6 (1997)).
Integral membrane proteins that mediate the intake of a wide variety of molecules with the concomitant uptake of sodium ions are grouped into a number of distinct families. One of these families, known as the SSF, consists of integral membrane proteins that are predicted to comprise , at least ten membrane spanning domains. Members of the SSF catalyze solute:Na+ symport (Reizer et al,. Biochem. Biophys. Acta, 1197: 133-166 (1994)) can transport sugars, amino acids, nucleosides, inositols, vitamins, urea or anions, depending on the system. Members of the SSF family have been identified in bacteria, archaea and animals, and all functionally well characterized members catalyze solute uptake via Na+ symport.
The NON30 nucleic acids and polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in the mediation of metabolism and immune function and renal physiology . As such, the ΝON30 nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat metabolic, immune and renal disorders, e.g., metabolic diseases such as diabetes and hypertension, cancer, trauma, regeneration (in vitro and in vivo), viral/bacterial/parasitic infections, hyperparathyroidism, hypoparathyroidism,inflammatory bowel disease, diverticular disease, Non Hippel-Lindau (NHL) syndrome, Alzheimer's disease, stroke, tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, cerebral palsy, epilepsy, Lesch-Νyhan syndrome, multiple sclerosis, ataxia-telangiectasia, leukodystrophies, behavioral disorders, addiction, anxiety, pain, neurodegeneration, systemic lupus erythematosus, autoimmune disease, asthma, emphysema, scleroderma, allergy, ARDS, diabetes, autoimmune disease, renal artery stenosis, interstitial nephritis, glomerulonephritis, polycystic kidney disease, systemic lupus erythematosus, renal tubular acidosis, IgA nephropathy, hypercalceimia, inflammatory bowel disease, diverticular disease and other diseases, disorders and conditions of the like. For example, expression analysis has demonstrated that a NON30 nucleic acid is expressed in Kidney, Parathyroid, Brain, Hippocampus, Hypothalamus, Lung, Small Intestine, Spinal Chord, Substantia Νigra, Whole Organism.
Additional utilities for ΝON30 nucleic acids and polypeptides according to the invention are disclosed herein.
ΝOV31
A NOV31 polypeptide has been identified as a Olfactory Receptor (GPCR)-like protein (also referred to as CG56616-01). The disclosed novel NOV31 nucleic acid (SEQ ID NO:118) of 1201 nucleotides is shown in Table 31 A. An ORF begins with Kozak consensus ATG initiation codon at nucleotides 86-88 and ends with a TAA codon at nucleotides 1040-1042. A putative untranslated region and/or downstream from the termination codon is underlined in Table 31 A, and the start and stop codons are in bold letters.
Table 31A. NOV31 Nucleotide Sequence (SEQ ID NO:118)
TCATTGACACATGCTTGAAAGTAATCAGAGTAAGATAAATATTTGTCTTAACATGCTCTGTCTTACA AGCTAAAGAGGGAGTAAAATGGAATGGGAAAACCACACCATTCTGGTGGAATTTTTTCTGAAGGGAC TTTCTGGTCACCCAAGACTTGAGTTACTCTTTTTTGTGCTCATCTTCATAATGTATGTGGTCATCCT TCTGGGGAATGGTACTCTCATTTTAATCAGCATCTTGGACCCTCACCTTCACACCCCTATGTACTTC TTTCTGGGGAACCTCTCCTTCTTGGACATCTGCTACACCACCACCTCTATTCCCTCCACGCTAGTGA GCTTCCTTTCAGAAAGAAAGACCATTTCCCTTTCTGGCTGTGCAGTGCAGATGTTCCTCAGCTTGGC CATGGGGACAACAGAGTGTGTGCTTCTGGGCGTGATGGCCTTTGACCGCTATGTGGCTATCTGCAAC CCTCTGAGATATCCCATCATCATGAGTAAGGATGCCTATGTACCCATGGCAGCTGGGTCCTGGATCA TAGGAGCTGTCAATTCTGCAGTACAAACAGTGTTTGTGGTACAATTGCCTTTCTGCAGGAATAACAT CATCAATCATTTCACCTGTGAAATTCTAGCTGTCATGAAACTGGCCTGTGCTGACATCTCAGGCAAT GAGTTCATCCTGCTTGTGACCACAACATTGTTCCTATTGACACCTTTGTTATTAATTATTGTCTCTT ACACGTTAATCATTTTGAGCATCTTCAAAATTAGCTCTTCGGAGGGGAGAAGCAAACCTTCCTCTAC CTGCTCAGCTCGTCTGACTGTGGTGATAACATTCTGTGGGACCATCTTCCTCATGTACATGAAGCCC AAGTCTCAAGAGACACTTAATTCAGATGACTTGGATGCCACTGACAAACTTATATTCATATTCTACA GGGTGATGACTCCCATGATGAATCCTTTAATCTACAGTCTTAGAAACAAGGATGTGAAGGAGGCAGT AAAACACCTACTGAGAAGAAAAAATTTTAACAAGTAAATGAGAAAGGTGAGAGTAATTTTATAATCA CAATATGGAAATCAATTAGAGAAACCAAGGTTAAACAGATAGGTTCTCGTTGCTGTTTCACATTCAT CTCTCGAAGTTCTAAAGCTCCAAAATACACTTCTCTGATTGCGATACATAATGAAAAGAAGT
TheNOV31 protein (SEQ ID NO:119) encoded by SEQ ID NO:118 is 318 amino acid residues in length and is presented using the one-letter amino acid code in Table 3 IB. NON31 has at least 10 SΝP variants, whose variant positions for its nucleotide and amino acid sequences is numbered according to SEQ ID ΝOS:118 and 119, respectively. A variant sequence can include a single nucleotide polymorphism (SNP). A SNP can, in some instances, be referred to as a "cSNP" to denote that the nucleotide sequence containing the SNP originates as a cDNA. Variant sequences of NON31 are included in Example 2.
Psort analysis predicts the ΝON31 protein of the invention to be localized to the plasma membrane with a certainty of 0.6000. The Signal P predicts a likely cleavage site for a ON29b peptide is between positions 41 and 42, i.e., at the dash in the sequence LLG-ΝG.
Table 31B. Encoded ΝOV31 protein sequence (SEQ ID NO.-119)
ME ENHTILVEFFLKGLSGHPRLELLFFVLIFIMYWILLGNGTLILISILDPHLHTPMYFFLGN LSFLDICYTTTSIPSTLVSFLSERKTISLSGCAVQMFLSLAMGTTΞCVLLGVMAFDRYVAICNPL RYPIIMSKDAYVPIVLAAGS IIGAVNSAVQTVFVVQLPFCRNNIINHFTCEILAVMKLACADISGN EFILLVTTTLFLLTPLLLIIVSYTLIILSIFKISSSEGRSKPSSTCSARLTWITFCGTIFLMYM KPKSQETLNSDDLDATD LIFIFYRVMTPMMNPLIYSLRNKDVKEAVKHLLRRKNFNK
A search against the Patp database, a proprietary database that contains sequences published in patents and patent publications, yielded several homologous proteins shown in Table 31C.
Table 31C. Patp results for NOV31
Smallest Sum
Reading High Prob
Sequences producing High- scoring Segment Pairs: Frame Score P(N)
>patp:AAG71953 Human olfactory receptor polypeptide +1 1620 2.7e-166
>patp:AAU24703 Human olfactory receptor AOLFR202 +1 1620 2.7e-166
>patp:AAG71431 Human olfactory receptor polypeptide +1 1504 5.2e-154
>patp:AAU24702 Human olfactory receptor AOLFR201 +1 1504 5.2e-154
>patp:AAG72024 Human olfactory receptor polypeptide +1 1399 7.0e-143
In a BLAST search of public sequence databases, it was found, for example, that the NOV31 nucleic acid sequence of this invention has 702 of 991 bases (70%) identical to a gb:GENBANK-ID:MMU133426|acc:AJ133426.1 mRNA from Mus musculus (or37c gene). The full amino acid sequence of the protein of the invention was found to have 209 of 314 amino acid residues (66%) identical to, and 246 of 314 amino acid residues (78%) similar to, the 318 amino acid residue ptnr:SPTREMBL-ACC:Q9QZ21 protein from Mus musculus (OLFACTORY RECEPTOR). Also 100% similarity to Genbank_AL450426.3 sequence that is not annotated.
NOV31 also has homology to the proteins shown in the BLASTP data in Table 3 ID. Table 31D. BLAST results for NOV31
Gene Index/ Protein/ Organism Length Identity Positives Expect Identifier (aa) (%) (%) gi 117452276 I ref |XP_ similar to 318 291/318 291/318 le-150 071094.11 olfactory (91%) (91%) (XM 071094) receptor 37b [Homo sapiens] g 1174522711 ref |XP_ similar to 318 272/318 279/318 le-137 071093. l| olfactory (85%) (87%) (XM 071093) receptor 37b [Homo sapiens] gi 117452269 I ref |XP_ similar to 318 255/318 268/318 le-127
071092.1] olfactory (80%) (84%)
(XM 071092) receptor 37b [Homo sapiens] gi 111276077 I ref |NP_ olfactory 318 200/314 230/314 le-101 062347. l| receptor 37b [Mus (63%) (72%) (NM 019474) musculus] gi 111276079 | ref | P_ olfactory 318 198/310 224/310 8e-99 062348. l| receptor 37c [Mus (63%) (71%) (NM 019475) musculus]
A multiple sequence alignment is given in Table 3 IE, with the NOV31 protem being shown on line 1 in Table 3 IE in a ClustalW analysis, and comparing the NOV31 protein with the related protein sequences shown in Table 3 ID. This BLASTP data is displayed graphically in the ClustalW in Table 3 IE.
Table 31E. ClustalW Analysis of NOV31
1) > NOV31; SEQ ID NO:119
2) > gi| 1745227/ similar to Olfactory receptor 37b [Homo sapiens] ; SEQ ID NO:322
3) > gi|1745227/ similar to olfactory receptor 37b [Homo sapiens] ; SEQ ID NO:323
4) > gi|1745226/ similar to olfactory receptor 37b [Homo sapiens] ; SEQ ID NO:324
5) > gijl 127607/ olfactory receptor 37b [Mus musculus] ; SEQ ED NO:325
6) > gijl 127607/ olfactory receptor 37c [Mus musculus] ; SEQ ED NO:326
Figure imgf000291_0001
60 70 80 90 100
NOV31 LDPHLHTPMYFFLGNLSFLDICYTTTSIPSTLVSFLSERKTISfflSGCAVQ gi | l745227 LDPHLHTPMYFFLGNLSFLDICYTTTSIPSTLVSFLSERKTISfflSGCAVQ gi j 1745227 LDPHLHTPMYFFLGNLSFLDICYTTTSIPSTLVSFLSERKTISfflSGCAVQ gi j 1745226 LDPHLHTPMYFFLGNLSFLDICYTTTSIPSTLVSFLSERKTIsBsGCAVQ gi j ll27607 gi l ll27607 110 120 130 140 150
N0V31 iiaiM-tjiMι- aw<aι;tn iι!l jaw;y*vt ι>ivιa-ιry«a>» iaι ' tiai > eM'? gi 1745227 gi 1745227 gi 1745226 IFLffiLAMGTTECVLLGMMAFDRYVAI CNPLRYPI IMSKSlAYVPI gi 1127607 MΑ.ri-Wtfia-Wrøli.feS: gi 1127607 ffiiS-ElFl » «(!l9«*4
Figure imgf000292_0001
The presence of identifiable domains in the protein disclosed herein was determined by searches using algorithms such as PROSITE, Blocks, Pfam, ProDomain, Prints and then determining the Interpro number by crossing the domain match (or numbers) using the Inteφro website (http:www.ebi.ac.uk/inteφro/). The DOMAIN analysis results indicate that the NOV31 protein contains the following protein domain (as defined by Interpro): domain name 7tm_l 7 transmembrane receptor (rhodopsin family). DOMAIN results for NOV31 were collected from the Conserved Domain Database (CDD) with Reverse Position Specific BLAST. This BLAST samples domains found in the Smart and Pfam collections. As discussed below, the NON31 protein of the invention contained significant homology to the 7tm_l domain. This indicates that the ΝON31 sequence has properties similar to those of other proteins known to contain this 7tm_l domain and similar to the properties of these domains. The 254 amino acid domain termed 7tm_l (SEQ ID ΝO:327; Pfam Ace. No. 00001) a seven transmembrane receptor (rhodopsin family), is shown in Table 3 IF.
Table 31F. 7tm_l, 7 transmembrane receptor domain (SEQ ID NO:327)
GNLLVILVILRTKKLRTPTNIFLLNLAVADLLFLLTLPP ALYYLVGGDWVFGDALCKLVGALFWNGYASI LLTAISIDRYL AIVHPLRYRRIRTPRRAKVLILLVVLALLLSLPPLLFS LRTVEEGNTTVCLIDFPEESVKRSYVLLSTLVGFVLPLLVILVC YTRILRTLRKRARSQRSLKRRSSSERKAAKMLLVVVVVFVLC LPYHIVLLLDSLCLLSI RVLPTALLITL LAYVNSCLNPI IY
The DOMAIN results are listed in Table 31G with the statistics and domain description.
An alignment of NOV31 residues 41-296 (SEQ ID NO:119) with the full 7tm_l domain, residues 1-254 (SEQ ID NO:327), are shown in Table 31G. This indicates that the NOV31 sequences have properties similar to those of other proteins known to contain this domain as well as to the 254 amino acid 7tm domain (SEQ ID NO:327). For Table 31G, fully conserved single residues are indicated by the vertical line and "strong" semi-conserved residues are indicated by the "plus sign." The "strong" group of conserved amino acid residues may be any one of the following groups of amino acids: STA, NEQK, NHQK, NDEQ, QHRK, MEN, MILF, HY, FYW.
Table 31G Domain Analysis of ΝOV31
PSSMs producing significant alignments: Score E
(bits) value gnl|Pfam|pfam00001 7tm_l, 7 transmembrane receptor (rhodopsin family) 128.7 7.9e-40
*->GNlLVilvilrtkklrtptnifilNLAvADLLflltlppwalyylvg ||+ ||+ ++ +|+| I +++| ++| |++ |+++++| I +|+ ++ NOV31 41 GNGTLILISILDPHLHTPMYFFLGNLSFLDICYTTTSIPSTLVSFLS 87 gsed pfGsalCklvtaldwnmyaSillLtalSiDRYlAIvhPlryrrr I ++ ++ +| ++++| ++ +++ + || ++++| I I + | |++| I I |+ + NOV31 88 --ERKTISLSGCAVQMFLSLAMGTTECVLLGV AFDRYVAICNPLRYPII 135 rtsprrAkwillvWvlalllslPpllfswvktveegngtlnvnvtvCli ++ + + + ++ |+++++ I + +| I ++++++ I + +|+++| I NOV31 136 MS-KDAYVPMAAGSWIIGAVNSAVQTVF-WQLPFCRNNI--INHFTCEI 181 dfpeestas .vstwlrsyvllstlvgFllPllvilvcYtrllrtlr . . . .
+ ++ + I +++ |+ i+++ i III+I+I I |+|+ +++ ++
N0V31 182 LAVMKLACAdlSGN-EFILLVTTTLFLLTPLLLIIVSYTLIILSIFkiss 230 kaaktllvwwFvlCWlPyfivllldtlc. IsiimsstCel
++++++++ ++ I+M++I+ +++|++ ++ I + I + N0V31 231 segrskpsSTCSARLTWITFC GTIFLMYMKpKS QETLNS 270 ervlptallvtlwLayvNsclNPiIY<-* (SEQ ID NO: 327) ++ I + I +++++ + +++ I I + M
N0V31 271 DDLDATD LIFIFYRV TPMMNPLIY 296 (SEQ ID NO : 119)
Consistent with other known members of the GPCR family of proteins, NON31 contains 7tm_l 7 transmembrane receptor (rhodopsin family) domain as illustrated in Table 31 G as well as homology and cellular localization, i.e. plasma membrane.
ΝON31 nucleic acids, and the encoded polypeptides, according to the invention are useful in a variety of applications and contexts. For example, ΝON31 nucleic acids and polypeptides can be used to identify proteins that are members of the GPCR family of proteins. The ΝON31 nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance ΝON31 activity or function. Specifically, the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., cellular signal transduction. These molecules can be used to treat, e.g., cancer, immune disorders, and endocrine disorders. In addition, various ΝON31 nucleic acids and polypeptides according to the invention are useful, inter alia, as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins. For example, the ΝON31 nucleic acids and their encoded polypeptides include 7tm_l 7 transmembrane receptor (rhodopsin family) domain and sequence homology that are characteristic of proteins belonging to the family of GPCR such as the G protein-coupled olfactory receptor. The ΝON31 protein of the invention has a high homology to the 7tm_l domain (PFam Ace. No. pfamOOOOl). The 7tm_l domain is from the 7 transmembrane receptor family, which includes a number of different proteins, including, for example, serotonin receptors, dopamine receptors, histamine receptors, andrenergic receptors, cannabinoid receptors, angiotensin II receptors, chemokine receptors, opioid receptors, G-protein coupled receptor (GPCR) proteins, olfactory receptors (OR), and the like.
G-Protein Coupled Receptor proteins ("GPCRs") have been identified as a large family of G protein-coupled receptors in a number of species. These receptors share a seven transmembrane domain structure with many neurotransmitter and hormone receptors, and are likely to underlie the recognition and G-protein-mediated transduction of various signals. Human GPCR generally do not contain introns and belong to four different gene subfamilies, displaying great sequence variability. These genes are dominantly expressed in olfactory epithelium. See, e.g., Ben-Arie et al, Hum. Mol. Genet. 3:229-235 (1994); and, Online Mendelian Inheritance in Man ("OMIM") entry # 164342 (http://www.ncbi.nlm.nih.gov/ entrez/ dispomim.cgi?).
The NON31 nucleic acids and polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in the mediation of cellular signal transduction. As such the ΝON31 nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat a wide range of disorders such as cancer, immune disorders, endocrine disorders and other diseases, e.g., developmental diseases; MHCπ and III diseases (immune diseases); taste and scent detectability disorders; Burkitt's lymphoma; corticoneurogenic disease; signal transduction pathway disorders; metabolic pathway disorders; retinal diseases including those involving photoreception; cell growth rate disorders; cell shape disorders; metabolic disorders; feeding disorders; control of feeding; the metabolic syndrome X; wasting disorders associated with chronic diseases; obesity; potential obesity due to over-eating or metabolic disturbances; potential disorders due to starvation (lack of appetite); diabetes; noninsulin-dependent diabetes mellitus (ΝEDDM); infectious disease; bacterial, fungal, protozoal and viral infections (particularly infections caused by HIV-1 or HIV-2); pain; cancer (including but not limited to neoplasm; adenocarcinoma; lymphoma; prostate cancer; uterus cancer); cancer-associated cachexia; anorexia; bulimia; asthma; Parkinson's disease; acute heart failure; hypotension; hypertension; urinary retention; osteoporosis; Crohn's disease; multiple sclerosis; Albright Hereditary Ostoeodystrophy; angina pectoris; myocardial infarction; ulcers; allergies; benign prostatic hypertrophy; and psychotic and neurological disorders; including anxiety; schizophrenia; manic depression; delirium; dementia; neurodegenerative disorders;
Alzheimer's disease; severe mental retardation; Dentatorubro-pallidoluysian atrophy DRPLA); Hypophosphatemic rickets; autosomal dominant (2) Acrocallosal syndrome and dyskinesias, such as Huntington's disease or Gilles de la Tourette syndrome; immune disorders; Adrenoleukodystrophy; Congenital Adrenal Hypeφlasia; Hemophilia; Hypercoagulation; Idiopathic thrombocytopemc puφura; autoimmume disease; immunodeficiencies; transplantation; Non Hippel-Lindau (NHL) syndrome; Stroke; Tuberous sclerosis; hypercalceimia; Cerebral palsy; Epilepsy; Lesch-Nyhan syndrome; Ataxia-telangiectasia; Leukodystrophies; Behavioral disorders;
Addiction; Neuroprotection; Cirrhosis; Transplantation; Systemic lupus erythematosus;
Emphysema; Scleroderma; ARDS; Renal artery stenosis; Interstitial nephritis;
Glomerulonephritis; Polycystic kidney disease; Systemic lupus erythematosus; Renal tubular acidosis; IgA nephropathy; Cardiomyopathy; Atherosclerosis; Congenital heart defects; Aortic stenosis ; Atrial septal defect (ASD); Atrioventricular (A-V) canal defect; Ductus arteriosus;
Pulmonary stenosis ; Subaortic stenosis; Ventricular septal defect (VSD); valve diseases;
Scleroderma; fertility; Pancreatitis; Endocrine dysfunctions; Growth and reproductive disorders;
Inflammatory bowel disease; Diverticular disease; Leukodystrophies; Graft vesus host; Hyperthyroidism; Endometriosis; and hematopoietic disorders.
The NON31 nucleic acids and polypeptides are useful for detecting specific cell types. For example, expression analysis has demonstrated that a ΝOV31 nucleic acid is expressed in Apical microvilli of the retinal pigment epithelium, arterial (aortic), basal forebrain, brain, Burkitt lymphoma cell lines, coφus callosum, cardiac (atria and ventricle), caudate nucleus, CNS and peripheral tissue, cerebellum, cerebral cortex, colon, cortical neurogenic cells, endothelial
(coronary artery and umbilical vein) cells, palate epithelia, eye, neonatal eye, frontal cortex, fetal hematopoietic cells, heart, hippocampus, hypothalamus, leukocytes, liver, fetal liver, lung, lung lymphoma cell lines, fetal lymphoid tissue, adult lymphoid tissue, Those that express MHC II and
III nervous, medulla, subthalamic nucleus, ovary, pancreas, pituitary, placenta, pons, prostate, putamen, serum, skeletal muscle, small intestine, smooth muscle (coronary artery in aortic) spinal cord, spleen, stomach, taste receptor cells of the tongue, testis, thalamus, and thymus tissue.
Additional utilities for NOV31 nucleic acids and polypeptides according to the invention are disclosed herein.
NOV32
A NON32 polypeptide has been identified as a Phosphoenolpyruvate Carboxykinase (PCK)-like protein (also referred to as 153065222). The disclosed novel ΝOV32 nucleic acid (SEQ ID NO:120) of 2069 nucleotides is shown in Table 32A. The cDNA coding for the NOV32 was cloned by polymerase chain reaction (PCR) using the following primers: CCTTCCATACCTCCCCGGCTC (SEQ ID NO:328) and
TGTGGGAAGGTCTATGGCACATTGA (SEQ ID NO:329) on the following pools of human cDNAs: Pool 1 - adrenal gland, bone marrow, brain - amygdala, brain - cerebellum, brain - hippocampus, brain - substantia nigra, brain - thalamus, brain -whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma - Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea, uterus.
An ORF begins with Kozak consensus ATG initiation codon at nucleotides 67-69 and ends with a TGA codon at nucleotides 1891-1893. A putative untranslated region and/or downstream from the termination codon is underlined in Table 32A, and the start and stop codons are in bold letters.
Table 32A. NOV32 Nucleotide Sequence (SEQ ID NO:120)
CCCGCCTTCCATACCTCCCCGGCTCCGCTCGGTTCCTGGCCACCCCGCAGCCCCTGCCCAGGTGCCA TGGCCGCATTGTACCGCCCTGGCCTGCGGCTTAACTGGCATGGGCTGAGCCCCTTGGGCTGGCCATC ATGCCGTAGCATCCAGACCCTGCGAGTGCTTAGTGGAGATCTGGGCCAGCTTCCCACTGGCATTCGA GATTTTGTAGAGCACAGTGCCCGCCTGTGCCAACCAGAGGGCATCCACATCTGTGATGGAACTGAGG CTGAGAATACTGCCACACTGACCCTGCTGGAGCAGCAGGGCCTCATCCGAAAGCTCCCCAAGTACAA TAACTGCTGGCTGGCCCGCACAGACCCCAAGGATGTGGCACGAGTAGAGAGCAAGACGGTGATTGTA ACTCCTTCTCAGCGGGACACGGTACCACTCCCGCCTGGTGGGGCCTGTGGGCAGCTGGGCAACTGGA TGTCCCCAGCTGATTTCCAGCGAGCTGTGGATGAGAGGTTTCCAGGCTGCATGCAGGGCCGCACCAT GTATGTGCTTCCATTCAGCATGGGTCCTGTGGGCTCCCCGCTGTCCCGCATCGGGGTGCAGCTCACT GACTCAGCCTATGTGGTGGCAAGCATGCGTATTATGACCCGACTGGGGACACCTGTGCTTCAGGCCC TGGGAGATGGTGACTTTGTCAAGTGTCTGCACTCCGTGGGCCAGCCCCTGACAGGACAAGGGGAGCC AGTGAGCCAGTGGCCGTGCAACCCAGAGAAAACCCTGATTGGCCACGTGCCCGACCAGCGGGAGATC ATCTCCTTCGGCAGCGGCTATGGTGGCAACTCCCTGCTGGGCAAGAAGTGCTTTGCCCTACGCATCG CCTCTCGGCTGGCCCGGGATGAGGGCTGGCTGGCAGAGCACATGCTGATCCTGGGCATCACCAGCCC TGCAGGGAAGAAGGCGCTATGTGCAGCCGCCTTCCCTAGTGCCTGTGGCAAGACCAACCTGGCTATG ATGCGGCCTGCACTGCCAGGCTGGAAAGTGGAGTGTGTGGGGGATGATATTGCTTGGATGAGGTTTG ACAGTGAAGGTCGACTCCGGGCCATCAACCCTGAGAACGGCTTCTTTGGGGTTGCCCCTGGTACCTC TGCCACCACCAATCCCAACGCCATGGCTACAATCCAGAGTAACACTATTTTTACCAATGTGGCTGAG ACCAGTGATGGTGGCGTGTACTGGGAGGGCATTGACCAGCCTCTTCCACCTGGTGTTACTGTGACCT CCTGGCTGGGCAAACCCTGGAAACCTGGTGACAAGGAGCCCTGTGCACATCCCAACTCTCGATTTTG TGCCCCGGCTCGCCAGTGCCCCATCATGGACCCAGCCTGGGAGGCCCCAGAGGGTGTCCCCATTGAC GCCATCATCTTTGGTGGCCGCAGACCCAAAGGGAAGATCATCATGCACGACCCATTTGCCATGCGGC CCTTTTTTGGCTACAACTTCGGGCACTACCTGGAACACTGGCTGAGCATGGAAGGGCGCAAGGGGGC CCAGCTGCCCCGTATCTTCCATGTCAACTGGTTCCGGCGTGACGAGGCAGGGCACTTCCTGTGGCCA GGCTTTGGGGAGAATGCTCGGGTGCTAGACTGGATCTGCCGGCGGTTAGAGGGGGAGGACAGTGCCC GAGAGACACCCATTGGGCTGGTGCCAAAGGAAGGAGCCTTGGATCTCAGCGGCCTCAGAGCTATAGA CACCACTCAGCTGTTCTCCCTCCCCAAGGACTTCTGGGAACAGGAGGTTCGTGACATTCGGAGCTAC CTGACAGAGCAGGTCAACCAGGATCTGCCCAAAGAGGTGTTGGCTGAGCTTGAGGCCCTGGAGAGAC GTGTGCACAAAATGTGACCTGAGGCCTAGTCTAGCAAGAGGACATAGCACCCTCATCTGGGAATAGG GAAGGCACCTTGCAGAAAATATGAGCAATTGATATTAACTAACATCTTCAATGTGCCATAGACCTTC CCACAAAGACTGTCCAATAATAAGAGATGCTTATCTATTTTAAAAAAAAAAAAAAAAAA
The NOV32 protein (SEQ ID NO:121) encoded by SEQ ID NO:120 is 608 amino acid residues in length and is presented using the one-letter amino acid code in Table 32B. NON32 has two SNP variants, whose variant positions for its nucleotide and amino acid sequences is numbered according to SEQ ID NOS:120 and 121, respectively. A variant sequence can include a single nucleotide polymoφhism (SNP). A SNP can, in some instances, be referred to as a "cSNP" to denote that the nucleotide sequence containing the SNP originates as a cDNA.
NOV32 variant 13376584 is a G to A SNP at 116 bp of the nucleotide sequence that results in a Ser to Asn change at amino acid 17 of protein sequence, and variant 13376583 is a C to T SNP at 1297 bp of the nucleotide sequence that results in a Pro to Ser change at amino acid 411 of protein sequence.
Psort analysis predicts the NOV32 protein of the invention to be localized in the mitochondria with a certainty of 0.5801.
Table 32B. Encoded NOV32 protein sequence (SEQ ID NO:121)
MAALYRPGLRLNWHGLSPLG PSCRSIQTLRVLSGDLGQLPTGIRDFVEHSARLCQPEGIHICDGTEAENTAT LTLLEQQGLIRKLPKYNNCWLARTDPKDVARVESKTVIVTPSQRDTVPLPPGGACGQLGNMSPADFQRAVDE RFPGCMQGRTMYVLPFSMGPVGSPLSRIGVQLTDSAYWASMRIMTRLGTPVLQALGDGDFVKCLHSVGQPLT GQGEPVSQ PCNPEKTLIGHVPDQREIISFGSGYGGNSLLGKKCFALRIASRLARDEGWLAEHMLILGITSPA GKKALCAAAFPSACGKTNLAMMRPALPGWKVECVGDDIAMRFDSEGRLRAINPENGFFGVAPGTSATTNPNA MATIQSNTIFTNVAETSDGGVYWEGIDQPLPPGVTVTSWLGKP KPGDKEPCAHPNSRFCAPARQCPIMDPAW EAPEGVPIDAIIFGGRRPKGKIIMHDPFAMRPFFGYNFGHYLEHWLSMEGRKGAQLPRIFHVNWFRRDEAGHF LWPGFGENARVLDWICRRLEGEDSARETPIGLVPKEGALDLSGLRAIDTTQLFSLPKDFWEQEVRDIRSYLTE QVNQDLPKEVLAELEALERRVHKM
A search against the Patp database, a proprietary database that contains sequences published in patents and patent publications, yielded several homologous proteins shown in Table 32C.
Table 32C. Patp results for NOV32
Smallest Sum
Reading High Prob
Sequences producing High-scoring Segment Pairs: Frame Score P(N)
>patp:AAY80296 Human mitochondrial PEPCK +1 2494 0.0
>patp:AAB71890 Mouse PCK-cytosolic protein +1 1765 3.9e-238
>patp:AAB71880 Human PCK-cytosolic protein +1 1763 1.4e-235
>patp:AAR15144 Hae onchus contortus PEPCK +1 1410 1.2e-194
>patp:AAY35500 Chlamydia pneumoniae transmembrane prot ≥in +1 1251 1.4e-161
In a BLAST search of public sequence databases, it was found, for example, that the
NOV32 nucleic acid sequence of this invention has 1557 of 1636 bases (95%) identical to a gb:GENBANK-ID:HSPPPCK|acc:X92720.1 mRNA from Homo sapiens (mRNA for phosphoenolpyruvate carboxykinase). The full amino acid sequence of the protein of the invention was found to have 459 of 469 amino acid residues (97%) identical to, and 463 of 469 amino acid residues (98%) similar to, the 640 amino acid residue ptnr:SWISSPROT- ACC:Q16822 protein from Homo sapiens (PHOSPHOENOLPYRUVATE CARBOXYKINASE, MITOCHONDRIAL PRECURSOR [GTP] (EC 4.1.1.32) (PHOSPHOENOLPYRUVATE CARBOXYLASE) (PEPCK-M)).
NOV32 also has homology to the proteins shown in the BLASTP data in Table 32D.
Table 32D. BLAST results for NOV32
Gene Index/ Protein/ Organism Length Identity Positives Expect Identifier (aa) (%) (%) gi 114750965 I ref |XP_ phosphoenolpyruva 640 577/640 577/640 0.0 033337. l| te carboxykinase (90%) (90%) (XM 033337) 2 (mitochondrial) [Homo sapiens] gi|3287892|sp|Q1682 PPCM_HUMAN 640 581/640 581/640 0.0 2| PHOSPHOENOLPYRUVA (90%) (90%) TE CARBOXYKINASE, MITOCHONDRIAL PRECURSOR [GTP]
(PHOSPHOENOLPYRUV ATE CARBOXYLASE)
(PEPCK-M) gi|16307539|gb|AAHl Similar to 640 530/624 549/624 0.0 0318.11AAH10318 phosphoenolpyruva (84%) (87%) (BC010318) te carboxykinase 2 (mitochondrial) [Mus musculus] gι|12655193 |gb|AAH0 phosphoenolpyruva 640 578/640 578/640 0.0 1454.11AAH01454 te carboxykinase (90%) . (90%) (BC001454) 2 (mitochondrial) [Homo sapiens] gi I 4758886 I ef |NP_0 phosphoenolpyruva 640 582/640 582/640 0.0 04554. l| te carboxykinase (90%) (90%) (NM 004563) 2 (mitochondrial) [Homo sapiens]
A multiple sequence alignment is given in Table 32E, with the NON32 protein being shown on line 1 in Table 32E in a ClustalW analysis, and comparing the ΝOV32 protein with the related protein sequences shown in Table 32D. This BLASTP data is displayed graphically in the ClustalW in Table 32E. Table 32E. ClustalW Analysis of NON32
1) > ΝOV32; SEQ ID NO:121
2) > gi|1475096/ Phosphoenolpyruvate carboxykinase 2 (mitochondrial) [Homo sapiens]; SEQ ID NO:330
3) > giJ3287892/ PPCM_human phospoenolpyruvate carboxykinase, mitochondrial precursor [GTP]; SEQ ID NO:331
4) > gi| 1630753/ Similar to phosphoenolpyruvate carboxykinase 2 (mitochondrial) [Mus musculus]; SEQ ID NO:332
5) > gi| 1265519/ phosphoenolpyruvate carboxykinase 2 (mitochondrial) [Homo sapiens] ; SEQ ID NO:333
6) > gi|4758886/ phosphoenolpyruvate carboxykinase 2 (mitochondrial) [Homo sapiens]; SEQ ID NO:334
10 20 30 40 50
NOV32 YIAALYRPGLRLNWHGLSPLGWPSCRSIQTLRVLSGDLGQLPTGIRDFVEI gi 1 1475096 LAALYRPGLRLN HGLSPLGWPSCRSIQTLRVLSGDLGQLPTGIRDFVEH gi | 3287892 YIAALYRPGLRLNWHGLSPLGWPSCRSIQTLRVLSGDLGQLPTGIRDFVEI gi j l630753 gij 1265519 LYRPGLRLNWHGLSPLGWPSCRSIQTLRVLSGDLGQLPTGIRDFVEI gi|4758886 LYRPGLRLNWHGLSPLGWPSCRSIQTLRVLSGDLGOLPTGIRDFVEH
60 70 80 90 100
NOV32 ARLCQPEGIHICDGTEAENTATLTLLEQQGLIRKLPKYNNC LARTDPE gi I 1475096 3ARLCQPEGIHICDGTEAENTATLTLLEQQGLIRKLPKYNNC LARTDPK gi|3287892 3ARLCQPEGIHICDGTEAENTATLTLLEQQGLIRKLPKYNNCWLARTDPP gi|l630753 AgJLCQPEGIHICDGTEAENTAgLgLLEJ QGLIRKLPKYENCWLARTDPB gijl265519 ARLCQPEGIHICDGTEAENTATLTLLEQQGLIRKLPKYNNCWLARTDPB giJ4758886 SARLCQPEGIHICDGTEAENTATLTLLEQQGLIRKLPKYNNC LARTDPE
110 120 130 140 150
N0V32 DVARVESKTVIVTPSQRDTVPLPPGGAHGQLGN MSPADFG ΪVDERFPG
1475096 DVARVESKTVIVTPSQRDTV@LPPGGARGQLGN MSPADF gi 3287892 DVARVESKTVIVTPSQRDTVPLPPGGARGQLGNWMSPADFI gi 1630753 DVARVESKTVIVTPSQRDTVPLJEQGARGQLGNWMSP[3F gi 1265519 DVARVESKTVIVTPSQRDTVPLPPGGARGQLGNMSPADF gi 4758886 DVARVESKTVIVTPSQRDTVPLPPGGABGQLGN MSPADF
The NOV32 Clustal W alignment shown in Table 32E was modified to end at amino residue 150. The data in Table 32E includes all of the regions overlapping with the NOV32 protein sequences.
The presence of identifiable domains in the protein disclosed herein was determined by searches using algorithms such as PROSITE, Blocks, Pfam, ProDomain, Prints and then determimng the Inteφro number by crossing the domain match (or numbers) using the Inteφro website (http:www.ebi.ac.uk/inteφro/). Table 32F lists the domain description from DOMAIN analysis results against NON32.
Figure imgf000300_0001
Consistent with other known members of the PCK family of proteins, NON32 contains phosphoenolpyruvate carboxykinase domains as illustrated in Table32F. ΝOV32 nucleic acids, and the encoded polypeptides, according to the invention are useful in a variety of applications and contexts. For example, NOV32 nucleic acids and polypeptides can be used to identify proteins that are members of the PCK family of proteins. The NOV32 nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance NOV32 activity or function. Specifically, the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., gluconeogenesis. These molecules can be used to treat, e.g. , hypoglycemia and other diseases, disorders and conditions of the like.
In addition, various NOV32 nucleic acids and polypeptides according to the invention are useful, inter alia, as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins. For example, the NOV32 nucleic acids and their encoded polypeptides include structural motifs and homology that are characteristic of proteins belonging to the family of PCK proteins which
Phosphoenolpyruvate carboxykinase (GTP) (PCK; EC 4.1.1.32) catalyzes the formation of phosphoenolpyruvate by decarboxylation of oxaloacetate while hydrolyzing GTP, a rate limiting step in gluconeogenesis (the biosynthesis of glucose), hi vertebrates there are two isozymes: a cytosolic form whose activity is affected by hormones regulating this metabolic process (such as glucagon, or insulin) and a mitochondrial form. The activity is about equally distributed between cytosol and mitochondria in human liver. In contrast, PCK is essentially a cytosolic enzyme in rat liver. See also PCK1 (261680), the human cytosolic PCK enzyme. Modaressi et al. (1996) cloned and sequenced the cDNA of the mitochondrial form of hepatic PCK (Biochem J., 315 ( Pt 3):807- 14 (1996)). The gene encodes a 640-amino acid polypeptide. The gene has has an overall 68% DNA sequence identity and a 70% deduced amino acid sequence identity with human cytosolic PCK cDNA. Expression studies were also reported.
Deficiencies in PKC2 PEPCK2) have been documented. In 2 unrelated children, Hommes et al. (1976) observed hypoglycemia and liver impairment, with deficiency of PEPCK in liver tissue taken immediately after death (Acta Paediatr Scand., 65(2):233-40 (1976)). Massive fatty deposition in liver and kidneys was found at autopsy. Fiser et al. (1974) also observed hypoglycemia caused by deficiency of PEPCK (Am J Obstet Gynecol., 120(7):944-50 (1974)). Other enzymatic causes of hypoglycemia include deficiency of glucose-6-phosphatase (232200), fructose- 1,6-diphosphatase (229700), and pyruvate carboxylase (266150). Vidnes and Sovik (1976) described a case of persistent neonatal hypoglycemia in which only the extramitochondrial (i.e., cytosolic) form of hepatic phosphoenolpyruvate carboxykinase (PCKl) was deficient (Acta Paediatr Scand., 65(3):307-12 (1976)). Phosphoenolpyruvate carboxykinase can be measured in fϊbrob lasts, which are said to contain only mitochondrial PEPCK (Clayton etal, Eur J Pediatr., 145(l-2):46-50 (1986)). Clayton et al. (1986) reported this disorder in a female child who died of liver failure at 6 months and probably in her brother who died a crib death at 4 weeks (Eur J Pediatr., 145(l-2):46-50 (1986)). Leonard et al. (1991) studied the next child in this family, a boy who developed a similar illness with liver failure (Eur J Pediatr., 150(3): 198-9 (1991)). PEPCK activity in leukocytes and fibroblasts was normal, however, leading Leonard et al. (1991) to conclude that the primary defect in this family does not reside in this enzyme (Eur J Pediatr., 150(3):198-9 (1991)). Subsequent studies of a third affected child in this family by Bodnar et al. (1993) suggested that the sibs suffered from the mitochondrial DNA depletion syndrome (251880) and that this depletion is controlled by the nuclear genome (Am J Hum Genet., 53(3):663-9 (1993).
The NOV32 nucleic acids and polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in regulating glucose metabolism. As such the NOV32 nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat metabolic disorders, e.g., hypoglycemia.
The NOV32 nucleic acids and polypeptides are useful for detecting specific cell types. For example, expression analysis has demonstrated that a NOV32 nucleic acid is expressed in Liver, Adipose, Adrenal Gland/Suprarenal gland, Bone, Bone Marrow, Brain, Brown adipose, Cartilage, Cervix, Colon, Duodenum, Heart, Kidney, Kidney Cortex, Left cerebellum, Lung, Lymphoid tissue, Mammary gland/Breast, Ovary, Pancreas, Placenta, Prostate, Retina, Skin, Small Intestine, Spinal Chord, Stomach, Substantia Nigra, Synovium/Synovial membrane, Testis, Tonsils, Uterus, Vulva, Whole Organism. Additional utilities for NOV32 nucleic acids and polypeptides according to the invention are disclosed herein. NOV33
A NOV33 polypeptide has been identified as a G Protein-Coupled Receptor (GPCR)-like protein (also referred to as CG56610-01). The disclosed novel NOV33 nucleic acid (SEQ ID NO:122) of924 nucleotides is shown in Table 33A. An ORF begins with an AAA codon which codes for the amino acid lysine at nucleotides 3-5 and ends with a TGA codon atnucleotides 912- 914. A putative untranslated region and/or downstream from the termination codon is underlined in Table 33A, and the start and stop codons are inbold letters.
Table 33A. NON33 Nucleotide Sequence (SEQ ID NO:122)
CTAAATTTCCAACCTTCTTGTTGACCGGCATTCCTGGCCTAGAGTCTGCCCATGTCTGGATCTCCAT TCCTTTCTGTTGTTTTTATGCCATTGCCCTCTCTGGGAACAGCGTGATCCTGTTTGTCATCATTACC CAGCAGAGTCTCCATGAΆCCCATGTATTATTTCCTCTTCAGGCTATCAGCCACTGATCTGGACTTGA CTGTTTCTTCATTGTCAACAACATTAGGTATTCTCTGGTTTGAGGCACGTGAAATCAGTCTATATAG CTGCATTGTCCAGATGTTTTTTCTTCATGGATTCACTTTTATGGAATCTGGAGTGCTGGTGGCTACA GCCTTTGACCGTTATGCGGCCATCTGTGACCCTCTGAGGTACACTACCATTCTCACTAATTCCAGAΆ TCATTCAAATGGGTCTTCTGATGATTACACGTGCTATAGTACTAATATTGCCACTACTTTTGCTCCT TAAGCCTCTCTATTTCTGTAGAATGAATGCCCTTTCTCACTCCTATTGTTACCATCCAGATGTGATT CAATTAGCATGTTCAGACATTCGGGCAAATAGCATCTGTGGATTAACTGATCTCATCCTGACCACTG GAATAGATACACCATGCATTGTCCTGTCATATATCTTAATTATTCACTCTGTCCTCAGAATTGCCTC CCCTGAAGAATGGCACAAGGTCTTCAGCACCTGTGTCTCCCATGTGGGAGCAGTTGCTTTCTTCTAC ATCCACATGCTGAGCCTGTCCTTGGTGTATCGCTATGGTCGGTCAGCCCCCAGAGTAGTCCATTCAG TGATGGCTAATGTATACCTGCTTTTACCCCCTGTGCTCAACCCCATCATCGACAGTGTAAAAACAAA ACAAATCCGCAAGGCTATGCTCAGTCTGCTGCTTACAAAATGAACAGACATAG
The NOV33 protein (SEQ ID NO:123) encoded by SEQ ID NO:122 is 303 amino acid residues in length and is presented using the one-letter amino acid code in Table 33B. Psort analysis predicts the NOV33 protein ofthe invention to be localized at the plasma membrane with a certainty of0.6000. The Signal P predicts a likely cleavage site for aNOV33 peptide is between positions 33 and 34, i.e., at the dash in the sequence ALS-GN.
Table 33B. Encoded NOV33 protein sequence (SEQ ID NO:123)
KFPTFLLTGIPGLESAHV ISIPFCCFYAIALSGNSVILFVIITQQSLHEPMYYFLFRLSATDLD LTVSSLSTTLGILWFEAREISLYSCIVQMFFLHGFTFMESGVLVATAFDRYAAICDPLRYTTILT NSRIIQMGLLMITRAIVLILPLLLLLKPLYFCRMNALSHSYCYHPDVIQLACSDIRANSICGLTD LILTTGIDTPCIVLSYILIIHSVLRIASPEEWHKVFSTCVSHVGAVAFFYIHMLSLSLVYRYGRS APRWHSVMANVYLLLPPVLNPIIDSVKTKQIRKAMLSLLLTK A search against the Patp database, a proprietary database that contains sequences published in patents and patent publications, yielded several homologous proteins shown in Table 33C.
Table 33C. Patp results for NOV33
Smallest Sum
Reading High Prob
Sequences producing High-scoring Segment Pairs: Frame Score P(N)
>patp:AAG71696 Human olfactory receptor polypeptide +1 1501 l.le-153
>patp:AAG71724 Human olfactory receptor polypeptide +1 1028 1.4e-103
>patp:AAG71510 Human olfactory receptor polypeptide +1 935 1.0e-93
>patp:AAG72487 Human OR-like polypeptide query sequence +1 935 1.0e-93
>patp:AAG71698 Human olfactory receptor polypeptide +1 819 2.0e-81
In a BLAST search of public sequence databases, it was found, for example, that the NOV33 nucleic acid sequence of this invention has 579 of 905 bases (63%) identical to a gb:GENBANK-ID:AF133300|acc:AF133300.2 mRNA from as musculus (MOR 3'Betal, MOR 3'Beta2, MOR 3'Beta3, and MOR 3'Beta4 genes, complete eds; Cbx3 pseudogene, complete sequence; and MOR 3'Beta5 and MOR 3'Beta6 genes, complete eds). NOV33 polypeptide of the invention was found to have 155 of 295 amino acid residues (52%) identical to, and 208 of 295 amino acid residues (70%) similar to, the 312 amino acid residue ptππTREMBLNEW- ACC:AAG41678 protein from Homo sapiens (HOR5ΕETA12).
NOV33 also has homology to the proteins shown in the BLASTP data in Table 33D.
Figure imgf000304_0001
Figure imgf000305_0002
A multiple sequence alignment is given in Table 33E, with the NOV33 protein being shown on line 1 in Table 33E in a ClustalW analysis, and comparing the NOV33 protein with the related protein sequences shown in Table 33D. This BLASTP data is displayed graphically in the ClustalW in Table 33E.
Table 33E. ClustalW Analysis of NOV33
1) > NOV33; SEQ ID NO:123
2) > gi|1745680/ similar to olfactory receptor 5112 [Homo sapiens]; SEQ TD NO:335
3) > gi|1745676/ similar to prostate specific G-protein coupled receptor [Homo sapiens]; SEQ TD NO:336
4) > gij 1745677/ similar to olfactory receptor-like protein COR3beta [Homo sapiens] ; SEQ TD NO:337
5) > gijl 199186/ odorant receptor HOR'3betal [Homo sapiens] ; SEQ ID NO:338
6) > gij 1747278/ similar to olfactory receptor 5112 [Homo sapiens]; SEQ TD NO:339
Figure imgf000305_0001
Figure imgf000306_0001
The NOV33 Clustal W alignment shown in Table 33E was modified to begin at amino residue 551. The data in Table 33E includes all of the regions overlapping with the NOV33 protein sequences.
The presence of identifiable domains in the protein disclosed herein was determined by searches using algorithms such as PROSITE, Blocks, Pfam, ProDomain, Prints and then determimng the Inteφro number by crossing the domain match (or numbers) using the Interpro website (http:www.ebi.ac.uk/interpro/). The DOMAIN analysis results indicate that the NOV33 protein contains the following protein domain (as defined by Interpro): domain name 7tm_l 7 transmembrane receptor (rhodopsin family). DOMAIN results for NOV33 were collected from the Conserved Domain Database (CDD) with Reverse Position Specific BLAST. This BLAST samples domains found in the Smart and Pfam collections.
As discussed below, the NOV33 protein of the invention contained significant homology to the 7tm_l domain. This indicates that the NOV33 sequence has properties similar to those of other proteins known to contain this 7tm_l domain and similar to the properties of these domains. The 254 amino acid domain termed 7tm_l (SEQ ID NO:340; Pfam Ace. No. 00001) a seven transmembrane receptor (rhodopsin family), is shown in Table 33F.
Table 33F. 7tm_l, 7 transmembrane receptor domain (SEQ ID NO:340)
GNLLVILVILRTKKLRTPTNIFLLNLAVADLLFLLTLPP ALYYLVGGDVFGDALCKLVGALFWNGYASILLLTAISIDRYL AIVHPLRYRRIRTPRRAKVLILLVVLALLLSLPPLLFS LRTVEEGNTTVCLIDFPEESVKRSYVLLSTLVGFVLPLLVILVC YTRILRTLRKRARSQRSLKRRSSSERKAAKMLLVVVVVFVLC LPYHIVLLLDSLCLLSIWRVLPTALLITL LAYVNSCLNPI IY
The DOMAIN results are listed in Table 33G with the statistics and domain description. An alignment of NOV33 residues 34-133 (SEQ ID NO:123) with the full 7tm_l domain, residues 1 -254 (SEQ ID NO:340), are shown in Table 33G. This indicates that the NOV33 sequences have properties similar to those of other proteins known to contain this domain as well as to the 254 amino acid 7tm domain (SEQ ID NO:340). For Table 33G, fully conserved single residues are indicated by the vertical line and "strong" semi-conserved residues are indicated by the "plus sign." The "strong" group of conserved amino acid residues maybe any one of the following groups of amino acids: STA, NEQK, NHQK, NDEQ, QHRK, MILV, MILF, HY, FYW.
Table 33G Domain Analysis of NOV33
PSSMs producing significant alignments: Score E (bits) value gnl|Pfam|pfam00001 7tm_l, 7 transmembrane receptor (rhodopsin family) 42.1 1.5e-12
*->GNlLVilvilrtkklrtptnifilNLAvADLLflltlppwalyylvg GN+++++vi+ +++1+ p+++f++ L+ +DL ++++ + +1 +1++
NOV33 34 GNSVILFVIITQQSLHEPMYYFLFRLSATDLDLTVSSLSTTLGILWF 80 gsedWpfGsalCklvtald nmyaSillLtalSiDRYlAIvhPlryrrr e ++ + C +++++ ++++ L+a ++DRY AI++Plry ++
NOV33 81 --EAREISLYSCIVQMFFLHGFTFMESGVLVATAFDRYAAICDPLRYTTI 12 t rtsprr<-* (SEQ ID NO: 340) t r N0V33 129 LT-NSR 133 (SEQ ID NO: 123) Consistent with other known members of the GPCR family of proteins, NOV33 contains 7tm_l 7 transmembrane receptor (rhodopsin family) domain as illustrated in Table 33G as well as homology and cellular localization, i.e. plasma membrane.
NOV33 nucleic acids, and the encoded polypeptides, according to the invention are useful in a variety of applications and contexts. For example, NOV33 nucleic acids and polypeptides can be used to identify proteins that are members of the GPCR family of proteins. The NOV33 nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance NOV33 activity or function. Specifically, the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., cellular signal transduction. These molecules can be used to treat, e.g., cancer, immune disorders, and endocrine disorders. hi addition, various NOV33 nucleic acids and polypeptides according to the invention are useful, ter alia, as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins. For example, the NOV33 nucleic acids and their encoded polypeptides include 7tm_l 7 transmembrane receptor (rhodopsin family) domain and sequence homology that are characteristic of proteins belonging to the family of GPCR such as the G protein-coupled olfactory receptor. The NOV33 protein of the invention has a high homology to the 7tm_l domain (PFam Ace. No. pfamOOOOl). The 7tm_l domain is from the 7 transmembrane receptor family, which includes a number of different proteins, including, for example, serotonin receptors, dopamine receptors, histamine receptors, andrenergic receptors, cannabinoid receptors, angiotensin II receptors, chemokine receptors, opioid receptors, G-protein coupled receptor (GPCR) proteins, olfactory receptors (OR), and the like.
G-Protein Coupled Receptor proteins ("GPCRs") have been identified as a large family of G protein-coupled receptors in a number of species. These receptors share a seven transmembrane domain structure with many neurotransmitter and hormone receptors, and are likely to underlie the recognition and G-protein-mediated transduction of various signals. Human GPCR generally do not contain introns and belong to four different gene subfamilies, displaying great sequence variability. These genes are dominantly expressed in olfactory epithelium. See, e.g., Ben-Arie et al, Hum. Mol. Genet. 3:229-235 (1994); and, Online Mendelian Inheritance in Man ("OMIM") entry # 164342 (http://www.ncbi.nlm.nih.gov/ entrez/ dispomim.cgi?). The NOV33 nucleic acids and polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in the mediation of cellular signal transduction. As such the NOV33 nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat a wide range of disorders such as cancer, immune disorders, endocrine disorders and other diseases, e.g., developmental diseases; MHCπ and III diseases (immune diseases); taste and scent detectability disorders; Burkitt's lymphoma; corticoneurogenic disease; signal transduction pathway disorders; metabolic pathway disorders; retinal diseases including those involving photoreception; cell growth rate disorders; cell shape disorders; metabolic disorders; feeding disorders; control of feeding; the metabolic syndrome X; wasting disorders associated with chronic diseases; obesity; potential obesity due to over-eating or metabolic disturbances; potential disorders due to starvation (lack of appetite); diabetes; noninsulin-dependent diabetes mellitus (NIDDM); infectious disease; bacterial, fungal, protozoal and viral infections (particularly infections caused by HIV-1 or HIV-2); pain; cancer (including but not limited to neoplasm; adenocarcinoma; lymphoma; prostate cancer; uterus cancer); cancer- associated cachexia; anorexia; bulimia; asthma; Parkinson's disease; acute heart failure; hypotension; hypertension; urinary retention; osteoporosis; Crohn's disease; multiple sclerosis; Albright Hereditary Ostoeodystrophy; angina pectoris; myocardial infarction; ulcers; allergies; benign prostatic hypertrophy; and psychotic and neurological disorders; including anxiety; schizophrenia; manic depression; delirium; dementia; neurodegenerative disorders; Alzheimer's disease; severe mental retardation; Dentatorubro-pallidoluysian atrophy (DRPLA); Hypophosphatemic rickets; autosomal dominant (2) Acrocallosal syndrome and dyskinesias, such as Huntington's disease or Gilles de la Tourette syndrome; immune disorders; Adrenoleukodystrophy; Congenital Adrenal Hyperplasia; Hemophilia; Hypercoagulation; Idiopathic thrombocytopenic purpura; autoimmume disease; immunodeficiencies; transplantation; Von Hippel-Lindau (VHL) syndrome; Stroke; Tuberous sclerosis; hypercalceimia; Cerebral palsy; Epilepsy; Lesch-Nyhan syndrome; Ataxia-telangiectasia; Leukodystrophies; Behavioral disorders; Addiction; Neuroprotection; Cirrhosis; Transplantation; Systemic lupus erythematosus; Emphysema; Scleroderma; ARDS; Renal artery stenosis; Interstitial nephritis; Glomerulonephritis; Polycystic kidney disease; Systemic lupus erythematosus; Renal tubular acidosis; IgA nephropathy; Cardiomyopathy; Atherosclerosis; Congenital heart defects; Aortic stenosis ; Atrial septal defect (ASD); Atrioventricular (A-V) canal defect; Ductus arteriosus; Pulmonary stenosis ; Subaortic stenosis; Ventricular septal defect (VSD); valve diseases; Scleroderma; fertility; Pancreatitis; Endocrine dysfunctions; Growth and reproductive disorders; Inflammatory bowel disease; Diverticular disease; Leukodystrophies; Graft vesus host; Hyperthyroidism; Endometriosis; and hematopoietic disorders. The NOV33 nucleic acids and polypeptides are useful for detecting specific cell types. For example, expression analysis has demonstrated that a NOV33 nucleic acid is expressed in MHC It and III nervous, medulla, subthalamic nucleus, ovary, pancreas, pituitary, placenta, pons, prostate, putamen, serum, skeletal muscle, small intestine, smooth muscle (coronary artery in aortic) spinal cord, spleen, stomach, taste receptor cells of the tongue, testis, thalamus, and thymus tissue. Additional utilities for NOV33 nucleic acids and polypeptides according to the invention are disclosed herein.
NOVX Nucleic Acids and Polypeptides
One aspect of the invention pertains to isolated nucleic acid molecules that encode NOVX polypeptides or biologically active portions thereof. Also included in the invention are nucleic acid fragments sufficient for use as hybridization probes to identify NOVX-encoding nucleic acids (e.g., NOVX mRNAs) and fragments for use as PCR primers for the amplification and/or mutation of NOVX nucleic acid molecules. As used herein, the term "nucleic acid molecule" is intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNA generated using nucleotide analogs, and derivatives, fragments and homologs thereof. The nucleic acid molecule may be single-stranded or double- stranded, but preferably is comprised double-stranded DNA.
A NOVX nucleic acid can encode a mature NOVX polypeptide. As used herein, a "mature" form of a polypeptide or protein disclosed in the present invention is the product of a naturally occurring polypeptide or precursor form or proprotein. The naturally occurring polypeptide, precursor or proprotein includes, by way of nonlimiting example, the full-length gene product, encoded by the corresponding gene. Alternatively, it may be defined as the polypeptide, precursor or proprotein encoded by an ORF described herein. The product "mature" form arises, again by way of nonlimiting example, as a result of one or more naturally occurring processing steps as they may take place within the cell, or host cell, in which the gene product arises. Examples of such processing steps leading to a "mature" form of a polypeptide or protein include the cleavage of the N-terminal methionine residue encoded by the imtiation codon of an ORF, or the proteolytic cleavage of a signal peptide or leader sequence. Thus a mature form arising from a precursor polypeptide or protein that has residues 1 to N, where residue 1 is the N- terminal methionine, would have residues 2 through N remaining after removal of the N-terminal methionine. Alternatively, a mature form arising from a precursor polypeptide or protein having residues 1 to N, in which an N-terminal signal sequence from residue 1 to residue M is cleaved, would have the residues from residue M+l to residue N remaining. Further as used herein, a "mature" form of a polypeptide or protein may arise from a step of post-translational modification other than a proteolytic cleavage event. Such additional processes include, by way of non- limiting example, glycosylation, myristoylation or phosphorylation. hi general, a mature polypeptide or protein may result from the operation of only one of these processes, or a combination of any of them.
The term "probes", as utilized herein, refers to nucleic acid sequences of variable length, preferably between at least about 10 nucleotides (nt), 100 nt, or as many as approximately, e.g., 6,000 nt, depending upon the specific use. Probes are used in the detection of identical, similar, or complementary nucleic acid sequences. Longer length probes are generally obtained from a natural or recombinant source, are highly specific, and much slower to hybridize than shorter- length oligomer probes. Probes may be single- or double-stranded and designed to have specificity in PCR, membrane-based hybridization technologies, or ELISA-like technologies. The term "isolated" nucleic acid molecule, as utilized herein, is one, which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid. Preferably, an "isolated" nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5'- and 3'-termini of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated NOVX nucleic acid molecules can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell/tissue from which the nucleic acid is derived (e.g., brain, heart, liver, spleen, etc.). Moreover, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material or culture medium when produced by recombinant techniques, or of chemical precursors or other chemicals when chemically synthesized. A nucleic acid molecule of the invention, e.g., a nucleic acid molecule having the nucleotide sequence SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, and 122, or a complement of this aforementioned nucleotide sequence, can be isolated using standard molecular biology techniques and the sequence information provided herein. Using all or a portion of the nucleic acid sequence of SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, and 122 as a hybridization probe, NOVX molecules can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook, et al, (eds.), MOLECULAR CLONING: A LABORATORY MANUAL 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; and Ausubel, et al., (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, NY, 1993.) A nucleic acid of the invention can be amplified using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to NOVX nucleotide sequences can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
As used herein, the term "oligonucleotide" refers to a series of linked nucleotide residues, which oligonucleotide has a sufficient number of nucleotide bases to be used in a PCR reaction. A short oligonucleotide sequence may be based on, or designed from, a genomic or cDNA sequence and is used to amplify, confirm, or reveal the presence of an identical, similar or complementary DNA or RNA in a particular cell or tissue. Oligonucleotides comprise portions of a nucleic acid sequence having about 10 nt, 50 nt, or 100 nt in length, preferably about 15 nt to 30 nt in length. In one embodiment of the invention, an oligonucleotide comprising a nucleic acid molecule less than 100 nt in length would further comprise at least 6 contiguous nucleotides SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, and 122, or a complement thereof. Oligonucleotides may be chemically synthesized and may also be used as probes.
In another embodiment, an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule that is a complement of the nucleotide sequence shown in SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, and 122, or a portion of this nucleotide sequence (e.g., a fragment that can be used as a probe or primer or a fragment encoding a biologically-active portion of an NOVX polypeptide). A nucleic acid molecule that is complementary to the nucleotide sequence shown SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, and 122 is one that is sufficiently complementary to the nucleotide sequence shown SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, . 108, 110, 112, 114, 116, 118, 120, and 122 that it can hydrogen bond with little or no mismatches to the nucleotide sequence shown SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, and 122 thereby forming a stable duplex.
As used herein, the term "complementary" refers to Watson-Crick or Hoogsteen base pairing between nucleotides units of a nucleic acid molecule, and the term "binding" means the physical or chemical interaction between two polypeptides or compounds or associated polypeptides or compounds or combinations thereof. Binding includes ionic, non-ionic, van der Waals, hydrophobic interactions, and the like. A physical interaction can be either direct or indirect. Indirect interactions may be through or due to the effects of another polypeptide or compound. Direct binding refers to interactions that do not take place through, or due to, the effect of another polypeptide or compound, but instead are without other substantial chemical intermediates. Fragments provided herein are defined as sequences of at least 6 (contiguous) nucleic acids or at least 4 (contiguous) amino acids, a length sufficient to allow for specific hybridization in the case of nucleic acids or for specific recognition of an epitope in the case of amino acids, respectively, and are at most some portion less than a full length sequence. Fragments may be derived from any contiguous portion of a nucleic acid or amino acid sequence of choice. Derivatives are nucleic acid sequences or amino acid sequences formed from the native compounds either directly or by modification or partial substitution. Analogs are nucleic acid sequences or amino acid sequences that have a structure similar to, but not identical to, the native compound but differs from it in respect to certain components or side chains. Analogs may be synthetic or from a different evolutionary origin and may have a similar or opposite metabolic activity compared to wild type. Homologs are nucleic acid sequences or amino acid sequences of a particular gene that are derived from different species.
Derivatives and analogs may be full length or other than full length, if the derivative or analog contains a modified nucleic acid or amino acid, as described below. Derivatives or analogs of the nucleic acids or proteins of the invention include, but are not limited to, molecules comprising regions that are substantially homologous to the nucleic acids or proteins of the invention, in various embodiments, by at least about 70%, 80%, or 95% identity (with a preferred identity of 80-95%) over a nucleic acid or amino acid sequence of identical size or when compared to an aligned sequence in which the alignment is done by a computer homology program known in the art, or whose encoding nucleic acid is capable of hybridizing to the complement of a sequence encoding the aforementioned proteins under stringent, moderately stringent, or low stringent conditions. See e.g. Ausubel, et al, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, NY, 1993, and below.
A "homologous nucleic acid sequence" or "homologous amino acid sequence," or variations thereof, refer to sequences characterized by a homology at the nucleotide level or amino acid level as discussed above. Homologous nucleotide sequences encode those sequences coding for isoforms of NOVX polypeptides. Isoforms can be expressed in different tissues of the same organism as a result of, for example, alternative splicing of RNA. Alternatively, isoforms can be encoded by different genes. In the invention, homologous nucleotide sequences include nucleotide sequences encoding for an NOVX polypeptide of species other than humans, including, but not limited to: vertebrates, and thus can include, e.g., frog, mouse, rat, rabbit, dog, cat cow, horse, and other organisms. Homologous nucleotide sequences also include, but are not limited to, naturally occurring allelic variations and mutations of the nucleotide sequences set forth herein. A homologous nucleotide sequence does not, however, include the exact nucleotide sequence encoding human NOVX protein. Homologous nucleic acid sequences include those nucleic acid sequences that encode conservative amino acid substitutions (see below) in SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, and 122, as well as a polypeptide possessing NOVX biological activity. Various biological activities of the NOVX proteins are described below.
An NOVX polypeptide is encoded by the open reading frame ("ORF") of an NOVX nucleic acid. An ORF corresponds to a nucleotide sequence that could potentially be translated into a polypeptide. A stretch of nucleic acids comprising an ORF is uninterrupted by a stop codon. An ORF that represents the coding sequence for a full protein begins with an ATG "start" codon and terminates with one of the three "stop" codons, namely, TAA, TAG, or TGA. For the purposes of this invention, an ORF may be any part of a coding sequence, with or without a start codon, a stop codon, or both. For an ORF to be considered as a good candidate for coding for a bonafide cellular protein, a minimum size requirement is often set, e.g., a stretch of DNA that would encode a protein of 50 amino acids or more.
The nucleotide sequences determined from the cloning of the human NOVX genes allows for the generation of probes and primers designed for use in identifying and/or cloning NOVX homologues in other cell types, e.g. from other tissues, as well as NOVX homologues from other vertebrates. The probe/primer typically comprises substantially purified oligonucleotide. The oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, 25, 50, 100, 150, 200, 250, 300, 350 or 400 consecutive sense strand nucleotide sequence SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, and 122; or an anti-sense strand nucleotide sequence of SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, and 122; or of a naturally occurring mutant of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, and 122.
Probes based on the human NOVX nucleotide sequences can be used to detect transcripts or genomic sequences encoding the same or homologous proteins. In various embodiments, the probe further comprises a label group attached thereto, e.g. the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. Such probes can be used as a part of a diagnostic test kit for identifying cells or tissues which mis-express an NOVX protein, such as by measuring a level of an NOVX-encoding nucleic acid in a sample of cells from a subject e.g., detecting NOVX mRNA levels or determining whether a genomic NOVX gene has been mutated or deleted.
"A polypeptide having a biologically-active portion of an NOVX polypeptide" refers to polypeptides exhibiting activity similar, but not necessarily identical to, an activity of a polypeptide of the invention, including mature forms, as measured in a particular biological assay, with or without dose dependency. A nucleic acid fragment encoding a "biologically-active portion of NOVX" can be prepared by isolating a portion SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, and 122, that encodes a polypeptide having an NOVX biological activity (the biological activities of the NOVX proteins are described below), expressing the encoded portion of NOVX protein (e.g., by recombinant expression in vitro) and assessing the activity of the encoded portion of NOVX.
NOVX Nucleic Acid and Polypeptide Variants
The invention further encompasses nucleic acid molecules that differ from the nucleotide sequences shown in SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, and 122 due to degeneracy of the genetic code and thus encode the same NOVX proteins as that encoded by the nucleotide sequences shown in SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, and 122.
In another embodiment, an isolated nucleic acid molecule of the invention has a nucleotide sequence encoding a protein having an amino acid sequence shown in SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111,
113, 115, 117, 119, 121, and 123. In addition to the human NOVX nucleotide sequences shown in SEQ ID NOS:l, 3, 5, 7, 9,
11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, and 122, it will be appreciated by those skilled in the art that DNA sequence polymorphisms that lead to changes in the amino acid sequences of the NOVX polypeptides may exist within a population (e.g., the human population). Such genetic polymorphism in the NOVX genes may exist among individuals within a population due to natural allelic variation. As used herein, the terms "gene" and "recombinant gene" refer to nucleic acid molecules comprising an open reading frame (ORF) encoding an NOVX protein, preferably a vertebrate NOVX protein. Such natural allelic variations can typically result in 1-5% variance in the nucleotide sequence of the NOVX genes. Any and all such nucleotide variations and resulting amino acid polymorphisms in the NOVX polypeptides, which are the result of natural allelic variation and that do not alter the functional activity of the NOVX polypeptides, are intended to be within the scope of the invention.
Moreover, nucleic acid molecules encoding NOVX proteins from other species, and thus that have a nucleotide sequence that differs from the human SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112,
114, 116, 118, 120, and 122 are intended to be within the scope of the invention. Nucleic acid molecules corresponding to natural allelic variants and homologues of the NOVX cDNAs of the invention can be isolated based on their homology to the human NOVX nucleic acids disclosed herein using the human cDNAs, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions.
Accordingly, in another embodiment, an isolated nucleic acid molecule of the invention is at least 6 nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, and 122. In another embodiment, the nucleic acid is at least 10, 25, 50, 100, 250, 500, 750, 1000, 1500,'or 2000 or more nucleotides in length. In yet another embodiment, an isolated nucleic acid molecule of the invention hybridizes to the coding region. As used herein, the term "hybridizes under stringent conditions" is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60% homologous to each other typically remain hybridized to each other.
Homologs (i.e., nucleic acids encoding NOVX proteins derived from species other than human) or other related sequences (e.g., paralogs) can be obtained by low, moderate or high stringency hybridization with all or a portion of the particular human sequence as a probe using methods well known in the art for nucleic acid hybridization and cloning.
As used herein, the phrase "stringent hybridization conditions" refers to conditions under which a probe, primer or oligonucleotide will hybridize to its target sequence, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures than shorter sequences. Generally, stringent conditions are selected to be about 5 °C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present at excess, at Tm, 50% of the probes are occupied at equilibrium. Typically, stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes, primers or oligonucleotides (e.g., 10 nt to 50 nt) and at least about 60°C for longer probes, primers and oligonucleotides. Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide.
Stringent conditions are known to those skilled in the art and can be found in Ausubel, et al., (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Preferably, the conditions are such that sequences at least about 65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99% homologous to each other typically remain hybridized to each other. A non-limiting example of stringent hybridization conditions are hybridization in a high salt buffer comprising 6X SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02%) BSA, and 500 mg/ml denatured salmon sperm DNA at 65°C, followed by one or more washes in 0.2X SSC, 0.01% BSA at 50°C. An isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to the sequences SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, and 122, corresponds to a naturally-occurring nucleic acid molecule. As used herein, a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).
In a second embodiment, a nucleic acid sequence that is hybridizable to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, and 122, or fragments, analogs or derivatives thereof, under conditions of moderate stringency is provided. A non-limiting example of moderate stringency hybridization conditions are hybridization in 6X SSC, 5X Denhardt's solution, 0.5% SDS and 100 mg/ml denatured salmon sperm DNA at 55°C, followed by one or more washes in IX SSC, 0.1% SDS at 37°C. Other conditions of moderate stringency that may be used are well-known within the art. See, e.g., Ausubel, et al. (eds.), 1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY, and Kriegler, 1990; GENE TRANSFER AND EXPRESSION, A LABORATORY MANUAL, Stockton Press, NY.
In a third embodiment, a nucleic acid that is hybridizable to the nucleic acid molecule comprising the nucleotide sequences SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, and 122, or fragments, analogs or derivatives thereof, under conditions of low stringency, is provided. A non-limiting example of low stringency hybridization conditions are hybridization in 35% formamide, 5X SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 mg/ml denatured salmon sperm DNA, 10% (wt/vol) dextran sulfate at 40°C, followed by one or more washes in 2X SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS at 50°C. Other conditions of low stringency that may be used are well known in the art (e.g., as employed for cross-species hybridizations). See, e.g., Ausubel, et al. (eds.), 1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY, and Kriegler, 1990, GENE TRANSFER AND EXPRESSION, A LABORATORY MANUAL, Stockton Press, NY; Shilo and Weinberg, 1981. Proc Natl Acad Sci USA 78: 6789-6792.
Conservative Mutations
In addition to naturally-occurring allelic variants of NOVX sequences that may exist in the population, the skilled artisan will further appreciate that changes can be introduced by mutation into the nucleotide sequences SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, and 122, thereby leading to changes in the amino acid sequences of the encoded NOVX proteins, without altering the functional ability of said NOVX proteins. For example, nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues can be made in the sequence SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, and 123. A "non-essential" amino acid residue is a residue that can be altered from the wild-type sequences of the NOVX proteins without altering their biological activity, whereas an "essential" amino acid residue is required for such biological activity. For example, amino acid residues that are conserved among the NOVX proteins of the invention are predicted to be particularly non-amenable to alteration. Amino acids for which conservative substitutions can be made are well-known within the art.
Another aspect of the invention pertains to nucleic acid molecules encoding NOVX proteins that contain changes in amino acid residues that are not essential for activity. Such
NOVX proteins differ in amino acid sequence from SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, and 122 yet retain biological activity. In one embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence at least about 45% homologous to the amino acid sequences SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, and 123. Preferably, the protein encoded by the nucleic acid molecule is at least about 60% homologous to SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, and 123; more preferably at least about 70% homologous SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, and 123; still more preferably at least about 80% homologous to SEQ ID
NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, and 123; even more preferably at least about 90% homologous to SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, and 123; and most preferably at least about 95%> homologous to SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, and 123.
An isolated nucleic acid molecule encoding an NOVX protein homologous to the protein of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99,
101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, and 123 can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100,
102, 104, 106, 108, 110, 112, 114, 116, 118, 120, and 122, such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced into SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,
27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, and 122 by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted, non-essential amino acid residues. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined within the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted non-essential amino acid residue in the NOVX protein is replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of an NOVX coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for NOVX biological activity to identify mutants that retain activity. Following mutagenesis SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, and 122, the encoded protein can be expressed by any recombinant technology known in the art and the activity of the protein can be determined. The relatedness of amino acid families may also be determined based on side chain interactions. Substituted amino acids may be fully conserved "strong" residues or fully conserved "weak" residues. The "strong" group of conserved amino acid residues may be any one of the following groups: STA, NEQK, NHQK, NDEQ, QHRK, MILV, MILF, HY, FYW, wherein the single letter amino acid codes are grouped by those amino acids that may be substituted for each other. Likewise, the "weak" group of conserved residues may be any one of the following: CSA, ATV, SAG, STNK, STPA, SGND, SNDEQK, NDEQHK, NEQHRK, VLLVI, HFY, wherein the letters within each group represent the single letter amino acid code.
In one embodiment, a mutant NOVX protein can be assayed for (i) the ability to form proteimprotein interactions with other NOVX proteins, other cell-surface proteins, or biologically-active portions thereof, (ii) complex formation between a mutant NOVX protein and an NOVX ligand; or (iii) the ability of a mutant NOVX protein to bind to an intracellular target protein or biologically-active portion thereof; (e.g. avidin proteins).
In yet another embodiment, a mutant NOVX protein can be assayed for the ability to regulate a specific biological function (e.g., regulation of insulin release). Antisense Nucleic Acids
Another aspect of the invention pertains to isolated antisense nucleic acid molecules that are hybridizable to or complementary to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, and 122, or fragments, analogs or derivatives thereof. An "antisense" nucleic acid comprises a nucleotide sequence that is complementary to a "sense" nucleic acid encoding a protein (e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence). In specific aspects, antisense nucleic acid molecules are provided that comprise a sequence complementary to at least about 10, 25, 50, 100, 250 or 500 nucleotides or an entire NOVX coding strand, or to only a portion thereof. Nucleic acid molecules encoding fragments, homologs, derivatives and analogs of an NOVX protein of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, and 123, or antisense nucleic acids complementary to an NOVX nucleic acid sequence of SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, and 122, are additionally provided. In one embodiment, an antisense nucleic acid molecule is antisense to a "coding region" of the coding strand of a nucleotide sequence encoding an NOVX protein. The term "coding region" refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues. In another embodiment, the antisense nucleic acid molecule is antisense to a "noncoding region" of the coding strand of a nucleotide sequence encoding the NOVX protein. The term "noncoding region" refers to 5' and 3' sequences which flank the coding region that are not translated into amino acids (i.e., also referred to as 5' and 3' untranslated regions). Given the coding strand sequences encoding the NOVX protein disclosed herein, antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick or Hoogsteen base pairing. The antisense nucleic acid molecule can be complementary to the entire coding region of NOVX mRNA, but more preferably is an oligonucleotide that is antisense to only a portion of the coding or noncoding region of NOVX mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of NOVX mRNA. An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. An antisense nucleic acid of the invention can be constructed using chemical synthesis or enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally-occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids (e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used). Examples of modified nucleotides that can be used to generate the antisense nucleic acid include: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil,
3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection). The antisense nucleic acid molecules of the invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding an NOVX protein to thereby inhibit expression of the protein (e.g., by inhibiting transcription and/or translation). The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule that binds to DNA duplexes, through specific interactions in the major groove of the double helix. An example of a route of administration of antisense nucleic acid molecules of the invention includes direct injection at a tissue site. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For example, for systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface (e.g., by linking the antisense nucleic acid molecules to peptides or antibodies that bind to cell surface receptors or antigens). The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient nucleic acid molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.
In yet another embodiment, the antisense nucleic acid molecule of the invention is an α-anomeric nucleic acid molecule. An α-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual β-units, the strands run parallel to each other. See, e.g., Gaultier, et al, 1987. Nucl. Acids Res. 15: 6625-6641. The antisense nucleic acid molecule can also comprise a 2'-o-methylribonucleotide (See, e.g.,
Inoue, et al. 1987. Nucl. Acids Res. 15: 6131-6148) or a chimeric RNA-DNA analogue (See, e.g., frioue, et al, 1987. FEBS Lett. 215: 327-330).
Ribozymes and PNA Moieties Nucleic acid modifications include, by way of non-limiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability of the modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject. In one embodiment, an antisense nucleic acid of the invention is a ribozyme. Ribozymes are catalytic RNA molecules with ribonuclease activity that are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region. Thus, ribozymes (e.g., hammerhead ribozymes as described in Haselhoff and Gerlach 1988. Nature 334: 585-591) can be used to catalytically cleave NOVX mRNA transcripts to thereby inhibit translation of NOVX mRNA. A ribozyme having specificity for an NOVX-encoding nucleic acid can be designed based upon the nucleotide sequence of an NOVX cDNA disclosed herein (i.e., SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, and 122). For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in an
NOVX-encoding mRNA. See, e.g., U.S. Patent 4,987,071 to Cech, et al. and U.S. Patent 5,116,742 to Cech, et al. NOVX mRNA can also be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel et al, (1993) Science 261:1411-1418. Alternatively, NOVX gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the NOVX nucleic acid (e.g., the NOVX promoter and/or enhancers) to form triple helical structures that prevent transcription of the NOVX gene in target cells. See, e.g., Helene, 1991. Anticancer Drug Des. 6: 569-84; Helene, et al. 1992. Ann. N.Y. Acad. Sci. 660: 27-36; Maher, 1992. Bioassays 14: 807-15. In various embodiments, the NOVX nucleic acids can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule. For example, the deoxyribose phosphate backbone of the nucleic acids can be modified to generate peptide nucleic acids. See, e.g., Hyrup, et al, 1996. Bioorg Med Chem 4: 5-23. As used herein, the terms "peptide nucleic acids" or "PNAs" refer to nucleic acid mimics (e.g., DNA mimics) in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained. The neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup, et al, 1996. supra; Perry-O'Keefe, et al, 1996. Proc. Natl. Acad. Sci. USA 93: 14670-14675. PNAs of NONX can be used in therapeutic and diagnostic applications. For example, PΝAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, e.g., inducing transcription or translation arrest or inhibiting replication. PΝAs of ΝONX can also be used, for example, in the analysis of single base pair mutations in a gene (e.g., PΝA directed PCR clamping; as artificial restriction enzymes when used in combination with other enzymes, e.g., Si nucleases (See, Hyrup, et al, \996.supra); or as probes or primers for DΝA sequence and hybridization (See, Hyrup, et al, 1996, supra; Perry-O'Keefe, et al, 1996. supra). hi another embodiment, PΝAs of ΝONX can be modified, e.g., to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to PΝA, by the formation of PΝA-DΝA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art. For example, PΝA-DΝA chimeras of ΝONX can be generated that may combine the advantageous properties of PΝA and DΝA. Such chimeras allow DΝA recognition enzymes (e.g., RΝase H and DΝA polymerases) to interact with the DΝA portion while the PΝA portion would provide high binding affinity and specificity. PΝA-DΝA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (see, Hyrup, et al., 1996. supra). The synthesis of PΝA-DΝA chimeras can be performed as described in Hyrup, et al, 1996. supra and Finn, et al, 1996. Nucl Acids Res 24: 3357-3363. For example, a DΝA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry, and modified nucleoside analogs, e.g. ,
5'-(4-methoxytrityl)amino-5'-deoxy-thymidine phosphoramidite, can be used between the PΝA and the 5' end of DΝA. See, e.g., Mag, et al, 1989. Nucl Acid Res 17: 5973-5988. PΝA monomers are then coupled in a stepwise manner to produce a chimeric molecule with a 5' PΝA segment and a 3' DΝA segment. See, e.g., Finn, et al, 1996. supra. Alternatively, chimeric molecules can be synthesized with a 5' DΝA segment and a 3' PΝA segment. See, e.g., Petersen, et al, 1975. Bioorg. Med. Chem. Lett. 5: 1119-11124.
In other embodiments, the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger, et al, 1989. Proc. Natl. Acad. Sci. U.S.A. 86: 6553-6556; Lemaifre, et al, 1987. Proc. Natl. Acad. Sci. 84: 648-652; PCT Publication No. WO88/09810) or the blood-brain barrier (see, e.g., PCT Publication No. WO 89/10134). hi addition, oligonucleotides can be modified with hybridization triggered cleavage agents (see, e.g., Krol, et al, 1988. BioTechniques 6:958-976) or intercalating agents (see, e.g., Zon, 1988. Pharm. Res. 5: 539-549). To this end, the oligonucleotide may be conjugated to another molecule, e.g., a peptide, a hybridization triggered cross-linking agent, a transport agent, a hybridization-triggered cleavage agent, and the like.
NOVX Polypeptides
A polypeptide according to the invention includes a polypeptide including the amino acid sequence of NOVX polypeptides whose sequences are provided in SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, and 123. The invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residues shown in SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107,
109, 111, 113, 115, 117, 119, 121, and 123 while still encoding a protein that maintains its NOVX activities and physiological functions, or a functional fragment thereof.
In general, an NOVX variant that preserves NOVX-like function includes any variant in which residues at a particular position in the sequence have been substituted by other amino acids, and further include the possibility of inserting an additional residue or residues between two residues of the parent protein as well as the possibility of deleting one or more residues from the parent sequence. Any amino acid substitution, insertion, or deletion is encompassed by the invention, hi favorable circumstances, the substitution is a conservative substitution as defined above. One aspect of the invention pertains to isolated NOVX proteins, and biologically-active portions thereof, or derivatives, fragments, analogs or homologs thereof. Also provided are polypeptide fragments suitable for use as immunogens to raise anti-NOVX antibodies. In one embodiment, native NOVX proteins can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques, hi another embodiment, NOVX proteins are produced by recombinant DNA techniques. Alternative to recombinant expression, an NOVX protein or polypeptide can be synthesized chemically using standard peptide synthesis techniques.
An "isolated" or "purified" polypeptide or protein or biologically-active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the NOVX protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. The language "substantially free of cellular material" includes preparations of NOVX proteins in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly-produced. hi one embodiment, the language "substantially free of cellular material" includes preparations of NOVX proteins having less than about 30% (by dry weight) of non-NOVX proteins (also referred to herein as a "contaminating protein"), more preferably less than about 20% of non-NOVX proteins, still more preferably less than about 10% of non-NOVX proteins, and most preferably less than about 5% of non-NOVX proteins. When the NOVX protein or biologically-active portion thereof is recombinantly-produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%>, more preferably less than about 10%, and most preferably less than about 5% of the volume of the NOVX protein preparation.
The language "substantially free of chemical precursors or other chemicals" includes preparations of NOVX proteins in which the protein is separated from chemical precursors or other chemicals that are involved in the synthesis of the protein. In one embodiment, the language "substantially free of chemical precursors or other chemicals" includes preparations of NOVX proteins having less than about 30% (by dry weight) of chemical precursors or non-NOVX chemicals, more preferably less than about 20%> chemical precursors or non-NOVX chemicals, still more preferably less than about 10% chemical precursors or non-NOVX chemicals, and most preferably less than about 5% chemical precursors or non-NOVX chemicals. Biologically-active portions of NOVX proteins include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequences of the NOVX proteins (e.g., the amino acid sequence shown in SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, and 123) that include fewer amino acids than the full-length NOVX proteins, and exhibit at least one activity of an NOVX protein. Typically, biologically-active portions comprise a domain or motif with at least one activity of the NOVX protein. A biologically- active portion of an NOVX protein can be a polypeptide which is, for example, 10, 25, 50, 100 or more amino acid residues in length.
Moreover, other biologically-active portions, in which other regions of the protein are deleted, can be prepared by recombinant techniques and evaluated for one or more of the functional activities of a native NOVX protein.
In an embodiment, the NOVX protein has an amino acid sequence shown SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, and 123. In other embodiments, the NOVX protein is substantially homologous to SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, and 123, and retains the functional activity of the protein of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, and 123, yet differs in amino acid sequence due to natural allelic variation or mutagenesis, as described in detail, below. Accordingly, in another embodiment, the NOVX protein is a protein that comprises an amino acid sequence at least about 45%> homologous to the amino acid sequence SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, and 123, and retains the functional activity of the NOVX proteins of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, and 123.
Determining Homology Between Two or More Sequences
To determine the percent homology of two amino acid sequences or of two nucleic acids, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are homologous at that position (i.e., as used herein amino acid or nucleic acid "homology" is equivalent to amino acid or nucleic acid "identity"). The nucleic acid sequence homology may be determined as the degree of identity between two sequences. The homology may be determined using computer programs known in the art, such as GAP software provided in the GCG program package. See, Needleman and Wunsch, 1970. JMol Biol 48: 443-453. Using GCG GAP software with the following settings for nucleic acid sequence comparison: GAP creation penalty of 5.0 and GAP extension penalty of 0.3, the coding region of the analogous nucleic acid sequences referred to above exhibits a degree of identity preferably of at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, with the CDS (encoding) part of the DNA sequence shown in SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, and 122. The term "sequence identity" refers to the degree to which two polynucleotide or polypeptide sequences are identical on a residue-by-residue basis over a particular region of comparison. The term "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over that region of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I, in the case of nucleic acids) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number ofpositions in the region of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. The term "substantial identity" as used herein denotes a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 80 percent sequence identity, preferably at least 85 percent identity and often 90 to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence over a comparison region. Chimeric and Fusion Proteins
The invention also provides NOVX chimeric or fusion proteins. As used herein, an NOVX "chimeric protein" or "fusion protein" comprises an NOVX polypeptide operatively- linked to a non-NOVX polypeptide. An "NOVX polypeptide" refers to a polypeptide having an amino acid sequence corresponding to an NOVX protein SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, and 123, whereas a "non-NOVX polypeptide" refers to a polypeptide having an amino acid sequence corresponding to a protein that is not substantially homologous to the
NOVX protein, e.g., a protein that is different from the NOVX protein and that is derived from the same or a different organism. Within an NOVX fusion protein the NOVX polypeptide can correspond to all or a portion of an NOVX protein. In one embodiment, an NOVX fusion protem comprises at least one biologically-active portion of an NOVX protein, hi another embodiment, an NOVX fusion protein comprises at least two biologically-active portions of an NOVX protein, hi yet another embodiment, an NOVX fusion protein comprises at least three biologically-active portions of an NOVX protein. Within the fusion protein, the term "operatively-linked" is intended to indicate that the NOVX polypeptide and the non-NOVX polypeptide are fused in-frame with one another. The non-NOVX polypeptide can be fused to the N-terminus or C-terminus of the NOVX polypeptide.
In one embodiment, the fusion protein is a GST-NO VX fusion protein in which the NOVX sequences are fused to the C-terminus of the GST (glutathione S-transferase) sequences. Such fusion proteins can facilitate the purification of recombinant NOVX polypeptides.
In another embodiment, the fusion protein is an NOVX protein containing a heterologous signal sequence at its N-terminus. In certain host cells (e.g., mammalian host cells), expression and/or secretion of NOVX can be increased through use of a heterologous signal sequence, hi yet another embodiment, the fusion protein is an NOVX-immunoglobulin fusion protein in which the NOVX sequences are fused to sequences derived from a member of the immunoglobulin protein family. The NOVX-immunoglobulin fusion proteins of the invention can be incorporated into pharmaceutical compositions and admimstered to a subject to inhibit an interaction between an NOVX ligand and an NOVX protein on the surface of a cell, to thereby suppress NOVX-mediated signal transduction in vivo. The NOVX-immunoglobulin fusion proteins can be used to affect the bioavailability of an NOVX cognate ligand. Inliibition of the NOVX ligand/NOVX interaction may be useful therapeutically for both the treatment of proliferative and differentiative disorders, as well as modulating (e.g. promoting or inhibiting) cell survival. Moreover, the NOVX-immunoglobulin fusion proteins of the invention can be used as immunogens to produce anti-NOVX antibodies in a subject, to purify NOVX ligands, and in screening assays to identify molecules that inhibit the interaction of NOVX with an NOVX ligand.
An NOVX chimeric or fusion protein of the invention can be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, e.g., by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
Alternatively, PCR amplification of gene fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, e.g., Ausubel, et al. (eds.) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide). An NOVX-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the NOVX protein.
NOVX Agonists and Antagonists The invention also pertains to variants of the NOVX proteins that function as either
NOVX agonists (i.e., mimetics) or as NOVX antagonists. Variants of the NOVX protein can be generated by mutagenesis (e.g., discrete point mutation or truncation of the NOVX protein). An agonist of the NOVX protein can retain substantially the same, or a subset of, the biological activities of the naturally occurring form of the NOVX protein. An antagonist of the NOVX protein can inhibit one or more of the activities of the naturally occurring form of the NOVX protein by, for example, competitively binding to a downstream or upstream member of a cellular signaling cascade which includes the NOVX protein. Thus, specific biological effects can be elicited by treatment with a variant of limited function, hi one embodiment, treatment of a subject with a variant having a subset of the biological activities of the naturally occurring form of the protein has fewer side effects in a subject relative to treatment with the naturally occurring form of the NOVX proteins.
Variants of the NOVX proteins that function as either NOVX agonists (i.e., mimetics) or as NOVX antagonists can be identified by screening combinatorial libraries of mutants (e.g., truncation mutants) of the NOVX proteins for NOVX protein agonist or antagonist activity. In one embodiment, a variegated library of NOVX variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library. A variegated library of NOVX variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential NOVX sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of NOVX sequences therein. There are a variety of methods which can be used to produce libraries of potential NOVX variants from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be performed in an automatic DNA synthesizer, and the synthetic gene then ligated into an appropriate expression vector. Use of a degenerate set of genes allows for the provision, in one mixture, of all of the sequences encoding the desired set of potential NOVX sequences. Methods for synthesizing degenerate oligonucleotides are well-known within the art. See, e.g., Narang, 1983. Tetrahedron 39: 3; Itakura, et al, 1984. Annu. Rev. Biochem. 53: 323; Itakura, et al, 1984. Science 198: 1056; Ike, et al, 1983. Nucl. Acids Res. 11: 477.
Polypeptide Libraries
In addition, libraries of fragments of the NONX protein coding sequences can be used to generate a variegated population of ΝONX fragments for screening and subsequent selection of variants of an ΝONX protein. In one embodiment, a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of an ΝONX coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double-stranded DNA that can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with Si nuclease, and ligating the resulting fragment library into an expression vector. By this method, expression libraries can be derived which encodes N-terminal and internal fragments of various sizes of the NONX proteins.
Various techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDΝA libraries for gene products having a selected property. Such techniques are adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of ΝOVX proteins. The most widely used techniques, which are amenable to high throughput analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates isolation of the vector encoding the gene whose product was detected. Recursive ensemble mutagenesis (REM), a new technique that enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify ΝOVX variants. See, e.g., Arkin and Yourvan, 1992. Proc. Natl. Acad. Sci. USA 89: 7811-7815; Delgrave, et al, 1993. Protein Engineering 6:327-331.
Anti-ΝOVX Antibodies
The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (lg) molecules, i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen. Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fab, Fab' and F(ab')2 fragments, and an Fab expression library. In general, antibody molecules obtained from humans relates to any of the classes IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature of the heavy chain present in the molecule. Certain classes have subclasses as well, such as IgGu IgG2, and others. Furthermore;, in humans, the light chain may be a kappa chain or a lambda chain. Reference herein to antibodies includes a reference to all such classes, subclasses and types of human antibody species. An isolated protein of the invention intended to serve as an antigen, or a portion or fragment thereof, can be used as an immunogen to generate antibodies that immunospecifically bind the antigen, using standard techniques for polyclonal and monoclonal antibody preparation. The full-length protein can be used or, alternatively, the invention provides antigenic peptide fragments of the antigen for use as immunogens. An antigenic peptide fragment comprises at least 6 amino acid residues of the amino acid sequence of the full length protein, such as an amino acid sequence shown in SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, and 123, and encompasses an epitope thereof such that an antibody raised against the peptide forms a specific immune complex with the full length protein or with any fragment that contains the epitope. Preferably, the antigenic peptide comprises at least 10 amino acid residues, or at least 15 amino acid residues, or at least 20 amino acid residues, or at least 30 amino acid residues. Preferred epitopes encompassed by the antigenic peptide are regions of the protein that are located on its surface; commonly these are hydrophilic regions.
In certain embodiments of the invention, at least one epitope encompassed by the antigenic peptide is a region of SECX that is located on the surface of the protein, e.g., a hydrophilic region. A hydrophobicity analysis of the human SECX protein sequence will indicate which regions of a SECX polypeptide are particularly hydrophilic and, therefore, are likely to encode surface residues useful for targeting antibody production. As a means for targeting antibody production, hydropathy plots showing regions of hydrophilicity and hydrophobicity may be generated by any method well known in the art, including, for example, the Kyte Doolittle or the Hopp Woods methods, either with or without Fourier transformation. See, e.g., Hopp and Woods, 1981, Proc. Nat. Acad. Sci. USA 78: 3824-3828; Kyte and Doolittle 1982, J. Mol. Biol. 157: 105-142, each incorporated herein by reference in their entirety. Antibodies that are specific for one or more domains within an antigenic protein, or derivatives, fragments, analogs or homologs thereof, are also provided herein.
A protein of the invention, or a derivative, fragment, analog, homolog or ortholog thereof, may be utilized as an immunogen in the generation of antibodies that immunospecifically bind these protein components. Various procedures known within the art may be used for the production of polyclonal or monoclonal antibodies directed against a protein of the invention, or against derivatives, fragments, analogs homologs or orthologs thereof (see, for example, Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, incorporated herein by reference). Some of these antibodies are discussed below.
1. Polyclonal Antibodies
For the production of polyclonal antibodies, various suitable host animals (e.g., rabbit, goat, mouse or other mammal) may be immunized by one or more injections with the native protein, a synthetic variant thereof, or a derivative of the foregoing. An appropriate immunogenic preparation can contain, for example, the naturally occurring immunogenic protein, a chemically synthesized polypeptide representing the immunogenic protein, or a recombinantly expressed immunogenic protein. Furthermore, the protein may be conjugated to a second protein known to be immunogenic in the mammal being immunized. Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. The preparation can further include an adjuvant. Various adjuvants used to increase the immunological response include, but are not limited to, Freund's (complete and incomplete), mineral gels (e.g., aluminum hydroxide), surface active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, etc.), adjuvants usable in humans such as Bacille Calmette-Guerin and Corynebacterium parvum, or similar immunostimulatory agents. Additional examples of adjuvants which can be employed include MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).
The polyclonal antibody molecules directed against the immunogenic protein can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as affinity chromatography using protein A or protein G, which provide primarily the IgG fraction of immune serum. Subsequently, or alternatively, the specific antigen which is the target of the immunoglobulin sought, or an epitope thereof, may be immobilized on a column to purify the immune specific antibody by immunoaffmity chromatography. Purification of immunoglobulins is discussed, for example, by D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia PA, Vol. 14, No. 8 (April 17, 2000), pp. 25-28). 2. Monoclonal Antibodies
The term "monoclonal antibody" (MAb) or "monoclonal antibody composition", as used herein, refers to a population of antibody molecules that contain only one molecular species of antibody molecule consisting of a unique light chain gene product and a unique heavy chain gene product. In particular, the complementarity determining regions (CDRs) of the monoclonal antibody are identical in all the molecules of the population. MAbs thus contain an antigen binding site capable of immunoreacting with a particular epitope of the antigen characterized by a unique binding affinity for it.
Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes can be immunized in vitro.
The immunizing agent will typically include the protein antigen, a fragment thereof or a fusion protein thereof. Generally, either peripheral blood lymphocytes are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell [Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103]. Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells can be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl tiansferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances prevent the growth of HGPRT-deficient cells.
Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego,
California and the American Type Culture Collection, Manassas, Virginia. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies [Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al, Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63]. The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980). It is an objective, especially important in therapeutic applications of monoclonal antibodies, to identify antibodies having a high degree of specificity and a high binding affinity for the target antigen.
After the desired hybridoma cells are identified, the clones can be subcloned by limiting dilution procedures and grown by standard methods (Goding, 1986). Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells can be grown in vivo as ascites in a mammal.
The monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
The monoclonal antibodies can also be made by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567. DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells of the invention serve as a preferred source of such DNA. Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The DNA also can be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences (U.S. Patent No. 4,816,567; Morrison, Nature 368, 812-13 (1994)) or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combimng site of an antibody of the invention to create a chimeric bivalent antibody.
3. Humanized Antibodies
The antibodies directed against the protein antigens of the invention can further comprise humanized antibodies or human antibodies. These antibodies are suitable for administration to humans without engendering an immune response by the human against the administered immunoglobulin. Humanized forms of antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen- binding subsequences of antibodies) that are principally comprised of the sequence of a human immunoglobulin, and contain minimal sequence derived from a non-human immunoglobulin. Humanization can be performed following the method of Winter and co-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science. 239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. (See also U.S. Patent No. 5,225,539.) In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies can also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., 1986; Riechmann et al., 1988; and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)).
Human Antibodies Fully human antibodies essentially relate to antibody molecules in which the entire sequence of both the light chain and the heavy chain, including the CDRs, arise from human genes. Such antibodies are termed "human antibodies", or "fully human antibodies" herein. Human monoclonal antibodies can be prepared by the trioma technique; the human B-cell hybridoma technique (see Kozbor, et al., 1983 Immunol Today 4: 72) and the EBV hybridoma technique to produce human monoclonal antibodies (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). Human monoclonal antibodies may be utilized in the practice of the present invention and may be produced by using human hybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA 80: 2026-2030) or by transforming human B-cells with Epstein Barr Virus in vitro (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).
In addition, human antibodies can also be produced using additional techniques, including phage display libraries (Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., L Mol. Biol., 222:581 (1991)). Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in Marks et al. (Bio/Technology 10, 779-783 (1992)); Lonberg et al. (Nature 368 856-859 (1994)); Morrison ( Nature 368, 812-13 (1994)); Fishwild et al,( Nature Biotechnology 14, 845- 51 (1996)); Neuberger (Nature Biotechnology 14, 826 (1996)); and Lonberg and Huszar (Intern. Rev. Immunol. 13 65-93 (1995)).
Human antibodies may additionally be produced using transgenic nonhuman animals which are modified so as to produce fully human antibodies rather than the animal's endogenous antibodies in response to challenge by an antigen. (See PCT publication WO94/02602). The endogenous genes encoding the heavy and light immunoglobulin chains in the nonhuman host have been incapacitated, and active loci encoding human heavy and light chain immunoglobulins are inserted into the host's genome. The human genes are incorporated, for example, using yeast artificial chromosomes containing the requisite human DNA segments. An animal which provides all the desired modifications is then obtained as progeny by crossbreeding intermediate transgenic animals containing fewer than the full complement of the modifications. The preferred embodiment of such a nonhuman animal is a mouse, and is termed the Xenomouse™ as disclosed in PCT publications WO 96/33735 and WO 96/34096. This animal produces B cells which secrete fully human immunoglobulins. The antibodies can be obtained directly from the animal after immunization with an immunogen of interest, as, for example, a preparation of a polyclonal antibody, or alternatively from immortalized B cells derived from the animal, such as hybridomas producing monoclonal antibodies. Additionally, the genes encoding the immunoglobulins with human variable regions can be recovered and expressed to obtain the antibodies directly, or can be further modified to obtain analogs of antibodies such as, for example, single chain Fv molecules. An example of a method of producing a nonhuman host, exemplified as a mouse, lacking expression of an endogenous immunoglobulin heavy chain is disclosed in U.S. Patent No. 5,939,598. It can be obtained by a method including deleting the J segment genes from at least one endogenous heavy chain locus in an embryonic stem cell to prevent rearrangement of the locus and to prevent formation of a transcript of a rearranged immunoglobulin heavy chain locus, the deletion being effected by a targeting vector containing a gene encoding a selectable marker; and producing from the embryonic stem cell a transgenic mouse whose somatic and germ cells contain the gene encoding the selectable marker.
A method for producing an antibody of interest, such as a human antibody, is disclosed in U.S. Patent No. 5,916,771. It includes introducing an expression vector that contains a nucleotide sequence encoding a heavy chain into one mammalian host cell in culture, introducing an expression vector containing a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell. The hybrid cell expresses an antibody containing the heavy chain and the light chain.
In a further improvement on this procedure, a method for identifying a clinically relevant epitope on an immunogen, and a conelative method for selecting an antibody that binds immunospecifically to the relevant epitope with high affinity, are disclosed in PCT publication WO 99/53049.
5. Fgh Fragments and Single Chain Antibodies According to the invention, techniques can be adapted for the production of single-chain antibodies specific to an antigenic protein of the invention (see e.g., U.S. Patent No. 4,946,778). In addition, methods can be adapted for the construction of Fa expression libraries (see e.g., Huse, et al., 1989 Science 246: 1275-1281) to allow rapid and effective identification of monoclonal Fab fragments with the desired specificity for a protein or derivatives, fragments, analogs or homologs thereof. Antibody fragments that contain the idiotypes to a protein antigen may be produced by techniques known in the art including, but not limited to: (i) an F(a ')2 fragment produced by pepsin digestion of an antibody molecule; (ii) an Fab fragment generated by reducing the disulfide bridges of an F(ab-)2 fragment; (iii) an Fab fragment generated by the treatment of the antibody molecule with papain and a reducing agent and (iv) Fv fragments.
6. Bispecific Antibodies
Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens. In the present case, one of the binding specificities is for an antigenic protein of the invention. The second binding target is any other antigen, and advantageously is a cell-surface protein or receptor or receptor subunit.
Methods for making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305:537-539 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture often different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule is usually accomplished by affinity chromatography steps. Similar procedures are disclosed in WO 93/08829, published 13 May 1993, and in Traunecker et al., EMBO J., 10:3655-3659 (1991).
Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CHI) containing the site necessary for light-chain binding present in at least one of the fusions.
DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism. For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology. 121:210 (1986).
According to another approach described in WO 96/27011, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture. The preferred interface comprises at least a part of the CH3 region of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g. tyrosine or tryptophan). Compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.
Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g. F(ab')2 bispecific antibodies). Techniques for generating bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science 229:81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab')2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes. Additionally, Fab' fragments can be directly recovered from E. coli and chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med. 175:217-225 (1992) describe the production of a fully humanized bispecific antibody F(ab')2 molecule. Each Fab' fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody. The bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets. Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol. 148(5):1547-1553 (1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers. The "diabody" technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See, Gruber et al., J. Immunol. 152:5368 (1994).
Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60 (1991).
Exemplary bispecific antibodies can bind to two different epitopes, at least one of which originates in the protein antigen of the invention. Alternatively, an anti-antigenic arm of an immunoglobulin molecule can be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g. CD2, CD3, CD28, or B7), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD 16) so as to focus cellular defense mechanisms to the cell expressing the particular antigen. Bispecific antibodies can also be used to direct cytotoxic agents to cells which express a particular antigen. These antibodies possess an antigen-binding arm and an arm which binds a cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOT A, or TETA. Another bispecific antibody of interest binds the protein antigen described herein and further binds tissue factor (TF).
7. Heteroconjugate Antibodies Heteroconjugate antibodies are also within the scope of the present invention.
Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Patent No. 4,676,980), and for treatment of HIV infection (WO 91/00360; WO 92/200373; EP 03089). It is contemplated that the antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S. Patent No. 4,676,980.
8. Effector Function Engineering It can be desirable to modify the antibody of the invention with respect to effector function, so as to enhance, e.g., the effectiveness of the antibody in treating cancer. For example, cysteine residue(s) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated can have improved internalization capability and/or increased complement-mediated cell killing and antibody- dependent cellular cytotoxicity (ADCC). See Caron et al., J. Exp Med., 176: 1191-1195 (1992) and Shopes, J. Immunol., 148: 2918-2922 (1992). Homodimeric antibodies with enhanced anti- tumor activity can also be prepared using heterobifunctional cross-linkers as described in Wolff et al. Cancer Research, 53: 2560-2565 (1993). Alternatively, an antibody can be engineered that has dual Fc regions and can thereby have enhanced complement lysis and ADCC capabilities. See Stevenson et al., Anti-Cancer Drug Design. 3: 219-230 (1989).
9. Immunoconiugates
The invention also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).
Chemotherapeutic agents useful in the generation of such iinmunoconjugates have been described above. Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. A variety of radionuclides are available for the production of radioconjugated antibodies. Examples include 212Bi, 131L 131In, 90Y, and 186Re. Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis- (p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as l,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science, 238: 1098 (1987). Carbon- 14-labeled l-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See WO94/11026.
In another embodiment, the antibody can be conjugated to a "receptor" (such streptavidin) for utilization in tumor pretargeting wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then admimstration of a "ligand" (e.g., avidin) that is in turn conjugated to a cytotoxic agent.
10. Immunoliposomes
The antibodies disclosed herein can also be formulated as immunoliposomes. Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl Acad. Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Patent No. 5,013,556.
Particularly useful liposomes can be generated by the reverse-phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter. Fab' fragments of the antibody of the present invention can be conjugated to the liposomes as described in Martin et al ., J. Biol. Chem., 257: 286-288 (1982) via a disulfide-interchange reaction. A chemotherapeutic agent (such as Doxorubicin) is optionally contained within the liposome. See Gabizon et al., J. National Cancer hist.. 81(19): 1484 (1989).
11. Diagnostic Applications of Antibodies Directed Against the Proteins of the Invention
Antibodies directed against a protein of the invention may be used in methods known within the art relating to the localization and/or quantitation of the protein (e.g., for use in measuring levels of the protein within appropriate physiological samples, for use in diagnostic methods, for use in imaging the protein, and the like). In a given embodiment, antibodies against the proteins, or derivatives, fragments, analogs or homologs thereof, that contain the antigen binding domain, are utilized as pharmacologically-active compounds (see below).
An antibody specific for a protein of the invention can be used to isolate the protein by standard techniques, such aaimmunoaffinity chromatography or immunoprecipitation. Such an antibody can facilitate the purification of the natural protein antigen from cells and of recombinantly produced antigen expressed in host cells. Moreover, such an antibody can be used to detect the antigenic protein (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the antigenic protein. Antibodies directed against the protein can be used diagnostically to momtor protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelhferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 1251, 1311, 35S or 3H.
12. Antibody Therapeutics Antibodies of the invention, including polyclonal, monoclonal, humanized and fully human antibodies, may used as therapeutic agents. Such agents will generally be employed to treat or prevent a disease or pathology in a subject. An antibody preparation, preferably one having high specificity and high affinity for its target antigen, is administered to the subject and will generally have an effect due to its binding with the target. Such an effect may be one of two kinds, depending on the specific nature of the interaction between the given antibody molecule and the target antigen in question. In the first instance, administration of the antibody may abrogate or inhibit the binding of the target with an endogenous ligand to which it naturally binds. In this case, the antibody binds to the target and masks a binding site of the naturally occurring ligand, wherein the ligand serves as an effector molecule. Thus the receptor mediates a signal transduction pathway for which ligand is responsible.
Alternatively, the effect may be one in which the antibody elicits a physiological result by virtue of binding to an effector binding site on the target molecule. In this case the target, a receptor having an endogenous ligand which may be absent or defective in the disease or pathology, binds the antibody as a surrogate effector ligand, initiating a receptor-based signal transduction event by the receptor.
A therapeutically effective amount of an antibody of the invention relates generally to the amount needed to achieve a therapeutic objective. As noted above, this may be a binding interaction between the antibody and its target antigen that, in certain cases, interferes with the functioning of the target, and in other cases, promotes a physiological response. The amount required to be administered will furthermore depend on the binding affinity of the antibody for its specific antigen, and will also depend on the rate at which an administered antibody is depleted from the free volume other subject to which it is administered. Common ranges for therapeutically effective dosing of an antibody or antibody fragment of the invention may be, by way of nonlimiting example, from about 0.1 mg/kg body weight to about 50 mg/kg body weight. Common dosing frequencies may range, for example, from twice daily to once a week.
13. Pharmaceutical Compositions of Antibodies Antibodies specifically binding a protein of the invention, as well as other molecules identified by the screening assays disclosed herein, can be administered for the treatment of various disorders in the form of pharmaceutical compositions. Principles and considerations involved in preparing such compositions, as well as guidance in the choice of components are provided, for example, in Remington : The Science And Practice Of Pharmacy 19th ed. (Alfonso R. Gennaro, et al., editors) Mack Pub. Co., Easton, Pa. : 1995; Drug Absorption Enhancement : Concepts, Possibilities, Limitations, And Trends, Harwood Academic Publishers, Langhorne, Pa., 1994; and Peptide And Protein Drug Delivery (Advances In Parenteral Sciences, Vol. 4), 1991, M. Dekker, New York.
If the antigenic protein is intracellular and whole antibodies are used as inhibitors, internalizing antibodies are preferred. However, liposomes can also be used to deliver the antibody, or an antibody fragment, into cells. Where antibody fragments are used, the smallest inhibitory fragment that specifically binds to the binding domain of the target protein is preferred. For example, based upon the variable-region sequences of an antibody, peptide molecules can be designed that retain the ability to bind the target protein sequence. Such peptides can be synthesized chemically and/or produced by recombinant DNA technology. See, e.g., Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993). The formulation herein can also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Alternatively, or in addition, the composition can comprise an agent that enhances its function, such as, for example, a cytotoxic agent, cytokine, chemotherapeutic agent, or growth-inhibitory agent. Such molecules are suitably present in combination in amounts that are effective for the purpose intended.
The active ingredients can also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacrylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano- particles, and nanocapsules) or in macroemulsions.
The formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.
Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2- hydroxyethyl-methacrylate), or polyfvinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γ ethyl-L-glutamate, non-degradable ethylene- vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT ™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly- D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid- glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods.
ELISA Assay
An agent for detecting an analyte protein is an antibody capable of binding to an analyte protein, preferably an antibody with a detectable label. Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab)2) can be used. The term "labeled", with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently-labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently-labeled streptavidin. The term "biological sample" is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. Included within the usage of the term "biological sample", therefore, is blood and a fraction or component of blood including blood serum, blood plasma, or lymph. That is, the detection method of the invention can be used to detect an analyte mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo. For example, in vitro techniques for detection of an analyte mRNA include Northern hybridizations and in situ hybridizations. In vitro techniques for detection of an analyte protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations, and immunofluorescence. In vitro techniques for detection of an analyte genomic DNA include Southern hybridizations. Procedures for conducting immunoassays are described, for example in "ELISA: Theory and Practice: Methods in Molecular Biology", Vol. 42, J. R. Crowther (Ed.) Human Press, Totowa, NJ, 1995; "Immunoassay", E. Diamandis and T. Christopoulus, Academic Press, Inc., San Diego, CA, 1996; and "Practice and Thory of Enzyme Immunoassays", P. Tijssen, Elsevier Science Publishers, Amsterdam, 1985. Furthermore, in vivo techniques for detection of an analyte protein include introducing into a subject a labeled anti-an analyte protein antibody. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.
NOVX Recombinant Expression Vectors and Host Cells
Another aspect of the invention pertains to vectors, preferably expression vectors, containing a nucleic acid encoding an NOVX protein, or derivatives, fragments, analogs or homologs thereof. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g. , bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively-linked. Such vectors are referred to herein as "expression vectors". In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. h the present specification, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
The recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, "operably-lmked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
The term "regulatory sequence" is intended to includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., NOVX proteins, mutant forms of NOVX proteins, fusion proteins, etc.). The recombinant expression vectors of the invention can be designed for expression of
NOVX proteins in prokaryotic or eukaryotic cells. For example, NOVX proteins can be expressed in bacterial cells such as Escherichia coli, insect cells (using baculovirus expression vectors) yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS ΓN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
Expression of proteins in prokaryotes is most often carried out in Escherichia coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: (i) to increase expression of recombinant protein; (ii) to increase the solubility of the recombinant protein; and (iii) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988. Gene 67: 31-40), pMAL (New England Biolabs, Beverly, Mass.) and ρRIT5 (Pharmacia, Piscataway, N. J.) that fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein. Examples of suitable inducible non-fusion E. coli expression vectors include pTrc
(Arnrann et al, (1988) Gene 69:301-315) and pET lid (Studier et al, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89).
One strategy to maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein. See, e.g., Gottesman, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 119-128. Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (see, e.g., Wada, et al, 1992. Nucl Acids Res. 20: 2111-2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.
In another embodiment, the NOVX expression vector is a yeast expression vector. Examples of vectors for expression in yeast Saccharomyces cerivisae include pYepSecl (Baldari, et al, 1987. EMBOJ. 6: 229-234), pMFa (Kurjan and Herskowitz, 1982. Cell 30: 933-943), pJRY88 (Schultz et al, 1987. Gene 54: 113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ (InNitrogen Corp, San Diego, Calif).
Alternatively, ΝONX can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., SF9 cells) include the pAc series (Smith, et al, 1983. Mol. Cell. Biol. 3: 2156-2165) and the pNL series (Lucklow and Summers, 1989. Virology 170: 31-39). In yet another embodiment, a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, 1987. Nature 329: 840) and pMT2PC (Kaufman, et al, 1987. EMBO J. 6: 187-195). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, and simian virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al, MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.
In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert, et al, 1987. Genes Dev. 1: 268-277), lymphoid-specific promoters (Calame and Eaton, 1988. Adv. Immunol. 43: 235-275), in particular promoters of T cell receptors (Winoto and Baltimore, 1989. EMBO J. 8: 729-733) and immunoglobulins (Banerji, et al, 1983. Cell 33: 729-740; Queen and Baltimore, 1983. Cell 33: 741-748), neuron-specific promoters (e.g., the neuro filament promoter; Byrne and Ruddle, 1989. Proc. Natl. Acad. Sci. USA 86: 5473-5477), pancreas-specific promoters (Edlund, et al, 1985. Science 230: 912-916), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166). Developmentally-regulated promoters are also encompassed, e.g., the murine hox promoters (Kessel and Grass, 1990. Science 249: 374-379) and the D -fetoprotein promoter (Campes and Tilghman, 1989. Genes Dev. 3: 537-546).
The invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively-linked to a regulatory sequence in a manner that allows for expression (by transcription of the DNA molecule) of an RNA molecule that is antisense to
NONX mRΝA. Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen that direct the continuous expression of the antisense RΝA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen that direct constitutive, tissue specific or cell type specific expression of antisense RΝA. The antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced. For a discussion of the regulation of gene expression using antisense genes .see, e.g., Weintraub, et al, "Antisense RΝA as a molecular tool for genetic analysis," Reviews-Trends in Genetics, Vol. 1(1) 1986. Another aspect of the invention pertains to host cells into which a recombinant expression vector of the invention has been introduced. The terms "host cell" and "recombinant host cell" are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
A host cell can be any prokaryotic or eukaryotic cell. For example, NOVX protein can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art.
Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), and other laboratory manuals. For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Various selectable markers include those that confer resistance to drugs, such as G418, hygromycin and methotrexate. Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding NOVX or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die). A host cell of the invention, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) NOVX protein. Accordingly, the invention further provides , methods for producing NOVX protein using the host cells of the invention, hi one embodiment, the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding NOVX protein has been introduced) in a suitable medium such that NOVX protein is produced. In another embodiment, the method further comprises isolating NOVX protein from the medium or the host cell.
Transgenic NOVX Animals
The host cells of the invention can also be used to produce non-human transgenic animals. For example, in one embodiment, a host cell of the invention is a fertilized oocyte or an embryonic stem cell into which NOVX protein-coding sequences have been introduced. Such host cells can then be used to create non-human transgenic animals in which exogenous NOVX sequences have been introduced into their genome or homologous recombinant animals in which endogenous NONX sequences have been altered. Such animals are useful for studying the function and/or activity of ΝONX protein and for identifying and/or evaluating modulators of ΝONX protein activity. As used herein, a "transgenic animal" is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more of the cells of the animal includes a transgene. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, etc. A transgene is exogenous DΝA that is integrated into the genome of a cell from which a transgenic animal develops and that remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal. As used herein, a
"homologous recombinant animal" is a non-human animal, preferably a mammal, more preferably a mouse, in which an endogenous ΝONX gene has been altered by homologous recombination between the endogenous gene and an exogenous DΝA molecule introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal. A transgenic animal of the invention can be created by introducing ΝONX-encoding nucleic acid into the male pronuclei of a fertilized oocyte (e.g., by microinjection, retroviral infection) and allowing the oocyte to develop in a pseudopregnant female foster animal. The human ΝONX cDΝA sequences SEQ ID ΝOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, and 122 can be introduced as a transgene into the genome of a non-human animal. Alternatively, a non- human homologue of the human NONX gene, such as a mouse ΝONX gene, can be isolated based on hybridization to the human ΝONX cDΝA (described further supra) and used as a transgene. Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene. A tissue-specific regulatory sequence(s) can be operably-linked to the ΝONX transgene to direct expression of ΝONX protein to particular cells. Methods for generating transgenic animals via embryo manipulation and microinjection, particularly animals such as mice, have become conventional in the art and are described, for example, in U.S. Patent Νos. 4,736,866; 4,870,009; and 4,873,191; and Hogan, 1986. In: MANIPULATING THE MOUSE EMBRYO, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Similar methods are used for production of other transgenic animals. A transgenic founder animal can be identified based upon the presence of the NOVX transgene in its genome and/or expression of NOVX mRNA in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene-encoding NOVX protein can further be bred to other transgenic animals carrying other transgenes.
To create a homologous recombinant animal, a vector is prepared which contains at least a portion of an NOVX gene into which a deletion, addition or substitution has been introduced to thereby alter, e.g., functionally disrupt, the NONX gene. The ΝONX gene can be a human gene (e.g., the cDΝA of SEQ ID ΝOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, and 122), but more preferably, is a non-human homologue of a human NOVX gene. For example, a mouse homologue of human NOVX gene of SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, and 122 can be used to construct a homologous recombination vector suitable for altering an endogenous NOVX gene in the mouse genome. In one embodiment, the vector is designed such that, upon homologous recombination, the endogenous NOVX gene is functionally disrupted (i.e., no longer encodes a functional protein; also referred to as a "knock out" vector). Alternatively, the vector can be designed such that, upon homologous recombination, the endogenous NONX gene is mutated or otherwise altered but still encodes functional protein (e.g., the upstream regulatory region can be altered to thereby alter the expression of the endogenous NONX protein), h the homologous recombination vector, the altered portion of the ΝONX gene is flanked at its 5'- and 3 '-termini by additional nucleic acid of the ΝONX gene to allow for homologous recombination to occur between the exogenous ΝONX gene carried by the vector and an endogenous ΝONX gene in an embryonic stem cell. The additional flanking ΝONX nucleic acid is of sufficient length for successful homologous recombination with the endogenous gene. Typically, several kilobases of flanking DΝA (both at the 5'- and 3'-termini) are included in the vector. See, e.g., Thomas, et al, 1987. Cell 51: 503 for a description of homologous recombination vectors. The vector is ten introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced ΝONX gene has homologously-recombined with the endogenous ΝONX gene are selected. See, e.g., Li, et al., 1992. Cell 69: 915.
The selected cells are then injected into a blastocyst of an animal (e.g., a mouse) to form aggregation chimeras. See, e.g., Bradley, 1987. In: TERATOCARCΓΝOMAS AND EMBRYONIC STEM CELLS: A PRACTICAL APPROACH, Robertson, ed. URL, Oxford, pp. 113-152. A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term. Progeny harboring the homologously-recombined DNA in their germ cells can be used to breed animals in which all cells of the animal contain the homologously-recombined DNA by germline transmission of the transgene. Methods for constructing homologous recombination vectors and homologous recombinant animals are described further in Bradley, 1991. Curr. Opin. Biotechnol. 2: 823-829; PCT International Publication Nos.: WO 90/11354; WO 91/01140; WO 92/0968; and WO 93/04169.
In another embodiment, transgenic non-humans animals can be produced that contain selected systems that allow for regulated expression of the transgene. One example of such a system is the cre/loxP recombinase system of bacteriophage PI. For a description of the cre/loxP recombinase system, See, e.g., Lakso, et al, 1992. Proc. Natl. Acad. Sci. USA 89: 6232-6236. Another example of a recombinase system is the FLP recombinase system of Saccharomyces cerevisiae. See, O'Gorman, et al, 1991. Science 251:1351-1355. If a cre/loxP recombinase system is used to regulate expression of the transgene, animals containing transgenes encoding both the Cre recombinase and a selected protein are required. Such animals can be provided through the construction of "double" transgenic animals, e.g. , by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.
Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut, et al, 1997. Nature 385: 810-813. hi brief, a cell (e.g., a somatic cell) from the transgenic animal can be isolated and induced to exit the growth cycle and enter G0 phase. The quiescent cell can then be fused, e.g. , through the use of electrical pulses, to an enucleated oocyte from an animal of the same species from which the quiescent cell is isolated. The reconstructed oocyte is then cultured such that it develops to morula or blastocyte and then transfened to pseudopregnant female foster animal. The offspring borne of this female foster animal will be a clone of the animal from which the cell (e.g., the somatic cell) is isolated.
Pharmaceutical Compositions
The NONX nucleic acid molecules, ΝONX proteins, and anti-ΝONX antibodies (also refened to herein as "active compounds") of the invention, and derivatives, fragments, analogs and homologs thereof, can be incorporated into pharmaceutical compositions suitable for admimstration. Such compositions typically comprise the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference. Prefened examples of such carriers or diluents include, but are not limited to, water, saline, finger's solutions, dextrose solution, and 5%> human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incoφorated into the compositions.
A pharmaceutical composition of the invention is formulated to be compatible with its intended route of admimstration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermai (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N. J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absoφtion, for example, aluminum monostearate and gelatin.
Sterile injectable solutions can be prepared by incoφorating the active compound (e.g., an NOVX protein or anti-NOVX antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incoφorating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the puφose of oral therapeutic administration, the active compound can be incoφorated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
Systemic administration can also be by transmucosal or transdermai means. For transmucosal or transdermai admimstration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermai administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Coφoration and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.
, It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of admimstration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
The nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see, e.g., U.S. Patent No. 5,328,470) or by stereotactic injection (see, e.g., Chen, et al, 1994. Proc. Natl. Acad. Sci. USA 91: 3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells that produce the gene delivery system.
The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration. Screening and Detection Methods
The isolated nucleic acid molecules of the invention can be used to express NOVX protein (e.g., via a recombinant expression vector in a host cell in gene therapy applications), to detect NOVX mRNA (e.g. , in a biological sample) or a genetic lesion in an NOVX gene, and to modulate NOVX activity, as described further, below, hi addition, the NOVX proteins can be used to screen drags or compounds that modulate the NOVX protein activity or expression as well as to treat disorders characterized by insufficient or excessive production of NOVX protein or production of NOVX protein forms that have decreased or abenant activity compared to NOVX wild-type protein (e.g.; diabetes (regulates insulin release); obesity (binds and transport lipids); metabolic disturbances associated with obesity, the metabolic syndrome X as well as anorexia and wasting disorders associated with chronic diseases and various cancers, and infectious disease(possesses anti-microbial activity) and the various dyslipidemias. In addition, the anti-NOVX antibodies of the invention can be used to detect and isolate NOVX proteins and modulate NOVX activity, hi yet a further aspect, the invention can be used in methods to influence appetite, absoφtion of nutrients and the disposition of metabolic substrates in both a positive and negative fashion.
The invention further pertains to novel agents identified by the screening assays described herein and uses thereof for freatments as described, supra. Screening Assays
The invention provides a method (also refened to herein as a "screening assay") for identifying modulators, i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) that bind to NOVX proteins or have a stimulatory or inhibitory effect on, e.g., NOVX protein expression or NOVX protein activity. The invention also includes compounds identified in the screening assays described herein. In one embodiment, the invention provides assays for screening candidate or test compounds which bind to or modulate the activity of the membrane-bound form of an NOVX protein or polypeptide or biologically-active portion thereof. The test compounds of the invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the "one-bead one-compound" library method; and synthetic library methods using affinity chromatography selection. The biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds. See, e.g., Lam, 1997 '. Anticancer Drug Design 12: 145.
A "small molecule" as used herein, is meant to refer to a composition that has a molecular weight of less than about 5 kD and most preferably less than about 4 kD. Small molecules can be, e.g., nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids or other organic or inorganic molecules. Libraries of chemical and/or biological mixtures, such as fungal, bacterial, or algal extracts, are known in the art and can be screened with any of the assays of the invention.
Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt, et al, 1993. Proc. Natl. Acad. Sci. U.S.A. 90: 6909; Erb, et al, 1994. Proc. Natl. Acad. Sci. U.S.A. 91: 11422; Zuckermann, et al, 1994. J. Med. Chem. 37: 2678; Cho, et al, 1993. Science 261: 1303; Canell, et al, 1994. Angew. Chem. Int. Ed. Engl. 33: 2059; Carell, et al, 1994. Angew. Chem. Int. Ed. Engl. 33: 2061; and Gallop, et al, 1994. J. Med. Chem. 37: 1233.
Libraries of compounds may be presented in solution (e.g., Houghten, 1992. Biotechniques 13: 412-421), or on beads (Lam, 1991. Nature 354: 82-84), on chips (Fodor, 1993. Nature 364: 555-556), bacteria (Ladner, U.S. Patent No. 5,223,409), spores (Ladner, U.S. Patent 5,233,409), plasmids (Cull, et al, 1992. Proc. Natl. Acad. Sci. USA 89: 1865-1869) or on phage (Scott and Smith, 1990. Science 249: 386-390; Devlin, 1990. Science 249: 404-406; Cwirla, et al, 1990. Proc. Natl. Acad. Sci. U.S.A. 87: 6378-6382; Felici, 1991. J. Mol. Biol. 222: 301-310; Ladner, U.S. Patent No. 5,233,409.). hi one embodiment, an assay is a cell-based assay in which a cell which expresses a membrane-bound form of NONX protein, or a biologically-active portion thereof, on the cell surface is contacted with a test compound and the ability of the test compound to bind to an ΝONX protein determined. The cell, for example, can of mammalian origin or a yeast cell. Determining the ability of the test compound to bind to the ΝONX protein can be accomplished, for example, by coupling the test compound with a radioisotope or enzymatic label such that binding of the test compound to the ΝONX protein or biologically-active portion thereof can be determined by detecting the labeled compound in a complex. For example, test compounds can be labeled with 1251, 35S, 14C, or 3H, either directly or indirectly, and the radioisotope detected by direct counting of radioemission or by scintillation counting. Alternatively, test compounds can be enzymatically-labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product. In one embodiment, the assay comprises contacting a cell which expresses a membrane-bound form of NONX protein, or a biologically-active portion thereof, on the cell surface with a known compound wliich binds ΝONX to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with an ΝONX protein, wherein determining the ability of the test compound to interact with an ΝONX protein comprises determining the ability of the test compound to preferentially bind to ΝONX protein or a biologically-active portion thereof as compared to the known compound.
In another embodiment, an assay is a cell-based assay comprising contacting a cell expressing a membrane-bound form of ΝONX protein, or a biologically-active portion thereof, on the cell surface with a test compound and determining the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the ΝONX protein or biologically- active portion thereof. Determimng the ability of the test compound to modulate the activity of ΝONX or a biologically-active portion thereof can be accomplished, for example, by determining the ability of the ΝOVX protein to bind to or interact with an ΝOVX target molecule. As used herein, a "target molecule" is a molecule with which an ΝOVX protein binds or interacts in nature, for example, a molecule on the surface of a cell which expresses an ΝOVX interacting protein, a molecule on the surface of a second cell, a molecule in the extracellular milieu, a molecule associated with the internal surface of a cell membrane or a cytoplasmic molecule. An ΝOVX target molecule can be a non-ΝOVX molecule or an ΝOVX protein or polypeptide of the invention, hi one embodiment, an ΝOVX target molecule is a component of a signal fransduction pathway that facilitates transduction of an extracellular signal (e.g. a signal generated by binding of a compound to a membrane-bound ΝOVX molecule) through the cell membrane and into the cell. The target, for example, can be a second intercellular protein that has catalytic activity or a protein that facilitates the association of downstream signaling molecules with ΝOVX. Determining the ability of the ΝOVX protein to bind to or interact with an ΝOVX target molecule can be accomplished by one of the methods described above for determining direct binding. In one embodiment, determining the ability of the NOVX protein to bind to or interact with an NOVX target molecule can be accomplished by determining the activity of the target molecule. For example, the activity of the target molecule can be determined by detecting induction of a cellular second messenger of the target (i.e. intracellular Ca2+, diacylglycerol, IP3, etc.), detecting catalytic/enzymatic activity of the target an appropriate substrate, detecting the induction of a reporter gene (comprising an NOVX-responsive regulatory element operatively linked to a nucleic acid encoding a detectable marker, e.g., luciferase), or detecting a cellular response, for example, cell survival, cellular differentiation, or cell proliferation.
In yet another embodiment, an assay of the invention is a cell-free assay comprising contacting an NOVX protein or biologically-active portion thereof with a test compound and determining the ability of the test compound to bind to the NOVX protein or biologically-active portion thereof. Binding of the test compound to the NOVX protein can be determined either directly or indirectly as described above. In one such embodiment, the assay comprises contacting the NOVX protein or biologically-active portion thereof with a known compound which binds NOVX to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with an NOVX protein, wherein determimng the ability of the test compound to interact with an NOVX protein comprises determimng the ability of the test compound to preferentially bind to NOVX or biologically- active portion thereof as compared to the known compound. In still another embodiment, an assay is a cell-free assay comprising contacting NOVX protein or biologically-active portion thereof with a test compound and determining the ability of the test compound to modulate (e.g. stimulate or inhibit) the activity of the NOVX protein or biologically-active portion thereof. Determining the ability of the test compound to modulate the activity of NOVX can be accomplished, for example, by determining the ability of the NOVX protein to bind to an NOVX target molecule by one of the methods described above for determimng direct binding. In an alternative embodiment, determining the ability of the test compound to modulate the activity of NOVX protein can be accomplished by determimng the ability of the NOVX protein further modulate an NOVX target molecule. For example, the catalytic/enzymatic activity of the target molecule on an appropriate substrate can be determined as described, supra. hi yet another embodiment, the cell-free assay comprises contacting the NOVX protein or biologically-active portion thereof with a known compound which binds NOVX protein to form an assay mixture, contacting the assay mixture with a test compound, and determimng the ability of the test compound to interact with an NOVX protein, wherein determimng the ability of the test compound to interact with an NOVX protein comprises determining the ability of the NOVX protein to preferentially bind to or modulate the activity of an NOVX target molecule.
The cell-free assays of the invention are amenable to use of both the soluble form or the membrane-bound form of NOVX protein. In the case of cell-free assays comprising the membrane-bound form of NOVX protein, it may be desirable to utilize a solubilizing agent such that the membrane-bound form of NOVX protein is maintained in solution. Examples of such solubilizing agents include non-ionic detergents such as n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton® X-100, Triton® X-114, Thesit®, Isotridecypoly(ethylene glycol ether)n, N-dodecyl-- N,N-dimethyl-3-ammonio- 1-propane sulfonate, 3-(3-cholamidopropyl) dimethylamminiol- 1-propane sulfonate (CHAPS), or 3 -(3 -cholamidopropyl)dimethylarnminiol-2-hydroxy- 1-propane sulfonate (CHAPSO).
In more than one embodiment of the above assay methods of the invention, it may be desirable to immobilize either NOVX protein or its target molecule to facilitate separation of complexed from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay. Binding of a test compound to NOVX protein, or interaction of NOVX protein with a target molecule in the presence and absence of a candidate compound, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided that adds a domain that allows one or both of the proteins to be bound to a matrix. For example, GST-NO VX fusion proteins or GST-target fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtiter plates, that are then combined with the test compound or the test compound and either the non-adsorbed target protein or NOVX protein, and the mixture is incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described, supra. Alternatively, the complexes can be dissociated from the matrix, and the level of NOVX protein binding or activity determined using standard techniques.
Other techniques for immobilizing proteins on matrices can also be used in the screening assays of the invention. For example, either the NOVX protein or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin. Biotinylated NOVX protein or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques well-known within the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, III), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). Alternatively, antibodies reactive with NOVX protein or target molecules, but which do not interfere with binding of the NOVX protein to its target molecule, can be derivatized to the wells of the plate, and unbound target or NOVX protein trapped in the wells by antibody conjugation. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the NOVX protein or target molecule, as well as enzyme-linked assays that rely on detecting an enzymatic activity associated with the NOVX protein or target molecule.
In another embodiment, modulators of NOVX protein expression are identified in a method wherein a cell is contacted with a candidate compound and the expression of NOVX mRNA or protein in the cell is determined. The level of expression of NOVX mRNA or protein in the presence of the candidate compound is compared to the level of expression of NOVX mRNA or protein in the absence of the candidate compound. The candidate compound can then be identified as a modulator of NOVX mRNA or protein expression based upon this comparison. For example, when expression of NOVX mRNA or protein is greater (i.e., statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of NOVX mRNA or protein expression. Alternatively, when expression of NOVX mRNA or protein is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of NOVX mRNA or protein expression. The level of NOVX mRNA or protein expression in the cells can be determined by methods described herein for detecting NOVX mRNA or protein. In yet another aspect of the invention, the NOVX proteins can be used as "bait proteins" in a two-hybrid assay or three hybrid assay (see, e.g., U.S. Patent No. 5,283,317; Zervos, et al, 1993. Cell 72: 223-232; Madura, et al, 1993. J. Biol. Chem. 268: 12046-12054; Bartel, et al, \992>. Biotechniques 14: 920-924; Iwabuchi, et al, 1993. Oncogene 8: 1693-1696; and Brent WO 94/10300), to identify other proteins that bind to or interact with NOVX ("NOVX-binding proteins" or "NOVX-bp") and modulate NOVX activity. Such NOVX-binding proteins are also likely to be involved in the propagation of signals by the NOVX proteins as, for example, upstream or downstream elements of the NOVX pathway.
The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. In one construct, the gene that codes for NOVX is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g. , GAL-4). In the other construct, a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein ("prey" or "sample") is fused to a gene that codes for the activation domain of the known transcription factor. If the "bait" and the "prey" proteins are able to interact, in vivo, forming an NOVX-dependent complex, the DNA-binding and activation domains of the franscription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g. , LacZ) that is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene that encodes the protein which interacts with NOVX. The invention further pertains to novel agents identified by the aforementioned screening assays and uses thereof for treatments as described herein.
Detection Assays
Portions or fragments of the cDNA sequences identified herein (and the conesponding complete gene sequences) can be used in numerous ways as polynucleotide reagents. By way of example, and not of limitation, these sequences can be used to: (i) map their respective genes on a chromosome; and, thus, locate gene regions associated with genetic disease; (ii) identify an individual from a minute biological sample (tissue typing); and (iii) aid in forensic identification of a biological sample. Some of these applications are described in the subsections, below. Chromosome Mapping
Once the sequence (or a portion of the sequence) of a gene has been isolated, this sequence can be used to map the location of the gene on a chromosome. This process is called chromosome mapping. Accordingly, portions or fragments of the NOVX sequences, SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, and 122, or fragments or derivatives thereof, can be used to map the location of the NOVX genes, respectively, on a chromosome. The mapping of the NOVX sequences to chromosomes is an important first step in conelating these sequences with genes associated with disease.
Briefly, NOVX genes can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp in length) from the NOVX sequences. Computer analysis of the NOVX, sequences can be used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers can then be used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene conesponding to the NOVX sequences will yield an amplified fragment.
Somatic cell hybrids are prepared by fusing somatic cells from different mammals (e.g., human and mouse cells). As hybrids of human and mouse cells grow and divide, they gradually lose human chromosomes in random order, but retain the mouse chromosomes. By using media in which mouse cells cannot grow, because they lack a particular enzyme, but in which human cells can, the one human chromosome that contains the gene encoding the needed enzyme will be retained. By using various media, panels of hybrid cell lines can be established. Each cell line in a panel contains either a single human chromosome or a small number of human chromosomes, and a full set of mouse chromosomes, allowing easy mapping of individual genes to specific human chromosomes. See, e.g., D'Eustachio, et al, 1983. Science 220: 919-924. Somatic cell hybrids containing only fragments of human chromosomes can also be produced by using human chromosomes with franslocations and deletions. PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular sequence to a particular chromosome. Three or more sequences can be assigned per day using a single thermal cycler. Using the NOVX sequences to design oligonucleotide primers, sub- localization can be achieved with panels of fragments from specific chromosomes.
Fluorescence in situ hybridization (FISH) of a DNA sequence to a metaphase chromosomal spread can further be used to provide a precise chromosomal location in one step. Chromosome spreads can be made using cells whose division has been blocked in metaphase by a chemical like colcemid that disrupts the mitotic spindle. The chromosomes can be treated briefly with trypsin, and then stained with Giemsa. A pattern of light and dark bands develops on each chromosome, so that the chromosomes can be identified individually. The FISH technique can be used with a DNA sequence as short as 500 or 600 bases. However, clones larger than 1,000 bases have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection. Preferably 1,000 bases, and more preferably 2,000 bases, will suffice to get good results at a reasonable amount of time. For a review of this technique, see, Verma, et al, HUMAN CHROMOSOMES: A MANUAL OF BASIC TECHNIQUES (Pergamon Press, New York 1988). Reagents for chromosome mapping can be used individually to mark a single chromosome or a single site on that chromosome, or panels of reagents can be used for marking multiple sites and/or multiple chromosomes. Reagents conesponding to noncoding regions of the genes actually are prefened for mapping puφoses. Coding sequences are more likely to be conserved within gene families, thus increasing the chance of cross hybridizations during chromosomal mapping.
Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be conelated with genetic map data. Such data are found, e.g., in McKusick, MENDELIAN INHERITANCE IN MAN, available on-line through Johns Hopkins University Welch Medical Library). The relationship between genes and disease, mapped to the same chromosomal region, can then be identified through linkage analysis
(co-inheritance of physically adjacent genes), described in, e.g., Egeland, et al., 1987. Nature, 325: 783-787.
Moreover, differences in the DNA sequences between individuals affected and unaffected with a disease associated with the NOVX gene, can be determined. If a mutation is observed in some or all of the affected individuals but not in any unaffected individuals, then the mutation is likely to be the causative agent of the particular disease. Comparison of affected and unaffected individuals generally involves first looking for structural alterations in the chromosomes, such as deletions or franslocations that are visible from chromosome spreads or detectable using PCR based on that DNA sequence. Ultimately, complete sequencing of genes from several individuals can be performed to confirm the presence of a mutation and to distinguish mutations from polymoφhisms.
Tissue Typing
The NOVX sequences of the invention can also be used to identify individuals from minute biological samples, hi this technique, an individual's genomic DNA is digested with one or more restriction enzymes, and probed on a Southern blot to yield unique bands for identification. The sequences of the invention are useful as additional DNA markers for RFLP ("restriction fragment length polymoφhisms," described in U.S. Patent No. 5,272,057).
Furthermore, the sequences of the invention can be used to provide an alternative technique that determines the actual base-by-base DNA sequence of selected portions of an individual's genome. Thus, the NOVX sequences described herein can be used to prepare two PCR primers from the 5'- and 3'-termini of the sequences. These primers can then be used to amplify an individual's DNA and subsequently sequence it.
Panels of conesponding DNA sequences from individuals, prepared in this manner, can provide unique individual identifications, as each individual will have a unique set of such DNA sequences due to allelic differences. The sequences of the invention can be used to obtain such identification sequences from individuals and from tissue. The NOVX sequences of the invention uniquely represent portions of the human genome. Allelic variation occurs to some degree in the coding regions of these sequences, and to a greater degree in the noncoding regions. It is estimated that allelic variation between individual humans occurs with a frequency of about once per each 500 bases. Much of the allelic variation is due to single nucleotide polymoφhisms (SNPs), which include restriction fragment length polymoφhisms (RFLPs).
Each of the sequences described herein can, to some degree, be used as a standard against which DNA from an individual can be compared for identification puφoses. Because greater numbers of polymoφhisms occur in the noncoding regions, fewer sequences are necessary to differentiate individuals. The noncoding sequences can comfortably provide positive individual identification with a panel of perhaps 10 to 1,000 primers that each yield a noncoding amplified sequence of 100 bases. If predicted coding sequences, such as those in SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, and 122 are used, a more appropriate number of primers for positive individual identification would be 500-2,000.
Predictive Medicine
The invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, pharmacogenomics, and monitoring clinical trials are used for prognostic
(predictive) puφoses to thereby treat an individual prophylactically. Accordingly, one aspect of the invention relates to diagnostic assays for determining NOVX protein and/or nucleic acid expression as well as NOVX activity, in the context of a biological sample (e.g., blood, serum, cells, tissue) to thereby determine whether an individual is afflicted with a disease or disorder, or is at risk of developing a disorder, associated with abenant NOVX expression or activity. The disorders include metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer- associated cachexia, cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, and hematopoietic disorders, and the various dyslipidemias, metabolic disturbances associated with obesity, the metabolic syndrome X and wasting disorders associated with chronic diseases and various cancers. The invention also provides for prognostic (or predictive) assays for determining whether an individual is at risk of developing a disorder associated with NOVX protein, nucleic acid expression or activity. For example, mutations in an NOVX gene can be assayed in a biological sample. Such assays can be used for prognostic or predictive puφose to thereby prophylactically treat an individual prior to the onset of a disorder characterized by or associated with NOVX protein, nucleic acid expression, or biological activity. Another aspect of the invention provides methods for determimng NOVX protein, nucleic acid expression or activity in an individual to thereby select appropriate therapeutic or prophylactic agents for that individual (refened to herein as "pharmacogenomics"). Pharmacogenomics allows for the selection of agents (e.g., drugs) for therapeutic or prophylactic treatment of an individual based on the genotype of the individual (e.g., the genotype of the individual examined to determine the ability of the individual to respond to a particular agent.) Yet another aspect of the invention pertains to momtoring the influence of agents (e.g., drags, compounds) on the expression or activity of NOVX in clinical trials. These and other agents are described in further detail in the following sections.
Diagnostic Assays
An exemplary method for detecting the presence or absence of NOVX in a biological sample involves obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting NOVX protein or nucleic acid (e.g., mRNA, genomic DNA) that encodes NOVX protein such that the presence of NOVX is detected in the biological sample. An agent for detecting NOVX mRNA or genomic DNA is a labeled nucleic acid probe capable of hybridizing to NOVX mRNA or genomic DNA. The nucleic acid probe can be, for example, a full-length NOVX nucleic acid, such as the nucleic acid of SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, and 122, or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to NOVX mRNA or genomic DNA. Other suitable probes for use in the diagnostic assays of the invention are described herein. An agent for detecting NOVX protein is an antibody capable of binding to NOVX protein, preferably an antibody with a detectable label. Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab')2) can be used. The term "labeled", with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently-labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently-labeled sfreptavidin. The term "biological sample" is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. That is, the detection method of the invention can be used to detect NOVX mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo. For example, in vitro techniques for detection of NOVX mRNA include Northern hybridizations and in situ hybridizations. In vitro techniques for detection of NOVX protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations, and immunofluorescence. In vitro techniques for detection of NOVX genomic DNA include Southern hybridizations. Furthermore, in vivo techniques for detection of NOVX protein include introducing into a subject a labeled anti-NOVX antibody. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.
In one embodiment, the biological sample contains protein molecules from the test subject. Alternatively, the biological sample can contain mRNA molecules from the test subject or genomic DNA molecules from the test subject. A prefened biological sample is a peripheral blood leukocyte sample isolated by conventional means from a subject.
In another embodiment, the methods further involve obtaining a control biological sample from a control subject, contacting the control sample with a compound or agent capable of detecting NOVX protein, mRNA, or genomic DNA, such that the presence of NOVX protein, mRNA or genomic DNA is detected in the biological sample, and comparing the presence of NOVX protein, mRNA or genomic DNA in the control sample with the presence of NOVX protein, mRNA or genomic DNA in the test sample.
The invention also encompasses kits for detecting the presence of NOVX in a biological sample. For example, the kit can comprise: a labeled compound or agent capable of detecting NOVX protein or mRNA in a biological sample; means for determining the amount of NOVX in the sample; and means for comparing the amount of NOVX in the sample with a standard. The compound or agent can be packaged in a suitable container. The kit can further comprise instructions for using the kit to detect NOVX protein or nucleic acid. Prognostic Assays
The diagnostic methods described herein can furthermore be utilized to identify subjects having or at risk of developing a disease or disorder associated with abenant NOVX expression or activity. For example, the assays described herein, such as the preceding diagnostic assays or the following assays, can be utilized to identify a subject having or at risk of developing a disorder associated with NOVX protein, nucleic acid expression or activity. Alternatively, the prognostic assays can be utilized to identify a subject having or at risk for developing a disease or disorder. Thus, the invention provides a method for identifying a disease or disorder associated with abenant NOVX expression or activity in which a test sample is obtained from a subject and NOVX protein or nucleic acid (e.g., mRNA, genomic DNA) is detected, wherein the presence of NOVX protein or nucleic acid is diagnostic for a subject having or at risk of developing a disease or disorder associated with abenant NOVX expression or activity. As used herein, a "test sample" refers to a biological sample obtained from a subject of interest. For example, a test sample can be a biological fluid (e.g., serum), cell sample, or tissue.
Furthermore, the prognostic assays described herein can be used to determine whether a subject can be admimstered an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate) to treat a disease or disorder associated with abenant NOVX expression or activity. For example, such methods can be used to determine whether a subject can be effectively treated with an agent for a disorder. Thus, the invention provides methods for determining whether a subject can be effectively treated with an agent for a disorder associated with abenant NOVX expression or activity in which a test sample is obtained and NOVX protein or nucleic acid is detected (e.g. , wherein the presence of NOVX protein or nucleic acid is diagnostic for a subject that can be administered the agent to treat a disorder associated with abenant NOVX expression or activity).
The methods of the invention can also be used to detect genetic lesions in an NOVX gene, thereby determining if a subject with the lesioned gene is at risk for a disorder characterized by abenant cell proliferation and/or differentiation. In various embodiments, the methods include detecting, in a sample of cells from the subject, the presence or absence of a genetic lesion characterized by at least one of an alteration affecting the integrity of a gene encoding an NOVX-protein, or the misexpression of the NOVX gene. For example, such genetic lesions can be detected by ascertaining the existence of at least one of: (i) a deletion of one or more nucleotides from an NOVX gene; (ii) an addition of one or more nucleotides to an NOVX gene; (n't) a substitution of one or more nucleotides of an NOVX gene, (iv) a chromosomal reanangement of an NOVX gene; (v) an alteration in the level of a messenger RNA transcript of an NOVX gene, (vi) abenant modification of an NOVX gene, such as of the methylation pattern of the genomic DNA, (vii) the presence of a non- wild-type splicing pattern of a messenger RNA transcript of an NOVX gene, (viii) a non-wild-type level of an NOVX protein, (ix) allelic loss of an NOVX gene, and (x) inappropriate post-translational modification of an NOVX protein. As described herein, there are a large number of assay techniques known in the art which can be used for detecting lesions in an NOVX gene. A prefened biological sample is a peripheral blood leukocyte sample isolated by conventional means from a subject. However, any biological sample containing nucleated cells may be used, including, for example, buccal mucosal cells. In certain embodiments, detection of the lesion involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Patent Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran, et al, 1988. Science 241: 1077-1080; and Nakazawa, et al, 1994. Proc. Natl. Acad. Sci. USA 91 : 360-364), the latter of which can be particularly useful for detecting point mutations in the NOVX-gene (see, Abravaya, et al, 1995. Nucl. Acids Res. 23: 675-682). This method can include the steps of collecting a sample of cells from a patient, isolating nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers that specifically hybridize to an NOVX gene under conditions such that hybridization and amplification of the NOVX gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. It is anticipated that PCR and/or LCR may be desirable to use as a preliminary amplification step in conjunction with any of the techniques used for detecting mutations described herein.
Alternative amplification methods include: self sustained sequence replication (see, Guatelli, et al, 1990. Proc. Natl. Acad. Sci. USA 87: 1874-1878), transcriptional amplification system (see, Kwoh, et al, 1989. Proc. Natl. Acad. Sci. USA 86: 1173-1177); Qβ Replicase (see, Lizardi, et al, 1988. BioTechnology 6: 1197), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers. hi an alternative embodiment, mutations in an NOVX gene from a sample cell can be identified by alterations in restriction enzyme cleavage patterns. For example, sample and control DNA is isolated, amplified (optionally), digested with one or more restriction endonucleases, and fragment length sizes are determined by gel electrophoresis and compared. Differences in fragment length sizes between sample and control DNA indicates mutations in the sample DNA. Moreover, the use of sequence specific ribozymes (see, e.g., U.S. Patent No. 5,493,531) can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site.
In other embodiments, genetic mutations in NOVX can be identified by hybridizing a sample and control nucleic acids, e.g., DNA or RNA, to high-density anays containing hundreds or thousands of oligonucleotides probes. See, e.g., Cronin, et al., 1996. Human Mutation 7: 244-255; Kozal, et al, 1996. Nat. Med. 2: 753-759. For example, genetic mutations in ΝONX can be identified in two dimensional anays containing light-generated DΝA probes as described in Cronin, et al, supra. Briefly, a first hybridization anay of probes can be used to scan through long stretches of DΝA in a sample and control to identify base changes between the sequences by making linear arrays of sequential overlapping probes. This step allows the identification of point mutations. This is followed by a second hybridization anay that allows the characterization of specific mutations by using smaller, specialized probe anays complementary to all variants or mutations detected. Each mutation array is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene. In yet another embodiment, any of a variety of sequencing reactions known in the art can be used to directly sequence the ΝONX gene and detect mutations by comparing the sequence of the sample ΝOVX with the conesponding wild-type (control) sequence. Examples of sequencing reactions include those based on techniques developed by Maxim and Gilbert, 1977. Proc. Natl. Acad. Sci. USA 74: 560 or Sanger, 1977. Proc. Natl. Acad. Sci. USA 74: 5463. It is also contemplated that any of a variety of automated sequencing procedures can be utilized when performing the diagnostic assays (see, e.g., Νaeve, et al, 1995. Biotechniques 19: 448), including sequencing by mass specfromefry (see, e.g., PCT International Publication No. WO 94/16101; Cohen, et al, 1996. Adv. Chromatography 36: 127-162; and Griffin, et al, 1993. Appl. Biochem. Biotechnol. 38: 147-159). Other methods for detecting mutations in the NOVX gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes. See, e.g., Myers, et al, 1985. Science 230: 1242. In general, the art technique of "mismatch cleavage" starts by providing heteroduplexes of formed by hybridizing (labeled) RNA or DNA containing the wild-type NOVX sequence with potentially mutant RNA or DNA obtained from a tissue sample. The double-stranded duplexes are treated with an agent that cleaves single-stranded regions of the duplex such as which will exist due to basepair mismatches between the confrol and sample strands. For instance, RNA DNA duplexes can be treated with RNase and DNA/DNA hybrids treated with Si nuclease to enzymatically digesting the mismatched regions. In other embodiments, either DNA/DNA or RNA DNA duplexes can be treated with hydroxylamine or osmium tetroxide and with piperidine in order to digest mismatched regions. After digestion of the mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine the site of mutation. See, e.g., Cotton, et al, 1988. Proc. Natl. Acad. Sci. USA 85: 4397; Saleeba, et al, 1992. Methods Enzymol. 217: 286-295. hi an embodiment, the confrol DNA or RNA can be labeled for detection.
In still another embodiment, the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-sfranded DNA (so called "DNA mismatch repair" enzymes) in defined systems for detecting and mapping point mutations in NOVX cDNAs obtained from samples of cells. For example, the mutY enzyme of E. coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T at G/T mismatches. See, e.g., Hsu, et al, 1994. Carcinogenesis 15: 1657-1662. According to an exemplary embodiment, a probe based on an NOVX sequence, e.g., a wild-type NOVX sequence, is hybridized to a cDNA or other DNA product from a test cell(s). The duplex is treated with a DNA mismatch repair enzyme, and the cleavage products, if any, can be detected from electrophoresis protocols or the like. See, e.g., U.S. Patent No. 5,459,039.
In other embodiments, alterations in electrophoretic mobility will be used to identify mutations in NOVX genes. For example, single strand conformation polymoφhism (SSCP) may be used to detect differences in electrophoretic mobility between mutant and wild type nucleic acids. See, e.g., Orita, et al, 1989. Proc. Natl. Acad. Sci. USA: 86: 2766; Cotton, 1993. Mutat. Res. 285: 125-144; Hayashi, 1992. Genet. Anal. Tech. Appl. 9: 73-79. Single-stranded DNA fragments of sample and control NOVX nucleic acids will be denatured and allowed to renature. The secondary structure of single-stranded nucleic acids varies according to sequence, the resulting alteration in electrophoretic mobility enables the detection of even a single base change. The DNA fragments may be labeled or detected with labeled probes. The sensitivity of the assay may be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in sequence. In one embodiment, the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility. See, e.g., Keen, et al, 1991. Trends Genet. 7: 5. In yet another embodiment, the movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE). See, e.g., Myers, et al, 1985. Nature 313: 495. When DGGE is used as the method of analysis, DNA will be modified to insure that it does not completely denature, for example by adding a GC clamp of approximately 40 bp of high-melting GC-rich DNA by PCR. In a further embodiment, a temperature gradient is used in place of a denaturing gradient to identify differences in the mobility of confrol and sample DNA. See, e.g., Rosenbaum and Reissner, 1987. Biophys. Chem. 265: 12753.
Examples of other techniques for detecting point mutations include, but are not limited to, selective oligonucleotide hybridization, selective amplification, or selective primer extension. For example, oligonucleotide primers may be prepared in which the known mutation is placed centrally and then hybridized to target DNA under conditions that permit hybridization only if a perfect match is found. See, e.g., Saiki, et al, 1986. Nature 324: 163; Saiki, et al, 1989. Proc. Natl. Acad. Sci. USA 86: 6230. Such allele specific oligonucleotides are hybridized to PCR amplified target DNA or a number of different mutations when the oligonucleotides are attached to the hybridizing membrane and hybridized with labeled target DNA.
Alternatively, allele specific amplification technology that depends on selective PCR amplification may be used in conjunction with the instant invention. Oligonucleotides used as primers for specific amplification may carry the mutation of interest in the center of the molecule (so that amplification depends on differential hybridization; see, e.g., Gibbs, et al, 1989. Nucl. Acids Res. 17: 2437-2448) or at the extreme 3'-terminus of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (see, e.g., Prossner, 1993. Tibtech. 11 : 238). In addition it may be desirable to introduce a novel restriction site in the region of the mutation to create cleavage-based detection. See, e.g., Gasparini, et al, 1992. Mol. Cell Probes 6: 1. It is anticipated that in certain embodiments amplification may also be performed using Taq ligase for amplification. See, e.g., Barany, 1991. Proc. Natl. Acad. Sci. USA 88: 189. hi such cases, ligation will occur only if there is a perfect match at the 3'-terminus of the 5' sequence, making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification. The methods described herein may be performed, for example, by utilizing pre-packaged diagnostic kits comprising at least one probe nucleic acid or antibody reagent described herein, which may be conveniently used, e.g., in clinical settings to diagnose patients exhibiting symptoms or family history of a disease or illness involving an NONX gene.
Furthermore, any cell type or tissue, preferably peripheral blood leukocytes, in which ΝONX is expressed may be utilized in the prognostic assays described herein. However, any biological sample containing nucleated cells may be used, including, for example, buccal mucosal cells.
Pharmacogenomics
Agents, or modulators that have a stimulatory or inhibitory effect on ΝONX activity (e.g., ΝONX gene expression), as identified by a screening assay described herein can be administered to individuals to treat (prophylactically or therapeutically) disorders (The disorders include metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer-associated cachexia, cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, and hematopoietic disorders, and the various dyslipidemias, metabolic disturbances associated with obesity, the metabolic syndrome X and wasting disorders associated with chronic diseases and various cancers.) In conjunction with such treatment, the pharmacogenomics (i.e., the study of the relationship between an individual's genotype and that individual's response to a foreign compound or drag) of the individual may be considered. Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration of the pharmacologically active drug. Thus, the pharmacogenomics of the individual permits the selection of effective agents (e.g. , drags) for prophylactic or therapeutic treatments based on a consideration of the individual's genotype. Such pharmacogenomics can further be used to determine appropriate dosages and therapeutic regimens. Accordingly, the activity of ΝONX protein, expression of ΝOVX nucleic acid, or mutation content of ΝOVX genes in an individual can be determined to thereby select appropriate agent(s) for therapeutic or prophylactic treatment of the individual.
Pharmacogenomics deals with clinically significant hereditary variations in the response to drags due to altered drag disposition and abnormal action in affected persons. See e.g., Eichelbaum, 1996. Clin. Exp. Pharmacol. Physiol, 23: 983-985; Linder, 1997. Clin. Chem., 43: 254-266. In general, two types of pharmacogenetic conditions can be differentiated. Genetic conditions transmitted as a single factor altering the way drugs act on the body (altered drag action) or genetic conditions transmitted as single factors altering the way the body acts on drags (altered drag metabolism). These pharmacogenetic conditions can occur either as rare defects or as polymoφhisms. For example, glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common inherited enzymopathy in which the main clinical complication is hemolysis after ingestion of oxidant drags (anti-malarials, sulfonamides, analgesics, nitrofurans) and consumption of fava beans.
As an illustrative embodiment, the activity of drag metabolizing enzymes is a major determinant of both the intensity and duration of drag action. The discovery of genetic polymoφhisms of drag metabolizing enzymes (e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymes CYP2D6 and CYP2C19) has provided an explanation as to why some patients do not obtain the expected drag effects or show exaggerated drug response and serious toxicity after taking the standard and safe dose of a drag. These polymoφhisms are expressed in two phenotypes in the population, the extensive metabolizer (EM) and poor metabolizer (PM). The prevalence of PM is different among different populations. For example, the gene coding for CYP2D6 is highly polymoφhic and several mutations have been identified in PM, which all lead to the absence of functional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C 19 quite frequently experience exaggerated drag response and side effects when they receive standard doses. If a metabolite is the active therapeutic moiety, PM show no therapeutic response, as demonstrated for the analgesic effect of codeine mediated by its CYP2D6-formed metabolite moφbine. At the other extreme are the so called ultra-rapid metabolizers who do not respond to standard doses. Recently, the molecular basis of ultra-rapid metabolism has been identified to be due to CYP2D6 gene amplification.
Thus, the activity of NOVX protein, expression of NOVX nucleic acid, or mutation content of NOVX genes in an individual can be determined to thereby select appropriate agent(s) for therapeutic or prophylactic treatment of the individual, hi addition, pharmacogenetic studies can be used to apply genotyping of polymoφhic alleles encoding drag-metabolizing enzymes to the identification of an individual's drag responsiveness phenotype. This knowledge, when applied to dosing or drag selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic or prophylactic efficiency when treating a subject with an NOVX modulator, such as a modulator identified by one of the exemplary screening assays described herein. Monitoring of Effects During Clinical Trials
Monitoring the influence of agents (e.g., drags, compounds) on the expression or activity of NOVX (e.g., the ability to modulate abenant cell proliferation and/or differentiation) can be applied not only in basic drug screening, but also in clinical trials. For example, the effectiveness of an agent determined by a screening assay as described herein to increase NOVX gene expression, protein levels, or upregulate NOVX activity, can be monitored in clinical frails of subjects exhibiting decreased NOVX gene expression, protein levels, or downregulated NOVX activity. Alternatively, the effectiveness of an agent determined by a screening assay to decrease NOVX gene expression, protein levels, or downregulate NOVX activity, can be monitored in clinical trails of subjects exhibiting increased NOVX gene expression, protein levels, or upregulated NOVX activity. In such clinical trials, the expression or activity of NOVX and, preferably, other genes that have been implicated in, for example, a cellular proliferation or immune disorder can be used as a "read out" or markers of the immune responsiveness of a particular cell. By way of example, and not of limitation, genes, including NOVX, that are modulated in cells by treatment with an agent (e.g., compound, drag or small molecule) that modulates NOVX activity (e.g., identified in a screening assay as described herein) can be identified. Thus, to study the effect of agents on cellular proliferation disorders, for example, in a clinical trial, cells can be isolated and RNA prepared and analyzed for the levels of expression of NOVX and other genes implicated in the disorder. The levels of gene expression (i.e., a gene expression pattern) can be quantified by Northern blot analysis or RT-PCR, as described herein, or alternatively by measuring the amount of protein produced, by one of the methods as described herein, or by measuring the levels of activity of NOVX or other genes, hi this manner, the gene expression pattern can serve as a marker, indicative of the physiological response of the cells to the agent. Accordingly, this response state may be determined before, and at various points during, treatment of the individual with the agent.
In one embodiment, the invention provides a method for monitoring the effectiveness of treatment of a subject with an agent (e.g., an agonist, antagonist, protein, peptide, peptidomimetic, nucleic acid, small molecule, or other drug candidate identified by the screening assays described herein) comprising the steps of (i) obtaining a pre-administration sample from a subject prior to administration of the agent; (ii) detecting the level of expression of an NOVX protein, mRNA, or genomic DNA in the preadmimstration sample; (iii) obtaining one or more post-administration samples from the subject; (iv) detecting the level of expression or activity of the NOVX protein, mRNA, or genomic DNA in the post-administration samples; (v) comparing the level of expression or activity of the NOVX protein, mRNA, or genomic DNA in the pre-administration sample with the NOVX protein, mRNA, or genomic DNA in the post administration sample or samples; and (vi) altering the administration of the agent to the subject accordingly. For example, increased admimstration of the agent may be desirable to increase the expression or activity of NOVX to higher levels than detected, i.e., to increase the effectiveness of the agent. Alternatively, decreased admimstration of the agent may be desirable to decrease expression or activity of NOVX to lower levels than detected, i.e., to decrease the effectiveness of the agent.
Methods of Treatment
The invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with abenant NOVX expression or activity. The disorders include cardiomyopathy, atherosclerosis, hypertension, congenital heart defects, aortic stenosis, atrial septal defect (ASD), atrioventricular (A-V) canal defect, ductus arteriosus, pulmonary stenosis, subaortic stenosis, ventricular septal defect (VSD), valve diseases, tuberous sclerosis, scleroderma, obesity, transplantation, adrenoleukodystrophy, congenital adrenal hypeφlasia, prostate cancer, neoplasm; adenocarcinoma, lymphoma, uterus cancer, fertility, hemophilia, hypercoagulation, idiopathic thrombocytopemc puφura, immunodeficiencies, graft versus host disease, AIDS, bronchial asthma, Crohn's disease; multiple sclerosis, treatment of Albright Hereditary Ostoeodystrophy, and other diseases, disorders and conditions of the like.
These methods of treatment will be discussed more fully, below. Disease and Disorders
Diseases and disorders that are characterized by increased (relative to a subject not suffering from the disease or disorder) levels or biological activity may be treated with Therapeutics that antagonize (i.e., reduce or inhibit) activity. Therapeutics that antagonize activity may be administered in a therapeutic or prophylactic manner. Therapeutics that may be utilized include, but are not limited to: (i) an aforementioned peptide, or analogs, derivatives, fragments or homologs thereof; (ii) antibodies to an aforementioned peptide; (iii) nucleic acids encoding an aforementioned peptide; (iv) admimstration of antisense nucleic acid and nucleic acids that are "dysfunctional" (i.e., due to a heterologous insertion within the coding sequences of coding sequences to an aforementioned peptide) that are utilized to "knockout" endogenous function of an aforementioned peptide by homologous recombination (see, e.g., Capecchi, 1989. Science 244: 1288-1292); or (v) modulators ( i.e., inhibitors, agonists and antagonists, including additional peptide mimetic of the invention or antibodies specific to a peptide of the invention) that alter the interaction between an aforementioned peptide and its binding partner.
Diseases and disorders that are characterized by decreased (relative to a subject not suffering from the disease or disorder) levels or biological activity may be treated with
Therapeutics that increase (i.e., are agonists to) activity. Therapeutics that upregulate activity may be administered in a therapeutic or prophylactic manner. Therapeutics that may be utilized include, but are not limited to, an aforementioned peptide, or analogs, derivatives, fragments or homologs thereof; or an agonist that increases bio availability. Increased or decreased levels can be readily detected by quantifying peptide and/or RNA, by obtaining a patient tissue sample (e.g., from biopsy tissue) and assaying it in vitro for RNA or peptide levels, structure and/or activity of the expressed peptides (or mRNAs of an aforementioned peptide). Methods that are well-known within the art include, but are not limited to, immunoassays (e.g., by Western blot analysis, immunoprecipitation followed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, immunocytochemistry, etc.) and/or hybridization assays to detect expression of mRNAs (e.g., Northern assays, dot blots, in situ hybridization, and the like).
Prophylactic Methods hi one aspect, the invention provides a method for preventing, in a subject, a disease or condition associated with an abenant NONX expression or activity, by administering to the subject an agent that modulates ΝONX expression or at least one ΝONX activity. Subjects at risk for a disease that is caused or contributed to by abenant ΝONX expression or activity can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein. Admimstration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the ΝONX abenancy, such that a disease or disorder is prevented or, alternatively, delayed in its progression. Depending upon the type of ΝONX abenancy, for example, an NOVX agonist or NOVX antagonist agent can be used for treating the subject. The appropriate agent can be determined based on screening assays described herein. The prophylactic methods of the invention are further discussed in the following subsections.
Therapeutic Methods Another aspect of the invention pertains to methods of modulating NOVX expression or activity for therapeutic puφoses. The modulatory method of the invention involves contacting a cell with an agent that modulates one or more of the activities of NOVX protein activity associated with the cell. An agent that modulates NOVX protein activity can be an agent as described herein, such as a nucleic acid or a protein, a naturally-occurring cognate ligand of an NOVX protein, a peptide, an NOVX peptidomimetic, or other small molecule. In one embodiment, the agent stimulates one or more NOVX protein activity. Examples of such stimulatory agents include active NOVX protein and a nucleic acid molecule encoding NOVX that has been introduced into the cell, hi another embodiment, the agent inhibits one or more NOVX protein activity. Examples of such inhibitory agents include antisense NOVX nucleic acid molecules and anti-NOVX antibodies. These modulatory methods can be performed in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g., by administering the agent to a subject). As such, the invention provides methods of treating an individual afflicted with a disease or disorder characterized by abenant expression or activity of an NOVX protein or nucleic acid molecule. In one embodiment, the method involves administering an agent (e.g., an agent identified by a screening assay described herein), or combination of agents that modulates (e.g., up-regulates or down-regulates) NOVX expression or activity. In another embodiment, the method involves administering an NOVX protein or nucleic acid molecule as therapy to compensate for reduced or abenant NOVX expression or activity.
Stimulation of NOVX activity is desirable in sttwations in which NOVX is abnormally downregulated and/or in which increased NOVX activity is likely to have a beneficial effect. One example of such a situation is where a subject has a disorder characterized by abenant cell proliferation and/or differentiation (e.g., cancer or immune associated disorders). Another example of such a situation is where the subject has a gestational disease (e.g., preclampsia). Determination of the Biological Effect of the Therapeutic
In various embodiments of the invention, suitable in vitro or in vivo assays are performed to determine the effect of a specific Therapeutic and whether its administration is indicated for treatment of the affected tissue. In various specific embodiments, in vitro assays may be performed with representative cells of the type(s) involved in the patient's disorder, to determine if a given Therapeutic exerts the desired effect upon the cell type(s). Compounds for use in therapy may be tested in suitable animal model systems including, but not limited to rats, mice, chicken, cows, monkeys, rabbits, and the like, prior to testing in human subjects. Similarly, for in vivo testing, any of the animal model system known in the art may be used prior to administration to human subjects.
Prophylactic and Therapeutic Uses of the Compositions of the Invention
The NOVX nucleic acids and proteins of the invention are useful in potential prophylactic and therapeutic applications implicated in a variety of disorders including, but not limited to: metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer-associated cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, hematopoietic disorders, and the various dyslipidemias, metabolic disturbances associated with obesity, the metabolic syndrome X and wasting disorders associated with chronic diseases and various cancers. As an example, a cDNA encoding the NOVX protein of the invention may be useful in gene therapy, and the protein may be useful when administered to a subject in need thereof. By way of non-limiting example, the compositions of the invention will have efficacy for treatment of patients suffering from: metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer-associated cachexia, cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, hematopoietic disorders, and the various dyslipidemias. Both the novel nucleic acid encoding the NOVX protein, and the NOVX protein of the invention, or fragments thereof, may also be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein are to be assessed. A further use could be as an anti-bacterial molecule (i.e., some peptides have been found to possess anti-bacterial properties). These materials are further useful in the generation of antibodies, which immunospecifically-bind to the novel substances of the invention for use in therapeutic or diagnostic methods.
The invention will be further described in the following examples, wliich do not limit the scope of the invention described in the claims.
Examples
Example 1. Quantitative expression analysis of clones in various cells and tissues
The quantitative expression of various clones was assessed using microtiter plates containing RNA samples from a variety of normal and pathology-derived cells, cell lines and tissues using real time quantitative PCR (RTQ PCR). RTQ PCR was performed on an Applied Biosystems ABI PRISM® 7700 or an ABI PRISM® 7900 HT Sequence Detection System. Various collections of samples are assembled on the plates, and refened to as Panel 1 (containing normal tissues and cancer cell lines), Panel 2 (containing samples derived from tissues from normal and cancer sources), Panel 3 (containing cancer cell lines), Panel 4 (containing cells and cell lines from normal tissues and cells related to inflammatory conditions), Panel 5D/5I (containing human tissues and cell lines with an emphasis on metabolic diseases), AI_comprehensive_panel (containing normal tissue and samples from autoimmune diseases), Panel CNSD.01 (containing central nervous system samples from normal and diseased brains) and CNS_neurodegeneration_panel (containing samples from normal and Alzheimer's diseased brains).
RNA integrity from all samples is confrolled for quality by visual assessment of agarose gel electropherograms using 28S and 18S ribosomal RNA staining intensity ratio as a guide (2:1 to 2.5:1 28s:18s) and the absence of low molecular weight RNAs that would be indicative of degradation products. Samples are confrolled against genomic DNA contamination by RTQ PCR reactions run in the absence of reverse transcriptase using probe and primer sets designed to amplify across the span of a single exon.
First, the RNA samples were normalized to reference nucleic acids such as constitutively expressed genes (for example, β-actin and GAPDH). Normalized RNA (5 ul) was converted to cDNA and analyzed by RTQ-PCR using One Step RT-PCR Master Mix Reagents (Applied Biosystems; Catalog No. 4309169) and gene-specific primers according to the manufacturer's instructions.
In other cases, non-normalized RNA samples were converted to single strand cDNA (sscDNA) using Superscript II (Invitrogen Coφoration; Catalog No. 18064-147) and random hexamers according to the manufacturer's instructions. Reactions containing up to 10 μg of total RNA were performed in a volume of 20 μl and incubated for 60 minutes at 42°C. This reaction can be scaled up to 50 μg of total RNA in a final volume of 100 μl. sscDNA samples are then normalized to reference nucleic acids as described previously, using IX TaqMan® Universal Master mix (Applied Biosystems; catalog No. 4324020), following the manufacturer's instructions.
Probes and primers were designed for each assay according to Applied Biosystems Primer Express Software package (version I for Apple Computer's Macintosh Power PC) or a similar algorithm using the target sequence as input. Default settings were used for reaction conditions and the following parameters were set before selecting primers: primer concentration = 250 nM, primer melting temperature (Tm) range = 58°-60°C, primer optimal Tm = 59°C, maximum primer difference = 2°C, probe does not have 5'G, probe Tm must be 10°C greater than primer Tm, amplicon size 75bp to lOObp. The probes and primers selected (see below) were synthesized by Synthegen (Houston, TX, USA). Probes were double purified by HPLC to remove uncoupled dye and evaluated by mass spectroscopy to verify coupling of reporter and quencher dyes to the 5' and 3' ends of the probe, respectively. Their final concentrations were: forward and reverse primers, 900nM each, and probe, 200nM.
PCR conditions: When working with RNA samples, normalized RNA from each tissue and each cell line was spotted in each well of either a 96 well or a 384-well PCR plate (Applied Biosystems). PCR cocktails included either a single gene specific probe and primers set, or two multiplexed probe and primers sets (a set specific for the target clone and another gene-specific set multiplexed with the target probe). PCR reactions were set up using TaqMan® One-Step RT- PCR Master Mix (Applied Biosystems, Catalog No. 4313803) following manufacturer's instructions. Reverse transcription was performed at 48°C for 30 minutes followed by amplification/PCR cycles as follows: 95°C 10 min, then 40 cycles of 95°C for 15 seconds, 60°C for 1 minute. Results were recorded as CT values (cycle at which a given sample crosses a threshold level of fluorescence) using a log scale, with the difference in RNA concenfration between a given sample and the sample with the lowest CT value being represented as 2 to the power of delta CT. The percent relative expression is then obtained by taking the reciprocal of this RNA difference and multiplying by 100.
When working with sscDNA samples, normalized sscDNA was used as described previously for RNA samples. PCR reactions containing one or two sets of probe and primers were set up as described previously, using IX TaqMan® Universal Master mix (Applied Biosystems; catalog No. 4324020), following the manufacturer's instructions. PCR amplification was performed as follows: 95°C 10 min, then 40 cycles of 95°C for 15 seconds, 60°C for 1 minute. Results were analyzed and processed as described previously.
Panels 1, 1.1, 1.2, and 1.3D
The plates for Panels 1, 1.1, 1.2 and 1.3D include 2 control wells (genomic DNA control and chemistry control) and 94 wells containing cDNA from various samples. The samples in these panels are broken into 2 classes: samples derived from cultured cell lines and samples derived from primary normal tissues. The cell lines are derived from cancers of the following types: lung cancer, breast cancer, melanoma, colon cancer, prostate cancer, CNS cancer, squamous cell carcinoma, ovarian cancer, liver cancer, renal cancer, gastric cancer and pancreatic cancer. Cell lines used in these panels are widely available through the American Type Culture Collection
(ATCC), a repository for cultured cell lines, and were cultured using the conditions recommended by the ATCC. The normal tissues found on these panels are comprised of samples derived from all major organ systems from single adult individuals or fetuses. These samples are derived from the following organs: adult skeletal muscle, fetal skeletal muscle, adult heart, fetal heart, adult kidney, fetal kidney, adult liver, fetal liver, adult lung, fetal lung, various regions of the brain, the spleen, bone marrow, lymph node, pancreas, salivary gland, pituitary gland, adrenal gland, spinal cord, thymus, stomach, small intestine, colon, bladder, trachea, breast, ovary, uterus, placenta, prostate, testis and adipose.
In the results for Panels 1, 1.1, 1.2 and 1.3D, the following abbreviations are used: ca. = carcinoma, * = established from metastasis, met = metastasis, s cell var = small cell variant, non-s = non-sm = non-small, squam = squamous, pi. eff = pi effusion = pleural effusion, glio = glioma, astro = astrocytoma, and neuro = neuroblastoma.
General_screening_panel_vl .4
The plates for Panel 1.4 include 2 control wells (genomic DNA control and chemistry control) and 94 wells containing cDNA from various samples. The samples in Panel 1.4 are broken into 2 classes: samples derived from cultured cell lines and samples derived from primary normal tissues. The cell lines are derived from cancers of the following types: lung cancer, breast cancer, melanoma, colon cancer, prostate cancer, CNS cancer, squamous cell carcinoma, ovarian , cancer, liver cancer, renal cancer, gastric cancer and pancreatic cancer. Cell lines used in Panel 1.4 are widely available through the American Type Culture Collection (ATCC), a repository for cultured cell lines, and were cultured using the conditions recommended by the ATCC. The normal tissues found on Panel 1.4 are comprised of pools of samples derived from all major organ systems from 2 to 5 different adult individuals or fetuses. These samples are derived from the following organs: adult skeletal muscle, fetal skeletal muscle, adult heart, fetal heart, adult kidney, fetal kidney, adult liver, fetal liver, adult lung, fetal lung, various regions of the brain, the spleen, bone marrow, lymph node, pancreas, salivary gland, pituitary gland, adrenal gland, spinal cord, thymus, stomach, small intestine, colon, bladder, trachea, breast, ovary, uteras, placenta, prostate, testis and adipose. Abbreviations are as described for Panels 1, 1.1, 1.2, and 1.3D.
Panels 2D and 2.2
The plates for Panels 2D and 2.2 generally include 2 control wells and 94 test samples composed of RNA or cDNA isolated from human tissue procured by surgeons working in close cooperation with the National Cancer Institute's Cooperative Human Tissue Network (CHTN) or the National
Disease Research Initiative (NDRI). The tissues are derived from human malignancies and in cases where indicated many malignant tissues have "matched margins" obtained from noncancerous tissue just adjacent to the tumor. These are termed normal adjacent tissues and are denoted "NAT" in the results below. The tumor tissue and the "matched margins" are evaluated by two independent pathologists (the surgical pathologists and again by a pathologist at NDRI or CHTN). This analysis provides a gross histopathological assessment of tumor differentiation grade. Moreover, most samples include the original surgical pathology report that provides information regarding the clinical stage of the patient. These matched margins are taken from the tissue sunounding (i.e. immediately proximal) to the zone of surgery (designated "NAT", for normal adjacent tissue, in Table RR). In addition, RNA and cDNA samples were obtained from various human tissues derived from autopsies performed on elderly people or sudden death victims (accidents, etc.). These tissues were ascertained to be free of disease and were purchased from various commercial sources such as Clontech (Palo Alto, CA), Research Genetics, and Invitrogen.
Panel 3D
The plates of Panel 3D are comprised of 94 cDNA samples and two control samples. Specifically, 92 of these samples are derived from cultured human cancer cell lines, 2 samples of human primary cerebellar tissue and 2 controls. The human cell lines are generally obtained from ATCC (American Type Culture Collection), NCI or the German tumor cell bank and fall into the following tissue groups: Squamous cell carcinoma of the tongue, breast cancer, prostate cancer, melanoma, epidermoid carcinoma, sarcomas, bladder carcinomas, pancreatic cancers, kidney cancers, leukemias/lymphomas, ovarian uterine/cervical, gastric, colon, lung and CNS cancer cell lines, hi addition, there are two independent samples of cerebellum. These cells are all cultured under standard recommended conditions and RNA extracted using the standard procedures. The cell lines in panel 3D and 1.3D are of the most common cell lines used in the scientific literature.
Panels 4D, 4R, and 4.1D
Panel 4 includes samples on a 96 well plate (2 confrol wells, 94 test samples) composed of RNA (Panel 4R) or cDNA (Panels 4D/4.1D) isolated from various human cell lines or tissues related to inflammatory conditions. Total RNA from control normal tissues such as colon and lung (Stratagene, La Jolla, CA) and thymus and kidney (Clontech) was employed. Total RNA from liver tissue from cinhosis patients and kidney from lupus patients was obtained from BioChain (Biochain Institute, h e, Hayward, CA). Intestinal tissue for RNA preparation from patients diagnosed as having Crohn's disease and ulcerative colitis was obtained from the National Disease Research Interchange (NDRI) (Philadelphia, PA).
Astrocytes, lung fibroblasts, dermal fibroblasts, coronary artery smooth muscle cells, small airway epithelium, bronchial epithelium, microvascular dermal endothelial cells, microvascular lung endothelial cells, human pulmonary aortic endothelial cells, human umbilical vein endothelial cells were all purchased from Clonetics (Walkersville, MD) and grown in the media supplied for these cell types by Clonetics. These primary cell types were activated with various cytokines or combinations of cytokines for 6 and/or 12-14 hours, as indicated. The following cytokines were used; EL-1 beta at approximately l-5ng/ml, TNF alpha at approximately 5-lOng/ml, IFN gamma at approximately 20-50ng/ml, IL-4 at approximately 5-lOng/ml, IL-9 at approximately 5-lOng/ml, EL- 13 at approximately 5-lOng/ml. Endothelial cells were sometimes starved for various times by culture in the basal media from Clonetics with 0.1% serum.
Mononuclear cells were prepared from blood of employees at CuraGen Coφoration, using Ficoll. LAK cells were prepared from these cells by culture in DMEM 5% FCS (Hyclone), lOOμM non essential amino acids (Gibco/Life Technologies, Rockville, MD), ImM sodium pyravate (Gibco), mercaptoethanol 5.5xlO"5M (Gibco), and lOmM Hepes (Gibco) and Interleukin 2 for 4-6 days. Cells were then either activated with 10-20ng/ml PMA and l-2μg/ml ionomycin, IL-12 at 5-lOng/ml, IFN gamma at 20-50ng/ml and EL-18 at 5-lOng/ml for 6 hours. In some cases, mononuclear cells were cultured for 4-5 days in DMEM 5% FCS (Hyclone), lOOμM non essential amino acids (Gibco), ImM sodium pyravate (Gibco), mercaptoethanol 5.5xlO"5M (Gibco), and lOmM Hepes (Gibco) with PHA (phytohemagglutinin) or PWM (pokeweed mitogen) at approximately 5 μg/ml. Samples were taken at 24, 48 and 72 hours for RNA preparation. MLR (mixed lymphocyte reaction) samples were obtained by taking blood from two donors, isolating the mononuclear cells using Ficoll and mixing the isolated mononuclear cells 1:1 at a final concentration of approximately 2x106cells/ml in DMEM 5%> FCS (Hyclone), 1 OOμM non essential amino acids (Gibco), ImM sodium pyravate (Gibco), mercaptoethanol (5.5xlO"5M) (Gibco), and lOmM Hepes (Gibco). The MLR was cultured and samples taken at various time points ranging from 1- 7 days for RNA preparation. Monocytes were isolated from mononuclear cells using CD 14 Miltenyi Beads, +ve VS selection columns and a Vario Magnet according to the manufacturer's instructions. Monocytes were differentiated into dendritic cells by culture in DMEM 5% fetal calf serum (FCS) (Hyclone, Logan, UT), lOOμM non essential amino acids (Gibco), ImM sodium pyravate (Gibco), mercaptoethanol 5.5xlO"5M (Gibco), and lOmM Hepes (Gibco), 50ng/ml GMCSF and 5ng/ml IX- 4 for 5-7 days. Macrophages were prepared by culture of monocytes for 5-7 days in DMEM 5% FCS (Hyclone), lOOμM non essential amino acids (Gibco), ImM sodium pyravate (Gibco), mercaptoethanol 5.5xlO"5M (Gibco), lOmM Hepes (Gibco) and 10% AB Human Serum or MCSF at approximately 50ng/ml. Monocytes, macrophages and dendritic cells were stimulated for 6 and 12-14 hours with lipopolysaccharide (LPS) at lOOng/ml. Dendritic cells were also stimulated with anti-CD40 monoclonal antibody (Pharmingen) at 10 μg/ml for 6 and 12-14 hours.
CD4 lymphocytes, CD8 lymphocytes and NK cells were also isolated from mononuclear cells using CD4, CD8 and CD56 Miltenyi beads, positive VS selection columns and a Vario Magnet according to the manufacturer's instructions. CD45RA and CD45RO CD4 lymphocytes were isolated by depleting mononuclear cells of CD8, CD56, CD 14 and CD 19 cells using CD8, CD56, CD 14 and CD 19 Miltenyi beads and positive selection. CD45RO beads were then used to isolate the CD45RO CD4 lymphocytes with the remaining cells being CD45RA CD4 lymphocytes. CD45RA CD4, CD45RO CD4 and CD8 lymphocytes were placed in DMEM 5% FCS (Hyclone), lOOμM non essential amino acids (Gibco), ImM sodium pyravate (Gibco), mercaptoethanol 5.5x10" M (Gibco), and lOmM Hepes (Gibco) and plated at 10 cells/ml onto Falcon 6 well tissue culture plates that had been coated overnight with 0.5 μg/ml anti-CD28 (Pharmingen) and 3ug/ml anti-CD3 (OKT3, ATCC) in PBS. After 6 and 24 hours, the cells were harvested for RNA preparation. To prepare chronically activated CD8 lymphocytes, we activated the isolated CD8 lymphocytes for 4 days on anti-CD28 and anti-CD3 coated plates and then harvested the cells and expanded them in DMEM 5% FCS (Hyclone), lOOμM non essential amino acids (Gibco), ImM sodium pyravate (Gibco), mercaptoethanol 5.5xlO"5M (Gibco), and lOmM Hepes (Gibco) and IL-2. The expanded CD8 cells were then activated again with plate bound anti-CD3 and anti-CD28 for 4 days and expanded as before. RNA was isolated 6 and 24 hours after the second activation and after 4 days of the second expansion culture. The isolated NK cells were cultured in DMEM 5% FCS (Hyclone), lOOμM non essential amino acids (Gibco), ImM sodium pyruvate (Gibco), mercaptoethanol 5.5x10"5M (Gibco), and lOmM Hepes (Gibco) and IL- 2 for 4-6 days before RNA was prepared.
To obtain B cells, tonsils were procured from NDRI. The tonsil was cut up with sterile dissecting scissors and then passed through a sieve. Tonsil cells were then spun down and resupended at 106cells/ml in DMEM 5%> FCS (Hyclone), lOOμM non essential amino acids (Gibco), ImM sodium pyravate (Gibco), mercaptoethanol 5.5xlO"5M (Gibco), and lOmM Hepes (Gibco). To activate the cells, we used PWM at 5 μg/ml or anti-CD40 (Pharmingen) at approximately 1 Oμg/ml and IL-4 at 5-lOng/ml. Cells were harvested for RNA preparation at 24,48 and 72 hours.
To prepare the primary and secondary Thl/Th2 and Trl cells, six-well Falcon plates were coated overnight with 1 Oμg/ml anti-CD28 (Pharmingen) and 2μg/ml OKT3 (ATCC), and then washed twice with PBS. Umbilical cord blood CD4 lymphocytes (Poietic Systems, German Town, MD) were cultured at 105-106cells/ml in DMEM 5% FCS (Hyclone), lOOμM non essential amino acids (Gibco), ImM sodium pyravate (Gibco), mercaptoethanol 5.5xlO"5M (Gibco), lOmM Hepes (Gibco) and IL-2 (4ng/ml). IL-12 (5ng/ml) and anti-IL4 (1 μg/ml) were used to direct to Thl, while IL-4 (5ng/ml) and anti-IFN gamma (lμg/ml) were used to direct to Th2 and IL-10 at 5ng/ml was used to direct to Trl. After 4-5 days, the activated Thl, Th2 and Trl lymphocytes were washed once in DMEM and expanded for 4-7 days in DMEM 5% FCS (Hyclone), lOOμM non essential amino acids (Gibco), ImM sodium pyravate (Gibco), mercaptoethanol 5.5x10" M (Gibco), lOmM Hepes (Gibco) and IL-2 (lng/ml). Following this, the activated Thl, Th2 and Trl lymphocytes were re-stimulated for 5 days with anti-CD28/OKT3 and cytokines as described above, but with the addition of anti-CD95L (lμg/ml) to prevent apoptosis. After 4-5 days, the Thl, Th2 and Trl lymphocytes were washed and then expanded again with IL-2 for 4-7 days. Activated Thl and Th2 lymphocytes were maintained in this way for a maximum of three cycles. RNA was prepared from primary and secondary Thl, Th2 and Trl after 6 and 24 hours following the second and third activations with plate bound anti-CD3 and anti-CD28 mAbs and 4 days into the second and third expansion cultures in Interleukin 2.
The following leukocyte cells lines were obtained from the ATCC: Ramos, EOL-1, KU-812. EOL cells were further differentiated by culture in O.lmM dbcAMP at 5xl05cells/ml for 8 days, changing the media every 3 days and adjusting the cell concenfration to 5xl05cells/ml. For the culture of these cells, we used DMEM or RPMI (as recommended by the ATCC), with the addition of 5% FCS (Hyclone), lOOμM non essential amino acids (Gibco), ImM sodium pyruvate (Gibco), mercaptoethanol 5.5xlO"5M (Gibco), lOmM Hepes (Gibco). RNA was either prepared from resting cells or cells activated with PMA at lOng/ml and ionomycin at lμg/ml for 6 and 14 hours. Keratinocyte line CCD106 and an airway epithelial tumor line NCI-H292 were also obtained from the ATCC. Both were cultured in DMEM 5% FCS (Hyclone), lOOμM non essential amino acids (Gibco), ImM sodium pyravate (Gibco), mercaptoethanol 5.5xlO"5M (Gibco), and lOmM Hepes (Gibco). CCD1106 cells were activated for 6 and 14 hours with approximately 5 ng/ml TNF alpha and lng/ml IL-1 beta, while NCI-H292 cells were activated for 6 and 14 hours with the following cytokines: 5ng/ml IL-4, 5ng/ml IL-9, 5ng/ml IL-13 and 25ng/ml EFN gamma.
For these cell lines and blood cells, RNA was prepared by lysing approximately 107cells/ml using Trizol (Gibco BRL). Briefly, 1/10 volume of bromochloropropane (Molecular Research Coφoration) was added to the RNA sample, vortexed and after 10 minutes at room temperature, the tubes were spun at 14,000 rpm in a Sorvall SS34 rotor. The aqueous phase was removed and placed in a 15ml Falcon Tube. An equal volume of isopropanol was added and left at -20°C overnight. The precipitated RNA was spun down at 9,000 φm for 15 min in a Sorvall SS34 rotor and washed in 70% ethanol. The pellet was redissolved in 300μl of RNAse-free water and 35μl buffer (Promega) 5μl DTT, 7μl RNAsin and 8μl DNAse were added. The tube was incubated at 37°C for 30 minutes to remove contaminating genomic DNA, extracted once with phenol chloroform and re-precipitated with 1/10 volume of 3M sodium acetate and 2 volumes of 100% ethanol. The RNA was spun down and placed in RNAse free water. RNA was stored at -80°C.
Al comprehensive panel vl.O
The plates for Al_comprehensive panel_vl.O include two control wells and 89 test samples comprised of cDNA isolated from surgical and postmortem human tissues obtained from the Backus Hospital and Clinomics (Frederick, MD). Total RNA was extracted from tissue samples from the Backus Hospital in the Facility at CuraGen. Total RNA from other tissues was obtained from Clinomics.
Joint tissues including synovial fluid, synovium, bone and cartilage were obtained from patients undergoing total knee or hip replacement surgery at the Backus Hospital. Tissue samples were immediately snap frozen in liquid nitrogen to ensure that isolated RNA was of optimal quality and not degraded. Additional samples of osteoarthritis and rheumatoid arthritis joint tissues were obtained from Clinomics. Normal control tissues were supplied by Clinomics and were obtained during autopsy of trauma victims.
Surgical specimens of psoriatic tissues and adjacent matched tissues were provided as total
RNA by Clinomics. Two male and two female patients were selected between the ages of 25 and 47. None of the patients were taking prescription drugs at the time samples were isolated.
Surgical specimens of diseased colon from patients with ulcerative colitis and Crohns disease and adjacent matched tissues were obtained from Clinomics. Bowel tissue from three female and three male Crohn's patients between the ages of 41-69 were used. Two patients were not on prescription medication while the others were taking dexamethasone, phenobarbital, or tylenol. Ulcerative colitis tissue was from three male and four female patients. Four of the patients were taking lebvid and two were on phenobarbital.
Total RNA from post mortem lung tissue from trauma victims with no disease or with emphysema, asthma or COPD was purchased from Clinomics. Emphysema patients ranged in age from 40-70 and all were smokers, this age range was chosen to focus on patients with cigarette- linked emphysema and to avoid those patients with alpha- lanti-trypsin deficiencies. Asthma patients ranged in age from 36-75, and excluded smokers to prevent those patients that could also have COPD. COPD patients ranged in age from 35-80 and included both smokers and non- smokers. Most patients were taking corticosteroids, and bronchodilators.
In the labels employed to identify tissues in the AI_comprehensive panel_vl.O panel, the following abbreviations are used:
At = Autoimmunity
Syn = Synovial Normal = No apparent disease
Rep22 /Rep20 = individual patients
RA = Rheumatoid artliritis
Backus = From Backus Hospital
OA = Osteoarthritis (SS) (BA) (MF) = Individual patients
Adj = Adjacent tissue Match confrol = adjacent tissues
-M = Male
-F = Female
COPD = Chronic obstructive pulmonary disease
Panels 5D and 51
The plates for Panel 5D and 51 include two confrol wells and a variety of cDNAs isolated from human tissues and cell lines with an emphasis on metabolic diseases. Metabolic tissues were obtained from patients enrolled in the Gestational Diabetes study. Cells were obtained during different stages in the differentiation of adipocytes from human mesenchymal stem cells. Human pancreatic islets were also obtained.
In the Gestational Diabetes study subjects are young (18 - 40 years), otherwise healthy women with and without gestational diabetes undergoing routine (elective) Caesarean section. After delivery of the infant, when the surgical incisions were being repaired/closed, the obstetrician removed a small sample (<1 cc) of the exposed metabolic tissues during the closure of each surgical level. The biopsy material was rinsed in sterile saline, blotted and fast frozen within 5 minutes from the time of removal. The tissue was then flash frozen in liquid nitrogen and stored, individually, in sterile screw-top tubes and kept on dry ice for shipment to or to be picked up by CuraGen. The metabolic tissues of interest include uterine wall (smooth muscle), visceral adipose, skeletal muscle (rectus) and subcutaneous adipose. Patient descriptions are as follows:
Patient 2: Diabetic Hispanic, overweight, not on insulin
Patient 7-9: Nondiabetic Caucasian and obese (BMI>30)
Patient 10: Diabetic Hispanic, overweight, on insulin
Patient 11 : Nondiabetic African American and overweight Patient 12: Diabetic Hispanic on insulin
Adipocyte differentiation was induced in donor progenitor cells obtained from Osiras (a division of Clonetics/BioWhittaker) in triplicate, except for Donor 3U which had only two replicates. Scientists at Clonetics isolated, grew and differentiated human mesenchymal stem cells (HuMSCs) for CuraGen based on the published protocol found in Mark F. Pittenger, et al., Multilineage Potential of Adult Human Mesenchymal Stem Cells Science Apr 2 1999: 143-147. Clonetics provided Trizol lysates or frozen pellets suitable for mRNA isolation and ds cDNA production. A general description of each donor is as follows: Donor 2 and 3 U: Mesenchymal Stem cells, Undifferentiated Adipose Donor 2 and 3 AM: Adipose, AdiposeMidway Differentiated Donor 2 and 3 AD: Adipose, Adipose Differentiated
Human cell lines were generally obtained from ATCC (American Type Culture Collection), NCI or the German tumor cell bank and fall into the following tissue groups: kidney proximal convoluted tubule, uterine smooth muscle cells, small intestine, liver HepG2 cancer cells, heart primary stromal cells, and adrenal cortical adenoma cells. These cells are all cultured under standard recommended conditions and RNA extracted using the standard procedures. All samples were processed at CuraGen to produce single stranded cDNA.
Panel 51 contains all samples previously described with the addition of pancreatic islets from a 58 year old female patient obtained from the Diabetes Research Institute at the University of Miami School of Medicine. Islet tissue was processed to total RNA at an outside source and delivered to CuraGen for addition to panel 51.
In the labels employed to identify tissues in the 5D and 51 panels, the following abbreviations are used:
GO Adipose = Greater Omentum Adipose SK = Skeletal Muscle UT = Uteras PL = Placenta AD = Adipose Differentiated
AM = Adipose Midway Differentiated U = Undifferentiated Stem Cells
Panel CNSD.01
The plates for Panel CNSD.01 include two control wells and 94 test samples comprised of cDNA isolated from postmortem human brain tissue obtained from the Harvard Brain Tissue
Resource Center. Brains are removed from calvaria of donors between 4 and 24 hours after death, sectioned by neuroanatomists, and frozen at -80°C in liquid nitrogen vapor. All brains are sectioned and examined by neuropathologists to confirm diagnoses with clear associated neuropathology.
Disease diagnoses are taken from patient records. The panel contains two brains from each of the following diagnoses: Alzheimer's disease, Parkinson's disease, Huntington's disease, Progressive Supernuclear Palsy, Depression, and "Normal controls". Within each of these brains, the following regions are represented: cingulate gyras, temporal pole, globus palladus, substantia nigra, Brodman Area 4 (primary motor strip), Brodman Area 7 (parietal cortex), Brodman Area 9 (prefrontal cortex), and Brodman area 17 (occipital cortex). Not all brain regions are represented in all cases; e.g., Huntington's disease is characterized in part by neurodegeneration in the globus palladus, thus this region is impossible to obtain from confirmed Huntington's cases. Likewise Parkinson's disease is characterized by degeneration of the substantia nigra making this region more difficult to obtain. Normal control brains were examined for neuropathology and found to be free of any pathology consistent with neurodegeneration.
In the labels employed to identify tissues in the CNS panel, the following abbreviations are used:
PSP = Progressive supranuclear palsy Sub Nigra = Substantia nigra Glob Palladus= Globus palladus Temp Pole = Temporal pole Cing Gyr = Cingulate gyras
BA 4 = Brodman Area 4
Panel CNSJNeurodegeneration Vl.O
The plates for Panel CNS_Neurodegeneration_V1.0 include two control wells and 47 test samples comprised of cDNA isolated from postmortem human brain tissue obtained from the Harvard Brain Tissue Resource Center (McLean Hospital) and the Human Brain and Spinal Fluid Resource Center (NA Greater Los Angeles Healthcare System). Brains are removed from calvaria of donors between 4 and 24 hours after death, sectioned by neuroanatomists, and frozen at -80°C in liquid nitrogen vapor. All brains are sectioned and examined by neuropathologists to confirm diagnoses with clear associated neuropathology.
Disease diagnoses are taken from patient records. The panel contains six brains from
Alzheimer's disease (AD) patients, and eight brains from "Normal controls" who showed no evidence of dementia prior to death. The eight normal control brains are divided into two categories: Controls with no dementia and no Alzheimer's like pathology (Controls) and controls with no dementia but evidence of severe Alzheimer's like pathology, (specifically senile plaque load rated as level 3 on a scale of 0-3; 0 = no evidence of plaques, 3 = severe AD senile plaque load). Within each of these brains, the following regions are represented: hippocampus, temporal cortex (Brodman Area 21), parietal cortex (Brodman area 7), and occipital cortex (Brodman area 17). These regions were chosen to encompass all levels of neurodegeneration in AD. The hippocampus is a region of early and severe neuronal loss in AD; the temporal cortex is known to show neurodegeneration in AD after the hippocampus; the parietal cortex shows moderate neuronal death in the late stages of the disease; the occipital cortex is spared in AD and therefore acts as a "confrol" region within AD patients. Not all brain regions are represented in all cases.
hi the labels employed to identify tissues in the CNS_Neurodegeneration_N1.0 panel, the following abbreviations are used:
AD = Alzheimer's disease brain; patient was demented and showed AD-like pathology upon autopsy
Control - Control brains; patient not demented, showing no neuropathology
Control (Path) = Control brains; pateint not demented but showing sever AD-like pathology
SupTemporal Ctx = Superior Temporal Cortex
Inf Temporal Ctx = Inferior Temporal Cortex
NO VI: CG56181-01: Neurotrophin - isoform 1
Expression of gene CG56181-01 was assessed using the primer-probe set Ag2943, described in Table AA.
Table AA. Probe Name Ag2943
Figure imgf000402_0001
CNS_neurodegeneration_vl.O Summary: Ag2943 Expression of the CG56181-01 gene is low/undetectable in all samples on this panel (CTs>35).
Panel 1.3D Summary: Ag2943 Expression of the CG56181-01 gene is low/undetectable in all samples on this panel (CTs>35).
Panel 4D Summary: Ag2943 Expression of the CG56181-01 gene is low/undetectable in all samples on this panel (CTs>35).
NOV2: CG56275-01: guanylate kinase Expression of gene CG56275-01 was assessed using the primer-probe set Ag2944, described in Table BA. Results of the RTQ-PCR runs are shown in Tables BB, BC, BD and BE.
Table BA. Probe Name Ag2944
Figure imgf000403_0001
Table BB. Panel 1.3D
Figure imgf000403_0002
Figure imgf000404_0001
Figure imgf000405_0001
Figure imgf000406_0001
Figure imgf000407_0001
Table BD. Panel 3D
Figure imgf000408_0001
Figure imgf000409_0001
Figure imgf000410_0001
Figure imgf000411_0001
Figure imgf000412_0001
Panel 1.3D Summary: Ag2944 Two experiments with the same probe and primer set produce results that are in excellent agreement, with significant expression of the CG56275-01 gene restricted to cancer cell lines. Highest expression of this gene is seen in a cluster of brain cancer lines (CTs=32-34). Moderate levels of expression are also detected in a melanoma cell line. Thus, expression of this gene could be used to differentiate these samples from other samples on this panel and as a marker to detect the presence of these cancers. The protein encoded by this gene is homologous to guanylate kinase, an important enzyme in nucleotide metabolic pathways. This molecule is a known target for several chemotherapeutic agents. Therefore, therapeutic modulation of the expression or function of this novel gene could be effective in the treatment of brain cancer and melanoma.
References:
Stolworthy TS, Black ME. The mouse guanylate kinase double mutant E72Q/D103N is a functional adenylate kinase. Protein Eng 2001 Nov;14(ll):903-909.
Guanylate kinase catalyzes the phosphorylation of either GMP to GDP or dGMP to dGDP and is an important enzyme in nucleotide metabolic pathways. Because of its essential intracellular role, guanylate kinase is a target for a number of cancer chemotherapeutic agents such as 6-thioguanine and 8-azaguanine and is involved in antiviral drug activation. Guanylate kinase shares a similarity in function and structure to other nucleoside monophosphate kinases especially with that of the well-studied adenylate kinase. Amino acid substitutions were made within the GMP binding site of mouse guanylate kinase to alter the polarity of the side chains that interact with GMP as a means of evaluating the role that these residues play on substrate interaction. One of these mutants, E72Q/D103N, was shown by functional complementation and enzyme assays to embody both guanylate kinase activity and a novel adenylate kinase activity.
PMID: 11742110 Hoover KB, Liao SY, Bryant PJ. Loss of the tight junction MAGUK ZO-1 in breast cancer: relationship to glandular differentiation and loss of heterozygosity. Am J Pathol 1998 Dec;153(6):1767-73
Membrane-associated guanylate kinase homologs (MAGUKs) may play a role in cellular functions preventing tumorigenesis as indicated by the neoplastic phenotype caused by genetic loss of the MAGUK Dig in Drosophila. To test this possibility, we examined the expression and subcellular localization of the tight junction MAGUK ZO-1, as well as the cell adhesion molecule E-cadherin, in paraffin-embedded breast cancer samples, using immunohistochemistry and confocal microscopy. As expected, normal tissue showed intense staining for ZO-1 at the position of the epithelial tight junctions, but this staining was reduced or lost in 69% of breast cancers analyzed (n = 48). hi infiltrating ductal carcinomas (n = 38) there was a reduction in staining in 42% of well differentiated, in 83% of moderately differentiated and 93% of poorly differentiated tumors. ZO-1 staining was positively conelated with tumor differentiation (P = .011) and more specifically with the glandular differentiation of tumors (P = .0019). Reduction in ZO-1 staining was strongly conelated with reduced E-cadherin staining (P = 4.9 x 10(-5)). The results suggest that down-regulation of ZO-1 expression and its failure to accumulate at cell junctions maybe causally related to cancer progression. To detect loss of heterozygosity, the ZO-1 gene tjp-1 was mapped relative to other markers in 15ql3 and polymorphic markers flanking tjp-1 were identified. The marker D15S1019 showed loss of heterozygosity in 23% of informative tumors (n = 13). Loss of a tjp-1 -linked marker suggests that genetic loss may, in some cases, be responsible for the reduction in ZO-1 expression in breast cancer.
PMID: 9846967
Panel 2D Summary: Ag2944 Significant expression of the CG56275-01 gene is restricted to a sample derived from an ocular melanoma metastasis to the liver (CT=34). Thus, expression of this gene could be used to differentiate between this sample and other samples on this panel and as a marker to detect this form of cancer.
Panel 3D Summary: Ag2944 Highest expression of the CG56275-01 gene in this panel is seen in an ovarian cancer cell line (CT=30.10). Significant expression is also seen in a gastric cancer cell line and a lung cancer cell line. Thus, expression of this gene could be used to differentiate between these samples and other samples on this panel.
Panel 4D Summary: Ag2944 The CG56275-01 transcript is expressed in fibroblasts and endothelial cells regardless of freatment. This transcript encodes a putative guanylate kinase that may be needed for the normal function of the cells that express this protein. Thus, the transcript or the protein it encodes could be used to identify endothelium or fibroblasts. Furthermore, regulation of the transcript or the protem it encodes could be important in maintaining normal cellular homeostasis and in the treatment of inflammation, asthma, emphysema, arthritis, IBD or psoriasis.
NOV3a: CG53400-01 : Hypothetical 85.6 kDa human protein
Expression of gene CG53400-01 was assessed using the primer-probe set Ag2579, described in Table CA. Results of the RTQ-PCR runs are shown in Tables CB, CC, CD and CE.
Table CA. Probe Name Ag2579
Figure imgf000414_0001
Table CB. CNS_neurodegeneration_vl.O
Figure imgf000414_0002
Figure imgf000415_0001
Table CC. Panel 1.3D
Figure imgf000415_0002
Figure imgf000416_0001
Figure imgf000417_0001
Table CD. Panel 2.2
Figure imgf000417_0002
Figure imgf000418_0001
Figure imgf000419_0001
Figure imgf000420_0001
Figure imgf000421_0001
CNS_neurodegeneration_vl.O Summary: Ag2579 Expression of the CG53400-01 gene does not appear to show an association with Alzheimer's disease in this panel. However, this panel confirms the expression of this gene in the brain. Please see Panel 1.3D for discussion of utility of this gene in the cenfral nervous system.
Panel 1.3D Summary: Ag2579 Expression of the CG53400-01 gene is ubiquitous in this panel, with highest expression in the ascites derived ovarian cancer cell line SK-OV-3 (CT=28.5). There is also significant expression of this gene in a cluster of cell lines derived from renal cancer and melanoma. The widespread expression of this gene suggests that the gene product may be involved in cell differentiation and growth. Thus, expression of this gene could be used to differentiate between the samples mentioned above and other samples on this panel. Expression of this gene could also potentially be used as a marker for ascites derived ovarian cancer, ascites derived tissue samples, melanoma and renal cancer. Furthermore, therapeutic modulation of the expression or function of this gene may be effective in the treatment of these cancers.
This gene is also widely expressed among tissues with metabolic function, including adipose, adult and fetal skeletal muscle and heart, the pancreas, fetal liver, and the adrenal, thyroid, and pituitary glands. This expression profile suggests that this gene product may also be involved in metabolic function and that therapeutic modulation of the expression or function of this gene may be effective in the treatment of metabolic disorders, such as obesity and diabetes.
In addition, this gene appears to be expressed at much higher levels in fetal liver (CT=32) than in adult liver (CT=35). Thus, expression of this gene could be used to differentiate between adult and fetal sources of liver tissue.
The expression profile of this gene also shows widespread expression of this gene in the brain. This suggests that the protein encoded by this gene may be important for normal neurological function. Therefore, modulation of the function or expression of this gene maybe effective in the treatment of neurodegenerative disorders, such as Alzheimer's disease and Parkinson's disease.
Panel 2.2 Summary: Ag2579 Expression of the CG53400-01 gene is widespread in this panel, with highest expression in prostate cancer (CT=30.6). Furthermore, expression in prostate cancer is significantly higher than expression in the conesponding normal adjacent tissue. Conversely, expression of this gene is higher in normal kidney than in adjacent kidney tumor. Thus, expression of this gene could be used as a marker for kidney or prostate cancer. In addition, therapeutic modulation of the expression or function of this gene could be used in the treatment of kidney or prostate cancer. Panel 4D Summary: Ag2579 The CG53400-01 gene is ubiquitously expressed in this panel, with highest in dermal fibroblasts treated with TNF-alpha (CT=26.3). Significant expression is also seen in untreated dermal fibroblasts and PBMC treated with the B the B cell mitogen, PWM. The expression of this gene in activated dermal fibroblast combined with moderate expression in the mucoepidermoid cell line H292, often used as a model for airway epithelium, suggest that therapeutic modulation of this gene might also be useful in the treatment of asthma and emphysema. In addition, the high levels of expression of this gene in activated B cells are significant because B cells represent a principle component of immunity and contribute to the immune response in a number of important functional roles, including antibody production. Furthermore, production of antibodies against self-antigens is a major component in autoimmune disorders
Since B cells play an important role in autoimmunity, inflammatory processes and inflammatory cascades, therapeutic modulation of this gene product may therefore, reduce or eliminate the symptoms of patients suffering from asthma, allergies, chronic obstructive pulmonary disease, emphysema, Crohn's disease, ulcerative colitis, rheumatoid arthritis, psoriasis, osteoarthritis, and other autoimmune disorders including systemic lupus erythematosus.
NOV4a: CG56209-01: MYTONIC DYSTROPHY KINASE-RELATED CDC42-BINDING KINASE
Expression of gene CG56209-01 was assessed using the primer-probe set Ag4976, described in Table DA.
Table DA. Probe Name Ag4976
Figure imgf000423_0001
CNS_neurodegeneration_vl.0 Summary: Ag4976 Expression of the CG56209-01 gene is low/undetectable in all samples on this panel. (CTs>35). (Data not shown.) The amp plot indicates that there is a high probability of a probe failure in this experiment. General_screening_panel_vl.5 Summary: Ag4976 Expression of the CG56209-01 gene is low/undetectable in all samples on this panel. (CTs>35). (Data not shown.) The amp plot indicates that there is a high probability of a probe failure in this experiment.
Panel 4.1D Summary: Ag4976 Expression of the CG56209-01 gene is low/undetectable in all samples on this panel. (CTs>35). (Data not shown.) The amp plot indicates that there is a high probability of a probe failure in this experiment.
NOV7a and NOV7b: CG50365-01 and CG50365-02: Carbonate Dehydratase
Expression of gene CG50365-01 and variant CG50365-02 was assessed using the primer- probe sets Ag2644 and Ag2575, described in Tables EA and EB. Results of the RTQ-PCR runs are shown in Tables EC, ED, EE, EF, and EG.
Table EA. Probe Name Ag2644
Figure imgf000424_0001
Table EB. Probe Name Ag2575
Figure imgf000424_0002
Table EC. CNS_neurodegeneration_vl.O
Figure imgf000424_0003
Figure imgf000425_0001
Table ED. Panel 1.3D
Tissue Name Rel. Exp.(%) Rel. Exp.(%) Rel. Exp.(%) Rel. Exp.(%)
Tissue Name Ag2575, Run Ag2575, Run Ag2575, Run Ag2575, Run
Figure imgf000426_0001
Figure imgf000427_0001
Figure imgf000428_0001
Table EE. Panel 2D
Figure imgf000428_0002
Figure imgf000429_0001
Figure imgf000430_0001
Table EF. Panel 3D
Figure imgf000430_0002
Figure imgf000431_0001
Figure imgf000432_0001
Figure imgf000433_0001
Figure imgf000434_0001
CNS_neurodegeneration_vl.0 Summary: Ag2644 This panel does not show differential expression of the CG50365-01 gene in Alzheimer's disease. However, this expression profile confirms the presence of this gene in the brain. Please see Panel 1.3D for discussion of utility of this gene in the cenfral nervous system.
Panel 1.3D Summary: Ag2575 The expression of the CG50365-01 gene was assessed in two independent runs on panel 1.3D with excellent concordance between runs. The expression of this gene appears to be highest in a sample derived from a gastric cancer cell line (NCI- H87)(CTs=31). hi addition, there is substantial expression in several colon cancer cell lines, ovarian cancer cell lines and brain cancer cell lines. Thus, the expression of this gene could be used to distinguish NCI-H87 cells from other samples in the panel. Moreover, therapeutic modulation of this gene, through the use of small molecule drags, antibodies or protein therapeutics might be of benefit in the treatment of colon cancer, brain cancer or ovarian cancer.
hi addition, this gene is expressed at low levels in the cerebral cortex. Carbonate dehydratase may play an important role in modulating excitatory synaptic transmission in brain. Therefore, this molecule maybe of use in the treatment of schizophrenia, epilepsy, Alzheimer's disease, bipolar disorder, depression, or any clinical condition associated with impaired or altered neurotransmission.
References:
Parkkila S, Parkkila AK, Rajaniemi H, Shah GN, Grabb JH, Waheed A, Sly WS. Expression of membrane-associated carbonic anhydrase XIV on neurons and axons in mouse and human brain. Proc Natl Acad Sci U S A 2001 Feb 13;98(4):1918-23
Although long suspected from histochemical evidence for carbonic anhydrase (CA) activity on neurons and observations that CA inhibitors enhance the extracellular alkaline shifts associated with synaptic transmission, an extracellular CA in brain had not been identified. A candidate for this CA was suggested by the recent discovery of membrane CA (CA XIV) whose mRNA is expressed in mouse and human brain and in several other tissues. For immunolocalization of CA XIV in mouse and human brain, we developed two antibodies, one against a secretory form of enzymatically active recombinant mouse CA XIV, and one against a synthetic peptide conesponding to the 24 C-terminal amino acids in the human enzyme, hnmunostaining for CA XIV was found on neuronal membranes and axons in both mouse and human brain. The highest expression was seen on large neuronal bodies and axons in the anterolateral part of pons and medulla oblongata. Other CA XIN-positive sites included the hippocampus, corpus callosum, cerebellar white matter and peduncles, pyramidal tract, and choroid plexus. Mouse brain also showed a positive reaction in the molecular layer of the cerebral cortex and granular cellular layer of the cerebellum. These observations make CA XIV a likely candidate for the extracellular CA postulated to have an important role in modulating excitatory synaptic transmission in brain. Panel 2D Summary: Ag2644 The expression of the CG50365-01 gene appears to be highest in a sample derived from a gastric cancer. In addition there is substantial expression associated with other gastric cancers, when compared to their adjacent normal tissues, as well as expression associated with ovarian cancer, breast cancer, thyroid cancer and colon cancer. This expression conforms with expression in Panel 1.3D. Thus, the expression of this gene could be used to used to distinguish this gastric cancer sample from other samples in the panel. Moreover, therapeutic modulation of this gene, through the use of small molecule drugs, antibodies or protein therapeutics might be of benefit in the treatment of colon cancer, breast cancer, ovarian cancer, gastric cancer or thyroid cancer.
Panel 3D Summary: Ag2644 The expression of the CG50365-01 gene appears to be highest in a sample derived from a lung cancer cell line (DMS-79). In addition there appears to be expression associated with a colon cancer cell line, a gastric cancer cell line and a pancreatic cancer cell line. Thus, the expression of this gene could be used to distinguish DMS-79 cells from other samples in the panel. Moreover, therapeutic modulation of this gene, through the use of small molecule drugs, antibodies or protein therapeutics might be of benefit in the treatment of colon cancer, pancreatic cancer, gastric cancer or lung cancer.
Panel 4D Summary: Ag2644 The CG50365-01 transcript is expressed in lung fibroblasts treated with gamma interferon, NCI-H292 cells regardless of treatment, activated basophil cell line, and gamma interferon treated HUVECs. It is also expressed in normal colon and thymus. The regulation of the transcript expression in fibroblasts and HUVECs suggests that the protein encoded by this transcript may be contribute to the inflammatory changes due to gamma interferon. Therefore, therapies designed with the protein encoded by this transcript could be important for the treatment of emphysema, psoriasis, arthritis and IBD.
Panel 5 Islet Summary: Ag2575 Expression of the CG50365-01 gene is low/undetectable in all samples on this panel (CTs>35).
NOV8a, NOV8b, and NOV8c: CG55794-01 and CG55794-03 and CG55794- 06: Carboxypeptidase
Expression of gene CG55794-01, variant CG55794-03, and splice variant CG55794-06 was assessed using the primer-probe sets Ag2622, Ag3953 arid Ag6049, described in Tables FA, FB and FC. Results of the RTQ-PCR runs are shown in Tables FD, FE, FF, FG, FH, FI, FJ and FK. Please note that the probe/primer set Ag6049 matches only the CG55794-06 variant. This does not change the results presented below.
Table FA. Probe Name Ag2622
Figure imgf000437_0001
Table FB. Probe Name Ag3953
Figure imgf000437_0002
Table FC. Probe Name Ag6049
Figure imgf000437_0003
Table FD. CNS_neurodegeneration_vl.O
Figure imgf000437_0004
Figure imgf000438_0001
Table FE. General_screening_panel_vl.4
Figure imgf000438_0002
Figure imgf000439_0001
Figure imgf000440_0001
Table FF. Panel 1.3D
Figure imgf000440_0002
Figure imgf000441_0001
Figure imgf000442_0001
Figure imgf000443_0001
Table FG. Panel 2D
Figure imgf000443_0002
Figure imgf000444_0001
Figure imgf000445_0001
Table FH. Panel 4D
Figure imgf000445_0002
Figure imgf000446_0001
Figure imgf000447_0001
Figure imgf000448_0001
Table FI. Panel 5 Islet
Figure imgf000448_0002
Figure imgf000449_0001
CNS_neurodegeneration_vl.O Summary: Ag2622 This panel confirms the expression of the CG55794-01 gene in the CNS; See panel 1.3d for a discussion of utility. Ag6049 Results from one experiment with the CG55794-06 gene are not included. The amp plot indicates that there were experimental difficulties with this run.
General_screening_panel_vl.4 Summary: Ag3953 Highest expression of the CG55794-01 gene is seen in an ovarian cancer cell ine (CT=33.1). Thus, expression of this gene could be used to differentiate between this sample and other samples on this panel. Please see Panel 1.3D for further discussion of the utility of this gene in cancer.
As in the previous panel, this gene is also expressed in the brain, including the cerebral cortex, substantia nigra and thalamus. Please see Panel 1.3D for discussion of utility of this gene in the cenfral nervous system.
General_screening_panel_vl.5 Summary: Ag6049 Results from one experiment with the CG55794-06 gene are not included. The amp plot indicates that there were experimental difficulties with this run.
Panel 1.3D Summary: Ag2622 Two experiments with the same probe and primer sets produce results that are in reasonable agreement, with highest expression of the CG55794-01 gene in the brain and the kidney, interestingly, there is significantly lower expression in the brain cancer cell lines than normal brain samples. This suggests that absence of this gene might be involved in cell proliferation. Hence this might be used as a diagnostic marker for brain cancer.
As seen in previous panels, the CG55794-01 gene is also expressed at low levels in the CNS. Carboxypeptidase is believed to have a role in the degradation of APP and A-beta, the major component of senile plaques in Alzheimer's disease. Therapeutic upregulation of this gene or its protein product may therefore be of benefit in the treatment of Alzheimer's disease.
References:
Matsumoto A, Itoh K, Matsumoto R. A novel carboxypeptidase B that processes native beta- amyloid precursor protein is present in human hippocampus. Eur J Neurosci 2000 Jan; 12(1):227- 38
The processing of beta- amyloid precursor protein (APP) and generation of beta-amyloid (Abeta) are associated with the pathophysiology of Alzheimer's disease (AD). As the proteases responsible for the process in the human brain have yet to be clarified, we have searched for activities capable of cleaving native brain APP in the human hippocampus. A 40-kDa protein with proteolytic activity that degrades native brain APP in vitro was purified and characterized; molecular analysis identified it as a novel protease belonging to the carboxypeptidase B (CPB) family. PC12 cells overexpressing the cDNA encoding this protease generate a major 12-kDa beta-amyloid-bearing peptide in cytosol, a peptide which has also been detected in a cell-free system using purified brain APP as substrate. Although the protease is homologous to plasma CPB synthesized in liver, it has specific domains such as C-terminal 14 amino acid residues. Western analysis, cDNA-cloning process and Northern analysis suggested a brain-specific expression of this protease. An immunohistochemical study showed that the protease is expressed in various neuronal perikarya, including those of pyramidal neurons of the hippocampus and ependymal-choroid plexus cells, and in a portion of the microglia of normal brains, h brains of patients with sporadic AD, there is decreased neuronal expression of the protease, and clusters of microglia with protease immunoreactivity associated with its extracellular deposition are detected. These findings suggest that brain CPB has a physiological function in APP processing and may have significance in AD pathophysiology.
Panel 2D Summary: Ag2622 The CG55794-01 gene is expressed at low levels in the tissues used for panel 2D, with reasonable concordance between the runs. There is increased expression in normal prostate and kidney compared to the adjacent tumor tissues. There is also increased expression in breast cancer tissues compared to normal breast tissue. Hence, expression of this gene can be used as a diagnostic marker in breast, prostate and kidney cancer. Furthermore, therapeutic modulation of the gene product might be of use in the treatment of these cancers.
Panel 3D Summary: Ag2622 Expression of the CG55794-01 gene is low/undetectable in all samples on this panel (CTs>35).
Panel 4D Summary: Ag2622 The CG55794-01 gene, which encodes a putative carboxypeptidase, is expressed in the colon and down regulated in colon tissue isolated from Crohn's and colitis patients. The carboxypeptidase family of enzymes has been found in the colon and is associated with colon disease (ref. below). Thus, the expression of the transcript or the protein it encodes could be used to detect normal colon tissue. Furthermore, therapeutics designed with the protein encoded for by this transcript could be important in the treatment of IBD. Ag6049 Results from one experiment with the CG55794-06 gene showed low/undetectable in all samples on Panel 4. ID. (CTs>35).
References: Sommer H, Schweisfurth H, Schulz M. Serum angiotensin-I-converting enzyme and carboxypeptidase N in Crohn's disease and ulcerative colitis. Enzyme 1986;35(4):181-8
Angiotensin-I-converting enzyme (ACE) and carboxypeptidase Nl and N2 (CPN1, CPN2) inactivate kinins and might therefore play a role in the development of inflammatory reactions via an influence on the release of prostaglandins and inactivation of anaphylatoxic peptides of the complement system. In the present study, the serum activity of these enzymes was determined in 60 patients with Crohn's disease, 18 patients with ulcerative colitis and 70 healthy control subjects. ACE was significantly lowered in active Crohn's disease (CDAI greater than 150) and in ulcerative colitis (p less than 0.01), as long as the ileum or cecum was affected. Since ACE was detected in high concentrations in the human intestinal mucosa, decreased values may be explained by damage to the site of its production. CPN1 and CPN2 were raised in both diseases (p less than 0.005), inespective of their activity and location. These alterations in the activity of the kininases investigated may play a role in the pathogenesis of inflammatory bowel diseases.
Panel 5 Islet Summary: Ag3953 The CG55794-01 gene, a carboxypeptidase homolog, has little to no expression in any of the endocrine/metabolically-related tissues except for small intestine. This expression profile is in agreement with the results from Panel 4D. Carboxypeptidase-B processing of GI peptides (e.g. GLP-2 and CCK) is critical for bioactivity. Thus, a therapeutic modulator of this gene and/or gene-product may prove useful in treating diseases associated with the GI tract and metabolism.
References:
Orskov C, Buhl T, Rabenhoj L, Kofod H, Hoist JJ. Carboxypeptidase-B-like processing of the C- terminus of glucagon-like peptide-2 in pig and human small intestine.
FEBS Lett 1989 Apr 24;247(2): 193-6
We developed specific, C-terminal radioimmunoassays for three proglucagon (PG) fragments: PG 151-158, PG 151-160 and PG 126-159 (glucagon-like peptide-2 (GLP-2] in order to determine the exact C-terminal sequence of the newly isolated GLP-2 in man and pig. The antigens and the antisera showed no mutual cross-reactivity. By gel filtration of extracts of pig and human small intestine, the immunoreactivity eluting at the position of GLP-2 was identified by the radioimmunoassays for glucagon-like peptide-2 (PG 126-159) and for PG 151-158, whereas the assay for PG 151-160 was completely negative. We conclude that the C-terminal amino acid residue of pig and human ileal GLP-2 is PG 158. Thus the basic residues, PG 159 and 160 are removed during its processing in the small intestine.
PMJO: 2714431
Blanke SE, Johnsen AH, Rehfeld JF. N-terminal fragments of intestinal cholecystokinin: evidence for release of CCK-8 by cleavage on the carboxyl side of Arg74 of proCCK. Regul Pept 1993 Jul 23;46(3):575-82
From porcine duodenal mucosa we have identified three maj or procholecystokinin (proCCK) fragments: desoctaCCK-33, desnonaCCK-33 and desnonaCCK-39. (DesoctaCCK-33 means CCK-33 devoid of the 8 C-terminal amino acids, etc.). The fragments were purified by immunoaffinity chromatography and three steps of reverse phase HPLC monitored by a radioimmunoassay specific for the N-terminal part of CCK-33. The structures could be deduced from the proCCK sequence by N-terminal sequence determination and mass spectromefry. Whereas desnona-fragments of CCK have been described before, this is the first finding of a desoctaCCK, and it indicates that CCK-8 is released from the longer forms by endogenous cleavage of the Arg- Asp-bond. A carboxypeptidase B-like exopeptidase subsequently must produce the desnona-fragments by removing the arginine residue.
NOV8d: CG55794-07: Splice variant of CG55794-01
Expression of gene CG55794-07 was assessed using the primer-probe sets Ag2622, Ag6050 and Ag3953, described in Tables GA, GB and GC. Results of the RTQ-PCR runs are shown in Tables GD, GE, GF, GG, GH, GI, GJ and GK.
Table GA. Probe Name Ag2622
Figure imgf000453_0001
Figure imgf000454_0001
Table GB. Probe Name Ag6050
Figure imgf000454_0002
Table GC. Probe Name Ag3953
Figure imgf000454_0003
Table GD. CNS_neurodegeneration_vl.O
Figure imgf000454_0004
Figure imgf000455_0001
Table GE. General_screening_panel_vl.4
Figure imgf000456_0001
Figure imgf000457_0001
Figure imgf000458_0001
Figure imgf000459_0001
Table GG. Panel 1.3D
Figure imgf000459_0002
Figure imgf000460_0001
Figure imgf000461_0001
Table GH. Panel 2D
Figure imgf000462_0001
Figure imgf000463_0001
Figure imgf000464_0001
Table GI. Panel 4. ID
Figure imgf000464_0002
Figure imgf000465_0001
Figure imgf000466_0001
Table GJ. Panel 4D
Figure imgf000466_0002
Figure imgf000467_0001
Figure imgf000468_0001
Figure imgf000469_0001
Table GK. Panel 5 Islet
Figure imgf000469_0002
Figure imgf000470_0001
CNS_neurodegeneration_vl.0 Summary: Ag2622/Ag6050 Two experiments with two different probe and primer sets confirm the expression of the CG55794-07 gene in the CNS; See panel 1.3d for a discussion of utility of this gene in the central nervous system.
General_screening_panel_vl.4 Summary/General_screening_panel_vl.5
Summary: Ag3953/Ag6050 Two experiments with two different probe and primer sets show highest expression of the CG55794-07 gene is seen in an ovarian cancer cell line (CT=33.1).
Thus, expression of this gene could be used to differentiate between this sample and other samples on this panel. Please see Panel 1.3D for further discussion of the utility of this gene in cancer.
As in the previous panel, this gene is also expressed in the brain, including the cerebral cortex, substantia nigra and thalamus. Please see Panel 1.3D for discussion of utility of this gene in the cenfral nervous system. Panel 1.3D Summary: Ag2622 Two experiments with the same probe and primer sets produce results that are in reasonable agreement, with highest expression of the CG55794-07 gene in the brain and the kidney. Interestingly, there is significantly lower expression in the brain cancer cell lines than normal brain samples. This suggests that absence of this gene might be involved in cell proliferation. Hence this might be used as a diagnostic marker for brain cancer.
As seen in previous panels, the CG55794-07 gene is also expressed at low levels in the CNS. Carboxypeptidase is believed to have a role in the degradation of APP and A-beta, the major component of senile plaques in Alzheimer's disease. Therapeutic upregulation of this gene or its protein product may therefore be of benefit in the treatment of Alzheimer's disease.
References:
Matsumoto A, Itoh K, Matsumoto R. A novel carboxypeptidase B that processes native beta- amyloid precursor protein is present in human hippocampus. Eur J Neurosci 2000 Jan; 12(1) :227- 38
The processing of beta-amyloid precursor protein (APP) and generation of beta-amyloid (Abeta) are associated with the pathophysiology of Alzheimer's disease (AD). As the proteases responsible for the process in the human brain have yet to be clarified, we have searched for activities capable of cleaving native brain APP in the human hippocampus. A 40-kDa protein with proteolytic activity that degrades native brain APP in vitro was purified and characterized; molecular analysis identified it as a novel protease belonging to the carboxypeptidase B (CPB) family. PC12 cells overexpressing the cDNA encoding this protease generate a major 12-kDa beta-amyloid-bearing peptide in cytosol, a peptide which has also been detected in a cell-free system using purified brain APP as substrate. Although the protease is homologous to plasma
CPB synthesized in liver, it has specific domains such as C-terminal 14 amino acid residues.
Western analysis, cDNA-cloning process and Northern analysis suggested a brain-specific expression of this protease. An immunohistochemical study showed that the protease is expressed in various neuronal perikarya, including those of pyramidal neurons of the hippocampus and ependymal-choroid plexus cells, and in a portion of the microglia of normal brains. In brains of patients with sporadic AD, there is decreased neuronal expression of the protease, and clusters of microglia with protease immunoreactivity associated with its extracellular deposition are detected. These findings suggest that brain CPB has a physiological function in APP processing and may have significance in AD pathophysiology.
Panel 2D Summary: Ag2622 The CG55794-07 gene is expressed at low levels in the tissues used for panel 2D, with reasonable concordance between the runs. There is increased expression in normal prostate and kidney compared to the adjacent tumor tissues. There is also increased expression in breast cancer tissues compared to normal breast tissue. Hence, expression of this gene can be used as a diagnostic marker in breast, prostate and kidney cancer. Furthermore, therapeutic modulation of the gene product might be of use in the treatment of these cancers.
Panel 3D Summary: Ag2622 Expression of the CG55794-07 gene is low/undetectable in all samples on this panel (CTs>35).
Panel 4.1D Summary: Ag6050 The CG55794-07 transcript is expressed in EOL cells, fibroblasts and in normal kidney, thymus and colon. Low expression is noted in T cells, LAK cells, and B cells. The expression pattern with this set of primers and probe, which is specific to this gene, is different than that seen with the Ag2622 probe and primers, particularly in the colon, wheere expression of the transcript is comparatively low. Thus, this transcript or the protein it encodes could be used to identify the tissues where it is expressed, including kidney, and thymus.
Panel 4D Summary: Ag 2622 In two experiments with the same probe and primer set, the CG55794-07 gene, which encodes a putative carboxypeptidase, is expressed in the colon and down regulated in colon tissue isolated from Crohn's and colitis patients. The carboxypeptidase family of enzymes has been found in the colon and is associated with colon disease (ref. below). Thus, the expression of the transcript or the protein it encodes could be used to detect normal colon tissue. Furthennore, therapeutics designed with the protein encoded for by this transcript could be important in the freatment of IBD.
References:
Sommer H, Schweisfurth H, Schulz M. Serum angiotensin-I-converting enzyme and carboxypeptidase N in Crohn's disease and ulcerative colitis. Enzyme 1986;35(4): 181-8 Angiotensin-I-converting enzyme (ACE) and carboxypeptidase Nl and N2 (CPN1, CPN2) inactivate kinins and might therefore play a role in the development of inflammatory reactions via an influence on the release of prostaglandins and inactivation of anaphylatoxic peptides of the complement system. In the present study, the serum activity of these enzymes was determined in 60 patients with Crohn's disease, 18 patients with ulcerative colitis and 70 healthy confrol subjects. ACE was significantly lowered in active Crohn's disease (CDAI greater than 150) and in ulcerative colitis (p less than 0.01), as long as the ileum or cecum was affected. Since ACE was detected in high concentrations in the human intestinal mucosa, decreased values may be explained by damage to the site of its production. CPN1 and CPN2 were raised in both diseases (p less than 0.005), inespective of their activity and location. These alterations in the activity of the kininases investigated may play a role in the pathogenesis of inflammatory bowel diseases.
Panel 5 Islet Summary: Ag3953 The CG55794-07 gene, a carboxypeptidase homolog, has little to no expression in any of the endocrine/metabolically-related tissues except for small intestine. This expression profile is in agreement with the results from Panel 4D. Carboxypeptidase-B processing of GI peptides (e.g. GLP-2 and CCK) is critical for bioactivity. Thus, a therapeutic modulator of this gene and/or gene-product may prove useful in treating diseases associated with the GI tract and metabolism.
References:
Orskov C, Buhl T, Rabenhoj L, Kofod H, Hoist JJ. Carboxypeptidase-B-like processing of the C- terminus of glucagon-like peptide-2 in pig and human small intestine.
FEBS Lett 1989 Apr 24;247(2): 193-6
We developed specific, C-terminal radioimmunoassays for three proglucagon (PG) fragments: PG 151-158, PG 151-160 and PG 126-159 (glucagon-like peptide-2 (GLP-2] in order to determine the exact C-terminal sequence of the newly isolated GLP-2 in man and pig. The antigens and the antisera showed no mutual cross-reactivity. By gel filfration of exfracts of pig and human small intestine, the immunoreactivity eluting at the position of GLP-2 was identified by the radioimmunoassays for glucagon-like peptide-2 (PG 126-159) and for PG 151-158, whereas the assay for PG 151-160 was completely negative. We conclude that the C-terminal amino acid residue of pig and human ileal GLP-2 is PG 158. Thus the basic residues, PG 159 and 160 are removed during its processing in the small intestine.
PMID: 2714431
Blanke SE, Johnsen AH, Rehfeld JF. N-terminal fragments of intestinal cholecystokinin: evidence for release of CCK-8 by cleavage on the carboxyl side of Arg74 of proCCK. Regul Pept 1993 Jul 23;46(3):575-82
From porcine duodenal mucosa we have identified three major procholecystokinin (proCCK) fragments: desoctaCCK-33, desnonaCCK-33 and desnonaCCK-39. (DesoctaCCK-33 means CCK-33 devoid of the 8 C-terminal amino acids, etc.). The fragments were purified by immunoaffinity chromatography and three steps of reverse phase HPLC monitored by a radioimmunoassay specific for the N-terminal part of CCK-33. The structures could be deduced from the proCCK sequence by N-terminal sequence determination and mass spectrometry. Whereas desnona-fragments of CCK have been described before, this is the first finding of a desoctaCCK, and it indicates that CCK-8 is released from the longer forms by endogenous cleavage of the Arg- Asp-bond. A carboxypeptidase B-like exopeptidase subsequently must produce the desnona-fragments by removing the arginine residue.
NOV10: CG56321-01: novel human MAF-like protein
Expression of gene CG56321-01 was assessed using the primer-probe set Ag3095, described in Table HA. Results of the RTQ-PCR runs are shown in Tables HB, HC, HD and HE.
Table HA. Probe Name Ag3095
Figure imgf000474_0001
Table HB. Panel 1.3D
Figure imgf000475_0001
Figure imgf000476_0001
Table HC. Panel 2.2
Figure imgf000476_0002
Figure imgf000477_0001
Figure imgf000478_0001
Figure imgf000479_0001
Figure imgf000480_0001
Table HE. Panel 5D
Figure imgf000481_0001
Figure imgf000482_0001
Panel 1.3D Summary: Ag3095 The CG56321-01 gene is expressed predominantly in skeletal muscle and pancreas (CTs=30-33)as well as in several lung and ovarian cancer cell lines. MAF- like proteins are known to be involved in regulating differentiation. Thus, expression of this gene could be used to differentiate between these samples and other samples on this panel. Furthermore, therapeutic modulation of the expression or function of this gene product may be effective in the treatment of cancers that affects these tissues.
Panel 2.2 Summary: Ag3095 Expression of the CG56321-01 gene is restricted to samples derived from liver cancer cell lines (CT=34.4). Thus, expression of this gene could be used to differentiate between this sample and other samples on this panel and as a marker to detect the presence of liver cancer. Furthermore, therapeutic modulation of the expression or function of this gene may be effective in the treatment of liver cancer.
Panel 4D Summary: Ag3095 Highest expression of the CG56321-01 gene is seen in LAK cells stimulated with PMA/ionomycin (CT=31.3). Significant levels of expression are also seen in activated B lymphocytes and eosinophils. Owing to the importance of eosinophils and T cells in lung pathology, inflammatory bowel disease and autoimmune disorders, including rheumatoid arthritis, antibody or small molecule therapies designed with the protein encoded by this gene could block or inhibit inflammation or tissue damage due to lung conditions including asthma, allergies, hypersensitivity reactions, inflammatory bowel disease, viral infections and autoimmune disease. Detection of this gene product in LAK cells also suggests that modulation of the function of this gene product with a small molecule drag or antibody may lead to improvement of symptoms associated with tumor immunology and tumor cell clearance, as well as removal of virally and bacterial infected cells. Panel 5D Summary: Ag3095 The CG56321-01 gene is expressed exclusively in skeletal muscle of an individual who is diagnosed with gestational diabetes and is being treated with insulin (CT=33). Thus, the physiological role of this gene product may extend beyond regulating differentiation and also include regulating the physiology of skeletal muscle under conditions of metabolic stress.
NOVlla and NOVllb: CG56381-01 and CG56381-02: lysyl oxidase
Expression of gene CG56381-01 and variant CG56381-02 was assessed using the primer-probe sets Ag2916 and Ag2921, described in Tables IA and IB. Results of the RTQ-PCR runs are shown in Tables IC, ID and IE.
Table IA. Probe Name Ag2916
Figure imgf000483_0001
Table IB. Probe Name Ag2921
Figure imgf000483_0002
Table IC. Panel 1.3D
Figure imgf000483_0003
Figure imgf000484_0001
Figure imgf000485_0001
Figure imgf000486_0001
Figure imgf000487_0001
Figure imgf000488_0001
Table IE. Panel 4D
Figure imgf000488_0002
Figure imgf000489_0001
Figure imgf000490_0001
Figure imgf000491_0001
Panel 1.3D Summary: Ag2916/Ag2921 The expression of the CG56381-01 gene was assessed in two independent runs with the same probe and primer set, with good concordance between the rans. Highest expression is seen in a pancreatic cancer cell line CAPAN2 (CTs=26). Additionally, moderate expression is seen in a liver cell line as well as brain, colon, gastric, renal, lung, ovarian cancer cell lines as well as some melanoma cell lines. Thus, the expression of this gene might be associated with these forms of cancer and therapeutic modulation of this gene might be of use in the freatment of these cancers.
References:
Csiszar K.; Lysyl oxidases: a novel multifunctional amine oxidase family. Prog Nucleic Acid Res Mol Biol 2001;70:1-32.
Lysyl oxidase (LOX), a copper-containing amine oxidase, belongs to a heterogeneous family of enzymes that oxidize primary amine substrates to reactive aldehydes. LOX has been traditionally known for one function, the extracellular catalysis of lysine-derived cross-links in fibrillar collagens and elastin. More recently, diverse roles have been attributed to lysyl oxidase and these novel activities cover a spectrum of diverse biological functions such as developmental regulation, tumor suppression, cell motility, and cellular senescence. Lysyl oxidase has also been shown to have both intracellular and intranuclear locations. The multifunctional properties of lysyl oxidase (LOX) and our recent discovery of three novel members of this amine oxidase family, LOX-like (LOXL), LOXL2, and LOXL3, indicate the possibility that these varied functions are performed in both intracellular and extracellular environments by individual novel members of the LOX amine-oxidase family. Structural similarities of the highly conserved copper-binding and lysyl-tyrosylquinone cofactor sites among the LOX and LOX-like proteins may result in similar amine oxidase activities. However, specific novel functions, such as a potential role in cell adhesion and cell growth control, will be determined by other, conserved domains such as the cytokine receptor-like domain that is shared by all LOXs and by multiple scavenger receptor cysteine-rich (SRCR) domains present in LOXL2 and LOXL3. Furthermore, these functions may be carried out in a temporally and spatially regulated fashion.
PMID: 11642359
Panel 2.2 Summary: Ag2916/Ag2921 Two experiments with the same probe and primer set produce results that are in excellent agreement, with highest expression of the CG56381-01 gene in liver cancer (CTs=30). hi addition, liver cancers express this gene at a higher level than the normal adjacent liver tissue. Conversely nonnal ovary and colon tissue express higher level of this gene than the adjacent tumor tissue. Thus, expresseion of this gene can be used as a diagnostic marker for the presence of these cancers. Furthermore, therapetic modulation of this gene using antibodies and small molecule may be useful in the treatment of liver cancer.
Panel 4D Summary: Ag2916/Ag2921 Two experiments with the same probe and primer set produce results that are in excellent agreement, with highest expression of the CG56381-01 gene dermal fibroblasts treated with the proinflammatory cytokines IL-4 and gamma interferon
(CTs=26). The transcript, which encodes a putative lysyl oxidase, is expressed at low levels in most tissues in the panel. This enzyme is associated with dermal fibroblasts and is increased in scleroderma. Thus, the transcript or the protein it encodes could be used to identify activated dermal fibroblasts and as a diagnositic reagent for scleroderma. In addition, therapeutics designed with the protein encoded by this transcript could be important for the treatment of scleroderma and other skin diseases such as psoriasis.
References:
Chanoki M, Ishii M, Kobayasbi H, Fushida H, Yashiro N, Hamada T, Ooshima A. Increased expression of lysyl oxidase in skin with scleroderma. Br J Dermatol 1995 Nov;133(5):710-5
Lysyl oxidase initiates cross-linkage of collagen and elastin by catalysing the formation of a lysine-derived aldehyde. In order to study cross-linking in scleroderma, we used monoclonal antibodies to lysyl oxidase to determine the localization of this enzyme in systemic and localized scleroderma, and compared the distributions obtained with that in normal skin. Using an indirect immuno fluorescent antibody method and an avidin-biotinylated enzyme complex method, 11 cases of diffuse type of systemic scleroderma and seven cases of localized scleroderma were studied. In the oedematous stage of systemic scleroderma, intracellular and extracellular lysyl oxidase were remarkably increased in the dermis, particularly in groups around blood vessels, hi the sclerotic stage of systemic scleroderma, lysyl oxidase was detected intracellularly in fibroblasts and extracellularly among collagen bundles between the lower dermis and the subcutaneous fat tissue. In localized scleroderma, a marked increase in lysyl oxidase was observed in mononuclear cells and fibroblasts near blood vessels in the lower dermis and in the subcutaneous fat tissue, in addition to the extracellular deposits between collagen bundles. The increase in lysyl oxidase in localized scleroderma was much more common than in the oedematous stage of systemic scleroderma. These findings indicated that intracellular and extracellular expression of lysyl oxidase expression was greater in sclerodermatous skin than in normal skin.
NOV12a and NOV12b: CG56436-01 and CG56436-02: Phosphatase like
Expression of gene CG56436-01 and variant CG56436-02 was assessed using the primer-probe set Ag2927, described in Table JA. Results of the RTQ-PCR runs are shown in Tables JB and JC.
Table JA. Probe Name Ag2927
|Primers| Sequences [Length Start
Figure imgf000494_0001
Table JB. Panel 1.3D
Figure imgf000494_0002
Figure imgf000495_0001
Figure imgf000496_0001
Figure imgf000497_0001
Panel 1.3D Summary: Ag2927 Highest expression of the CG56436-01 gene is seen in the fetal heart (CT=27). Significant levels of expression are also seen in fetal liver and kidney (CTs=29- 30). Furthermore, the levels of expression in fetal tissue are much higher than the expression in their adult counterparts. This gene encodes a putative phosphatase and could potentially be used to differentiate between fetal and adult liver, kidney and heart tissue. Furthermore, the higher levels of expression in fetal tissue suggests that this gene may be involved in regulating phosphorylation states in proteins involved in cell growth and proliferation. This conclusion is further supported by the low expression in the tissues originating in the central nervous system, which are primarily composed of post-mitotic cells. Thus, this gene play a role in the cell cycle or possibly in the inhibition of cell differentiation. Therefore, this gene may be of use in stem cell research or therapy intended to confrol the fate of the stem cells.
Panel 4D Summary: Ag2927 The CG56436-01 transcript is highly expressed in dendritic cells (DC) and is upregulated in response to LPS or CD40 (CT=30). This gene, which encodes a phosphatase homolog, is also expressed in activated EOL cells and TNFalpha induced dermal fibroblasts. Thus, this putative phosphatase may be involved in signalling important in cellular differentiation. This is consistant with the low expression in monocytes, monocytes differentiated into dendritic cells, and monocytes differentiated into macrophages and the upregulation of this transcript in dendritic cells after activation with CD40. Furthermore, phosphatase involvement in DC maturation has been documented (see reference), hi addition, colon and lung expression of the transcript is may also result from dendritic cells present in these tissue. Therefore, therapeutic utilization of the protein encoded by this transcript may be important in immune modulation, organ/bone marrow transplantation, and the treatment of diseases where antigen presentation, a function of mature dendritic cells, plays an important role such as asthma, rheumatoid arthrtis, IBD, and psoriasis.
References:
Faries MB, Bedrosian I, Xu S, Koski G, Roros JG, Moise MA, Nguyen HQ, Engels FH, Cohen PA, Czemiecki BJ. Calcium signaling inhibits interleukin-12 production and activates CD83(+) dendritic cells that induce Th2 cell development. Blood 2001 Oct 15;98(8):2489-97
Mature dendritic cells (DCs), in addition to providing costimulation, can define the Thl, in contrast to the Th2, nature of a T-cell response through the production of cytokines and chemokines. Because calcium signaling alone causes rapid DC maturation of both normal and transformed myeloid cells, it was evaluated whether calcium-mobilized DCs polarize T cells toward a Thl or a Th2 phenotype. After human monocytes were cultured for 24 hours in serum- free medium and granulocyte-macrophage colony-stimulating factor to produce immature DCs, additional overnight culture with either calcium ionophore (Cl) or interferon gamma (IFN- gamma), tumor necrosis factor-alpha (TNF-alpha), and soluble CD40L resulted in phenotypically mature DCs that produced interleukin-8 (IL-8) and displayed marked expression of CD80, CD86, CD40, CD54, CD83, DC-LAMP, and RelB. DCs matured by IFN-gamma, TNF-alpha, and soluble CD40L were additionally distinguished by undetectable CD4 expression, marked secretion of IL-12, IL-6, and MlP-lbeta, and preferential ability to promote Thl/Tcl characteristics during T-cell sensitization. In contrast, DCs matured by Cl treatment were distinguished by CD4 expression, modest or absent levels of IL-12, IL-6, and MlP-lbeta, and preferential ability to promote Th2/Tc2 characteristics. Calcium signaling selectively antagomzed IL-12 production by mature DCs activated with IFN-gamma, TNF-alpha, and soluble CD40L. Although the activation of DCs by calcium signals is largely mediated through calcineurin phosphatase, the inhibition of IL-12 production by calcium signaling was independent of this enzyme. Naturally occuning calcium fluxes in immature DCs, therefore, negatively regulate Del differentiation while promoting Dc2 characteristics and Th2/Tc2 polarization. Calcium-mobilized DCs may have clinical usefulness in treating disease states with excessive Thl/Tcl activity, such as graft-versus-host disease or autoimmumty.
PMID: 11588047
NOV14: CG56443-01: MAST CELL FUNCTION-ASSOCIATED ANTIGEN- like protein
Expression of gene CG56443-01 was assessed using the primer-probe set Ag2928, described in Table KA. Table KA. Probe Name Ag2928
Figure imgf000499_0001
CNS_neurodegeneration_vl.0 Summary: Ag2928 Expression of the CG56443-01 gene is low/undetectable in all samples on this panel (CTs>35).
Panel 1.3D Summary: Ag2928 Expression of the CG56443-01 gene is low/undetectable in all samples on this panel (CTs>35).
Panel 4D Summary: Ag2928 Expression of the CG56443-01 gene is low/undetectable in all samples on this panel (CTs>35).
NOV15a, NOV15b, NOV15c, NOV15e, and NOV15f: CG56449-01, CG56449- 02, CG56449-03, CG56449-06, and CG56449-08: MEGF6
Expression of gene CG56449-02 and variants CG56449-01, CG56449-03, CG56449-06, and CG56449-08 was assessed using the primer-probe sets Ag252, Ag252b, Ag422, Agl513 and Agl937, described in Tables LA, LB, LC, LD and LE. Results of the RTQ-PCR runs are shown in Tables LF, LG, LH, LI, and LJ. Please note that the probe/primer set Aga422 does not conespond to the CG56449-01, CG56449-06, and CG56449-08 variants. This does not impact the results presented below.
Table LA. Probe Name Ag252
Figure imgf000500_0001
Table LB. Probe Name Ag252b
Figure imgf000500_0002
Table LC. Probe Name Ag422
Figure imgf000500_0003
Figure imgf000501_0001
Table LD. Probe Name Agl513
Figure imgf000501_0002
Table LE. Probe Name Agl937
Figure imgf000501_0003
Table LF. Panel 1
Figure imgf000501_0004
Figure imgf000502_0001
Figure imgf000503_0001
Figure imgf000504_0001
Table LG. Panel 1.3D
Figure imgf000504_0002
Figure imgf000505_0001
Figure imgf000506_0001
Table LH. Panel 2D
Figure imgf000506_0002
Figure imgf000507_0001
Figure imgf000508_0001
Table LI. Panel 4D
Rel.
Tissue Name Rel. Rel. Rel. Rel. Rel.
Tissue Name
Exp.(%) Exp.(%) Exp.(%) Exp.(%) Exp.(%) Exp.(%.
Figure imgf000509_0001
Figure imgf000510_0001
Figure imgf000511_0001
Table LJ. Panel 4R
Figure imgf000511_0002
Figure imgf000512_0001
Figure imgf000513_0001
Panel 1 Summary: Ag252/252b/Ag422 Multiple experiments with three different probe and primer sets produce results that are in excellent agreement, with highest expression of the CG56449-02 gene in a breast cancer cell line BT-549 (CTs=24) and the fetal lung. Based on homology, the protein encoded by this gene contains numerous EGF-motifs and may be required for cell growth and proliferation. The expression profile suggests that this gene product may be involved in brain, colon, renal, lung, ovarian and prostate cancer as well as melanomas. Thus, expression of this gene could be used as a diagnostic marker for the presence of these cancers. Furthermore, therapeutic inhibition of the expression or function of this gene product through the use of antibodies or small molecule drags might be of use in the treatment of these cancers.
Among tissues with metabolic function, this gene is expressed at moderate to low levels in pancreas, adrenal, thyroid, pituitary, heart, skeletal muscle, and adult and fetal liver. This widespread expression suggests that this gene product may be important for the pathogenesis, diagnosis, and/or freatment of metabolic and endocrine diseases, including obesity and Types 1 and 2 diabetes.
hi addition, this gene shows consistent low/moderate levels of expression in the brain. Please see Panel 1.3D for discussion of utility of this gene in the central nervous system.
Panel 1.3D Summary: Ag252 Highest levels of expression of the CG56449-02 gene are seen in a liver cell line HepG2 (CT=30.27). Based on expression in this panel, this gene may be involved in brain, colon, renal, lung, ovarian and prostate cancer as well as melanomas. Thus, expression of this gene could be used as a diagnostic marker for the presence of these cancers. Furthermore, therapeutic inhibition using antibodies or small molecule drags might be of use in the treatment of these cancers.
This gene product also shows low but significant levels of expression in pancreas, adrenal, thyroid, pituitary, adult and fetal heart, and adult and fetal liver. This widespread expression in tissues with metabolic function is in agreement with results from Panel 1 and suggests that this gene product may be important for the pathogenesis, diagnosis, and/or treatment of metabolic and endocrine diseases, including obesity and Types 1 and 2 diabetes. Furthermore, this gene is more highly expressed in fetal (CT=34) skeletal muscle when compared to expression in the adult (CT=40) and may be useful for the differentiation of the fetal and adult sources of this tissue.
In addition, this gene is expressed at moderate levels in the CNS, again consistent with Panel 1. This gene encodes a mouse epidermal growth factor homolog, and thus may increase axonal or dendritic outgrowth and synaptogenesis. Therefore, this gene may be of use in the freatment of clinical conditions associated with neuron loss such as head or spinal cord trauma, stroke, or any neurodegenerative disease.
Panel 2D Summary: Ag252 The CG56449-02 gene is expressed at low levels in all the samples on this panel, with highest expression in a kidney cancer sample (CT=31.1). Gastric, liver and colon cancers express this gene at a higher level than the normal adjacent tissue from these organs. There also appears to be increased expression in normal lung and ovarian tissue when compared to the adjacent tumor samples. These data indicate that the expression of this gene might be associated with gastric, liver and colon cancer and thus, therapeutic modulation of this gene product might be of use in the treatment of these cancers. Conversely, absence of expression is associated with ovarian and lung cancer and could potentially be used as a diagnostic marker for the presence of these cancers. Furthermore, therapeutic modulation of this gene might be of use in the freatment of these cancers.
Panel 3D Summary: Ag252 Data from one experiment with this probe and primer and the CG56449-02 gene is not included because the amp plot suggests that there were experimental difficulties with this run.
Panels 4D/4R Summary: Agl513/Agl937/Ag422 Multiple experiments with different probe and primer sets produce results that are in excellent agreement. The CG56449-02 franscript is expressed at low levels in T cells, fibroblasts, endothelium, smooth muscle cells and T cells regardless of treatment. The transcript is also expressed in normal colon, lung and thymus.
However, TNFalpha and IL-lbeta induce the expression of the transcript in asfrocytes. Thus, the franscript encodes a Notch like protein which may function in astrocyte differentiation and activation. Therefore, therapeutic regulation of this franscript or the design of therapeutics with the encoded protein could be important in the treatment of multiple scelrosis or other inflammatory diseases of the CNS.
References:
Tanigaki K, Nogaki F, Takahashi J, Tashiro K, Kurooka H, Honjo T. Notchl and Notch3 instructively restrict bFGF-responsive multipotent neural progenitor cells to an asfroglial fate. Neuron 2001 Jan;29(l):45-55
Notchl has been shown to induce glia in the peripheral nervous system. However, it has not been known whether Notch can direct commitment to glia from multipotent progenitors of the cenfral nervous system. Here we present evidence that activated Notchl and Notch3 promotes the differentiation of asfroglia from the rat adult hippocampus-derived multipotent progenitors
(AHPs). Quantitative clonal analysis indicates that the action of Notch is likely to be instructive. Transient activation of Notch can direct commitment of AHPs ineversibly to asfroglia. Asfroglial induction by Notch signaling was shown to be independent of STAT3, which is a key regulatory transcriptional factor when ciliary neurotrophic factor (CNTF) induces asfroglia. These data suggest that Notch provides a CNTF-independent instructive signal of asfroglia differentiation in CNS multipotent progenitor cells.
PMID: 11182080
Irvin DK, Zurcher SD, Nguyen T, Weinmaster G, Kornblum HI. Expression patterns of Notchl, Notch2, and Notch3 suggest multiple functional roles for the Notch-DSL signaling system during brain development. J Comp Neural 2001 Jul 23;436(2):167-81
The Notch-DSL signaling system consists of multiple receptors and ligands, and plays many roles in development. The function of Notch receptors and ligands in mammalian brain, however, is poorly understood, hi the cunent study, we examined the expression patterns for three receptors of this system, Notchl, 2, and 3, in late embryonic and postnatal rat brain by in situ hybridization. The three receptors have overlapping but different patterns of expression. Messenger RNA for all three proteins is found in postnatal central nervous system (CNS) germinal zones and, in early postnatal life, within numerous cells throughout the CNS. Within zones of cellular proliferation of the postnatal brain, Notchl mRNA is found in both the subventricular and the ventricular germinal zones, whereas Notclι2 and Notch3 mRNAs are more highly localized to the ventricular zones. Both Notchl and Notch3 mRNAs are expressed along the inner aspect of the dentate gyras, a site of adult neurogenesis. Notch2 mRNA is expressed in the external granule cell layer of the developing cerebellum. In several brain areas, Notchl and Notch2 mRNAs are relatively concentrated in white matter, whereas Notch3 mRNA is not. Neurosphere cultures (which contain CNS stem cells), purified asfrocyte cultures, and striatal neuron-enriched cultures express Notchl mRNA. However, in these latter cultures, Notchl mRNA is produced by nestin-containing cells, rather than by postmitotic neurons. Taken together, these results support multiple roles for Notchl, 2, and 3 receptor activation during CNS development, particularly during gliogenesis. Copyright 2001 Wiley-Liss, Inc.
PMLD: 11438922 Colombatti M, Moretto G, Tommasi M, Fiorini E, Poffe O, Colombara M, Tanel R, Tridente G, Ramarli D. Human MBP-specific T cells regulate IL-6 gene expression in asfrocytes through cell- cell contacts and soluble factors. Glia 2001 Sep;35(3):224-33
One of the distinctive features of multiple sclerosis (MS) attacks is homing to the CNS of activated T cells able to orchesfrate humoral and cell-based events, resulting in immune-mediated injury to myelin and oligodendrocytes. Of the complex interplay occurring between T cells and CNS constituents, we have examined some aspects of T-cell interactions with asfrocytes, the major components of the glial cells. Specifically, we focused on the ability of T cells to regulate the gene expression of interleukin-6 (IL-6) in asfrocytes, based on previous evidence showing the involvement of this cytokine in CNS disorders. We found that T-cell adhesion and T-cell soluble factors induce IL-6 gene expression in U251 asfrocytes through distinct signaling pathways, respectively, resulting in the activation of NF-kappaB and IRF-1 transcription factors. In a search for effector molecules at the asfrocyte surface, we found that alpha3betal integrins play a role in NF-kappaB activation induced by T-cell contact, whereas interferon-gamma (IFN-gamma) receptors dominate in IRF-1 induction brought about by T-cell-derived soluble factors. Similar phenomena were observed also in normal fetal asfrocyte cultures. We therefore propose that through asfrocyte induction, T cells may indirectly regulate the availability of a cytokine which is crucial in modulating fate and behavior of cell populations involved in the pathogenesis of MS inflammatory lesions.
PMID: 11494413
NOV16: AL359846_A_dal: GPCR
Expression of gene AL359846_A_dal was assessed using the primer-probe sets Agl851, Ag2544 and Agl706, described in Tables MA, MB and MC. Results of the RTQ-PCR runs are shown in Tables MD, ME, MF, MG and MH.
Table MA. Probe Name Agl851
Figure imgf000517_0001
Figure imgf000518_0001
Table MB. Probe Name Ag2544
Figure imgf000518_0002
Table MC. Probe Name Agl706
Figure imgf000518_0003
Table MD. CNS_neurodegeneration_vl.0
Figure imgf000518_0004
Figure imgf000519_0001
Table ME. Panel 1.3D
Figure imgf000520_0001
Figure imgf000521_0001
Figure imgf000522_0001
Table MF. Panel 2.2
Figure imgf000522_0002
Figure imgf000523_0001
Figure imgf000524_0001
Figure imgf000525_0001
Table MG. Panel 4D
Figure imgf000525_0002
Figure imgf000526_0001
Figure imgf000527_0001
Figure imgf000528_0001
Table MH. Panel CΝS 1
Figure imgf000528_0002
Figure imgf000529_0001
Figure imgf000530_0001
CNS_neurodegeneration_vl.O Summary: Agl851/Ag2544 Two experiments with two different probe and primer sets both show significant expression of the AL359846_A_dal gene in the brain. While no specific association with Alzheimer's disease is evident from the results of these experiments, the expression of this GPCR homolog in the brain is confirmed. Please see Panel 1.3D for discussion of potential utility in the central nervous system.
Panel 1.3D Summary: Agl706/Agl851/Ag2544 Three experiments with two different probe and primer sets show significant expression of the AL359846_A_dal gene, which encodes a novel G-protein coupled receptor (GPCR), in the brain. The GPCR family of receptors contains a large number of neurotransmitter receptors, including the dopamine, serotonin, a and b- adrenergic, acetylcholine muscarinic, histamine, peptide, and metabofropic glutamate receptors. GPCRs are excellent drug targets in various neurologic and psychiatric diseases. All antipsychotics have been shown to act at the dopamine D2 receptor; similarly novel antipsychotics also act at the serotonergic receptor, and often the muscarinic and adrenergic receptors as well. While the majority of antidepressants can be classified as selective serotonin reuptake inhibitors, blockade of the 5-HT1A and a2 adrenergic receptors increases the effects of these drugs. The GPCRs are also of use as drug targets in the freatment of stroke. Blockade of the glutamate receptors may decrease the neuronal death resulting from excitotoxicity; further more the purinergic receptors have also been implicated as drug targets in the treatment of cerebral ischemia. The b-adrenergic receptors have been implicated in the treatment of ADHD with Ritalin, while the a-adrenergic receptors have been implicated in memory. Therefore this gene may be of use as a small molecule target for the treatment of any of the described diseases.
References:
El Yacoubi M, Ledent C, Parmentier M, Bertorelli R, Ongini E, Costentin J, Vaugeois JM. Adenosine A2A receptor antagonists are potential antidepressants: evidence based on pharmacology and A2A receptor knockout mice. Br J Pharmacol 2001 Sep;134(l):68-77 1. Adenosine, an ubiquitous neuromodulator, and its analogues have been shown to produce 'depressant' effects in animal models believed to be relevant to depressive disorders, while adenosine receptor antagonists have been found to reverse adenosine-mediated 'depressant' effect.
2. We have designed studies to assess whether adenosine A2A receptor antagonists, or genetic inactivation of the receptor would be effective in established screening procedures, such as tail suspension and forced swim tests, which are predictive of clinical antidepressant activity. 3. Adenosine A2A receptor knockout mice were found to be less sensitive to 'depressant' challenges than their wildtype littermates. Consistently, the adenosine A2A receptor blockers SCH 58261 (1 - 10 mg kg(-l), i.p.) and KW 6002 (0.1 - 10 mg kg(-l), p.o.) reduced the total immobility time in the tail suspension test. 4. The efficacy of adenosine A2A receptor antagonists in reducing immobility time in the tail suspension test was confirmed and extended in two groups of mice. Specifically, SCH 58261 (1 - 10 mg kg(-l)) and ZM 241385 (15 - 60 mg kg(-l)) were effective in mice previously screened for having high immobility time, while SCH 58261 at 10 mg kg(-l) reduced immobility of mice that were selectively bred for their spontaneous 'helplessness' in this assay. 5. Additional experiments were carried out using the forced swim test. SCH 58261 at 10 mg kg(-l) reduced the immobility time by 61%, while KW 6002 decreased the total immobility time at the doses of 1 and 10 mg kg(-l) by 75 and 79%, respectively. 6. Administration of the dopamine D2 receptor antagonist haloperidol (50 - 200 microg kg(-l) i.p.) prevented the antidepressant-like effects elicited by SCH 58261 (10 mg kg(-l) i.p.) in forced swim test whereas it left unaltered its stimulant motor effects. 7. In conclusion, these data support the hypothesis that A2A receptor antagonists prolong escape-directed behaviour in two screening tests for antidepressants. Altogether the results support the hypothesis that blockade of the adenosine A2A receptor might be an interesting target for the development of effective antidepressant agents.
Blier P. Pharmacology of rapid-onset antidepressant treatment strategies. Clin Psychiatry 2001;62 Suppl 15:12-7
Although selective serotonin reuptake inhibitors (SSRIs) block serotonin (5-HT) reuptake rapidly, their therapeutic action is delayed. The increase in synaptic 5-HT activates feedback mechanisms mediated by 5-HT1A (cell body) and 5-HT1B (terminal) autoreceptors, which, respectively, reduce the firing in 5-HT neurons and decrease the amount of 5-HT released per action potential resulting in attenuated 5-HT neurofransmission. Long-term treatment desensitizes the inhibitory 5-HT1 autoreceptors, and 5-HT neurofransmission is enhanced. The time course of these events is similar to the delay of clinical action. The addition of pindolol, which blocks 5-HT1A receptors, to SSRI freatment decouples the feedback inhibition of 5-HT neuron firing and accelerates and enhances the antidepressant response. The neuronal circuitry of the 5-HT and norepinephrine (NE) systems and their connections to forebrain areas believed to be involved in depression has been dissected. The firing of 5-HT neurons in the raphe nuclei is driven, at least partly, by alphal- adrenoceptor-rnediated excitatory inputs from NE neurons. Inhibitory alpha2-adrenoceptors on the NE neuroterminals form part of a feedback confrol mechanism. Mirtazapine, an antagonist at alpha2-adrenoceptors, does not enhance 5-HT neurofransmission directly but disinhibits the NE activation of 5-HT neurons and thereby increases 5-HT neurofransmission by a mechamsm that does not require a time-dependent desensitization of receptors. These neurobiological phenomena may underlie the apparently faster onset of action of mirtazapine compared with the SSRIs.
Tranquillini ME, Reggiani A. Glycine-site antagonists and sfroke. Expert Opin hivestig Drugs 1999 Nov;8(l l):1837-1848
The excitatory amino acid, (S)-glutamic acid, plays an important role in controlling many neuronal processes. Its action is mediated by two main groups of receptors: the ionofropic receptors (wliich include NMD A, AMPA and kainic acid subtypes) and the metabofropic receptors (mGluR(l-8)) mediating G-protein coupled responses. This review, focuses on the strychnine insensitive glycine binding site located on the NMDA receptor channel, and on the possible use of selective antagonists for the treatment of stroke. Stroke is a devastating disease caused by a sudden vascular accident. Neurochemically, a massive release of glutamate occurs in neuronal tissue; this overactivates the NMDA receptor, leading to increased intracellular calcium influx, which causes neuronal cell death through necrosis. NMDA receptor activation strongly depends upon the presence of glycine as a co-agonist. Therefore, the administration of a glycine antagonist can block overactivation of NMDA receptors, thus preserving neurones from damage. The glycine antagonists cunently identified can be divided into five main categories depending on their chemical structure: indoles, tefrahydroquinolines, benzoazepines, quinoxalinediones and pyrida-zinoquinolines. Monopoli A, Lozza G, Forlani A, Mattavelli A, Ongini E. Blockade of adenosine A2A receptors by SCH 58261 results in neuroprotective effects in cerebral ischaemia in rats. Neuroreport 1998 Dec l;9(17):3955-9 Related Articles, Books, LinkOut
Blockade of adenosine receptors can reduce cerebral infarct size in the model of global ischaemia. Using the potent and selective A2A adenosine receptor antagonist, SCH 58261, we assessed whether A2A receptors are involved in the neuronal damage following focal cerebral ischaemia as induced by occluding the left middle cerebral artery. SCH 58261 (0.01 mg/kg either i.p. or i.v.) administered to normotensive rats 10 min after ischaemia markedly reduced cortical infarct volume as measured 24 h later (30% vs controls, p < 0.05). Similar effects were observed when SCH 58261 (0.01 mg/kg, i.p.) was admimstered to hypertensive rats (28% infarct volume reduction vs controls, p < 0.05). Neuroprotective properties of SCH 58261 administered after ischaemia indicate that blockade of A2A adenosine receptors is a potentially useful biological target for the reduction of brain injury.
Panel 2.2 Summary: Agl706/Agl851 Results from two experiments using the identical probe/primer set are in reasonable agreement. Expression of the AL359846_A_dal gene is highest in a normal liver sample. Lower levels of expression are also seen in several kidney and breast samples, both from tumor and normal adjacent tissue. Therefore, expression of this gene may be used to distinguish liver, kidney and breast from the other samples on this panel.
Panel 4D Summary: Agl706/1851/2544: Results from three experiments are in reasonable agreement. Expression of the AL359846_A_dal gene is detected in LAK cells, Ramos B cells, thymus and kidney. Expression does not appear to be dependent upon activation in the cell types tested. The expression of the franscript may be dependent upon the proliferation status of cells, since it is expressed in specific types of proliferating cells including LAK cells, B cells and cells in the thymus and kidney. Thus, the franscript or the protein it encodes may be important for detecting LAK cells or thymic and kidney tissue.
Panel CNS_1 Summary: Agl851 The results of this experiment further confirm the expression of the AL359846_A_dal gene in the brain. Please see Panel 1.3D for discussion of potential utility in the cenfral nervous system. NOV19 a and NOV19b: CG56574-01 and CG56574-02: Dystrophin
Expression of gene CG56574-01 and variant CG565724-02 was assessed using the primer-probe set Agl409, described in Table NA. Results of the RTQ-PCR runs are shown in Table NB.
Table NA. Probe Name Agl409
Figure imgf000534_0001
Table NB. Panel 1.2
Figure imgf000534_0002
Figure imgf000535_0001
Figure imgf000535_0002
Figure imgf000536_0001
Panel 1.2 Summary: Agl409 The CG56574-01 gene has moderate levels of expression (CT values = 28-31) in pancreas, thyroid and pituitary. This gene is highly expressed (CT values = 22- 27) in adult and fetal heart, adult and fetal liver, and adrenal gland. This widespread expression in tissues with metabolic function suggests that this putative cytoskeletal protein may be important for the pathogenesis, diagnosis, and/or freatment of metabolic diseases including obesity and Types 1 and 2 diabetes.
This gene is also expressed at moderate to high levels in most cancer cells in this panel. Thus, this gene may be involved in cell structure, binding to glycoproteins associated with the cell membrane. Hence, this gene would be required for cell survival and proliferation.
In addition, this gene, a homolog of dystrophin, is expressed at high levels in the CΝS and skeletal muscle. Dystrophin is associated with major Duchenne musclular dystrophy. Thus, therapeutic modulation of this gene or its protein product may be of clinical benefit in the freatment of muscular dystrophy.
ΝOV21: CG56500-01: TFIIIC BOX B-BINDING SUBUNIT Expression of gene CG56500-01 was assessed using the primer-probe set Ag4856, described in Table OA. Results of the RTQ-PCR runs are shown in Table OB.
Table OA. Probe Name Ag4856
Figure imgf000536_0002
Reverse 5 ' -gaacttcatctggcaggagtt-3 ' (SEQ ID N0 : 421) 21 5071
Table OB. General_screening_panel_vl.5
Figure imgf000537_0001
Figure imgf000538_0001
General_screening_panel_vl.5 Summary: Ag4856 This gene, which represents a novel transcription factor, is expressed ubiquitously in this panel, with highest expression in a colon cancer cell line (CT=26). This expression profile suggests that the gene product may be required for cell growth and proliferation and is required for tumor growth. This gene is also expressed at low-to-moderate levels in many metabolic tisssues including adipose, adult and fetal liver, heart, and skeletal muscle, adrenal, pituitary, and pancreas. This gene product represents a novel franscription factor and is an excellent drug target for metabolic and endocrine diseases, including obesity and Types 1 and 2 diabetes.
Among tissues originating in the CNS, this gene is expressed at moderate levels. Because this gene encodes a putative transcription factore, this gene is an excellent drug target for neurological diseases in which franscription of a disease protein (e.g., Huntington's disease) is believed to be central to the progression of the disease.
NON22: CG56475-01: NUCLEOSIDE DIPHOSPHATE KINASE B Expression of gene CG56475-01 was assessed using the primer-probe set Ag2946, described in Table PA. Results of the RTQ-PCR runs are shown in Table PB.
Table PA. Probe Name Ag2946
Figure imgf000539_0001
Table PB. Panel 4D
Figure imgf000539_0002
Figure imgf000540_0001
Figure imgf000541_0001
CNS_neurodegeneration_vl.O Summary: Ag2946 Expression of the CG56475-01 gene is low/undetectable in all samples on this panel (CTs>35).
Panel 1.3D Summary: Ag2946 Expression of the CG56475-01 gene is low/undetectable in all samples on this panel (CTs>35).
Panel 4D Summary: Ag2946 Expression of the CG56475-01 franscript is exclusive to B cells and a B cell line (Ramos). Activation of RAMOS cells with PMA and ionomycin increases the expression level of the franscript. The gene encodes a putative nucleoside diphosphate kinase B like protein. These proteins may be involved in inducing franscription and preferentially bind single strand DNA. Thus, the protein encoded by this franscript may play a role in B cell differentiation and/or isotype switching. Thus, expression of this gene or the encoded protein could be used to identify B cells. Furthermore, therapeutics could be designed with the protein encoded by this franscript that might regulate B cell function, and potentially reduce symptoms in diseases where B cells play an important role, including systemic lupus erythematosus and rheumatoid arthritis. References:
Agou F, Raveh S, Mesnildrey S, Veron M. Single strand DNA specificity analysis of human nucleoside diphosphate kinase B. J Biol Chem 1999 Jul 9;274(28): 19630-8
Nucleoside diphosphate kinases (NDP kinases) form a family of oligomeric enzymes present in all organisms. Eukaryotic NDP kinases are hexamers composed of identical subunits
(approximately 17 kDa). A distinctive property of human NDPK-B encoded by the gene nm23- H2 is its ability to stimulate the gene transcription. This property is independent of its catalytic activity and is possibly related to the role of this protein in cellular events including differentiation and tumor metastasis. In this paper, we report the first characterization of human NDPK-B.DNA complex formation using a filter-binding assay and fluorescence spectroscopy. We analyzed the binding of several oligonucleotides mimicking the promoter region of the c-myc oncogene including variants in sequence, structure, and length of both strands. We show that NDPK-B binds to single-stranded oligonucleotides in a nonsequence specific manner, but that it exhibits a poor binding activity to double-sfranded oligonucleotides. This indicates that the specificity of recognition to DNA is a function of the structural conformation of DNA rather than of its specific sequence. Moreover, competition experiments performed with all nucleotides provide evidence for the contribution of the six active sites in the DNA.protein complex formation. We propose a mechanism through which human NDPK-B could stimulate franscription of c-myc or possibly other genes involved in cellular differentiation.
PMID: 10391900
NOV23: CG56352-02: T-cell-Immunoglobulin
Expression of gene CG56352-02 was assessed using the primer-probe sets Ag3865, Ag3864 and Ag2918, described in Tables QA, QB and QC. Results of the RTQ-PCR runs are shown in Tables QD, QE, QF and QG.
Table OA. Probe Name Ag3865
Figure imgf000542_0001
Figure imgf000543_0001
Table OB. Probe Name Ag3864
Figure imgf000543_0002
Table QC. Probe Name Ag2918
Figure imgf000543_0003
Table QD. CNS_neurodegeneration_vl.O
Figure imgf000543_0004
Figure imgf000544_0001
Table QE. General_screening_panel_vl.4
Figure imgf000544_0002
Figure imgf000545_0001
Figure imgf000546_0001
Table OF. Panel 2.2
Figure imgf000547_0001
Figure imgf000548_0001
Figure imgf000549_0001
Table OG. Panel 4. ID
Figure imgf000549_0002
Figure imgf000550_0001
Figure imgf000551_0001
CNS_neurodegeneration_vl.O Summary: Ag3865 This panel confirms the expression of the CG56352-02 gene in the CNS. See General_screening_panel_vl.4 for a discussion of utility. Ag2918 Expression of the CG56352-02 gene is low/undetectable in all samples on this panel (CTs>35).
General_screening_panel_vl.4 Summary: Ag3864/Ag3865 Two experiments produce results that are in very good agreement, with highest expression of the CG56352-02 gene in fetal lung and spleen (CT=28-32). Furthermore, expression of this gene is higher in fetal lung (CTs=28- 30)when compared to expression in the adult (CTs=33-40). Thus, expression of this gene could be used to differentiate between fetal and adult lung tissue.
Low, but significant expression of this gene is also seen in colon, breast, renal and CNS cancer cell lines on this panel. Thus, expression of this gene may be associated with these cancers and modulation of expression might be used for freatment of colon, breast, renal and brain cancers.
Among tissues with metabolic function, this gene is expressed at low levels in adipose, adult and fetal liver, adult and fetal heart, adult and fetal skeletal muscle, adrenal, thyroid and pancreas. Based on its tissue distribution, this gene product may be important for the pathogenesis, diagnosis, and/or treatment of endocrine and metabolic disease, including obesity and Types 1 and 2 diabetes.
This gene is expressed at moderate levels in the CNS. Therapeutic modulation of this gene or its protein product may be of use in controlling the inflammatory response and be of benefit in any clinical condition associated with neuroinflammation, such as sfroke, head or spinal cord trauma, multiple sclerosis, Alzheimer's disease, and viral infections of the CNS.
Panel 1.3D Summary: Ag2918 Expression of the CG56352-02 gene is low/undetectable in all samples on this panel (CTs>35).
Panel 2.2 Summary: Ag3864 The CG56352-02 gene is expressed at low level in the tissues used for panel 2.2. The highest expression is seen in a normal kidney sample (CT= 29.7). In addition, there appears to be increased expression in 5 of 6 samples of normal lung tissue when compared to lung cancers and in 7 of 9 samples of normal kidney compared to the adjacent kidney cancer tissue. Thus, loss of expression of this gene maybe associated with these cancers and therapeutic modulation of this gene may therefore be of use in the freatment of these cancers.
Panel 2D Summary: Ag2918 Expression of the CG56352-02 gene is low/undetectable in all samples on this panel (CTs>35). Panel 4.1D Summary: Ag 3864 The CG56352-02 franscript is found in T cells, particularly chronically activated Thl, Th2 and Trl cells. LAK cells, macrophages and dendritic cells also express the franscript. The only non-hematopoietic cell type that expresses the franscript detected by these primers and probe are dermal fibroblasts. Lung, thymus and kidney also express low levels of the franscript. Thus, this franscript or the protein it encodes could be used to detect hematopoietically-derived cells. Furthermore, therapeutics designed with the protein encoded by this transcript could be important in the regulation the function of antigen presenting cells (macrophages and dendritic cells)or T cells and be important in the treatment of asthma, emphysema, psoriasis, arthrtis, and IBD.
Ag3865 The CG56352-02 franscript is expressed at low levels in many tissues and at high levels in Th2 cells. The expression profile in this panel using this probe and primer set differs from the Ag3864 results in the expression seen in many nonhematopoitic tissues. Therapeutics designed with the protein encoded by this transcript could be important in the regulation of T cell function.
Panel 4D Summary: Ag2918 Expression of the CG56352-02 gene is low/undetectable in all samples on this panel (CTs>35).
NON24a and ΝON24b: CG56062-01 and CG56062-02: Organic Anion Transporter 3
Expression of gene CG56062-01 and variant CG56062-02 was assessed using the primer-probe sets Ag3948, Ag2874 and Ag3532, described in Tables RA, RB and RC. Results of the RTQ-PCR runs are shown in Tables RD, RE, RF, RG, RH, RI and RJ.
Table RA. Probe Name Ag3948
Figure imgf000553_0001
Table RC. Probe Name Ag3532
Figure imgf000554_0001
Table RD. CNS_neurodegeneration_vl.O
Figure imgf000554_0002
Figure imgf000555_0001
Table RE. General_screening_panel_vl.4
Figure imgf000555_0002
Figure imgf000556_0001
Figure imgf000557_0001
Table RF. Panel 1.3D
Figure imgf000557_0002
Figure imgf000558_0001
Figure imgf000559_0001
Table RG. Panel 2D
Figure imgf000559_0002
Figure imgf000560_0001
Figure imgf000561_0001
Table RH. Panel 3D
Figure imgf000561_0002
Figure imgf000562_0001
Figure imgf000563_0001
Figure imgf000564_0001
Figure imgf000565_0001
Table RJ. Panel 4D
Figure imgf000566_0001
Figure imgf000567_0001
Figure imgf000568_0001
CNS_neurodegeneration_vl.0 Summary: Ag3948 This panel does not show differential expression of the CG56062-01 gene in Alzheimer's disease. However, this expression profile confirms the presence of this gene in the brain. Please see Panel General_screening_panel_vl.4 for discussion of utility of this gene in the cenfral nervous system. Ag3532 Expression of the CG56062-01 gene is low/undetectable in all samples on this panel (CTs>35).
General_jscreening_panel_vl.4 Summary: Ag3948 The expression of the CG56062-01 gene, an organic anion transporter homolog, is highest in a small cell lung cancer line LX-1 (CT= 28.2). This gene is also expressed in some ovarian, breast, CNS, gastric, pancreatic, renal and colon cancer cell lines. Therefore, expression of this gene maybe associated with these forms of cancer and therapeutic modulation of this gene might be of use in the freatment or diagnosis of these cancers.
This gene is also expressed at low levels in the cerebellum and fetal brain. The organic anion transporters are involved in transport across the blood brain barrier. This gene may therefore be of use in drug delivery to the CNS, specifically for compounds such as nerve growth factors protein therapeutics which are believed to have numerous uses in the CNS, but lack a delivery system. Ag3532 Results from one experiment with this gene are not included. The amp plot indicates that there were instrumental difficulties with this run.
References:
Sugiyama D, Kusuhara H, Shitara Y, Abe T, Meier PJ, Sekine T, Endou H, Suzuki H, Sugiyama Y. Characterization of the efflux transport of 17beta-estradiol-D-17beta-glucuronide from the brain across the blood-brain barrier. J Pharmacol Exp Ther 2001 Jul;298(l):316-22
The contribution of organic anion transporters to the total efflux of 17beta-esfradiol-D-17beta- glucuronide (E(2) 17betaG) through the blood-brain barrier (BBB) was investigated using the Brain Efflux Index method by examining the inhibitory effects of probenecid, taurocholate (TCA), p-aminohippurate (PAH), and digoxin. E(2)17betaG was eliminated through the BBB with a rate constant of 0.037 min(-l) after the microinjection into the brain. Probenecid and TCA inhibited this elimination with an IC50 value of 34 and 1.8 nmol/0.5 microl of injectate, respectively, whereas PAH and digoxin reduced the total efflux to about 80 and 60% of the control value, respectively. The selectivity of these inhibitors was confirmed by examining their inhibitory effects on the transport via organic anion transporting polypeptide 1 (Oatpl), Oatp2, organic anion transporter 1 (Oatl), and Oat3 fransfectants using LLC-PK1 cells as hosts. Digoxin specifically inhibited the transport via Oatp2 (K(i) = 0.037 microM). The K(i) values of TCA for Oatpl and Oatp2 (11 and 39 microM, respectively) were about 20 times lower than those for Oatl and Oat3 (2.8 and 0.8 mM, respectively). PAH did not affect the transport via the Oatp family, but had a similar affinity for Oatl and Oat3 (85 and 300 microM, respectively). Probenecid had a similar affinity for these transporters (Oatpl, Oatp2, Oatl, and Oat3) examined in this study. Taking the selectivity of these inhibitors into consideration, the maximum contribution made by the Oatp2 and Oat family to the total efflux of E(2)17betaG from the brain appears to be about 40 and 20%, respectively.
Panel 1.3D Summary: Ag2874 The expression of the CG56062-01 gene was assessed in two independent runs on this panel with reasonable concordance between the runs. The highest expression is seen in a small cell lung cancer line LX-1 (CTs=31-32), consistent with expression in Panel 1.3D. This gene is also expressed in some ovarian, breast, CNS, gastric and colon cancer cell lines. Therefore, expression of this gene might be associated with these forms of cancer and therapeutic modulation of this gene might be of use in the freatment or diagnosis of these cancers.
Panel 2D Summary: Ag2874 The CG56062-01 gene is expressed at low levels in the tissues used for panel 2D. The highest expression is seen in a breast cancer sample (CT=34.2). Significant expression is also seen in single samples of ovarian, bladder, prostate and colon cancers compared with the normal adjacent tissue. This indicates that the expression of this gene might be associated with these forms of cancer and therapeutic modulation of this gene might be of use in the freatment or diagnosis of these cancers.
Panel 3D Summary: Ag2874 Highest expression of the CG56062-01 gene is seen in a pancreatic cancer cell line (CT=31.6). Thus, expression of this gene could be used to differentiate between this sample and other samples on this panel.
Panels 4D and 4.1D Summary: Ag2874/Ag3498 The highest expression of the CG56062-01 transcript is found in the kidney and in the pulmonary muco-epidermoid cell line NCI-H292. The expression of this transcript, although constitutive in the H292 cell line, is up regulated upon treatment with IL-4, 11-9 and IL-13, cytokines that have been linked to the pathogenesis of asthma and/or COPD. This franscript is also found in small airway epithelium and keratinocytes treated with the inflammatory cytokines TNF-a and IL-lb. Therefore, modulation of the expression or activity of the protein encoded by this transcript through the application of small molecule therapeutics may be useful in the treatment of asthma, COPD, emphysema, psoriasis and wound healing.
NOV25f, NOV25b, NOV25e, NON25g, ΝON25a, and ΝOV26: CG56653-01, CG56653-02, CG56653-06, CG56653-09, 152736829, and 152736833: Ficolin
Expression of gene CG56653-01 and CG56653-02 and CG56653-06 and CG56653-09 and 152736829 and 152736833 was assessed using the primer-probe sets Agl446, Ag5126 and
Ag4934, described in Tables SA, SB and SC. Results of the RTQ-PCR runs are shown in Tables
SD, SE, SF, SG, SH and SI. Please note that CG56653-09, a splice variant of CG56653-01, is the only variant that corresponds to the Ag5126 probe/primer set. This does not impact the results presented below. Table SA. Probe Name Agl446
Figure imgf000571_0001
Table SB. Probe Name Ag5126
Figure imgf000571_0002
Table SC. Probe Name Ag4934
Figure imgf000571_0003
Table SD. AI_comprehensive panel_vl.O
Figure imgf000571_0004
Figure imgf000572_0001
Figure imgf000573_0001
Figure imgf000574_0001
Table SE. CNS_neurodegeneration_vl.O
Figure imgf000574_0002
Figure imgf000575_0001
Figure imgf000576_0001
Table SF. General_screening_panel_vl.5
Figure imgf000576_0002
Figure imgf000577_0001
Figure imgf000578_0001
Table SG. Panel 1.2
Figure imgf000578_0002
Figure imgf000579_0001
Figure imgf000580_0001
Table SH. Panel 4. ID
Figure imgf000580_0002
Figure imgf000581_0001
Figure imgf000582_0001
Figure imgf000583_0001
Table SL Panel 4D
Figure imgf000583_0002
Figure imgf000584_0001
Figure imgf000585_0001
AI_comprehensive panel vl.O Summary: Ag 1446 Two experiments with the same probe and primer set produce results that are in excellent agreement, with expression of the CG56653-01 gene essentially limited to bone from OA and RA patients. Low to undetectable expression is found in normal bone. Low expression is also found in colon. This franscript encodes a putative ficolin 1 precursor. Ficolins are multimeric lectins that are capable of binding to bacteria. It has been reported to function as a monocyte cell surface molecule important for binding to bacteria, elastin and monocyte adhesion. Therefore, ficolin may play a role in the inflammation of joints in patients suffering from osteoarthritis (OA) and/or rheumatoid arthritis (RA). Antibodies against proteins encoded by this franscript may thus prevent tissue destruction mediated by ficolin activity during osteoarthritis and arthritis.
CNS_neurodegeneration_vl.O Summary: Agl446/Ag5126 Expression of the CG56653-01 gene is low/undetectable in all samples on this panel (CTs>35).
General_screening_panel_vl.5 Summary: Ag4934/Ag5126 Two experiments with different probe and primer sets show highest expression of the CG56653-01 gene in the fetal lung (CTs=31-34). Expression of this gene is also higher in fetal lung and fetal liver (CT-33) than in their adult counterparts (CTs=38-40). Thus expression of this gene could be used to differentiate between the two sources of lung and liver tissue. In addition, low but significant levels of expression in adipose, heart, skeletal muscle, thyroid and pancreas suggest that modulation of this gene product may be a treatment for metabolic or endocrine disease including obesity and Types 1 and 2 diabetes.
Panel 1.2 Summary: Agl446 The CG56653-01 gene is most highly expressed in bone marrow (CT=22). Thus, expression of this gene could be used to differentiate between this sample and other samples on this panel. In addition, this gene has low-to-moderate levels of expression (CT values = 27-33) in many metabolic tissues including liver, heart, skeletal muscle, thyroid, pancreas, adrenal and pituitary, as seen in General j3creerιmg_panel_v 1.5. Thus, modulation of this gene product may be a freatment for metabolic or endocrine disease including obesity and Types 1 and 2 diabetes.
Panel 4D/4.1D Summary: Agl446/Ag4934/Ag5126 Multiple experiments show the CG56653- 01 gene highly and selectively expressed in resting monocytes and to a lesser extent in macrophages and granulocytes (neutrophils and EOL cell line), in agreement with published expression profiles. This transcript encodes a putative ficolin 1 precursor. Ficolins are multimeric lectins that are capable of binding to bacteria. It has been reported to function as a monocyte cell surface molecule that is important for binding to bacteria, elastin and monocyte adhesion. Ficolin may also play a role in alleviating inflammation in joints and other sites of inflammation. Therefore, protein therapeutics designed with the protein encoded by this transcript could function as an opsinin to target and eliminate bacteria by complement-mediated destruction. These proteins could also be important for the treatment of bacterial septicemia. In addition, ficolins may have the ability to bind to elastins. Elastins are functionally important for lung alveolar development and inactivation of these proteins can lead to emphysema-like disease. Therefore, antibodies against proteins encoded by this transcript may prevent tissue destruction mediated by ficolin activity during emphysema, asthma and arthritis.
References:
Harumiya S, Takeda K, Sugiura T, Fukumoto Y, Tachikawa H, Miyazono K, Fujimoto D, Ichijo H. Characterization of ficolins as novel elastin-binding proteins and molecular cloning of human ficolin-1 J Biochem (Tokyo) 1996 Oct;120(4):745-51
A novel elastin-binding protein, EBP-37, was recently identified and purified from human plasma. Its partial amino acid sequences showed significant homology to porcine ficolins, wliich were originally purified from porcine uterus membranes as multimeric proteins with fibrinogen- and collagen-like domains. Here we report the presence of ficolins in an elastin-binding fraction of porcine plasma and the direct binding of recombinant porcine ficolin-alpha to elastin. In addition, a cDNA encoding a human counterpart of porcine ficolins that is composed of 319 amino acids and is different from EBP-37 was cloned and named human ficolin-1. Northern blotting of various human tissues revealed that human ficolin-1 mRNA is highly expressed in peripheral blood leukocytes. These data suggested that there are at least two kinds of ficolin-related proteins in both pig and human, and they may function as plasma proteins with elastin-binding activities.
PMID: 8947836
Teh C, Le Y, Lee SH, Lu J. Immunology 2000 Oct;101(2):225-32 M-ficolin is expressed on monocytes and is a lectin binding to N-acetyl-D-glucosamine and mediates monocyte adhesion and phagocytosis of Escherichia coli.
Ficolins are a group of multimeric proteins that contain collagen-like and fibrinogen-like (FBG) sequences. Three types of ficolins have been characterized: H-, L- and M-ficolins. Both H- and L- ficolins have demonstrated lectin activities. In the present study, the FBG domain of M-ficolin was expressed and shown to bind to N-acetyl-D-glucosamine. M-ficolin mRNA was expressed in monocytes but not in the more differentiated macrophages and dendritic cells. By flow cytometry, surface biotinylation and immunoprecipitation, we showed that M-ficolin was associated with the surface of promonocytic U937 cells. M-ficolin transiently expressed in COS-7 cells was also clearly detected on the cell surface by immunoprecipitation. By flow cytometry, M-ficolin was detected on peripheral blood monocytes but not on lymphocytes or granulocytes. Immobilized rabbit anti-M-ficolin F(ab')2 mediated U937 cell adhesion, and the antibody also inhibited phagocytosis of Escherichia coli K-12 by U937 cells. Therefore, M-ficolin might act as a phagocytic receptor or adaptor on circulating monocytes for micro-organism recognition and may potentially mediate monocyte adhesion.
PMID: 11012776
NOV27: CG56262-01: Ca-binding transporter
Expression of gene CG56262-01 was assessed using the primer-probe sets Ag2896 and Ag2920, described in Tables TA and TB. Results of the RTQ-PCR runs are shown in Tables TC, TD, TE and TF.
Table TA. Probe Name Ag2896
Figure imgf000588_0001
Table TB. Probe Name Ag2920
Figure imgf000588_0002
Table TC. CNS_neurodegeneration_vl.O
Figure imgf000588_0003
Figure imgf000589_0001
Figure imgf000590_0001
Table TD. Panel 1.3D
Figure imgf000590_0002
Figure imgf000591_0001
Figure imgf000592_0001
Table TE. Panel 4D
Figure imgf000592_0002
Figure imgf000593_0001
Figure imgf000594_0001
Figure imgf000595_0001
Table TF. Panel CNS 1
Figure imgf000595_0002
Figure imgf000596_0001
CNS_neurodegeneration_vl.0 Summary: Ag2896/Ag2920 This panel does not show differential expression of the CG56153-01 gene in Alzheimer's disease. However, this expression profile confirms the presence of this gene in the brain. Please see Panel 1.3D for discussion of utility of this gene in the central nervous system.
Panel 1.3D Summary: Ag2896/Ag2920 Two experiments produce results that are in excellent agreement, with highest expression of the CG56262-01 gene in the brain. This gene encodes a Ca binding transporter. Ca++ is critical for synaptic vesicle release. Thus, this gene would be an excellent small molecule target for any disease believed to result from altered/inappropriate synaptic transmission such as epilepsy, schizophrenia, bipolar disorder, depression, and mania.
This gene also has moderate levels of expression adult and fetal heart, skeletal muscle and liver, and adipose. This gene product is homologous to a mitochondrial calcium-dependent transporter. Since intracellular calcium homeostasis is critically important for energy metabolism and signal fransduction, modulation of this gene product may therefore be a therapeutic for metabolic and endocrine diseases.
Moderate expression in also seen in almost all cell lines on this panel. This suggests that expression of this gene product is required for cell growth and proliferaton in almost all cell types.
References:
Kovacs I, Szarics E, Nyitrai G, Blandl T, Kardos J. Matching kinetics of synaptic vesicle recycling and enhanced neurotransmitter influx by Ca2+ in brain plasma membrane vesicles. Neurochem hit 1998 Nov;33(5):399-405
Using native plasma membrane vesicle suspensions from the rat cerebral cortex under conditions designed to alter intravesicular [Ca2+], we found that Ca2+ induced 47 +/- 5% more influx of [3H]GABA, [3H]D-aspartate and [3H]glycine at 37 degrees C with half-times 1.7 +/- 0.5, 1.3 +/- 0.4 and 1.3 +/- 0.4 min, respectively. We labelled GABA transporter sites with the uptake inhibitor, [3H]-(R,S)-N-[4,4-bis(3-methyl-2-thienyl)but-3-en-l-yl]nipecotic acid and found that Ca2+ induced a partial dissociation of the bound inhibitor from GABA transporter sites with a similar half-time. By means of rapid kinetic techniques applied to native plasma membrane vesicle suspensions, containing synaptic vesicles stained with the amphipathic fluorescent styryl membrane probe N-(3-triethylammoniumpropyl)-4-[4-(dibutylamino)styryl]pyrid inium dibromide, we have measured the progress of the release and reuptake of synaptic vesicles in response to Ca2+ and high-[K+] depolarization in the 0.0004-100 s range of time. Synaptic vesicle exocytosis, strongly influenced by external [Ca2+], appeared with the kinetics accelerated by depolarization. These results are consistent with the potential involvement of Ca2+ in taking low-affinity transporters to the plasma membrane surface via exocytosis.
Panel 4D Summary: Ag2896/Ag2920 Two experiments show moderate to low expression of the CG56262-01 transcript across a wide range of cells of this panel including epithelium, fibroblasts, and endothelial cells. Lower but still significant levels of expression are also seen in the key players of innate and adaptive immunity: monocytes/macrophages, T and B cells. However, the expression of this franscript is highest in the B lymphoma cell line, and NCI H292, a mucoepidermoid cell line (CTs=26.4-27). Thus, inhibition of the function of the protein encoded by this transcript with a small molecule drug, could lead to improvement of the symptoms of patients suffering from autoimmune and inflammatory diseases such as asthma, COPD, emphysema, psoriasis, inflammatory bowel disease, lupus erythematosus, or rheumatoid arthritis.
Panel CNS_1 Summary: Ag2896 This expression profile confirms the presence of this gene in the brain. Please see Panel 1.3D for discussion of utility of this gene in the cenfral nervous system.
NOV28: CG56559-01 : Na(+)/glucose cotransporter
Expression of gene CG56559-01 was assessed using the primer-probe sets Ag2950 and Ag2966, described in Tables UA and UB. Results of the RTQ-PCR runs are shown in Tables UC, UD, UE an UF.
Table UA. Probe Name Ag2950
Figure imgf000598_0001
Reverse 5 ' -ggtcactgactgcatgtagatg-3 ' (SEQ ID NO : 460) 22 1379
Table UB. Probe Name Ag2966
Figure imgf000599_0001
Table UC. Panel 1.3D
Figure imgf000599_0002
Figure imgf000600_0001
Figure imgf000601_0001
Table UP. Panel 2D
Figure imgf000602_0001
Figure imgf000603_0001
Figure imgf000604_0001
Table UE. Panel 3D
Figure imgf000604_0002
Figure imgf000605_0001
Figure imgf000606_0001
Table UF. Panel 4D
Figure imgf000606_0002
Figure imgf000607_0001
Figure imgf000608_0001
Figure imgf000609_0001
Panel 1.3D Summary: Ag2950/Ag2966 Three experiments both show expression of the CG56559-01 gene, a sodium-glucose cotransporter homolog, limited to the kidney ( Ts=:29). This restricted expression is in agreement with published data that shows secondary active transport of glucose in the kidney is mediated by sodium glucose cotransporter. (See ref. 1). Thus, expression of this gene could be used as a marker for kidney tissue. Furthermore, the protein product may be important for normal function of the kidney. Thus, therapeutic modulation of the expression or function of this protein may be useful in treating diseases that affect the kidney, including diabetes.
References:
Bissonnette P, Noel J, Coady M J, Lapointe JY. Functional expression of tagged human Na+- glucose cotransporter in Xenopus laevis oocytes. J Physiol 1999 Oct 15;520 Pt 2:359-71
1. High-affinity, secondary active transport of glucose in the intestine and kidney is mediated by an integral membrane protein named SGLTl (sodium glucose cotransporter). Though basic properties of the transporter are now defined, "many questions regarding the structure- function relationship of the protein, its biosynthesis and targeting remain unanswered, h order to better address these questions, we produced a functional hSGLTl protein (from human) containing a reporter tag. 2. Six constructs, made from three tags (myc, haemaglutinin and poly-His) inserted at both the C- and N-terminal positions, were thus tested using the Xenopus oocyte expression system via electrophysiology and immunohistochemistry. Of these, only the hSGLTl construct with the myc tag inserted at the N-terminal position proved to be of interest, all other constructs showing no or little transport activity. A systematic comparison of transport properties was therefore performed between the myc-tagged and the untagged hSGLTl proteins. 3. On the basis of both steady-state (affinities for substrate (glucose) and inhibitor (phlorizin) as well as expression levels) and presteady-state parameters (transient currents) we conclude that the two proteins are functionally indistinguishable, at least under these criteria. Immunological detection confirmed the appropriate targeting of the tagged protein to the plasma membrane of the oocyte with the epitope located at the extracellular side. 4. The myc-tagged hSGLTl was also successfully expressed in polarized MDCK cells. alpha-Methylglucose uptake studies on fransfected cells showed an exclusively apical uptake pathway, thus indicating that the expressed protein was correctly targeted to the apical domain of the cell. 5. These comparative studies demonstrate that the myc epitope inserted at the N-tenninus of hSGLTl produces a fully functional protein while other epitopes of similar size inserted at either end of the protein inactivated the final protein.
PMID: 10523405
Panel 2D Summary: Ag2966 Expression of the CG56559-01 gene is predominantly limited to the kidney. This result is in agreement with the expression seen in Panel 1.3D. Thus, expression of this gene might be used as a marker of normal kidney tissue.
Panel 3D Summary: Ag2966 Results from one experiment with the CG56559-01 gene are not included. The amp plot indicates that there were experimental difficulties with this run.
Panel 4D Summary: Ag2950/Ag2966 Expression of the CG56559-01 gene is predominantly found in normal tissue from thymus, lung, colon and kidney. This expression profile suggests that the protein product may be involved in glucose transport and normal homeostasis in these tissues. Therefore, therapeutic modulation of the expression or function of this protein may be useful in for maintaining or restoring normal function to these organs during inflammation.
NOV29a: CG56557-01: Na(+)/glucose cotransporter
Expression of gene CG56557-01 was assessed using the primer-probe set Ag2931 , described in Table NA. Results of the RTQ-PCR runs are shown in Table NB.
Table NA. Probe Name Ag2931
Figure imgf000610_0001
Table VB.Panel 4D
Figure imgf000611_0001
Figure imgf000612_0001
CNS_neurodegeneration_vl.0 Summary: Ag2931 Data from one experiment with this probe and primer set and the CG56557-01 gene show low/undetectable levels of expression in all samples on this panel. (CTs>35). (Data not shown.) The amp plot indicates that there is a high probability of a probe failure in this experiment. Panel 1.3D Summary: Ag2931 Data from one experiment with this probe and primer set and the CG56557-01 gene show low/undetectable levels of expression in all samples on this panel. (CTs>35). (Data not shown.) The amp plot indicates that there is a high probability of a probe failure in this experiment.
Panel 2D Summary: Ag2931 Data from one experiment with this probe and primer set and the CG56557-01 gene show low/undetectable levels of expression in all samples on this panel. (CTs>35). (Data not shown.) The amp plot indicates that there is a high probability of a probe failure in this experiment.
Panel 4D Summary: Ag2931 This CG56557-01 transcript, a Na/glucose cotransporter homolog, is expressed at low levels in small airway epithelium, bronchial epithelium, keratinocytes and lung micro vasculature. Furthermore, expression of this transcript is up-regulated by the proinflammatory cytokines TNF-a and IL-lb in all these samples. Modulation of the expression and/or activity of this putative protein by antibodies or small molecules may reduce or eliminate inflammatory reactions that occurs in the lung or skin as a result of astyhma, COPD, emphysema, psoriasis or other skin inflammatory diseases. Ag2931 Data from one experiment on this panel with the CG56557-01 gene, designated Run 164300130, is not included. The amp plot indicates that there were experimental difficulties with this run.
NOV29d and NOV29f: CG56557-04 and CG56557-06: Na(+)/glucose cotransporter Expression of gene CG56557-04 and variant CG56557-06 was assessed using the primer-probe set Ag6054, described in Table WA. Please note that CG56557-04 is a splice variant of CG56557- 01 (presented in section V above)
Table WA. Probe Name Ag6054
Figure imgf000613_0001
CNS_neurodegeneration_vl.0 Summary: Ag6054 Expression of the CG56557-06 gene is low/undetectable in all samples on this panel (CTs>35).
General_screening_panel_vl.5 Summary: Ag6054 Expression of the CG56557-06 gene is low/undetectable in all samples on this panel (CTs>35). Panel 4.1D Summary: Ag6054 Expression of the CG56557-06 gene is low/undetectable in all samples on this panel (CTs>35).
NOV29c: CG56557-03: splice variant of CG56557-01
Expression of gene CG56557-03 was assessed using the primer-probe set Ag6053, described in Table XA. Results of the RTQ-PCR runs are shown in Tables XB and XC.
Table XA. Probe Name Ag6053
Figure imgf000614_0001
Table XB General_screening_panel_vl.5
Figure imgf000614_0002
Figure imgf000615_0001
Figure imgf000616_0002
Table XC Panel 4. ID
Figure imgf000616_0001
Figure imgf000617_0001
Figure imgf000618_0001
CNS_neurodegeneration_vl.O Summary: Ag6053 Expression of the CG56557-03 gene is low/undetectable in all samples on this panel.
General_screening_panel_vl.5 Summary: Ag6053 The CG56557-03 gene is expressed at low levels in most samples in this panel, with highest expression in CaCo-2 colon cancer cells (CT=30). Significant expression is also seen in some ovarian, colon, renal, CNS cancer cell lines. Hence, expression of this gene could be used as a diagnostic marker and/or for freatment of similar cancers.
In addition, this gene is expressed at low levels in pancreas and adrenal (CT values = 33-34). Thus, this gene product may be a small molecule target for the treatment of metabolic diseases including obesity and Types 1 and 2 diabetes. Furthermore, this gene is expressed at higher levels in fetal liver(CT value = 31) when compared to expression in adult liver (CT value = 36) and may be useful for the differentiation between the two sources of liver tissue.
Panel 4.1D Summary: Ag6053 The CG56557-03 franscript is mostly expressed in kidney (CT=29.5). Low expression of this transcript is also found in colon and thymus. The protein encoded by this transcript may thus be involved in normal tissue/cellular functions in the kidney and colon. Therefore, therapeutics designed with the protein encoded by this franscript may be important in maintaining or restoring nonnal function to these organs during inflammation.
NON29e: CG56557-05: splice variant of CG56557-01
Expression of gene CG56557-05 was assessed using the primer-probe set Ag6055, described in Table YA. Results of the RTQ-PCR runs are shown in Tables YB and YC.
Table YA. Probe Name Ag6055
Figure imgf000619_0001
Table YB General_screeningjpanel_vl.5
Figure imgf000619_0002
Figure imgf000620_0001
Table YC. Panel 4. ID
Figure imgf000621_0001
Figure imgf000622_0001
CNS_neurodegeneration_vl.0 Summary: Ag6055 Expression of the CG56557-05 gene is low/undetectable in all samples on this panel (CTs>35).
General_screeningjpanel_vl.5 Summary: Ag6055 Highest expression of the CG56557-05 gene is in fetal liver (CT value=31). Furthermore, this gene is expressed at much higher levels in the fetal liver when compared to expression in the adult liver (CT=36). Thus, this gene product may be useful for the differentiation of between the adult and fetal sources of this tissue. Significant expression is also seen in CaCo-2 colon cancer cells, TK-10 renal cells and HepG2 liver cells. Hence, expression of this gene can be used as a diagnostic marker and/or as freatment for related kidney and colon cancers.
Panel 4.1D Summary: Ag6055 The CG56557-05 transcript is selectively expressed at low levels in colon (CT=32.7) and kidney. Thus, the protein encoded for this franscript may be involved in normal tissue/cellular functions. Therefore, therapeutics designed with the protein encoded by this franscript may be important for maintaining or restoring normal function to these organs during inflammation.
NOV30: CG56398-01: Na/glucose cotransporter
Expression of gene CG56398-01 was assessed using the primer-probe set Ag2925, described in Table ZA. Results of the RTQ-PCR runs are shown in Tables ZB, ZC, ZD, ZE and ZF.
Table ZA. Probe Name Ag2925
Figure imgf000623_0001
Table ZB. CNS_neurodegeneration_vl.O
Figure imgf000623_0002
Figure imgf000624_0001
Table ZC. Panel 1.3D
Tissue Name Rel. Exp.(%) Ag2925, Rel. Exp.(%) Ag2925,
Tissue Name Run 158046924 Run 158046924
Liver adenocarcinoma 0.4 Kidney (fetal) 1.2
Pancreas 0.0 Renal ca. 786-0 0.0
Pancreatic ca. CAPAN 2 0.0 Renal ca. A498 0.1
Figure imgf000625_0001
Figure imgf000626_0001
Figure imgf000627_0001
Figure imgf000628_0001
Table ZE. Panel 4D
Figure imgf000629_0001
Figure imgf000630_0001
Table ZF. Panel CNS 1
Figure imgf000630_0002
Figure imgf000631_0001
Figure imgf000632_0001
CNS_neurodegeneration_vl.O Summary: Ag2925 This panel does not show differential expression of the CG56398-01 gene in Alzheimer's disease. However, this expression profile confirms the presence of this gene in the brain. Please see Panel 1.3D for discussion of utility of this gene in the cenfral nervous system.
Panel 1.3D Summary: Ag2925 Expression of the CG56398-01 gene appears to be brain- specific. Highest expression is detected in the hippocampus (CT=28) a region that degenerates in Alzheimer's disease. Thus, this gene would be useful for distinguishing brain tissue from non- neural tissue, and may be beneficial as a drug target in neurodegenerative disease. Panel 2D Summary: Ag2925 The CG56398-01 gene is most highly expressed in a normal kidney sample (CT= 28.95). Interestingly, expression of this gene is lost in the adjacent cancer samples. Hence, the loss of expression could potentially be used as a diagnostic marker for kidney cancer. This gene is also expressed at low levels in breast and bladder cancer samples and is absent or exfremely low in normal adjacent tissue. Therefore, therapeutic inhibition of the activity of this protein product, through the use of small molecule drugs or antibodies, may be useful in the treatment of breast and bladder cancer or as a diagnostic marker for the presence of these cancers.
Panel 4D Summary: Ag2925 Expression of the CG56398-01 transcript is almost exclusively restricted to colon and thymus, with highest expression in normal colon (CT=29). Furthermore, it is expressed at much lower levels in IBD colon. Therefore, the protein encoded by this transcript may be involved in normal tissue/cellular functions in the kidney and colon. Loss-of-expression of this protein may serve as a diagnostic marker for lupus or IBD.
Panel CNS_1 Summary: Ag2925 This panel confirms expression of the CG56398-01 gene in the brain. Please see Panel 1.3D for discussion of utility of this gene in the central nervous system.
NOV31: CG56616-01: Olfactory Receptor
Expression of gene CG56616-01 was assessed using the primer-probe sets Agl371 and Ag2014, described in Tables AAA, AAB, and AAC. Results of the RTQ-PCR runs are shown in Tables AAD, AAE, AAF and AAG.
Table AAA. Probe Name Agl371
Figure imgf000633_0001
Table AAB. Probe Name Agl656
Figure imgf000634_0001
Table AAC. Probe Name Ag2014
Figure imgf000634_0002
Table AAD. Panel 1.2
Figure imgf000634_0003
Figure imgf000635_0001
Figure imgf000636_0001
Figure imgf000637_0001
Figure imgf000638_0001
Figure imgf000639_0001
Figure imgf000640_0001
Table AAG. Panel 4D
Figure imgf000640_0002
Figure imgf000641_0001
Figure imgf000642_0001
Panel 1.2 Summary: Agl371 Expression of the CG56616-01 gene in this panel is seen in a number of normal tissues including colon, small intestine, bone marrow, thymus, spleen, lymph node, bladder, fetal kidney, ovary, and testis.
In addition, the CG56616-01 transcript is present in a number of metabolically relevant tissues, with low expression in adrenal gland (CT = 33) and skeletal muscle (CT = 32.6), and moderate expression in thyroid (CT = 30.8), pituitary (CT = 28) and liver (CT = 29.2). Therefore, this gene product may be involved in signal transduction pathways in thyroid, pituitary and liver, and may be a drug target for any disease involving one or more of these tissues. For example, this GPCR shows high expression in the pituitary, which controls much endocrine secretion through response to hypophysiotrophic hormones (such as thyrotropin-releasing hormone, somatostatin, somatocrinin, gonadotropin-releasing hormone, corticofropin-releasing hormone) in the posterior pituitary, and response to peripheral hormones (e.g., estrogen, testosterone, etc) in the anterior pituitary. There are a number of diseases associated with pituitary pathophysiology, including hyper- and hypothyroidism, gigantism, dwarfism, acromegaly, Addison's disease, Cushing's disease, diabetes insipidus. Therefore, therapeutic modulation, blockade, freatment with antagonists, or stimulation of the GPCR encoded by the CG56616-01 gene may be useful in the freatment of one or more of these diseases.
The CG56616-01 gene is expressed at low to moderate levels throughout the CNS, including in amygdala, cerebellum, hippocampus, thalamus, spinal cord and developing brain, with highest expression in cerebral cortex (CT = 26.6). The CG56616-01 gene encodes a putative GPCR. Several neurotransmitter receptors are GPCRs, including the dopamine receptor family, the serotonin receptor family, the GABAB receptor, muscarinic acetylcholine receptors, and others; thus this GPCR may represent a novel neurotransmitter receptor. Targeting various neurotransmitter receptors (dopamine, serotonin) has proven to be an effective therapy in psychiatric illnesses such as schizophrenia, bipolar disorder and depression. Furthermore, the cerebral cortex and hippocampus are regions of the brain that are known to play critical roles in Alzheimer's disease, seizure disorders, and in the normal process of memory formation. Therefore, therapeutic modulation of the CG56616-01 gene or its protein product may be beneficial in the freatment of one or more of these diseases, as may stimulation and/or blockade of the receptor coded for by the gene. Levels of this gene are high, however, in areas outside of the cenfral nervous system (such as. the liver), suggesting the possibility of a wider role in intercellular signaling.
interestingly, the CG56616-01 gene appears to be expressed by a cluster of cell lines derived from melanoma, prostate cancer, lung cancer and ovarian cancer. In addition, this gene seems to be more highly expressed by adult liver when compared to fetal liver and expressed more highly in fetal kidney when compared to adult kidney. Thus, these data indicate that expression of the CG56616-01 gene might be useful in the distinction of adult vs. fetal liver or kidney tissue. Therapeutic application of the CG56616-01 protein might be of use in the treatment of diseases involving the liver in which the diseased state resembles fetal liver. In contrast this gene seems to be expressed by fetal kidney when compared to adult kidney. Thus, application of the CG56616- 01 protein might be useful in the treatment of kidney disorders that require tissue regeneration. Also, therapeutic modulation of the CG56616-01 gene product, through the use of small molecule drugs or antibodies might be of use in the treatment of ovarian cancer, prostate cancer, lung cancer or melanoma.
Panel 1.3D Summary: Ag2014 Significant expression of the CG56616-01 gene is detected in pituitary gland (CT = 34.7), cerebral cortex (CT =34.5), fetal skeletal muscle (CT = 34.6), and an ovarian cancer cell line (CT = 34.3). These results are consistent with what is observed in other panels and the potential implications are discussed above and below. A second experiment with the probe/primer set Agl656 shows low/undetectable levels of expression in all the samples on this panel (CTs>35).
Panel 2.2 Summary: Ag2014 Expression of the CG56616-01 gene in panel 2.2 is generally low. However, there is significant expression in samples from kidney cancer, thyroid cancer, liver derived tissue (both normal and malignant) and ovarian derived tissue (both normal and malignant). Of particular interest is the comparison of CG56616-01 gene expression between samples of kidney and thyroid cancers and their respective normal adjacent tissue samples. In both cases the CG56616-01 gene is overexpressed in the malignant tissue when compared to the normal adjacent tissue. Thus, based on these data, therapeutic modulation of the activity of the CG56616-01 gene product, through the use of small molecule drugs or antibodies, maybe of use in the freatment of kidney or thyroid cancer. A second experiment with the probe/primer set Agl656 shows low/undetectable levels of expression in all the samples on this panel (CTs>35).
Panel 4D Summary: Ag2014 The H292 lung epithelial cell line expresses the CG56616-01 gene after IL-9-stimulation. Therefore, the putative GPCR encoded by the CG56616-01 gene may be involved in lung inflammation and mucus secretion (ref. 1). Antibodies or small molecule therapeutics that block the function of this membrane protein may thus be useful as anti- inflammatory therapeutics for the treatment of asthma and emphysema. Very low expression is also detected in a number of other samples including IBD colitis 2 (CT = 34.1), Crohn's (CT = 34.9), thymus (CT = 34.8), kidney (CT = 34.8), monocytes freated with LPS (CT = 34.7) and asfrocytes freated with TNFalpha + IL-lbeta (CT =34). A second experiment with the probe/primer set Agl656 shows low/undetectable levels of expression in all the samples on this panel (CTs>35).
References:
1. Louahed J., Toda M., Jen J., Hamid Q., Renauld J.C., Levitt R.C., Nicolaides N.C. (2000)
Interleukin-9 upregulates mucus expression in the airways. Am. J. Respir. Cell. Mol. Biol. 22: 649-656. 2. Interleukin (IL)-9 has recently been shown to play an important role in allergic disease because its expression is strongly associated withthe degree of airway responsiveness and the asthmatic-like phenotype. IL-9 is a pleiofropic cytokine that is active on many cell typesinvolved in the allergic immune response. Mucus hypersecretion is a clinical feature of chronic airway diseases; however, themechanisms underlying the induction of mucin are poorly understood, hi this report, we show that IL-9 regulates the expression of asubset of mucin genes in lung cells both in vivo and in vitro. In vivo, the constitutive expression of IL-9 in transgenic mice results inelevated MUC2 and MUC5AC gene expression in airway epithelial cells and periodic acid-Schiff-positive staining (reflecting mucousglycogenates). Similar results were observed in C57BL/6J mice after IL-9 infratracheal instillation. In contrast, instillation of the Thelper 1 -associated cytokine interferon gamma failed to induce mucin production, hi vifro, our studies showed that IL-9 also inducesexpression of MUC2 and MUC5AC in human primary lung cultures and in the human muccoepidermoid NCI-H292 cell line, indicatinga direct effect of IL-9 on inducing mucin expression in these cells. Altogether, these results suggest that upregulation of mucin by IL-9might contribute to the pathogenesis of human inflammatory airway disorders, such as asthma. These data extend the role of thebiologic processes that IL-9 has on regulating the many clinical features of asthma and further supports the IL-9 pathway as a keymediator of the asthmatic response.
NOV32: 153065222/CG56234-02: Splice variant of PCK2
Expression of gene CG56234-02 was assessed using the primer-probe set Ag5111, described in Table ABA. Results of the RTQ-PCR runs are shown in Tables ABB and ABC.
Table ABA. Probe Name Ag5111
Figure imgf000645_0001
Table ABB. General_screening_panel_vl.5
Figure imgf000645_0002
Figure imgf000646_0001
Figure imgf000647_0001
Table ABC. Panel 4. ID
Figure imgf000647_0002
Figure imgf000648_0001
Figure imgf000649_0001
CNS_neurodegeneration_vl.0 Summary: Ag5111 Expression of the CG56234-02 gene is low/undetectable in all samples on this panel (CTs>35).
General_screening_panel_vl.5 Summary: Ag5111 Highest expression of the CG56234-02 gene is seen in an ovarian cancer cell line (CT=30). This gene encodes a splice variant of PEPCK2, the rate-limiting enzyme in gluconeogenesis that has been shown to be regulated in response to hormones and environmental stress. In addition, to the ovarian cancer cell line, this gene is expressed at a moderate level in most of the cancer cell lines used in this panel. Therefore, modulation of the gene product using small molecule drugs may affect the growth and survival of cancer cells. Expression of this gene could potentially be used as a diagnostic marker of the metabolic status of cells and inhibition of activity of this gene product might be used for therapeutic freatment of cancers.
This gene is also moderately expressed (CT values = 34) in adult and fetal liver. Inhibition of this enzyme could potentially decrease hepatic glucose production and thus serve as an effective freatment for Type 2 diabetes, which is characterized by excess hepatic glucose production.
Panel 4.1D Summary: Ag 5111 The CG56234-02 transcript is expressed at low levels in a wide range of cell across this panel (CTs=33.3-34.4), including CD4 T cells (naive and memory T cells), CD8 T cells, B cells and macrophages. Expression of this franscript is also found in dermal fibroblasts and kidney. This transcript encodes a homolog of a key enzyme in glucogenesis and therefore may be important for the metabolic status of all these cell types that contribute to the inflammatory response. Therefore, modulation of the activity or expression of this putative protein by small molecules could affect the activity of these cells and be useful for the treatment of autoimmune diseases such as inflammatory bowel diseases, rheumatoid arthritis, asthma, COPD, psoriasis and lupus.
Example 2. SNP analysis of NOVX clones
SeqCallingTM Technology: cDNA was derived from various human samples representing multiple tissue types, normal and diseased states, physiological states, and developmental states from different donors. Samples were obtained as whole tissue, cell lines, primary cells or tissue cultured primary cells and cell lines. Cells and cell lines may have been freated with biological or chemical agents that regulate gene expression for example, growth factors, chemokines, steroids. The cDNA thus derived was then sequenced using CuraGen's proprietary SeqCalling technology. Sequence traces were evaluated manually and edited for corrections if appropriate. cDNA sequences from all samples were assembled with themselves and with public ESTs using bioinformatics programs to generate CuraGen's human SeqCalling database of SeqCalling assemblies. Each assembly contains one or more overlapping cDNA sequences derived from one or more human samples. Fragments and ESTs were included as components for an assembly when the extent of identity with another component of the assembly was at least 95% over 50 bp. Each assembly can represent a gene and/or its variants such as splice forms and/or single nucleotide polymorphisms (SNPs) and their combinations. Variant sequences are included in this application. A variant sequence can include a single nucleotide polymorphism (SNP). A SNP can, in some instances, be refened to as a "cSNP" to denote that the nucleotide sequence containing the SNP originates as a cDNA. A SNP can arise in several ways. For example, a SNP may be due to a substitution of one nucleotide for another at the polymorphic site. Such a substitution can be either a transition or a transversion. A SNP can also arise from a deletion of a nucleotide or an insertion of a nucleotide, relative to a reference allele. In this case, the polymorphic site is a site at which one allele bears a gap with respect to a particular nucleotide in another allele. SNPs occurring within genes may result in an alteration of the amino acid encoded by the gene at the position of the SNP. fritragenic SNPs may also be silent, however, in the case that a codon including a SNP encodes the same amino acid as a result of the redundancy of the genetic code. SNPs occurring outside the region of a gene, or in an intron within a gene, do not result in changes in any amino acid sequence of a protein but may result in altered regulation of the expression pattern for example, alteration in temporal expression, physiological response regulation, cell type expression regulation, intensity of expression, stability of franscribed message.
Method of novel SNP Identification: SNPs are identified by analyzing sequence assemblies using CuraGen's proprietary SNPTool algorithm. SNPTool identifies variation in assemblies with the following criteria: SNPs are not analyzed within 10 base pairs on both ends of an alignment; Window size (number of bases in a view) is 10; The allowed number of mismatches in a window is 2; Minimum SNP base quality (PHRED score) is 23; Minimum number of changes to score an SNP is 2/assembly position. SNPTool analyzes the assembly and displays SNP positions, associated individual variant sequences in the assembly, the depth of the assembly at that given position, the putative assembly allele frequency, and the SNP sequence variation. Sequence traces are then selected and brought into view for manual validation. The consensus assembly sequence is imported into CuraTools along with variant sequence changes to identify potential amino acid changes resulting from the SNP sequence variation. Comprehensive SNP data analysis is then exported into the SNPCalling database. Method of novel SNP Confirmation :
SNPs are confirmed employing a validated method know as Pyrosequencing (Pyrosequencing, Westborough, MA). Detailed protocols for Pyrosequencing can be found in: Alderborn et al. Determination of Single Nucleotide Polymorphisms by Real-time Pyrophosphate DNA Sequencing. (2000). Genome Research. 10, Issue 8, August. 1249-1265. In brief, Pyrosequencing is a real time primer extension process of genotyping. This protocol takes double- sfranded, biotinylated PCR products from genomic DNA samples and binds them to sfreptavidin beads. These beads are then denatured producing single stranded bound DNA. SNPs are characterized utilizing a technique based on an indirect bioluminometric assay of pyrophosphate (PPi) that is released from each dNTP upon DNA chain elongation. Following Klenow polymerase-mediated base incorporation, PPi is released and used as a subsfrate, together with adenosine 5'-phosphosulfate (APS), for ATP sulfurylase, which results in the formation of ATP. Subsequently, the ATP accomplishes the conversion of fuciferin to its oxi-derivative by the action of luciferase. The ensuing light output becomes proportional to the number of added bases, up to about four bases. To allow processivity of the method dNTP excess is degraded by apyrase, which is also present in the starting reaction mixture, so that only dNTPs are added to the template during the sequencing. The process has been fully automated and adapted to a 96-well format, which allows rapid screening of large SNP panels. The DNA and protein sequences for the novel single nucleotide polymorphic variants are reported. Variants are reported individually but any combination of all or a select subset of variants are also included, hi addition, the positions of the variant bases and the variant amino acid residues are underlined.
RESULTS
Variants are reported individually but any combination of all or a select subset of variants are also included as contemplated NOVX embodiments of the invention.
NON4a SΝP data
The DΝA and protein sequences for the novel single nucleotide polymorphic variants of the Myotonic dystrophy kinase-related CDC42-binding kinase-like gene of ΝON4a are reported in Table 41. Variants are reported individually but any combination of all or a select subset of variants are also included, hi summary, there are 4 variants reported .
Figure imgf000652_0001
ΝOV6 SNP data The DNA and protein sequences for the novel single nucleotide polymorphic variants of the GPCR-like gene of NOV6 are reported in Table 6H. Variants are reported individually but any combination of all or a select subset of variants are also included. In summary, there are 2 variants reported .
Figure imgf000653_0001
NOV8a SNP data
The DNA and protein sequences for the novel single nucleotide polymorphic variants of the Carboxypeptidase-like gene of NON8a are reported in Table 8M. Variants are reported individually but any combination of all or a select subset of variants are also included. In summary, there are 14 variants reported.
Figure imgf000653_0002
Figure imgf000654_0001
NON8b SΝP data: hi the following positions, one or more consensus positions (Cons. Pos.) of the nucleotide sequence have been identified as SΝPs. ΝOV8b has 7 SNP variants, whose variant positions for its nucleotide and amino acid sequences is numbered according to SEQ ID NOS:21 and 22, respectively. The nucleotide sequence of the NOV8b variant differs as shown in Table 8N.
Figure imgf000654_0002
NON9 SΝP data
The DΝA and protein sequences for the novel single nucleotide polymorphic variants of the Neurotransmitter Receptor-like gene of NOV9 are reported in Table 9G. Variants are reported individually but any combination of all or a select subset of variants are also included. In summary, there is 1 variant reported.
Figure imgf000654_0003
NOVlla SNP data
The DNA and protein sequences for the novel single nucleotide polymorphic variants of the Lysyl oxidase-like gene of NOVl la are reported in Table 111. Variants are reported individually but any combination of all or a select subset of variants are also included. In summary, there are 3 variants reported.
Figure imgf000655_0001
NOVllb SNP data: hi the following positions, one or more consensus positions (Cons. Pos.) of the nucleotide sequence have been identified as SNPs. NOVl lb has 11 SNP variants, whose variant positions for its nucleotide and amino acid sequences is numbered according to SEQ ID NOS:33 and 34, respectively. The nucleotide sequence of the NOVl lb variant differs as shown in Table 11 J.
Figure imgf000655_0002
NOV12a SNP data
The DNA and protein sequences for the novel single nucleotide polymorphic variants of the Phosphatase-like gene of NOV12a are reported in Table 12 I. Variants are reported individually but any combination of all or a select subset of variants are also included. In summary, there is 1 variant reported.
Figure imgf000656_0001
NOV13 SNP data
The DNA and protein sequences for the novel single nucleotide polymorphic variants of the Chloride Channel Protein CLC-KA-like gene of NOVl 3 are reported in Table 13G. Variants are reported individually but any combination of all or a select subset of variants are also included, hi summary, there is 1 variant reported. hi Figure 3, the positions of the variant bases and the variant amino acid residues are underlined, in figure 3. Variant is a T to C SNP at 425 bp of the nucleotide sequence that results in no change in the protein sequence (silent).
Figure imgf000656_0002
NOV15a SNP data
The DNA and protein sequences for the novel single nucleotide polymorphic variants of the MEGF6-like gene of NOVl 5a are reported in Table 15Q. Variants are reported individually but any combination of all or a select subset of variants are also included. In summary, there are 4 variants reported.
Figure imgf000657_0001
NOV16 SNP data:
In the following positions, one or more consensus positions (Cons. Pos.) of the nucleotide sequence have been identified as SNPs. NOVl 6 has 3 SNP variants, whose variant positions for its nucleotide and amino acid sequences is numbered according to SEQ ID NOS:55 and 56, respectively. The nucleotide sequence of the NOV16 variant differs as shown in Table 16G.
Figure imgf000657_0002
NOVl 7a SNP data
The DNA and protein sequences for the novel single nucleotide polymorphic variants of the PEST-containing fransporter-like gene of NOVl 7a are reported in Table 171. Variants are reported individually but any combination of all or a select subset of variants are also included. In summary, there are 2 variants reported.
Figure imgf000657_0003
Figure imgf000658_0001
NOV18a SNP data
The DNA and protein sequences for the novel single nucleotide polymorphic variants of the GPCR-like gene of NOVl 8a are reported in Table 18H. Variants are reported individually but any combination of all or a select subset of variants are also included. In summary, there are 1 variants reported.
Figure imgf000658_0002
NOV18b SNP data: h the following positions, one or more consensus positions (Cons. Pos.) of the nucleotide sequence have been identified as SNPs. NOVl 8b has 1 SNP variants, whose variant positions for its nucleotide and amino acid sequences is numbered according to SEQ ID NOS: 63 and 62, respectively. The nucleotide sequence of the NOVl 8b variant differs as shown in Table 181.
Figure imgf000658_0003
NOV19b SNP data: In the following positions, one or more consensus positions (Cons. Pos.) of the nucleotide sequence have been identified as SNPs. NOVl 9b has one SNP variant, whose variant positions for its nucleotide and amino acid sequences is numbered according to SEQ ID NOS:66 and 67, respectively. The nucleotide sequence of the NOVl 9b variant differs as shown in Table 191.
Figure imgf000659_0001
NOV29f SNP data:
In the following positions, one or more consensus positions (Cons. Pos.) of the nucleotide sequence have been identified as SNPs. NOV29f has nine SNP variants, whose variant positions for its nucleotide and amino acid sequences is numbered according to SEQ ID NOS:114 and 115, respectively. The nucleotide sequence of the NO V29f variant differs as shown in Table 29Q.
Figure imgf000659_0002
NOV31 SNP data:
In the following positions, one or more consensus positions (Cons. Pos.) of the nucleotide sequence have been identified as SNPs. NOV31 has ten SNP variants, whose variant positions for its nucleotide and amino acid sequences is numbered according to SEQ ID NOS:118 and 119, respectively. The nucleotide sequence of the NOV31 variant differs as shown in Table 31H.
Table 31H. cSNP and Coding Variants for NOV31
NT Position Wild Type Variant NT of cSNP NT
Figure imgf000660_0001
Example 3. Identification of NOVX clones
5 The novel NOVX target sequences identified in the present invention were subjected to the exon linking process to confirm the sequence. PCR primers were designed by starting at the most upstream sequence available, for the forward primer, and at the most downstream sequence available for the reverse primer. Table 34A shows the sequences of the PCR primers used for obtaining different clones for NOVl -18, if any. PCR primers for NOVl 9-33, if any, are disclosed
10 separately within their respective section above. In each case, the sequence was examined, walking inward from the respective termini toward the coding sequence, until a suitable sequence that is either unique or highly selective was encountered, or, in the case of the reverse primer, until the stop codon was reached. Such primers were designed based on in silico predictions for the full length cDNA, part (one or more exons) of the DNA or protein sequence of the target
15 sequence, or by translated homology of the predicted exons to closely related human sequences from other species. These primers were then employed in PCR amplification based on the following pool of human cDNAs: adrenal gland, bone marrow, brain - amygdala, brain - cerebellum, brain - hippocampus, brain - substantia nigra, brain - thalamus, brain -whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma - Raji, mammary gland,
20 pancreas, pituitary gland, placenta, prostate, sahvary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea, uterus.
Figure imgf000660_0002
Figure imgf000661_0001
Usually the resulting amplicons were gel purified, cloned and sequenced to high redundancy. The PCR product derived from exon linking was cloned into the pCR2.1 vector from Invitrogen. The resulting bacterial clone has an insert covering the entire open reading frame cloned into the pCR2.1 vector. Table 34B shows a list of these bacterial clones for NOV1-18, if any. Bacterial clones for NOV19-33, if any, are treated in their respective sections above. The resulting sequences from all clones were assembled with themselves, with other fragments in CuraGen Corporation's database and with public ESTs. Fragments and ESTs were included as 0 components for an assembly when the extent of their identity with another component of the assembly was at least 95% over 50 bp. In addition, sequence traces were evaluated manually and edited for corrections if appropriate. These procedures provide the sequence reported herein.
Figure imgf000661_0002
Figure imgf000662_0001
OTHER EMBODIMENTS
Although particular embodiments have been disclosed herein in detail, this has been done by way of example for purposes of illustration only, and is not intended to be limiting with respect to the scope of the appended claims, which follow. In particular, it is contemplated by the inventors that various substitutions, alterations, and modifications may be made to the invention without departing from the spirit and scope of the invention as defined by the claims. The choice of nucleic acid starting material, clone of interest, or library type is believed to be a matter of routine for a person of ordinary skill in the art with knowledge of the embodiments described herein. Other aspects, advantages, and modifications considered to be within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:
1. An isolated polypeptide comprising an amino acid sequence selected from the group consisting of: (a) a mature form of an amino acid sequence selected from the group consisting of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, and 123; (b) a variant of a mature form of an amino acid sequence selected from the group consisting of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, and 123, wherein one or more amino acid residues in said variant differs from the amino acid sequence of said mature form, provided that said variant differs in no more than 15% of the amino acid residues from the amino acid sequence of said mature form;
(c) an amino acid sequence selected from the group consisting of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 73, 75, 77, 79, 81,
83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, and 123; and
(d) a variant of an amino acid sequence selected from the group consisting of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 73, 75,
77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, and 123 wherein one or more amino acid residues in said variant differs from the amino acid sequence of said mature form, provided that said variant differs in no more than 15% of amino acid residues from said amino acid sequence.
2 The polypeptide of claim 1, wherein said polypeptide comprises the amino acid sequence of a naturally-occurring allelic variant of an amino acid sequence selected from the group consisting of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36,
38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, and 123.
3. The polypeptide of claim 2, wherein said allelic variant comprises an amino acid sequence that is the translation of a nucleic acid sequence differing by a single nucleotide from a nucleic acid sequence selected from the group consisting of SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 64, 66, 68, 70, 72, 74, 76,(78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104,
106, 108, 110, 112, 114, 116, 118, 120, and 122.
4. The polypeptide of claim 1 , wherein the amino acid sequence of said variant comprises a conservative amino acid substitution.
5. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence selected from the group consisting of:
(a) a mature form of an amino acid sequence selected from the group consisting of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, and 123;
(b) a variant of a mature form of an amino acid sequence selected from the group consisting of SEQ TD NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, and 123, wherein one or more amino acid residues in said variant differs from the amino acid sequence of said mature form, provided that said variant differs in no more than 15% of the amino acid residues from the amino acid sequence of said mature form;
(c) an amino acid sequence selected from the group consisting of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, and 123;
(d) a variant of an amino acid sequence selected from the group consisting of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34,
36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, and 123, wherein one or more amino acid residues in said variant differs from the amino acid sequence of said mature form, provided that said variant differs in no more than 15% of amino acid residues from said amino acid sequence;
(e) a nucleic acid fragment encoding at least a portion of a polypeptide comprising an amino acid sequence chosen from the group consisting of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 73, 75,
77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, and 123, or a variant of said polypeptide, wherein one or more amino acid residues in said variant differs from the amino acid sequence of said mature form, provided that said variant differs in no more than 15% of amino acid residues from said amino acid sequence; and
(f) a nucleic acid molecule comprising the complement of (a), (b), (c), (d) or (e).
6. The nucleic acid molecule of claim 5, wherein the nucleic acid molecule comprises the nucleotide sequence of a natin ally-occurring allelic nucleic acid variant.
7. The nucleic acid molecule of claim 5, wherein the nucleic acid molecule encodes a polypeptide comprising the amino acid sequence of a naturally-occurring polypeptide variant.
8. The nucleic acid molecule of claim 5, wherein the nucleic acid molecule differs by a single nucleotide from a nucleic acid sequence selected from the group consisting of SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, and 122.
9. The nucleic acid molecule of claim 5, wherein said nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of (a) a nucleotide sequence selected from the group consisting of SEQ ID
NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, and 122; (b) a nucleotide sequence differing by one or more nucleotides from a nucleotide sequence selected from the group consisting of SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, and 122, provided that no more than 20% of the nucleotides differ from said nucleotide sequence; (c) a nucleic acid fragment of (a); and (d) a nucleic acid fragment of (b).
10. The nucleic acid molecule of claim 5, wherein said nucleic acid molecule hybridizes under stringent conditions to a nucleotide sequence chosen from the group consisting of SEQ ID
NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, ' 49, 51, 53, 55, 57, 59, 61, 63, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, and 122, or a complement of said nucleotide sequence.
11. The nucleic acid molecule of claim 5, wherein the nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of
(a) a first nucleotide sequence comprising a coding sequence differing by one or more nucleotide sequences from a coding sequence encoding said amino acid sequence, provided that no more than 20% of the nucleotides in the coding sequence in said first nucleotide sequence differ from said coding sequence; (b) an isolated second polynucleotide that is a complement of the first polynucleotide; and (c) a nucleic acid fragment of (a) or (b).
12. A vector comprising the nucleic acid molecule of claim 11.
13. The vector of claim 12, further comprising a promoter operably-linked to said nucleic acid molecule.
14. A cell comprising the vector of claim 12.
15. An antibody that immunospecifically-binds to the polypeptide of claim 1.
16. The antibody of claim 15, wherein said antibody is a monoclonal antibody.
17. The antibody of claim 15, wherein the antibody is a humanized antibody.
18. A method for determimng the presence or amount of the polypeptide of claim 1 in a sample, the method comprising: (a) providing the sample;
(b) contacting the sample with an antibody that binds immunospecifically to the polypeptide; and
(c) determimng the presence or amount of antibody bound to said polypeptide, thereby determining the presence or amount of polypeptide in said sample.
19. A method for determimng the presence or amount of the nucleic acid molecule of claim 5 in a sample, the method comprising:
(a) providing the sample;
(b) contacting the sample with a probe that binds to said nucleic acid molecule; and (c) determining the presence or amount of the probe bound to said nucleic acid molecule, thereby determining the presence or amount of the nucleic acid molecule in said sample.
20. A method of identifying an agent that binds to a polypeptide of claim 1 , the method comprising:
(a) contacting said polypeptide with said agent; and
(b) determining whether said agent binds to said polypeptide.
21. A method for identifying an agent that modulates the expression or activity of the polypeptide of claim 1, the method comprising:
(a) providing a cell expressing said polypeptide;
(b) contacting the cell with said agent; and
(c) determimng whether the agent modulates expression or activity of said polypeptide, whereby an alteration in expression or activity of said peptide indicates said agent modulates expression or activity of said polypeptide.
22. A method for modulating the activity of the polypeptide of claim 1 , the method comprising contacting a cell sample expressing the polypeptide of said claim with a compound that binds to said polypeptide in an amount sufficient to modulate the activity of the polypeptide.
23. A method of treating or preventing a NONX-associated disorder, said method comprising administering to a subject in which such freatment or prevention is desired the polypeptide of claim 1 in an amount sufficient to treat or prevent said ΝONX-associated disorder in said subject.
24. The method of claim 23, wherein said subject is a human.
25. A method of treating or preventing a ΝONX-associated disorder, said method comprising administering to a subject in which such freatment or prevention is desired the nucleic acid of claim 5 in an amount sufficient to treat or prevent said ΝONX-associated disorder in said subject.
26. The method of claim 25, wherein said subject is a human.
27. A method of treating or preventing a NONX-associated disorder, said method comprising admimstering to a subject in which such treatment or prevention is desired the antibody of claim 15 in an amount sufficient to freat or prevent said ΝONX-associated disorder in said subject.
28. The method of claim 27, wherein the subject is a human.
29. A pharmaceutical composition comprising the polypeptide of claim 1 and a pharmaceutically-acceptable carrier.
30. A pharmaceutical composition comprising the nucleic acid molecule of claim 5 and a pharmaceutically-acceptable carrier.
31. A pharmaceutical composition comprising the antibody of claim 15 and a pharmaceutically-acceptable carrier.
32. A kit comprising in one or more containers, the pharmaceutical composition of claim 29.
33. A kit comprising in one or more containers, the pharmaceutical composition of claim 30.
34. A kit comprising in one or more containers, the pharmaceutical composition of claim 31.
35. The use of a therapeutic in the manufacture of a medicament for treating a syndrome associated with a human disease, the disease selected from a ΝONX-associated disorder, wherein said therapeutic is selected from the group consisting of a ΝONX polypeptide, a ΝONX nucleic acid, and a ΝONX antibody.
36. A method for screening for a modulator of activity or of latency or predisposition to a ΝONX-associated disorder, said method comprising:
(a) administering a test compound to a test animal at increased risk for a ΝONX-associated disorder, wherein said test animal recombinantly expresses the polypeptide of claim 1;
(b) measuring the activity of said polypeptide in said test animal after admimstering the compound of step (a); (c) comparing the activity of said protein in said test animal with the activity of said polypeptide in a confrol animal not admimstered said polypeptide, wherein a change in the activity of said polypeptide in said test animal relative to said control animal indicates the test compound is a modulator of latency of or predisposition to a NOVX-associated disorder.
37. The method of claim 36, wherein said test animal is a recombinant test animal that expresses a test protein transgene or expresses said fransgene under the control of a promoter at an increased level relative to a wild-type test animal, and wherein said promoter is not the native gene promoter of said transgene.
38. A method for determimng the presence of or predisposition to a disease associated with altered levels of the polypeptide of claim 1 in a first mammalian subject, the method comprising:
(a) measuring the level of expression of the polypeptide in a sample from the first mammalian subject; and
(b) comparing the amount of said polypeptide in the sample of step (a) to the amount of the polypeptide present in a confrol sample from a second mammalian subject known not to have, or not to be predisposed to, said disease, wherein an alteration in the expression level of the polypeptide in the first subject as compared to the confrol sample indicates the presence of or predisposition to said disease.
39. A method for determimng the presence of or predisposition to a disease associated with altered levels of the nucleic acid molecule of claim 5 in a first mammalian subject, the method comprising:
(a) measuring the amount of the nucleic acid in a sample from the first mammalian subject; and
(b) comparing the amount of said nucleic acid in the sample of step (a) to the amount of the nucleic acid present in a confrol sample from a second mammalian subject known not to have or not be predisposed to, the disease; wherein an alteration in the level of the nucleic acid in the first subject as compared to the confrol sample indicates the presence of or predisposition to the disease.
40. A method of treating a pathological state in a mammal, the method comprising admimstering to the mammal a polypeptide in an amount that is sufficient to alleviate the pathological state, wherein the polypeptide is a polypeptide having an amino acid sequence at least 95% identical to a polypeptide comprising an amino acid sequence of at least one of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36,
38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, and 123, or a biologically active fragment thereof.
41. A method of treating a pathological state in a mammal, the method comprising administering to the mammal the antibody of claim 15 in an amount sufficient to alleviate the pathological state.
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