US20030186863A1 - Nck SH3 binding peptides - Google Patents

Nck SH3 binding peptides Download PDF

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US20030186863A1
US20030186863A1 US10/161,791 US16179102A US2003186863A1 US 20030186863 A1 US20030186863 A1 US 20030186863A1 US 16179102 A US16179102 A US 16179102A US 2003186863 A1 US2003186863 A1 US 2003186863A1
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seq
peptide
amino acid
domain
binding
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Andrew Sparks
Brian Kay
Judith Thorn
Lawrence Quilliam
Channing Der
Dana Fowlkes
James Rider
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B30/00—Methods of screening libraries
    • C40B30/04—Methods of screening libraries by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5041—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects involving analysis of members of signalling pathways
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6845—Methods of identifying protein-protein interactions in protein mixtures
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to SH3 binding peptides having a broad range of binding specificities. That is, certain members of the SH3 binding peptides disclosed bind with approximately the same facility with SH3 domains derived from different SH3 domain-containing proteins. Other members, in contrast, bind with a much greater degree of affinity for specific SH3 domains.
  • the SH3 binding peptides are obtained from random peptide libraries that are also phage-displayed. Methods are described of obtaining the phage clones that bind to the SH3 domain targets and of determining their relevant nucleotide sequences and consequent primary amino acid sequence of the binding peptides.
  • the resulting SH3 binding proteins are useful in a number of ways, including, but not limited to, providing a method of modulating signal transduction pathways at the cellular level, of modulating oncogenic protein activity or of providing lead compounds for development of drugs with the ability to modulate broad classes, as well as specific classes, of proteins involved in signal transduction.
  • SH3 domain a common sequence motif that influences the expression of proteins involved in eukaryotic cell signaling. It is 50-70 amino acids in length, moderately conserved in primary structure, and can be present from one to several times in a large number of proteins involved in signal transduction and in cytoskeletal proteins.
  • the protein pp60c-src represents a family of at least nine non-receptor protein tyrosine kinases (NR-PTKs). Members of this family share an overall structural organization comprising a series of catalytic and non-catalytic domains.
  • Src a 14-amino-acid myristylation signal resides at the extreme amino-terminus, and is followed by a unique region that is not highly conserved among family members. Following this region are two highly conserved 60-and 100-amino-acid regions, the Src homology (SH) domains 3 and 2, respectively.
  • SH2 and SH3 domains have been shown to play an important role in mediating protein-protein interactions in a variety of signaling pathways. Koch, C.
  • Src-related tyrosine kinases are expressed in a variety of cell types including those of the immune system (lymphocytes, T cells, B cells, and natural killer cells) and the central nervous system (neural cells, neurons, oligodendrocytes, parts of the cerebellum, and the like).
  • lymphocytes those of the immune system
  • central nervous system neural cells, neurons, oligodendrocytes, parts of the cerebellum, and the like.
  • Umemori H. et al., in Brain Res. Mol. Brain Res . (1992) Dec. 16(3-4):303-310.
  • mice with disruptions in their Src-like genes, Hck and Fgr possess macrophages with impaired phagocytic activity or exhibit a novel immunodeficiency characterized by an increased susceptibility to infection with Listeria monocytogenes.
  • LPS bacterial lipopolysaccharide
  • lymphokines such as TNF-alpha, IL-1, IL-6, and IL-8.
  • Inhibition of the protein tyrosine kinases blocks production of TNF-alpha and IL-1.
  • SH3 domains are sites of protein-protein interaction, but it has been unclear what SH3 domains actually bind. Efforts to identify ligands for SH3 domains have led to the characterization of a number of SH3-binding proteins, including 3BP1 and 2 (Ren, R., Mayer, et al., in Science (1993) 259:1157-61), SOS (Olivier, J.
  • PI-3′ Kinase phosphatidylinositol 3-kinase
  • a “biased” library is to be distinguished from a “random” library in that the amino acid residue at certain positions of the synthetic peptide are fixed, i.e., not allowed to vary in a random fashion. Indeed, as stated by these research workers, screening of a “random” combinatorial library failed to yield suitable ligands for a PI-3′ Kinase SH3 domain probe. The binding affinities of these unsuitable ligands was described as weak, >100 ⁇ M, based on dissociation constants measured by the Biosensor System (BIAcore).
  • Yu, et al. (Yu, H., et al., in Cell (1994) 76:933-945) used a “biased” synthetic peptide library of the form XXXPPXPXX (SEQ ID NO:2), wherein X represents any amino acid other than cysteine, to identify a series of peptides which bind the Src and PI-3′ Kinase SH3 domains.
  • the bias was accomplished by fixing the proline residues at the specific amino acid positions indicated for the “random” peptide.
  • the technique disclosed fails to identify any SH3 domain-binding peptides.
  • RXLPPRPXX SEQ ID NO:3
  • X tends to be a basic residue (like R, K or H).
  • the binding affinities of several SH3 binding peptides were disclosed as ranging from 8.7 to 30 ⁇ M.
  • RKLPPRPRR SEQ ID NO:4
  • RKLPPRPRR SEQ ID NO:4
  • VPPPVPPRRR SEQ ID NO:5
  • the present invention relates to such SH3 binding peptides, methods for their identification, and compositions comprising same.
  • peptides comprising particular sequences of amino acid residues are disclosed which were isolated from random peptide libraries.
  • clones were isolated from a phage-displayed random peptide library which exhibited strong binding affinities for SH3 domain-containing protein targets.
  • Some of these protein targets include Abl, Src, Grb2, PLC- ⁇ , PLC- ⁇ , Ras GAP, Nck, and p85 PI-3′ Kinase. From the nucleotide sequence of the binding phage, the amino acid sequence of the peptide inserts has been deduced.
  • Synthetic peptides having the desired amino acid sequences are shown to bind the SH3 domain of the target proteins.
  • synthetic peptides combining a core consensus sequence and additional amino acid residues flanking the core sequence are especially effective at binding to particular target protein SH3 domains.
  • the SH3 binding peptides disclosed herein can be utilized in a number of ways, including the potential modulation of oncogenic protein activity in vivo. These peptides also serve as useful leads in the production of peptidomimetic drugs that modulate a large class of proteins involved in signal transduction pathways and oncogenesis.
  • the synthesized peptides bind quite well to the Src SH3 domain and act as potent competitors of natural Src SH3 interactions in cell lysates. For instance, these peptides can compete with radiolabelled proteins from cell lysates in binding to immobilized Src-GST, with an apparent IC 50 of 1-10 ⁇ M.
  • RPLPPLP consensus sequence
  • peptides having at least nine and up to forty-five amino acid residues, including an amino acid sequence of the formula, R-2-L-P-5-6-P-8-9 (SEQ ID NO:10), positioned anywhere along the peptide, in which each number represents an amino acid residue, such that 2 represents any amino acid residue except cysteine, 5 and 6 each represents a hydrophobic amino acid residue, 8 represents any amino acid residue except cysteine, and 9 represents a hydrophilic amino acid residue except cysteine, each letter being the standard one-letter symbol for the corresponding amino acid, said peptide exhibiting a binding affinity for the SH3 domain of Src, provided that said peptide is not R-P-L-P-P-L-P-T-S (SEQ ID NO:11).
  • the peptides also exhibit a binding affinity for the SH3 domain of Src-related proteins, including Yes, Fyn, Lyn, Lck, Hck
  • the present invention also contemplates SH3 domain-binding peptides that further comprise a C-terminal-flanking amino acid sequence of the formula 10, 10-11, 10-11-12, 10-11-12-13 (SEQ ID NO:12) or 10-11-12-13-14 (SEQ ID NO:13), in which each number represents any amino acid residue except cysteine, such that 10 is bound to 9 by a peptide bond.
  • peptides are also provided which further comprise an N-terminal-flanking amino acid sequence of the formula 1′, 2′-1′, 3′-2′-1′ or 4′-3′-2′-1′ (SEQ ID NO:14) in which each number represents any amino acid residue except cysteine, such that 1′ is bound to R by a peptide bond.
  • a peptide having at least thirteen and up to forty-five amino acid residues, including an amino acid sequence of the formula, 3′-2′-1′-R-2-L-P-5-6-P-8-9-10 (SEQ ID NO:15), positioned anywhere along the peptide, in which each number represents an amino acid residue, such that 3′, 2′, 1′, 2, 8, and 10 each represents any amino acid residue except cysteine, 5 and 6 each represents a hydrophobic amino acid residue, and 9 represents a hydrophilic amino acid residue except cysteine, each letter being the standard one-letter symbol for the corresponding amino acid, said peptide exhibiting a binding affinity for the SH3 domain of Src.
  • SEQ ID NO:15 amino acid sequence of the formula, 3′-2′-1′-R-2-L-P-5-6-P-8-9-10
  • the present invention also seeks to provide new consensus sequences or motifs that reflect variations in SH3 domain binding selectivities or specificities.
  • the present invention also contemplates conjugates of the SH3 binding peptides and a second molecule or chemical moiety.
  • This second molecule may be any desired substance whose delivery to the region of the SH3 domain of a particular protein (or cell containing the protein) is sought.
  • Possible target cells include, but are not limited to, neural cells, immune cells (e.g., T cells, B cells, natural killer cells, and the like), osteoclasts, platelets, epidermal cells, and the like, which cells express Src, Src-related proteins, and potentially, other SH3 domain-containing proteins. In this manner, the modulation of the biological activity of proteins bearing an SH3 domain can be accomplished.
  • a method is disclosed of modulating the activity of Src or Src-related proteins comprising administering a composition comprising an effective amount of a peptide of the present invention and a carrier, preferably a pharmaceutically acceptable carrier.
  • the contemplated method results in the inhibition of the activity of Src or Src-related proteins.
  • the method is effective to activate Src or Src-related proteins.
  • a method is disclosed of identifying a peptide having a region that binds to an SH3 domain comprising: (a) providing an immobilized target protein comprising an SH3 domain; (b) incubating the immobilized target protein with an aliquot taken from a random peptide library; (c) washing unbound library peptides from the immobilized target protein; (d) recovering the peptide bound to the immobilized target protein; and (e) determining the primary sequence of the SH3 domain-binding peptide.
  • a method is disclosed of imaging cells, tissues, and organs in which Src or Src-related proteins are expressed, which comprises administering an effective amount of a composition comprising an SH3 domain-binding peptide conjugated to detectable label or an imaging agent.
  • the invention also provides assays for identifying a compound that affects the binding between a first molecule comprising an SH3 domain and a second molecule that binds to the SH3 domain comprising incubating one or more candidate compounds from which it is desired to select such a compound with the first molecule and the second molecule under conditions conducive to binding and detecting the one or more compounds that affect binding of the first molecule to the second molecule.
  • kits for performing such assays comprising a first molecule comprising an SH3 domain and a second molecule that binds to the SH3 domain.
  • FIG. 1 illustrates a scheme for the generation of a random 36 amino acid peptide library (TSAR-9; e.g., SEQ ID NO:16).
  • Oligonucleotides were synthesized (SEQ ID NOS:17-18), converted into double-stranded DNA, cleaved with restriction enzymes (SEQ ID NOS:19-20), and cloned into the M13 vector, m663.
  • the random peptide region encoded by the oligonucleotides is shown in the box (SEQ ID NO:16) and is situated at the N-terminus of mature protein III (SEQ ID NO:21).
  • SEQ ID NO:22 includes the three amino acids preceding the signal peptidase cleavage site.
  • FIG. 2 illustrates a scheme for the generation of a random 22 amino acid peptide library (TSAR-12; e.g., SEQ ID NO:23).
  • Oligonucleotides were synthesized (SEQ ID NOS:24-25), converted into double-stranded DNA, cleaved with restriction enzymes (SEQ ID NOS:26-27), and cloned into the M13 vector, m663.
  • the random peptide region encoded by the oligonucleotides is shown in the box (SEQ ID NO:23) and is situated at the N-terminus of mature protein III (SEQ ID NO:28).
  • SEQ ID NO:29 includes the three amino side preceding the signal peptidase cleavage site.
  • FIG. 3 illustrates a scheme for the generation-of a random 8 amino acid peptide library (R8C; SEQ ID NO:30).
  • oligonucleotides were synthesized (SEQ ID NOS:31-32), converted into double-stranded DNA, cleaved with restriction enzymes (SEQ ID NOS:33-34), and cloned into the M13 vector, m663.
  • the random peptide region (SEQ ID NO:30) is flanked by cysteine residues and is situated at the N-terminus of mature protein III (SEQ ID NO:35).
  • FIG. 4 illustrates the possible origin of one class of double-insert R8C recombinants (e.g., encoding SEQ ID NO:36).
  • Double-stranded oligonucleotides e.g., SEQ ID NO:37
  • SEQ ID NO:37 may have ligated in a head-to-head fashion at the Xba I site prior to cloning in the Xho I- Xba I cleaved M13 vector.
  • FIG. 5 shows a list of random peptide recombinants (SEQ ID NOS:38-61 and 106) isolated by the method of the present invention and the displayed peptide sequence.
  • the amino acid sequences are aligned to highlight the core sequences.
  • the flanking sequences are shown to the N-terminal and C-terminal ends of the core sequence.
  • SEQ ID NOS:38-61 are shown in order from top to bottom except that SSCDHTLGLGWCGSRSTRQLPIPP TTTRPSR is SEQ ID NO:106 and RPLPPLP is SEQ ID NO:9.
  • T12.Src3.1 is a Class II ligand (See Section 6.14.5).
  • FIG. 6 graphically illustrates the relative binding affinities of selected phage clones for various SH3 domains. The results indicate that certain amino acid sequences provide generic SH3 domain binding, while others can provide greater selectivity for the SH3 domain of Src. Still other clones exhibit Src SH3 domain preferential binding.
  • FIG. 7 shows the binding of synthetic peptides (SEQ ID NOS:9 and 62-70) representing Src SH3-selected phage inserts to Src SH3-GST fusion target (shaded columns) over background GST binding (unshaded columns) relative to the core peptide RPLPPLP (SEQ ID NO:9) and proline-rich peptide segments derived from naturally occurring proteins. Bound biotinylated peptide was detected with streptavidin-alkaline phosphatase ELISA. Each point was performed in triplicate; average absorbance at 405 nm is presented. Error bars represent SD. SEQ ID NOS:62-70 are shown in order from top to bottom except that RPLPPLP is SEQ ID NO:9.
  • FIG. 8 illustrates the relative specificity of selected peptides (SEQ ID NOS:9 and 62-70) for SH3 domains derived from different proteins.
  • Src SH3-GST Yes SH3-GST, Grb2-GST, Crk SH3-GST, Abl SH3-GST, PLC ⁇ 1 SH2SH3-GST.
  • Bound biotinylated peptide was detected with streptavidin-alkaline phosphatase. Each point was performed in triplicate; values are average signal (absorbance at 405 nm) above GST background, with error bars representing standard deviation. Hatched bars indicate saturation of the ELISA signal.
  • SEQ ID NOS:62-70 are shown in order from top to bottom except that RPLPPLP is SEQ ID NO:9.
  • FIG. 9 presents the results of competition experiments in which selected peptides were found to inhibit the binding of proteins from cell lysates to immobilized Src SH3-GST or Abl SH3-GST protein fusion targets.
  • FIG. 10 presents a graph illustrating the increased rate of progesterone-induced maturation of oocytes injected with an SH3 domain-binding peptide, VLKRPLPIPPVTR (SEQ ID NO:64), of the present invention.
  • Stage VI oocyted were prepared and injected as previously described (see, Kay, B. K., in Methods in Cell Biol . (1991) 36:663-669).
  • Oocytes were injected with 40 nL of 100 ⁇ M test peptide or water. After injection, the oocytes were placed in 2 ⁇ g/mL progesterone (Sigma, St. Louis, Mo.) and scored hourly for germinal vesicle breakdown (GVBD).
  • LAPPKPPLPEGEV is SEQ ID NO:70.
  • FIG. 12 illustrates a scheme for the generation of a biased peptide library.
  • Oligorncleotides were synthesized (SEQ ID NOS:162-163), converted into double-stranded DNA (SEQ ID NO:454), cleaved with restriction enzymes XhoI and XbaI (SEQ ID NOs:455-456), and cloned into the mBAX vector (SEQ ID NOs:457-458), described further below in the Examples section.
  • the biased peptide region (SEQ ID NO:459) is situated at the N-terminus of mature pIII protein.
  • CTAGACGTGTCAGT is a portion of SEQ ID NO:162.
  • ACTGACACGT is a portion of SEQ ID NO:454.
  • TCGAGGCACAG is a portion of SEQ ID NO:454.
  • FIG. 13 illustrates the peptide sequence encoded in the mBAX vector situated at the N-terminus of mature pIII protein.
  • TCCTCGAGTATCGACATGCCTTAGACTGCTAGCACTATGTACAACATGCTT CATCGCAACGAGCCA is SEQ ID NO:460.
  • SSIDMP*TASTMYNM LHRNEP is SEQ ID NO:461.
  • CATGCCGCCCGCCCTCCTGAAGAGGTCTAGA is SEQ ID NO:462.
  • GGRKLSPPANDMPPALLKRSR is SEQ ID NO:463.
  • FIG. 14 illustrates the relative binding of SH3-selected phage clones to various SH3 domains.
  • the present invention relates to peptides that exhibit a binding affinity for an SH3 domain, which domain has been found to be present in a number of physiologically significant proteins.
  • peptides are disclosed which exhibit general binding characteristics to the SH3 domains found in a group of proteins, including but not limited to Abl, Src, Grb2, PLC- ⁇ , PLC- ⁇ , Ras GAP, Nck, and p85 PI-3′ Kinase.
  • Preferred peptides exhibit selective, if not specific, binding affinity for the SH3 domain of Src.
  • the peptides of the present invention include a core sequence, preferably a consensus seqeunce, and additional amino acid residues that flank the core sequence. These peptides, including the methods for their identification, are described in greater detail, below.
  • peptides which have at least nine and up to about forty-five amino acid residues, including an amino acid sequence resembling the formula,
  • R-2-L-P-5-6-P-8-9 (SEQ ID NO:10)
  • each number represents an amino acid residue, such that 2 represents any amino acid residue except cysteine, 5 and 6 each represents a hydrophobic amino acid residue, 8 represents any amino acid residue except cysteine, and 9 represents a hydrophilic amino acid residue except cysteine.
  • Each letter used in the formulas herein represent the standard one-letter symbol for the corresponding amino acid.
  • the peptides of particular interest are those that exhibit a binding affinity for the SH3 domain of Src and Src-related proteins, including Yes, Fyn, Lyn, Lck, Hck and Fgr.
  • the peptides of the invention exhibit a binding affinity for the SH3 domain of Src, which is at least three-fold, more preferably at least four-fold, most preferably at least about five-fold greater than that exhibited by the peptide RPLPPLP (SEQ ID NO:9).
  • the peptides exhibit a binding affinity for the SH3 domain of Src which is at least ten-fold greater than that exhibited by the peptide RPLPPLP (SEQ ID NO:9).
  • peptides are disclosed in which the various amino acid residues at the indicated positions may independently have the following preferred identities: 2 is a P, R, A, L, Q, E or S, more preferably P or R; 5 represents a P, M, I or L, more preferably P or M; 6 is a P, L, I or V, more preferably P or L; 8 is a T, R, P, I, N, E, V, S, A, G or L, more preferably T or R; and 9 is a T, R, S, H or D, more preferably T or R.
  • hydrophobic amino acid residues at 5 and 6 in some cases it may be desirable to have hydrophilic amino acid residues at these positions.
  • amino acid residue 5 may be a T, R or S
  • amino acid residue 6 may be a T or R
  • a hydrophilic amino acid residue is preferred at position 9, in some instances a hydrophobic residue, such as a P or A, may be desirable.
  • the present invention also contemplates SH3 domain-binding peptides with a minimum length of 10, 11, 12, 13, 14, 15 or more amino acids. Such peptides contain additional amino acid residues flanking the core sequence of R-2-L-P-5-6-P (SEQ ID NO:71) either at the C-terminal end, the N-terminal end or both.
  • such peptides include those that further comprise a C-terminal-flanking amino acid sequence of the formula 10, 10-11, 10-11-12, 10-11-12-13 (SEQ ID NO:12) or 10-11-12-13-14 (SEQ ID NO:13), in which each number represents any amino acid residue except cysteine, such that the amino acid residue 10 is bound to the amino acid residue 9 by a peptide bond.
  • specific embodiments include an amino acid residue 10 which is T, R, L, S, D, P, A or N, preferably T or R, an amino acid residue 11 which is R, P, A, Q, S or T, preferably R or P, an amino acid residue 12 which is P, S, R or T, preferably P or S, an amino acid residue 13 which is P, S, R, F, H or T, preferably P or S, and an amino acid residue 14 which is S, R, G or T, preferably, S or R.
  • peptides are also provided which further comprise an N-terminal-flanking amino acid sequence of the formula 1′, 2′-1′, 3′-2′-1′ or 4′-3′-2′-1′ (SEQ ID NO:14) in which each number represents any amino acid residue except cysteine, such that 1′ is bound to R by a peptide bond.
  • amino acid residue 1′ is T, P, S, N, F, W, K, H, Q or G, preferably T or P
  • amino acid residue 2′ is S, T, G, P, R, Q, L, A or H, preferably S or T
  • amino acid residue 3′ is R, S, P, G, A, V, Y or L, preferably S or T
  • amino acid residue 4′ is R, S, V, T, G, L or F, preferably R or S.
  • a peptide having at least thirteen and up to forty-five amino acid residues, including an amino acid sequence of the formula, 3′-2′-1′-R-2-L-P-5-6-P-8-9-10 (SEQ ID NO:15), positioned anywhere along the peptide, in which each number represents an amino acid residue, such that 3′, 2′, 1′, 2, 8, and 10 each represents any amino acid residue except cysteine, 5 and 6 each represents a hydrophobic amino acid residue, and 9 represents a hydrophilic amino acid residue except cysteine, each letter being the standard one-letter symbol for the corresponding amino acid, said peptide exhibiting a binding affinity for the SH3 domain of Src.
  • SEQ ID NO:15 amino acid sequence of the formula, 3′-2′-1′-R-2-L-P-5-6-P-8-9-10
  • Preferred 13-mers include, but are not limited to, those having an amino acid residue 5 which is a P or M, an amino acid residue 1′ which is T, P, S or N, an amino acid residue 2′ which is S or T, an amino acid residue 3′ which is R or S, and an amino acid residue 10 which is T or R.
  • the prohibition against the use of the hydrophilic amino acid residue cysteine (C) does not extend beyond the 7-mer “core” sequence and the additional amino acid residues flanking the core up to a total (core+flanking) of about 20 amino acids. That is, the occasional use of a cysteine is not absolutely prohibited.
  • cyclized structures appear to be disfavored, at least with potential binding peptides of less than about 15 amino acid residues in length.
  • the concern for the formation of cyclized structures comprising the core sequence diminishes with increasing size of the peptide. Presumably, a large enough structure, though cyclic, may allow the critical core sequence to adopt a more or less linear conformation.
  • RSTPRPLPMLPTTR SEQ ID NO. 62
  • RSTPRPLPPLPTTR SEQ ID NO. 67
  • GILAPPVPPRNTR SEQ ID NO. 63
  • VLKRPLPIPPVTR SEQ ID NO. 64
  • GPHRRLPPTPATR SEQ ID NO. 65
  • ANPSPATRPLPTR SEQ ID NO. 66
  • Phage clones are also disclosed, along with the amino acid sequences that are responsible for SH3 domain binding. These phage clones are identified in FIG. 5.
  • SH3 domain-binding peptides are contemplated which have a total of 11, 13, 14, 18, 20, 22, 23, 25, 30, 36, 38 or 45 amino acid residues.
  • the peptides of the present invention may be prepared by any number of practicable methods, including but not limited to solution-phase synthesis, solid-phase synthesis, protein expression by a transformed host, cleavage from a naturally-derived, synthetic or semi-synthetic polypeptide, or a combination of these techniques.
  • the SH3 binding peptides exhibit a wide range of biological activity which includes the enhancement (or inhibition, depending on the particular peptide or the nature of the peptide's target molecule, in this case a protein bearing an SH3 domain) of the natural function or biological activity of the peptide's target molecule.
  • the interaction of the binding peptide of the present invention could result in the modulation of the oncogenic activity of the target molecule bearing the SH3 domain.
  • the target molecule has, in turn, a natural binding partner or ligand
  • the peptides of the present invention may also exhibit antagonistic or agonistic activity in relation to the biological activity of the natural binding partner.
  • the intensity of the immune response can thus be stimulated for example, by the increased production of certain lymphokines, such as TNF-alpha and interleukin-1.
  • lymphokines such as TNF-alpha and interleukin-1.
  • a more intense immune response may be in order in certain conditions, such as in combating a particularly tenacious infection, viral or otherwise, or a malignancy.
  • a conjugate compound which comprises the peptide of the present invention and a second chemical moiety.
  • the second chemical moiety can be selected from a wide variety of chemical compounds including the peptide itself.
  • the second chemical moiety is selected to be other than the peptide of the present invention, including but not limited to an amino acid, a peptide other than an SH3 binding peptide of the present invention, a polypeptide or protein (i.e., the conjugate is a fusion protein), a nucleic acid, a nucleoside, a glycosidic residue (i.e., any sugar or carbohydrate), a label or image-enhancing agent (including metals, isotopes, radioisotopes, chromophores, fluorophores (such as FITC, TRITC, and the like), and enzyme substrates), a drug (including synthetic, semisynthetic, and naturally-occurring compounds), small molecules (e.g., biotin, hormones, factors) and the like.
  • a polypeptide or protein i.e., the conjugate is a fusion protein
  • a nucleic acid i.e., a nucleoside, a glycosidic
  • the peptide of the present invention can be conjugated to the second chemical moiety either directly (e.g., through appropriate functional groups, such as an amine or carboxylic acid group to form, for example, an amine, imine, amide, ester, acyl or other carbon-carbon bond) or indirectly through the intermediacy of a linker group (e.g., an aliphatic or aromatic polyhydroxy, polyamine, polycarboxylic acid, polyolefin or appropriate combinations thereof).
  • a linker group e.g., an aliphatic or aromatic polyhydroxy, polyamine, polycarboxylic acid, polyolefin or appropriate combinations thereof.
  • conjugate is also meant to encompass non-covalent interactions, including but not limited to ionic, affinity or other complexation interactions. Preferably, such other non-covalent interactions provide definable, most preferably, isolatable chemical conjugate species.
  • the peptides of the present invention have been shown to localize within certain cellular compartments which contain Src or Src-related proteins. Consequently, the above-described conjugate can be utilized as a delivery system for introduction of a drug to cells, tissues or organs that include SH3 domain-containing proteins.
  • the present invention seeks to provide a recombinant construct comprising a nucleic acid or its complement that includes codons or nucleotide and sequences encoding a peptide having a region that binds to an SH3 domain, preferably the Src SH3 domain.
  • the recombinant nucleic acid may be a DNA or RNA polynucleotide.
  • the present invention contemplates a recombinant construct which is a transforming vector.
  • a transforming vector include those well known to those of ordinary skill in the art, which effect the transfer or expression of the nucleotide sequence after introduction to a host, such as recombinant plasmid, phage or yeast artificial chromosome. These vectors may be closed circular loops or they may be linearized.
  • the vectors contemplated include those that exist extrachromosomally after host transformation or transfection, as well as those that integrate within or even displace portions of the host chromosome.
  • the vectors may be introduced to the cell with the help of transfection aids or techniques well-known in the art.
  • these aids or techniques may take the form of electroporation, use of calcium chloride, calcium phosphate, DEAE dextran, liposomes or polar lipid reagents known as LIPOFECTIN or LIPOFECTAMINE.
  • the present invention contemplates the direct introduction of the desired nucleic acid to the host cell, for instance, by injection.
  • Transformed host cells are also obtained by the methods of the present invention which are capable of reproducing the polynucleotide sequences of interest and/or expressing the corresponding peptide products.
  • a variety of hosts are contemplated, including prokaryotic and eukaryotic hosts.
  • bacterial, viral, yeast, animal, and plant cells are potentially transformable hosts.
  • a method is disclosed to obtain a transformed host cell that can produce, preferably secrete, a peptide having a region that binds to an SH3 domain comprising (a) providing an expression vector, preferably a secretory expression vector, comprising a nucleotide sequence encoding at least one copy of a peptide having a region that binds to an SH3 domain; and (b) introducing the vector to a competent host cell.
  • compositions which comprise an SH3 domain-binding peptide, including a core sequence and flanking sequences, and a suitable carrier.
  • compositions contemplated by the present invention may also include other components, from those that facilitate the introduction or administration of the compositions to those that have their own innate activity, such as a prophylactic, a diagnostic or a therapeutic action. Such innate activity may be distinct from that of the peptides of the present invention or be complementary thereto.
  • the compositions of the present invention include those that are suitable for administration into mammals, including humans.
  • the compositions (including necessarily the carrier) of the present invention are sterile, though others may need only be cosmetically, agriculturally or pharmaceutically acceptable. Still other compositions may be adapted for veterinary use.
  • compositions including the drug delivery systems described herein, are contemplated to be administered in a variety of ways, such as parenterally, orally, enterally, topically or by inhalation.
  • the compositions may also be adminstered intranasally, opthalmically or intravaginally.
  • the compositions of the invention can take several forms, such as solids, gels, liquids, aerosols or patches.
  • a method is provided of identifying a peptide having a region that binds to an SH3 domain comprising: (a) providing an immobilized target protein comprising an SH3 domain; (b) incubating the immobilized target protein with an aliquot taken from a phage-displayed random peptide library, which library includes peptides having a random sequence of ⁇ 8 amino acid residues; (c) washing unbound phage from the immobilized target protein; (d) recovering the phage bound to the immobilized target protein; and (e) determining the relevant nucleotide sequence of said binding phage nucleic acid and deducing the primary sequence corresponding to the SH3 domain-binding peptide.
  • the method further comprises amplifying the titer of the recovered phage and repeating the steps of incubation, washing and recovery to provide SH3 domain-binding peptide-enriched phage.
  • any other mode by which the peptide library, random or otherwise, can be “displayed” can be utilized in the present invention, however.
  • longer random peptide sequences e.g., >6 amino acid residues, preferably >10, and most preferably, >12
  • Phage display offers several conveniences: first, the expressed peptides are on the surface of the viral particles and accessible for interactions; second, the recombinant viral particles are stable (i.e., can be frozen, exposed to pH extremes); third, the viruses can be amplified; and fourth, each viral particle contains the DNA encoding the recombinant genome. Consequently, these libraries can be screened by isolating viral particles that bind to targets. The isolates can be grown up overnight, and the displayed peptide sequence responsible for binding can be deduced by DNA sequencing.
  • TSAR libraries have approximately >108 different recombinants, and nucleotide sequencing of the inserts suggests that the expressed peptides are indeed random in amino acid sequence.
  • TSAR libraries where TSAR stands for Totally Synthetic Affinity Reagents. The preparation of the TSAR libraries are described further below.
  • peptides have been isolated from an unconstrained random peptide library which exhibit a binding affinity for SH3 domains. Furthermore, the binding affinities exhibited by the disclosed peptides differ in their selectivities with certain peptides showing comparable binding affinities for SH3 domains derived from different proteins, while others manifest greater affinities for specific SH3 domains.
  • FIG. 5 The amino acid sequence of various peptides isolated by the present method are listed in FIG. 5. As can be seen from this list, certain groups of SH3 domain binding peptides are isolated from three separate random peptide libraries, each based on a different type of random peptide insert, all displayed at the amino-terminus of the pIII protein on the surface of M13 viral particles. Ten clones were isolated from the R8C library, seven from the TSAR-12 library, and seven from the TSAR-9 library. The sequences are presented to highlight the particular amino acid residues believed to bind directly to the SH3 domain, as well as to point out the remaining amino acid resiudes of the random insert and the viral flanking sequences and complementary site amino acid residues common to each group of clones. The frequency with which each particular clone is found in each library is also indicated in FIG. 5. Thus, clones T12.SRC3.1 and T12.SRC3.2 are by far the most abundant clones found among the three libraries.
  • a synthetic peptide bearing only the core consensus sequence RPLPPLP was less effective in binding to target SH3 domains than synthetic peptides that also included additional amino acid residues flanking the core sequences.
  • 13-mers and 14-mers having the sequences RSTPRPLPMLPTTR (SEQ ID NO:62), RSTPRPLPPLPTTR (SEQ ID NO:67), GILAPPVPPRNTR (SEQ ID NO:63), GPHRRLPPTPATR (SEQ ID NO:65), and VLKRPLPIPPVTR (SEQ ID NO:64) have been prepared and shown to bind to SH3 domains, such as those of Src and Yes, much more avidly than the 7-mer, RPLPPLP (SEQ ID NO:9).
  • the 13-mer ANPSPATRPLPTR (SEQ ID NO:66) has been shown to have binding affinities comparable to the core consensus sequence.
  • the 13-mers comprise a 7-mer “core” sequence plus additional amino acid residues flanking same, some of which additional amino acid residues are contributed by the viral flanking sequences.
  • a 7-mer core includes a consensus motif of the formula RXLP ⁇ P (SEQ ID NO:71), wherein R is arginine, L is leucine, P is proline, X represents any amino acid except cysteine and ⁇ represents a hydrophobic amino acid residue.
  • R is arginine
  • L leucine
  • P proline
  • X represents any amino acid except cysteine
  • ⁇ represents a hydrophobic amino acid residue.
  • hydrophobic amino acid residue the applicants mean to include F, Y, W, V, A, I, L, P or M, each letter representing the standard one-letter designation for the corresponding amino acid residue.
  • a preferred 9-mer peptide comprising two additional amino acids on the C-terminal end of the core sequence is envisioned having a consensus motif of the formula RXLP ⁇ PX ⁇ (SEQ ID NO:10).
  • the symbol ⁇ represents a hydrophilic amino acid residue, except cysteine.
  • hydrophilic amino acid residue the applicants mean to include K, R, H, D, E, N, Q, T, S or C, and the other symbols are as defined above.
  • a glycine residue (G) may be considered either a hydrophobic or a hydrophilic amino acid residue.
  • the one-letter symbols B and Z, which stand for N or D and Q or E, respectively, are considered hydrophilic amino acid residues.
  • Particular 13-mer peptides of the present invention include those listed, below. It is noted, however, that not all the following 13-mer peptides correlate strictly to or comply with the preferred 9-mer consensus motif, described above. Those peptides that do not comply (indicated in italics, with the non-complying amino acid residues underscored) can, thus, be described as “resembling” those that do comply (indicated in normal type) with the preferred 9-mer consensus motif: PGFRELPPLPPSR (SEQ ID NO:72),
  • PPN S PLPPLPTHL SEQ ID NO:74
  • TGR G PLPPLPNDS SEQ ID NO:75
  • PATRPLP TR PSRT (SEQ ID NO:81), YSTRPLP SR PSRT (SEQ ID NO:82),
  • RSTRPLPILPRTT SEQ ID NO:85
  • STPRPLPMLPTTR SEQ ID NO:86
  • STNRPLPMIPTTR SEQ ID NO:87
  • RSTRPLP S LPITT SEQ ID NO:88
  • STSRPLP S LPTTR SEQ ID NO:89
  • RSTRSLPPLPPTT SEQ ID NO:90
  • RSTRQLPIPPTTT SEQ ID NO:91
  • STPRPLPLIPTTP SEQ ID NO:92
  • RSTRPLPP T PLTT SEQ ID NO:93
  • RSTRPQPPPPITT SEQ ID NO:94
  • the binding peptides disclosed have a greater relative binding affinity for one SH3 domain over another.
  • FIG. 8 the relative binding affinities of the various peptides described above toward different SH3 domain targets are graphically presented. As one can see, the relative binding affinities of the respective peptides can differ by orders of magnitude.
  • the peptide GPHRRLPPTPATR SEQ ID NO:65
  • the relevant sequence of the phage clone identified as T12.SRC3.3 is specific to Src family SH3 domains, including, but not limited to, Src, Yes, Lck, Hck, Fgr, Fyn, and Lyn.
  • This SH3 binding peptide has little affinity for SH3 domains derived from PLC ⁇ or Grb2.
  • the peptide GILAPPVPPRNTR (SEQ ID NO:63), corresponding to the relevant sequence of the phage clone T12.SRC3.1, which is one of the most abundant binding clones found by the present method, binds generically to a broad range of SH3 domains, including Src, PLC ⁇ , and Grb2.
  • the present invention has also uncovered a peptide, VLKRPLPIPPVTR (SEQ ID NO:64), corresponding to the relevant sequence of the phage clone T12.SRC3.6, which is Src preferential; that is, this peptide exhibits strong binding affinities for members of the Src family, some binding affinities for Grb2 proteins, but little binding affinities for PLC ⁇ domains.
  • the peptide ANPSPATRPLPTR (SEQ ID NO:66), corresponding to the relevant sequence of the phage clone T12.SRC3.2, also exhibits Src family specificity similar to GPHRRLPPTPATR (SEQ ID NO:65).
  • RSTPRPLPMLPTTR R8C.YES3.5; SEQ ID NO:62
  • RSTPRPLPPLPTTR R8C.YES3.5
  • SEQ ID NO:67 RSTPRPLPPLPTTR
  • GILAPPVPPRNTR SEQ ID NO:63
  • VLKRPLPIPPVTR SEQ ID NO:64
  • GPHRRLPPTPATR SEQ ID NO:65
  • ANPSPATRPLPTR sport core sequences and flanking sequences that do not closely adhere to the preferred core consensus sequences.
  • the binding characteristics of Src SH3-selected peptides was determined using synthetic biotinylated peptides corresponding to the sequences displayed by Src SH3-selected phage. These biotinylated peptides were assayed for direct binding to immobilized Src SH3-GST. Each of the five library-derived peptides tested were found to bind to Src SH3-GST and Yes SH3-GST over background (FIG. 8). Furthermore, a strong correlation was observed between the similarity of a given peptide to the preferred core consensus sequence RPLP(P/M)LP (SEQ ID NO:96) and the peptidets affinity for Src SH3-GST. The core sequence of the clone T12.SRC3.1 (GILAPPVPPRNTR; SEQ ID NO:63) appears to provide more generic SH3 domain-binding characteristics.
  • Xenopus laevis oocytes injected with mRNA encoding constitutively active Src undergo progesterone-induced maturation at an accelerated rate relative to oocytes injected with water or c-Src MRNA.
  • stage VI oocytes were injected with peptide, exposed to progesterone, and scored for germinal vesicle breakdown.
  • FIG. 10 shows that the rate of maturation was accelerated by approximately one hour when oocytes were injected with the SH3-binding peptide consisting of RPLPPLP (SEQ ID NO:9) flanked by residues from clone T12.SRC3.6 (VLKRPLPIPPVTR; SEQ ID NO:64), but not with water or a peptide corresponding to a proline-rich segment of vinculin (LAPPKPPLPEGEV; SEQ ID NO:70) as controls.
  • the magnitude of this effect is roughly equivalent to that seen with injection of mRNA encoding constituitively active Src.
  • the present invention also seeks to provide diagnostic, prophylactic, and therapeutic agents based on the SH3 binding peptides described herein.
  • diagnostic agents are provided, preferably in the form of kits, comprising an SH3 domain-binding peptide and a detectable label conjugated to said peptide directly, indirectly or by complexation, said peptide comprising: (i) a core sequence motif of the formula RXLP ⁇ P (SEQ ID NO:71), wherein X represents any amino acid except cysteine and ⁇ represents a hydrophobic amino acid residue, including F, Y, W, V, A, I, L, P, M or G, each letter representing the standard one-letter designation for the corresponding amino acid residue; and (ii) two or more additional amino acid residues flanking said core sequence at its C-terminal end, N-terminal end or both.
  • RXLP ⁇ P SEQ ID NO:71
  • the diagnostic agents of the present invention can be used to detect the presence of SH3 domains of a generic or specific type in cells, tissues or organs either in vitro or in vivo.
  • the diagnostic agent is preferably mixed with a pharmaceutically acceptable carrier for administration, either enterally, parenterally or by some other route dictated by the needs of the particular application.
  • an assay based on a fusion product which comprises a Src SH3 domain-binding peptide of the invention and a substrate for deregulated or “activated” Src.
  • a muscle biopsy taken from a subject suspected of being infected by the Rous sarcoma virus, can be treated with an effective amount of the fusion product.
  • the degree of conversion of the substrate By subsequent analysis of the degree of conversion of the substrate, one can potentially detect infection by the Rous sarcoma virus in the subject, particularly mammals, especially chickens.
  • the presence of the retrovirus, which causes the expression of deregulated or “activated” Src may thus be indicated by unusually high levels of Src as revealed by large amounts of the converted substrate.
  • the enzyme can be isolated by selective binding to a form of the SH3 domain-binding peptides of the present invention (e.g., biotin-peptide conjugate). After isolation of the protein-peptide conjugate complex (e.g., on a column comprising streptavidin), the activity of the enzyme can then be assayed by conventional methods to determine its level of protein kinase activity which can be taken as an indication of the presence of the deregulated or “activated” form of the enzyme.
  • An assay for Src kinase has been described by Klinz and Maness, in Neuroprotocols (a companion to Neuroscience ) (1992) 1(3):224-231.
  • the diagnostic agents of the invention can also serve as imaging agents of cells, tissues or organs, especially those that contain proteins with an SH3 domain.
  • neural cells e.g., neurons, other areas of the brain
  • osteoclasts e.g., osteoblasts, platelets, immune cells, and other dividing cells are known to express or contain proteins with SH3 domains.
  • an image can be taken of portions of the body to serve as a baseline for subsequent images to detect physiologic or biochemical changes in the subject's body. For instance, changes in the condition of cellular levels of Src or a transformation of the cellular Src to an “activated” form may be detected using the diagnostic or imaging agents of the present invention.
  • an SH3-binding peptide tagged with a fluorescence emitter can provide an image of the cytoskeleton.
  • the images are presented in FIG. 11.
  • panels A, B, and C show the fluorescence image that is obtained on treating NIH 3T3 fibroblasts with SH3 domain-binding peptides modified to include a fluorescent tag.
  • panel D shows only a dark image that is produced when the cells are treated with a proline-rich segment of vinculin as a control.
  • an SH3 domain-binding peptide-horseradish immunoperoxidase complex or related immunohistochemical agent could be used to detect and quantitate specific receptor molecules in tissues, serum or body fluids.
  • the present invention provides useful diagnostic reagents for use in immunoassays, Southern or Northern hybridization, and in situ assays. Accordingly, the diagnostic agents described herein may be suitable for use in vitro or in vivo.
  • the diagnostic or imaging agent of the present invention is not limited by the nature of the detectable label.
  • the diagnostic agent may contain one or more such labels including, but not limited to, radioisotope, fluorescent tags, paramagnetic substances, heavy metals, or other image-enhancing agents.
  • labels including, but not limited to, radioisotope, fluorescent tags, paramagnetic substances, heavy metals, or other image-enhancing agents.
  • Those of ordinary skill in the art would be familiar with the range of label and methods to incorporate or conjugate them into the SH3 domain-binding peptide to form diagnostic agents.
  • compositions comprising an SH3 domain-binding peptide and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition is useful for the modulation of the activity of SH3 domain-containing proteins.
  • modulation is meant either inhibition or enhancement of the activity of the protein target.
  • a pharmaceutical composition comprising an SH3 domaih-binding peptide and a pharmaceutically acceptable carrier, said peptide comprising: (i) a 9-mer sequence motif of the formula RXLP ⁇ PX ⁇ (SEQ ID NO:10), wherein X represents any amino acid except cysteine, ⁇ represents a hydrophobic amino acid residue, and wherein ⁇ is a hydrophilic amino acid residue except cysteine, each letter representing the standard one-letter designation for the corresponding amino acid residue; and, optionally, (ii) additional amino acid residues flanking the 9-mer sequence at its C-terminal end, N-terminal end or both, up to a total of 45 amino acid residues, including said 9-mer sequence.
  • the peptide comprises at least one, more preferably at least two, and most preferably at least three additional amino acids flanking the 9-mer sequence.
  • the therapeutic or diagnostic agents of the invention may also contain appropriate pharmaceutically acceptable carriers, diluents and adjuvants.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium carbonate, magnesium stearate, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. These compositions can take the form of solutions, suspensions, tablets, pills, capsules, powders, sustained-release formulations and the like. Suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin.
  • compositions will contain an effective therapeutic amount of the active compound together with a suitable amount of carrier so as to provide the form for proper administration to the subject.
  • intravenous injection is a very effective form of administration
  • other modes can be employed, including but not limited to intramuscular, intraperitoneal, and subcutaneous infection, and oral, nasal, enteral, and parenteral administration.
  • the therapeutic agents and diagnostic agents of the instant invention are used for the treatment and/or diagnosis of animals, and more preferably, mammals including humans, as well as dogs, cats, horses, cows, pigs, guinea pigs, mice and rats. Accordingly, other methods contemplated in the present invention, include, but are not limited to, a method of modulating, i.e., inhibiting or enhancing, bone resorption in a mammal (see, e.g., Hall, T. J., in Biochem. Biophys. Res. Commun .
  • the diagnostic or therapeutic agents of the present invention can be modified by attachment to soluble macromolecules such as proteins, polysaccharides, or synthetic polymers.
  • the peptide could be coupled to styrene-maleic acid copolymers (see, e.g., Matsumura and Maeda, Cancer Res . (1986) 46:6387), methacrylamide copolymers (Kopececk and Duncan, J. Controlled Release (1987) 6:315), or polyethylene glycol (PEG) (e.g., Hershfield and Buckley, N. Engl. J. Med . (1987) 316:589; Ho et al., Drug Metab. Discos .
  • agents are further targeted by attachment to an antibody, especially a monoclonal antibody.
  • antibodies include but are not limited to chimeric, single chain, Fab fragments, and Fab expression libraries.
  • the agent is coupled to the macromolecule via a degradable linkage so that it will be released in vivo in its active form.
  • the therapeutic or diagnostic agent may be delivered in a vesicle, in particular a liposome.
  • a vesicle in particular a liposome. See, Langer, Science (1990) 249:1527-1533; Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer , Lopez-Berestein and Fidler (eds.), Liss, N.Y. (1989) pp. 353-365; Lopez-Berestein, ibid ., pp. 317-327.
  • the therapeutic or in vivo diagnostic agent can be delivered in a controlled release system.
  • a pump may be used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng . (1987) 14:201; Buchwald et al., Surgery (1980) 88:507; Saudek et al., N. Engl. J. Med . (1989) 321:574).
  • polymeric materials may be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla., 1974; Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.) Wiley, N.Y. 1984; Raner and Peppas, J. Macromol. Sci. Rev. Macromol. Chem . (1983) 23:61; see, also, Levy et al., Science (1985) 228:190; During et al., Ann. Neurol . (1989) 25:351; Howard et al., J. Neurosurg . (1989) 71:105).
  • a controlled release system may be placed next to the therapeutic target, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release , supra, (1984) 2:115-138). It will be recognized by one of ordinary skill in the art that a particular advantage of the invention is that a peptide will not be subject to the problems of denaturation and aggregation associated with proteins held in the warm, most environment of a body in a controlled release system.
  • a common problem in the development of new drugs is that of identifying a single, or a small number, of compounds that possess a desirable characteristic from among a background of a large number of compounds that lack that desired characteristic. This problem arises both in the testing of compounds that are natural products from plant, animal, or microbial sources and in the testing of man-made compounds. Typically, hundreds, or even thousands, of compounds are randomly screened by the use of in vitro assays such as those that monitor the compound's effect on some enzymatic activity or its ability to bind to a reference substance such as a receptor or other protein.
  • the present invention provides methods of identifying a compound that affects the binding of a molecule comprising an SH3 domain and a ligand of the SH3 domain.
  • the effect on binding can be an increase or decrease in total amount of binding or in affinity of bidning.
  • the effect is an inhibition (reduction in or loss of binding).
  • the invention provides a method of identifying an inhibitor of the binding between a first molecule comprising an SH3 domain and a second molecule that binds to the SH3 domain comprising incubating one or more compounds from which it is desired to select such an inhibitor with the first molecule and the second molecule under conditions conducive to binding and detecting the one or more compounds that inhibit binding of the first molecule to the second molecule.
  • the second molecule is obtained by:
  • the second molecule comprises the peptide comprising the consensus sequence.
  • the second molecule is obtained by:
  • step (iv) producing a peptide comprising an amino acid sequence identified in step (iii);
  • the second molecule comprises the peptide comprising an amino acid sequence identified in step (iii).
  • Second molecules that bind SH3 domains can be obtained by, e.g., the use of diversity libraries, such as random or combinatorial peptide or nonpeptide libraries which can be screened for molecules that specifically bind to SH3 domains.
  • diversity libraries such as random or combinatorial peptide or nonpeptide libraries which can be screened for molecules that specifically bind to SH3 domains.
  • Many libraries are known in the art that can be used, e.g., chemically synthesized libraries, recombinant (e.g., phase display libraries), and in vitro translation-based libraries.
  • phage display libraries are described in Scott and Smith, 1990, Science 249:386-390; Devlin et al., 1990, Science, 249:404-406; Christian, R. B., et al., 1992, J. Mol. Biol. 227:711-718); Lenstra, 1992, J. Immunol. Meth. 152:149-157; Kay et al., 1993, Gene 128:59-65; and PCT Publication No. WO 94/18318 dated Aug. 18, 1994.
  • a benzodiazepine library (see e.g., Bunin et al., 1994, Proc. Natl. Acad. Sci. USA 91:4708-4712) can be adapted for use.
  • Peptoid libraries (Simon et al., 1992, Proc. Natl. Acad. Sci. USA 89:9367-9371) can also be used.
  • Another example of a library that can be used, in which the amide functionalities in peptides have been permethylated to generate a chemically transformed combinatorial library, is described by Ostresh et al. (1994, Proc. Natl. Acad. Sci. USA 91:11138-11142).
  • Screening the libraries can be accomplished by any of a variety of commonly known methods. See, e.g., the following references, which disclose screening of peptide libraries: Parmley and Smith, 1989, Adv. Exp. Med. Biol. 251:215-218; Scott and Smith, 1990, Science 249:386-390; Fowlkes et al., 1992; BioTechniques 13:422-427; Oldenburg et al., 1992, Proc. Natl. Acad. Sci.
  • screening can be carried out by contacting the library members with an SH3 domain immobilized on a solid phase and harvesting those library members that bind to the SH3 domain.
  • panning techniques are described by way of example in Parmley and Smith, 1988, Gene 73:305-318; Fowlkes et al., 1992, BioTechniques 13:422-427; PCT Publication No. WO 94/18318; and in references cited hereinabove.
  • the two-hybrid system for selecting interacting proteins in yeast can be used to identify molecules that specifically bind to SH3 domains.
  • a typical assay of the present invention consists of at least the following components: (1) a molecule (e.g., protein or polypeptide) comprising an SH3 domain; (2) a ligand of the SH3 domain; (3) a candidate compound, suspected of having the capacity to affect the binding between the protein containing the SH3 domain and the ligand.
  • the assay components may further comprise (4) a means of detecting the binding of the protein comprising the SH3 domain and the ligand.
  • Such means can be e.g., a detectable label affixed to the protein, the ligand, or the candidate compound.
  • the invention provides a method of identifying a compound that affects the binding of a molecule comprising an SH3 domain and a ligand of the SH3 domain comprising:
  • step (b) comparing the amount of binding in step (a) with the amount of binding known or determined to occur between the molecule and the ligand in the absence of the candidate compound, where a difference in the amount of binding between step (a) and the amount of binding known or determined to occur between the molecule and the ligand in the absence of the candidate compound indicates that the candidate compound is a compound that affects the binding of the molecule comprising an SH3 domain and the ligand.
  • a kit comprises, in one or more containers, one or more components of the assay of the invention, e.g., a first molecule comprising an SH3 domain and a second molecule that binds to the SH3 domain.
  • the assay comprises allowing the protein or polypeptide containing an SH3 domain to contact the ligand of the SH3 domain in the presence and in the absence of the candidate compound under conditions such that binding of the ligand to the protein containing an SH3 domain will occur unless that binding is disrupted or prevented by the candidate compound.
  • One version of an assay suitable for use in the present invention comprises binding the protein containing an SH3 domain to a solid support such as the wells of a microtiter plate.
  • the wells contain a suitable buffer and other substances to ensure that conditions in the wells permit the binding of the protein or polypeptide containing an SH3 domain to its ligand.
  • the ligand and a candidate compound are then added to the wells.
  • the ligand is preferably labeled, e.g., it might be biotinylated or labeled with a radioactive moiety, or it might be linked to an enzyme, e.g., alkaline phosphatase.
  • the wells are washed to remove any unbound ligand and compound. If the candidate compound does not interfere with the binding of the protein or polypeptide containing an SH3 domain to the labeled ligand, the labeled ligand will bind to the protein or polypeptide containing an SH3 domain in the well. This binding can then be detected. If the candidate compound interferes with the binding of the protein or polypeptide containing an SH3 domain and the labeled ligand, label will not be present in the wells, or will be present to a lesser degree than is the case when compared to control wells that contain the protein or polypeptide containing an SH3 domain and the labeled ligand but to which no candidate compound is added. Of course, it is possible that the presence of the candidate compound will increase the binding between the protein or polypeptide containing an SH3 domain and the labeled ligand. Alternatively, the ligand can be affixed to solid substrate during the assay.
  • the present invention provides ligands capable of binding SH3 domains that are suitable for incorporation into assays such as those described above.
  • Ligands provided by the present invention include those SH3 domain-binding amino acid sequences disclosed in Tables 1-13 below and proteins or polypeptides containing those amino acid sequences. Also provided are nucleic acids encoding the SH3 domain-binding amino acid sequences disclosed in Tables 1-13 below.
  • FIG. 1 shows the formula of the oligonucleotides and the assembly scheme used in construction of the TSAR-9 library.
  • the oligonucleotides were synthesized with an applied Biosystems 380a synthesizer (Foster City, Calif.), and the full-length oligonucleotides were purified by HPLC.
  • oligonucleotides Approximately 5% of the assembled oligonucleotides can be expected to have internal Xho I or Xba I sites; however, only the full-length molecules were used in the ligation step of the assembly scheme.
  • concentration of the synthetic oligonucleotide fragments was estimated by comparing the intensity on an ethidium bromide stained gel run along with appropriate quantitated markers. All DNA manipulations not described in detail were performed according to Maniatis, supra.
  • M13 derived phage vectors useful for expressing a TSAR library has been recently described (Fowlkes, D. et al. BioTech. (1992) 13:422-427).
  • an M13 derived vector, m663 was constructed as described in Fowlkes.
  • the m663 vector contains the pIII gene having a c-myc-epitope, i.e., as a stuffer fragment, introduced at the mature N-terminal end, flanked by Xho I and Xba I restriction sites (see also, FIG. I of Fowlkes).
  • the synthesized oligonucleotides were then ligated to Xho I and Xba I double-digested m663 RF DNA containing, the pIII gene (Fowlkes) by incubation with ligase overnight at 12° C. More particularly, 50 ng of vector DNA and 5 ng of the digested synthesized DNA and was mixed together in 50 ⁇ L ligation buffer (50 mM Tris, pH 8.0, 10 mM MgCl 2 , 20 mM DTT, 0.1 mM ATP) with T4 DNA ligase. After overnight ligation at 12° C., the DNA was concentrated by ethanol precipitation and washed with 70% ethanol. The ligated DNA was then introduced into E. coli (DH5 ⁇ F′; GIBCO BRL, Gaithersburg, Md.) by electroporation.
  • E. coli DH5 ⁇ F′
  • GIBCO BRL Gaithersburg, Md.
  • a small aliquot of the electroporated cells was plated and the number of plaques counted to determine that 10 8 recombinants were generated.
  • the library of E. coli cells containing recombinant vectors was plated at a high density ( ⁇ 400,000 per 150 mM petri plate) for a single amplification of the recombinant phage.
  • the recombinant bacteriophage were recovered by washing each plate for 18 hr with SMG buffer (100 mM NaCl, 10 mM Tris-HCl, pH 7.5, 10 mM MgCl 2 , 0.05% gelatin) and after the addition of glycerol to 50% were frozen at ⁇ 80° C.
  • the TSAR-9 library thus formed had a working titer of ⁇ 2 ⁇ 10 11 pfu/ml.
  • FIG. 2 shows the formula for the synthetic oligonucleotides and the assembly scheme used in the construction of the TSAR-12 library.
  • the TSAR-12 library was prepared substantially the same as the TSAR-9 library described in Section 6.1 above with the following exceptions: (1) each of the variant non-predicted oligonucleotide sequences, i.e., NNB, was 30 nucleotides in length, rather than 54 nucleotides; (2) the restriction sites included at the 5′ termini of the variant, non-predicted sequences were Sal I and Spe 1, rather than Xho I and Xba I; and (3) the invariant sequence at the 3′ termini to aid annealing of the two strands was GCGGTG and CGCCAC rather than CCAGGT and GGTCCA (5′ to 3′).
  • coli cells were plated at high density ( ⁇ 400,000 per 150 mm petri late) for amplification of the recombinant phage. After bout 8 hr, the recombinant bacteriophage were recovered by ashing, for 18 hr with SMG buffer and after the addition of lycerol to 50% were frozen at ⁇ 80° C.
  • the TSAR-12 library thus formed had a working titer of ⁇ 2 ⁇ 10 11 pfu/mL.
  • Inserted synthetic oligonucleotide fragments of 23 randomly chosen isolates were examined from the TSAR-9 library. Individual plaques were used to inoculate I ml of 2XYT broth containing E. coli (DH5 ⁇ F′) cells and the cultures were allowed to grow overnight at 37° C. with aeration. DNA was isolated from the culture supernatants according to Maniatis, supra. Twenty-three individual isolates were sequenced according to the method of Sanger ( Proc. Natl. Acad. Sci . USA (1979) 74:5463-5467) using as a primer the oligonucleotide 5′-AGCGTAACGATCTCCCG (SEQ ID NO. 99), which is 89 nucleotides downstream of the pIII gene cloning site of the m663 vector used to express the TSARS.
  • Nucleotide sequences and their encoded amino acid sequences were analyzed with the MacVector computer program (IBI, New Haven, Conn.). The Microsoft EXCEL program was used to evaluate amino acid frequencies. Such analyses showed that the nucleotide codons coding for and hence most amino acids, occurred at the expected frequency in the TSAR-9 library of expressed proteins. The notable exceptions were glutamine and tryptophan, which were over- and under-represented, respectively.
  • TAG stop codons in the inserts i.e., only 2 of ⁇ 200 isolates characterized contained a TAG stop codon.
  • About half [1-(47/48) 36 ] of the phage inserts were expected to have at least one TAG codon in view of the assembly scheme used.
  • most of the TAG-bearing phage appear to have been lost from the library, even though the bacterial host was supE. This may be a consequence of suppression being less than 100% effective.
  • amino acids encoded by the inserted double stranded synthesized oligonucleotide sequences were concatenated into a single sequence and the usage frequency determined for each amino acid using the Microsoft EXCEL program. These frequencies were compared to that expected from the assembly scheme of the oligonucleotides, and the divergence from expected values represented by the size of the bars above and below the baseline. Chi square analysis was used to determine the significance of the deviations. The majority of amino acids were found to occur at the expected frequency, with the notable exceptions that glutamine and tryptophan were somewhat over- and under-represented, respectively. Thus, except for the invariant Pro-Gly, any position could have any amino acid; hence, the sequences are unpredicted or random.
  • two oligonucleotides were synthesized on an Applied Biosystems Model 380a machine with the sequence 5′-TGACGTCTCGAGTTGTNNKNNKNNKNNKNNKNNKNNKNNKNNKNNKNNKTGTGGATCTAGAAGGATC-3′ (SEQ ID NO:31) and 5′-GATCCTTCTAGATCC-3′ (SEQ ID NO:32), where N is an equimolar ratio of deoxynucleotides A, C, G, and T, and K is an equimolar ratio of G and T. Fifty pmol of each oligonucleotide was incubated at 42° C. for 5 min, then 37° C.
  • SequenaseTM buffer U.S. Biochemicals, Cleveland, Ohio
  • SequenaseTM buffer U.S. Biochemicals, Cleveland, Ohio
  • 10 units of SequenaseTM U.S. Biochemicals
  • 0.2 mM of each dNTP were added and incubated at 37° C. for 15 min.
  • the sample was then heated at 65° C. for 2 hr, digested with 100 units of both Xho I and Xba I (New England BioLabs, Beverly, Mass.), phenol extracted, ethanol precipitated, and resolved on a 15% non-denaturing polyacrylamide gel.
  • the assembled, digested fragment was gel purified prior to ligation.
  • The. vector, m663 (Fowlkes, D. et al. Biotech . (1992) 13:422-427), was prepared by digestion with Xho I and Xba I, calf alkaline phosphatase (Boehringer Mannheim, Indianapolis, Ind.) treatment, phenol extracted, and purified by agarose gel electrophoresis.
  • 20 ⁇ g vector was combined with 0.2 ⁇ g insert in 3 mL with T4 DNA ligase (Boehringer Mannheim), according to the manufacturer.
  • the ligated DNA was electroporated into XL1-Blue E. coli (Stratagene, San Diego, Calif.) and plated for eight hours at 37° C.
  • XL1-Blue E. coli (Stratagene, San Diego, Calif.)
  • the top agar was collected with a spatula, mixed with an equal volume of 100 mM NaCl, 10 mM MgCl 2 , and 50 mM Tris-HCI (pH7.5), and disrupted by two passes through an 18-gauge syringe needle.
  • the bacterial cells were removed by centrifugation, and phage particles were collected by polyethylene glycol precipitation and stored at ⁇ 70° C. in 25% glycerol.
  • the library had 10 8 total recombinants and a working titer of 6 ⁇ 10 13 pfu/mL.
  • phage supernatant Five microliters of the phage supernatant were then transferred to new tubes containing buffer (67 mM Tris-HCl, pH 8.8/10 mM ⁇ - mercaptoethanol/16.6 mM ammonium sulfate/6.7 mM EDTA/50 ⁇ g bovine serum albumin per mL), 0.1 mM deoxynucleotide triphosphates, and 1.25 units of Taq DNA polymerase (Boehringer Mannheim, Indianapolis, Ind.) with 100 pmoles of oligonucleotide primers.
  • the primers flanked the cloning site in gene III of m663 (5′-TTCACCTCGAAAGCAAGCTG-3′ (SEQ ID NO:100) and
  • fusion proteins were prepared as per the manufacturer's instructions, and quantified by Coomassie Blue staining of SDS-polyacrylamide gels. Microtiter wells were coated with 5-20 ⁇ g GST-SH3 fusion protein in 100 mM NaHCO 3 , pH 8.5, blocked with 100 mM NaHCO 3 (pH 8.5) 1% BSA, and washed.
  • Src-GST Bacterially expressed Src SH3 glutathione-S-transferase (Src-GST) fusion protein was purified from bacterial lysates using glutathione agarose 4B (Pharmacia), according to the manufacturer's instructions. Bourd Src-GST fusion protein was eluted from the glutathione agarose with 10 mM glutathione in PBS. Microtiter wells were then coated with Src-GST fusion protein (1-10 ⁇ g/well, in 50 mM NaHCO 3 , pH 8.5) overnight at 4° C.
  • Clones were confirmed as binding the SH3 domain by applying equal titers of phage to wells containing Src SH3-GST or GST alone, and titering the number of eluted particles from each well, or detecting bound phage with an anti-phage antibody-based ELISA (Pharmacia).
  • Bound phage are eluted with 100 AL 50 mM glycine (pH 2.2), transferred to a new well, and neutralized with 100 ⁇ L 200 mM NaHPO 4 (pH 7.0). The phage are diluted 10 3 - to 10 ⁇ 6 -fold, and aliquots are plated onto lawns of DH5 ⁇ F′ E. coli cells to establish the number of plaque forming units in the output sample. From these experiments, the relative specificity of different Src SH3 binding clones for SH3 domains derived from other proteins is determined.
  • ELISA enzyme-linked-immuno-assays
  • the phage are incubated in the well with 25 ⁇ L of PBS-0.1% Tween 20 at RT for 2 hr. The wells are then washed multiple times over 30 minutes. To each well is added 50 ⁇ L of polyclonal anti-phage antibody conjugated to horseradish peroxidase. The antibody is diluted 1:3000 in BSA-PBS-Tween 20; it was obtained from Pharmacia (Piscataway, N.J.; catalog number 27-9402-01). After minutes, the wells are washed again with BSA-PBS-Tween 20 for ⁇ 20 minutes. Finally, 100 AL of ABTS reagent (Pharmacia, with H 2 O 2 ) are added to each well for the development of color. Plates are read with a plate reader (Molecular Devices, Menlo Park, Calif.) at 405 nm wavelength.
  • nucleotide sequence of the relevant segments of the Src SH3 binding clones were sequenced using standard methods. Sanger, F., et al., in Proc. Natl. Acad. Sci. USA (1977) 74:5463-5467.
  • the oligo primer 5′-AGCGTAACGATCTAAA-3′ was used, which is 89 nucleotides downstream of the gene III cloning site of M13 m666.
  • the nucleotide sequences were analyzed with the MacVector computer program (IBI, New Haven, Conn., USA).
  • Peptides were obtained from Research Genetics (Birmingham, Ala.), Chiron Mimotopes (Victoria, Australia), or synthesized by conventional techniques by Dr. J. Mark Carter of Cytogen Corporation (Princeton, N.J.). Peptide purity was assessed by HPLC and/or mass spectrometry. Biotinylated peptides were synthesized with either a KSGSG (SEQ ID NO:103) or a GSGS (SEQ ID NO:104) peptide linker (a spacer) between the biotin and the N-terminus of the peptide. Binding experiments were performed as above, excepting the use of 10 AM peptide instead of phage.
  • Bound biotinylated peptide was detected with streptavidin conjugated to alkaline phosphatase (Sigma Chemical Co., St. Louis, Mo.). After one hour incubation period at room temperature, the wells were washed, and a solution of 3 mM p-nitrophenyl-phosphate (US Biochemicals, Cleveland, Ohio) in 50 mM NaCO 3 (pH 9.8), and 50 mM MgCl 2 was added and color allowed to develop. Signals were read with an ELISA plate reader (Molecular Devices, Menlo Park, Calif.) at 405 nm wavelength. Binding experiments were performed in triplicate. The results are presented in FIGS. 7 and 8.
  • Labeled proteins are-prepared by incubating a culture of HeLa cells overnight with ⁇ 100 ⁇ Ci/mL 35 S-methionine. The cells are then washed and lysed with mild detergent. This mixture of radioactive proteins is incubated with Src-GST fusion protein that has been immobilized on glutathione-linked Sepharose beads (Pharmacia, Piscataway, N.J.). After several hours of tumbling, the beads are pelleted gently by low-speed centrifugation, and the supernatant is discarded. The beads are then resuspended into a slurry in PBS-0.1% Tween 20, pelleted, and washed several additional times.
  • a 2% SDS solution is added to the sample, which is then boiled at 100° C. for 3 minutes. Afterward, the sample is centrifuged, and the supernatant loaded on a 10% polyacrylamide SDS gel for electrophoresis. After the proteins have been resolved, the gel is fixed, dried down, and exposed to X-ray film for autoradiography or phosphor plates for scanning by a Molecular Dynamics PhosphorImager.
  • Src SH3 The ability of Src SH3 to bind certain 35 S-labeled proteins is examined for competability with exogenous peptides. Synthetic peptides corresponding to phage-displayed inserts and motifs are added at the time that the lysate is incubated with the Src-GST fusion protein immobilized on glutathione-linked sepharose beads. The SH3 inding peptides block binding of all or some of the labeled roteins while negative control peptides (unrelated peptide sequences) do not. The amount of competition is quantified and correlated with the amount of added SH3-domain binding peptides.
  • NIH 3T3 cells were grown in Dulbecco's Modified Eagle Medium (DME)+10% fetal calf serum (FCS)+80 ⁇ Ci/mL Tran 35 Slabel (ICN), washed with PBS, lysed in RIPA buffer, and pelleted. Supernatant from 1.5 ⁇ 10 6 cells was recleared with 100 ⁇ g glutathione-agarose-immobilized GST. The supernatant was then incubated with 10 ⁇ g glutathione-agarose-immobilized GST-SH3 fusion protein with or without added test peptide in a final volume of 250 ⁇ L.
  • DME Dulbecco's Modified Eagle Medium
  • FCS fetal calf serum
  • ICN Tran 35 Slabel
  • the sample is centrifuged, and the supernatant is loaded on a 10% polyacrylamide SDS gel for electrophoresis. After the proteins have been resolved, the gel is blotted to nitrocellulose or nylon (i.e., western blot). The filter is then probed with a PI-3′ Kinase antibody (monoclonal and polyclonal antibodies are available from Upstate Biotechnology Incorporated, Lake Placid, N.Y.) and an enzyme-linked secondary antibody. The amount of PI-3′ Kinase is then quantitated.
  • PI-3′ Kinase antibody monoclonal and polyclonal antibodies are available from Upstate Biotechnology Incorporated, Lake Placid, N.Y.
  • the SH3-binding peptides are tagged and localized in cells.
  • Bar-Sagi et al., in Cell (1993) 74:83-91 have shown that SH3-binding proteins localize to the cytoskeleton when expressed in cells.
  • the SH3 domain-binding peptides of the present invention can serve as cellular targetting signals (e.g., to the cytoskeleton). Accordingly, the peptides are tagged with biotin and, subsequently, injected into cells.
  • the location of the biotinylated peptide or the GFP fusion protein is then assayed with FITC-labeled streptavidin in paraformaldehyde-fixed cells or by direct fluorescence in living cells, respectively.
  • Localization of the SH3-binding peptides to the cytoskeleton demonstrates that the SH3-binding peptides can bind SH3-domain proteins in vivo.
  • focal adhesions which are rich in Src, are also sites of potential subcellular localization of SH3-binding peptides.
  • NIH 3T3 fibroblasts were cultured in vitro on glass coverslips coated with fibronectin. After two days of growth at 37° C., the cells were fixed for one hour at room temperature in the presence of 2% paraformaldehyde (pH 7.5). The coverslips were washed with PBS-0.1% Tween 20 several times to remove the fixative. Next, the coverslips were dipped into acetone (chilled at ⁇ 20° C.) for approximately 20 seconds and allowed to air-dry.
  • acetone chilled at ⁇ 20° C.
  • the coverslips were washed again with PBS-0.1% Tween 20, containing BSA (1 mg/mL), and incubated for 2 hours at room temperature with different biotinylated peptides in PBS-0.1% Tween 20.
  • the coverslips were washed and then incubated with 1 mg/mL streptavidin-Cy3 (Jackson Immunoresearch Co., West Grove, Pa.) for 1 hour at room temperature.
  • streptavidin-Cy3 Jackson Immunoresearch Co., West Grove, Pa.
  • FIG. 11 The results are presented in FIG. 11, in which panel A displays cells stained with the conjugate biotin-spacer-VLKRPLPIPPVTR (SEQ ID NO:64); panel B exhibits cells stained with the conjugate, biotin-spacer-GILAPPVPPRNTR (SEQ ID NO:63); panel C shows cells stained with the long consensus peptide, biotin-spacer-RSTPRPLPPLPTTR (SEQ ID NO:67); and panel D shows cells stained with the proline-rich vinculin peptide conjugate, biotin-spacer-LAPPKPPLPEGEV (SEQ ID NO:70).
  • the “spacer” sequence is KSGSG (SEQ ID NO:103).
  • the panels in which SH3 domain-binding peptides were used present a bright display of fluorescence activity that is in sharp contrast to the relatively “dark” features of panel D (non-SH3 domain binding vinculin segment).
  • the state of the cytoskeleton is inspected visually by observing the arrangement of cortical pigment granules under a dissecting microscope.
  • the state of phosphorylation of several proteins is examined by western blotting with an anti-phosphotryosine monoclonal antibody (4G10; Upstate Biotechnology Incorporated), as described in Unger and Steele, above.
  • oocytes After injection, the oocytes were placed in OR2 with 2 mg/mL progesterone (Sigma, St Louis, Mo.) and incubated at 20° C. Oocytes were scored at hourly time points for germinal vesicle breakdown (GVBD).
  • GVBD germinal vesicle breakdown
  • FIG. 10 presents the results of this experiment.
  • oocytes injected with the SH3 domain-binding peptide VLKRPLPIPPVTR exhibit a faster rate of progesterone-induced germinal vesicle breakdown relative to oocytes that had been injected with water or with the proline-rich vinculin peptide, LAPPKPPLPEGEV (SEQ ID NO:70).
  • LAPPKPPLPEGEV proline-rich vinculin peptide
  • Src SH3 domain-binding peptides modulate the biochemical activity of “cellular” Src; in particular, it is proposed that at least some of the Src SH3 domain-binding peptides of the present invention upregulate the biochemical activity of “cellular” Src, which may be downregulated or inhibited in its normal state.
  • the administration of the SH3 domain-binding peptides of the present invention can constitute a novel method of modulating the activity of Src or Src-related proteins. Specifically, certain of these peptides are able to activate Src-family proteins.
  • the coding regions for SH3-binding peptides are cloned into vectors that direct their expression in animal cells.
  • a bipartite gene is constructed, encoding a protein with c-myc epitope and SH3-binding peptide, which is transcribed from a strong constitutive promoter (e.g., SV40, CMV, HSV TK, calmodulin).
  • the vector is introduced into either normal or Src-transformed cells via transfection (e.g., electroporation, calcium phosphate, liposomes, DEAE dextran). Transfected cells express the bipartite gene transiently in culture.
  • the vector carries a selectable marker (e.g., neomycin resistance) or transfection is performed in the presence of excess plasmid carrying a selectable marker (e.g., neomycin resistance) and cells selected for the marker.
  • Transfected cells are stained by immunofluorescence to detect expression of the bipartite protein.
  • the hybridoma 9E10 secretes a monoclonal antibody that is highly specific for the c-myc epitope (EQKLISEEDLN [SEQ ID NO:105]; see, Evan, G.A. et al., in Mol. Cell. Biol . (1985) 5:3610-3616). This antibody is sed in immunofluorescence experiments to demonstrate that he bipartite protein is expressed inside the cells, and in some cases, localized to subcellular structures enriched in SH3 domain bearing proteins.
  • Transfected cells are examined for any changes in cell shape, behavior, and metabolism as a consequence of expressing the SH3 binding peptides.
  • Cell shape is examined by phase contrast microscope at several times after transfection; in particular, the flatness of the cells, their adhesion to the substrate, and the degree of cell ruffling are monitored. Cell division rates, cell migration, and contact inhibition are also observed over time. Finally, the amount of phosphorylated tyrosine in transfected cells is quantitated by phosphoaminoacid analysis and with an anti- phosphotryosine monoclonal antibody (4G10; Upstate Biotechnology Incorporated) in western blotting experiments.
  • 4G10 anti- phosphotryosine monoclonal antibody
  • oligonucleotide inserts were constructed according to the schematic provided in FIG. 12.
  • the mBAX vector was created by generating cloning sites in gene III of the M13mpl8 vector (Messing, J., 1991, “Cloning in M13 phage or how to use biology at its best,” Gene 100, 3-12) in the manner of Fowlkes et al., 1992, Biotechniques 13, 422-427.
  • the mBAX vector displays a peptide sequence at the N-terminus of the mature pIII protein that encodes the epitope for the mouse monoclonal antibody 7E11 (see FIG. 13); it includes the stop codon TAG in the coding region, which is suppressed in E. coli carrying suppressor tRNA gene mutations known as supE or supF.
  • the mBAX vector also carries a segment of the alpha fragment of ⁇ -galactosidase; bacterial cells expressing the omega fragment of ⁇ -galactosidase that are infected by a bacteriophage that expresses the alpha fragment convert the clear XGal substrate into an insoluble blue precipitate. Thus, plaques of such bacteriophage on such cells appear blue.
  • Recombinant mBAX molecules can be distinguished easily from non-recombinant molecules due to the TAG codon in the XhoI-XbaI segment in gene III of mBAX.
  • the recombinants can be grown in bacteria with (e.g., DH5 ⁇ F′) or without (e.g., JS5) suppressor tRNA mutant genes.
  • the non-recombinant mBAX molecules fail to produce plaques on bacterial lawns where the bacteria (e.g., JS5) lack such suppressor genes. This is because in JS5, the TAG codon serves as a stop codon to yield a truncated pIII molecule during translation; since pIII is an essential protein component of viable M13 viral particles, no plaques will form.
  • Phage particles were harvested from each fraction by diffusion into 100 ml PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na 2 HPO 4 , 1.4 mM KH 2 PO 4 ), concentrated by polyethylene glycol precipitation as in Sambrook et al. (1989, supra), and resuspended in 10 ml PBS+10% glycerol. Each fraction contained approximately 5 ⁇ 10 7 unique recombinants, for a total library complexity of approximately 3 ⁇ 10 8 .
  • the resulting phage-displayed library contained peptides of the form X 6 PXXPX 6 (SEQ ID NO:164), where X represents any amino acid.
  • phage were eluted with 50 mM glycine-HCl (pH 2.2). Recovered phage were propagated in 10 ml 2xYT media and 100 ⁇ l of a saturated DH5 ⁇ F′ E. coli culture and affinity purified twice more as above. Affinity purified phage were plated onto 2xYT+0.8% agar plates to yield isolated plaques from which clonal phage stocks and DNA were produced. Phage binding was confirmed by incubating equal amounts of a clonal phage stock in wells coated with 1 ⁇ g GST-SH3 or GST.
  • pGEX-derived constructs expressing GST fusions to the SH3 domains of Yes, Cortactin, Crk, Abl, and PLC ⁇ were kindly provided by M. Sudol (Rockefeller University), J. T. Parsons (University of Virginia at Charlottsville), M. Matsuda (Tokyo, Japan), A. M. Pendergast (Duke University), and S. Earp (University of North Carolina at Chapel Hill), respectively.
  • the GST-SH3 fusion proteins for Yes, Cortactin, Crk, Abl, and PLC ⁇ could have been prepared as above for Grb2 N, Nck N, Nck M, Nck C, p53bp2, and Src, using published sequence information for these proteins. See, e.g., Suen et al., (1993) Mol. Cell. Biol. 13, 5500-5512 (Grb2); Lehmann et al., (1990) Nucleic Acids Res. 18, 1048 (Nck); Iwabuchi et al., (1994) Proc. Natl. Acad. Sci.
  • GST-SH3 fusion proteins were prepared as described in Smith, D. B., & Johnson, K. S. (1988) Gene 67, 31-40. The integrity and purity of the fusion proteins were confirmed by SDS-PAGE. Protein concentrations were determined using a the BioRad protein assay (BioRad, Hercules, Calif.).
  • Tables 1-5 list some of the relevant amino acid sequences obtained when the biased peptide library described in Section 6.14.1 was screened with GST-SH3 fusion proteins.
  • the underscored amino acid residues in Tables 1-5 indicate the fixed positions.
  • a “consensus” sequence which seeks to include the additional features gleaned from the new binding peptides.
  • the symbol “ ⁇ ” in the consensus sequences of Tables 1-5 represents a hydrophobic residue.
  • the symbol x in the consensus sequences of Tables 1-5 represents any amino acid.
  • Nck SH3 domain binding clones a GST-SH3 fusion protein containing the middle SH3 domain of Nck was used.
  • ⁇ represents aliphatic amino acid residues (A, V, L, I, P); X represents any amino acid; ⁇ putative class II peptide (see Section 6.14.5). Invariant proline residues are underlined.
  • ⁇ represents aliphatic amino acid residues (A, V, L, I, P) and X 1 , X 2 , X 3 , X 4 , and X 5 represent any amino acid; except that if
  • X 1 S
  • X 2 is not R, H, A, N, T, G, V, M, or W; or
  • X 1 A
  • X 2 is not R, Q, N, S, or L;
  • ⁇ represents aromatic amino acid residues
  • ⁇ represents aliphatic amino acid residues (A, V, L, I, P)
  • X represents any amino acid. Invariant proline residues are underlined.
  • the PXXP (SEQ ID NO:161) biased peptide library described in Section 6.14.1 was also used to obtain phage clones that specifically bound the SH3 domain from the p53bp2 rotein.
  • the amino acid sequences of the peptides expressed by the p53bp2 SH3 domain-binding phage are shown in Table 10 below.
  • the PXXP (SEQ ID NO:161) biased peptide library described in Section 6.14.1 was also used to obtain phage clones that specifically bound the SH3 domain from the N terminal portion of the Crk protein.
  • the amino acid sequences of the peptides expressed by the Crk N terminal SH3 domain-binding phage are shown in Table 11 below.
  • ⁇ represents aliphatic amino acid residues (A, V, L, I, P). Invariant proline residues are underlined.
  • the present invention provides a purified peptide that binds to the SH3 domain of Crk, the purified peptide comprising the amino acid sequence ⁇ P ⁇ LP ⁇ K (SEQ ID NO:210), where ⁇ represents aliphatic amino acid residues (A, V, L, I, P), with the proviso that the peptide does not comprise the amino acid sequence WNERQPAPALPPKPPKPT (SEQ ID NO:456).
  • the PXXP (SEQ ID NO:161) biased peptide library described in Section 6.14.1 was also used to obtain phage clones that specifically bound the SH3 domain from the Yes protein.
  • the amino acid sequences of the peptides expressed by the Yes SH3 domain-binding phage are shown in Table 12 below.
  • ⁇ represents aliphatic amino acid residues (A, V, L, I, P); X represents any amino acid. Invariant proline residues are underlined.
  • Another consensus sequence that can be derived from the amino acid sequences of the inserts from the Yes SH3 domain-binding phage is:
  • ⁇ represents aliphatic amino acid residues (A, V, L, I, P) and X 1 , X 2 , X 3 , X 4 , and X 5 represent any amino acid; except that if
  • X 1 S
  • X 2 is not R, H, A, N, T, G, V, M, or W; or
  • the present invention also provides a purified peptide that binds to the SH3 domain of Yes, the purified peptide comprising the amino acid sequence ⁇ X 1 X 2 RPLPX 3 LPX 4 X 5 (SEQ ID NO:455), where ⁇ represents aliphatic amino acid residues (A, V, L, I, P) and X 1 , X 2 , X 3 , X 4 , and X 5 represent any amino acid, with the proviso that the peptide does not comprise the amino acid sequence AGDRPLPPLPYNPKS (SEQ ID NO:457).
  • the PXXP (SEQ ID NO:161) biased peptide library described in Section 6.14.1 was also used to obtain phage clones that specifically bound the SH3 domain from the N terminal portion of the Grb2 protein.
  • the amino acid sequences of the peptides expressed by the Grb2 N terminal SH3 domain-binding phage are shown in Table 13 below. These sequences can be arranged into three groups of sequences that have different, but related, consensus sequences. An overall consensus sequence, + ⁇ DXPLPXLP (SEQ ID NO:223), can be derived for the three groups.
  • Peptide ligands bound to SH3 domains have been shown to assume a left-handed polyproline type II (PPII) helix conformation (Yu, H., Chen, J. K., Feng, S., Dalgarno, D. C., Brauer, A. W., & Schreiber, S. L. (1994) Cell 76, 933-45).
  • SH3 ligands are pseudo-symmetrical and may therefore bind in one of two opposite orientations (Feng, S., Chen, J. K., Yu, H., Simon, J. A., & Schreiber, S. L. (1994) Science 266, 1241-7) (Feng et al.).
  • ligands that bind in the Class I or Class II orientation conform to the consensus +pYPpYP (SEQ ID NO:244) or YPpYPp+ (SEQ ID NO:245) respectively, where uppercase positions represent conserved residues that contact the SH3 domain and confer specificity, and lowercase positions represent scaffolding residues that tend to be proline.
  • each SH3 ligand consensus motif was assigned to class I or II based on its agreement with the class I or II consensus motif. Highly (>90%) conserved positions in each SH3 ligand consensus motif are listed in boldface and were interpreted as SH3 contact residues.
  • the Src SH3 domain is capable of binding both Class I and Class II peptides Feng et al., supra.
  • Class I peptides predominate in the population of Src SH3 ligands selected from the PXXP (SEQ ID NO:161) library, one clone conforms well to the Class II consensus (see Table 6).
  • PXXP SEQ ID NO:161
  • Each instance of cross-reactivity may be explained by similarities between the sequences of the peptides and the ligand preferences of the cross-reactive SH3 domains. For example, Crk SH3 cross-reacted with three phage clones selected with other SH3 domains; each of these clones coincidentally expressed peptides conforming to the Crk SH3 preferred ligand consensus motif. Similarly, the cross-reactivity observed between the Src, Yes, and Grb2 SH3 domains and clones selected by other SH3 domains within this group may be a consequence of the fact that these SH3 domains prefer the same proline-rich core.
  • * represents amino acid sequences previously demonstrated to bind their respective SH3 domains. Residues within the sequences that agree with the most highly conserved residues of the consensus motifs are shown in bold. Each entry shows an abbreviation of the name of the SH3 domain binding protein and the species from which it was derived. The amino acid positions in the mature proteins of the sequences shown are indicated in parentheses. For more details, see the reference listed for each protein.
  • Reference 1 is Flynn, D. C., Leu, T. H., Reynolds, A. B., & Parsons, J. T. (1993) Mol Cell Biol 13, 7892-7900.
  • Reference 2 is Barfod, E. T., Zheng, Y., Kuang, W, J., Hart, M. J., Evans, T., Cerione, R. A., & Ashkenazi, A. (1993) J Biol Chem 268, 26059-62.
  • Reference 3 is Weng, Z., Thomas, S. M., Rickles, R. J., Taylor, J. A., Brauer, A. W., Seidel-Dugan, C., Michael, W. M., Dreyfuss, G., & Brugge, J. S. (1994) Mol Cell Biol 14, 4509-21.
  • Reference 6 is Gout, I., Dhand, R., Hiles, I. D., Fry, M. J., Panayotou, G., Das, P., Truong, O., Totty, N. F., Hsuan, J., Booker, G, W. & et al. (1993) Cell 75, 25-36.
  • Reference 7 is Rivero-Lezcano, O. M., Sameshima, J. H., 30 Marcilla, A., & Robbins, K. C. (1994) J Biol Chem 269, 17363-6.
  • Reference 8 is Odai, H., Sasaki, K., Iwamatsu, A., Hanazono, Y., Tanaka, T., Mitani, K., Yazaki, Y. & Hirai, H. (1995) J Biol Chem 270, 10800-5.
  • Reference 11 is Karlsson, T., Songyang, Z., Landgren, E., Lavergne, C., Di-Fiore, P. P., Anafi, M., Pawson, T., Cantley, L. C., Claesson-Welsh, L., & Welsh, M. (1995) Oncogene 10, 1475-83.
  • Reference 12 is Ren, R., Mayer, B. J., Cicchetti, P., & Baltimore, D. (1993) Science 259, 1157-61.
  • Reference 13 is Ren, R., Ye, Z. S., & Baltimore, D. (1994) Genes Dev 8, 783-95.
  • Reference 15 is Rozakis-Adcock, M., Fernley, R., Wade, J., Pawson, T., & Bowtell, D. (1993) Nature 363, 83-5.
  • Reference 16 is McPherson, P, S., Czernik, A, J., Chilcote, T, J., Onofri, F., Benfenati, F., Greengard, P., Schlessinger, J., & De-Camilli, P. (1994) Proc Natl Acad Sci USA 91, 6486-90.
  • the consensus sequences shown above in Tables 1-13 can be used to search databases (e.g., GenBank) containing the amino acid sequences of proteins that are not known to bind to SH3 domains. In this way, a large number of proteins not previously suspected of containing amino acid sequences that bind SH3 domains have been shown to contain such sequences. The portions of the amino acid sequences of these proteins that resemble one or more of the consensus motifs of Tables 1-13 are shown below in Table 16.
  • the SH3 domain-binding sequences of the proteins shown in Table 16 can be used as ligands in the assays for the identification of compounds that affect binding of SH3 domain-containing proteins and their ligands that are described above in Section 5.6.
  • locus and accession number refer to the entries' names and accession numbers in GenBank or the Swiss-Prot database.
  • the two numbers immediately to the left of the displayed sequences refer to the amino acid positions of the displayed sequences in the mature proteins. The leftmost of these two numbers refers to the starting amino acid number of the displayed sequence in the mature protein.
  • the numbers in parentheses immediately to the right of the displayed sequences refer to the sequences' SEQ ID NOs:.
  • the eight columns to the extreme right of Table 16 show the discrepancies between the displayed sequences and the consensus motifs of Tables 6-15.
  • the leftmost Src column refers to Class I motifs; the rightmost Src column refers to Class II motifs.

Abstract

Peptides having general and specific binding affinities for the Src homology region 3 (SH3) domains of proteins are disclosed in the present invention. In particular, SH3 binding peptides have been isolated from phage-displayed random peptide libraries which had been screened for isolates that bind to bacterial fusion proteins comprising SH3 and glutathione S-transferase (GST). Preferred peptides are disclosed which comprise a core 7-mer sequence (preferably, a consensus motif) and two or more, preferably at least six, additional amino acid residues flanking the core sequence, for a total length of 9, preferably at least 13, amino acid residues and no more than about 45 amino acid residues. Such peptides manifest preferential binding affinities for certain SH3 domains. The preferred peptides exhibit specific binding affinities for the Src-family of proteins. In vitro and in vivo results are presented which demonstrate the biochemical activity of such peptides.

Description

  • This application is a continuation-in-part of co-pending U.S. patent application Ser. No. 08/483,555 filed Jun. 7, 1995 which in turn is a continuation-in-part of U.S. patent application Ser. No. 08/278,865 filed Jul. 22, 1994, the entire contents of each of which are incorporated herein by reference.[0001]
    • 1. FIELD OF THE INVENTION
  • The present invention relates to SH3 binding peptides having a broad range of binding specificities. That is, certain members of the SH3 binding peptides disclosed bind with approximately the same facility with SH3 domains derived from different SH3 domain-containing proteins. Other members, in contrast, bind with a much greater degree of affinity for specific SH3 domains. The SH3 binding peptides are obtained from random peptide libraries that are also phage-displayed. Methods are described of obtaining the phage clones that bind to the SH3 domain targets and of determining their relevant nucleotide sequences and consequent primary amino acid sequence of the binding peptides. The resulting SH3 binding proteins are useful in a number of ways, including, but not limited to, providing a method of modulating signal transduction pathways at the cellular level, of modulating oncogenic protein activity or of providing lead compounds for development of drugs with the ability to modulate broad classes, as well as specific classes, of proteins involved in signal transduction. [0002]
    • 2. BACKGROUND OF THE INVENTION
  • 2.1. Src and the SH3 Domain [0003]
  • Among a number of proteins involved in eukaryotic cell signaling, there is a common sequence motif called the SH3 domain. It is 50-70 amino acids in length, moderately conserved in primary structure, and can be present from one to several times in a large number of proteins involved in signal transduction and in cytoskeletal proteins. [0004]
  • The protein pp60c-src represents a family of at least nine non-receptor protein tyrosine kinases (NR-PTKs). Members of this family share an overall structural organization comprising a series of catalytic and non-catalytic domains. In Src, a 14-amino-acid myristylation signal resides at the extreme amino-terminus, and is followed by a unique region that is not highly conserved among family members. Following this region are two highly conserved 60-and 100-amino-acid regions, the Src homology (SH) [0005] domains 3 and 2, respectively. SH2 and SH3 domains have been shown to play an important role in mediating protein-protein interactions in a variety of signaling pathways. Koch, C. A., et al., in Science (1991) 252:668-74. The carboxy-terminal half of Src contains the PTK catalytic domain, as well as a negative regulatory tyrosine (Y527) near the carboxy terminus. Phosphorylation of this residue (e.g., by Csk) results in the inhibition of PTK activity. Cooper, J. A., et al., in Science (1986) 231:1431-1434. Mutation of Y527→F generates forms of Src with increased PTK and oncogenic activity. Cartwright, C. A., et al., in Cell (1987) 49:83-91; Kmiecik, T. E., et al., in Cell (1987) 49:65-73; and Piwicna-Worms, H., et al., in Cell (1987) 75-82.
  • The fact that some mutations which result in increased Src PTK and transforming activity map to the Src SH2 (Seidel-Dugan, C., et al., in [0006] Mol. Cell. Biol. (1992) 12:1835-45; and Hirai, H. and Varmus, H. E. in Mol. Cell. Biol. (1990) 10:1307-1318) and SH3 domains (Seidel-Dugan, C., et al., supra; Hirai, H. and Varmus, H.E., supra; Superti-Furga, G., et al., in Embo. J. (1993) 12:2625-34; and Potts, W. M., et al., in Oncogene Res. (1988) 3:343-355) suggests a negative regulatory role for these domains. That phosphotyrosine residues within specific sequence contexts represent high affinity ligands for SH2 domains suggests a model in which the SH2 domain participates in Y527-mediated inhibition of PTK activity by binding phosphorylated Y527, thereby locking the kinase domain in an inactive configuration. Matsuda, M., Mayer, B. J., et al., in Science (1990) 248:1537-1539. This model is supported by the observation that phosphopeptides corresponding to the carboxy-terminal tail of Src bind active, but not inactive, variants of Src. Roussel, R. R., et al., in Proc. Natl. Acad. Sci. U S A (1991) 88:10696-700; and Liu, X., et al., in Oncogene (1993) 8:1119-1126.
  • The mechanism of SH3-mediated inhibition of Src PTK activity remains unclear. There is evidence that pY527-mediated inhibition of Src PTK activity involves the SH3 domain as well as the SH2 domain. Okada, M., Howell, et al., in [0007] J. Biol. Chem. (1993) 268:18070-5; Murphy, S. M., et al., in Mol. Cell. Biol. (1993) 13:5290-300; and Superti-Furga, G., et al., supra. Although these effects are thought to be a consequence of SH3-mediated protein-protein interactions, precisely how the Src SH3 domain exerts its negative regulatory effect is unclear. Identification of high affinity ligands for the Src SH3 domain could help resolve these issues.
  • 2.2. Protein Tyrosine Kinases and The Immune Response [0008]
  • Src-related tyrosine kinases are expressed in a variety of cell types including those of the immune system (lymphocytes, T cells, B cells, and natural killer cells) and the central nervous system (neural cells, neurons, oligodendrocytes, parts of the cerebellum, and the like). Umemori, H. et al., in [0009] Brain Res. Mol. Brain Res. (1992) Dec. 16(3-4):303-310. Their presence in these cells and tissues and their interaction with specific cell surface receptors and immunomodulatory proteins (such as T cell antigen receptor, CD14, CD2, CD4, CD40 or CD45) suggest that these kinases serve an important role in the signalling pathways of not only the central nervous system but of the immune system, as well. See, e.g., Ren, C. L. et al., in J. Exp. Med. (1994) 179(2):673-680 (signal transduction via CD40 involves activation of Lyn kinase); Donovan, J. A. and Koretzky, G. A., in J. Am. Soc. Nephrol. (1993) 4(4):976-985 (CD45, the immune response, and regulation of Lck and Fyn kinases); and Carmo, A. M. et al., in Eur. J. Immunol. (1993) 23(9):2196-2201 (physical association of the cytoplasmic domain of CD2 with p56lck and p59fyn).
  • For instance, mice with disruptions in their Src-like genes, Hck and Fgr, possess macrophages with impaired phagocytic activity or exhibit a novel immunodeficiency characterized by an increased susceptibility to infection with Listeria monocytogenes. Lowell, C. A. et al., in [0010] Genes Dev. (1994) 8(4):387-398. Also, it has been shown that bacterial lipopolysaccharide (LPS) activates CD14-associated p56lyn, p68hck, and p59c-fgr, while inducing the production of lymphokines, such as TNF-alpha, IL-1, IL-6, and IL-8. Inhibition of the protein tyrosine kinases blocks production of TNF-alpha and IL-1.
  • 2.3. SH3 Binding Peptides [0011]
  • As mentioned above, it has long been suspected that SH3 domains are sites of protein-protein interaction, but it has been unclear what SH3 domains actually bind. Efforts to identify ligands for SH3 domains have led to the characterization of a number of SH3-binding proteins, including 3BP1 and 2 (Ren, R., Mayer, et al., in [0012] Science (1993) 259:1157-61), SOS (Olivier, J. P., et al., in Cell (1993) 73:179-91; and Rozakis-Adcock, M., et al., in Nature (1993) 363:83-5), p85 PI-3′ Kinase (Xingquan, L., et al., in Mol. Cell. Biol. (1993) 13:5225-5232), dynamin (Gout, I., et al., in Cell (1993) 75:25-36), AFAP-110 (Flynn, D. C., et al., in Mol. Cell. Biol. (1993) 13:7892-7900), and CD42 (Barfod, E. T., et al., in J. Biol. Chem. (1993) 268:26059-26062). These proteins tend to possess short, proline-rich stretches of amino acids, some of which have been directly implicated in SH3 binding. A variety of consensus sequences have been proposed, although the similarity among proline-rich regions of different SH3-binding proteins tends to be fairly low. Also, attempts to build consensus sequences are likely complicated by the incorporation of data from proteins that bind different SH3 domains.
  • Thus, Cicchetti, P., et al., in [0013] Science (1992) 257:803-806, published their work relating to the isolation and sequencing of two naturally-occurring proteins that could be bound in vitro by the SH3 domain of the abl oncogene product. These workers found that SH3 domains bind short, proline-rich regions of such proteins. Subsequently, this same group disclosed further results (Ren, R. et al., supra) in which the SH3 binding sites of the SH3 binding proteins were localized to “a nine- or ten-amino acid stretch rich in proline residues.” A consensus sequence incorporating the features of the SH3 binding sites of four SH3 binding proteins was proposed: XPXXPPP
    Figure US20030186863A1-20031002-P00900
    XP (SEQ ID NO:1), wherein X indicates a position in the amino acid sequence which is not conserved among the four SH3 binding proteins, P represents proline, and
    Figure US20030186863A1-20031002-P00900
    indicates a hydrophobic amino acid residue, such as P or L.
  • The screening of complex random peptide libraries has been used to identify peptide epitopes for monoclonal (Scott, J. K. and Smith, G. P. in [0014] Science (1990) 249:386-390) and polyclonal (Kay, B. K., et al., in Gene (1993) 128:59-65) antibodies, as well as peptide ligands for a variety of proteins, including streptavidin (Devlin, J. J., et al., in Science (1990) 249:404-406; and Lam, K., et al., in Nature (1991) 354:82-84), the endoplasmic reticulum chaperone BiP (Blond-Elguindi, S., et al., in Cell (1993) 75:717-728), and CaM (Dedman, J. R., et al., in J. Biol. Chem. (1993) 268:23025-23030).
  • Recently, Chen, J. K. et al., in [0015] J. Am. Chem. Soc. (1993) 115:12591-12592, described ligands for the SH3 domain of phosphatidylinositol 3-kinase (PI-3′ Kinase) which were isolated from a biased combinatorial library. A “biased” library is to be distinguished from a “random” library in that the amino acid residue at certain positions of the synthetic peptide are fixed, i.e., not allowed to vary in a random fashion. Indeed, as stated by these research workers, screening of a “random” combinatorial library failed to yield suitable ligands for a PI-3′ Kinase SH3 domain probe. The binding affinities of these unsuitable ligands was described as weak, >100 μM, based on dissociation constants measured by the Biosensor System (BIAcore).
  • More recently, Yu, et al. (Yu, H., et al., in [0016] Cell (1994) 76:933-945) used a “biased” synthetic peptide library of the form XXXPPXPXX (SEQ ID NO:2), wherein X represents any amino acid other than cysteine, to identify a series of peptides which bind the Src and PI-3′ Kinase SH3 domains. The bias was accomplished by fixing the proline residues at the specific amino acid positions indicated for the “random” peptide. As stated previously, without this bias, the technique disclosed fails to identify any SH3 domain-binding peptides.
  • A consensus sequence, based on 13 binding peptides was suggested: RXLPPRPXX (SEQ ID NO:3), where X tends to be a basic residue (like R, K or H). The binding affinities of several SH3 binding peptides were disclosed as ranging from 8.7 to 30 μM. A “composite” peptide, RKLPPRPRR (SEQ ID NO:4), was reported to have a binding affinity of 7.6 μM. This value compares favorably to the binding affinity of the peptide, VPPPVPPRRR (SEQ ID NO:5), to the N-terminal SH3 domain of Grb2. See, Kraulis, P.J. [0017] J. Appl. Crystalloqr. (1991) 24:946. Recognizing the limitations of their technique, Chen and co-workers, supra, stated that their results “illustrate the utility of biased combinatorial libraries for ligand discovery in systems where there is some general knowledge of the ligand-binding characteristics of the receptor” (emphasis added).
  • Yu and co-workers, supra, further described an SH3 binding site consensus sequence, XpφPpXP (SEQ ID NO:6), wherein X represents non-conserved residues, φ represents hydrophobic residues, P is proline, and p represents residues that tend to be proline. A consensus motif of RXLPPRPXX (SEQ ID NO:7), where X represents any amino acid other than cysteine, was proposed for ligands of PI-3′ Kinase SH3 domain. A consensus motif of RXLPPLPRφ (SEQ ID NO:8), where φ represents hydrophobic residues, was proposed for ligands of Src SH3 domain. Still, the dissociation constants reported for the 9-mer peptides ranged only from about 8-70 μM and selectivity between one type of SH3 domain and another was relatively poor, the KDS differing by only about a factor of four. [0018]
  • Hence, there remains a need to develop techniques for the identification of Src SH3 binding peptides which do not rely on such “biased” combinatorial peptide libraries that are limited to a partially predetermined set of amino acid sequences. Indeed, the isolation of SH3 binding peptides from a “random” peptide library has not been achieved successfully before now. Furthermore, particular peptides having much greater binding affinities, whether general or more selective binding for specific SH3 domains, remain to be identified. Binding peptides specific for particular SH3 domains are useful, for example, in modulating the activity of a particular SH3 domain-containing protein, while leaving others bearing an SH3 domain unaffected. Still, the more promiscuous general binding peptides are useful for the modulation of a broad spectrum of SH3 domain-containing proteins. [0019]
  • The present invention relates to such SH3 binding peptides, methods for their identification, and compositions comprising same. In particular, peptides comprising particular sequences of amino acid residues are disclosed which were isolated from random peptide libraries. In the present invention, clones were isolated from a phage-displayed random peptide library which exhibited strong binding affinities for SH3 domain-containing protein targets. Some of these protein targets, include Abl, Src, Grb2, PLC-δ, PLC-γ, Ras GAP, Nck, and p85 PI-3′ Kinase. From the nucleotide sequence of the binding phage, the amino acid sequence of the peptide inserts has been deduced. Synthetic peptides having the desired amino acid sequences are shown to bind the SH3 domain of the target proteins. In particular, synthetic peptides combining a core consensus sequence and additional amino acid residues flanking the core sequence are especially effective at binding to particular target protein SH3 domains. The SH3 binding peptides disclosed herein can be utilized in a number of ways, including the potential modulation of oncogenic protein activity in vivo. These peptides also serve as useful leads in the production of peptidomimetic drugs that modulate a large class of proteins involved in signal transduction pathways and oncogenesis. [0020]
    • 3. SUMMARY OF THE INVENTION
  • Accordingly, three phage-displayed random peptide libraries were screened for isolates that bind to bacterial fusion proteins consisting of the Src homology region 3 (SH3) and glutathione S-transferase (GST). DNA sequencing of the isolates showed that they contained sequences that resemble the consensus motif, RPLPPLP (SEQ ID NO:9), within their 8, 22, or 36 amino acid long random regions. When peptides were synthesized corresponding to the pIII inserts of the SH3-binding phage, they bound to the GST fusions of the SH3 domains of Src and the Src-related proteins, such as Yes, but not of Grb2, Crk, Abl, or PLCγ1. The synthesized peptides bind quite well to the Src SH3 domain and act as potent competitors of natural Src SH3 interactions in cell lysates. For instance, these peptides can compete with radiolabelled proteins from cell lysates in binding to immobilized Src-GST, with an apparent IC[0021] 50 of 1-10 μM. When a peptide, bearing the consensus sequence RPLPPLP (SEQ ID NO:9) was injected into Xenopus laevis oocytes, it accelerated the rate of progesterone-induced maturation. These results demonstrate the utility of phage-displayed random peptide libraries in identifying SH3-binding peptide sequences and that such identified peptides exhibit both in vivo and in vitro biological activity.
  • Thus, it is an object of the present invention to provide peptides having at least nine and up to forty-five amino acid residues, including an amino acid sequence of the formula, R-2-L-P-5-6-P-8-9 (SEQ ID NO:10), positioned anywhere along the peptide, in which each number represents an amino acid residue, such that 2 represents any amino acid residue except cysteine, 5 and 6 each represents a hydrophobic amino acid residue, 8 represents any amino acid residue except cysteine, and 9 represents a hydrophilic amino acid residue except cysteine, each letter being the standard one-letter symbol for the corresponding amino acid, said peptide exhibiting a binding affinity for the SH3 domain of Src, provided that said peptide is not R-P-L-P-P-L-P-T-S (SEQ ID NO:11). In a particular embodiment of the present invention, the peptides also exhibit a binding affinity for the SH3 domain of Src-related proteins, including Yes, Fyn, Lyn, Lck, Hck and Fgr. [0022]
  • The present invention also contemplates SH3 domain-binding peptides that further comprise a C-terminal-flanking amino acid sequence of the [0023] formula 10, 10-11, 10-11-12, 10-11-12-13 (SEQ ID NO:12) or 10-11-12-13-14 (SEQ ID NO:13), in which each number represents any amino acid residue except cysteine, such that 10 is bound to 9 by a peptide bond. Furthermore, peptides are also provided which further comprise an N-terminal-flanking amino acid sequence of the formula 1′, 2′-1′, 3′-2′-1′ or 4′-3′-2′-1′ (SEQ ID NO:14) in which each number represents any amino acid residue except cysteine, such that 1′ is bound to R by a peptide bond.
  • Thus, in a particular embodiment, a peptide is disclosed having at least thirteen and up to forty-five amino acid residues, including an amino acid sequence of the formula, 3′-2′-1′-R-2-L-P-5-6-P-8-9-10 (SEQ ID NO:15), positioned anywhere along the peptide, in which each number represents an amino acid residue, such that 3′, 2′, 1′, 2, 8, and 10 each represents any amino acid residue except cysteine, 5 and 6 each represents a hydrophobic amino acid residue, and 9 represents a hydrophilic amino acid residue except cysteine, each letter being the standard one-letter symbol for the corresponding amino acid, said peptide exhibiting a binding affinity for the SH3 domain of Src. [0024]
  • The present invention also seeks to provide new consensus sequences or motifs that reflect variations in SH3 domain binding selectivities or specificities. The present invention also contemplates conjugates of the SH3 binding peptides and a second molecule or chemical moiety. This second molecule may be any desired substance whose delivery to the region of the SH3 domain of a particular protein (or cell containing the protein) is sought. Possible target cells include, but are not limited to, neural cells, immune cells (e.g., T cells, B cells, natural killer cells, and the like), osteoclasts, platelets, epidermal cells, and the like, which cells express Src, Src-related proteins, and potentially, other SH3 domain-containing proteins. In this manner, the modulation of the biological activity of proteins bearing an SH3 domain can be accomplished. [0025]
  • Other methods and compositions consistent with the objectives of the present invention are likewise disclosed. in particular, a method is disclosed of modulating the activity of Src or Src-related proteins comprising administering a composition comprising an effective amount of a peptide of the present invention and a carrier, preferably a pharmaceutically acceptable carrier. In a specific embodiment, the contemplated method results in the inhibition of the activity of Src or Src-related proteins. Alternatively, the method is effective to activate Src or Src-related proteins. [0026]
  • In yet another embodiment, a method is disclosed of identifying a peptide having a region that binds to an SH3 domain comprising: (a) providing an immobilized target protein comprising an SH3 domain; (b) incubating the immobilized target protein with an aliquot taken from a random peptide library; (c) washing unbound library peptides from the immobilized target protein; (d) recovering the peptide bound to the immobilized target protein; and (e) determining the primary sequence of the SH3 domain-binding peptide. [0027]
  • Moreover, a method is disclosed of imaging cells, tissues, and organs in which Src or Src-related proteins are expressed, which comprises administering an effective amount of a composition comprising an SH3 domain-binding peptide conjugated to detectable label or an imaging agent. [0028]
  • Other objectives of the present invention will become apparent to one of ordinary skill in the art after consideration of the above disclosure and the following detailed description of the preferred embodiments. [0029]
  • The invention also provides assays for identifying a compound that affects the binding between a first molecule comprising an SH3 domain and a second molecule that binds to the SH3 domain comprising incubating one or more candidate compounds from which it is desired to select such a compound with the first molecule and the second molecule under conditions conducive to binding and detecting the one or more compounds that affect binding of the first molecule to the second molecule. [0030]
  • Also provided are kits for performing such assays comprising a first molecule comprising an SH3 domain and a second molecule that binds to the SH3 domain.[0031]
    • 4. BRIEF DESCRIPTION OF THE FIGURES
  • FIG. 1 illustrates a scheme for the generation of a random 36 amino acid peptide library (TSAR-9; e.g., SEQ ID NO:16). Oligonucleotides were synthesized (SEQ ID NOS:17-18), converted into double-stranded DNA, cleaved with restriction enzymes (SEQ ID NOS:19-20), and cloned into the M13 vector, m663. The random peptide region encoded by the oligonucleotides is shown in the box (SEQ ID NO:16) and is situated at the N-terminus of mature protein III (SEQ ID NO:21). SEQ ID NO:22 includes the three amino acids preceding the signal peptidase cleavage site. [0032]
  • FIG. 2 illustrates a scheme for the generation of a random 22 amino acid peptide library (TSAR-12; e.g., SEQ ID NO:23). Oligonucleotides were synthesized (SEQ ID NOS:24-25), converted into double-stranded DNA, cleaved with restriction enzymes (SEQ ID NOS:26-27), and cloned into the M13 vector, m663. The random peptide region encoded by the oligonucleotides is shown in the box (SEQ ID NO:23) and is situated at the N-terminus of mature protein III (SEQ ID NO:28). SEQ ID NO:29 includes the three amino side preceding the signal peptidase cleavage site. [0033]
  • FIG. 3 illustrates a scheme for the generation-of a random 8 amino acid peptide library (R8C; SEQ ID NO:30). oligonucleotides were synthesized (SEQ ID NOS:31-32), converted into double-stranded DNA, cleaved with restriction enzymes (SEQ ID NOS:33-34), and cloned into the M13 vector, m663. The random peptide region (SEQ ID NO:30) is flanked by cysteine residues and is situated at the N-terminus of mature protein III (SEQ ID NO:35). [0034]
  • FIG. 4 illustrates the possible origin of one class of double-insert R8C recombinants (e.g., encoding SEQ ID NO:36). Double-stranded oligonucleotides (e.g., SEQ ID NO:37) may have ligated in a head-to-head fashion at the Xba I site prior to cloning in the Xho I- Xba I cleaved M13 vector. [0035]
  • FIG. 5 shows a list of random peptide recombinants (SEQ ID NOS:38-61 and 106) isolated by the method of the present invention and the displayed peptide sequence. The amino acid sequences are aligned to highlight the core sequences. The flanking sequences are shown to the N-terminal and C-terminal ends of the core sequence. SEQ ID NOS:38-61 are shown in order from top to bottom except that SSCDHTLGLGWCGSRSTRQLPIPP TTTRPSR is SEQ ID NO:106 and RPLPPLP is SEQ ID NO:9. T12.Src3.1 is a Class II ligand (See Section 6.14.5). [0036]
  • FIG. 6 graphically illustrates the relative binding affinities of selected phage clones for various SH3 domains. The results indicate that certain amino acid sequences provide generic SH3 domain binding, while others can provide greater selectivity for the SH3 domain of Src. Still other clones exhibit Src SH3 domain preferential binding. [0037]
  • FIG. 7 shows the binding of synthetic peptides (SEQ ID NOS:9 and 62-70) representing Src SH3-selected phage inserts to Src SH3-GST fusion target (shaded columns) over background GST binding (unshaded columns) relative to the core peptide RPLPPLP (SEQ ID NO:9) and proline-rich peptide segments derived from naturally occurring proteins. Bound biotinylated peptide was detected with streptavidin-alkaline phosphatase ELISA. Each point was performed in triplicate; average absorbance at 405 nm is presented. Error bars represent SD. SEQ ID NOS:62-70 are shown in order from top to bottom except that RPLPPLP is SEQ ID NO:9. [0038]
  • FIG. 8 illustrates the relative specificity of selected peptides (SEQ ID NOS:9 and 62-70) for SH3 domains derived from different proteins. In particular, the binding affinities of the peptides for the SH3 domains of the following protein fusion targets were tested: Src SH3-GST, Yes SH3-GST, Grb2-GST, Crk SH3-GST, Abl SH3-GST, PLCγ1 SH2SH3-GST. Bound biotinylated peptide was detected with streptavidin-alkaline phosphatase. Each point was performed in triplicate; values are average signal (absorbance at 405 nm) above GST background, with error bars representing standard deviation. Hatched bars indicate saturation of the ELISA signal. SEQ ID NOS:62-70 are shown in order from top to bottom except that RPLPPLP is SEQ ID NO:9. [0039]
  • FIG. 9 presents the results of competition experiments in which selected peptides were found to inhibit the binding of proteins from cell lysates to immobilized Src SH3-GST or Abl SH3-GST protein fusion targets. [0040]
  • FIG. 10 presents a graph illustrating the increased rate of progesterone-induced maturation of oocytes injected with an SH3 domain-binding peptide, VLKRPLPIPPVTR (SEQ ID NO:64), of the present invention. Briefly, Stage VI oocyted were prepared and injected as previously described (see, Kay, B. K., in [0041] Methods in Cell Biol. (1991) 36:663-669). Oocytes were injected with 40 nL of 100 μM test peptide or water. After injection, the oocytes were placed in 2 μg/mL progesterone (Sigma, St. Louis, Mo.) and scored hourly for germinal vesicle breakdown (GVBD). LAPPKPPLPEGEV is SEQ ID NO:70.
  • FIG. 11 shows the results of fluorescence experiments in which certain peptides, Panel A=VRPLPIPPVTR (SEQ ID NO:64), Panel B=GILAPPVPPRNTR (SEQ ID NO:63), Panel C=RSTPRPLPPLPTTR (SEQ ID NO:67), of the invention were shown to localize within cellular compartments thought to contain Src or Src-related proteins. [0042]
  • FIG. 12 illustrates a scheme for the generation of a biased peptide library. Oligorncleotides were synthesized (SEQ ID NOS:162-163), converted into double-stranded DNA (SEQ ID NO:454), cleaved with restriction enzymes XhoI and XbaI (SEQ ID NOs:455-456), and cloned into the mBAX vector (SEQ ID NOs:457-458), described further below in the Examples section. The biased peptide region (SEQ ID NO:459) is situated at the N-terminus of mature pIII protein. CTAGACGTGTCAGT is a portion of SEQ ID NO:162. ACTGACACGT is a portion of SEQ ID NO:454. TCGAGGCACAG is a portion of SEQ ID NO:454. [0043]
  • FIG. 13 illustrates the peptide sequence encoded in the mBAX vector situated at the N-terminus of mature pIII protein. TCCTCGAGTATCGACATGCCTTAGACTGCTAGCACTATGTACAACATGCTT CATCGCAACGAGCCA is SEQ ID NO:460. SSIDMP*TASTMYNM LHRNEP is SEQ ID NO:461. GGTGGGAGGAAGTTGAGCCCGCCCGCCAACGA CATGCCGCCCGCCCTCCTGAAGAGGTCTAGA is SEQ ID NO:462. GGRKLSPPANDMPPALLKRSR is SEQ ID NO:463. [0044]
  • FIG. 14 illustrates the relative binding of SH3-selected phage clones to various SH3 domains. Two clones (A and B) representing each consensus motif were assayed for binding to 1 μg of each immobilized GST-SH3 fusion protein. Bound phage were detected by anti-phage ELISA. Sequences of peptides displayed by each clone are aligned with their respective consensus motifs. Invariant proline residues are underlined. Solid bars, specific binding; open bars, cross-reactive binding. Values are average OD[0045] 405±SD (N=3).
    • 5. DETIALED DESCRIPTION OF THE INVENTION
  • 5.1. General Considerations [0046]
  • The present invention relates to peptides that exhibit a binding affinity for an SH3 domain, which domain has been found to be present in a number of physiologically significant proteins. In particular, peptides are disclosed which exhibit general binding characteristics to the SH3 domains found in a group of proteins, including but not limited to Abl, Src, Grb2, PLC-δ, PLC-γ, Ras GAP, Nck, and p85 PI-3′ Kinase. Preferred peptides exhibit selective, if not specific, binding affinity for the SH3 domain of Src. As described herein, the peptides of the present invention include a core sequence, preferably a consensus seqeunce, and additional amino acid residues that flank the core sequence. These peptides, including the methods for their identification, are described in greater detail, below. [0047]
  • Thus, in a specific embodiment of the invention, peptides are provided which have at least nine and up to about forty-five amino acid residues, including an amino acid sequence resembling the formula, [0048]
  • R-2-L-P-5-6-P-8-9 (SEQ ID NO:10), [0049]
  • positioned anywhere along the peptide. In the above-mentioned formula, each number represents an amino acid residue, such that 2 represents any amino acid residue except cysteine, 5 and 6 each represents a hydrophobic amino acid residue, 8 represents any amino acid residue except cysteine, and 9 represents a hydrophilic amino acid residue except cysteine. Each letter used in the formulas herein represent the standard one-letter symbol for the corresponding amino acid. When the peptide is a 9-mer, the peptide R-P-L-P-P-L-P-T-S (SEQ ID NO:ll) is excluded. The peptides of particular interest are those that exhibit a binding affinity for the SH3 domain of Src and Src-related proteins, including Yes, Fyn, Lyn, Lck, Hck and Fgr. Preferably, the peptides of the invention exhibit a binding affinity for the SH3 domain of Src, which is at least three-fold, more preferably at least four-fold, most preferably at least about five-fold greater than that exhibited by the peptide RPLPPLP (SEQ ID NO:9). In still other embodiments, the peptides exhibit a binding affinity for the SH3 domain of Src which is at least ten-fold greater than that exhibited by the peptide RPLPPLP (SEQ ID NO:9). [0050]
  • In specific embodiments, peptides are disclosed in which the various amino acid residues at the indicated positions may independently have the following preferred identities: 2 is a P, R, A, L, Q, E or S, more preferably P or R; 5 represents a P, M, I or L, more preferably P or M; 6 is a P, L, I or V, more preferably P or L; 8 is a T, R, P, I, N, E, V, S, A, G or L, more preferably T or R; and 9 is a T, R, S, H or D, more preferably T or R. Despite the preference for hydrophobic amino acid residues at 5 and 6, in some cases it may be desirable to have hydrophilic amino acid residues at these positions. Specifically, [0051] amino acid residue 5 may be a T, R or S, and amino acid residue 6 may be a T or R. Likewise, while a hydrophilic amino acid residue is preferred at position 9, in some instances a hydrophobic residue, such as a P or A, may be desirable.
  • The present invention also contemplates SH3 domain-binding peptides with a minimum length of 10, 11, 12, 13, 14, 15 or more amino acids. Such peptides contain additional amino acid residues flanking the core sequence of R-2-L-P-5-6-P (SEQ ID NO:71) either at the C-terminal end, the N-terminal end or both. Thus, for example, such peptides include those that further comprise a C-terminal-flanking amino acid sequence of the [0052] formula 10, 10-11, 10-11-12, 10-11-12-13 (SEQ ID NO:12) or 10-11-12-13-14 (SEQ ID NO:13), in which each number represents any amino acid residue except cysteine, such that the amino acid residue 10 is bound to the amino acid residue 9 by a peptide bond. In that case, specific embodiments include an amino acid residue 10 which is T, R, L, S, D, P, A or N, preferably T or R, an amino acid residue 11 which is R, P, A, Q, S or T, preferably R or P, an amino acid residue 12 which is P, S, R or T, preferably P or S, an amino acid residue 13 which is P, S, R, F, H or T, preferably P or S, and an amino acid residue 14 which is S, R, G or T, preferably, S or R.
  • Furthermore, peptides are also provided which further comprise an N-terminal-flanking amino acid sequence of the [0053] formula 1′, 2′-1′, 3′-2′-1′ or 4′-3′-2′-1′ (SEQ ID NO:14) in which each number represents any amino acid residue except cysteine, such that 1′ is bound to R by a peptide bond. In such a case, specific embodiments are provided in which the amino acid residue 1′ is T, P, S, N, F, W, K, H, Q or G, preferably T or P, wherein the amino acid residue 2′ is S, T, G, P, R, Q, L, A or H, preferably S or T, wherein the amino acid residue 3′ is R, S, P, G, A, V, Y or L, preferably S or T, and wherein the amino acid residue 4′ is R, S, V, T, G, L or F, preferably R or S.
  • In a particular embodiment, a peptide is disclosed having at least thirteen and up to forty-five amino acid residues, including an amino acid sequence of the formula, 3′-2′-1′-R-2-L-P-5-6-P-8-9-10 (SEQ ID NO:15), positioned anywhere along the peptide, in which each number represents an amino acid residue, such that 3′, 2′, 1′, 2, 8, and 10 each represents any amino acid residue except cysteine, 5 and 6 each represents a hydrophobic amino acid residue, and 9 represents a hydrophilic amino acid residue except cysteine, each letter being the standard one-letter symbol for the corresponding amino acid, said peptide exhibiting a binding affinity for the SH3 domain of Src. Preferred 13-mers include, but are not limited to, those having an [0054] amino acid residue 5 which is a P or M, an amino acid residue 1′ which is T, P, S or N, an amino acid residue 2′ which is S or T, an amino acid residue 3′ which is R or S, and an amino acid residue 10 which is T or R. In all the SH3 domain-binding peptides described herein, the prohibition against the use of the hydrophilic amino acid residue cysteine (C) does not extend beyond the 7-mer “core” sequence and the additional amino acid residues flanking the core up to a total (core+flanking) of about 20 amino acids. That is, the occasional use of a cysteine is not absolutely prohibited. What should be kept in mind is that the potential for the formation of intramolecular disulfide bonds, to form a cyclic structure, be minimized as much as possible. Applicants have found that cyclized structures appear to be disfavored, at least with potential binding peptides of less than about 15 amino acid residues in length. The concern for the formation of cyclized structures comprising the core sequence diminishes with increasing size of the peptide. Presumably, a large enough structure, though cyclic, may allow the critical core sequence to adopt a more or less linear conformation.
  • In particular, specific peptides are disclosed which exhibit binding affinities to SH3 domains. These include the peptides, RSTPRPLPMLPTTR (SEQ ID NO. 62), RSTPRPLPPLPTTR (SEQ ID NO. 67), GILAPPVPPRNTR (SEQ ID NO. 63), VLKRPLPIPPVTR (SEQ ID NO. 64), GPHRRLPPTPATR (SEQ ID NO. 65), and ANPSPATRPLPTR (SEQ ID NO. 66). [0055]
  • Phage clones are also disclosed, along with the amino acid sequences that are responsible for SH3 domain binding. These phage clones are identified in FIG. 5. [0056]
  • In other embodiments of the present invention, SH3 domain-binding peptides are contemplated which have a total of 11, 13, 14, 18, 20, 22, 23, 25, 30, 36, 38 or 45 amino acid residues. [0057]
  • The peptides of the present invention, having been disclosed herein, may be prepared by any number of practicable methods, including but not limited to solution-phase synthesis, solid-phase synthesis, protein expression by a transformed host, cleavage from a naturally-derived, synthetic or semi-synthetic polypeptide, or a combination of these techniques. [0058]
  • The SH3 binding peptides exhibit a wide range of biological activity which includes the enhancement (or inhibition, depending on the particular peptide or the nature of the peptide's target molecule, in this case a protein bearing an SH3 domain) of the natural function or biological activity of the peptide's target molecule. For example, the interaction of the binding peptide of the present invention could result in the modulation of the oncogenic activity of the target molecule bearing the SH3 domain. If the target molecule has, in turn, a natural binding partner or ligand, the peptides of the present invention may also exhibit antagonistic or agonistic activity in relation to the biological activity of the natural binding partner. [0059]
  • Thus, it is an object of the present invention to provide a method of activating Src or Src-related protein tyrosine kinases by administering an effective amount of the SH3 domain-binding peptides generally described herein. The intensity of the immune response can thus be stimulated for example, by the increased production of certain lymphokines, such as TNF-alpha and interleukin-1. As is generally known to those of ordinary skill in the art, a more intense immune response may be in order in certain conditions, such as in combating a particularly tenacious infection, viral or otherwise, or a malignancy. [0060]
  • Furthermore, in a specific embodiment of the present invention, a conjugate compound is contemplated which comprises the peptide of the present invention and a second chemical moiety. The second chemical moiety can be selected from a wide variety of chemical compounds including the peptide itself. Typically, however, the second chemical moiety is selected to be other than the peptide of the present invention, including but not limited to an amino acid, a peptide other than an SH3 binding peptide of the present invention, a polypeptide or protein (i.e., the conjugate is a fusion protein), a nucleic acid, a nucleoside, a glycosidic residue (i.e., any sugar or carbohydrate), a label or image-enhancing agent (including metals, isotopes, radioisotopes, chromophores, fluorophores (such as FITC, TRITC, and the like), and enzyme substrates), a drug (including synthetic, semisynthetic, and naturally-occurring compounds), small molecules (e.g., biotin, hormones, factors) and the like. [0061]
  • The peptide of the present invention can be conjugated to the second chemical moiety either directly (e.g., through appropriate functional groups, such as an amine or carboxylic acid group to form, for example, an amine, imine, amide, ester, acyl or other carbon-carbon bond) or indirectly through the intermediacy of a linker group (e.g., an aliphatic or aromatic polyhydroxy, polyamine, polycarboxylic acid, polyolefin or appropriate combinations thereof). Moreover, the term “conjugate,” as used herein, is also meant to encompass non-covalent interactions, including but not limited to ionic, affinity or other complexation interactions. Preferably, such other non-covalent interactions provide definable, most preferably, isolatable chemical conjugate species. [0062]
  • As described further herein, the peptides of the present invention have been shown to localize within certain cellular compartments which contain Src or Src-related proteins. Consequently, the above-described conjugate can be utilized as a delivery system for introduction of a drug to cells, tissues or organs that include SH3 domain-containing proteins. [0063]
  • It should also be pointed out that the present invention seeks to provide a recombinant construct comprising a nucleic acid or its complement that includes codons or nucleotide and sequences encoding a peptide having a region that binds to an SH3 domain, preferably the Src SH3 domain. The recombinant nucleic acid may be a DNA or RNA polynucleotide. [0064]
  • In a specific embodiment, the present invention contemplates a recombinant construct which is a transforming vector. Such vectors include those well known to those of ordinary skill in the art, which effect the transfer or expression of the nucleotide sequence after introduction to a host, such as recombinant plasmid, phage or yeast artificial chromosome. These vectors may be closed circular loops or they may be linearized. The vectors contemplated include those that exist extrachromosomally after host transformation or transfection, as well as those that integrate within or even displace portions of the host chromosome. The vectors may be introduced to the cell with the help of transfection aids or techniques well-known in the art. For example, these aids or techniques may take the form of electroporation, use of calcium chloride, calcium phosphate, DEAE dextran, liposomes or polar lipid reagents known as LIPOFECTIN or LIPOFECTAMINE. In addition, the present invention contemplates the direct introduction of the desired nucleic acid to the host cell, for instance, by injection. [0065]
  • Transformed host cells are also obtained by the methods of the present invention which are capable of reproducing the polynucleotide sequences of interest and/or expressing the corresponding peptide products. A variety of hosts are contemplated, including prokaryotic and eukaryotic hosts. In particular, bacterial, viral, yeast, animal, and plant cells are potentially transformable hosts. Thus, a method is disclosed to obtain a transformed host cell that can produce, preferably secrete, a peptide having a region that binds to an SH3 domain comprising (a) providing an expression vector, preferably a secretory expression vector, comprising a nucleotide sequence encoding at least one copy of a peptide having a region that binds to an SH3 domain; and (b) introducing the vector to a competent host cell. [0066]
  • The peptides, thus produced, may then be introduced to cells, tissues, organs, or administered to the subject for the purpose of modulating the biochemical activity of the SH3 domain-containing proteins present therein. Accordingly, in specific embodiments of the present invention, compositions are provided which comprise an SH3 domain-binding peptide, including a core sequence and flanking sequences, and a suitable carrier. [0067]
  • The compositions contemplated by the present invention may also include other components, from those that facilitate the introduction or administration of the compositions to those that have their own innate activity, such as a prophylactic, a diagnostic or a therapeutic action. Such innate activity may be distinct from that of the peptides of the present invention or be complementary thereto. In any event, the compositions of the present invention include those that are suitable for administration into mammals, including humans. Preferably, the compositions (including necessarily the carrier) of the present invention are sterile, though others may need only be cosmetically, agriculturally or pharmaceutically acceptable. Still other compositions may be adapted for veterinary use. [0068]
  • The compositions, including the drug delivery systems described herein, are contemplated to be administered in a variety of ways, such as parenterally, orally, enterally, topically or by inhalation. The compositions may also be adminstered intranasally, opthalmically or intravaginally. Furthermore, the compositions of the invention can take several forms, such as solids, gels, liquids, aerosols or patches. [0069]
  • In another embodiment of the present invention a method is provided of identifying a peptide having a region that binds to an SH3 domain comprising: (a) providing an immobilized target protein comprising an SH3 domain; (b) incubating the immobilized target protein with an aliquot taken from a phage-displayed random peptide library, which library includes peptides having a random sequence of ≧8 amino acid residues; (c) washing unbound phage from the immobilized target protein; (d) recovering the phage bound to the immobilized target protein; and (e) determining the relevant nucleotide sequence of said binding phage nucleic acid and deducing the primary sequence corresponding to the SH3 domain-binding peptide. Preferably, the method further comprises amplifying the titer of the recovered phage and repeating the steps of incubation, washing and recovery to provide SH3 domain-binding peptide-enriched phage. [0070]
  • Any other mode by which the peptide library, random or otherwise, can be “displayed” can be utilized in the present invention, however. Moreover, the present applicants believe that longer random peptide sequences (e.g., >6 amino acid residues, preferably >10, and most preferably, >12) provide not only much greater diversity but also a richer degree of secondary structure conducive to binding activity. If the random region of the peptide is less than or equal to an 8-mer, it should preferably not be cyclized. [0071]
  • 5.2. Preparation of Random Peptide Libraries [0072]
  • The preparation and characterization of the preferred phage-displayed random peptide libraries have been described elsewhere. See, for example, Kay, B. K. et al. in [0073] Gene (1992) 128:59-65, for a description of the preparation of the phage-displayed random peptide library known as TSAR-9, more below. In particular, by cloning degenerate oligonucleotides of fixed length into bacteriophage vectors, recombinant libraries of random peptides can be generated which are expressed at the amino-terminus of the pIII protein on the surface of M13 viral particles. (There are 3-5 copies of the pIII-fusion on the surface of each particle.) Phage display offers several conveniences: first, the expressed peptides are on the surface of the viral particles and accessible for interactions; second, the recombinant viral particles are stable (i.e., can be frozen, exposed to pH extremes); third, the viruses can be amplified; and fourth, each viral particle contains the DNA encoding the recombinant genome. Consequently, these libraries can be screened by isolating viral particles that bind to targets. The isolates can be grown up overnight, and the displayed peptide sequence responsible for binding can be deduced by DNA sequencing.
  • These libraries have approximately >108 different recombinants, and nucleotide sequencing of the inserts suggests that the expressed peptides are indeed random in amino acid sequence. These libraries are referred to herein as TSAR libraries, where TSAR stands for Totally Synthetic Affinity Reagents. The preparation of the TSAR libraries are described further below. [0074]
  • 5.3. SH3 Binding Clones And Their Characteristics [0075]
  • Accordingly, peptides have been isolated from an unconstrained random peptide library which exhibit a binding affinity for SH3 domains. Furthermore, the binding affinities exhibited by the disclosed peptides differ in their selectivities with certain peptides showing comparable binding affinities for SH3 domains derived from different proteins, while others manifest greater affinities for specific SH3 domains. [0076]
  • The amino acid sequence of various peptides isolated by the present method are listed in FIG. 5. As can be seen from this list, certain groups of SH3 domain binding peptides are isolated from three separate random peptide libraries, each based on a different type of random peptide insert, all displayed at the amino-terminus of the pIII protein on the surface of M13 viral particles. Ten clones were isolated from the R8C library, seven from the TSAR-12 library, and seven from the TSAR-9 library. The sequences are presented to highlight the particular amino acid residues believed to bind directly to the SH3 domain, as well as to point out the remaining amino acid resiudes of the random insert and the viral flanking sequences and complementary site amino acid residues common to each group of clones. The frequency with which each particular clone is found in each library is also indicated in FIG. 5. Thus, clones T12.SRC3.1 and T12.SRC3.2 are by far the most abundant clones found among the three libraries. [0077]
  • Interestingly, all the binding peptides are found to have the proline-rich amino acid residue motif, which is apparently responsible for binding, the motif being located predominantly at the C-terminal end of the insert, although each clone also contains an insert at the N-terminal end. The significance of this observation is not presently understood, although this finding may indicate the possible importance of the C-terminal viral flanking sequences in SH3 domain binding. [0078]
  • Indeed, a synthetic peptide bearing only the core consensus sequence RPLPPLP (SEQ ID NO:9) was less effective in binding to target SH3 domains than synthetic peptides that also included additional amino acid residues flanking the core sequences. Thus, 13-mers and 14-mers having the sequences RSTPRPLPMLPTTR (SEQ ID NO:62), RSTPRPLPPLPTTR (SEQ ID NO:67), GILAPPVPPRNTR (SEQ ID NO:63), GPHRRLPPTPATR (SEQ ID NO:65), and VLKRPLPIPPVTR (SEQ ID NO:64) have been prepared and shown to bind to SH3 domains, such as those of Src and Yes, much more avidly than the 7-mer, RPLPPLP (SEQ ID NO:9). The 13-mer ANPSPATRPLPTR (SEQ ID NO:66) has been shown to have binding affinities comparable to the core consensus sequence. In each case, the 13-mers comprise a 7-mer “core” sequence plus additional amino acid residues flanking same, some of which additional amino acid residues are contributed by the viral flanking sequences. [0079]
  • Thus, in one embodiment of the present invention, a 7-mer core includes a consensus motif of the formula RXLPφφP (SEQ ID NO:71), wherein R is arginine, L is leucine, P is proline, X represents any amino acid except cysteine and φ represents a hydrophobic amino acid residue. By “hydrophobic amino acid residue,” the applicants mean to include F, Y, W, V, A, I, L, P or M, each letter representing the standard one-letter designation for the corresponding amino acid residue. [0080]
  • Furthermore, a preferred 9-mer peptide comprising two additional amino acids on the C-terminal end of the core sequence is envisioned having a consensus motif of the formula RXLPφφPXψ (SEQ ID NO:10). In this preferred 9-mer consensus motif, the symbol ψ represents a hydrophilic amino acid residue, except cysteine. By “hydrophilic amino acid residue,” the applicants mean to include K, R, H, D, E, N, Q, T, S or C, and the other symbols are as defined above. For the purposes of the present invention, a glycine residue (G) may be considered either a hydrophobic or a hydrophilic amino acid residue. The one-letter symbols B and Z, which stand for N or D and Q or E, respectively, are considered hydrophilic amino acid residues. [0081]
  • Particular 13-mer peptides of the present invention include those listed, below. It is noted, however, that not all the following 13-mer peptides correlate strictly to or comply with the preferred 9-mer consensus motif, described above. Those peptides that do not comply (indicated in italics, with the non-complying amino acid residues underscored) can, thus, be described as “resembling” those that do comply (indicated in normal type) with the preferred 9-mer consensus motif: PGFRELPPLPPSR (SEQ ID NO:72), [0082]
  • AQSRPLPIPPETR (SEQ ID NO:73), VLKRPLPIPPVTR (SEQ ID NO:64), [0083]
  • PPN[0084] SPLPPLPTHL (SEQ ID NO:74), TGRGPLPPLPNDS (SEQ ID NO:75),
  • YSTRP[0085] VPPITRPS (SEQ ID NO:76), SHKSRLPPLPTRP (SEQ ID NO:77),
  • YRFRALP[0086] SPPSAS (SEQ ID NO:78), GPHRRLPPTPATR (SEQ ID NO:65),
  • LAQRQLPP[0087] TPGRD (SEQ ID NO:79), ALQRRLPRTPPPA (SEQ ID NO:80),
  • PATRPLP[0088] TRPSRT (SEQ ID NO:81), YSTRPLPSRPSRT (SEQ ID NO:82),
  • XPGRIL[0089] LLPSEPR (SEQ ID NO:83), SGGILAPPVPPRN (SEQ ID NO:84),
  • RSTRPLPILPRTT (SEQ ID NO:85), STPRPLPMLPTTR (SEQ ID NO:86), [0090]
  • STNRPLPMIPTTR (SEQ ID NO:87), RSTRPLP[0091] SLPITT (SEQ ID NO:88),
  • STSRPLP[0092] SLPTTR (SEQ ID NO:89), RSTRSLPPLPPTT (SEQ ID NO:90),
  • RSTRQLPIPPTTT (SEQ ID NO:91), STPRPLPLIPTTP (SEQ ID NO:92), [0093]
  • RSTRPLPP[0094] TPLTT (SEQ ID NO:93), and RSTRPQPPPPITT (SEQ ID NO:94). Accordingly, other peptides not specifically disclosed, which either comply with or “resemble” the preferred 9-mer consensus motif, can be readily envisioned by those of ordinary skill in the art and are considered to be equivalent to those that are specifically disclosed above. In particular, non-compliance at positions 1 (S, G, and I, in place of R, are tolerated), 3 (V, A, and Q, in place of L, are tolerated), 4 (L, in place of P, is tolerated), 5 (hydrophilic amino acid residues, S, R, and T, are tolerated in place of a hydrophobic amino acid residue), 6 (hydrophilic amino acid residues, R and T, are tolerated in place of a hydrophobic amino acid residue), 7 (T, and S, in place of P, are tolerated), and 9 (P and A are tolerated in place of a hydrophilic amino acid residue) have been observed.
  • 5.3.1. Binding specificities [0095]
  • It has been discovered that certain of the binding peptides disclosed have a greater relative binding affinity for one SH3 domain over another. Referring now to FIG. 8, the relative binding affinities of the various peptides described above toward different SH3 domain targets are graphically presented. As one can see, the relative binding affinities of the respective peptides can differ by orders of magnitude. Thus, as shown in FIG. 8, the peptide GPHRRLPPTPATR (SEQ ID NO:65), having the relevant sequence of the phage clone identified as T12.SRC3.3, is specific to Src family SH3 domains, including, but not limited to, Src, Yes, Lck, Hck, Fgr, Fyn, and Lyn. This SH3 binding peptide has little affinity for SH3 domains derived from PLCγ or Grb2. On the other hand, the peptide GILAPPVPPRNTR (SEQ ID NO:63), corresponding to the relevant sequence of the phage clone T12.SRC3.1, which is one of the most abundant binding clones found by the present method, binds generically to a broad range of SH3 domains, including Src, PLCγ, and Grb2. [0096]
  • On an intermediate level, the present invention has also uncovered a peptide, VLKRPLPIPPVTR (SEQ ID NO:64), corresponding to the relevant sequence of the phage clone T12.SRC3.6, which is Src preferential; that is, this peptide exhibits strong binding affinities for members of the Src family, some binding affinities for Grb2 proteins, but little binding affinities for PLCγ domains. The peptide ANPSPATRPLPTR (SEQ ID NO:66), corresponding to the relevant sequence of the phage clone T12.SRC3.2, also exhibits Src family specificity similar to GPHRRLPPTPATR (SEQ ID NO:65). The peptides RSTPRPLPMLPTTR (R8C.YES3.5; SEQ ID NO:62) and RSTPRPLPPLPTTR (representative consensus motif; SEQ ID NO:67) are highly specific for SH3 domain of Src, Yes, and other Src-related proteins. [0097]
  • 5.4. Further Discussion of Binding Experiments [0098]
  • At the outset it is apparent that the binding affinity of certain peptides to the SH3 domain of Src and Src-related proteins is governed by more than just the presence of the preferred core consensus sequences, RPLPPLP (SEQ ID NO:9) or RPLPMLP (SEQ ID NO:95; i.e., RPLP(P/M)LP, SEQ ID NO:96). Thus, while the synthetic peptides RSTPRPLPMLPTTR (R8C.YES3.5; SEQ ID NO:62) and RSTPRPLPPLPTTR (consensus; (SEQ ID NO:67) exhibit a strong specific binding affinity for Src SH3, the other synthetic peptides tested also exhibited an avid binding affinity to SH3 domains relative to the 7-mer, RPLPPLP (SEQ ID NO:9). These other peptides, GILAPPVPPRNTR (SEQ ID NO:63), VLKRPLPIPPVTR (SEQ ID NO:64), GPHRRLPPTPATR (SEQ ID NO:65), and ANPSPATRPLPTR (SEQ ID NO:66), sport core sequences and flanking sequences that do not closely adhere to the preferred core consensus sequences. Thus, these results suggest that binding affinity to SH3 domains is governed to a large extent by the nature of the amino acid residues flanking the core 7-mer sequence. [0099]
  • The binding characteristics of Src SH3-selected peptides was determined using synthetic biotinylated peptides corresponding to the sequences displayed by Src SH3-selected phage. These biotinylated peptides were assayed for direct binding to immobilized Src SH3-GST. Each of the five library-derived peptides tested were found to bind to Src SH3-GST and Yes SH3-GST over background (FIG. 8). Furthermore, a strong correlation was observed between the similarity of a given peptide to the preferred core consensus sequence RPLP(P/M)LP (SEQ ID NO:96) and the peptidets affinity for Src SH3-GST. The core sequence of the clone T12.SRC3.1 (GILAPPVPPRNTR; SEQ ID NO:63) appears to provide more generic SH3 domain-binding characteristics. [0100]
  • Experiments comparing the relative binding of various phage clones to SH3 domains taken from a variety of proteins demonstrated the preference of these clones for Src and Src-related SH3 domains over SH3 domains taken from other proteins. [0101]
  • It was further found that while the 7-mer having the consensus sequence RPLPPLP (SEQ ID NO:9) bound to Src SH3-GST only weakly, peptides comprising the consensus sequence flanked by residues encoded by one of the Src SH3-selected clones (R8C.YES3.5), RSTP (SEQ ID NO:97) at the N-terminal end and TTR at the C-terminal end, bound significantly better than any of the peptides tested (FIG. 7). Thus, as stated previously, sequences that flank the RPLP(P/M)LP (SEQ ID NO:96) core appear to be important contributors to SH3 binding. It is further surmised that a peptide having or resembling the sequence RSTPAPPVPPRTTR (SEQ ID NO:98) should exhibit strong but generic binding to a variety of SH3 domains. [0102]
  • Similarly, it is observed that most of the Src SH3-binding motifs are located near the carboxy-terminus of the random peptides, adjacent to sequences which are fixed in every clone (FIG. 5). The exceptional clones tend to possess sequences that resemble motifs that include fixed flanking sequences. This clustering contrasts with previous results, in which binding motifs are distributed throughout the random peptide. Kay, B. K., et al., in [0103] Gene (1993) 128:59-65.
  • The binding of the library-derived Src SH3-binding peptides was compared to that of peptides corresponding to proline-rich regions of natural proteins. Peptides corresponding to SH3-binding regions in human PI-3′ Kinase (KISPPTPKPRPPRPLPV; SEQ ID NO:69) and human SOS1.20 (GTVEPVPPPVPPRRRPESA; SEQ ID NO:68), as well as a proline-rich region of the cytoskeletal protein vinculin (LAPPKPPLPEGEV; SEQ ID NO:70), bound Src SH3 much less well than the library-derived peptides (FIG. 7). [0104]
  • As mentioned above, the relative specificity of binding was explored. Thus, the relative binding of Src SH3-selected peptides to equal amounts of GST fusions to SH3 domains from different proteins was determined (FIG. 8). While all of the library-derived peptides bound the Src and Yes SH3 domains almost equally well, none of the peptides (with the exception of peptide T12.SRC3.1, the most divergent peptide tested) bound the SH3 domains of Grb2, Crk, Abl or PLCγ1 appreciably. Thus, the library-derived peptides, in contrast with a peptide derived from SOS1, exhibit SH3 binding that is relatively specific for Src-family members. [0105]
  • Next, it was determined whether the binding to the Src SH3 domain was qualitatively like the interactions of the SH3 domain and natural proteins found in cell lysates. Thus, radiolabeled proteins were prepared from NIH 3T3 cell lysates and chromatographed over Src SH3-GST immobilized on glutathione linked Sepharose. SDS-PAGE shows that a number of proteins can be affinity purified in this manner. The synthesized peptides bind quite well to the Src SH3 domain, as they can compete the binding of radiolabeled proteins from cell lysates to immobilized Src-GST, with an IC[0106] 50 of 1-10 mM (FIG. 9). In conclusion, the peptides can efficiently block the interaction of cellular proteins with Src SH3 in vitro.
  • Moreover, [0107] Xenopus laevis oocytes injected with mRNA encoding constitutively active Src undergo progesterone-induced maturation at an accelerated rate relative to oocytes injected with water or c-Src MRNA. Unger, T. F. and Steele, R. E. in Mol. Cell. Biol. (1992) 12:5485-5498. To explore the ability of the library-derived Src SH3-binding peptides to exert a biochemical effect in vivo, the influence of the peptides on the maturation of Xenopus laevis oocytes was examined. Hence, stage VI oocytes were injected with peptide, exposed to progesterone, and scored for germinal vesicle breakdown. FIG. 10 shows that the rate of maturation was accelerated by approximately one hour when oocytes were injected with the SH3-binding peptide consisting of RPLPPLP (SEQ ID NO:9) flanked by residues from clone T12.SRC3.6 (VLKRPLPIPPVTR; SEQ ID NO:64), but not with water or a peptide corresponding to a proline-rich segment of vinculin (LAPPKPPLPEGEV; SEQ ID NO:70) as controls. The magnitude of this effect is roughly equivalent to that seen with injection of mRNA encoding constituitively active Src. See, e.g., FIG. 3B in Unger, T. F. and Steele, R. E., supra. This result suggests that the library-derived Src SH3-binding peptide is effectively relieving an inhibitory effect of the Src SH3 domain upon Src PTK activity. This model is consistent with a number of studies which have demonstrated an inhibitory effect of the Src SH3 domain upon Src kinase and transforming activity. See, e.g., Okada, M., et al., supra; Murphy, S. M., et al., supra; and Superti-Furga, G., et al., supra.
  • 5.5. Diagnostic And Therapeutic Agents Based on SH3 Binding Peptides and Additional Methods of Their Use [0108]
  • As already indicated above, the present invention also seeks to provide diagnostic, prophylactic, and therapeutic agents based on the SH3 binding peptides described herein. [0109]
  • In one embodiment, diagnostic agents are provided, preferably in the form of kits, comprising an SH3 domain-binding peptide and a detectable label conjugated to said peptide directly, indirectly or by complexation, said peptide comprising: (i) a core sequence motif of the formula RXLPφφP (SEQ ID NO:71), wherein X represents any amino acid except cysteine and φ represents a hydrophobic amino acid residue, including F, Y, W, V, A, I, L, P, M or G, each letter representing the standard one-letter designation for the corresponding amino acid residue; and (ii) two or more additional amino acid residues flanking said core sequence at its C-terminal end, N-terminal end or both. [0110]
  • The diagnostic agents of the present invention can be used to detect the presence of SH3 domains of a generic or specific type in cells, tissues or organs either in vitro or in vivo. For in vivo applications, the diagnostic agent is preferably mixed with a pharmaceutically acceptable carrier for administration, either enterally, parenterally or by some other route dictated by the needs of the particular application. [0111]
  • In a particular embodiment, for example, an assay based on a fusion product is contemplated which comprises a Src SH3 domain-binding peptide of the invention and a substrate for deregulated or “activated” Src. For instance, a muscle biopsy, taken from a subject suspected of being infected by the Rous sarcoma virus, can be treated with an effective amount of the fusion product. By subsequent analysis of the degree of conversion of the substrate, one can potentially detect infection by the Rous sarcoma virus in the subject, particularly mammals, especially chickens. The presence of the retrovirus, which causes the expression of deregulated or “activated” Src, may thus be indicated by unusually high levels of Src as revealed by large amounts of the converted substrate. See, for example, Paxton, W. G. et al., in [0112] Biochem. Biophys. Res. Commun. (1994) 200(1):260-267 (detection of phosphorylated tyrosine and serine residues of angiotensin II AT1 receptor, a substrate of Src family tyrosine kinases); another suitable substrate may be the protein p68 (Fumagalli, S. et al., in Nature (1994) 368(6474):871-874; Taylor, S. J. and Shalloway, D., in Ibid. at 867-871.
  • Alternatively, the enzyme can be isolated by selective binding to a form of the SH3 domain-binding peptides of the present invention (e.g., biotin-peptide conjugate). After isolation of the protein-peptide conjugate complex (e.g., on a column comprising streptavidin), the activity of the enzyme can then be assayed by conventional methods to determine its level of protein kinase activity which can be taken as an indication of the presence of the deregulated or “activated” form of the enzyme. An assay for Src kinase has been described by Klinz and Maness, in [0113] Neuroprotocols (a companion to Neuroscience) (1992) 1(3):224-231.
  • Moreover, the diagnostic agents of the invention can also serve as imaging agents of cells, tissues or organs, especially those that contain proteins with an SH3 domain. For example, neural cells (e.g., neurons, other areas of the brain), osteoclasts, osteoblasts, platelets, immune cells, and other dividing cells are known to express or contain proteins with SH3 domains. Thus, an image can be taken of portions of the body to serve as a baseline for subsequent images to detect physiologic or biochemical changes in the subject's body. For instance, changes in the condition of cellular levels of Src or a transformation of the cellular Src to an “activated” form may be detected using the diagnostic or imaging agents of the present invention. [0114]
  • Accordingly, it has been demonstrated that an SH3-binding peptide tagged with a fluorescence emitter can provide an image of the cytoskeleton. The images are presented in FIG. 11. As can be seen from FIG. 11, panels A, B, and C show the fluorescence image that is obtained on treating NIH 3T3 fibroblasts with SH3 domain-binding peptides modified to include a fluorescent tag. In sharp contrast, panel D shows only a dark image that is produced when the cells are treated with a proline-rich segment of vinculin as a control. [0115]
  • In another embodiment, an SH3 domain-binding peptide-horseradish immunoperoxidase complex or related immunohistochemical agent could be used to detect and quantitate specific receptor molecules in tissues, serum or body fluids. In particular, the present invention provides useful diagnostic reagents for use in immunoassays, Southern or Northern hybridization, and in situ assays. Accordingly, the diagnostic agents described herein may be suitable for use in vitro or in vivo. [0116]
  • In addition, the diagnostic or imaging agent of the present invention is not limited by the nature of the detectable label. Hence, the diagnostic agent may contain one or more such labels including, but not limited to, radioisotope, fluorescent tags, paramagnetic substances, heavy metals, or other image-enhancing agents. Those of ordinary skill in the art would be familiar with the range of label and methods to incorporate or conjugate them into the SH3 domain-binding peptide to form diagnostic agents. [0117]
  • In yet a further embodiment, pharmaceutical compositions are provided comprising an SH3 domain-binding peptide and a pharmaceutically acceptable carrier. In a specific embodiment of the invention, the pharmaceutical composition is useful for the modulation of the activity of SH3 domain-containing proteins. By “modulation” is meant either inhibition or enhancement of the activity of the protein target. Accordingly, a pharmaceutical composition is disclosed comprising an SH3 domaih-binding peptide and a pharmaceutically acceptable carrier, said peptide comprising: (i) a 9-mer sequence motif of the formula RXLPφφPXψ (SEQ ID NO:10), wherein X represents any amino acid except cysteine, φ represents a hydrophobic amino acid residue, and wherein ψ is a hydrophilic amino acid residue except cysteine, each letter representing the standard one-letter designation for the corresponding amino acid residue; and, optionally, (ii) additional amino acid residues flanking the 9-mer sequence at its C-terminal end, N-terminal end or both, up to a total of 45 amino acid residues, including said 9-mer sequence. Preferably, the peptide comprises at least one, more preferably at least two, and most preferably at least three additional amino acids flanking the 9-mer sequence. [0118]
  • As stated above, the therapeutic or diagnostic agents of the invention may also contain appropriate pharmaceutically acceptable carriers, diluents and adjuvants. Such pharmaceutical carriers can be sterile liquids, such as water and oils including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium carbonate, magnesium stearate, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. These compositions can take the form of solutions, suspensions, tablets, pills, capsules, powders, sustained-release formulations and the like. Suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin. [0119]
  • Such compositions will contain an effective therapeutic amount of the active compound together with a suitable amount of carrier so as to provide the form for proper administration to the subject. While intravenous injection is a very effective form of administration, other modes can be employed, including but not limited to intramuscular, intraperitoneal, and subcutaneous infection, and oral, nasal, enteral, and parenteral administration. [0120]
  • The therapeutic agents and diagnostic agents of the instant invention are used for the treatment and/or diagnosis of animals, and more preferably, mammals including humans, as well as dogs, cats, horses, cows, pigs, guinea pigs, mice and rats. Accordingly, other methods contemplated in the present invention, include, but are not limited to, a method of modulating, i.e., inhibiting or enhancing, bone resorption in a mammal (see, e.g., Hall, T. J., in [0121] Biochem. Biophys. Res. Commun. (1994) 199(3):1237-44), a method of disrupting protein tyrosine kinase-mediated signal transduction pathways or a method of regulating the processing, trafficking or translation of RNA in a cell by introducing or administering an effective amount of an SH3 domain-binding peptide of the present invention (see, e.g., Taylor, S. J. and Shalloway, D., supra).
  • The diagnostic or therapeutic agents of the present invention can be modified by attachment to soluble macromolecules such as proteins, polysaccharides, or synthetic polymers. For example, the peptide could be coupled to styrene-maleic acid copolymers (see, e.g., Matsumura and Maeda, [0122] Cancer Res. (1986) 46:6387), methacrylamide copolymers (Kopececk and Duncan, J. Controlled Release (1987) 6:315), or polyethylene glycol (PEG) (e.g., Hershfield and Buckley, N. Engl. J. Med. (1987) 316:589; Ho et al., Drug Metab. Discos. (1986) 14:349; Chua et al., Ann. Intern. Med. (1988) 109:114). The agents, if desired, are further targeted by attachment to an antibody, especially a monoclonal antibody. Such antibodies include but are not limited to chimeric, single chain, Fab fragments, and Fab expression libraries. In one embodiment the agent is coupled to the macromolecule via a degradable linkage so that it will be released in vivo in its active form.
  • In another embodiment, the therapeutic or diagnostic agent may be delivered in a vesicle, in particular a liposome. See, Langer, Science (1990) 249:1527-1533; Treat et al., in [0123] Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, N.Y. (1989) pp. 353-365; Lopez-Berestein, ibid., pp. 317-327.
  • In yet another embodiment, the therapeutic or in vivo diagnostic agent can be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton, [0124] CRC Crit. Ref. Biomed. Eng. (1987) 14:201; Buchwald et al., Surgery (1980) 88:507; Saudek et al., N. Engl. J. Med. (1989) 321:574). In another embodiment, polymeric materials may be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla., 1974; Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.) Wiley, N.Y. 1984; Raner and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. (1983) 23:61; see, also, Levy et al., Science (1985) 228:190; During et al., Ann. Neurol. (1989) 25:351; Howard et al., J. Neurosurg. (1989) 71:105). In a preferred embodiment, a controlled release system may be placed next to the therapeutic target, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, (1984) 2:115-138). It will be recognized by one of ordinary skill in the art that a particular advantage of the invention is that a peptide will not be subject to the problems of denaturation and aggregation associated with proteins held in the warm, most environment of a body in a controlled release system.
  • Other controlled release systems are discussed in the review by Langer, in Science (1990) 249:1527-1533. [0125]
  • 5.6 Identification of Compounds that Affect Binding of SH3 Domain-containing Proteins and their Ligands [0126]
  • A common problem in the development of new drugs is that of identifying a single, or a small number, of compounds that possess a desirable characteristic from among a background of a large number of compounds that lack that desired characteristic. This problem arises both in the testing of compounds that are natural products from plant, animal, or microbial sources and in the testing of man-made compounds. Typically, hundreds, or even thousands, of compounds are randomly screened by the use of in vitro assays such as those that monitor the compound's effect on some enzymatic activity or its ability to bind to a reference substance such as a receptor or other protein. [0127]
  • The compounds which pass this original screening test are known as “lead” compounds. These lead compounds are then put through further testing, including, eventually, in vivo testing in animals and humans, from which the promise shown by the lead compounds in the original in vitro tests is either confirmed or refuted. See [0128] Remington's Pharmaceutical Sciences, 1990, A. R. Gennaro, ed., Chapter 8, pages 60-62, Mack Publishing Co., Easton, PA; Ecker and Crooke, 1995, Bio/Technology 13:351-360.
  • There is, of course, a continual need for new compounds to be tested in the in vitro assays that make up the first testing step described above. There is also a continual need for new assays by which the pharmacological activities of these compounds may be tested. It is an object of the present invention to provide such new assays to determine whether a candidate compound is capable of affecting the binding between a protein or polypeptide containing an SH3 domain and a ligand of the SH3 domain. A compound capable of affecting this binding would be useful as a means of modulating the pharmacological activity of proteins or polypeptides containing the SH3 domain. The present invention provides suitable ligands for SH3 domains for use in such assays. Such assays can be performed where the SH3 domains include, but are not limited to, SH3 domains from Cortactin, Nck, Abl, PLCγ, Src, p53bp2, Crk, Yes, and Grb2. [0129]
  • The present invention provides methods of identifying a compound that affects the binding of a molecule comprising an SH3 domain and a ligand of the SH3 domain. The effect on binding can be an increase or decrease in total amount of binding or in affinity of bidning. Preferably, the effect is an inhibition (reduction in or loss of binding). [0130]
  • Accordingly, the invention provides a method of identifying an inhibitor of the binding between a first molecule comprising an SH3 domain and a second molecule that binds to the SH3 domain comprising incubating one or more compounds from which it is desired to select such an inhibitor with the first molecule and the second molecule under conditions conducive to binding and detecting the one or more compounds that inhibit binding of the first molecule to the second molecule. [0131]
  • In a particular embodiment of the above-described method, the second molecule is obtained by: [0132]
  • (i) screening a peptide library with the SH3 domain to obtain peptides that bind the SH3 domain; [0133]
  • (ii) determining a consensus sequence for the peptides obtained in step (i); [0134]
  • (iii) producing a peptide comprising the consensus sequence; [0135]
  • wherein the second molecule comprises the peptide comprising the consensus sequence. [0136]
  • In another embodiment, the second molecule is obtained by: [0137]
  • (i) screening a peptide library with the SH3 domain to obtain peptides that bind the SH3 domain; [0138]
  • (ii) determining a consensus sequence for the peptides obtained in step (i); [0139]
  • (iii) searching a database to identify amino acid sequences that resemble the consensus sequence of step (ii); [0140]
  • (iv) producing a peptide comprising an amino acid sequence identified in step (iii); [0141]
  • wherein the second molecule comprises the peptide comprising an amino acid sequence identified in step (iii). [0142]
  • Second molecules that bind SH3 domains can be obtained by, e.g., the use of diversity libraries, such as random or combinatorial peptide or nonpeptide libraries which can be screened for molecules that specifically bind to SH3 domains. Many libraries are known in the art that can be used, e.g., chemically synthesized libraries, recombinant (e.g., phase display libraries), and in vitro translation-based libraries. [0143]
  • Examples of chemically synthesized libraries are described in Fodor et al., 1991, Science 251:767-773; Houghten et al., 1991, Nature 354:84-86; Lam et al., 1991, Nature 354:82-84; Medynski, 1994, Bio/Technology 12:709-710; Gallop et al., 1994, J. Medicinal Chemistry 37(9):1233-1251; ohlmeyer et al., 1993, Proc. Natl. Acad. Sci. USA 90:10922-10926; Erb et al., 1994, Proc. Natl. Acad. Sci. USA 91:11422-11426; Houghten et al., 1992, Biotechniques 13:412; Jayawickreme et al., 1994, Proc. Natl. Acad. Sci. USA 91:1614-1618; Salmon et al., 1993, Proc. Natl. Acad. Sci. USA 90:11708-11712; PCT Publication No. WO 93/20242; and Brenner and Lerner, 1992, Proc. Natl. Acad. Sci. USA 89:5381-5383. [0144]
  • Examples of phage display libraries are described in Scott and Smith, 1990, Science 249:386-390; Devlin et al., 1990, Science, 249:404-406; Christian, R. B., et al., 1992, J. Mol. Biol. 227:711-718); Lenstra, 1992, J. Immunol. Meth. 152:149-157; Kay et al., 1993, Gene 128:59-65; and PCT Publication No. WO 94/18318 dated Aug. 18, 1994. [0145]
  • In vitro translation-based libraries include but are not limited to those described in PCT Publication No. WO 91/05058 dated Apr. 18, 1991; and Mattheakis et al., 1994, Proc. Natl. Acad. Sci. USA 91:9022-9026. [0146]
  • By way of examples of nonpeptide libraries, a benzodiazepine library (see e.g., Bunin et al., 1994, Proc. Natl. Acad. Sci. USA 91:4708-4712) can be adapted for use. Peptoid libraries (Simon et al., 1992, Proc. Natl. Acad. Sci. USA 89:9367-9371) can also be used. Another example of a library that can be used, in which the amide functionalities in peptides have been permethylated to generate a chemically transformed combinatorial library, is described by Ostresh et al. (1994, Proc. Natl. Acad. Sci. USA 91:11138-11142). [0147]
  • Screening the libraries can be accomplished by any of a variety of commonly known methods. See, e.g., the following references, which disclose screening of peptide libraries: Parmley and Smith, 1989, Adv. Exp. Med. Biol. 251:215-218; Scott and Smith, 1990, Science 249:386-390; Fowlkes et al., 1992; BioTechniques 13:422-427; Oldenburg et al., 1992, Proc. Natl. Acad. Sci. USA 89:5393-5397; Yu et al., 1994, Cell 76:933-945; Staudt et al., 1988, Science 241:577-580; Bock et al., 1992, Nature 355:564-566; Tuerk et al., 1992, Proc. Natl. Acad. Sci. USA 89:6988-6992; Ellington et al., 1992, Nature 355:850-852; U.S. Pat. No. 5,096,815, U.S. Pat. No. 5,223,409, and U.S. Pat. No. 5,198,346, all to Ladner et al.; Rebar and Pabo, 1993, Science 263:671-673; and PCT Publication No. WO 94/18318. [0148]
  • In a specific embodiment, screening can be carried out by contacting the library members with an SH3 domain immobilized on a solid phase and harvesting those library members that bind to the SH3 domain. Examples of such screening methods, termed “panning” techniques are described by way of example in Parmley and Smith, 1988, Gene 73:305-318; Fowlkes et al., 1992, BioTechniques 13:422-427; PCT Publication No. WO 94/18318; and in references cited hereinabove. [0149]
  • In another embodiment, the two-hybrid system for selecting interacting proteins in yeast (Fields and Song, 1989, Nature 340:245-246; Chien et al., 1991, Proc. Natl. Acad. Sci. USA 88:9578-9582) can be used to identify molecules that specifically bind to SH3 domains. [0150]
  • A typical assay of the present invention consists of at least the following components: (1) a molecule (e.g., protein or polypeptide) comprising an SH3 domain; (2) a ligand of the SH3 domain; (3) a candidate compound, suspected of having the capacity to affect the binding between the protein containing the SH3 domain and the ligand. The assay components may further comprise (4) a means of detecting the binding of the protein comprising the SH3 domain and the ligand. Such means can be e.g., a detectable label affixed to the protein, the ligand, or the candidate compound. [0151]
  • In another specific embodiment, the invention provides a method of identifying a compound that affects the binding of a molecule comprising an SH3 domain and a ligand of the SH3 domain comprising: [0152]
  • (a) contacting the SH3 domain and the ligand under conditions conducive to binding in the presence of a candidate compound and measuring the amount of binding between the SH3 domain and the ligand; [0153]
  • (b) comparing the amount of binding in step (a) with the amount of binding known or determined to occur between the molecule and the ligand in the absence of the candidate compound, where a difference in the amount of binding between step (a) and the amount of binding known or determined to occur between the molecule and the ligand in the absence of the candidate compound indicates that the candidate compound is a compound that affects the binding of the molecule comprising an SH3 domain and the ligand. [0154]
  • A kit is provided that comprises, in one or more containers, one or more components of the assay of the invention, e.g., a first molecule comprising an SH3 domain and a second molecule that binds to the SH3 domain. [0155]
  • In one embodiment, the assay comprises allowing the protein or polypeptide containing an SH3 domain to contact the ligand of the SH3 domain in the presence and in the absence of the candidate compound under conditions such that binding of the ligand to the protein containing an SH3 domain will occur unless that binding is disrupted or prevented by the candidate compound. By detecting the amount of binding of the ligand to the protein containing an SH3 domain in the presence of the candidate compound and comparing that amount of binding to the amount of binding of the ligand to the protein or polypeptide containing an SH3 domain in the absence of the candidate compound, it is possible to determine whether the candidate compound affects the binding and thus is a useful lead compound for the modulation of the activity of proteins containing the SH3 domain. The effect of the candidate compound may be to either increase or decrease the binding. [0156]
  • One version of an assay suitable for use in the present invention comprises binding the protein containing an SH3 domain to a solid support such as the wells of a microtiter plate. The wells contain a suitable buffer and other substances to ensure that conditions in the wells permit the binding of the protein or polypeptide containing an SH3 domain to its ligand. The ligand and a candidate compound are then added to the wells. The ligand is preferably labeled, e.g., it might be biotinylated or labeled with a radioactive moiety, or it might be linked to an enzyme, e.g., alkaline phosphatase. After a suitable period of incubation, the wells are washed to remove any unbound ligand and compound. If the candidate compound does not interfere with the binding of the protein or polypeptide containing an SH3 domain to the labeled ligand, the labeled ligand will bind to the protein or polypeptide containing an SH3 domain in the well. This binding can then be detected. If the candidate compound interferes with the binding of the protein or polypeptide containing an SH3 domain and the labeled ligand, label will not be present in the wells, or will be present to a lesser degree than is the case when compared to control wells that contain the protein or polypeptide containing an SH3 domain and the labeled ligand but to which no candidate compound is added. Of course, it is possible that the presence of the candidate compound will increase the binding between the protein or polypeptide containing an SH3 domain and the labeled ligand. Alternatively, the ligand can be affixed to solid substrate during the assay. [0157]
  • The present invention provides ligands capable of binding SH3 domains that are suitable for incorporation into assays such as those described above. Ligands provided by the present invention include those SH3 domain-binding amino acid sequences disclosed in Tables 1-13 below and proteins or polypeptides containing those amino acid sequences. Also provided are nucleic acids encoding the SH3 domain-binding amino acid sequences disclosed in Tables 1-13 below. [0158]
    • 6. EXAMPLES
  • 6.1. Preparation of the TSAR-9 Library [0159]
  • 6.1.1. synthesis and Assembly of Oligonucleotides [0160]
  • FIG. 1 shows the formula of the oligonucleotides and the assembly scheme used in construction of the TSAR-9 library. The oligonucleotides were synthesized with an applied Biosystems 380a synthesizer (Foster City, Calif.), and the full-length oligonucleotides were purified by HPLC. [0161]
  • Five micrograms of each of the pair of oligonucleotides were mixed together in buffer (10 mM Tris-HCl, pH 8.3, 15 mM KCl, 0.001% gelatin, 1.5 mM magnesium chloride), with 0.1% Triton X-100, 2 mM dNTP's, and 20 units of Tag DNA polymerase. The assembly reaction mixtures were incubated at 72° C. for 30 seconds and then 30° C. for 30 seconds; this cycle was repeated 60 times. It should be noted that the assembly reaction is not PCR, since a denaturation step was not used. Fill-in reactions were carried out in a thermal cycling, device (Ericomp, LaJolla, Calif.) with the following protocol: 30 seconds at 72° C., 30 seconds at 30° C., repeated for 60 cycles. The lower temperature allows for annealing of the six base complementary region between the two sets of the oligonucleotide pairs. The reaction products were phenol/chloroform extracted and ethanol precipitated. Greater than 90% of the nucleotides were found to have been converted to double stranded synthetic oligonucleotides. [0162]
  • After resuspension in 300 AL of buffer containing 10 mM Tris-HCI, pH 7.5, 1 mM EDTA (TE buffer), the ends of the oligonucleotide fragments were cleaved with Xba I and Xho I (New England BioLabs, Beverly, Mass.) according to the supplier's recommendations. The fragments were purified by 4% agarose gel electrophoresis. The band of correct size was removed and electroeluted, concentrated by ethanol precipitation and resuspended in 100 μL TE buffer. Approximately 5% of the assembled oligonucleotides can be expected to have internal Xho I or Xba I sites; however, only the full-length molecules were used in the ligation step of the assembly scheme. The concentration of the synthetic oligonucleotide fragments was estimated by comparing the intensity on an ethidium bromide stained gel run along with appropriate quantitated markers. All DNA manipulations not described in detail were performed according to Maniatis, supra. [0163]
  • To demonstrate that the assembled enzyme digested oligonucleotides could be ligated, the synthesized DNA fragments were examined for their ability to self-ligate. The digested fragments were incubated overnight at 18° C. in ligation buffer with T4 DNA ligase. When the ligation products were examined by agarose gel electrophoresis, a concatamer of bands was visible upon ethidium bromide staining. As many as five different unit length concatamer bands (i.e., dimer, trimer, tetramer, pentamer, hexamer) were evident, suggesting that the synthesized DNA fragments were efficient substrates for ligation. [0164]
  • 6.1.2. Construction of Vectors [0165]
  • The construction of the M13 derived phage vectors useful for expressing a TSAR library has been recently described (Fowlkes, D. et al. BioTech. (1992) 13:422-427). To express the TSAR-9 library, an M13 derived vector, m663, was constructed as described in Fowlkes. The m663 vector contains the pIII gene having a c-myc-epitope, i.e., as a stuffer fragment, introduced at the mature N-terminal end, flanked by Xho I and Xba I restriction sites (see also, FIG. I of Fowlkes). [0166]
  • 6.1.3. Expression of the TSAR-9 Library [0167]
  • The synthesized oligonucleotides were then ligated to Xho I and Xba I double-digested m663 RF DNA containing, the pIII gene (Fowlkes) by incubation with ligase overnight at 12° C. More particularly, 50 ng of vector DNA and 5 ng of the digested synthesized DNA and was mixed together in 50 μL ligation buffer (50 mM Tris, pH 8.0, 10 mM MgCl[0168] 2, 20 mM DTT, 0.1 mM ATP) with T4 DNA ligase. After overnight ligation at 12° C., the DNA was concentrated by ethanol precipitation and washed with 70% ethanol. The ligated DNA was then introduced into E. coli (DH5αF′; GIBCO BRL, Gaithersburg, Md.) by electroporation.
  • A small aliquot of the electroporated cells was plated and the number of plaques counted to determine that 10[0169] 8 recombinants were generated. The library of E. coli cells containing recombinant vectors was plated at a high density (˜400,000 per 150 mM petri plate) for a single amplification of the recombinant phage. After 8 hr, the recombinant bacteriophage were recovered by washing each plate for 18 hr with SMG buffer (100 mM NaCl, 10 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 0.05% gelatin) and after the addition of glycerol to 50% were frozen at −80° C. The TSAR-9 library thus formed had a working titer of −2×1011 pfu/ml.
  • 6.2. Preparation of the TSAR-12 Library [0170]
  • FIG. 2 shows the formula for the synthetic oligonucleotides and the assembly scheme used in the construction of the TSAR-12 library. As shown in FIG. 2, the TSAR-12 library was prepared substantially the same as the TSAR-9 library described in Section 6.1 above with the following exceptions: (1) each of the variant non-predicted oligonucleotide sequences, i.e., NNB, was 30 nucleotides in length, rather than 54 nucleotides; (2) the restriction sites included at the 5′ termini of the variant, non-predicted sequences were Sal I and [0171] Spe 1, rather than Xho I and Xba I; and (3) the invariant sequence at the 3′ termini to aid annealing of the two strands was GCGGTG and CGCCAC rather than CCAGGT and GGTCCA (5′ to 3′).
  • After synthesis including numerous rounds of annealing and chain extension in the presence of dNTP's and Taq DNA polymerase, and purification as described above in Section 6.1.1, the synthetic double stranded, oligonucleotide fragments were digested with Sal I and Spe I restriction enzymes and ligated with T4 DNA ligase to the nucleotide sequence encoding the M13 pIII gene contained in the m663 vector to yield a library of TSAR-expression vectors as escribed in Sections 6.1.2 and 6.1.3. The ligated DNA was hen introduced into [0172] E. coli (DH5αF′; GIBCO BRL, aithersburg, MD by electroporation. The library of E. coli cells were plated at high density (˜400,000 per 150 mm petri late) for amplification of the recombinant phage. After bout 8 hr, the recombinant bacteriophage were recovered by ashing, for 18 hr with SMG buffer and after the addition of lycerol to 50% were frozen at −80° C.
  • The TSAR-12 library thus formed had a working titer of ˜2×10[0173] 11 pfu/mL.
  • 6.3. Characterization of the TSAR-9 and -12 Libraries [0174]
  • The inserted synthetic oligonucleotides for each of the TSAR libraries, described in Sections 6.1 and 6.2 above, ad a potential coding complexity of 20[0175] 36 (˜1047) and 2020, respectively, and since ˜1014 molecules were used in each transformation experiment, each member of these TSAR libraries should be unique. After plate amplification the library solution or stock has 104 copies of each member/mL.
  • It was observed that very few (<10%) of the inserted oligonucleotide sequences characterized so far in both of the libraries have exhibited deletions or insertions. This is likely a reflection of the accuracy assembling the oligonucleotides under the conditions used and the fact that certain types of mutations (i.e., frame-shifts) would not be tolerated as pIII an essential protein for phage propagation. [0176]
  • In order to determine whether any coding bias existed in the variant non-predicted peptides expressed by these libraries, perhaps due to biases imposed in vitro during synthesis of the oligonucleotides or in vivo during expression by the reproducing phage, inserts were sequenced as set forth below. [0177]
  • 6.3.1. Characterization of TSAR-9 Library [0178]
  • Inserted synthetic oligonucleotide fragments of 23 randomly chosen isolates were examined from the TSAR-9 library. Individual plaques were used to inoculate I ml of 2XYT broth containing [0179] E. coli (DH5αF′) cells and the cultures were allowed to grow overnight at 37° C. with aeration. DNA was isolated from the culture supernatants according to Maniatis, supra. Twenty-three individual isolates were sequenced according to the method of Sanger (Proc. Natl. Acad. Sci. USA (1979) 74:5463-5467) using as a primer the oligonucleotide 5′-AGCGTAACGATCTCCCG (SEQ ID NO. 99), which is 89 nucleotides downstream of the pIII gene cloning site of the m663 vector used to express the TSARS.
  • Nucleotide sequences and their encoded amino acid sequences were analyzed with the MacVector computer program (IBI, New Haven, Conn.). The Microsoft EXCEL program was used to evaluate amino acid frequencies. Such analyses showed that the nucleotide codons coding for and hence most amino acids, occurred at the expected frequency in the TSAR-9 library of expressed proteins. The notable exceptions were glutamine and tryptophan, which were over- and under-represented, respectively. [0180]
  • It is of interest to note the paucity of TAG stop codons in the inserts, i.e., only 2 of ˜200 isolates characterized contained a TAG stop codon. About half [1-(47/48)[0181] 36] of the phage inserts were expected to have at least one TAG codon in view of the assembly scheme used. However, most of the TAG-bearing phage appear to have been lost from the library, even though the bacterial host was supE. This may be a consequence of suppression being less than 100% effective.
  • The amino acids encoded by the inserted double stranded synthesized oligonucleotide sequences, excluding the fixed PG-encoding centers, were concatenated into a single sequence and the usage frequency determined for each amino acid using the Microsoft EXCEL program. These frequencies were compared to that expected from the assembly scheme of the oligonucleotides, and the divergence from expected values represented by the size of the bars above and below the baseline. Chi square analysis was used to determine the significance of the deviations. The majority of amino acids were found to occur at the expected frequency, with the notable exceptions that glutamine and tryptophan were somewhat over- and under-represented, respectively. Thus, except for the invariant Pro-Gly, any position could have any amino acid; hence, the sequences are unpredicted or random. [0182]
  • 6.3.2. Characterization of TSAR-12 Library [0183]
  • Approximately 10 randomly chosen inserted oligonucleotides from the TSAR-12 library were examined by DNA sequencing as described above in Section 6.3.1. The isolates were chosen at random from the TSAR-12 library and prepared for sequencing, as were the TSAR-9 isolates. Analysis showed that except for the invariant Gly any position could have any amino acid; hence, the sequences are unpredicted or random. [0184]
  • 6.4. Preparation of R8C Library [0185]
  • Referring now to FIG. 3, two oligonucleotides were synthesized on an Applied Biosystems Model 380a machine with the [0186] sequence 5′-TGACGTCTCGAGTTGTNNKNNKNNKNNKNNKNNKNNKNNKTGTGGATCTAGAAGGATC-3′ (SEQ ID NO:31) and 5′-GATCCTTCTAGATCC-3′ (SEQ ID NO:32), where N is an equimolar ratio of deoxynucleotides A, C, G, and T, and K is an equimolar ratio of G and T. Fifty pmol of each oligonucleotide was incubated at 42° C. for 5 min, then 37° C. for 15 min, in 50 μL of Sequenase™ buffer (U.S. Biochemicals, Cleveland, Ohio) with 0.1 μg/μL acetylated BSA, and 10 mM DTT. After annealing, 10 units of Sequenase™ (U.S. Biochemicals) and 0.2 mM of each dNTP were added and incubated at 37° C. for 15 min. The sample was then heated at 65° C. for 2 hr, digested with 100 units of both Xho I and Xba I (New England BioLabs, Beverly, Mass.), phenol extracted, ethanol precipitated, and resolved on a 15% non-denaturing polyacrylamide gel. The assembled, digested fragment was gel purified prior to ligation. The. vector, m663 (Fowlkes, D. et al. Biotech. (1992) 13:422-427), was prepared by digestion with Xho I and Xba I, calf alkaline phosphatase (Boehringer Mannheim, Indianapolis, Ind.) treatment, phenol extracted, and purified by agarose gel electrophoresis. To ligate, 20 μg vector was combined with 0.2 μg insert in 3 mL with T4 DNA ligase (Boehringer Mannheim), according to the manufacturer. S After removal of the protein and buffer by phenol extraction and ethanol precipitation, the ligated DNA was electroporated into XL1-Blue E. coli (Stratagene, San Diego, Calif.) and plated for eight hours at 37° C. To recover the recombinant phage, the top agar was collected with a spatula, mixed with an equal volume of 100 mM NaCl, 10 mM MgCl2, and 50 mM Tris-HCI (pH7.5), and disrupted by two passes through an 18-gauge syringe needle. The bacterial cells were removed by centrifugation, and phage particles were collected by polyethylene glycol precipitation and stored at −70° C. in 25% glycerol. The library had 108 total recombinants and a working titer of 6×1013pfu/mL.
  • Members of the library were checked for inserts by the polymerase chain reaction (Saiki, et al. [0187] Science (1988) 239:487-491). Individual plaques on a petri plate were touched with a sterile toothpick and the tip was stirred into 2xYT with F+ E. coli bacteria and incubated overnight at 37° C. with aeration. Five microliters of the phage supernatant were then transferred to new tubes containing buffer (67 mM Tris-HCl, pH 8.8/10 mM β- mercaptoethanol/16.6 mM ammonium sulfate/6.7 mM EDTA/50 μg bovine serum albumin per mL), 0.1 mM deoxynucleotide triphosphates, and 1.25 units of Taq DNA polymerase (Boehringer Mannheim, Indianapolis, Ind.) with 100 pmoles of oligonucleotide primers. The primers flanked the cloning site in gene III of m663 (5′-TTCACCTCGAAAGCAAGCTG-3′ (SEQ ID NO:100) and
  • 5′-CCTCATAGTTAGCGTAACG-3′ (SEQ ID NO:101)). The assembly reactions were incubated at 94° C. for 1 min, 56° C. for 2 min, and 72° C. for 3 min; this cycle was repeated 24 times. The reaction products were then resolved by electrophoresis on a NuSieve 2.0% agarose gel (FMC, Rockland, Me.). Gels revealed that for 20 plaques tested, all were recombinant and had single inserts of the expected size. [0188]
  • Based on the sample size of the library, it was anticipated that 100% of the recombinants had single inserts. However, all of the SH3-binding phage isolated from the R8C library had double-inserts. Such phage are presumed rare (i.e., <5%) within the library, yet because the SH3-binding peptide appears to need to be linear they were selected for by our screening methods. Most likely they were formed during the generation of the library; one scenario is that the inserts ligated together to form head-to-head dimers and that they were subsequently cloned into m663 DNA by ligation with the vector's Xho I sticky end and by illegitimate ligation with the vector's Xba I site (see, FIG. 4). [0189]
  • 6.5. Preparation Of Target-Coated Microtiter Wells [0190]
  • 6.5.1. Preparation Of GST-SH3 Fusion Proteins [0191]
  • The preparation of Src-GST fusion protein was first described by Smith and Johnson, in [0192] Gene (1988) 67:31, the disclosure of which is incorporated by reference herein. Briefly, pGEX-derived (Pharmacia, Piscataway, N.J.) constructs expressing GST fusion proteins containing the SH3 domains of Src, Grb2, Crk, Abl, or PLCγ were obtained from Dr. Channing Der (University of North Carolina at Chapel Hill); a construct expressing the SH3 domain of Yes was obtained from Dr. Marius Sudol (Rockefeller University). The use of the pGEX bacterial expression vector for the production of GST-SH3 fusion proteins is well-known to those in the art. See, e.g., Cicchetti, P. et al., in Science (1992) 257:803-806. Briefly, the coding region for a particular SH3 domain can be fused in-frame at the Bam HI site of pGEX-2T. Thus, fusion proteins were prepared as per the manufacturer's instructions, and quantified by Coomassie Blue staining of SDS-polyacrylamide gels. Microtiter wells were coated with 5-20 μg GST-SH3 fusion protein in 100 mM NaHCO3, pH 8.5, blocked with 100 mM NaHCO3 (pH 8.5) 1% BSA, and washed. All washes consisted of five applications of 1XPBS, 0.1% Tween 20, 0.1% BSA (Buffer A). Where appropriate, the amount of protein bound to each well was quantified with an anti-GST antibody-based ELISA (Pharmacia, Piscataway, N.J.), and with a GST-binding phage, isolated during the course of this work.
  • 6.5.2. Coating of Microtiter Wells [0193]
  • Bacterially expressed Src SH3 glutathione-S-transferase (Src-GST) fusion protein was purified from bacterial lysates using glutathione agarose 4B (Pharmacia), according to the manufacturer's instructions. Bourd Src-GST fusion protein was eluted from the glutathione agarose with 10 mM glutathione in PBS. Microtiter wells were then coated with Src-GST fusion protein (1-10 μg/well, in 50 mM NaHCO[0194] 3, pH 8.5) overnight at 4° C. To block non-specific binding of phage, 100 μL 1% BSA in 100 mM NaHCO3, pH 8.5, was added to each well and allowed to incubate at room temperature for 1 hour. The wells were then washed five times with 200 μL PBS, 0.1% Tween 20, 0.1% BSA (Buffer A).
  • 6.6. Biopanning And Subsequent Characterization of Phage-Displayed Random Peptide Libraries With Src-GST Fusion Protein As Target Molecule [0195]
  • 6.6.1. Isolation of Src SH3-Binding Phage [0196]
  • Library screens were performed as previously described. Kay, B. K., et al., in Gene (1993) 128:59-65. Briefly, 1×10[0197] 11 pfu TSAR 9, TSAR 12, or R8C phage in Buffer A were incubated in a Src SH3-GST-coated well for 2 hours. The wells were washed, and bound phage were eluted with 100 μL 50 mM glycine.HCl (pH 2.2), transferred to a new well, and neutralized with 100 mL 200 mM NaHPO4 (pH 7.0). Recovered phage were used to infect 1×109 DH5αF′ E. coli cells in 20 mL 2×YT; the infected cells were grown overnight, resulting in a 1000- to 10,000-fold amplification of phage titer. Amplified phage were panned twice more, as above, excepting the amplification step. Binding phage recovered after the third round of panning were plated at a low density on a lawn of DH5αF′ E. coli cells to yield isolated plaques for clonal analysis. Isolated plaques were used to produce small cultures from which phage stocks and DNA were recovered for phage binding experiments and dideoxy sequencing (Sanger, F., et al., in Proc. Nati. Acad. Sci. USA (1977) 74:5463-5467), respectively. Clones were confirmed as binding the SH3 domain by applying equal titers of phage to wells containing Src SH3-GST or GST alone, and titering the number of eluted particles from each well, or detecting bound phage with an anti-phage antibody-based ELISA (Pharmacia).
  • Indeed, the ability of isolated phage clones to bind to several SH3 domains derived from a variety of different proteins can be investigated by the manner described above. GST-SH3 fusion proteins containing SH3 domains from a variety of different proteins are bound to microliter wells. An aliquot of the aforementioned phage stocks (50 μL) is introduced into wells containing the different GST-SH3 fusion proteins. After room temperature incubation for 1-2 hours, the liquid contents of the microtiter plates are removed, and the wells are washed 5 times with 200 AL Buffer A. Bound phage are eluted with 100 [0198] AL 50 mM glycine (pH 2.2), transferred to a new well, and neutralized with 100 μL 200 mM NaHPO4 (pH 7.0). The phage are diluted 103- to 10−6-fold, and aliquots are plated onto lawns of DH5αF′ E. coli cells to establish the number of plaque forming units in the output sample. From these experiments, the relative specificity of different Src SH3 binding clones for SH3 domains derived from other proteins is determined.
  • 6.6.2. Phage ELISA and Nucleotide Sequencing [0199]
  • To evaluate the binding of isolates to various targets proteins, enzyme-linked-immuno-assays (ELISA) were also performed. Bacterial cultures were infected with phage isolates and cultured overnight in 2XYT at 37° C. The cells were spun down and 25 mL of supernatant was added to microtiter plate wells coated with 50 AL of protein (1 mg/mL in 100 mM NaHCO[0200] 3, pH 8.4; overnight at 4° C. or for a few hours at room temperature) and blocked (1 mg/mL BSA in 100 mM NaHCO3, pH 8.4; for about one hour). The phage are incubated in the well with 25 μL of PBS-0.1% Tween 20 at RT for 2 hr. The wells are then washed multiple times over 30 minutes. To each well is added 50 μL of polyclonal anti-phage antibody conjugated to horseradish peroxidase. The antibody is diluted 1:3000 in BSA-PBS-Tween 20; it was obtained from Pharmacia (Piscataway, N.J.; catalog number 27-9402-01). After minutes, the wells are washed again with BSA-PBS-Tween 20 for −20 minutes. Finally, 100 AL of ABTS reagent (Pharmacia, with H2O2) are added to each well for the development of color. Plates are read with a plate reader (Molecular Devices, Menlo Park, Calif.) at 405 nm wavelength.
  • The nucleotide sequence of the relevant segments of the Src SH3 binding clones (or phage clones that bind to SH3 domains of other proteins) were sequenced using standard methods. Sanger, F., et al., in [0201] Proc. Natl. Acad. Sci. USA (1977) 74:5463-5467. The oligo primer 5′-AGCGTAACGATCTAAA-3′ (SEQ ID NO:102) was used, which is 89 nucleotides downstream of the gene III cloning site of M13 m666. The nucleotide sequences were analyzed with the MacVector computer program (IBI, New Haven, Conn., USA). From this nucleotide sequence information the primary sequence of each Src SH3 binding peptide was deduced. The corresponding synthetic peptides were then prepared by techniques well known in the art with or without flanking sequences. Indeed, these synthetic peptides have been shown to bind to SH3 domain targets, with those possessing the phage flanking amino acid residues exhibiting greater binding affinity.
  • 6.7 In Vitro Peptide Binding Assays [0202]
  • Peptides were obtained from Research Genetics (Birmingham, Ala.), Chiron Mimotopes (Victoria, Australia), or synthesized by conventional techniques by Dr. J. Mark Carter of Cytogen Corporation (Princeton, N.J.). Peptide purity was assessed by HPLC and/or mass spectrometry. Biotinylated peptides were synthesized with either a KSGSG (SEQ ID NO:103) or a GSGS (SEQ ID NO:104) peptide linker (a spacer) between the biotin and the N-terminus of the peptide. Binding experiments were performed as above, excepting the use of 10 AM peptide instead of phage. Bound biotinylated peptide was detected with streptavidin conjugated to alkaline phosphatase (Sigma Chemical Co., St. Louis, Mo.). After one hour incubation period at room temperature, the wells were washed, and a solution of 3 mM p-nitrophenyl-phosphate (US Biochemicals, Cleveland, Ohio) in 50 mM NaCO[0203] 3 (pH 9.8), and 50 mM MgCl2 was added and color allowed to develop. Signals were read with an ELISA plate reader (Molecular Devices, Menlo Park, Calif.) at 405 nm wavelength. Binding experiments were performed in triplicate. The results are presented in FIGS. 7 and 8.
  • 6.8. Peptide Competition of GST-SH3 Affinity Precipitations of Cell Lysates [0204]
  • Labeled proteins are-prepared by incubating a culture of HeLa cells overnight with ≧100 μCi/mL [0205] 35S-methionine. The cells are then washed and lysed with mild detergent. This mixture of radioactive proteins is incubated with Src-GST fusion protein that has been immobilized on glutathione-linked Sepharose beads (Pharmacia, Piscataway, N.J.). After several hours of tumbling, the beads are pelleted gently by low-speed centrifugation, and the supernatant is discarded. The beads are then resuspended into a slurry in PBS-0.1% Tween 20, pelleted, and washed several additional times. Finally, a 2% SDS solution is added to the sample, which is then boiled at 100° C. for 3 minutes. Afterward, the sample is centrifuged, and the supernatant loaded on a 10% polyacrylamide SDS gel for electrophoresis. After the proteins have been resolved, the gel is fixed, dried down, and exposed to X-ray film for autoradiography or phosphor plates for scanning by a Molecular Dynamics PhosphorImager.
  • The ability of Src SH3 to bind certain [0206] 35S-labeled proteins is examined for competability with exogenous peptides. Synthetic peptides corresponding to phage-displayed inserts and motifs are added at the time that the lysate is incubated with the Src-GST fusion protein immobilized on glutathione-linked sepharose beads. The SH3 inding peptides block binding of all or some of the labeled roteins while negative control peptides (unrelated peptide sequences) do not. The amount of competition is quantified and correlated with the amount of added SH3-domain binding peptides.
  • Alternatively, NIH 3T3 cells were grown in Dulbecco's Modified Eagle Medium (DME)+10% fetal calf serum (FCS)+80 μCi/mL Tran[0207] 35Slabel (ICN), washed with PBS, lysed in RIPA buffer, and pelleted. Supernatant from 1.5×106 cells was recleared with 100 μg glutathione-agarose-immobilized GST. The supernatant was then incubated with 10 μg glutathione-agarose-immobilized GST-SH3 fusion protein with or without added test peptide in a final volume of 250 μL. Pelleted beads were washed with 1 mL each of RIPA, RIPA+1% deoxycholate+0.1% SDS, and PBS, resuspended in 50 μL SDS-PAGE sample buffer, boiled, and subjected to SDS-PAGE (7.5%). Labeled proteins were detected by phosphorimaging (Molecular Dynamics). The results are presented in FIG. 9.
  • 6.9. Peptide Competition of GST-SH3 Affinity Precipitations of PI-3′ Kinase From Cell Lysates [0208]
  • It is possible to follow the precipitation of PI-3′ Kinase by Src from cell lysates in the presence or absence of SH3-binding peptides. HeLa cells are lysed with detergent and the protein mixtures are incubated for several hours with the Src-GST fusion protein immobilized on glutathione-linked Sepharose beads. After several hours of tumbling, the beads are pelleted gently by low-speed centrifugation and the supernatant is discarded. The beads are then resuspended into a slurry in PBS-0.1[0209] % Tween 20, pelleted, and washed several additional times. Finally, an SDS solution is added to the sample, which is then boiled at 100° C. for 3 minutes. Subsequently, the sample is centrifuged, and the supernatant is loaded on a 10% polyacrylamide SDS gel for electrophoresis. After the proteins have been resolved, the gel is blotted to nitrocellulose or nylon (i.e., western blot). The filter is then probed with a PI-3′ Kinase antibody (monoclonal and polyclonal antibodies are available from Upstate Biotechnology Incorporated, Lake Placid, N.Y.) and an enzyme-linked secondary antibody. The amount of PI-3′ Kinase is then quantitated.
  • The ability of Src SH3 to bind PI-3′ Kinase is examined for competability with exogenous peptides. Synthetic peptides corresponding to phage-displayed inserts and motifs are added at the time that the lysate is incubated with the Src-GST fusion protein that has been immobilized on glutathione-linked sepharose beads. Ten-fold and one hundred-fold molar excess of peptides are used relative to SH3 proteins. The SH3 binding peptides block binding of the PI-3′ Kinase as detected on western blots while negative control peptides (unrelated peptide sequences) do not. The amount of competition is quantified and correlated with the amount of added SH3-domain binding peptides. [0210]
  • 6.10. In Vivo Association Of SH3-Binding Peptides With SH3-Domains Of Proteins [0211]
  • To demonstrate association of the SH3-binding peptides with SH3-domains of proteins inside cells, the SH3-binding peptides are tagged and localized in cells. For example, Bar-Sagi et al., in [0212] Cell (1993) 74:83-91, have shown that SH3-binding proteins localize to the cytoskeleton when expressed in cells. Thus, the SH3 domain-binding peptides of the present invention can serve as cellular targetting signals (e.g., to the cytoskeleton). Accordingly, the peptides are tagged with biotin and, subsequently, injected into cells. Alternatively, one can transfect into cells a recombinant plasmid that expresses a fusion protein comprising of the SH3-binding peptide and the green fluorescent protein (GFP, Chalfie et al., in Science (1994) 263:802-805). The location of the biotinylated peptide or the GFP fusion protein is then assayed with FITC-labeled streptavidin in paraformaldehyde-fixed cells or by direct fluorescence in living cells, respectively. Localization of the SH3-binding peptides to the cytoskeleton demonstrates that the SH3-binding peptides can bind SH3-domain proteins in vivo. In addition, focal adhesions, which are rich in Src, are also sites of potential subcellular localization of SH3-binding peptides.
  • Thus, NIH 3T3 fibroblasts were cultured in vitro on glass coverslips coated with fibronectin. After two days of growth at 37° C., the cells were fixed for one hour at room temperature in the presence of 2% paraformaldehyde (pH 7.5). The coverslips were washed with PBS-0.1[0213] % Tween 20 several times to remove the fixative. Next, the coverslips were dipped into acetone (chilled at −20° C.) for approximately 20 seconds and allowed to air-dry. The coverslips were washed again with PBS-0.1% Tween 20, containing BSA (1 mg/mL), and incubated for 2 hours at room temperature with different biotinylated peptides in PBS-0.1% Tween 20. The coverslips were washed and then incubated with 1 mg/mL streptavidin-Cy3 (Jackson Immunoresearch Co., West Grove, Pa.) for 1 hour at room temperature. Finally, the coverslips were washed in PBS-0.1% Tween 20, mounted in a glycerol solution on a glass slide, and viewed with a Nikon Optiphot epifluorescence microscope and a 60× oil immersion lens.
  • The results are presented in FIG. 11, in which panel A displays cells stained with the conjugate biotin-spacer-VLKRPLPIPPVTR (SEQ ID NO:64); panel B exhibits cells stained with the conjugate, biotin-spacer-GILAPPVPPRNTR (SEQ ID NO:63); panel C shows cells stained with the long consensus peptide, biotin-spacer-RSTPRPLPPLPTTR (SEQ ID NO:67); and panel D shows cells stained with the proline-rich vinculin peptide conjugate, biotin-spacer-LAPPKPPLPEGEV (SEQ ID NO:70). The “spacer” sequence is KSGSG (SEQ ID NO:103). As shown in FIG. 11, the panels in which SH3 domain-binding peptides were used present a bright display of fluorescence activity that is in sharp contrast to the relatively “dark” features of panel D (non-SH3 domain binding vinculin segment). These results demonstrate further the ability of the SH3 domain-binding peptides of the present invention to localize to protein targets (e.g., Src and Src-related proteins) within cells and provide an image thereof. [0214]
  • 6.11. In Vivo Modulation of Src In Oocytes With SH3-Binding Peptides [0215]
  • When [0216] Xenopus laevis oocytes are injected with mRNA encoding deregulated Src, there are dramatic cytological and biochemical changes in the oocyte (Unger, T.F. and Steele, R. E., in Mol. Cell. Biol. (1992) 12:5485-5498). The applicants have obtained plasmids for generating wild type and deregulated Src mRNA, which are available from Dr. Robert Steele (University of California at Irvine). Synthetic SH3-binding peptides are injected into oocytes that have been previously injected with Src mRNA. The state of the cytoskeleton is inspected visually by observing the arrangement of cortical pigment granules under a dissecting microscope. The state of phosphorylation of several proteins is examined by western blotting with an anti-phosphotryosine monoclonal antibody (4G10; Upstate Biotechnology Incorporated), as described in Unger and Steele, above.
  • 6.12. Progesterone-induced [0217] X. laevis oocyte Maturation
  • Segments of adult ovary were removed surgically and incubated in 0.1% collagenase type D (Boehringer Mannheim, Indianapolis, Ind.) in Ca[0218] 2+-free OR2 (82.5 mM NaCl, 2.5 mM KCl, 1.0 mM MgCl2, 1.0 mM Na2HPO4, 5.0 mM HEPES, and 3.8 mM NaOH, pH 7.6). Oocytes were then washed 3-5 times with OR2 containing 1.0 mM CaCl2 and allowed to recover in OR2 overnight at 18° C. Stage VI oocytes were injected with 40 nL of 100 mM peptide or water. After injection, the oocytes were placed in OR2 with 2 mg/mL progesterone (Sigma, St Louis, Mo.) and incubated at 20° C. Oocytes were scored at hourly time points for germinal vesicle breakdown (GVBD).
  • FIG. 10 presents the results of this experiment. As shown by the graph, oocytes injected with the SH3 domain-binding peptide VLKRPLPIPPVTR (SEQ ID NO:64) exhibit a faster rate of progesterone-induced germinal vesicle breakdown relative to oocytes that had been injected with water or with the proline-rich vinculin peptide, LAPPKPPLPEGEV (SEQ ID NO:70). These results parallel those of Unger and Steele, supra, wherein oocytes injected with deregulated or active Src RNA matured at a faster rate than oocytes injected with water or wild-type Src mRNA (See FIG. 3B of the Unger and Steele article). [0219]
  • The present results obtained with Src SH3 domain-binding peptides suggest that these peptides modulate the biochemical activity of “cellular” Src; in particular, it is proposed that at least some of the Src SH3 domain-binding peptides of the present invention upregulate the biochemical activity of “cellular” Src, which may be downregulated or inhibited in its normal state. Hence, the administration of the SH3 domain-binding peptides of the present invention can constitute a novel method of modulating the activity of Src or Src-related proteins. Specifically, certain of these peptides are able to activate Src-family proteins. [0220]
  • 6.13. In Vivo Antagonism Of Src In src Transformed Cells With SH3-Binding Peptides [0221]
  • The coding regions for SH3-binding peptides are cloned into vectors that direct their expression in animal cells. A bipartite gene is constructed, encoding a protein with c-myc epitope and SH3-binding peptide, which is transcribed from a strong constitutive promoter (e.g., SV40, CMV, HSV TK, calmodulin). The vector is introduced into either normal or Src-transformed cells via transfection (e.g., electroporation, calcium phosphate, liposomes, DEAE dextran). Transfected cells express the bipartite gene transiently in culture. To create stable transformed cell lines, the vector carries a selectable marker (e.g., neomycin resistance) or transfection is performed in the presence of excess plasmid carrying a selectable marker (e.g., neomycin resistance) and cells selected for the marker. Transfected cells are stained by immunofluorescence to detect expression of the bipartite protein. The hybridoma 9E10 secretes a monoclonal antibody that is highly specific for the c-myc epitope (EQKLISEEDLN [SEQ ID NO:105]; see, Evan, G.A. et al., in [0222] Mol. Cell. Biol. (1985) 5:3610-3616). This antibody is sed in immunofluorescence experiments to demonstrate that he bipartite protein is expressed inside the cells, and in some cases, localized to subcellular structures enriched in SH3 domain bearing proteins.
  • There are several controls used in these experiments. First, cells are transfected with vectors that do not have he SH3-binding peptide coding region. Second, normal (non-ransformed) cells are transfected. Third, cells transformed by oncogenes other than Src are used in the transfection experiments. Fourth, cells are stained with other monoclonal antibodies that do not recognize the c-myc epitope. [0223]
  • Transfected cells are examined for any changes in cell shape, behavior, and metabolism as a consequence of expressing the SH3 binding peptides. Cell shape is examined by phase contrast microscope at several times after transfection; in particular, the flatness of the cells, their adhesion to the substrate, and the degree of cell ruffling are monitored. Cell division rates, cell migration, and contact inhibition are also observed over time. Finally, the amount of phosphorylated tyrosine in transfected cells is quantitated by phosphoaminoacid analysis and with an anti- phosphotryosine monoclonal antibody (4G10; Upstate Biotechnology Incorporated) in western blotting experiments. [0224]
  • 6.14 Distinct Ligand Preferences of Various 8H3 Domains [0225]
  • 6.14.1 Preparation of PXXP (SEQ ID NO: 161) Biased Peptide Libraries [0226]
  • Using procedures similar to those described in Sections 6.1 and 6.4 and also described in Sparks, A. B., et al., in [0227] Methods in Enzymology, (1995) 255:498-509, oligonucleotide inserts were constructed according to the schematic provided in FIG. 12. The two synthetic oligonucleotides (5′-ctgtgcctcgagk(nnk)6cca(nnk)2cca(nnk)6tctagacgtgtcagt-3′ (SEQ ID NO:162) and 5′-actgacacgtctaga-3′(SEQ ID NO:163), where k=g+t and n=g+a+t+c) were annealed and filled in with Sequenase (Amersham, Arlington Heights, Ill.). The inserts were then digested with Xho I and Xba I and were ligated into gene III of the mBAX vector.
  • The mBAX vector was created by generating cloning sites in gene III of the M13mpl8 vector (Messing, J., 1991, “Cloning in M13 phage or how to use biology at its best,” [0228] Gene 100, 3-12) in the manner of Fowlkes et al., 1992, Biotechniques 13, 422-427. The mBAX vector displays a peptide sequence at the N-terminus of the mature pIII protein that encodes the epitope for the mouse monoclonal antibody 7E11 (see FIG. 13); it includes the stop codon TAG in the coding region, which is suppressed in E. coli carrying suppressor tRNA gene mutations known as supE or supF. There are no other stop codons in the mBAX genome. The mBAX vector also carries a segment of the alpha fragment of β-galactosidase; bacterial cells expressing the omega fragment of β-galactosidase that are infected by a bacteriophage that expresses the alpha fragment convert the clear XGal substrate into an insoluble blue precipitate. Thus, plaques of such bacteriophage on such cells appear blue.
  • Recombinant mBAX molecules can be distinguished easily from non-recombinant molecules due to the TAG codon in the XhoI-XbaI segment in gene III of mBAX. When recombinants are generated by replacing the Xho I-Xba I fragment with oligonucleotides encoding random peptides, the recombinants can be grown in bacteria with (e.g., DH5αF′) or without (e.g., JS5) suppressor tRNA mutant genes. On the other hand, the non-recombinant mBAX molecules fail to produce plaques on bacterial lawns where the bacteria (e.g., JS5) lack such suppressor genes. This is because in JS5, the TAG codon serves as a stop codon to yield a truncated pIII molecule during translation; since pIII is an essential protein component of viable M13 viral particles, no plaques will form. [0229]
  • The ligated DNA was electroporated into JS5 [0230] E. coli and recombinant phage were propagated on two hundred 100 mm 2xYT +0.8% agar plates as described in Sambrook, J., Frisch, E, F., & Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, Plainview, N.Y.) (Sambrook et al.). To minimize the recovery of sibling clones during affinity purification of binding phage, six distinct library fractions were prepared by dividing the plates into six roughly equal groups. Each fraction was treated separately in all subsequent manipulations. Phage particles were harvested from each fraction by diffusion into 100 ml PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4), concentrated by polyethylene glycol precipitation as in Sambrook et al. (1989, supra), and resuspended in 10 ml PBS+10% glycerol. Each fraction contained approximately 5×107 unique recombinants, for a total library complexity of approximately 3×108. The resulting phage-displayed library contained peptides of the form X6PXXPX6 (SEQ ID NO:164), where X represents any amino acid.
  • 6.14.2 Affinity purification of SH3-binding phage [0231]
  • Library screens were performed as described in Sparks, A. B., et al., in [0232] Methods in Enzymology, (1995) 255:498-509. Briefly, wells of an ELISA microtiter plate were coated with 10 μg GST-SH3 fusion protein in 100 mM NaHCO3 (pH 8.5) for 3 hours and blocked with Superblock (Pierce, Rockford, Ill.) for 1 hour. Approximately 5×1011 infectious particles from each library fraction were diluted in 200 μl PBS+0.1% Tween 20 and incubated in a GST-SH3-coated well for 3 hours. The wells were washed five times with PBS+0.1% Tween 20, and bound phage were eluted with 50 mM glycine-HCl (pH 2.2). Recovered phage were propagated in 10 ml 2xYT media and 100 μl of a saturated DH5αF′ E. coli culture and affinity purified twice more as above. Affinity purified phage were plated onto 2xYT+0.8% agar plates to yield isolated plaques from which clonal phage stocks and DNA were produced. Phage binding was confirmed by incubating equal amounts of a clonal phage stock in wells coated with 1 μg GST-SH3 or GST. The wells were washed five times with PBS+0.1% Tween 20, and bound phage were detected by anti-phage ELISA according to the manufacturer's instructions (Pharmacia, Piscataway, N.J.). Clones with strong SH3-binding activity were selected for further analysis. The sequences of peptides displayed by these clones were determined by DNA sequencing of phage inserts.
  • 6.14.3 Preparation of GST-SH3 fusion proteins [0233]
  • Constructs encoding GST fusions to the Grb2 N-terminal (Grb2 N, aa 1-58), Nck N-terminal (Nck N, aa 1-68), Nck middle (Nck M, aa 101-166), Nck C-terminal (Nck C, aa 191-257), p53bp2 (aa 454-530), or Src (aa 87-143) SH3 domains were generated by PCR cloning of the appropriate cDNAs into pGEX-2T (Pharmacia, Piscataway, NJ; a general reference for the PGEX vectors is Smith, D. B., & Johnson, K. S. (1988) [0234] Gene 67, 31-40). The integrity of the constructs was confirmed by DNA sequencing. pGEX-derived constructs expressing GST fusions to the SH3 domains of Yes, Cortactin, Crk, Abl, and PLCγ were kindly provided by M. Sudol (Rockefeller University), J. T. Parsons (University of Virginia at Charlottsville), M. Matsuda (Tokyo, Japan), A. M. Pendergast (Duke University), and S. Earp (University of North Carolina at Chapel Hill), respectively. Alternatively, the GST-SH3 fusion proteins for Yes, Cortactin, Crk, Abl, and PLCγ could have been prepared as above for Grb2 N, Nck N, Nck M, Nck C, p53bp2, and Src, using published sequence information for these proteins. See, e.g., Suen et al., (1993) Mol. Cell. Biol. 13, 5500-5512 (Grb2); Lehmann et al., (1990) Nucleic Acids Res. 18, 1048 (Nck); Iwabuchi et al., (1994) Proc. Natl. Acad. Sci. USA 91, 6098-6102 (p53bp2); Takeya et al., (1983) Cell 32, 881-890 (Src); Sudol et al., (1988) Nucleic Acids Res. 16, 9876 (Yes); Wu et al., (1991) Mol. Cell. Biol. 11, 5113-5124 (Cortactin); Matsuda et al., (1992) Mol. Cell. Biol. 12, 3482-3489 (Crk); Shtivelman et al., (1986) Cell 47, 277-284 (Abl); Burgess et al., (1990) Mol. Cell. Biol. 10, 4770-4777 (PLCγ). GST-SH3 fusion proteins were prepared as described in Smith, D. B., & Johnson, K. S. (1988) Gene 67, 31-40. The integrity and purity of the fusion proteins were confirmed by SDS-PAGE. Protein concentrations were determined using a the BioRad protein assay (BioRad, Hercules, Calif.).
  • 6.14.4 SH3 Domain Binding Peptides and Consensus Sequences [0235]
  • The use of second generation or biased peptide libraries, which fix all or part of the PXXP (SEQ ID NO:161) consensus motif for SH3 domain binding peptides and randomize flanking residues, has defined additional sequence residues exhibiting selective SH3 domain binding. [0236]
  • Tables 1-5, below, list some of the relevant amino acid sequences obtained when the biased peptide library described in Section 6.14.1 was screened with GST-SH3 fusion proteins. The underscored amino acid residues in Tables 1-5 indicate the fixed positions. Also, indicated for each set of new binders is a “consensus” sequence, which seeks to include the additional features gleaned from the new binding peptides. The symbol “φ” in the consensus sequences of Tables 1-5 represents a hydrophobic residue. The symbol x in the consensus sequences of Tables 1-5 represents any amino acid. For the Nck SH3 domain binding clones, a GST-SH3 fusion protein containing the middle SH3 domain of Nck was used. [0237]
    TABLE 1
    CORTACTIN SH3-BINDING PEPTIDES
    SEQ.
    ID NO.
    PXXP.CORT.M1/2/3.PP    SSLLGPPVPPKPQTLFSFSR 107
    PXXP.CORT.M4.PP SRLGEFSKPPIPQKPTWMSR 108
    PXXP.CORT.N2.PP    SRTERPPLPQRPWLSYSSR 109
    PXXP.CORT.N3.PP.INC SREPDWLCPNCPLLLRSDSR 110
    PXXP.CORT.01/2/3.PP SSSSHNSRPPLPEKPSWLSR 111
    PXXP.CORT.04.PP SRLTPQSKPPLPPKPSAVSR 112
    CONSENSUS        KPPφPxKPxW 113
           R
  • [0238]
    TABLE 2
    NCK SH3-BINDING PEPTIDES
    SEQ.
    ID NO.
    PXXP.NCK.Q1/4.PP SSLGVGWKPLPPMRTASLSR 114
    PXXP.NCK.Q2/3.PP.INC SSVGFADRPRPPLRVESLSR 115
    PXXP.NCK.R1.PP.INC SSAGILRPPEKPXRSFSLSR 116
    PXXP.NCK.R2.PP SSPYTGDVPIPPLRGASLSR 117
    PXXP.NCK.R3.PP SSLMGSWPPVPPLRSDSLSR 118
    PXXP.NCK.R4.PP SSIGEDTPPSPPTRRASLSR 119
    PXXP.NCK.S1/4.PP SRSLSEVSPKPPIRSVSLSR 120
    PXXP.NCK.S2.PP.INC SSVSEGYSPPLPPRSTSLSR 121
    PXXP.NCK.S3.PP SSSFTLAAPTPPTRSLSLSR 122
    PXXP.NCK.T1.PP SSPPYELPPRPPNRTVSLSR 123
    PXXP.NCK.T2.PP SRVVDGLAPPPPVRLSSLSR 124
    PXXP.NCK.T3.PP.INC SSLGYSGAPVPPHRxSSLSR 125
    PXXP.NCK.T4.PP SSISDYSRPPPPVRTLSLSR 126
    CONSENSUS   φxxxxxPxPPφRSxSL 127
                T
  • [0239]
    TABLE 3
    ABL SH3 BINDING PEPTIDES
    SEQ.
    ID NO.
    PXXP.ABL.G1/2.PP   SRPPRWSPPPVPLPTSLDSR 128
    PXXP.ABL.G3/4.PP   SSPPDYAAPAIPSSLWVDSR 129
    PXXP.ABL.H1/3/4.PP   SSPPHWAPPAPPAMSPPISR 130
    PXXP.ABL.H2.PP.INC   SSDRCWECPPWPAGGQRGSR 131
    PXXP.ABL.I1/2/3.PP   SSPPKFSPPPPPYWQLHASR 132
    PXXP.ABL.I4.PP   SSPPSFAPPAAPPRHSFGSR 133
    PXXP.ABL.J1.PP   SSAPKKPAPPVPMMAHVMSR 134
    PXXP.ABL.J2.PP.INC    SSPTYPPPPPPDTAKGASR 135
    PXXP.ABL.J3.PP.INC   SSPPXXXPPPIPNSPQVLSR 136
    PXXP.ABL.J4.PP   SSPPTWTPPKPPGWGVVFSR 137
    PXXP.ABL.L1.PP   SSAPTWSPPALPNVAKYKSR 138
    PXXP.ABL.L2/3.PP SSIKGPRFPVPPVPLNGVSR 139
    PXXP.ABL.L4.PP   SSPPAWSPPHRPVAFGSTSR 140
    CONSENSUS     PPxWxPPPφP 141
  • [0240]
    TABLE 4
    PLCγ SH3-BINDING PEPTIDES
    SEQ.
    ID NO.
    PXXP.PLCγ.P1.PP SSMKVHNFPPLPSYETSR 142
    PXXP.PLCγ.P2.PP SRVPPLVAPRPPSTLNSLSR 143
    PXXP.PLCγ.PE.PP.INC SSLYWQHGPDPPVGAPQLSR 144
    PXXP.PLCγ.P4.PP SSHPLNSWPGGPFRHNLSSR 145
  • [0241]
    TABLE 5
    SRC SH3-BINDING PEPTIDES
    SEQ. ID NO.
    PXXP.SRC.A1.PP SSRALRVRPLPPVPGTSLSR 146
    PXXP.SRC.A2.PP SSFRALPLPPTPDNPFAGSR 147
    PXXP.SRC.A3.PP SRDAPGSLPFRPLPPVPTSR 148
    PXXP.SRC.A4.PP SSISQRALPPLPLHSDPASR 149
    PXXP.SRC.B1.PP SSPAYRPLPRLPDLSVIYSR 150
    PXXP.SRC.B2/3/PP SSFINRRLPALPPDNSLLSR 151
    PXXP.SRC.B4.PP SRLTGRPLPALPPPFSDFSR 152
    PXXP.SRC.C1.PP SRMKDRVLPPIPTVESAVSR 153
    PXXP.SRC.C2.PP.INC SSLYSAIAPDPPPRNSSSSR 154
    PXXP.SRC.C3.PP SSLASRPLPLLPNSAPGQSR 155
    PXXP.SRC.D1.PP SSLTSRPLPDIPVRPSKSSR 156
    PXXP.SRC.D2.PP.INC SSLKWRALPPLPETDTPYSR 157
    PXXP.SRC.D3.PP SSNTNRLPPPTPDGLDVRSR 158
    PXXP.SRC.D4.PP SSLQSRPLPLPPQSSYPISR 159
    CONSENSUS      RPLPPLP 9
  • In addition to the consensus sequence shown in Table 5, the amino acid sequences of the inserts from the Src SH3 domain-binding phage isolated from the PXXP (SEQ ID NO:161) biased peptide library described in Section 6.14.1 also give rise to the consensus sequence LXXRPLPXψP (SEQ ID NO:165), as shown in Table 6, below. In the consensus sequence LXXRPLPXψP (SEQ ID NO:165), ψ represents aliphatic amino acid residues (A, V, L, I, P); X represents any amino acid. [0242]
    TABLE 6
    Src SH3 Binding Peptides
         LASRPLPLLPNSAPGQ (a portion of
    SEQ ID NO:155)
         LTGRPLPALPPPFSDF (a portion of
    SEQ ID NO:152)
         PAYRPLPRLPDLSVIY (a portion of
    SEQ ID NO:150)
       RALRVRPLPPVPGTSL (a portion of
    SEQ ID NO:146)
    DAPGSLPFRPLPPVPT (a portion of
    SEQ ID NO:148)
         LKWRALPPLPETDTPY (a portion of
    SEQ ID NO:157)
         ISQRALPPLPLMSDPA (a portion of
    SEQ ID NO:149)
         LTSRPLPDIPVRPSKS (a portion of
    SEQ ID NO:156)
         NTNRPLPPTPDGLDVR (a portion of
    SEQ ID NO:158)
         MKDRVLPPIPTVESAV (a portion of
    SEQ ID NO:153)
         LQSRPLPLPPQSSYPI (a portion of
    SEQ ID NO:159)
         FINRRLPALPPDNSLL (a portion of
    SEQ ID NO:151)
           FRALPLPPTPDNPFAG (a portion of
    SEQ ID NO:147)
         LYSAIAPDPPPRNSSS♦ (a portion of SEQ
    ID NO:154)
         LXXRPLPXψP=CONSENSUS (SEQ. ID NO: 165)
  • In Table 6, ψ represents aliphatic amino acid residues (A, V, L, I, P); X represents any amino acid; ♦ putative class II peptide (see Section 6.14.5). Invariant proline residues are underlined. [0243]
  • Another consensus sequence that can be derived from the amino acid sequences of the inserts from the Src SH3 domain-binding phage is: [0244]
  • LX[0245] 1X2RPLPX3ψPX4 5 (SEQ ID NO:454)
  • where ψ represents aliphatic amino acid residues (A, V, L, I, P) and X[0246] 1, X2, X3, X4, and X5 represent any amino acid; except that if
  • X[0247] 3=P, ψ=L, X4=P, and X5=P, then:
  • where X[0248] 1=F, then X2 is not H or R; or
  • where X[0249] 1=S, then X2 is not R, H, A, N, T, G, V, M, or W; or
  • where X[0250] 1=C, then X2 is not S or G; or
  • where X[0251] 1=R, then X2 is not T or F; or
  • where X[0252] 1=A, then X2 is not R, Q, N, S, or L; or
  • where X[0253] 1=Q, then X2 is not M; or
  • where X[0254] 1=L, then X2 is not R; or
  • where X[0255] 1=I, then X2 is not A; or
  • where X[0256] 1=P, then X2 is not P, W, or R; or
  • where X[0257] 1=G, then X2 is not S or R; or
  • where X[0258] 1=T, then X2 is not T.
  • In addition to the consensus sequence shown in Table 1, the amino acid sequences of the inserts from the cortactin SH3 domain-binding phage isolated from the PXXP (SEQ ID NO:161) biased peptide library described in Section 6.14.1 also give rise to the consensus sequence +PPψPXKPXWL (SEQ ID NO:166), as shown in Table 7, below. [0259]
    TABLE 7
    Cortactin SH3 Binding Peptides
    LTPQSKPPLPPKPSAV (a portion of
    SEQ ID NO:112)
    SSHNSRPPLPEKPSWL (a portion of
    SEQ ID NO:111)
       PVKPPLPAKPWWLPPL (SEQ ID NO:167)
       TERPPLPQRPDWLSYS (a portion of
    SEQ ID NO:109)
    LGEFSKPPIPQKPTWM (a portion of
    SEQ ID NO:108)
    YPQFRPPVPPKPSLMQ (SEQ ID NO:168)
       VTRPPLPPKPGHMADF (SEQ ID NO:169)
    VSLGLKPPVPPKPMQL (SEQ ID NO:170)
       LLGPPVPPKPQTLFSF (a portion of
    SEQ ID NO:107)
        YKPEVPARPIWLSEL (SEQ ID NO:171)
    GAGAARPLVPKKPLFL (SEQ ID NO:172)
         +PPψPXKPXWL =CONSENSUS (SEQ ID NO:166)
  • In Table 7, +represents basic amino acid residues (R, K); ψrepresents aliphatic amino acid residues (A, V, L, I, P); X represents any amino acid. Invariant proline residues are underlined. [0260]
  • In addition to the consensus sequence shown in Table 3, the amino acid sequences of the inserts from the Abl SH3 domain-binding phage isolated from the PXXP (SEQ ID NO:161) biased peptide library described in Section 6.14.1 also give rise to the consensus sequence PPXθXPPPψP (SEQ ID NO:173), as shown in Table 8, below. [0261]
    TABLE 8
    Ab1 SH3 Binding Peptides
      PPWWAPPPIPNSPQVL (SEQ ID NO:174)
      PPKFSPPPPPYWQLHA (a portion of SEQ ID NO:132)
      PPHWAPPAPPAMSPPI (a portion of SEQ ID NO:130)
      PPTWTPPKPPGWGVVF (a portion of SEQ ID NO:137)
      PPSFAPPAAPPRHSFG (a portion of SEQ ID NO:133)
       PTYPPPPPPDTAKGA‡ (a portion of SEQ ID NO:135)
      GPRWSPPPVPLPTSLD (a portion of SEQ ID NO:128)
      APTWSPPALPNVAKYK (a portion of SEQ ID NO:138)
      PPDYAAPAIPSSLWVD (a portion of SEQ ID NO:129)
    IKGPRFPVPPVPLNGV (a portion of SEQ ID NO:139)
      PPAWSPPHRPVAFGST (a portion of SEQ ID NO:140)
      APKKPAPPVPMMAHVM (a portion of SEQ ID NO:134)
      PPXθXPPPψP = (SEQ ID NO:173)
    CONSENSUS
  • In Table 8, θ represents aromatic amino acid residues; ψ represents aliphatic amino acid residues (A, V, L, I, P); X represents any amino acid. Invariant proline residues are underlined. [0262]
  • ‡This clone contained a three nucleotide deletion in the random peptide coding sequence. [0263]
  • The amino acid sequences of the inserts from the PLCγ SH3 domain-binding phage isolated from the PXXP (SEQ ID NO:161) biased peptide library described in Section 6.14.1 give rise to the consensus sequence PPVPPRPXXTL (SEQ ID NO:175), as shown in Table 9, below. [0264]
    TABLE 9
    PLCγ SH3 Binding PeptideB
        MPPPVPPRPPGTLQVA (SEQ ID NO:176)
     LSYSPPPVPPRPDSTL (SEQ ID NO:177)
       VLAPPVPPRPGNTFFT (SEQ ID NO:178)
        YRPPVAPRPPSSLSVD (SEQ ID NO:179)
    LQCPDCPRVPPRPIPI (SEQ ID NO:180)
        VPPLVAPRPPSTLNSL (a portion of
    SEQ ID NO:143)
       LTPPPFPKRPRWTLPE (SEQ ID NO:181)
     YWPHRPPLAPPQTTLG (SEQ ID NO:182)
    PPVPPRPXXTL=CONSENSUS (SEQ ID NO:175)
  • In Table 9, the symbol X represents any amino acid. Invariant proline residues are underlined. [0265]
  • The PXXP (SEQ ID NO:161) biased peptide library described in Section 6.14.1 was also used to obtain phage clones that specifically bound the SH3 domain from the p53bp2 rotein. The amino acid sequences of the peptides expressed by the p53bp2 SH3 domain-binding phage are shown in Table 10 below. [0266]
    TABLE 10
    p53bp2 SH3 Binding Peptides
    YDASSAPQRPPLPVRKSRP (SEQ ID NO:183)
    EYVNASPERPPIPGRKSRP (SEQ ID NO:184)
    WNGIAIPGRPEIPPRASRP (SEQ ID NO:185)
    SMIFIYPERPSPPPRFSRP (SEQ ID NO:186)
    GVEEWNPERPQIPLRLSRP (SEQ ID NO:187)
       WVVDSRPDIPLRRSLP (SEQ ID NO:188)
       VVPLGRPEIPLRKSLP (SEQ ID NO:189)
       GGTVGRPPIPERKSVD (SEQ ID NO:190)
       YSHAGRPEVPPRQSKP (SEQ ID NO:191)
       FSAAARPDIPSRASTP (SEQ ID NO:192)
       LYIPKRPEVPPRRHEA (SEQ ID NO:193)
       NNISARPPLPSRQNPP (SEQ ID NO:194)
       MAGTPRPAVPQRMNPP (SEQ ID NO:195)
            RPXψPψR + SXP=CONSENSUS (SEQ ID NO:196)
  • In Table 10, + represents basic amino acid residues (R, K); ψ represents aliphatic amino acid residues (A, V, L, I, P); X represents any amino acid. Invariant proline or flanking residues are underlined. [0267]
  • The PXXP (SEQ ID NO:161) biased peptide library described in Section 6.14.1 was also used to obtain phage clones that specifically bound the SH3 domain from the N terminal portion of the Crk protein. The amino acid sequences of the peptides expressed by the Crk N terminal SH3 domain-binding phage are shown in Table 11 below. [0268]
    TABLE 11
    Crk N SH3 Binding Peptides
    GQPAGDPDPPPLPAKF (SEQ ID NO: 197)
       FEQTGVPLLPPKSFKY (SEQ ID NO:198)
       IFGDPPPPIPMKGRSL (SEQ ID NO:199)
       SNQGSIPVLPIKRVQY (SEQ ID NO:200)
    NYVNALPPGPPLPAKN (SEQ ID NO:201)
       SSDPERPVLPPKLWSV (SEQ ID NO:202)
       HFGPSKPPLPIKTRIT (SEQ ID NO:203)
    DWKVPEPPVPKLPLKQ (SEQ ID NO:204)
       ATSEGLPILPSKVGSY (SEQ ID NO:205)
    NANVSAPRAPAFPVKT (SEQ ID NO:206)
        EMVLGPPVPPKRGTVV (SEQ ID NO:207)
       AGSRHPPTLPPKESGG (SEQ ID NO:208)
       SVAADPPRLPAKSRPQ (SEQ ID NO:209)
             ψPψLPψK=CONSENSUS (SEQ ID NO:210)
  • In Table 11, ψ represents aliphatic amino acid residues (A, V, L, I, P). Invariant proline residues are underlined. [0269]
  • The present invention provides a purified peptide that binds to the SH3 domain of Crk, the purified peptide comprising the amino acid sequence ψPψLPψK (SEQ ID NO:210), where ψ represents aliphatic amino acid residues (A, V, L, I, P), with the proviso that the peptide does not comprise the amino acid sequence WNERQPAPALPPKPPKPT (SEQ ID NO:456). [0270]
  • The PXXP (SEQ ID NO:161) biased peptide library described in Section 6.14.1 was also used to obtain phage clones that specifically bound the SH3 domain from the Yes protein. The amino acid sequences of the peptides expressed by the Yes SH3 domain-binding phage are shown in Table 12 below. [0271]
    TABLE 12
    Yes SH3 Binding Peptides
    ITMRPLPALPGHGQIH (SEQ ID NO:211)
    LPRRPLPDLPMAAGKG (SEQ ID NO:212)
    LGSRPLPPTPRQWPEV (SEQ ID NO:213)
    STIRPLPAIPRDTLLT (SEQ ID NO:214)
    RSGRPLPPIPEVGHNV (SEQ ID NO:215)
    IGSRPLPWTPDDLGSA (SEQ ID NO:216)
    LAQRELPGLPAGAGVS (SEQ ID NO:217)
    IPGRALPELPPQRALP (SEQ ID NO:218)
    FVGRELPPTPRTVIPW (SEQ ID NO:219)
    DPRSALPALPLTPLQT (SEQ ID NO:220)
    SPHDVLPALPDSHSKS (SEQ ID NO:221)
    ψXXRPLPXLP =CONSENSUS (SEQ ID NO:222)
  • In Table 12, ψ represents aliphatic amino acid residues (A, V, L, I, P); X represents any amino acid. Invariant proline residues are underlined. [0272]
  • Another consensus sequence that can be derived from the amino acid sequences of the inserts from the Yes SH3 domain-binding phage is: [0273]
  • ψX[0274] 1X2RPLPX3LPX4X5 (SEQ ID NO:455)
  • where ψ represents aliphatic amino acid residues (A, V, L, I, P) and X[0275] 1, X2, X3, X4, and X5 represent any amino acid; except that if
  • X[0276] 3=P, X4=P, and X5=P, then:
  • when ψ=L, [0277]
  • where X[0278] 1=F, then X2 is not H or R; or
  • where X[0279] 1=S, then X2 is not R, H, A, N, T, G, V, M, or W; or
  • where X[0280] 1=C, then X2 is not S or G; or
  • where X[0281] 1=R, then X2 is not T or F; or
  • where X[0282] 1=A, then X2 is not R, Q, N, S, or L; or
  • where X[0283] 1=Q, then X2 is not M; or
  • where X[0284] 1=L, then X2 is not R; or
  • where X[0285] 1=I, then X2 is not A; or
  • where X[0286] 1=P, then X2 is not P, W, or R; or
  • where X[0287] 1=G, then X2 is not S or R; or
  • where X[0288] 1=T, then X2 is not T; and
  • when ψ=P, [0289]
  • where X[0290] 1=A, then X2 is not R; or
  • where X[0291] 1=S, then X2 is not R or Y; or
  • where X[0292] 1=M, then X2 is not S; or
  • where X[0293] 1=V, then X2 is not G; or
  • where X[0294] 1=R, then X2 is not S; or
  • where X[0295] 1=I, then X2 is not R; and
  • when ψ=A, [0296]
  • where X[0297] 1=A, then X2 is not K; and
  • when ψ=V, [0298]
  • where X[0299] 1=A, then X2 is not C or Q; or
  • where X[0300] 1=P, then X2 is not P; and
  • when ψ=I, [0301]
  • where X[0302] 1=G, then X2 is not H; or
  • where X[0303] 1=T, then X2 is not S; or
  • where X[0304] 1=R, then X2 is not S.
  • The present invention also provides a purified peptide that binds to the SH3 domain of Yes, the purified peptide comprising the amino acid sequence ψX[0305] 1X2RPLPX3LPX4X5 (SEQ ID NO:455), where ψ represents aliphatic amino acid residues (A, V, L, I, P) and X1, X2, X3, X4, and X5 represent any amino acid, with the proviso that the peptide does not comprise the amino acid sequence AGDRPLPPLPYNPKS (SEQ ID NO:457).
  • The PXXP (SEQ ID NO:161) biased peptide library described in Section 6.14.1 was also used to obtain phage clones that specifically bound the SH3 domain from the N terminal portion of the Grb2 protein. The amino acid sequences of the peptides expressed by the Grb2 N terminal SH3 domain-binding phage are shown in Table 13 below. These sequences can be arranged into three groups of sequences that have different, but related, consensus sequences. An overall consensus sequence, +θDXPLPXLP (SEQ ID NO:223), can be derived for the three groups. [0306]
    TABLE 13
    Grb2 N SH3 Binding Peptides
      KWDSLLPALPPAFTVE (SEQ ID NO:224)
      RWDQVLPELPTSKGQI (SEQ ID NO:225)
      RFDFPLPTHPNLQKAH (SEQ ID NO:226)
      RLDSPLPALPPTVMQN (SEQ ID NO:227)
      RWGAPLPPLPEYSWST (SEQ ID NO:228)
      YWDMPLPRLPGEEPSL (SEQ ID NO:229)
      RFDYNLPDVPLSLGTA (SEQ ID NO:230)
    TKKPNAPLPPLPAYMG (SEQ ID NO:231)
      KWDLDLPPEPMSLGNY (SEQ ID NO:232)
      θ+DXPLPXLP =CONSENSUS (SEQ ID NO:223)
      YYQRPLPPLPLSHFES (SEQ ID NO:234)
      YYRKPLPNLPRGQTDD (SEQ ID NO:235)
      YFDKPLPESPGALMSL (SEQ ID NO:236)
      YFSRALPGLPERQEAH (SEQ ID NO:237)
      YθX+PLPXLP =CONSENSUS (SEQ ID NO:238)
      SLWDPLPPIPQSKTSV (SEQ ID NO:239)
      SYYDPLPKLPDPGDLG (SEQ ID NO:240)
      KLYYPLPPVPFKDTKH (SEQ ID NO:241)
      DPYDALPETPSMKASQ (SEQ ID NO:242)
        θDPLPXLP =CONSENSUS (SEQ ID NO:243)
      θ+DXPLPXLP =OVERALL CONSENSUS (SEQ ID NO:223)
  • In Table 13, + represents basic amino acid residues (R, K); θ represents aromatic amino acid residues; X represents any amino acid. Invariant proline residues are underlined. [0307]
  • 6.14.5 SH3 Ligand Binding Orientation [0308]
  • Peptide ligands bound to SH3 domains have been shown to assume a left-handed polyproline type II (PPII) helix conformation (Yu, H., Chen, J. K., Feng, S., Dalgarno, D. C., Brauer, A. W., & Schreiber, S. L. (1994) [0309] Cell 76, 933-45). SH3 ligands are pseudo-symmetrical and may therefore bind in one of two opposite orientations (Feng, S., Chen, J. K., Yu, H., Simon, J. A., & Schreiber, S. L. (1994) Science 266, 1241-7) (Feng et al.). Feng et al., supra, have demonstrated that peptides that bind in one or the other orientation share different consensus motifs. Specifically, ligands that bind in the Class I or Class II orientation conform to the consensus +pYPpYP (SEQ ID NO:244) or YPpYPp+ (SEQ ID NO:245) respectively, where uppercase positions represent conserved residues that contact the SH3 domain and confer specificity, and lowercase positions represent scaffolding residues that tend to be proline.
  • According to this model, we predict that the peptides selected by the Src, Yes, Abl, and Grb2 N SH3 domains bind in the Class I orientation, whereas the peptides selected by the Cortactin, p53bp2, PLCγ, and Crk N SH3 domains bind in the Class II orientation (see Table 14). Interestingly, most of the SH3 ligand consensus motifs identified in this work contain additional conserved residues flanking the SH3-binding core defined by Feng et al., supra. Furthermore, these conserved residues are situated N- and C-terminal of the SH3-binding core in Class I and Class II motifs, respectively, and are therefore predicted to interact with equivalent regions of their target SH3 domains (see Table 14). [0310]
    TABLE 14
    Alignment of SH3 ligand consensus motifs
    SEQ ID NO:
    Class I +pψPpψP 244
    Src LXXRPLPXψP 165
    Yes ψXXRPLPXLP 222
    Abl PPXθXPPPψP 173
    Grb2 N +θDXPLPXLP 223
    YθXRPLPXLP 246
      θDPLPXLP 243
    Class II ψPpψPp+ 245
    Cortactin +PPψPXKPXWL 166
    p53bp2 RPXψPψR+SXP 196
    PLCγ XPPVPPRPXXTL 247
    Crk N ψPψLPψK 210
  • In Table 14, each SH3 ligand consensus motif was assigned to class I or II based on its agreement with the class I or II consensus motif. Highly (>90%) conserved positions in each SH3 ligand consensus motif are listed in boldface and were interpreted as SH3 contact residues. +represents basic amino acid residues (K, R); ψrepresents aliphatic amino acid residues (A, V, L, I, P); θrepresents aromatic amino acid residues; X represents any amino acid; lower case p represents residues that tend to be proline. [0311]
  • The Src SH3 domain is capable of binding both Class I and Class II peptides Feng et al., supra. Although Class I peptides predominate in the population of Src SH3 ligands selected from the PXXP (SEQ ID NO:161) library, one clone conforms well to the Class II consensus (see Table 6). Previously, Sparks, A. B., Quilliam, L. A., Thorn, J. M., Der, C. J., & Kay, B. K. (1994) J. Biol. Chem. 269, 23853-6 and Yu, H., Chen, J. K., Feng, S., Dalgarno, D. C., Brauer, 35 A. W., & Schreiber, S. L. (1994) Cell 76, 933-45 had isolated Class II Src SH3 ligands sharing the consensus PPψPPR (SEQ ID NO:248). Similarly, whereas the Grb2 N SH3 domain has been shown to bind peptides from SOS with the Class II consensus sequence PPψPPR (SEQ ID NO:248) (Rozakis-Adcock, M., Fernley, R., Wade, J., Pawson, T., & Bowtell, D. (1993) Nature 363, 83-5), we have isolated Grb2 N SH3 ligands that conform to the Class I consensus (see Table 14). Thus, both the Src and the Grb2 N SH3 domains apparently have the capacity to bind both Class I and Class II peptide ligands. [0312]
  • 6.14.6 SH3 Ligand Binding Characteristics [0313]
  • To explore further the capacity of SH3 domains to discriminate between different SH3 ligands, we investigated the binding of phage expressing various peptide ligands to a panel of SH3 domains. Equal titers of clonal phage stocks were incubated in microtiter wells coated with different GST-SH3 fusion proteins. The wells were washed several times, and bound phage were detected with an anti-phage antibody (see FIG. 14). Positive ELISA signals were equivalent to those obtained with previously characterized Src SH3-binding clones (Sparks, A. B., Quilliam, L. A., Thorn, J. M., Der, C. J., & Kay, B. K. (1994) J. Biol. Chem. 269, 23853-6) and are indicative of SH3:peptide affinities in the 5 to 75 μM range (Yu, H., Chen, J. K., Feng, S., Dalgarno, D. C., Brauer, A. W., & Schreiber, S. L. (1994) Cell 76, 933-945; Rickles, R. J., Botfield, M. C., Weng, Z., Taylor, J. A., Green, 0. M., Brugge, J. S., & Zoller, M. J. (1994) EMBO J. 13, 5598-604). Whereas the Src, Yes, Crk, and Grb2 N SH3 domains cross-reacted with a few phage clones selected with other SH3 domains, the Abl, Cortactin, p53bp2, and PLCγ SH3 domains displayed considerable specificity. Significantly, only 33 of 220 potential instances of cross-reactivity were observed, suggesting that SH3 selectivity is the rule rather than the exception. [0314]
  • Each instance of cross-reactivity may be explained by similarities between the sequences of the peptides and the ligand preferences of the cross-reactive SH3 domains. For example, Crk SH3 cross-reacted with three phage clones selected with other SH3 domains; each of these clones coincidentally expressed peptides conforming to the Crk SH3 preferred ligand consensus motif. Similarly, the cross-reactivity observed between the Src, Yes, and Grb2 SH3 domains and clones selected by other SH3 domains within this group may be a consequence of the fact that these SH3 domains prefer the same proline-rich core. Finally, the Src and Yes SH3 domains cross-reacted with the PLCγ SH3 ligand MPPPVPPRPPGTL (a portion of SEQ ID NO:176), which contains the Class II Src SH3-binding sequence PPVPPR (SEQ ID NO:249). Taken together, these data demonstrate the capacity of SH3 domains to discern subtle differences in the primary structure of potential ligands. [0315]
  • 6.15 Use of Consensus sequences to Determine the Amino Acid Sequences Responsible for Binding in Proteins that are Known to Bind SH3 Domains [0316]
  • There are many proteins that are known to bind SH3 domains but for which the specific sequences of those proteins that are responsible for binding to SH3 domains are not known. The consensus sequences shown above in Tables 1-13 can be used to search databases (e.g., GenBank) containing the amino acid sequences of those proteins in order to determine which sequences are responsible for the binding of those proteins to SH3 domains. This was done for a number of known SH3 domain binding proteins and sequences resembling the consensus sequences of Tables 1-13 were identified. The results are shown in Table 15. For comparison, also shown in Table 15 are the amino acid sequences that had previously been demonstrated to be responsible for SH3 domain binding for a number of proteins. [0317]
    TABLE 15
    SEQ
    ID Refer-
    NO: ence
    Src SH3 Class I LXXRPLPXψP 165
    Hs AFAP-110 (62-73) PPQMPLPEIPQQ 250 1
    (76-87) PPDNGPPPLPTS 251 1
    Hs CDC42 GAP (250-261) TAPKPMPPRPPL 252 2
    Hs hnRNP K  (302-313)* SRARNLPLPPPP 253 3
    Mm p62 (328-339) TVTRGVPPPPTV 254 3
    Hs P13K p85   (90-101)* RPPRPLPVAPGS 255 9
    Hs Shc p52 (296-307) VRKQMLPPPPCP 256 3
    Src SH3 Class II    PPψPPR 248
    Hs Dynamin (810-820) GGAPPVPSRPG 257 6
    (827-837) GPPPQVPSRPN 258 6
    (838-848) RAPPGVPSRSG 259 6
    Hs hnRNP K  (308-318)* PLPPPPPPRGG 260 3
    Mm p62 (294-304) APPPPPVPRGR 261 3
    Hs Paxillin (42-52) AVPPPVPPPPS 262 10
    Hs PI3K p85  (302-312)* QPAPALPPKPP 263 9
    Hs Shb (50-60) GGPPPGPGRRG 264 11
    (103-113) TKSPPQPPRPD 265 11
    Yes SH3 ψXXRPLPXLP 222
    Hs Yap65 (240-251) PVKQPPPLAPQS 266 4
    Ab1 SH3 PPXθXPPPψP 173
    Mm 3BP-1  (265-276)* RAPTMPPPLPPV 267 12
    Mm 3BP-2  (200-211)* YPPAYPPPPVPV 268 12
    Dm Ena (350-361) PGPGYGPPPVPP 269 5
    PLCγ SH3 PPVPPRPXXTL 175
    Hs Dynamin (812-823) APPVPSRPGASP 270 6
    (829-840) PPQVPSRPNRNR 271 6
    Hs c-Cb1 (493-504) LPPVPPRLDLLP 272 7
    Crk N SH3 PψLPψK 210
    Hs Ab1  (524-533)* QAPELPTKTR 273 13
     (568-577)* VSPLLPRKER 274 13
    (758-767) EKPALPRKRA 275 13
    Hs C3G  (282-291)* PPPALPPKKR 276 14
     (452-461)* TPPALPEKKR 277 14
     (539-548)* KPPPLPEKKN 278 14
     (607-616)* PPPALPPKQR 279 14
    Grb2 N SH3 Class I +θDXPLPXLP 233
    YθX+PLPXLP 238
      θDPLPXLP 243
    Hs c-Cb1 (560-571) PQRRPLPCTPGD 280 8
    (589-600) WLPRPIPKVPVS 281 8
    Grb2 N SH3 Class II PPPψPPR 282
    Hs Ab1  (523-533)* LQAPELPTKTR 283 13
     (567-577)* AVSPLLPRKER 284 13
     (609-619)* KTAPTPPKRSS 285 13
    Hs c-Cb1 (491-501) ASLPPVPPRLD 286 8
    Hs Dynamin (810-820) GGAPPVPSRPG 287 6
    (827-837) GPPPQVPSRPN 288 6
    (838-848) RAPPGVPSRSG 289 6
    Hs S0S1  (1148-1158)* PVPPPVPPRRR 290 15
    (1177-1187) DSPPAIPPRQP 291 15
     (1209-1219)* ESPPLLPPREP 292 15
     (1287-1297)* IAGPPVPPRQS 293 15
    Rn Synapsin I (592-602) NLPEPAPPRPS 294 16
    (670-680) PPGPAGPIRQA 295 16
  • In Table 15, + represents basic amino acid residues (R, K); ψ represents aliphatic amino acid residues (A, V, L, I, P); θ represents aromatic amino acid residues; X represents any amino acid. * represents amino acid sequences previously demonstrated to bind their respective SH3 domains. Residues within the sequences that agree with the most highly conserved residues of the consensus motifs are shown in bold. Each entry shows an abbreviation of the name of the SH3 domain binding protein and the species from which it was derived. The amino acid positions in the mature proteins of the sequences shown are indicated in parentheses. For more details, see the reference listed for each protein. [0318]
  • [0319] Reference 1 is Flynn, D. C., Leu, T. H., Reynolds, A. B., & Parsons, J. T. (1993) Mol Cell Biol 13, 7892-7900.
  • [0320] Reference 2 is Barfod, E. T., Zheng, Y., Kuang, W, J., Hart, M. J., Evans, T., Cerione, R. A., & Ashkenazi, A. (1993) J Biol Chem 268, 26059-62.
  • [0321] Reference 3 is Weng, Z., Thomas, S. M., Rickles, R. J., Taylor, J. A., Brauer, A. W., Seidel-Dugan, C., Michael, W. M., Dreyfuss, G., & Brugge, J. S. (1994) Mol Cell Biol 14, 4509-21.
  • [0322] Reference 4 is Sudol, M. (1994) Oncogene 9, 2145-52.
  • [0323] Reference 5 is Gertler, F. B., Comer, A. R., Juang, J. L., Ahern, S. M., Clark, M. J., Liebl, E. C., & Hoffmann, F. M. (1995) Genes Dev 9, 521-33.
  • [0324] Reference 6 is Gout, I., Dhand, R., Hiles, I. D., Fry, M. J., Panayotou, G., Das, P., Truong, O., Totty, N. F., Hsuan, J., Booker, G, W. & et al. (1993) Cell 75, 25-36.
  • [0325] Reference 7 is Rivero-Lezcano, O. M., Sameshima, J. H., 30 Marcilla, A., & Robbins, K. C. (1994) J Biol Chem 269, 17363-6.
  • [0326] Reference 8 is Odai, H., Sasaki, K., Iwamatsu, A., Hanazono, Y., Tanaka, T., Mitani, K., Yazaki, Y. & Hirai, H. (1995) J Biol Chem 270, 10800-5.
  • [0327] Reference 9 is Kapeller, R., Prasad, K. V., Janssen, O., Hou, W., Schaffhausen, B. S., Rudd, C. E., & Cantley, L. C. (1994) J Biol Chem 269, 1927-33.
  • [0328] Reference 10 is Weng, Z., Taylor, J. A., Turner, C. E., Brugge, J. S., & Seidel-Dugan, C. (1993) J Biol Chem 268, 14956-63.
  • Reference 11 is Karlsson, T., Songyang, Z., Landgren, E., Lavergne, C., Di-Fiore, P. P., Anafi, M., Pawson, T., Cantley, L. C., Claesson-Welsh, L., & Welsh, M. (1995) [0329] Oncogene 10, 1475-83.
  • [0330] Reference 12 is Ren, R., Mayer, B. J., Cicchetti, P., & Baltimore, D. (1993) Science 259, 1157-61.
  • Reference 13 is Ren, R., Ye, Z. S., & Baltimore, D. (1994) [0331] Genes Dev 8, 783-95.
  • [0332] Reference 14 is Knudsen, B. S., Feller, S. M., & Hanafusa, H. (1994) J Biol Chem 269, 32781-7.
  • Reference 15 is Rozakis-Adcock, M., Fernley, R., Wade, J., Pawson, T., & Bowtell, D. (1993) Nature 363, 83-5. [0333]
  • [0334] Reference 16 is McPherson, P, S., Czernik, A, J., Chilcote, T, J., Onofri, F., Benfenati, F., Greengard, P., Schlessinger, J., & De-Camilli, P. (1994) Proc Natl Acad Sci USA 91, 6486-90.
  • The sequences shown in Table 15 are useful in that they can be used as ligands in the assays for the identification of compounds that affect binding of SH3 domain-containing proteins and their ligands that is described above in Section 5.6. [0335]
  • 6.16 Use of Consensus Sequences to Identify Amino Acid Sequences Resembling SH3 Domain-binding Sequences in Proteins that are Not Known to Bind SH3 Domains [0336]
  • The consensus sequences shown above in Tables 1-13 can be used to search databases (e.g., GenBank) containing the amino acid sequences of proteins that are not known to bind to SH3 domains. In this way, a large number of proteins not previously suspected of containing amino acid sequences that bind SH3 domains have been shown to contain such sequences. The portions of the amino acid sequences of these proteins that resemble one or more of the consensus motifs of Tables 1-13 are shown below in Table 16. The SH3 domain-binding sequences of the proteins shown in Table 16 can be used as ligands in the assays for the identification of compounds that affect binding of SH3 domain-containing proteins and their ligands that are described above in Section 5.6. [0337]
    TABLE 16
    LOCUS ACCESSION #'S DESCRIPTION SEQUENCE SRC SRC ABL COR P53 PLC GRB CRK
    ABL_DROME P00522 TYROSINE-PROTEIN KINASE DRO 132 146 LLQSRPLPHIPAGST (296) 1
    DASH/AB
    1380 1395 QIQQKPAVPHKPPLND (297) 2 2
    ABP1_YEAST P15891 ACTIN BINDING PROTEIN SAC 514 528 SSAAPPPPPRRATPE (298) 1 2
    ACES_HUMAN P22303 ACETYLCHOLINESTERASE HOM 73 87 MGPRRFLPPEPKQPW (299) 2
    PRECURSOR
    ACM4_HUMAN P08173 MUSCARINIC HOM 276 290 PPPALPPPPRPVADK (300) 3 2
    ACETYLCHOLINE RECEPT
    ACRO_HUMAN P10323 ACROSIN PRECURSOR (EC HOM 329 343 QPPPRPLPPRPPAAQ (301) 1 2 1
    3 4 21 10
    AGIE_RAT Q00900 DNA-BINDING PROTEIN AGIE- RAT 642 656 PNLRRGLPQVPYFSL (302) 2
    BP1 (A
    ANDR_HUMAN P10275 ANDROGEN RECEPTOR HOM 368 385 ALAGPPPPPPPHPHARI (303)
    AOFB_HUMAN P27338 AMINE OXIDASE (FLAVIN- HOM 480 494 TFLERHLPSVPGLLR (304) 2
    CONTAINING)
    AP2_HUMAN P05549 TRANSCRIPTION FACTOR HOM 52 68 DFQPPYFPPPPYQPIYPQ (305) 2
    AP-2
    ATF3_HUMAN P18847 CYCLIC-AMP-DEPENDENT HOM 57 71 CFCHRPLPVPPGSLV (306) 1
    TRANSCRIPT
    B1AR_HUMAN P08588 BETA-1 ADRENERGIC HOM 282 296 APAPPPGPPRPAAAA (307) 0 0
    RECEPTOR
    B3AR_HUMAN P13945 BETA-3 ADRENERGIC HOM 361 375 CRCGRRLPPEPCAAA (308) 2
    RECEPTOR
    BCL2_CHICK Q00709 APOPTOSIS REGULATOR GAL 33 47 GEDRPPVPPAPAPAA (309)
    BCL-2
    BNI1_YEAST P41832 BNI1 PROTEIN (SYNTHETIC SAC 1242 1256 PPPPPPPVPAKLFGE (310) 4 0
    LETHAL
    CADM_MOUSE P33146 MUSCLE-CADHERIN (M- MUS 645 659 PQPHRVLPTSPSDIA (311) 3
    CADHERIN)
    CALR_PIG P25117 CALCITONIN RECEPTOR SUS 14 28 IFLNRPVLPDSAD (312) 1
    PRECURSOR
    CBL_HUMAN P22681 PROTO-ONCOGENE C-CBL HOM 490 504 QASSLPPVPPRLDLLP (313) 1 1
    536 555 PPTLRDLPPPPPPDRPYSVG 2 2 2
    (314)
    559 573 RPQRRPLPCTPGDCP (315) 2
    CCB3_RABIT Q02343 BRAIN CALCIUM CHANNEL ORY 19 33 SDQGRNLPGTPVPAS (316) 3
    BII-1 PR
    2100 2114 RHSRRQLPPVPPKPRPLL (317) 1 1 1 0
    CCB4_RABIT Q02344 BRAIN CALCIUM CHANNEL ORY 19 33 SDQGRNLPGTPVPAS (318) 3
    BII-2 PRO
    CG2A_BOVIN P30274 G2/MITOTIC-SPECIFIC BOS 56 70 NDEYVPVPPWKANNK (319) 5
    CYCLIN A
    CIC1_RAT P35524 CHLORIDE CHANNEL RAT 724 741 QTPTPPPPPPPPLPPQFP (320)
    PROTEIN SKELE
    CIK5_HUMAN P22460 POTASSIUM CHANNEL HOM 60 74 DSGVRPLPPLPDPGV (321) 0
    PROTIEN KV1 5
    71 85 DPGVRPLPPLPEELP (322) 0
    CINC_RAT P15389 SODIUM CHANNEL PROTEIN, RAT 1723 1739 LNTGPPYCDPNLPNSNG (323) 3
    CANDIAO
    CP12_RABIT P00187 CYTOCHROME P450 IA2 (EC ORY 238 252 FPILRYLPNRPLQRF (324) 3
    1 14 14
    CP75_SOLME P37120 CYTOCHROME P450 LXXVA SOL 30 44 SWRRRKLPPGPEGWP (325) 2
    (EC 1 14
    CPC7_RAT P05179 CYTOCHROME P450 IIC7 (EC RAT 23 37 SSRRRKLPPGPTPLP (326) 2
    1 14
    CPC8_HUMAN P10631 CYTOCHROME P450 IIC8 (EC HOM 23 37 SCRRRKLPPGPTPLP (327) 2
    1 14
    CPCK_MACFA P33262 CYTOCHROME P450 IIC20 (EC MAC 23 37 SSGRRKLPPGPTPLP (328) 2
    1 14
    CPCM_RAT P19225 CYTOCHROME P450 IIC22 (EC RAT 23 37 HHVRRKLPPGPTPLP (329) 2
    1 14
    CPT7_MOUSE P27786 CYTOCHROME P450 XVIIA1 MUS 25 39 AKFPRSLPFLPLVGS (330) 2
    (P450-C
    CR2_MOUSE P19070 COMPLEMENT RECEPTOR MUS 22 38 NARKPYYSLPIVPGTVL (331) 3
    TYPE 2 PREG
    CTK1_YEAST Q03957 CTD KINASE ALPHA SUBUNIT SAC 30 44 QSLARPPPPKRIRTD (332) 3 1 3
    (EC 2
    CXA3_BOVIN P41987 GAP JUNCTION ALPHA-3 BOS 287 301 ASPARALPGPPHPRR (333) 2 3 3
    PROTEIN
    CYA3_RAT P21932 ADENYLATE CYCLASE, RAT 829 843 TDSRLPLVPSKYSMT (334) 4 1
    OLFACTIVE TY
    CYGR_HUMAN Q02846 RETINAL GUANYLYL HOM 15 31 GLCGPAWWAPSLPRLPR (335) 3
    CYCLASE PRECUR
    CYLI_HUMAN P35663 CYLICIN (FRAGMENT) HOM 571 587 LCWCKMPPPPPKPRYAP (336) 2 3 2
    CYRG_MOUSE P34902 CYTOKINE RECEPTOR MUS 283 298 WLERMPPIPPIKNLED (337) 5 2
    COMMON GAMMA
    DCD_HUMAN P20711 AROMATIC-L-AMINO-ACID HOM 31 47 PDVEPGYLRPLIPAAAP (338) 3
    DECARBOXY
    DMD_HUMAN P11532 DYSTROPHIN HOM 700 714 QEELPPPPPQKKRQI (339) 1
    DPOD_BOVIN P28339 DNA POLYMERASE DELTA BOS 104 118 VAPARPLPGAPPPSQ (340) 1
    CATALYTIC
    DRA_HUMAN P40879 DRA PROTEIN (DOWN- HOM 319 335 GDMNPGFQPPITPDVET (341) 3
    REGULATED IN
    DY15_DROME P13496 150 KD DYNEIN-ASSOCIATED DRO 1250 1264 ARSARRLPSWPPTLD (342) 3
    POLYPE
    DYN1_HUMAN Q05193 DYNAMIN-1 HOM 809 823 LGGAPPVPSRPGASP (343) 1 1
    E75C_DROME P13055 ECDYSONE-INDUCIBLE DRO 398 413 VMRPPPPPPPPKVKHA (344) 3 3 1
    PROTEIN E75-
    587 601 MRHGRGLPSTPCHTS (345) 3
    EGR2_HUMAN P11161 EARLY GROWTH RESPONSE HOM 113 127 HLYSPPPPPPPYSGC (346)
    PROTEIN
    ELK1_MOUSE P41969 PROTEIN ELK-1 (FRAGMENT) MUS 164 178 PQPQPPIPPRPASVL (347) 1 1
    ENL_HUMAN Q03111 ENL PROTEIN HOM 272 286 PPPPPPPPPRASSKR (348) 1 2
    452 467 LPSREPPPPQKPPPN (349) 2
    EP15_HUMAN P42566 EPIDERMAL GROWTH HOM 763 778 KSEDEPPALPPKIGTP (350) 3 0
    FACTOR RECEPTOR
    ERB3_HUMAN P21860 ERBB-3 RECEPTOR PROTEIN- HOM 1204 1218 RRHSPPHPPRPSSLE (351) 4 2 1
    TYROSIN
    EZRI_HUMAN P15311 P23714 EZRLIN (P81) (CYTOVILLIN) HOM 465 479 VMTAPPPPPPPVYEP (352)
    (VILLI
    FAK_HUMAN Q05397 FOCAL ADHESION KINASE HOM 183 197 KEGERALPSIPKLAN (353) 2
    (EC 2 7 1
    FASL_MOUSE P41047 FAS ANTIGEN LIGAND MUS 41 55 DQRRPPPPPPPVSPL (354) 3
    FGR_FSVGR P00544 TYROSINE-PROTEINKINASE FEL 9 23 VCRPRPLPPLPPTAM (355) 0
    TRANSFO
    FOR4_MOUSE Q05859 FORMIN 4 (LIMB DEFORMITY MUS 655 669 PPLIPPPPPLPPGLG (356)
    PROTEIN
    681 700 CPVSPPPPPPPPPPTPVPPS (357)
    699 718 PSDGPPPPPPPPPPLPNVLA
    (358)
    721 740 NSGGPPPPPPPPPPPPGLAP
    (359)
    FOSB_MOUSE P13346 FOSB PROTEIN MUS 253 269 GWLLPPPPPPPLPFQSS (360)
    FOSB_CHICK P11939 P55-C-FOS PROTO-ONCOGENE GAL 239 254 LMTEAPPAVPPKEPSG (361) 3 0
    PROTEIN
    FSH_DROME P13709 P13710 FEMALE STERILE HOMEOTIC DRO 4 20 SEPPPRYEPPVEPVNGI (362) 2
    PROTEIN
    G33_RATE P05432 GENE 33 POLYPEPTIDE RAT 146 160 DRSSRPLPPLPISED (363) 0
    281 295 IPPRVPIPPRPAKPD (364) 3 3 1 3
    GLI3_HUMAN P10071 GLI3 PROTEIN HOM 789 804 MFPRLNPILPPKAPAV (365) 4 3 1
    986 1000 AAPPRLLPPLPTCYG (366) 1
    GTPA_BOVIN P09851 GTPASE-ACTIVATIGN BOS 127 141 GGGFPPLPPPPPQLP (367)
    PROTEIN (GAP)
    HME1_MOUSE P09065 HOMEOBOX PROTEIN MUS 72 91 LPHPPPPPPPPPPPPPQHLA
    ENGRAILED-1 (M (368)
    HMOC_DROME P22810 HOMEOTIC PROTEIN DRO 453 467 SAPQRPMPPNRPSPP (369) 4 1 2
    ORTHODENTICLE
    HS27_HUMAN P04792 HEAT SHOCK 27 KD PROTEIN HOM 48 64 GSSWPGYVRPLPPAAIE (370) 4
    (HSP 2
    HXA4_CHICK P17277 HOMEOBOX PROTEIN HOX- GAL 42 59 HPHAPPPPPPPPPPHLHA (371)
    A4 (CHOX-1
    127 141 GASPPPPPPAKGHPG (372) 3 5
    HXAA— P31260 HOMEOBOX PROTEIN HOX- HOM 223 237 PQQQPPPPPQPPQPA (373)
    HUMAN A10 (HOX-1
    HXB2_HUMAN P14652 P17485 P10913 HOMEOBOX PROTEIN HOX-B2 HOM 75 91 GPALPPPPPPPLPAAPP (374)
    (HOX-2H
    HXB3_HUMAN P14651 P17484 HOMEOBOX PROTEIN HOX-B3 HOM 280 296 HSMTPSYESPSPPAFGK (375) 4
    (HOX-2G
    HXB4_HUMAN P17483 HOMEOBOX PROTEIN HOX-B4 HOM 69 91 RDPGPPPPPPPPPPPPPPPGLSP
    (HOX-2 (376)
    HXC4_HUMAN P09017 HOMEOBOX PROTEIN HOX-C4 HOM 50 64 QELYPPPPPRPSYPE (377) 1
    (HOX-3
    IBP1_BOVIN P24591 INSULIN-LIKE GROWTH BOX 83 97 GLSCRALPGEPRPLH (378) 3
    FACTOR BIND
    IDE_HUMAN P14735 INSULIN-DEGRADING HOM 995 1009 TEFKRGLPLFPLVKP (379) 3
    ENZYME (EC 3
    IEFS_HUMAN P31948 TRANSFERMATION- HOM 195 211 EIATPPPPPPPKKETKP (380) 3 2
    SENSITIVE PROTEI
    IHBB_RAT P17491 INHIBIN BETA B CHAIN RAT 35 49 SPAAPPPPPPPGAPG (381)
    PRECURSOR
    IRS1_HUMAN P35568 INSULIN RECEPTOR HOM 1197 1211 PEPQPPPPPPPHQPL (382)
    SUBSTRATE-1 (I
    ISP3_SCHPO P40899 SEXUAL DIFFERENTIATION SCH 39 55 QHQQPTYWYPPPPPRHH (383) 2 3 2
    PROCESS
    JUND_CHICK P27921 TRANSCRIPTION FACTOR GAL 203 218 PRLPPPPPPPLKDEPQ (384) 4 4 2
    JUN-D
    KICH_HUMAN P35790 CHOLINE KINASE (EC 2 1 1 32) HOM 53 67 ALALPPPPPLPLPLP (385)
    KIJ5_YEAST P40494 PROBABLE SAC 744 759 KDKSRPPRPPPKPLHL (386) 2
    SERINE/THREONINE-PROTE
    KIR1_HUMAN Q04771 SERINE/THREONINE-PROTEIN HOM 450 464 VDQQRPNIPNRWFSD (387) 3 1
    KINASE
    KIR4_HUMAN P36897 SERINE/THREONINE-PROTEIN HOM 447 461 EQKLRPNIPNRWQSC (388) 1
    KINASE
    KRAF_CAEEL Q07292 RAF HOMOLOG CAE 458 473 LDAQRPRPPQKPHHED (389) 2
    SERINE/THREONINE-P
    MAPA_RAT P34926 MICROTUBULE-ASSOCIATED RAT 1812 1826 VPKDRPLPPAPLSPA (390) 0
    PROTEIN
    2421 2437 GELSPSFLNPPLPPSTD (391) 2
    MAPB_MOUSE P14873 MICROTUBULE-ASSOCIATED MUS 520 535 DLTGQVPTPPVKQVKL (392) 5
    PROTEIN
    MIS_HUMAN P03971 MUELLERIAN INHIBITING HOM 266 280 LDTVPFPPPRPSAEL (393) 2
    FACTOR
    387 401 AAELRSLPGLPPATA (394) 2
    MPK1_XENLA Q05116 DUAL SPECIFICITY MITOGEN- XEN 286 300 ELAPRPRPPGRPISS (395) 3 0 3
    ACTIVA
    MPK2_HUMAN P36507 DUAL SPECIFICITY MITOGEN- HOM 293 307 SISPRPRPPGRPVSG (396) 3 0 3
    ACTIVA
    MYBB_CHICK Q03237 MYB-RELATED PROTEIN B GAL 512 526 YGPIRPLPQTPHLEE (397) 2
    MYSA_CAEEL P12844 MYOSINE HEAVY CHAIN A CAE 561 577 LGKHPNFQKPKPPKGKQ (398) 4
    (MHC A)
    MYSB_CAEEL P02566 MYOSINE HEAVY CHAIN B CAE 559 575 LGKHPNFEKPKPPKGKQ (399) 4
    (MHC B)
    MYSC_CAEEL P12844 MYOSINE HEAVY CHAIN C CAE 562 578 LGKHPNFEKPKPPKGKQ (400) 4
    (MHC C)
    MYSD_CAEEL P02567 MYOSINE HEAVY CHAIN D CAE 556 572 LGKHPNFEKPKPPKGKQ (401) 4
    (MHC D)
    NCF1_HUMAN P14598 NEUTROPHIL CYTOSOL HOM 359 373 SKPQPAVPPRPSADL (402) 2 1
    FACTOR 1 (N
    NEU_RAT P06494 NEU ONCOGENE PRECURSOR RAT 560 574 VSDKRCLPCHPECQP (403) 3
    (EC 2 7
    NG3_DROME P40140 NEW-GLUE PROTEIN 3 DRO 33 47 LRLPPPLPPRPRQPL (404) 0 0
    PRECURSOR (
    NME4_MOUSE Q03391 GLUTAMATE (NMDA) MUS 901 915 PPAKPPPPPQPLPSP (405)
    RECEPTOR SUBU
    OIF_HUMAN PS0774 OSTEOINDUCTIVE FACTOR HOM 177 192 NQLLKLPVLPPKLTLF (406) 3
    PRECURSOR
    P11B_HUMAN P42338 PHOSPHATIDYLINOSITOL 3- HOM 309 323 SNLPLPLPPKKTRII (407) 4
    KINASE (
    P2B1_HUMAN P16298 SERINE/THREONINE PROTEIN HOM 7 25 ARAAPPPPPPPPPPPGADR 3
    PHOSPH (408)
    P53_CHICK P10360 CELLULAR TUMOR ANTIGEN GAL 45 62 EPSDPPPPPPPPPLPLAA (409)
    P53
    P85A_HUMAN P27986 PHOSPHATIDYLINOSITOL 3- HOM 89 103 PRPPRPLPVAPGSSK (410) 1
    KINASE
    P85B_BOVIN P23726 PHOSPHATIDYLINOSITOL 3- BOS 90 105 PRGPRPLPPARPRDGP (411) 2 3 0 3
    KINASE
    290 305 EQEVAPPALPPKPPKT (412) 2 0
    PFTA_RAT Q04631 PROTEIN RAT 18 34 QPEQPPPPPPPPPAQQP (413)
    FARNESYLTRANSFERASE AL
    PRGR_HUMAN P06401 PROGESTERONE RECEPTOR HOM 419 433 LGPPPPLPPRATPSR (414) 0 1
    (PR) (FOR
    PRO_DROME P29617 PROTEIN PROSPERO. DRO 1076 1090 YHPQPPPPPPPMMPV (415)
    PRPB_HUMAN P02814 PROLINE-RICH PEPTIDE P-B HOM 17 33 QPFGPGFVPPPPPPPYG (416) 2
    PTN1_HUMAN P18031 PROTEIN-TYROSINE HOM 302 316 PPEHIPPPPRPPKRI (417) 3 3 2 2
    PHOSPHATASE 1
    PTN3_HUMAN P26045 PROTEIN-TYROSINE HOM 860 874 CLTERNLPIYPLDIV (418) 3
    PHOSPHATASE P
    PTN4_HUMAN P29074 PROTEIN-TYROSINE HOM 457 472 PGDGKPPALPPKQSKK (419) 3
    PHOSPHATASE ME
    PTP1_DROME P35992 PROTEIN-TYROSINE DRO 1430 1446 FTTWPDFGVPNPPQTLV (420) 4
    PHOSPHATASE 10
    PTPK_MOUSE P35822 PROTEIN-TYROSINE MUS 60 76 SAQEPHYLPPEMPQGSY (421) 2
    PHOSPHATASE KA
    RADI_HUMAN P35241 RADIXIN HOM 466 481 VMSAPPPPPPPPVIPP (422)
    RB_HUMAN P06400 RETINOBLASTOMA- HOM 19 33 EPPAPPPPPPPEEDP (423)
    ASSOCIATED PROTE
    ROG_HUMAN P38159 HETEROGENEOUS NUCLEAR HOM 92 106 GRRGPPPPPPSRGPP (424) 4 1 2
    RIBONUCLE
    ROK_HUMAN Q07244 HETEROGENEOUS NUCLEAR HOM 267 281 GRGGRPMPPSRRDYD (425) 3 1
    RIBONUCLE
    HOM 301 321 GSRARNLPLPPPPPPRGGDL 3 1 1
    (426)
    ROL_HUMAN P14866 HETEROGENEOUS NUCLEAR HOM 326 346 SRYGPQYGHPPPPPPPPEYGP 3
    RIBONUCLE (427)
    RRG1_HUMAN P13631 RETINOIC ACID RECEPTOR HOM 76 90 SSPSPPPPPRVYKPC (428) 2 2
    GAMMA-1
    RRG2_HUMAN P22932 RETINOIC ACID RECEPTOR HOM 65 79 SSPSPPPPPRVYKPC (429) 2 2
    GAMMA-2
    RRXB_HUMAN P28702 RETINOIC ACID RECEPTOR HOM 95 109 GSGAPPPPPMPPPPL (430)
    RXR-BETA
    RRXC_HUMAN P28703 RETINOIC ACID RECEPTOR 115 129 GSGAPPPPPMPPPPL (431)
    RXR-BETA
    RYNR_HUMAN P21817 RYANODINE RECEPTOR, HOM 4516 4531 PKKQAPPSPPPKKEEA (432) 4
    SKELETAL MU
    SHC_HUMAN P29353 SHC TRANSFORMING HOM 297 311 RKQMPPPPPCPGREL (433)
    PROTEINS 46 8
    SLP1_DROME P32030 FORK HEAD DOMAIN DRO 242 258 GAPAPSYGYPAVPFAAA (434) 3
    TRANSCRIPTION
    SOS_DROME P36675 SON OF SEVENLESS PROTEIN DRO 1339 1353 RAVPPPLPPRRKERT (435) 0 1
    1377 1391 ELSPPPIPPRLNHST (436) 0 1
    ST20_YEAST Q03497 SERINE/THREONINE-PROTEIN SAC 533 547 EQPLPPIPPTKSKTS (437)
    KINASE
    SUF_DROME P25991 SUPPRESSOR OR FORKED DRO 229 243 KGLNRNLPAVPPTLT (438) 2
    PROTEIN
    SXLF_DROME P19339 SEX-LETHAL PROTEIN, DRO 308 322 PANVPPPPPQPPAHM (439)
    FEMALE-SPEC
    TACT_HUMAN P40200 T-CELL SURFACE PROTEIN HOM 538 553 PPPFKPPPPPIKYTCI (440) 1 4
    TACTILE
    TGFB_HUMAN P22064 TRANSFORMING GROWTH HOM 440 454 KSTHPPPLPAKEEPV (441) 3
    FACTOR BETA
    TIE2-MOUSE Q02858 TYROSINE-PROTEIN KINASE MUS 725 739 SHELRTLPHSPASAD (442) 3
    RECEPTOR
    TJ6_MOUSE P15920 IMMUNE SUPPRESSOR MUS 81 96 EGEASPPAPPLKHVLE (443)
    FACTOR J6B7
    TLL_DROME P18102 TAILLESS PROTEIN DRO 214 228 ALATRALPPTPPLMA (444) 2
    TOP1_HUMAN P11387 DNA TOPOISOMERASE I (EC HOM 221 237 EHKGPVFAPPYEPLPEN (445) 3
    5 99 1
    TOPA_HUMAN P11388 DNA TOPOISOMERASE II, HOM 833 849 QRVEPEWYIPIIPMVLI (446) 3
    ALPHA ISO
    TOPB_HUMAN Q02880 DNA TOPOISOMERASE II, HOM 855 871 QRVEPEWYIPIIPMVLI (447) 3
    BETA ISOZ
    TRA1_HUMAN P34708 SEX-DETERMINING CAE 1069 1090 PEDDPIYALPPPPPPPAPPRRR 1 3 1
    TRANSFORMER PRO (448)
    TRT1_HUMAN P13805 TROPONIN T, SLOW HOM 42 57 SRPVVPPLIPPKIPEG (449) 3
    SKELETAL MUSCLE
    XA1_XENLA P23507 XA-1 PROTEIN PRECURSOR XEN 23 39 GEDSPVFRPPSPPMGPS (450) 2
    121 136 FRTGRPLLPIKPEHGR (451) 2
    Z01_HUMAN Q07157 TIGHT JUNCTION PROTEIN HOM 1410 1424 IQATPPPPPLPSQYA (452)
    ZO-1
    ZYX_CHICK Q04584 ZYXIN GAL 120 134 AFPSPPPPPPPMFDE (453)
  • In Table 16, locus and accession number refer to the entries' names and accession numbers in GenBank or the Swiss-Prot database. The two numbers immediately to the left of the displayed sequences refer to the amino acid positions of the displayed sequences in the mature proteins. The leftmost of these two numbers refers to the starting amino acid number of the displayed sequence in the mature protein. The numbers in parentheses immediately to the right of the displayed sequences refer to the sequences' SEQ ID NOs:. The eight columns to the extreme right of Table 16 show the discrepancies between the displayed sequences and the consensus motifs of Tables 6-15. The leftmost Src column refers to Class I motifs; the rightmost Src column refers to Class II motifs. [0338]
  • It should be apparent to one of ordinary skill that many other embodiments of the present invention can be contemplated beyond the preferred embodiments described above but which other embodiments nevertheless fall within the scope and spirit of the present invention. Hence, the present invention should not be construed to be limited to the preferred embodiments described herein, which serve only to illustrate the present invention, but only by the claims that follow. [0339]
  • Also, numerous references are cited throughout the specification. The complete disclosures of these references are incorporated by reference herein. [0340]
  • 0
    SEQUENCE LISTING
    (1) GENERAL INFORMATION:
    (iii) NUMBER OF SEQUENCES: 467
    (2) INFORMATION FOR SEQ ID NO:1:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 10 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (ix) FEATURE:
    (A) NAME/KEY: Modified-site
    (B) LOCATION: 8
    (D) OTHER INFORMATION: /note= “X = a hydrophobic amino
    acid residue, such as Pro or Leu”
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
    Xaa Pro Xaa Xaa Pro Pro Pro Xaa Xaa Pro
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:2:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 9 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (ix) FEATURE:
    (A) NAME/KEY: Modified-site
    (B) LOCATION: 1..3
    (D) OTHER INFORMATION: /note= “X = any amino acid residue
    other than Cys”
    (ix) FEATURE:
    (A) NAME/KEY: Modified-site
    (B) LOCATION: 6
    (D) OTHER INFORMATION: /note= “X = any amino acid residue
    other than Cys”
    (ix) FEATURE:
    (A) NAME/KEY: Modified-site
    (B) LOCATION: 8..9
    (D) OTHER INFORMATION: /note= “X = any amino acid residue
    other than Cys”
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
    Xaa Xaa Xaa Pro Pro Xaa Pro Xaa Xaa
    1 5
    (2) INFORMATION FOR SEQ ID NO:3:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 9 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (ix) FEATURE:
    (A) NAME/KEY: Modified-site
    (B) LOCATION: 2
    (D) OTHER INFORMATION: /note= “X tends to be a basic amino
    acid residue (like Arg, Lys or His)”
    (ix) FEATURE:
    (A) NAME/KEY: Modified-site
    (B) LOCATION: 8..9
    (D) OTHER INFORMATION: /note= “X tends to be a basic amino
    acid residue (like Arg, Lys and His)”
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
    Arg Xaa Leu Pro Pro Arg Pro Xaa Xaa
    1 5
    (2) INFORMATION FOR SEQ ID NO:4:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 9 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
    Arg Lys Leu Pro Pro Arg Pro Arg Arg
    1 5
    (2) INFORMATION FOR SEQ ID NO:5:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 10 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
    Val Pro Pro Pro Val Pro Pro Arg Arg Arg
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:6:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 7 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (ix) FEATURE:
    (A) NAME/KEY: Modified-site
    (B) LOCATION: 2
    (D) OTHER INFORMATION: /note= “X tends to be Pro”
    (ix) FEATURE:
    (A) NAME/KEY: Modified-site
    (B) LOCATION: 3
    (D) OTHER INFORMATION: /note= “X = a hydrophobic amino
    acid residue”
    (ix) FEATURE:
    (A) NAME/KEY: Modified-site
    (B) LOCATION: 5
    (D) OTHER INFORMATION: /note= “X tends to be Pro”
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
    Xaa Xaa Xaa Pro Xaa Xaa Pro
    1 5
    (2) INFORMATION FOR SEQ ID NO:7:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 9 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (ix) FEATURE:
    (A) NAME/KEY: Modified-site
    (B) LOCATION: 2
    (D) OTHER INFORMATION: /note= “X= any amino acid residue
    other than Cys”
    (ix) FEATURE:
    (A) NAME/KEY: Modified-site
    (B) LOCATION: 8..9
    (D) OTHER INFORMATION: /note= “X = any amino acid residue
    other than Cys”
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
    Arg Xaa Leu Pro Pro Arg Pro Xaa Xaa
    1 5
    (2) INFORMATION FOR SEQ ID NO:8:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 9 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (ix) FEATURE:
    (A) NAME/KEY: Modified-site
    (B) LOCATION: 9
    (D) OTHER INFORMATION: /note= “X = any hydrophobic amino
    acid residue”
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
    Arg Xaa Leu Pro Pro Leu Pro Arg Xaa
    1 5
    (2) INFORMATION FOR SEQ ID NO:9:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 7 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
    Arg Pro Leu Pro Pro Leu Pro
    1 5
    (2) INFORMATION FOR SEQ ID NO:10:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 9 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (ix) FEATURE:
    (A) NAME/KEY: Modified-site
    (B) LOCATION: 2
    (D) OTHER INFORMATION: /note= “X = any amino acid residue
    except Cys”
    (ix) FEATURE:
    (A) NAME/KEY: Modified-site
    (B) LOCATION: 5..6
    (D) OTHER INFORMATION: /note= “X = any hydrophobic amino
    acid residue”
    (ix) FEATURE:
    (A) NAME/KEY: Modified-site
    (B) LOCATION: 8
    (D) OTHER INFORMATION: /note= “X = any amino acid residue
    except Cys”
    (ix) FEATURE:
    (A) NAME/KEY: Modified-site
    (B) LOCATION: 9
    (D) OTHER INFORMATION: /note= “X = any hydrophilic amino
    acid residue except Cys”
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
    Arg Xaa Leu Pro Xaa Xaa Pro Xaa Xaa
    1 5
    (2) INFORMATION FOR SEQ ID NO:11:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 9 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
    Arg Pro Leu Pro Pro Leu Pro Thr Ser
    1 5
    (2) INFORMATION FOR SEQ ID NO:12:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 4 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (v) FRAGMENT TYPE: C-terminal
    (ix) FEATURE:
    (A) NAME/KEY: Modified-site
    (B) LOCATION: 1..4
    (D) OTHER INFORMATION: /note= “X = any amino acid residue
    except Cys”
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
    Xaa Xaa Xaa Xaa
    1
    (2) INFORMATION FOR SEQ ID NO:13:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 5 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (v) FRAGMENT TYPE: C-terminal
    (ix) FEATURE:
    (A) NAME/KEY: Modified-site
    (B) LOCATION: 1..5
    (D) OTHER INFORMATION: /note= “X = any amino acid residue
    except Cys”
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
    Xaa Xaa Xaa Xaa Xaa
    1 5
    (2) INFORMATION FOR SEQ ID NO:14:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 4 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (v) FRAGMENT TYPE: N-terminal
    (ix) FEATURE:
    (A) NAME/KEY: Modified-site
    (B) LOCATION: 1..4
    (D) OTHER INFORMATION: /note= “X = any amino acid residue
    except Cys”
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
    Xaa Xaa Xaa Xaa
    1
    (2) INFORMATION FOR SEQ ID NO:15:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 13 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (ix) FEATURE:
    (A) NAME/KEY: Modified-site
    (B) LOCATION: 1..3
    (D) OTHER INFORMATION: /note= “X = any amino acid residue
    except Cys”
    (ix) FEATURE:
    (A) NAME/KEY: Modified-site
    (B) LOCATION: 5
    (D) OTHER INFORMATION: /note= “X = any amino acid residue
    except Cys”
    (ix) FEATURE:
    (A) NAME/KEY: Modified-site
    (B) LOCATION: 8..9
    (D) OTHER INFORMATION: /note= “X = any hydrophobic amino
    acid residue”
    (ix) FEATURE:
    (A) NAME/KEY: Modified-site
    (B) LOCATION: 11
    (D) OTHER INFORMATION: /note= “X = any amino acid residue
    except Cys”
    (ix) FEATURE:
    (A) NAME/KEY: Modified-site
    (B) LOCATION: 12
    (D) OTHER INFORMATION: /note= “X = any hydrophilic amino
    acid residue except Cys”
    (ix) FEATURE:
    (A) NAME/KEY: Modified-site
    (B) LOCATION: 13
    (D) OTHER INFORMATION: /note= “X = any amino acid residue
    except Cys”
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
    Xaa Xaa Xaa Arg Xaa Leu Pro Xaa Xaa Pro Xaa Xaa Xaa
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:16:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 42 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: peptide
    (ix) FEATURE:
    (A) NAME/KEY: Modified-site
    (B) LOCATION: 2
    (D) OTHER INFORMATION: /note= “X = Arg or Ser”
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
    Ser Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
    1 5 10 15
    Xaa Xaa Xaa Xaa Pro Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
    20 25 30
    Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser Arg
    35 40
    (2) INFORMATION FOR SEQ ID NO:17:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 69 bases
    (B) TYPE: nucleic acid
    (C) STRANDEDNESS: single
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: DNA
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
    GGCTCGAGNN NBNNBNNBNN BNNBNNBNNB NNBNNBNNBN NBNNBNNBNN BNNBNNBNNB 60
    NNBCCAGGT 69
    (2) INFORMATION FOR SEQ ID NO:18:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 68 bases
    (B) TYPE: nucleic acid
    (C) STRANDEDNESS: single
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: DNA
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
    GGTCTAGAVN NVNNVNNVNN VNNVNNVNNV NNVNNVNNVN NVNNVNNVNN VNNVNNVNNV 60
    NNACCTGG 68
    (2) INFORMATION FOR SEQ ID NO:19:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 66 bases
    (B) TYPE: nucleic acid
    (C) STRANDEDNESS: single
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: DNA
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
    TCGAGNNNBN NBNNBNNBNN BNNBNNBNNB NNBNNBNNBN NBNNBNNBNN BNNBNNBNNB 60
    CCAGGT 66
    (2) INFORMATION FOR SEQ ID NO:20:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 65 bases
    (B) TYPE: nucleic acid
    (C) STRANDEDNESS: single
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: DNA
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
    CTAGAVNNVN NVNNVNNVNN VNNVNNVNNV NNVNNVNNVN NVNNVNNVNN VNNVNNVNNA 60
    CCTGG 65
    (2) INFORMATION FOR SEQ ID NO:21:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 46 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: protein
    (v) FRAGMENT TYPE: N-terminal
    (ix) FEATURE:
    (A) NAME/KEY: Modified-site
    (B) LOCATION: 2
    (D) OTHER INFORMATION: /note= “X = Arg or Ser”
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
    Ser Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
    1 5 10 15
    Xaa Xaa Xaa Xaa Pro Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
    20 25 30
    Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser Arg Pro Ser Arg Thr
    35 40 45
    (2) INFORMATION FOR SEQ ID NO:22:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 49 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: protein
    (ix) FEATURE:
    (A) NAME/KEY: Modified-site
    (B) LOCATION: 5
    (D) OTHER INFORMATION: /note= “X = Arg or Ser”
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
    Ser His Ser Ser Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
    1 5 10 15
    Xaa Xaa Xaa Xaa Xaa Xaa Xaa Pro Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa
    20 25 30
    Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser Arg Pro Ser Arg
    35 40 45
    Thr
    (2) INFORMATION FOR SEQ ID NO:23:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 27 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: peptide
    (ix) FEATURE:
    (A) NAME/KEY: Modified-site
    (B) LOCATION: 2
    (D) OTHER INFORMATION: /note= “X = Ser or Thr”
    (ix) FEATURE:
    (A) NAME/KEY: Modified-site
    (B) LOCATION: 13
    (D) OTHER INFORMATION: /note= “X = Ser, Arg, Gly, Cys or
    Trp”
    (ix) FEATURE:
    (A) NAME/KEY: Modified-site
    (B) LOCATION: 15
    (D) OTHER INFORMATION: /note= “X = Val, Ala, Asp, Glu, or
    Gly”
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
    Ser Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly Xaa Xaa
    1 5 10 15
    Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Thr Arg
    20 25
    (2) INFORMATION FOR SEQ ID NO:24:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 48 bases
    (B) TYPE: nucleic acid
    (C) STRANDEDNESS: single
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: DNA
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
    TTTTGTCGAC NNNBNNBNNB NNBNNBNNBN NBNNBNNBNN BNGCGGTG 48
    (2) INFORMATION FOR SEQ ID NO:25:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 48 bases
    (B) TYPE: nucleic acid
    (C) STRANDEDNESS: single
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: DNA
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
    TTTTACTAGT VNNVNNVNNV NNVNNVNNVN NVNNVNNVNN VNCACCGC 48
    (2) INFORMATION FOR SEQ ID NO:26:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 43 bases
    (B) TYPE: nucleic acid
    (C) STRANDEDNESS: single
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: DNA
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:
    TCGACNNNBN NBNNBNNBNN BNNBNNBNNB NNBNNBNGCG GTG 43
    (2) INFORMATION FOR SEQ ID NO:27:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 43 bases
    (B) TYPE: nucleic acid
    (C) STRANDEDNESS: single
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: DNA
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:
    CTAGTVNNVN NVNNVNNVNN VNNVNNVNNV NNVNNVNCAC CGC 43
    (2) INFORMATION FOR SEQ ID NO:28:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 31 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: protein
    (v) FRAGMENT TYPE: N-terminal
    (ix) FEATURE:
    (A) NAME/KEY: Modified-site
    (B) LOCATION: 2
    (D) OTHER INFORMATION: /note= “X = Ser or Thr”
    (ix) FEATURE:
    (A) NAME/KEY: Modified-site
    (B) LOCATION: 13
    (D) OTHER INFORMATION: /note= “X = Ser, Arg, Gly, Cys or
    Trp”
    (ix) FEATURE:
    (A) NAME/KEY: Modified-site
    (B) LOCATION: 15
    (D) OTHER INFORMATION: /note= “X = Val, Ala, Asp, Glu or
    Gly”
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:
    Ser Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly Xaa Xaa
    1 5 10 15
    Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Thr Arg Pro Ser Arg Thr
    20 25 30
    (2) INFORMATION FOR SEQ ID NO:29:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 34 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: protein
    (ix) FEATURE:
    (A) NAME/KEY: Modified-site
    (B) LOCATION: 5
    (D) OTHER INFORMATION: /note= “X = Ser or Thr”
    (ix) FEATURE:
    (A) NAME/KEY: Modified-site
    (B) LOCATION: 16
    (D) OTHER INFORMATION: /note= “X = Ser, Arg, Gly, Cys or
    Trp”
    (ix) FEATURE:
    (A) NAME/KEY: Modified-site
    (B) LOCATION: 18
    (D) OTHER INFORMATION: /note= “X = Val, Ala, Asp, Glu or
    Gly”
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:
    Ser His Ser Ser Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
    1 5 10 15
    Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Thr Arg Pro Ser
    20 25 30
    Arg Thr
    (2) INFORMATION FOR SEQ ID NO:30:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 8 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: peptide
    (v) FRAGMENT TYPE: internal
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:
    Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
    1 5
    (2) INFORMATION FOR SEQ ID NO:31:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 58 bases
    (B) TYPE: nucleic acid
    (C) STRANDEDNESS: single
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: DNA
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:
    TGACGTCTCG AGTTGTNNKN NKNNKNNKNN KNNKNNKNNK TGTGGATCTA GAAGGATC 58
    (2) INFORMATION FOR SEQ ID NO:32:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 bases
    (B) TYPE: nucleic acid
    (C) STRANDEDNESS: single
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: DNA
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:
    GATCCTTCTA GATCC 15
    (2) INFORMATION FOR SEQ ID NO:33:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 40 bases
    (B) TYPE: nucleic acid
    (C) STRANDEDNESS: single
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: DNA
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:
    TCGAGTTGTN NKNNKNNKNN KNNKNNKNNK NNKTGTGGAT 40
    (2) INFORMATION FOR SEQ ID NO:34:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 40 bases
    (B) TYPE: nucleic acid
    (C) STRANDEDNESS: single
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: DNA
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:
    CTAGATCCAC AMNNMNNMNN MNNMNNMNNM NNMNNACAAC 40
    (2) INFORMATION FOR SEQ ID NO:35:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 19 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: protein
    (v) FRAGMENT TYPE: N-terminal
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:
    Ser Ser Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Gly Ser Arg Pro
    1 5 10 15
    Ser Arg Thr
    (2) INFORMATION FOR SEQ ID NO:36:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 28 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: protein
    (v) FRAGMENT TYPE: N-terminal
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:
    Ser Ser Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Gly Ser Arg Ser
    1 5 10 15
    Thr Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Thr Thr Arg
    20 25
    (2) INFORMATION FOR SEQ ID NO:37:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 80 bases
    (B) TYPE: nucleic acid
    (C) STRANDEDNESS: double
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: DNA
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:
    TCGAGTTGTN NKNNKNNKNN KNNKNNKNNK NNKTGTGGAT CTAGATCCAC AMNNMNNMNN 60
    MNNMNNMNNM NNMNNACAAC 80
    (2) INFORMATION FOR SEQ ID NO:38:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 45 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:
    Ser Ser Phe Asp Gln Gln Asp Trp Asp Tyr Ser Ile Ala Glu Lys Met
    1 5 10 15
    His Pro Ile Arg Pro Gly Phe Arg Glu Leu Pro Pro Leu Pro Pro Ser
    20 25 30
    Arg Ala Ser Phe Gly Gly Gly Ala Ser Arg Pro Ser Arg
    35 40 45
    (2) INFORMATION FOR SEQ ID NO:39:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 30 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:
    Ser Thr Asn Val Trp Val Thr Gly Ser Val Ile Ala Arg Gly Ala Gln
    1 5 10 15
    Ser Arg Pro Leu Pro Ile Pro Pro Glu Thr Arg Pro Ser Arg
    20 25 30
    (2) INFORMATION FOR SEQ ID NO:40:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 30 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:
    Ser Thr Ala Pro Trp Gly Leu Arg Val Ala His Glu Gly Gly Val Leu
    1 5 10 15
    Lys Arg Pro Leu Pro Ile Pro Pro Val Thr Arg Pro Ser Arg
    20 25 30
    (2) INFORMATION FOR SEQ ID NO:41:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 45 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:
    Ser Ser Ser Gly Tyr Val Val Pro Lys Arg Leu Gly Asp Met Arg Glu
    1 5 10 15
    Tyr Asn Ala His Pro Gly Leu His Val Pro Pro Asn Ser Pro Leu Pro
    20 25 30
    Pro Leu Pro Thr His Leu Gln Ser Ser Arg Pro Ser Arg
    35 40 45
    (2) INFORMATION FOR SEQ ID NO:42:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 44 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:
    Ser Ser Arg Gly Glu Gly Asn Asn Ile Ile Ser Ser Arg Pro Phe Leu
    1 5 10 15
    Ser Asn Ser Asp Pro Gly Val Ser Asn Lys Leu Thr Gly Arg Gly Pro
    20 25 30
    Leu Pro Pro Leu Pro Asn Asp Ser Arg Pro Ser Arg
    35 40
    (2) INFORMATION FOR SEQ ID NO:43:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 31 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:
    Ser Thr Ala Val Ser Phe Arg Phe Met Pro Gly Gly Gly Gly Ala Phe
    1 5 10 15
    Tyr Ser Thr Arg Pro Val Pro Pro Ile Thr Arg Pro Ser Arg Thr
    20 25 30
    (2) INFORMATION FOR SEQ ID NO:44:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 31 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:
    Ser Thr Ala His Ser Leu Trp Asp Trp Gly Thr Phe Ser Gly Val Ser
    1 5 10 15
    His Lys Ser Arg Leu Pro Pro Leu Pro Thr Arg Pro Ser Arg Thr
    20 25 30
    (2) INFORMATION FOR SEQ ID NO:45:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 26 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:
    Xaa Pro Gly Tyr Ala Arg Ile Val Ser Tyr Arg Phe Arg Ala Leu Pro
    1 5 10 15
    Ser Pro Pro Ser Ala Ser Arg Pro Ser Arg
    20 25
    (2) INFORMATION FOR SEQ ID NO:46:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 30 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:
    Ser Thr Asn Asp Val Asp Trp Met His Met Trp Asn Ser Gly Gly Pro
    1 5 10 15
    His Arg Arg Leu Pro Pro Thr Pro Ala Thr Arg Pro Ser Arg
    20 25 30
    (2) INFORMATION FOR SEQ ID NO:47:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 45 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:
    Ser Ser Asp Asn Trp Ala Arg Arg Val His Ala Ser Glu Leu Ile Tyr
    1 5 10 15
    Thr Asp Leu Ser Pro Gly Ile Leu Leu Ala Gln Arg Gln Leu Pro Pro
    20 25 30
    Thr Pro Gly Arg Asp Pro Ser His Ser Arg Pro Ser Arg
    35 40 45
    (2) INFORMATION FOR SEQ ID NO:48:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 45 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:
    Ser Ser Glu Ser Pro Leu Met Tyr Asn Arg Val Gly Ala Leu Gln Ser
    1 5 10 15
    Leu Thr Ser Val Pro Gly Ser Met Met His Phe Ala Leu Gln Arg Arg
    20 25 30
    Leu Pro Arg Thr Pro Pro Pro Ala Ser Arg Pro Ser Arg
    35 40 45
    (2) INFORMATION FOR SEQ ID NO:49:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 35 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:
    Ser Thr Arg Trp Ser His Ser Trp Pro Gly Tyr Val Gly Gly Ala Asn
    1 5 10 15
    Pro Ser Pro Ala Thr Arg Pro Leu Pro Thr Arg Pro Ser Arg Thr Val
    20 25 30
    Glu Ser Cys
    35
    (2) INFORMATION FOR SEQ ID NO:50:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 50 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:
    Ser Arg Tyr Asn Asp Leu Gly Thr Arg Pro Val Ser Glu Val Ile Lys
    1 5 10 15
    Tyr Asp Tyr Phe Pro Gly Tyr Ser Gln His Val Ile Thr Pro Asp Gly
    20 25 30
    Ser Tyr Ser Thr Arg Pro Leu Pro Ser Arg Pro Ser Arg Thr Val Glu
    35 40 45
    Ser Cys
    50
    (2) INFORMATION FOR SEQ ID NO:51:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 26 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:
    Xaa Pro Gly Arg Ile Leu Leu Leu Pro Ser Glu Pro Arg Thr Phe Tyr
    1 5 10 15
    Asn Tyr Gly His Asp Ser Arg Pro Ser Arg
    20 25
    (2) INFORMATION FOR SEQ ID NO:52:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 30 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:
    Ser Thr Met Tyr Gly Val Ser Trp Leu Ser Ser Gly Ser Gly Gly Ile
    1 5 10 15
    Leu Ala Pro Pro Val Pro Pro Arg Asn Thr Arg Pro Ser Arg
    20 25 30
    (2) INFORMATION FOR SEQ ID NO:53:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 31 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:
    Ser Ser Cys Thr Glu Lys Thr Val Ser Gly Trp Cys Gly Ser Arg Ser
    1 5 10 15
    Thr Arg Pro Leu Pro Ile Leu Pro Arg Thr Thr Arg Pro Ser Arg
    20 25 30
    (2) INFORMATION FOR SEQ ID NO:54:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 31 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:
    Ser Ser Cys Met Leu Pro Thr Asp Gly Trp Gln Cys Gly Ser Arg Ser
    1 5 10 15
    Thr Pro Arg Pro Leu Pro Met Leu Pro Thr Thr Arg Pro Ser Arg
    20 25 30
    (2) INFORMATION FOR SEQ ID NO:55:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 31 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:
    Ser Ser Cys Asp Gly Thr Gln Phe Arg Leu Asn Cys Gly Ser Arg Ser
    1 5 10 15
    Thr Asn Arg Pro Leu Pro Met Ile Pro Thr Thr Arg Pro Ser Arg
    20 25 30
    (2) INFORMATION FOR SEQ ID NO:56:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 31 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:
    Ser Ser Cys Met Gln Gly Gln Ala Gly Leu Lys Cys Gly Ser Arg Ser
    1 5 10 15
    Thr Arg Pro Leu Pro Ser Leu Pro Ile Thr Thr Arg Pro Ser Arg
    20 25 30
    (2) INFORMATION FOR SEQ ID NO:57:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 31 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:
    Ser Ser Cys Tyr Arg Glu Lys Asp Thr Trp Gly Cys Gly Ser Arg Ser
    1 5 10 15
    Thr Ser Arg Pro Leu Pro Ser Leu Pro Thr Thr Arg Pro Ser Arg
    20 25 30
    (2) INFORMATION FOR SEQ ID NO:58:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 31 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:
    Ser Ser Cys Leu Phe Glu Gln Gly Ala Gly Thr Cys Gly Ser Arg Ser
    1 5 10 15
    Thr Arg Ser Leu Pro Pro Leu Pro Pro Thr Thr Arg Pro Ser Arg
    20 25 30
    (2) INFORMATION FOR SEQ ID NO:59:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 44 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:
    Ser Ser Cys Asp Thr Gly Arg Ile Ala Pro Gly Cys Gly Ser Arg Ser
    1 5 10 15
    Thr Pro Arg Pro Leu Pro Leu Ile Pro Thr Thr Pro Arg Ser Thr Asn
    20 25 30
    Leu Asn Leu Thr Ser Thr Thr Thr Arg Pro Ser Arg
    35 40
    (2) INFORMATION FOR SEQ ID NO:60:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 31 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:
    Ser Ser Cys Gly Leu Asp Asn Ala Ala Lys Thr Cys Gly Ser Arg Ser
    1 5 10 15
    Thr Arg Pro Leu Pro Pro Thr Pro Leu Thr Thr Arg Pro Ser Arg
    20 25 30
    (2) INFORMATION FOR SEQ ID NO:61:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 31 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:
    Ser Ser Cys Ser Arg Ala His Glu Thr Glu Met Cys Gly Ser Arg Ser
    1 5 10 15
    Thr Arg Pro Gln Pro Pro Pro Pro Ile Thr Thr Arg Pro Ser Arg
    20 25 30
    (2) INFORMATION FOR SEQ ID NO:62:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 14 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:
    Arg Ser Thr Pro Arg Pro Leu Pro Met Leu Pro Thr Thr Arg
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:63:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 13 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:
    Gly Ile Leu Ala Pro Pro Val Pro Pro Arg Asn Thr Arg
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:64:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 13 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:
    Val Leu Lys Arg Pro Leu Pro Ile Pro Pro Val Thr Arg
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:65:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 13 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:
    Gly Pro His Arg Arg Leu Pro Pro Thr Pro Ala Thr Arg
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:66:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 13 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:66:
    Ala Asn Pro Ser Pro Ala Thr Arg Pro Leu Pro Thr Arg
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:67:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 14 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67:
    Arg Ser Thr Pro Arg Pro Leu Pro Pro Leu Pro Thr Thr Arg
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:68:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 19 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:68:
    Gly Thr Val Glu Pro Val Pro Pro Pro Val Pro Pro Arg Arg Arg Pro
    1 5 10 15
    Glu Ser Ala
    (2) INFORMATION FOR SEQ ID NO:69:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 17 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:69:
    Lys Ile Ser Pro Pro Thr Pro Lys Pro Arg Pro Pro Arg Pro Leu Pro
    1 5 10 15
    Val
    (2) INFORMATION FOR SEQ ID NO:70:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 13 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:70:
    Leu Ala Pro Pro Lys Pro Pro Leu Pro Glu Gly Glu Val
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:71:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 7 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (ix) FEATURE:
    (A) NAME/KEY: Modified-site
    (B) LOCATION: 2
    (D) OTHER INFORMATION: /note= “X = any amino acid residue
    except cysteine”
    (ix) FEATURE:
    (A) NAME/KEY: Modified-site
    (B) LOCATION: 5..6
    (D) OTHER INFORMATION: /note= “X = a hydrophobic amino
    acid residue”
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:71:
    Arg Xaa Leu Pro Xaa Xaa Pro
    1 5
    (2) INFORMATION FOR SEQ ID NO:72:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 13 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:72:
    Pro Gly Phe Arg Glu Leu Pro Pro Leu Pro Pro Ser Arg
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:73:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 13 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:73:
    Ala Gln Ser Arg Pro Leu Pro Ile Pro Pro Glu Thr Arg
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:74:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 13 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:74:
    Pro Pro Asn Ser Pro Leu Pro Pro Leu Pro Thr His Leu
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:75:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 13 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:75:
    Thr Gly Arg Gly Pro Leu Pro Pro Leu Pro Asn Asp Ser
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:76:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 13 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:76:
    Tyr Ser Thr Arg Pro Val Pro Pro Ile Thr Arg Pro Ser
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:77:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 13 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:77:
    Ser His Lys Ser Arg Leu Pro Pro Leu Pro Thr Arg Pro
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:78:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 13 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:78:
    Tyr Arg Phe Arg Ala Leu Pro Ser Pro Pro Ser Ala Ser
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:79:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 13 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:79:
    Leu Ala Gln Arg Gln Leu Pro Pro Thr Pro Gly Arg Asp
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:80:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 13 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:80:
    Ala Leu Gln Arg Arg Leu Pro Arg Thr Pro Pro Pro Ala
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:81:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 13 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:81:
    Pro Ala Thr Arg Pro Leu Pro Thr Arg Pro Ser Arg Thr
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:82:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 13 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:82:
    Tyr Ser Thr Arg Pro Leu Pro Ser Arg Pro Ser Arg Thr
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:83:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 13 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:83:
    Xaa Pro Gly Arg Ile Leu Leu Leu Pro Ser Glu Pro Arg
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:84:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 13 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:84:
    Ser Gly Gly Ile Leu Ala Pro Pro Val Pro Pro Arg Asn
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:85:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 13 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:85:
    Arg Ser Thr Arg Pro Leu Pro Ile Leu Pro Arg Thr Thr
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:86:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 13 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:86:
    Ser Thr Pro Arg Pro Leu Pro Met Leu Pro Thr Thr Arg
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:87:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 13 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:87:
    Ser Thr Asn Arg Pro Leu Pro Met Ile Pro Thr Thr Arg
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:88:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 13 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:88:
    Arg Ser Thr Arg Pro Leu Pro Ser Leu Pro Ile Thr Thr
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:89:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 13 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:89:
    Ser Thr Ser Arg Pro Leu Pro Ser Leu Pro Thr Thr Arg
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:90:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 13 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:90:
    Arg Ser Thr Arg Ser Leu Pro Pro Leu Pro Pro Thr Thr
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:91:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 13 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:91:
    Arg Ser Thr Arg Gln Leu Pro Ile Pro Pro Thr Thr Thr
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:92:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 13 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:92:
    Ser Thr Pro Arg Pro Leu Pro Leu Ile Pro Thr Thr Pro
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:93:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 13 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:93:
    Arg Ser Thr Arg Pro Leu Pro Pro Thr Pro Leu Thr Thr
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:94:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 13 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:94:
    Arg Ser Thr Arg Pro Gln Pro Pro Pro Pro Ile Thr Thr
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:95:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 7 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:95:
    Arg Pro Leu Pro Met Leu Pro
    1 5
    (2) INFORMATION FOR SEQ ID NO:96:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 7 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (ix) FEATURE:
    (A) NAME/KEY: Modified-site
    (B) LOCATION: 5
    (D) OTHER INFORMATION: /note= “X = Pro or Met”
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:96:
    Arg Pro Leu Pro Xaa Leu Pro
    1 5
    (2) INFORMATION FOR SEQ ID NO:97:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 4 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:97:
    Arg Ser Thr Pro
    1
    (2) INFORMATION FOR SEQ ID NO:98:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 14 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:98:
    Arg Ser Thr Pro Ala Pro Pro Val Pro Pro Arg Thr Thr Arg
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:99:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 17 bases
    (B) TYPE: nucleic acid
    (C) STRANDEDNESS: single
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: DNA
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:99:
    AGCGTAACGA TCTCCCG 17
    (2) INFORMATION FOR SEQ ID NO:100:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 20 bases
    (B) TYPE: nucleic acid
    (C) STRANDEDNESS: single
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: DNA
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:100:
    TTCACCTCGA AAGCAAGCTG 20
    (2) INFORMATION FOR SEQ ID NO:101:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 19 bases
    (B) TYPE: nucleic acid
    (C) STRANDEDNESS: single
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: DNA
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:101:
    CCTCATAGTT AGCGTAACG 19
    (2) INFORMATION FOR SEQ ID NO:102:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 bases
    (B) TYPE: nucleic acid
    (C) STRANDEDNESS: single
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: DNA
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:102:
    AGCGTAACGA TCTAAA 16
    (2) INFORMATION FOR SEQ ID NO:103:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 5 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:103:
    Lys Ser Gly Ser Gly
    1 5
    (2) INFORMATION FOR SEQ ID NO:104:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 4 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:104:
    Gly Ser Gly Ser
    1
    (2) INFORMATION FOR SEQ ID NO:105:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 11 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:105:
    Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:106:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 31 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:106:
    Ser Ser Cys Asp His Thr Leu Gly Leu Gly Trp Cys Gly Ser Arg Ser
    1 5 10 15
    Thr Arg Gln Leu Pro Ile Pro Pro Thr Thr Thr Arg Pro Ser Arg
    20 25 30
    (2) INFORMATION FOR SEQ ID NO:107:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 20 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:107:
    Ser Ser Leu Leu Gly Pro Pro Val Pro Pro Lys Pro Gln Thr Leu Phe
    1 5 10 15
    Ser Phe Ser Arg
    20
    (2) INFORMATION FOR SEQ ID NO:108:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 20 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:108:
    Ser Arg Leu Gly Glu Phe Ser Lys Pro Pro Ile Pro Gln Lys Pro Thr
    1 5 10 15
    Trp Met Ser Arg
    20
    (2) INFORMATION FOR SEQ ID NO:109:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 20 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:109:
    Ser Arg Thr Glu Arg Pro Pro Leu Pro Gln Arg Pro Asp Trp Leu Ser
    1 5 10 15
    Tyr Ser Ser Arg
    20
    (2) INFORMATION FOR SEQ ID NO:110:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 20 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:110:
    Ser Arg Glu Pro Asp Trp Leu Cys Pro Asn Cys Pro Leu Leu Leu Arg
    1 5 10 15
    Ser Asp Ser Arg
    20
    (2) INFORMATION FOR SEQ ID NO:111:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 20 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:111:
    Ser Ser Ser Ser His Asn Ser Arg Pro Pro Leu Pro Glu Lys Pro Ser
    1 5 10 15
    Trp Leu Ser Arg
    20
    (2) INFORMATION FOR SEQ ID NO:112:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 20 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:112:
    Ser Arg Leu Thr Pro Gln Ser Lys Pro Pro Leu Pro Pro Lys Pro Ser
    1 5 10 15
    Ala Val Ser Arg
    20
    (2) INFORMATION FOR SEQ ID NO:113:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 10 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (ix) FEATURE:
    (A) NAME/KEY: Modified-site
    (B) LOCATION: 4
    (D) OTHER INFORMATION: /note= “X = a hydrophobic amino
    acid residue”
    (ix) FEATURE:
    (A) NAME/KEY: Modified-site
    (B) LOCATION: 1
    (D) OTHER INFORMATION: /note= “X = Lys or Arg”
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:113:
    Xaa Pro Pro Xaa Pro Xaa Lys Pro Xaa Trp
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:114:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 20 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:114:
    Ser Ser Leu Gly Val Gly Trp Lys Pro Leu Pro Pro Met Arg Thr Ala
    1 5 10 15
    Ser Leu Ser Arg
    20
    (2) INFORMATION FOR SEQ ID NO:115:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 20 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:115:
    Ser Ser Val Gly Phe Ala Asp Arg Pro Arg Pro Pro Leu Arg Val Glu
    1 5 10 15
    Ser Leu Ser Arg
    20
    (2) INFORMATION FOR SEQ ID NO:116:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 20 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:116:
    Ser Ser Ala Gly Ile Leu Arg Pro Pro Glu Lys Pro Xaa Arg Ser Phe
    1 5 10 15
    Ser Leu Ser Arg
    20
    (2) INFORMATION FOR SEQ ID NO:117:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 20 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:117:
    Ser Ser Pro Tyr Thr Gly Asp Val Pro Ile Pro Pro Leu Arg Gly Ala
    1 5 10 15
    Ser Leu Ser Arg
    20
    (2) INFORMATION FOR SEQ ID NO:118:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 20 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:118:
    Ser Ser Leu Met Gly Ser Trp Pro Pro Val Pro Pro Leu Arg Ser Asp
    1 5 10 15
    Ser Leu Ser Arg
    20
    (2) INFORMATION FOR SEQ ID NO:119:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 20 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:119:
    Ser Ser Ile Gly Glu Asp Thr Pro Pro Ser Pro Pro Thr Arg Arg Ala
    1 5 10 15
    Ser Leu Ser Arg
    20
    (2) INFORMATION FOR SEQ ID NO:120:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 20 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:120:
    Ser Arg Ser Leu Ser Glu Val Ser Pro Lys Pro Pro Ile Arg Ser Val
    1 5 10 15
    Ser Leu Ser Arg
    20
    (2) INFORMATION FOR SEQ ID NO:121:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 20 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:121:
    Ser Ser Val Ser Glu Gly Tyr Ser Pro Pro Leu Pro Pro Arg Ser Thr
    1 5 10 15
    Ser Leu Ser Arg
    20
    (2) INFORMATION FOR SEQ ID NO:122:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 20 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:122:
    Ser Ser Ser Phe Thr Leu Ala Ala Pro Thr Pro Pro Thr Arg Ser Leu
    1 5 10 15
    Ser Leu Ser Arg
    20
    (2) INFORMATION FOR SEQ ID NO:123:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 20 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:123:
    Ser Ser Pro Pro Tyr Glu Leu Pro Pro Arg Pro Pro Asn Arg Thr Val
    1 5 10 15
    Ser Leu Ser Arg
    20
    (2) INFORMATION FOR SEQ ID NO:124:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 20 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:124:
    Ser Arg Val Val Asp Gly Leu Ala Pro Pro Pro Pro Val Arg Leu Ser
    1 5 10 15
    Ser Leu Ser Arg
    20
    (2) INFORMATION FOR SEQ ID NO:125:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 20 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:125:
    Ser Ser Leu Gly Tyr Ser Gly Ala Pro Val Pro Pro His Arg Xaa Ser
    1 5 10 15
    Ser Leu Ser Arg
    20
    (2) INFORMATION FOR SEQ ID NO:126:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 20 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:126:
    Ser Ser Ile Ser Asp Tyr Ser Arg Pro Pro Pro Pro Val Arg Thr Leu
    1 5 10 15
    Ser Leu Ser Arg
    20
    (2) INFORMATION FOR SEQ ID NO:127:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (ix) FEATURE:
    (A) NAME/KEY: Modified-site
    (B) LOCATION: 1
    (D) OTHER INFORMATION: /note= “X = a hydrophobic amino
    acid residue”
    (ix) FEATURE:
    (A) NAME/KEY: Modified-site
    (B) LOCATION: 11
    (D) OTHER INFORMATION: /note= “X = a hydrophobic amino
    acid residue”
    (ix) FEATURE:
    (A) NAME/KEY: Modified-site
    (B) LOCATION: 13
    (D) OTHER INFORMATION: /note= “X = Ser or Thr”
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:127:
    Xaa Xaa Xaa Xaa Xaa Xaa Pro Xaa Pro Pro Xaa Arg Xaa Xaa Ser Leu
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:128:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 20 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:128:
    Ser Arg Gly Pro Arg Trp Ser Pro Pro Pro Val Pro Leu Pro Thr Ser
    1 5 10 15
    Leu Asp Ser Arg
    20
    (2) INFORMATION FOR SEQ ID NO:129:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 20 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:129:
    Ser Ser Pro Pro Asp Tyr Ala Ala Pro Ala Ile Pro Ser Ser Leu Trp
    1 5 10 15
    Val Asp Ser Arg
    20
    (2) INFORMATION FOR SEQ ID NO:130:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 20 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:130:
    Ser Ser Pro Pro His Trp Ala Pro Pro Ala Pro Pro Ala Met Ser Pro
    1 5 10 15
    Pro Ile Ser Arg
    20
    (2) INFORMATION FOR SEQ ID NO:131:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 20 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:131:
    Ser Ser Asp Arg Cys Trp Glu Cys Pro Pro Trp Pro Ala Gly Gly Gln
    1 5 10 15
    Arg Gly Ser Arg
    20
    (2) INFORMATION FOR SEQ ID NO:132:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 20 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:132:
    Ser Ser Pro Pro Lys Phe Ser Pro Pro Pro Pro Pro Tyr Trp Gln Leu
    1 5 10 15
    His Ala Ser Arg
    20
    (2) INFORMATION FOR SEQ ID NO:133:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 20 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:133:
    Ser Ser Pro Pro Ser Phe Ala Pro Pro Ala Ala Pro Pro Arg His Ser
    1 5 10 15
    Phe Gly Ser Arg
    20
    (2) INFORMATION FOR SEQ ID NO:134:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 20 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:134:
    Ser Ser Ala Pro Lys Lys Pro Ala Pro Pro Val Pro Met Met Ala His
    1 5 10 15
    Val Met Ser Arg
    20
    (2) INFORMATION FOR SEQ ID NO:135:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 19 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:135:
    Ser Ser Pro Thr Tyr Pro Pro Pro Pro Pro Pro Asp Thr Ala Lys Gly
    1 5 10 15
    Ala Ser Arg
    (2) INFORMATION FOR SEQ ID NO:136:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 20 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:136:
    Ser Ser Pro Pro Xaa Xaa Xaa Pro Pro Pro Ile Pro Asn Ser Pro Gln
    1 5 10 15
    Val Leu Ser Arg
    20
    (2) INFORMATION FOR SEQ ID NO:137:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 20 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:137:
    Ser Ser Pro Pro Thr Trp Thr Pro Pro Lys Pro Pro Gly Trp Gly Val
    1 5 10 15
    Val Phe Ser Arg
    20
    (2) INFORMATION FOR SEQ ID NO:138:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 20 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:138:
    Ser Ser Ala Pro Thr Trp Ser Pro Pro Ala Leu Pro Asn Val Ala Lys
    1 5 10 15
    Tyr Lys Ser Arg
    20
    (2) INFORMATION FOR SEQ ID NO:139:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 20 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:139:
    Ser Ser Ile Lys Gly Pro Arg Phe Pro Val Pro Pro Val Pro Leu Asn
    1 5 10 15
    Gly Val Ser Arg
    20
    (2) INFORMATION FOR SEQ ID NO:140:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 20 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:140:
    Ser Ser Pro Pro Ala Trp Ser Pro Pro His Arg Pro Val Ala Phe Gly
    1 5 10 15
    Ser Thr Ser Arg
    20
    (2) INFORMATION FOR SEQ ID NO:141:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 10 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (ix) FEATURE:
    (A) NAME/KEY: Modified-site
    (B) LOCATION: 9
    (D) OTHER INFORMATION: /note= “X = a hydrophobic amino
    acid residue”
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:141:
    Pro Pro Xaa Trp Xaa Pro Pro Pro Xaa Pro
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:142:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 20 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:142:
    Ser Ser Met Lys Val His Asn Phe Pro Leu Pro Pro Leu Pro Ser Tyr
    1 5 10 15
    Glu Thr Ser Arg
    20
    (2) INFORMATION FOR SEQ ID NO:143:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 20 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:143:
    Ser Arg Val Pro Pro Leu Val Ala Pro Arg Pro Pro Ser Thr Leu Asn
    1 5 10 15
    Ser Leu Ser Arg
    20
    (2) INFORMATION FOR SEQ ID NO:144:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 20 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:144:
    Ser Ser Leu Tyr Trp Gln His Gly Pro Asp Pro Pro Val Gly Ala Pro
    1 5 10 15
    Gln Leu Ser Arg
    20
    (2) INFORMATION FOR SEQ ID NO:145:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 20 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:145:
    Ser Ser His Pro Leu Asn Ser Trp Pro Gly Gly Pro Phe Arg His Asn
    1 5 10 15
    Leu Ser Ser Arg
    20
    (2) INFORMATION FOR SEQ ID NO:146:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 20 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:146:
    Ser Ser Arg Ala Leu Arg Val Arg Pro Leu Pro Pro Val Pro Gly Thr
    1 5 10 15
    Ser Leu Ser Arg
    20
    (2) INFORMATION FOR SEQ ID NO:147:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 20 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:147:
    Ser Ser Phe Arg Ala Leu Pro Leu Pro Pro Thr Pro Asp Asn Pro Phe
    1 5 10 15
    Ala Gly Ser Arg
    20
    (2) INFORMATION FOR SEQ ID NO:148:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 20 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:148:
    Ser Arg Asp Ala Pro Gly Ser Leu Pro Phe Arg Pro Leu Pro Pro Val
    1 5 10 15
    Pro Thr Ser Arg
    20
    (2) INFORMATION FOR SEQ ID NO:149:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 20 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:149:
    Ser Ser Ile Ser Gln Arg Ala Leu Pro Pro Leu Pro Leu Met Ser Asp
    1 5 10 15
    Pro Ala Ser Arg
    20
    (2) INFORMATION FOR SEQ ID NO:150:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 20 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:150:
    Ser Ser Pro Ala Tyr Arg Pro Leu Pro Arg Leu Pro Asp Leu Ser Val
    1 5 10 15
    Ile Tyr Ser Arg
    20
    (2) INFORMATION FOR SEQ ID NO:151:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 20 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:151:
    Ser Ser Phe Ile Asn Arg Arg Leu Pro Ala Leu Pro Pro Asp Asn Ser
    1 5 10 15
    Leu Leu Ser Arg
    20
    (2) INFORMATION FOR SEQ ID NO:152:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 20 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:152:
    Ser Arg Leu Thr Gly Arg Pro Leu Pro Ala Leu Pro Pro Pro Phe Ser
    1 5 10 15
    Asp Phe Ser Arg
    20
    (2) INFORMATION FOR SEQ ID NO:153:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 20 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:153:
    Ser Arg Met Lys Asp Arg Val Leu Pro Pro Ile Pro Thr Val Glu Ser
    1 5 10 15
    Ala Val Ser Arg
    20
    (2) INFORMATION FOR SEQ ID NO:154:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 20 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:154:
    Ser Ser Leu Tyr Ser Ala Ile Ala Pro Asp Pro Pro Pro Arg Asn Ser
    1 5 10 15
    Ser Ser Ser Arg
    20
    (2) INFORMATION FOR SEQ ID NO:155:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 20 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:155:
    Ser Ser Leu Ala Ser Arg Pro Leu Pro Leu Leu Pro Asn Ser Ala Pro
    1 5 10 15
    Gly Gln Ser Arg
    20
    (2) INFORMATION FOR SEQ ID NO:156:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 20 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:156:
    Ser Ser Leu Thr Ser Arg Pro Leu Pro Asp Ile Pro Val Arg Pro Ser
    1 5 10 15
    Lys Ser Ser Arg
    20
    (2) INFORMATION FOR SEQ ID NO:157:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 20 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:157:
    Ser Ser Leu Lys Trp Arg Ala Leu Pro Pro Leu Pro Glu Thr Asp Thr
    1 5 10 15
    Pro Tyr Ser Arg
    20
    (2) INFORMATION FOR SEQ ID NO:158:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 20 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:158:
    Ser Ser Asn Thr Asn Arg Leu Pro Pro Pro Thr Pro Asp Gly Leu Asp
    1 5 10 15
    Val Arg Ser Arg
    20
    (2) INFORMATION FOR SEQ ID NO:159:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 20 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:159:
    Ser Ser Leu Gln Ser Arg Pro Leu Pro Leu Pro Pro Gln Ser Ser Tyr
    1 5 10 15
    Pro Ile Ser Arg
    20
    (2) INFORMATION FOR SEQ ID NO:160:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 12 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (ix) FEATURE:
    (A) NAME/KEY: Modified-site
    (B) LOCATION: 9
    (D) OTHER INFORMATION: /note= “Xaa represents an aliphatic
    amino acid”
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:160:
    Leu Xaa Xaa Arg Pro Leu Pro Xaa Xaa Pro Xaa Xaa
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:161:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 4 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:161:
    Pro Xaa Xaa Pro
    1
    (2) INFORMATION FOR SEQ ID NO:162:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 76 bases
    (B) TYPE: nucleic acid
    (C) STRANDEDNESS: single
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: DNA
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:162:
    CTGTGCCTCG AGKNNKNNKN NKNNKNNKNN KCCANNKNNK CCANNKNNKN NKNNKNNKNN 60
    KTCTAGACGT GTCAGT 76
    (2) INFORMATION FOR SEQ ID NO:163:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 bases
    (B) TYPE: nucleic acid
    (C) STRANDEDNESS: single
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: DNA
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:163:
    ACTGACACGT CTAGA 15
    (2) INFORMATION FOR SEQ ID NO:164:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 12 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (ix) FEATURE:
    (A) NAME/KEY: Modified-site
    (B) LOCATION: 1
    (D) OTHER INFORMATION: /note= “Xaa represents an aliphatic
    amino acid”
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:164:
    Xaa Xaa Xaa Arg Pro Leu Pro Xaa Leu Pro Xaa Xaa
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:165:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 10 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (ix) FEATURE:
    (A) NAME/KEY: Modified-site
    (B) LOCATION: 9
    (D) OTHER INFORMATION: /note= “X = an aliphatic amino acid
    residue like (Ala, Val, Leu, Ile, or Pro)”
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:165:
    Leu Xaa Xaa Arg Pro Leu Pro Xaa Xaa Pro
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:166:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 11 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (ix) FEATURE:
    (A) NAME/KEY: Modified-site
    (B) LOCATION: 1
    (D) OTHER INFORMATION: /note= “X = a basic amino acid
    residue”
    (ix) FEATURE:
    (A) NAME/KEY: Modified-site
    (B) LOCATION: 4
    (D) OTHER INFORMATION: /note= “X = an aliphatic amino acid
    residue”
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:166:
    Xaa Pro Pro Xaa Pro Xaa Lys Pro Xaa Trp Leu
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:167:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:167:
    Pro Val Lys Pro Pro Leu Pro Ala Lys Pro Trp Trp Leu Pro Pro Leu
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:168:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:168:
    Tyr Pro Gln Phe Arg Pro Pro Val Pro Pro Lys Pro Ser Leu Met Gln
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:169:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:169:
    Val Thr Arg Pro Pro Leu Pro Pro Lys Pro Gly His Met Ala Asp Phe
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:170:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:170:
    Val Ser Leu Gly Leu Lys Pro Pro Val Pro Pro Lys Pro Met Gln Leu
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:171:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:171:
    Tyr Lys Pro Glu Val Pro Ala Arg Pro Ile Trp Leu Ser Glu Leu
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:172:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:172:
    Gly Ala Gly Ala Ala Arg Pro Leu Val Pro Lys Lys Pro Leu Phe Leu
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:173:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 10 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (ix) FEATURE:
    (A) NAME/KEY: Modified-site
    (B) LOCATION: 4
    (D) OTHER INFORMATION: /note= “X = an aromatic amino acid
    residue”
    (ix) FEATURE:
    (A) NAME/KEY: Modified-site
    (B) LOCATION: 9
    (D) OTHER INFORMATION: /note= “X = an aliphatic amino acid
    residue”
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:173:
    Pro Pro Xaa Xaa Xaa Pro Pro Pro Xaa Pro
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:174:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:174:
    Pro Pro Trp Trp Ala Pro Pro Pro Ile Pro Asn Ser Pro Gln Val Leu
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:175:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 11 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:175:
    Pro Pro Val Pro Pro Arg Pro Xaa Xaa Thr Leu
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:176:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:176:
    Met Pro Pro Pro Val Pro Pro Arg Pro Pro Gly Thr Leu Gln Val Ala
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:177:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:177:
    Leu Ser Tyr Ser Pro Pro Pro Val Pro Pro Arg Pro Asp Ser Thr Leu
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:178:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:178:
    Val Leu Ala Pro Pro Val Pro Pro Arg Pro Gly Asn Thr Phe Phe Thr
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:179:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:179:
    Tyr Arg Pro Pro Val Ala Pro Arg Pro Pro Ser Ser Leu Ser Val Asp
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:180:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:180:
    Leu Gln Cys Pro Asp Cys Pro Arg Val Pro Pro Arg Pro Ile Pro Ile
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:181:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:181:
    Leu Thr Pro Pro Pro Phe Pro Lys Arg Pro Arg Trp Thr Leu Pro Glu
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:182:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:182:
    Tyr Trp Pro His Arg Pro Pro Leu Ala Pro Pro Gln Thr Thr Leu Gly
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:183:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 19 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:183:
    Tyr Asp Ala Ser Ser Ala Pro Gln Arg Pro Pro Leu Pro Val Arg Lys
    1 5 10 15
    Ser Arg Pro
    (2) INFORMATION FOR SEQ ID NO:184:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 19 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:184:
    Glu Tyr Val Asn Ala Ser Pro Glu Arg Pro Pro Ile Pro Gly Arg Lys
    1 5 10 15
    Ser Arg Pro
    (2) INFORMATION FOR SEQ ID NO:185:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 19 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:185:
    Trp Asn Gly Ile Ala Ile Pro Gly Arg Pro Glu Ile Pro Pro Arg Ala
    1 5 10 15
    Ser Arg Pro
    (2) INFORMATION FOR SEQ ID NO:186:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 19 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:186:
    Ser Met Ile Phe Ile Tyr Pro Glu Arg Pro Ser Pro Pro Pro Arg Phe
    1 5 10 15
    Ser Arg Pro
    (2) INFORMATION FOR SEQ ID NO:187:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 19 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:187:
    Gly Val Glu Glu Trp Asn Pro Glu Arg Pro Gln Ile Pro Leu Arg Leu
    1 5 10 15
    Ser Arg Pro
    (2) INFORMATION FOR SEQ ID NO:188:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:188:
    Trp Val Val Asp Ser Arg Pro Asp Ile Pro Leu Arg Arg Ser Leu Pro
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:189:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:189:
    Val Val Pro Leu Gly Arg Pro Glu Ile Pro Leu Arg Lys Ser Leu Pro
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:190:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:190:
    Gly Gly Thr Val Gly Arg Pro Pro Ile Pro Glu Arg Lys Ser Val Asp
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:191:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:191:
    Tyr Ser His Ala Gly Arg Pro Glu Val Pro Pro Arg Gln Ser Lys Pro
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:192:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:192:
    Phe Ser Ala Ala Ala Arg Pro Asp Ile Pro Ser Arg Ala Ser Thr Pro
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:193:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:193:
    Leu Tyr Ile Pro Lys Arg Pro Glu Val Pro Pro Arg Arg His Glu Ala
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:194:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:194:
    Asn Asn Ile Ser Ala Arg Pro Pro Leu Pro Ser Arg Gln Asn Pro Pro
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:195:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:195:
    Met Ala Gly Thr Pro Arg Pro Ala Val Pro Gln Arg Met Asn Pro Pro
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:196:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 11 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (ix) FEATURE:
    (A) NAME/KEY: Modified-site
    (B) LOCATION: 4
    (D) OTHER INFORMATION: /note= “X = an aliphatic amino
    acid”
    (ix) FEATURE:
    (A) NAME/KEY: Modified-site
    (B) LOCATION: 6
    (D) OTHER INFORMATION: /note= “X = an aliphatic amino
    acid”
    (ix) FEATURE:
    (A) NAME/KEY: Modified-site
    (B) LOCATION: 8
    (D) OTHER INFORMATION: /note= “X = a basic amino acid.”
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:196:
    Arg Pro Xaa Xaa Pro Xaa Arg Xaa Ser Xaa Pro
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:197:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:197:
    Gly Gln Pro Ala Gly Asp Pro Asp Pro Pro Pro Leu Pro Ala Lys Phe
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:198:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:198:
    Phe Glu Gln Thr Gly Val Pro Leu Leu Pro Pro Lys Ser Phe Lys Tyr
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:199:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:199:
    Ile Phe Gly Asp Pro Pro Pro Pro Ile Pro Met Lys Gly Arg Ser Leu
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:200:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:200:
    Ser Asn Gln Gly Ser Ile Pro Val Leu Pro Ile Lys Arg Val Gln Tyr
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:201:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:201:
    Asn Tyr Val Asn Ala Leu Pro Pro Gly Pro Pro Leu Pro Ala Lys Asn
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:202:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:202:
    Ser Ser Asp Pro Glu Arg Pro Val Leu Pro Pro Lys Leu Trp Ser Val
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:203:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:203:
    His Phe Gly Pro Ser Lys Pro Pro Leu Pro Ile Lys Thr Arg Ile Thr
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:204:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:204:
    Asp Trp Lys Val Pro Glu Pro Pro Val Pro Lys Leu Pro Leu Lys Gln
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:205:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:205:
    Ala Thr Ser Glu Gly Leu Pro Ile Leu Pro Ser Lys Val Gly Ser Tyr
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:206:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:206:
    Asn Ala Asn Val Ser Ala Pro Arg Ala Pro Ala Phe Pro Val Lys Thr
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:207:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:207:
    Glu Met Val Leu Gly Pro Pro Val Pro Pro Lys Arg Gly Thr Val Val
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:208:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:208:
    Ala Gly Ser Arg His Pro Pro Thr Leu Pro Pro Lys Glu Ser Gly Gly
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:209:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:209:
    Ser Val Ala Ala Asp Pro Pro Arg Leu Pro Ala Lys Ser Arg Pro Gln
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:210:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 7 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (ix) FEATURE:
    (A) NAME/KEY: Modified-site
    (B) LOCATION: 1
    (D) OTHER INFORMATION: /note= “X = an aliphatic amino acid
    residue”
    (ix) FEATURE:
    (A) NAME/KEY: Modified-site
    (B) LOCATION: 3
    (D) OTHER INFORMATION: /note= “X = an aliphatic amino acid
    residue”
    (ix) FEATURE:
    (A) NAME/KEY: Modified-site
    (B) LOCATION: 6
    (D) OTHER INFORMATION: /note= “X = an aliphatic acid
    residue”
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:210:
    Xaa Pro Xaa Leu Pro Xaa Lys
    1 5
    (2) INFORMATION FOR SEQ ID NO:211:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:211:
    Ile Thr Met Arg Pro Leu Pro Ala Leu Pro Gly His Gly Gln Ile His
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:212:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:212:
    Leu Pro Arg Arg Pro Leu Pro Asp Leu Pro Met Ala Ala Gly Lys Gly
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:213:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:213:
    Leu Gly Ser Arg Pro Leu Pro Pro Thr Pro Arg Gln Trp Pro Glu Val
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:214:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:214:
    Ser Thr Ile Arg Pro Leu Pro Ala Ile Pro Arg Asp Thr Leu Leu Thr
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:215:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:215:
    Arg Ser Gly Arg Pro Leu Pro Pro Ile Pro Glu Val Gly His Asn Val
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:216:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:216:
    Ile Gly Ser Arg Pro Leu Pro Trp Thr Pro Asp Asp Leu Gly Ser Ala
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:217:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:217:
    Leu Ala Gln Arg Glu Leu Pro Gly Leu Pro Ala Gly Ala Gly Val Ser
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:218:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:218:
    Ile Pro Gly Arg Ala Leu Pro Glu Leu Pro Pro Gln Arg Ala Leu Pro
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:219:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:219:
    Phe Val Gly Arg Glu Leu Pro Pro Thr Pro Arg Thr Val Ile Pro Trp
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:220:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:220:
    Asp Pro Arg Ser Ala Leu Pro Ala Leu Pro Leu Thr Pro Leu Gln Thr
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:221:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:221:
    Ser Pro His Asp Val Leu Pro Ala Leu Pro Asp Ser His Ser Lys Ser
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:222:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 10 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (ix) FEATURE:
    (A) NAME/KEY: Modified-site
    (B) LOCATION: 1
    (D) OTHER INFORMATION: /note= “X represents an aliphatic
    amino acid residue (Ala, Val, Leu, Ile or Pro)”
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:222:
    Xaa Xaa Xaa Arg Pro Leu Pro Xaa Leu Pro
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:223:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 10 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (ix) FEATURE:
    (A) NAME/KEY: Modified-site
    (B) LOCATION: 1
    (D) OTHER INFORMATION: /note= “X represents a basic amino
    acid residue (Arg or Lys).”
    (ix) FEATURE:
    (A) NAME/KEY: Modified-site
    (B) LOCATION: 2
    (D) OTHER INFORMATION: /note= “X represents an aromatic
    amino acid residue.”
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:223:
    Xaa Xaa Asp Xaa Pro Leu Pro Xaa Leu Pro
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:224:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:224:
    Lys Trp Asp Ser Leu Leu Pro Ala Leu Pro Pro Ala Phe Thr Val Glu
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:225:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:225:
    Arg Trp Asp Gln Val Leu Pro Glu Leu Pro Thr Ser Lys Gly Gln Ile
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:226:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:226:
    Arg Phe Asp Phe Pro Leu Pro Thr His Pro Asn Leu Gln Lys Ala His
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:227:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:227:
    Arg Leu Asp Ser Pro Leu Pro Ala Leu Pro Pro Thr Val Met Gln Asn
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:228:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:228:
    Arg Trp Gly Ala Pro Leu Pro Pro Leu Pro Glu Tyr Ser Trp Ser Thr
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:229:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:229:
    Tyr Trp Asp Met Pro Leu Pro Arg Leu Pro Gly Glu Glu Pro Ser Leu
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:230:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:230:
    Arg Phe Asp Tyr Asn Leu Pro Asp Val Pro Leu Ser Leu Gly Thr Ala
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:231:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:231:
    Thr Lys Lys Pro Asn Ala Pro Leu Pro Pro Leu Pro Ala Tyr Met Gly
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:232:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:232:
    Lys Trp Asp Leu Asp Leu Pro Pro Glu Pro Met Ser Leu Gly Asn Tyr
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:233:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 12 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (ix) FEATURE:
    (A) NAME/KEY: Modified-site
    (B) LOCATION: 2..3, 8..9, 11..12
    (D) OTHER INFORMATION: /note= “where Xaa at position 9
    represents aliphatic amino acid residues (Ala, Val, Leu,
    Ile, Pro) and Xaa represent any amino acid; except that if
    Xaa at position 8 = Pro, Xaa at position 9 = Leu, Xaa at
    position 11 = Pro, and Xaa at position 12 = Pro, then:
    where Xaa at position 2 = Phe, then Xaa at position 3 = is
    n or Arg; or where Xaa at position 2 = Ser, then Xaa at
    position 3 is not Arg, His, Ala, Asn, Thr, Gly, Val, Met,
    or Trp; or where Xaa at position 2 = Cys, then Xaa at
    position 3 is not Ser or Gly; or where Xaa at position 2 =
    Ar then Xaa at position 3 is not Thr or Phe; or where Xaa
    at position 2 = Ala, then Xaa at position 3 is not Arg,
    Gln, Asn, Ser, or Leu; or where Xaa at position 2 = Gln,
    then Xaa at position 3 is not M; or where Xaa at position
    = Leu, then Xaa at position 3 is not Arg; or where Xaa 2
    at position 2 = Ile, then Xaa at position 3 is not Ala; or
    where Xaa at position 2 = Pro, then Xaa at position 3 is
    not Pro, Trp, or Arg; or where Xaa at position 2 = Gly, th
    Xaa at position 3 is not Ser or Arg; or where Xaa at
    position 2 = Thr, then Xaa at position 3 is not Thr.”
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:233:
    Leu Xaa Xaa Arg Pro Leu Pro Xaa Xaa Pro Xaa Xaa
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:234:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:234:
    Tyr Tyr Gln Arg Pro Leu Pro Pro Leu Pro Leu Ser His Phe Glu Ser
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:235:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:235:
    Tyr Tyr Arg Lys Pro Leu Pro Asn Leu Pro Arg Gly Gln Thr Asp Asp
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:236:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:236:
    Tyr Phe Asp Lys Pro Leu Pro Glu Ser Pro Gly Ala Leu Met Ser Leu
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:237:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:237:
    Tyr Phe Ser Arg Ala Leu Pro Gly Leu Pro Glu Arg Gln Glu Ala His
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:238:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 10 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (ix) FEATURE:
    (A) NAME/KEY: Modified-site
    (B) LOCATION: 2
    (D) OTHER INFORMATION: /note= “X represents an aromatic
    amino acid residue.”
    (ix) FEATURE:
    (A) NAME/KEY: Modified-site
    (B) LOCATION: 4
    (D) OTHER INFORMATION: /note= “X represents basic amino
    acid residues (Arg or Lys).”
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:238:
    Tyr Xaa Xaa Xaa Pro Leu Pro Xaa Leu Pro
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:239:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:239:
    Ser Leu Trp Asp Pro Leu Pro Pro Ile Pro Gln Ser Lys Thr Ser Val
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:240:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:240:
    Ser Tyr Tyr Asp Pro Leu Pro Lys Leu Pro Asp Pro Gly Asp Leu Gly
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:241:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:241:
    Lys Leu Tyr Tyr Pro Leu Pro Pro Val Pro Phe Lys Asp Thr Lys His
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:242:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:242:
    Asp Pro Tyr Asp Ala Leu Pro Glu Thr Pro Ser Met Lys Ala Ser Gln
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:243:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 8 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (ix) FEATURE:
    (A) NAME/KEY: Modified-site
    (B) LOCATION: 1
    (D) OTHER INFORMATION: /note= “X represents an aromatic
    amino acid.”
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:243:
    Xaa Asp Pro Leu Pro Xaa Leu Pro
    1 5
    (2) INFORMATION FOR SEQ ID NO:244:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 7 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (ix) FEATURE:
    (A) NAME/KEY: Modified-site
    (B) LOCATION: 1
    (D) OTHER INFORMATION: /note= “X represents a basic
    aminoacid residue (Arg or Lys).”
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:244:
    Xaa Pro Tyr Pro Pro Tyr Pro
    1 5
    (2) INFORMATION FOR SEQ ID NO:245:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 7 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (ix) FEATURE:
    (A) NAME/KEY: Modified-site
    (B) LOCATION: 7
    (D) OTHER INFORMATION: /note= “X represents a basic amino
    acid (Arg or Lys).”
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:245:
    Tyr Pro Pro Tyr Pro Pro Xaa
    1 5
    (2) INFORMATION FOR SEQ ID NO:246:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 10 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (ix) FEATURE:
    (A) NAME/KEY: Modified-site
    (B) LOCATION: 2
    (D) OTHER INFORMATION: /note= “X represents an aromatic
    amino acid residue.”
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:246:
    Tyr Xaa Xaa Arg Pro Leu Pro Xaa Leu Pro
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:247:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 12 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:247:
    Xaa Pro Pro Val Pro Pro Arg Pro Xaa Xaa Thr Leu
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:248:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 6 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (ix) FEATURE:
    (A) NAME/KEY: Modified-site
    (B) LOCATION: 3
    (D) OTHER INFORMATION: /note= “X represents aliphatic
    amino acid residues (Ala, Val, Leu, Ile or Pro).”
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:248:
    Pro Pro Xaa Pro Pro Arg
    1 5
    (2) INFORMATION FOR SEQ ID NO:249:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 6 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:249:
    Pro Pro Val Pro Pro Arg
    1 5
    (2) INFORMATION FOR SEQ ID NO:250:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 12 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:250:
    Pro Pro Gln Met Pro Leu Pro Glu Ile Pro Gln Gln
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:251:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 12 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:251:
    Pro Pro Asp Asn Gly Pro Pro Pro Leu Pro Thr Ser
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:252:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 12 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:252:
    Thr Ala Pro Lys Pro Met Pro Pro Arg Pro Pro Leu
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:253:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 12 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:253:
    Ser Arg Ala Arg Asn Leu Pro Leu Pro Pro Pro Pro
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:254:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 12 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:254:
    Thr Val Thr Arg Gly Val Pro Pro Pro Pro Thr Val
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:255:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 12 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:255:
    Arg Pro Pro Arg Pro Leu Pro Val Ala Pro Gly Ser
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:256:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 12 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:256:
    Val Arg Lys Gln Met Leu Pro Pro Pro Pro Cys Pro
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:257:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 11 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:257:
    Gly Gly Ala Pro Pro Val Pro Ser Arg Pro Gly
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:258:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 11 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:258:
    Gly Pro Pro Pro Gln Val Pro Ser Arg Pro Asn
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:259:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 11 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:259:
    Arg Ala Pro Pro Gly Val Pro Ser Arg Ser Gly
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:260:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 11 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:260:
    Pro Leu Pro Pro Pro Pro Pro Pro Arg Gly Gly
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:261:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 11 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:261:
    Ala Pro Pro Pro Pro Pro Val Pro Arg Gly Arg
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:262:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 11 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:262:
    Ala Val Pro Pro Pro Val Pro Pro Pro Pro Ser
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:263:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 11 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:263:
    Gln Pro Ala Pro Ala Leu Pro Pro Lys Pro Pro
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:264:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 11 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:264:
    Gly Gly Pro Pro Pro Gly Pro Gly Arg Arg Gly
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:265:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 11 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:265:
    Thr Lys Ser Pro Pro Gln Pro Pro Arg Pro Asp
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:266:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 12 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:266:
    Pro Val Lys Gln Pro Pro Pro Leu Ala Pro Gln Ser
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:267:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 12 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:267:
    Arg Ala Pro Thr Met Pro Pro Pro Leu Pro Pro Val
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:268:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 12 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:268:
    Tyr Pro Pro Ala Tyr Pro Pro Pro Pro Val Pro Val
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:269:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 12 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:269:
    Pro Gly Pro Gly Tyr Gly Pro Pro Pro Val Pro Pro
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:270:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 12 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:270:
    Ala Pro Pro Val Pro Ser Arg Pro Gly Ala Ser Pro
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:271:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 12 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:271:
    Pro Pro Gln Val Pro Ser Arg Pro Asn Arg Asn Arg
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:272:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 12 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:272:
    Leu Pro Pro Val Pro Pro Arg Leu Asp Leu Leu Pro
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:273:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 10 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:273:
    Gln Ala Pro Glu Leu Pro Thr Lys Thr Arg
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:274:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 10 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:274:
    Val Ser Pro Leu Leu Pro Arg Lys Glu Arg
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:275:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 10 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:275:
    Glu Lys Pro Ala Leu Pro Arg Lys Arg Ala
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:276:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 10 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:276:
    Pro Pro Pro Ala Leu Pro Pro Lys Lys Arg
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:277:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 10 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:277:
    Thr Pro Pro Ala Leu Pro Glu Lys Lys Arg
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:278:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 10 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:278:
    Lys Pro Pro Pro Leu Pro Glu Lys Lys Asn
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:279:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 10 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:279:
    Pro Pro Pro Ala Leu Pro Pro Lys Gln Arg
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:280:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 12 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:280:
    Pro Gln Arg Arg Pro Leu Pro Cys Thr Pro Gly Asp
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:281:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 12 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:281:
    Trp Leu Pro Arg Pro Ile Pro Lys Val Pro Val Ser
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:282:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 7 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (ix) FEATURE:
    (A) NAME/KEY: Modified-site
    (B) LOCATION: 4
    (D) OTHER INFORMATION: /note= “X represents aliphatic
    amino acid residues (Ala, Val, Leu, Ile or Pro).”
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:282:
    Pro Pro Pro Xaa Pro Pro Arg
    1 5
    (2) INFORMATION FOR SEQ ID NO:283:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 11 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:283:
    Leu Gln Ala Pro Glu Leu Pro Thr Lys Thr Arg
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:284:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 11 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:284:
    Ala Val Ser Pro Leu Leu Pro Arg Lys Glu Arg
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:285:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 11 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:285:
    Lys Thr Ala Pro Thr Pro Pro Lys Arg Ser Ser
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:286:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 11 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:286:
    Ala Ser Leu Pro Pro Val Pro Pro Arg Leu Asp
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:287:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 11 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:287:
    Gly Gly Ala Pro Pro Val Pro Ser Arg Pro Gly
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:288:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 11 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:288:
    Gly Pro Pro Pro Gln Val Pro Ser Arg Pro Asn
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:289:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 11 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:289:
    Arg Ala Pro Pro Gly Val Pro Ser Arg Ser Gly
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:290:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 11 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:290:
    Pro Val Pro Pro Pro Val Pro Pro Arg Arg Arg
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:291:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 11 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:291:
    Asp Ser Pro Pro Ala Ile Pro Pro Arg Gln Pro
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:292:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 11 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:292:
    Glu Ser Pro Pro Leu Leu Pro Pro Arg Glu Pro
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:293:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 11 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:293:
    Ile Ala Gly Pro Pro Val Pro Pro Arg Gln Ser
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:294:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 11 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:294:
    Asn Leu Pro Glu Pro Ala Pro Pro Arg Pro Ser
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:295:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 11 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:295:
    Pro Pro Gly Pro Ala Gly Pro Ile Arg Gln Ala
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:296:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:296:
    Leu Leu Gln Ser Arg Pro Leu Pro His Ile Pro Ala Gly Ser Thr
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:297:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:297:
    Gln Ile Gln Gln Lys Pro Ala Val Pro His Lys Pro Pro Leu Asn Asp
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:298:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:298:
    Ser Ser Ala Ala Pro Pro Pro Pro Pro Arg Arg Ala Thr Pro Glu
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:299:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:299:
    Met Gly Pro Arg Arg Phe Leu Pro Pro Glu Pro Lys Gln Pro Trp
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:300:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:300:
    Pro Pro Pro Ala Leu Pro Pro Pro Pro Arg Pro Val Ala Asp Lys
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:301:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:301:
    Gln Pro Pro Pro Arg Pro Leu Pro Pro Arg Pro Pro Ala Ala Gln
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:302:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:302:
    Pro Asn Leu Arg Arg Gly Leu Pro Gln Val Pro Tyr Phe Ser Leu
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:303:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 18 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:303:
    Ala Leu Ala Gly Pro Pro Pro Pro Pro Pro Pro Pro His Pro His Ala
    1 5 10 15
    Arg Ile
    (2) INFORMATION FOR SEQ ID NO:304:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:304:
    Thr Phe Leu Glu Arg His Leu Pro Ser Val Pro Gly Leu Leu Arg
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:305:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 18 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:305:
    Asp Phe Gln Pro Pro Tyr Phe Pro Pro Pro Pro Tyr Gln Pro Ile Tyr
    1 5 10 15
    Pro Gln
    (2) INFORMATION FOR SEQ ID NO:306:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:306:
    Cys Phe Cys His Arg Pro Leu Pro Val Pro Pro Gly Ser Leu Val
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:307:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:307:
    Ala Pro Ala Pro Pro Pro Gly Pro Pro Arg Pro Ala Ala Ala Ala
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:308:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:308:
    Cys Arg Cys Gly Arg Arg Leu Pro Pro Glu Pro Cys Ala Ala Ala
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:309:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:309:
    Gly Glu Asp Arg Pro Pro Val Pro Pro Ala Pro Ala Pro Ala Ala
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:310:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:310:
    Pro Pro Pro Pro Pro Pro Pro Val Pro Ala Lys Leu Phe Gly Glu
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:311:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:311:
    Pro Gln Pro His Arg Val Leu Pro Thr Ser Pro Ser Asp Ile Ala
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:312:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:312:
    Ile Phe Leu Asn Arg Pro Leu Pro Val Leu Pro Asp Ser Ala Asp
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:313:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:313:
    Gln Ala Ser Ser Leu Pro Pro Val Pro Pro Arg Leu Asp Leu Leu Pro
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:314:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 20 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:314:
    Pro Pro Thr Leu Arg Asp Leu Pro Pro Pro Pro Pro Pro Asp Arg Pro
    1 5 10 15
    Tyr Ser Val Gly
    20
    (2) INFORMATION FOR SEQ ID NO:315:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:315:
    Arg Pro Gln Arg Arg Pro Leu Pro Cys Thr Pro Gly Asp Cys Pro
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:316:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:316:
    Ser Asp Gln Gly Arg Asn Leu Pro Gly Thr Pro Val Pro Ala Ser
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:317:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 18 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:317:
    Arg His Ser Arg Arg Gln Leu Pro Pro Val Pro Pro Lys Pro Arg Pro
    1 5 10 15
    Leu Leu
    (2) INFORMATION FOR SEQ ID NO:318:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:318:
    Ser Asp Gln Gly Arg Asn Leu Pro Gly Thr Pro Val Pro Ala Ser
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:319:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:319:
    Asn Asp Glu Tyr Val Pro Val Pro Pro Trp Lys Ala Asn Asn Lys
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:320:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 18 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:320:
    Gln Thr Pro Thr Pro Pro Pro Pro Pro Pro Pro Pro Leu Pro Pro Gln
    1 5 10 15
    Phe Pro
    (2) INFORMATION FOR SEQ ID NO:321:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:321:
    Asp Ser Gly Val Arg Pro Leu Pro Pro Leu Pro Asp Pro Gly Val
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:322:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:322:
    Asp Pro Gly Val Arg Pro Leu Pro Pro Val Pro Glu Glu Leu Pro
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:323:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 17 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:323:
    Leu Asn Thr Gly Pro Pro Tyr Cys Asp Pro Asn Leu Pro Asn Ser Asn
    1 5 10 15
    Gly
    (2) INFORMATION FOR SEQ ID NO:324:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:324:
    Phe Pro Ile Leu Arg Tyr Leu Pro Asn Arg Pro Leu Gln Arg Phe
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:325:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:325:
    Ser Trp Arg Arg Arg Lys Leu Pro Pro Gly Pro Glu Gly Trp Pro
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:326:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:326:
    Ser Ser Arg Arg Arg Lys Leu Pro Pro Gly Pro Thr Pro Leu Pro
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:327:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:327:
    Ser Cys Arg Arg Arg Lys Leu Pro Pro Gly Pro Thr Pro Leu Pro
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:328:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:328:
    Ser Ser Gly Arg Arg Lys Leu Pro Pro Gly Pro Thr Pro Leu Pro
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:329:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:329:
    His His Val Arg Arg Lys Leu Pro Pro Gly Pro Thr Pro Leu Pro
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:330:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:330:
    Ala Lys Phe Pro Arg Ser Leu Pro Phe Leu Pro Leu Val Gly Ser
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:331:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 17 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:331:
    Asn Ala Arg Lys Pro Tyr Tyr Ser Leu Pro Ile Val Pro Gly Thr Val
    1 5 10 15
    Leu
    (2) INFORMATION FOR SEQ ID NO:332:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:332:
    Gln Ser Leu Ala Arg Pro Pro Pro Pro Pro Lys Arg Ile Arg Thr Asp
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:333:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:333:
    Ala Ser Pro Ala Arg Ala Leu Pro Gly Pro Pro His Pro Arg Arg
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:334:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:334:
    Thr Asp Ser Arg Leu Pro Leu Val Pro Ser Lys Tyr Ser Met Thr
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:335:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 17 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:335:
    Gly Leu Cys Gly Pro Ala Trp Trp Ala Pro Ser Leu Pro Arg Leu Pro
    1 5 10 15
    Arg
    (2) INFORMATION FOR SEQ ID NO:336:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 17 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:336:
    Leu Cys Trp Cys Lys Met Pro Pro Pro Pro Pro Lys Pro Arg Tyr Ala
    1 5 10 15
    Pro
    (2) INFORMATION FOR SEQ ID NO:337:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:337:
    Trp Leu Glu Arg Met Pro Pro Ile Pro Pro Ile Lys Asn Leu Glu Asp
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:338:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 17 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:338:
    Pro Asp Val Glu Pro Gly Tyr Leu Arg Pro Leu Ile Pro Ala Ala Ala
    1 5 10 15
    Pro
    (2) INFORMATION FOR SEQ ID NO:339:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:339:
    Gln Glu Glu Leu Pro Pro Pro Pro Pro Gln Lys Lys Arg Gln Ile
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:340:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:340:
    Val Ala Pro Ala Arg Pro Leu Pro Gly Ala Pro Pro Pro Ser Gln
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:341:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 17 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:341:
    Gly Asp Met Asn Pro Gly Phe Gln Pro Pro Ile Thr Pro Asp Val Glu
    1 5 10 15
    Thr
    (2) INFORMATION FOR SEQ ID NO:342:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:342:
    Ala Arg Ser Ala Arg Arg Leu Pro Ser Trp Pro Pro Thr Leu Asp
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:343:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:343:
    Leu Gly Gly Ala Pro Pro Val Pro Ser Arg Pro Gly Ala Ser Pro
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:344:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:344:
    Val Met Arg Pro Pro Pro Pro Pro Pro Pro Pro Lys Val Lys His Ala
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:345:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:345:
    Met Arg His Gly Arg Gly Leu Pro Ser Thr Pro Cys His Thr Ser
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:346:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:346:
    His Leu Tyr Ser Pro Pro Pro Pro Pro Pro Pro Tyr Ser Gly Cys
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:347:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:347:
    Pro Gln Pro Gln Pro Pro Ile Pro Pro Arg Pro Ala Ser Val Leu
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:348:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:348:
    Pro Pro Pro Pro Pro Pro Pro Pro Pro Arg Ala Ser Ser Lys Arg
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:349:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:349:
    Leu Pro Ser Arg Glu Pro Pro Pro Pro Gln Lys Pro Pro Pro Asn
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:350:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:350:
    Lys Ser Glu Asp Glu Pro Pro Ala Leu Pro Pro Lys Ile Gly Thr Pro
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:351:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:351:
    Arg Arg His Ser Pro Pro His Pro Pro Arg Pro Ser Ser Leu Glu
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:352:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:352:
    Val Met Thr Ala Pro Pro Pro Pro Pro Pro Pro Val Tyr Glu Pro
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:353:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:353:
    Lys Glu Gly Glu Arg Ala Leu Pro Ser Ile Pro Lys Leu Ala Asn
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:354:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:354:
    Asp Gln Arg Arg Pro Pro Pro Pro Pro Pro Pro Val Ser Pro Leu
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:355:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:355:
    Val Cys Arg Pro Arg Pro Leu Pro Pro Leu Pro Pro Thr Ala Met
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:356:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:356:
    Pro Pro Leu Ile Pro Pro Pro Pro Pro Leu Pro Pro Gly Leu Gly
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:357:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 20 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:357:
    Cys Pro Val Ser Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Thr Pro
    1 5 10 15
    Val Pro Pro Ser
    20
    (2) INFORMATION FOR SEQ ID NO:358:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 20 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:358:
    Pro Ser Asp Gly Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Leu Pro
    1 5 10 15
    Asn Val Leu Ala
    20
    (2) INFORMATION FOR SEQ ID NO:359:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 20 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:359:
    Asn Ser Gly Gly Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro
    1 5 10 15
    Gly Leu Ala Pro
    20
    (2) INFORMATION FOR SEQ ID NO:360:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 17 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:360:
    Gly Trp Leu Leu Pro Pro Pro Pro Pro Pro Pro Leu Pro Phe Gln Ser
    1 5 10 15
    Ser
    (2) INFORMATION FOR SEQ ID NO:361:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:361:
    Leu Met Thr Glu Ala Pro Pro Ala Val Pro Pro Lys Glu Pro Ser Gly
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:362:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 17 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:362:
    Ser Glu Pro Pro Pro Arg Tyr Glu Pro Pro Val Glu Pro Val Asn Gly
    1 5 10 15
    Ile
    (2) INFORMATION FOR SEQ ID NO:363:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:363:
    Asp Arg Ser Ser Arg Pro Leu Pro Pro Leu Pro Ile Ser Glu Asp
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:364:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:364:
    Ile Pro Pro Arg Val Pro Ile Pro Pro Arg Pro Ala Lys Pro Asp
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:365:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:365:
    Met Phe Pro Arg Leu Asn Pro Ile Leu Pro Pro Lys Ala Pro Ala Val
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:366:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:366:
    Ala Ala Pro Pro Arg Leu Leu Pro Pro Leu Pro Thr Cys Tyr Gly
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:367:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:367:
    Gly Gly Gly Phe Pro Pro Leu Pro Pro Pro Pro Pro Gln Leu Pro
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:368:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 20 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:368:
    Leu Pro His Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro
    1 5 10 15
    Gln His Leu Ala
    20
    (2) INFORMATION FOR SEQ ID NO:369:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:369:
    Ser Ala Pro Gln Arg Pro Met Pro Pro Asn Arg Pro Ser Pro Pro
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:370:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 17 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:370:
    Gly Ser Ser Trp Pro Gly Tyr Val Arg Pro Leu Pro Pro Ala Ala Ile
    1 5 10 15
    Glu
    (2) INFORMATION FOR SEQ ID NO:371:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 18 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:371:
    His Pro His Ala Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro His Leu
    1 5 10 15
    His Ala
    (2) INFORMATION FOR SEQ ID NO:372:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:372:
    Gly Ala Ser Pro Pro Pro Pro Pro Pro Ala Lys Gly His Pro Gly
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:373:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:373:
    Pro Gln Gln Gln Pro Pro Pro Pro Pro Gln Pro Pro Gln Pro Ala
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:374:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 17 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:374:
    Gly Pro Ala Leu Pro Pro Pro Pro Pro Pro Pro Leu Pro Ala Ala Pro
    1 5 10 15
    Pro
    (2) INFORMATION FOR SEQ ID NO:375:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 17 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:375:
    His Ser Met Thr Pro Ser Tyr Glu Ser Pro Ser Pro Pro Ala Phe Gly
    1 5 10 15
    Lys
    (2) INFORMATION FOR SEQ ID NO:376:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 23 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:376:
    Arg Asp Pro Gly Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro
    1 5 10 15
    Pro Pro Pro Gly Leu Ser Pro
    20
    (2) INFORMATION FOR SEQ ID NO:377:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:377:
    Gln Glu Leu Tyr Pro Pro Pro Pro Pro Arg Pro Ser Tyr Pro Glu
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:378:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:378:
    Gly Leu Ser Cys Arg Ala Leu Pro Gly Glu Pro Arg Pro Leu His
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:379:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:379:
    Thr Glu Phe Lys Arg Gly Leu Pro Leu Phe Pro Leu Val Lys Pro
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:380:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 17 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:380:
    Glu Ile Ala Thr Pro Pro Pro Pro Pro Pro Pro Lys Lys Glu Thr Lys
    1 5 10 15
    Pro
    (2) INFORMATION FOR SEQ ID NO:381:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:381:
    Ser Pro Ala Ala Pro Pro Pro Pro Pro Pro Pro Gly Ala Pro Gly
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:382:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:382:
    Pro Glu Pro Gln Pro Pro Pro Pro Pro Pro Pro His Gln Pro Leu
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:383:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 17 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:383:
    Gln His Gln Gln Pro Thr Tyr Trp Tyr Pro Pro Pro Pro Pro Arg His
    1 5 10 15
    His
    (2) INFORMATION FOR SEQ ID NO:384:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:384:
    Pro Arg Leu Pro Pro Pro Pro Pro Pro Pro Leu Lys Asp Glu Pro Gln
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:385:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:385:
    Ala Leu Ala Leu Pro Pro Pro Pro Pro Leu Pro Leu Pro Leu Pro
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:386:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:386:
    Lys Asp Lys Ser Arg Pro Pro Arg Pro Pro Pro Lys Pro Leu His Leu
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:387:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:387:
    Val Asp Gln Gln Arg Pro Asn Ile Pro Asn Arg Trp Phe Ser Asp
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:388:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:388:
    Glu Gln Lys Leu Arg Pro Asn Ile Pro Asn Arg Trp Gln Ser Cys
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:389:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:389:
    Leu Asp Ala Gln Arg Pro Arg Pro Pro Gln Lys Pro His His Glu Asp
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:390:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:390:
    Val Pro Lys Asp Arg Pro Leu Pro Pro Ala Pro Leu Ser Pro Ala
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:391:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 17 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:391:
    Gly Glu Leu Ser Pro Ser Phe Leu Asn Pro Pro Leu Pro Pro Ser Thr
    1 5 10 15
    Asp
    (2) INFORMATION FOR SEQ ID NO:392:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:392:
    Asp Leu Thr Gly Gln Val Pro Thr Pro Pro Val Lys Gln Val Lys Leu
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:393:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:393:
    Leu Asp Thr Val Pro Phe Pro Pro Pro Arg Pro Ser Ala Glu Leu
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:394:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:394:
    Ala Ala Glu Leu Arg Ser Leu Pro Gly Leu Pro Pro Ala Thr Ala
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:395:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:395:
    Glu Leu Ala Pro Arg Pro Arg Pro Pro Gly Arg Pro Ile Ser Ser
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:396:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:396:
    Ser Ile Ser Pro Arg Pro Arg Pro Pro Gly Arg Pro Val Ser Gly
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:397:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:397:
    Tyr Gly Pro Ile Arg Pro Leu Pro Gln Thr Pro His Leu Glu Glu
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:398:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 17 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:398:
    Leu Gly Lys His Pro Asn Phe Gln Lys Pro Lys Pro Pro Lys Gly Lys
    1 5 10 15
    Gln
    (2) INFORMATION FOR SEQ ID NO:399:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 17 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:399:
    Leu Gly Lys His Pro Asn Phe Glu Lys Pro Lys Pro Pro Lys Gly Lys
    1 5 10 15
    Gln
    (2) INFORMATION FOR SEQ ID NO:400:
    (i) SEQUENCE CHARACTERISTICS:
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:400:
    THIS SEQUENCE HAS BEEN INTENTIONALLY SKIPPED
    (2) INFORMATION FOR SEQ ID NO:401:
    (i) SEQUENCE CHARACTERISTICS:
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:401:
    THIS SEQUENCE HAS BEEN INTENTIONALLY SKIPPED
    (2) INFORMATION FOR SEQ ID NO:402:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 14 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:402:
    Ser Lys Pro Gln Pro Ala Val Pro Pro Arg Ser Ala Asp Leu
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:403:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:403:
    Val Ser Asp Lys Arg Cys Leu Pro Cys His Pro Glu Cys Gln Pro
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:404:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:404:
    Leu Arg Leu Pro Pro Pro Leu Pro Pro Arg Pro Arg Gln Pro Leu
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:405:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:405:
    Pro Pro Ala Lys Pro Pro Pro Pro Pro Gln Pro Leu Pro Ser Pro
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:406:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:406:
    Asn Gln Leu Leu Lys Leu Pro Val Leu Pro Pro Lys Leu Thr Leu Phe
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:407:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:407:
    Ser Asn Leu Pro Leu Pro Leu Pro Pro Lys Lys Thr Arg Ile Ile
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:408:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 19 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:408:
    Ala Arg Ala Ala Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Gly
    1 5 10 15
    Ala Asp Arg
    (2) INFORMATION FOR SEQ ID NO:409:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 18 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:409:
    Glu Pro Ser Asp Pro Pro Pro Pro Pro Pro Pro Pro Pro Leu Pro Leu
    1 5 10 15
    Ala Ala
    (2) INFORMATION FOR SEQ ID NO:410:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:410:
    Pro Arg Pro Pro Arg Pro Leu Pro Val Ala Pro Gly Ser Ser Lys
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:411:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:411:
    Pro Arg Gly Pro Arg Pro Leu Pro Pro Ala Arg Pro Arg Asp Gly Pro
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:412:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:412:
    Glu Gln Glu Val Ala Pro Pro Ala Leu Pro Pro Lys Pro Pro Lys Thr
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:413:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 17 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:413:
    Gln Pro Glu Gln Pro Pro Pro Pro Pro Pro Pro Pro Pro Ala Gln Gln
    1 5 10 15
    Pro
    (2) INFORMATION FOR SEQ ID NO:414:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:414:
    Leu Gly Pro Pro Pro Pro Leu Pro Pro Arg Ala Thr Pro Ser Arg
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:415:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:415:
    Tyr His Pro Gln Pro Pro Pro Pro Pro Pro Pro Met Met Pro Val
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:416:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 17 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:416:
    Gln Pro Phe Gly Pro Gly Phe Val Pro Pro Pro Pro Pro Pro Pro Tyr
    1 5 10 15
    Gly
    (2) INFORMATION FOR SEQ ID NO:417:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:417:
    Pro Pro Glu His Ile Pro Pro Pro Pro Arg Pro Pro Lys Arg Ile
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:418:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:418:
    Cys Leu Thr Glu Arg Asn Leu Pro Ile Tyr Pro Leu Asp Ile Val
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:419:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:419:
    Pro Gly Asp Gly Lys Pro Pro Ala Leu Pro Pro Lys Gln Ser Lys Lys
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:420:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 17 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:420:
    Phe Thr Thr Trp Pro Asp Phe Gly Val Pro Asn Pro Pro Gln Thr Leu
    1 5 10 15
    Val
    (2) INFORMATION FOR SEQ ID NO:421:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 17 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:421:
    Ser Ala Gln Glu Pro His Tyr Leu Pro Pro Glu Met Pro Gln Gly Ser
    1 5 10 15
    Tyr
    (2) INFORMATION FOR SEQ ID NO:422:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:422:
    Val Met Ser Ala Pro Pro Pro Pro Pro Pro Pro Pro Val Ile Pro Pro
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:423:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:423:
    Glu Pro Pro Ala Pro Pro Pro Pro Pro Pro Pro Glu Glu Asp Pro
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:424:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:424:
    Gly Arg Arg Gly Pro Pro Pro Pro Pro Pro Ser Arg Gly Pro Pro
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:425:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:425:
    Gly Arg Gly Gly Arg Pro Met Pro Pro Ser Arg Arg Asp Tyr Asp
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:426:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 20 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:426:
    Gly Ser Arg Ala Arg Asn Leu Pro Leu Pro Pro Pro Pro Pro Pro Arg
    1 5 10 15
    Gly Gly Asp Leu
    20
    (2) INFORMATION FOR SEQ ID NO:427:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 21 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:427:
    Ser Arg Tyr Gly Pro Gln Tyr Gly His Pro Pro Pro Pro Pro Pro Pro
    1 5 10 15
    Pro Glu Tyr Gly Pro
    20
    (2) INFORMATION FOR SEQ ID NO:428:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:428:
    Ser Ser Pro Ser Pro Pro Pro Pro Pro Arg Val Tyr Lys Pro Cys
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:429:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:429:
    Ser Ser Pro Ser Pro Pro Pro Pro Pro Arg Val Tyr Lys Pro Cys
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:430:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:430:
    Gly Ser Gly Ala Pro Pro Pro Pro Pro Met Pro Pro Pro Pro Leu
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:431:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:431:
    Gly Ser Gly Ala Pro Pro Pro Pro Pro Met Pro Pro Pro Pro Leu
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:432:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:432:
    Pro Lys Lys Gln Ala Pro Pro Ser Pro Pro Pro Lys Lys Glu Glu Ala
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:433:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:433:
    Arg Lys Gln Met Pro Pro Pro Pro Pro Cys Pro Gly Arg Glu Leu
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:434:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 17 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:434:
    Gly Ala Pro Ala Pro Ser Tyr Gly Tyr Pro Ala Val Pro Phe Ala Ala
    1 5 10 15
    Ala
    (2) INFORMATION FOR SEQ ID NO:435:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:435:
    Arg Ala Val Pro Pro Pro Leu Pro Pro Arg Arg Lys Glu Arg Thr
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:436:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:436:
    Glu Leu Ser Pro Pro Pro Ile Pro Pro Arg Leu Asn His Ser Thr
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:437:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:437:
    Glu Gln Pro Leu Pro Pro Ile Pro Pro Thr Lys Ser Lys Thr Ser
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:438:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:438:
    Lys Gly Leu Asn Arg Asn Leu Pro Ala Val Pro Pro Thr Leu Thr
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:439:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:439:
    Pro Ala Asn Val Pro Pro Pro Pro Pro Gln Pro Pro Ala His Met
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:440:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:440:
    Pro Pro Pro Phe Lys Pro Pro Pro Pro Pro Ile Lys Tyr Thr Cys Ile
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:441:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:441:
    Lys Ser Thr His Pro Pro Pro Leu Pro Ala Lys Glu Glu Pro Val
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:442:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:442:
    Ser His Glu Leu Arg Thr Leu Pro His Ser Pro Ala Ser Ala Asp
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:443:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:443:
    Glu Gly Glu Ala Ser Pro Pro Ala Pro Pro Leu Lys His Val Leu Glu
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:444:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:444:
    Ala Leu Ala Thr Arg Ala Leu Pro Pro Thr Pro Pro Leu Met Ala
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:445:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 17 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:445:
    Glu His Lys Gly Pro Val Phe Ala Pro Pro Tyr Glu Pro Leu Pro Glu
    1 5 10 15
    Asn
    (2) INFORMATION FOR SEQ ID NO:446:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 17 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:446:
    Gln Arg Val Glu Pro Glu Trp Tyr Ile Pro Ile Ile Pro Met Val Leu
    1 5 10 15
    Ile
    (2) INFORMATION FOR SEQ ID NO:447:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 17 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:447:
    Gln Arg Val Glu Pro Glu Trp Tyr Ile Pro Ile Ile Pro Met Val Leu
    1 5 10 15
    Ile
    (2) INFORMATION FOR SEQ ID NO:448:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 22 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:448:
    Pro Glu Asp Asp Pro Ile Tyr Ala Leu Pro Pro Pro Pro Pro Pro Pro
    1 5 10 15
    Ala Pro Pro Arg Arg Arg
    20
    (2) INFORMATION FOR SEQ ID NO:449:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:449:
    Ser Arg Pro Val Val Pro Pro Leu Ile Pro Pro Lys Ile Pro Glu Gly
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:450:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 17 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:450:
    Gly Glu Asp Ser Pro Val Phe Arg Pro Pro Ser Pro Pro Met Gly Pro
    1 5 10 15
    Ser
    (2) INFORMATION FOR SEQ ID NO:451:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 16 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:451:
    Phe Arg Thr Gly Arg Pro Leu Leu Pro Ile Lys Pro Glu His Gly Arg
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:452:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:452:
    Ile Gln Ala Thr Pro Pro Pro Pro Pro Leu Pro Ser Gln Tyr Ala
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:453:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:453:
    Ala Phe Pro Ser Pro Pro Pro Pro Pro Pro Pro Met Phe Asp Glu
    1 5 10 15
    (2) INFORMATION FOR SEQ ID NO:454:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 76 bases
    (B) TYPE: nucleic acid
    (C) STRANDEDNESS: single
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: DNA
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:454:
    ACTGACACGT CTAGAMNNMN NMNNMNNMNN MNNTGGMNNM NNTGGMNNMN NMNNMNNMNN 60
    MNNMCTCGAG GCACAG 76
    (2) INFORMATION FOR SEQ ID NO:455:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 55 bases
    (B) TYPE: nucleic acid
    (C) STRANDEDNESS: single
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: DNA
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:455:
    TCGAGKNNKN NKNNKNNKNN KNNKCCANNK NNKCCANNKN NKNNKNNKNN KNNKT 55
    (2) INFORMATION FOR SEQ ID NO:456:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 55 bases
    (B) TYPE: nucleic acid
    (C) STRANDEDNESS: single
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: DNA
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:456:
    CTAGAMNNMN NMNNMNNMNN MNNTGGMNNM NNTGGMNNMN NMNNMNNMNN MNNMC 55
    (2) INFORMATION FOR SEQ ID NO:457:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 60 bases
    (B) TYPE: nucleic acid
    (C) STRANDEDNESS: single
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: DNA
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:457:
    TCGAGKNNKN NKNNKNNKNN KNNKCCANNK NNKCCANNKN NKNNKNNKNN KNNKTCTAGA 60
    (2) INFORMATION FOR SEQ ID NO:458:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 60 bases
    (B) TYPE: nucleic acid
    (C) STRANDEDNESS: single
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: DNA
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:458:
    CTCAGAMNNM NNMNNMNNMN NMNNTGGMNN MNNTGGMNNM NNMNNMNNMN NMNNMCTCGA 60
    (2) INFORMATION FOR SEQ ID NO:459:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 20 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (ix) FEATURE:
    (A) NAME/KEY: Modified-site
    (B) LOCATION: 2
    (D) OTHER INFORMATION: /note= “Xaa can be either Ser or
    Arg”
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:459:
    Ser Xaa Xaa Xaa Xaa Xaa Xaa Xaa Pro Xaa Xaa Pro Xaa Xaa Xaa Xaa
    1 5 10 15
    Xaa Xaa Ser Arg
    20
    (2) INFORMATION FOR SEQ ID NO:460:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 66 bases
    (B) TYPE: nucleic acid
    (C) STRANDEDNESS: single
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: DNA
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:460:
    TCCTCGAGTA TCGACATGCC TTAGACTGCT AGCACTATGT ACAACATGCT TCATCGCAAC 60
    GAGCCA 66
    (2) INFORMATION FOR SEQ ID NO:461:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 6 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:461:
    Ser Ser Ile Asp Met Pro
    1 5
    (2) INFORMATION FOR SEQ ID NO:462:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 63 bases
    (B) TYPE: nucleic acid
    (C) STRANDEDNESS: single
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: DNA
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:462:
    GGTGGGAGGA AGTTGAGCCC GCCCGCCAAC GACATGCCGC CCGCCCTCCT GAAGAGGTCT 60
    AGA 63
    (2) INFORMATION FOR SEQ ID NO:463:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 21 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:463:
    Gly Gly Arg Lys Leu Ser Pro Pro Ala Asn Asp Met Pro Pro Ala Leu
    1 5 10 15
    Leu Lys Arg Ser Arg
    20
    (2) INFORMATION FOR SEQ ID NO:464:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 14 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:464:
    Thr Ala Ser Thr Met Tyr Asn Met Leu His Arg Asn Glu Pro
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:465:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 18 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:465:
    Trp Asn Glu Arg Gln Pro Ala Pro Ala Leu Pro Pro Lys Pro Pro Lys
    1 5 10 15
    Pro Thr
    (2) INFORMATION FOR SEQ ID NO:466:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 12 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (ix) FEATURE:
    (A) NAME/KEY: Modified-site
    (B) LOCATION: 1..3, 8, 11..12
    (D) OTHER INFORMATION: /note= “where Xaa at position 1
    represents aliphatic amino acid residues (Ala, Val, Leu,
    Ile, Pro) and Xaa represent any amino acid; except that if
    Xaa at postion 8 = Pro, Xaa at position 11 = Pro, and Xaa
    at position 12 = Pro, then: when Xaa at position 1 = Leu,
    where Xaa at position 2 = Phe, then Xaa at position 3 is
    not His or Arg; or where Xaa at position 2 = Ser, then Xaa
    at position 3 is not Arg, His, Ala, Asn, Thr, Gly, Val,
    Met, or Trp; or where Xaa at position 2 = Cys, then Xaa at
    position 3 is not Ser or Gly; or where Xaa at position 2 =
    Arg, then Xaa at position 3 is not Thr or Phe; or where at
    position 2 = Ala, then Xaa at position 3 is not Arg, Gln
    or where Xaa at position 2 = Gln, then Xaa at position 3
    is not M; or where Xaa at position 2 = Leu, then Xaa at
    position 3 is not Arg; or where Xaa at position 2 = Ile,
    then Xaa at position 3 is not Ala; or where Xaa at
    position 2 = Pro, then Xaa at position 3 is not Pro, Trp,
    or Arg; or where Xaa at position 2 = Gly, then Xaa at
    position 3 is not Ser or Arg; or where Xaa at postion 2 =
    Thr, then Xaa at position 3 is not Thr; and when Xaa at p
    1 = Pro, where Xaa and position 2 = Ala, then Xaa at
    position 3 is not Arg; or where Xaa at position 2 = Ser,
    then Xaa at position 3 is not Arg or Tyr; or where Xaa at
    position 2 = Met, then Xaa at position 3 is not Ser; or w
    Xaa at position 2 = Val, then Xaa at position 3 is not
    Gly; or where Xaa at position 2 = Arg, then Xaa at
    position 3 is not Ser; or where Xaa at position 2 = Ile,
    then Xaa at position 3 is not Arg; and when Xaa at
    position then Xaa at position 3 is not Lys; and when Xaa
    at position 1 = Val, where Xaa at position 2 = Ala, then
    Xaa at position 3 is not Cys or Gln or where Xaa at
    position 2 = Pro, then Xaa at position 3 is not Pro; and
    when Xaa a 1 = Ile, where Xaa at position 2 = Gly, then
    Xaa at position 3 is not His; or where Xaa at position 2 =
    Thr, then Xaa at position 3 is not Ser; or where Xaa at
    position 2 = Arg, then Xaa at position 3 is not Ser.”
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:466:
    Xaa Xaa Xaa Arg Pro Leu Pro Xaa Leu Pro Xaa Xaa
    1 5 10
    (2) INFORMATION FOR SEQ ID NO:467:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 15 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: unknown
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:467:
    Ala Gly Asp Arg Pro Leu Pro Pro Leu Pro Tyr Asn Pro Lys Ser
    1 5 10 15

Claims (126)

What is claimed is:
1. A peptide having at least nine and up to forty-five amino acid residues, including an amino acid sequence of the formula, R-2-L-P-5-6-P-8-9 (SEQ ID NO:10), positioned anywhere along the peptide, in which each number represents an amino acid residue, such that 2 represents any amino acid residue except cysteine, 5 and 6 each represents a hydrophobic amino acid residue, 8 represents any amino acid residue except cysteine, and 9 represents a hydrophilic amino acid residue except cysteine, each letter being the standard one-letter symbol for the corresponding amino acid, said peptide exhibiting a binding affinity for the SH3 domain of Src, provided that said peptide is not R-P-L-P-P-L-P-T-S (SEQ ID NO:11).
2. The peptide of claim 1 in which 2 is a P, R, A, L, Q, E or S.
3. The peptide of claim 1 in which 5 is a P, M, I or L.
4. The peptide of claim 1 in which 6 is a P, L, I or V.
5. The peptide of claim 1 in which 8 is a T, R, P, I, N, E, V, S, A, G or L.
6. The peptide of claim 1 in which 9 is a T, R, S, H or D.
7. The peptide of claim 1 which further comprises a C-terminal-flanking amino acid sequence of the formula 10, 10-11, 10-11-12, 10-11-12-13 (SEQ ID NO:12) or 10-11-12-13-14 (SEQ ID NO:13), in which each number represents any amino acid residue except cysteine, such that 10 is bound to 9 by a peptide bond.
8. The peptide of claim 7 in which 10 is T, R, L, S, D, P, A or N.
9. The peptide of claim 7 in which 11 is R, P, A, Q, S or T.
10. The peptide of claim 7 in which 12 is P, S, R or T.
11. The peptide of claim 7 in which 13 is P, S, R, F, H or T.
12. The peptide of claim 7 in which 14 is S, R, G or T.
13. The peptide of claim 1 which further comprises an N-terminal-flanking amino acid sequence of the formula 1′, 2′-1′, 3′-2′-1′ or 4′-3′-2′-1′ (SEQ ID NO:14) in which each number represents any amino acid residue except cysteine, such that 1′ is bound to R by a peptide bond.
14. The peptide of claim 13 in which 1′ is T. P, S, N, F, W, K, H, Q or G.
15. The peptide of claim 13 in which 2′ is S, T, G, P, R, Q, L, A or H.
16. The peptide of claim 13 in which 3′ is R, S, P, G, A, V, Y or L.
17. The peptide of claim 13 in which 4′ is R, S, V, T, G, L or F.
18. A peptide having at least thirteen and up to forty-five amino acid residues, including an amino acid sequence of the formula, 3′-2′-1′-R-2-L-P-5-6-P-8-9-10 (SEQ ID NO:15), positioned anywhere along the peptide, in which each number represents an amino acid residue, such that 3′, 2′, 1′, 2, 8, and 10 each represents any amino acid residue except cysteine, 5 and 6 each represents a hydrophobic amino acid residue, and 9 represents a hydrophilic amino acid residue except cysteine, each letter being the standard one-letter symbol for the corresponding amino acid, said peptide exhibiting a binding affinity for the SH3 domain of Src.
19. The peptide of claim 18 in which 5 is a P or M.
20. The peptide of claim 18 in which 1′ is T, P, S or N.
21. The peptide of claim 18 in which 2′ is S or T.
22. The peptide of claim 18 in which 3′ is R or S.
23. The peptide of claim 18 in which 10 is T or R.
24. The peptide of claim 1 the binding affinity of which is at least three-fold greater than that exhibited by the peptide RPLPPLP for the SH3 domain of Src.
25. The peptide of claim 18 the binding affinity of which is at least three-fold greater than that exhibited by the peptide RPLPPLP (SEQ ID NO:9) for the SH3 domain of Src.
26. The peptide of claim 1 the binding affinity of which is at least four-fold greater than that exhibited by the peptide RPLPPLP (SEQ ID NO:9) for the SH3 domain of Src.
27. The peptide of claim 18 the binding affinity of which is at least four-fold greater than that exhibited by the peptide RPLPPLP (SEQ ID NO:9) for the SH3 domain of Src.
28. The peptide of claim 1 which further exhibits a general binding affinity for the SH3 domain of Abl, Grb2, PLC- , PLC- , Ras GAP, Nck, p85 PI-3′Kinase, and proteins related thereto.
29. The peptide of claim 1 which exhibits a selective binding affinity for the SH3 domain of Src and Src-related proteins.
30. The peptide of claim 18 which further exhibits a general binding affinity for the SH3 domain of Abl, Grb2, PLC- , PLC- , Ras GAP, Nck, p85 PI-3′Kinase, and proteins related thereto.
31. The peptide of claim 18 which exhibits a selective binding affinity for the SH3 domain of Src and Src-related proteins.
32. A peptide having the amino acid sequence RSTPRPLPMLPTTR (SEQ ID NO:62).
33. A peptide having the amino acid sequence RSTPRPLPPLPTTR (SEQ ID NO:67).
34. A peptide having the amino acid sequence GILAPPVPPRNTR (SEQ ID NO:63).
35. A peptide having the amino acid sequence VLKRPLPIPPVTR (SEQ ID NO:64).
36. A peptide having the amino acid sequence GPHRRLPPTPATR (SEQ ID NO:65).
37. A peptide having the amino acid sequence ANPSPATRPLPTR (SEQ ID NO:66).
38. A peptide having an amino acid sequence selected from the group consisting of RSTRPLPILPRTT, STPRPLPMLPTTR, STNRPLPMIPTTR, RSTRPLPSLPITT, STSRPLPSLPTTR, RSTRSLPPLPPTT, RSTRQLPIPPTTT, STPRPLPLIPTTP, RSTRPLPPTPLTT, and RSTRPQPPPPITT (SEQ ID NOS:85-94).
39. A peptide having the amino acid sequence selected from the group consisting of VLKRPLPIPPVTR (SEQ ID NO:64), YSTRPVPPITRPS (SEQ ID NO:76), SHKSRLPPLPTRP (SEQ ID NO:77), GPHRRLPPTPATR (SEQ ID NO:65), PATRPLPTRPSRT (SEQ ID NO:81), and SGGILAPPVPPRN (SEQ ID NO:84).
40. A peptide having the amino acid sequence selected from the group consisting of PPNSPLPPLPTHL (SEQ ID NO:72), TGRGPLPPLPNDS (SEQ ID NO:74), YRFRALPSPPSAS, LAQRQLPPTPGRD ALQRRLPRTPPPA (SEQ ID NOS:78-80), YSTRPLPSRPSRT, and XPGRILLLPSEPR (SEQ ID NOS:82-83).
41. A construct comprising a nucleic acid encoding a peptide of claim 1 or its complement.
42. The construct of claim 41 which is a DNA polynucleotide.
43. The construct of claim 41 which is a RNA polynucleotide.
44. A construct comprising a nucleic acid encoding a peptide of claim 18 or its complement.
45. The construct of claim 44 which is a DNA polynucleotide.
46. The construct of claim 44 which is a RNA polynucleotide.
47. The construct of claim 41 which is a transforming vector.
48. The construct of claim 44 which is a transforming vector.
49. A host cell transformed with the vector of claim 47.
50. A host cell transformed with the vector of claim 48.
51. A conjugate comprising a peptide of claim 1 and a second molecule.
52. The conjugate of claim 51 in which said second molecule is selected from the group consisting of an amino acid, a peptide, a protein, a nucleic acid, a nucleoside, a glycosidic residue, a label, a drug or a small molecule.
53. A diagnostic kit for the detection of SH3 domains comprising an SH3 domain-binding peptide and a detectable label conjugated to said peptide directly, indirectly or by complexation, said peptide comprising: (i) a core sequence motif of the formula RXLPφφP (SEQ ID NO:71), wherein X represents any amino acid except cysteine and φ represents a hydrophobic amino acid residue, each letter representing the standard one-letter designation for the corresponding amino acid residue; and (ii) two or more additional amino acid residues flanking said core sequence at its C-terminal end, N-terminal end or both.
54. A drug delivery system comprising an SH3 domain-binding peptide and a drug conjugated to said peptide directly, indirectly or by complexation, said peptide comprising: (i) a core sequence motif of the formula RXLPφφP (SEQ ID NO:71), wherein X represents any amino acid except cysteine and φ represents a hydrophobic amino acid residue, each letter representing the standard one-letter designation for the corresponding amino acid residue; and (ii) two or more additional amino acid residues flanking said core sequence at its C-terminal end, N-terminal end or both.
55. The drug delivery system of claim 54 which may be administered parenterally, orally, enterally, topically or by inhalation.
56. The drug delivery system of claim 54 which may be administered intranasally, opthalmically or intravaginally.
57. The drug delivery system of claim 54 which is in the form of a solid, gel, liquid or aerosol.
58. A method of modulating the activity of Src or Src-related proteins comprising administering a composition comprising an effective amount of a peptide of claim 1 and a carrier.
59. The method of claim 58 which inhibits the activity of Src or Src-related proteins.
60. The method of claim 58 which activates Src or Src-related proteins.
61. A method of identifying a peptide having a region that binds to an SH3 domain comprising:
(a) providing an immobilized target protein comprising an SH3 domain;
(b) incubating said immobilized target protein with an aliquot taken from a random peptide library;
(c) washing unbound peptide from said immobilized target protein;
(d) recovering the peptide bound to said immobilized target protein; and
(e) determining the primary sequence of the SH3 domain-binding peptide.
62. The method of claim 61 in which said library is a displayed random peptide library.
63. The method of claim 62 in which said library is a phage-displayed random peptide library.
64. The method of claim 62 in which said library is a phagemid-displayed random peptide library.
65. The method of claim 61 in which step (c) includes washing unbound phage from said immobilized target protein; step (d) includes recovering the phage bound to said immobilized target protein; and step (e) includes determining the relevant nucleotide sequence of said binding phage nucleic acid, from which the primary sequence corresponding to the SH3 domain-binding peptide is deduced.
66. A method of identifying a peptide having a region that binds to an SH3 domain comprising:
(a) providing an immobilized target protein comprising an SH3 domain;
(b) incubating said immobilized target protein with an aliquot taken from a phage-displayed random peptide library, which library includes peptides having a random sequence of 8 amino acid residues;
(c) washing unbound phage from said immobilized target protein;
(d) recovering the phage bound to said immobilized target protein; and
(e) determining the relevant nucleotide sequence of said binding phage nucleic acid and deducing the primary sequence corresponding to the SH3 domain-binding peptide.
67. The method of claim 66 which further comprises amplifying the titer of the recovered phage.
68. The method of claim 66 which further comprises repeating the steps of incubation, washing and recovery to provide SH3 domain-binding peptide-enriched phage.
69. A pharmaceutical composition comprising an SH3 domain-binding peptide and a pharmaceutically acceptable carrier, said peptide comprising: (i) a 9-mer sequence motif of the formula RXLPφφPXψ (SEQ ID NO:10), wherein X represents any amino acid except cysteine, φ represents a hydrophobic amino acid residue, and wherein ψ is a hydrophilic amino acid residue except cysteine, each letter representing the standard one-letter designation for the corresponding amino acid residue; and, optionally, (ii) additional amino acid residues flanking said 9-mer sequence at its C-terminal end, N-terminal end or both, up to a total of 45 amino acid residues, including said 9-mer sequence.
70. The composition of claim 69 in which at least one additional amino acid flanks said 9-mer sequence.
71. The composition of claim 69 in which at least two additional amino acids flank said 9-mer sequence.
72. The composition of claim 69 in which at least three additional amino acids flank said 9-mer sequence.
73. A method of disrupting protein tyrosine kinase-mediated signal transduction pathways comprising administering an effective amount of a peptide of claim 1.
74. A method of regulating the processing, trafficking or translation of RNA by administering an effective amount of a peptide of claim 1.
75. A purified peptide that binds to the SH3 domain of Cortactin, said peptide comprising the amino acid sequence ZPPφPxKPxW (SEQ ID NO:113), where Z represents K or R; φ represents a hydrophobic amino acid; and x represents any amino acid.
76. A purified peptide that binds to the middle SH3 domain of Nck, said peptide comprising the amino acid sequence φxxxxPxPPφRZxSL (SEQ ID NO:127), where Z represents S or T; φ represents a hydrophobic amino acid; and x represents any amino acid.
77. A purified peptide that binds to the SH3 domain of Abl, said peptide comprising the amino acid sequence PPxWxPPPφP (SEQ ID NO:141), where φ represents a hydrophobic amino acid; and x represents any amino acid.
78. A purified peptide that binds to the SH3 domain of Src, said peptide comprising the amino acid sequence LXXRPLPXψP (SEQ ID NO:165), where ψ represents an aliphatic amino acid; and X represents any amino acid.
79. A purified peptide that binds to the SH3 domain of Cortactin, said peptide comprising the amino acid sequence +PPψPXKPXWL (SEQ ID NO:166), where +represents a basic amino acid; ψ represents an aliphatic amino acid; and X represents any amino acid.
80. A purified peptide that binds to the SH3 domain of Abl, said peptide comprising the amino acid sequence PPXθXPPPψP (SEQ ID NO:173) , where θ represents an aromatic amino acid; ψ represents an aliphatic amino acid; and X represents any amino acid.
81. A purified peptide that binds to the SH3 domain of PLC said peptide comprising the amino acid sequence PPVPPRPXXTL (SEQ ID NO:175), where X represents any amino acid.
82. A purified peptide that binds to the SH3 domain of p53bp2, said peptide comprising the amino acid sequence RPXψPψR+SXP (SEQ ID NO:196), where + represents a basic amino acid; ψ represents an aliphatic amino acid; and X represents any amino acid.
83. A purified peptide that binds to the N terminal SH3 domain of Crk, said peptide comprising the amino acid sequence ψPψPψLPψK (SEQ ID NO:210), where ψ represents an aliphatic amino acid; and X represents any amino acid.
84. A purified peptide that binds to the SH3 domain of Yes, said peptide comprising the amino acid sequence ψXXRPLPXLP (SEQ ID NO:222), where ψ represents an aliphatic amino acid; and X represents any amino acid.
85. A purified peptide that binds to the N terminal SH3 domain of Grb2, said peptide comprising an amino acid sequence selected from the group consisting of: +θDXPLPXLP (SEQ ID NO:223), YθX+PLPXLP (SEQ ID NO:238), and 6DPLPXLP (SEQ ID NO:243), where θ represent an aromatic amino acid; +represents a basic amino acid; ψ represents an aliphatic amino acid; and X represents any amino acid.
86. A purified peptide that binds to the SH3 domain of Cortactin, said peptide comprising an amino acid sequence selected from the group consisting of:
LTPQSKPPLPPKPSAV; (a portion of SEQ ID NO:112) SSHNSRPPLPEKPSWL; (a portion of SEQ ID NO:111) PVKPPLPAKPWWLPPL; (SEQ ID NO:167) TERPPLPQRPDWLSYS; (a portion of SEQ ID NO:109) LGEFSKPPIPQKPTWM; (a portion of SEQ ID NO:108) YPQFRPPVPPKPSLMQ; (SEQ ID NO:168) VTRPPLPPKPGHMADF; (SEQ ID NO:169) VSLGLKPPVPPKPMQL; (SEQ ID NO:170) LLGPPVPPKPQTLFSF; (a portion of SEQ ID NO:107) YKPEVPARPIWLSEL; (SEQ ID NO:171) GAGAARPLVPKKPLFL; and (SEQ ID NO:172) SREPDWLCPNCPLLLRSDSR. (SEQ ID NO:110)
87. A purified peptide that binds to the middle SH3 domain of Nck, said peptide comprising an amino acid sequence selected from the group consisting of:
SSLGVGWKPLPPMRTASLSR; (SEQ ID NO:114) SSVGFADRPRPPLRVESLSR; (SEQ ID NO:115) SSAGILRPPEKPXRSFSLSR; (SEQ ID NO:116) SSPYTGDVPIPPLRGASLSR; (SEQ ID NO:117) SSLMGSWPPVPPLRSDSLSR; (SEQ ID NO:118) SSIGEDTPPSPPTRRASLSR; (SEQ ID NO:119) SRSLSEVSPKPPIRSVSLSR; (SEQ ID NO:120) SSVSEGYSPPLPPRSTSLSR; (SEQ ID NO:121) SSSFTLAAPTPPTRSLSLSR; (SEQ ID NO:122) SSPPYELPPRPPNRTVSLSR; (SEQ ID NO:123) SRVVDGLAPPPPVRLSSLSR; (SEQ ID NO:124) SSLGYSGAPVPPHRxSSLSR; and (SEQ ID NO:125) SSISDYSRPPPPVRTLSLSR. (SEQ ID NO:126)
88. A purified peptide that binds to the SH3 domain of Abl, said peptide comprising an amino acid sequence selected from the group consisting of:
PPWWAPPPIPNSPQVL; (SEQ ID NO:174); PPKFSPPPPPYWQLHA; (a portion of SEQ ID NO:132) PPHWAPPAPPAMSPPI; (a portion of SEQ ID NO:130) PPTWTPPKPPGWGVVF; (a portion of SEQ ID NO:137) PPSFAPPAAPPRHSFG; (a portion of SEQ ID NO:133) PTYPPPPPPPDTAKGA; (a portion of SEQ ID NO:135) GPRWSPPPVPLPTSLD; (a portion of SEQ ID NO:128) APTWSPPALPNVAKYK; (a portion of SEQ ID NO:138) PPDYAAPAIPSSLWVD; (a portion of SEQ ID NO:129) IKGPRFPVPPVPLNGV; (a portion of SEQ ID NO:139) PPAWSPPHRPVAFGST; (a portion of SEQ ID NO:140) APKKPAPPVPMMAHVM; (a portion of SEQ ID NO:134) SSDRCWECPPWPAGGQRGSR; (SEQ ID NO:131) and SSPPXXXPPPIPNSPQVLSR. (SEQ ID NO:136)
89. A purified peptide that binds to the SH3 domain of PLC said peptide comprising an amino acid sequence selected from the group consisting of:
MPPPVPPRPPGTLQVA; (SEQ ID NO:176) LSYSPPPVPPRPDSTL; (SEQ ID NO:177) VLAPPVPPRPGNTFFT; (SEQ ID NO:178) YRPPVAPRPPSSLSVD; (SEQ ID NO:179) LQCPDCPRVPPRPIPI; (SEQ ID NO:180) VPPLVAPRPPSTLNSL; (a portion of SEQ ID NO:143) LTPPPFPKRPRWTLPE; (SEQ ID NO:181) YWPHRPPLAPPQTTLG; (SEQ ID NO:182) SSMKVHNFPLPPLPSYETSR; (SEQ ID NO:142) SSLYWQHGPDPPVGAPQLSR; (SEQ ID NO:144) and SSHPLNSWPGGPFRHNLSSR. (SEQ ID NO:145)
90. A purified peptide that binds to the SH3 domain of Src, said peptide comprising an amino acid sequence selected from the group consisting of:
LASRPLPLLPNSAPGQ; (a portion of SEQ ID NO:155) LTGRPLPALPPPFSDF; (a portion of SEQ ID NO:152) PAYRPLPRLPDLSVIY; (a portion of SEQ ID NO:150) RALRVRPLPPVPGTSL; (a portion of SEQ ID NO:146) DAPGSLPFRPLPPVPT; (a portion of SEQ ID NO:148) LKWRALPPLPETDTPY; (a portion of SEQ ID NO:157) ISQRALPPLPLMSDPA; (a portion of SEQ ID NO:149) LTSRPLPDIPVRPSKS; (a portion of SEQ ID NO:156) NTNRPLPPTPDGLDVR; (a portion of SEQ ID NO:158) MKDRVLPPIPTVESAV; (a portion of SEQ ID NO:153) LQSRPLPLPPQSSYPI; (a portion of SEQ ID NO:159) FINRRLPALPPDNSLL; (a portion of SEQ ID NO:151) FRALPLPPTPDNPFAG; and (a portion of SEQ ID NO:147) LYSAIAPDPPPRNSSS. (a portion of SEQ ID NO:154)
91. A purified peptide that binds to the SH3 domain of p53bp2, said peptide comprising an amino acid sequence selected from the group consisting of:
YDASSAPQRPPLPVRKSRP; (SEQ ID NO:183) EYVNASPERPPIPGRKSRP; (SEQ ID NO:184) WNGIAIPGRPEIPPRASRP; (SEQ ID NO:185) SMIFIYPERPSPPPRFSRP; (SEQ ID NO:186) GVEEWNPERPQIPLRLSRP; (SEQ ID NO:187) WVVDSRPDIPLRRSLP; (SEQ ID NO:188) VVPLGRPEIPLRKSLP; (SEQ ID NO:189) GGTVGRPPIPERKSVD; (SEQ ID NO:190) YSHAGRPEVPPRQSKP; (SEQ ID NO:191) FSAAARPDIPSRASTP; (SEQ ID NO:192) LYIPKRPEVPPRRHEA; (SEQ ID NO:193) NNISARPPLPSRQNPP; and (SEQ ID NO:194) MAGTPRPAVPQRMNPP. (SEQ ID NO:195)
92. A purified peptide that binds to the N terminal SH3 domain of Crk, said peptide comprising an amino acid sequence selected from the group consisting of:
GQPAGDPDPPPLPAKF; (SEQ ID NO:197) FEQTGVPLLPPKSFKY; (SEQ ID NO:198) IFGDPPPPIPMKGRSL; (SEQ ID NO:199) SNQGSIPVLPIKRVQY; (SEQ ID NO:200) NYVNALPPGPPLPAKN; (SEQ ID NO:201) SSDPERPVLPPKLWSV; (SEQ ID NO:202) HFGPSKPPLPIKTRIT; (SEQ ID NO:203) DWKVPEPPVPKLPLKQ; (SEQ ID NO:204) ATSEGLPILPSKVGSY; (SEQ ID NO:205) NANVSAPRAPAFPVKT; (SEQ ID NO:206) EMVLGPPVPPKRGTVV; (SEQ ID NO:207) AGSRHPPTLPPKESGG; and (SEQ ID NO:208) SVAADPPRLPAKSRPQ. (SEQ ID NO:209)
93. A purified peptide that binds to the SH3 domain of Yes, said peptide comprising an amino acid sequence selected from the group consisting of:
ITMRPLPALPGHGQIH; (SEQ ID NO:211) LPRRPLPDLPMAAGKG; (SEQ ID NO:212) LGSRPLPPTPRQWPEV; (SEQ ID NO:213) STIRPLPAIPRDTLLT; (SEQ ID NO:214) RSGRPLPPIPEVGHNV; (SEQ ID NO:215) IGSRPLPWTPDDLGSA; (SEQ ID NO:216) LAQRELPGLPAGAGVS; (SEQ ID NO:217) IPGRALPELPPQRALP; (SEQ ID NO:218) FVGRELPPTPRTVIPW; (SEQ ID NO:219) DPRSALPALPLTPLQT; and (SEQ ID NO:220) SPHDVLPALPDSHSKS. (SEQ ID NO:221)
94. A purified peptide that binds to the N terminal SH3 domain of Grb2, said peptide comprising an amino acid sequence selected from the group consisting of:
KWDSLLPALPPAFTVE; (SEQ ID NO:224) RWDQVLPELPTSKGQI; (SEQ ID NO:225) RFDFPLPTHPNLQKAH; (SEQ ID NO:226) RLDSPLPALPPTVMQN; (SEQ ID NO:227) RWGAPLPPLPEYSWST; (SEQ ID NO:228) YWDMPLPRLPGEEPSL; (SEQ ID NO:229) RFDYNLPDVPLSLGTA; (SEQ ID NO:230) TKKPNAPLPPLPAYMG; (SEQ ID NO:231) KWDLDLPPEPMSLGNY; (SEQ ID NO:232) YYQRPLPPLPLSHFES; (SEQ ID NO:234) YYRKPLPNLPRGQTDD; (SEQ ID NO:235) YFDKPLPESPGALMSL; (SEQ ID NO:236) YFSRALPGLPERQEAH; (SEQ ID NO:237) SLWDPLPPIPQSKTSV; (SEQ ID NO:239) SYYDPLPKLPDPGDLG; (SEQ ID NO:240) KLYYPLPPVPFKDTKH; and (SEQ ID NO:241) DPYDALPETPSMKASQ. (SEQ ID NO:242)
95. A purified peptide having an amino acid sequence selected from the group consisting of: SEQ ID NOs: 250-252, 254, 256-259, 261, 262, 264-266, 269-272, 275, 280, 281, 286-289, 291, 294, and 295.
96. A purified peptide having an amino acid sequence selected from the group consisting of: SEQ ID NOs: 296-453.
97. A method of identifying an inhibitor of the binding between a first molecule comprising an SH3 domain and a second molecule that binds to the SH3 domain comprising incubating one or more compounds from which it is desired to select such an inhibitor with the first molecule and the second molecule under conditions conducive to binding and detecting the one or more compounds that inhibit binding of the first molecule to the second molecule
98. The method of claim 97 where the second molecule is obtained by:
(i) screening a peptide library with the SH3 domain to obtain peptides that bind the SH3 domain;
(ii) determining a consensus sequence for the peptides obtained in step (i);
(iii) producing a peptide comprising the consensus sequence;
wherein the second molecule comprises the peptide comprising the consensus sequence.
99. The method of claim 97 where the second molecule is obtained by:
(i) screening a peptide library with the SH3 domain to obtain peptides that bind the SH3 domain;
(ii) determining a consensus sequence for the peptides obtained in step (i);
(iii) searching a database to identify amino acid sequences that resemble the consensus sequence of step (ii);
(iv) producing a peptide comprising an amino acid sequence identified in step (iii);
wherein the second molecule comprises the peptide comprising an amino acid sequence identified in step (iii).
100. The method of claim 97 where the second molecule is a peptide that binds to the SH3 domain of Cortactin, said peptide comprising the amino acid sequence ZPPφPxKPxW (SEQ ID NO:113), where Z represents K or R; + represents a hydrophobic amino acid; and x represents any amino acid.
101. The method of claim 97 where the second molecule is a peptide that binds to the middle SH3 domain of Nck, said peptide comprising the amino acid sequence φxxxxxPxPPφRZxSL (SEQ ID NO:127), where Z represents S or T; φ represents a hydrophobic amino acid; and x represents any amino acid.
102. The method of claim 97 where the second molecule is a peptide that binds to the SH3 domain of Abl, said peptide comprising the amino acid sequence PPxWxPPPφP (SEQ ID NO:141), where φ represents a hydrophobic amino acid; and x represents any amino acid.
103. The method of claim 97 where the second molecule is a peptide that binds to the SH3 domain of Src, said peptide comprising the amino acid sequence LXXRPLPXψP (SEQ ID NO:165), where ψ represents an aliphatic amino acid; and X represents any amino acid.
104. The method of claim 97 where the second molecule is a peptide that binds to the SH3 domain of Cortactin, said peptide comprising the amino acid sequence +PPψPXKPXWL (SEQ ID NO:166), where + represents a basic amino acid; ψ represents an aliphatic amino acid; and X represents any amino acid.
105. The method of claim 97 where the second molecule is a peptide that binds to the SH3 domain of Abl, said peptide comprising the amino acid sequence PPXθXPPPψP (SEQ ID NO:173), where θrepresents an aromatic amino acid; ψ represents an aliphatic amino acid; and X represents any amino acid.
106. The method of claim 97 where the second molecule is a peptide that binds to the SH3 domain of PLCγ, said peptide comprising the amino acid sequence PPVPPRPXXTL (SEQ ID NO:175), where X represents any amino acid.
107. The method of claim 97 where the second molecule is a peptide that binds to the SH3 domain of p53bp2, said peptide comprising the amino acid sequence RPXψPψR+SXP (SEQ ID NO:196), where + represents a basic amino acid; ψ represents an aliphatic amino acid; and X represents any amino acid.
108. The method of claim 97 where the second molecule is a peptide that binds to the N terminal SH3 domain of Crk, said peptide comprising the amino acid sequence ψPψLPψK (SEQ ID NO:210), where ψ represents an aliphatic amino acid; and X represents any amino acid.
109. The method of claim 97 where the second molecule is a peptide that binds to the SH3 domain of Yes, said peptide comprising the amino acid sequence ψXXRPLPXLP (SEQ ID NO:222), where ψ represents an aliphatic amino acid; and X represents any amino acid.
110. The method of claim 97 where the second molecule is a peptide that binds to the N terminal SH3 domain of Grb2, said peptide comprising an amino acid sequence selected from the group consisting of: +θDXPLPXLP (SEQ ID NO:223), YθX+PLPXLP (SEQ ID NO:238), and θDPLPXLP (SEQ ID NO:243), where θ represent an aromatic amino acid; +represents a basic amino acid; ψ represents an aliphatic amino acid; and X represents any amino acid.
111. The method of claim 97 where the second molecule is a peptide that binds to the SH3 domain of Cortactin, said peptide comprising an amino acid sequence selected from the group consisting of:
LTPQSKPPLPPKPSAV; (a portion of SEQ ID NO:112) SSHNSRPPLPEKPSWL; (a portion of SEQ ID NO:111) PVKPPLPAKPWWLPPL;              (SEQ ID NO:167) TERPPLPQRPDWLSYS; (a portion of SEQ ID NO:109) LGEFSKPPIPQKPTWM; (a portion of SEQ ID NO:108) YPQFRPPVPPKPSLMQ;              (SEQ ID NO:168) VTRPPLPPKPGHMADF;              (SEQ ID NO:169) VSLGLKPPVPPKPMQL;              (SEQ ID NO:170) LLGPPVPPKPQTLFSF; (a portion of SEQ ID NO:107) YKPEVPARPIWLSEL;              (SEQ ID NO:171) GAGAARPLVPKKPLFL; and              (SEQ ID NO:172) SREPDWLCPNCPLLLRSDSR.              (SEQ ID NO:110)
112. The method of claim 97 where the second molecule is a peptide that binds to the middle SH3 domain of Nck, said peptide comprising an amino acid sequence selected from the group consisting of:
SSLGVGWKPLPPMRTASLSR; (SEQ ID NO:114) SSVGFADRPRPPLRVESLSR; (SEQ ID NO:115) SSAGILRPPEKPXRSFSLSR; (SEQ ID NO:116) SSPYTGDVPIPPLRGASLSR; (SEQ ID NO:117) SSLMGSWPPVPPLRSDSLSR; (SEQ ID NO:118) SSIGEDTPPSPPTRRASLSR; (SEQ ID NO:119) SRSLSEVSPKPPIRSVSLSR; (SEQ ID NO:120) SSVSEGYSPPLPPRSTSLSR; (SEQ ID NO:121) SSSFTLAAPTPPTRSLSLSR; (SEQ ID NO:122) SRVVDGLAPPPPVRLSSLSR; (SEQ ID NO:123) SRVVDGLAPPPPVRLSSLSR; (SEQ ID NO:124) SSLGYSGAPVPPHRxSSLSR; and (SEQ ID NO:125) SSISDYSRPPPPVRTLSLSR. (SEQ ID NO:126)
113. The method of claim 97 where the second molecule is a peptide that binds to the SH3 domain of Abl, said peptide comprising an amino acid sequence selected from the group consisting of:
PPWWAPPPIPNSPQVL;              (SEQ ID NO:174) PPKFSPPPPPYWQLHA; (a portion of SEQ ID NO:132) PPHWAPPAPPAMSPPI; (a portion of SEQ ID NO:130) PPTWTPPKPPGWGVVF; (a portion of SEQ ID NO:137) PPSFAPPAAPPRHSFG; (a portion of SEQ ID NO:133) PTYPPPPPPDTAKGA; (a portion of SEQ ID NO:135) GPRWSPPPVPLPTSLD; (a portion of SEQ ID NO:128) APTWSPPALPNVAKYK; (a portion of SEQ ID NO:138) PPDYAAPAIPSSLWVD; (a portion of SEQ ID NO:129) IKGPRFPVPPVPLNGV; (a portion of SEQ ID NO:139) PPAWSPPHRPVAFGST; (a portion of SEQ ID NO:140) APKKPAPPVPMMAHVM; (a portion of SEQ ID NO:134) SSDRCWECPPWPAGGQRGSR;              (SEQ ID NO:131) and SSPPXXXPPPIPNSPQVLSR.              (SEQ ID NO:136)
114. The method of claim 97 where the second molecule is a peptide that binds to the SH3 domain of PLCγ, said peptide comprising an amino acid sequence selected from the group consisting of:
MPPPVPPRPPGTLQVA;              (SEQ ID NO:176) LSYSPPPVPPRPDSTL;              (SEQ ID NO:177) VLAPPVPPRPGNTFFT;              (SEQ ID NO:178) YRPPVAPRPPSSLSVD;              (SEQ ID NO:179) LQCPDCPRVPPRPIPI;              (SEQ ID NO:180) VPPLVAPRPPSTLNSL; (a portion of SEQ ID NO:143) LTPPPFPKRPRWTLPE;              (SEQ ID NO:181) YWPHRPPLAPPQTTLG;              (SEQ ID NO:182) SSMKVHNFPLPPLPSYETSR;              (SEQ ID NO:142) SSLYWQHGPDPPVGAPQLSR;              (SEQ ID NO:144) and SSHPLNSWPGGPFRHNLSSR.              (SEQ ID NO:145)
115. The method of claim 97 where the second molecule is a peptide that binds to the SH3 domain of Src, said peptide comprising an amino acid sequence selected from the group consisting of:
LASRPLPLLPNSAPGQ; (a portion of SEQ ID NO:155) LTGRPLPALPPPFSDF; (a portion of SEQ ID NO:152) PAYRPLPRLPDLSVIY; (a portion of SEQ ID NO:150) RALRVRPLPPVPGTSL; (a portion of SEQ ID NO:146) DAPGSLPFRPLPPVPT; (a portion of SEQ ID NO:148) LKWRALPPLPETDTPY; (a portion of SEQ ID NO:157) ISQRALPPLPLMSDPA; (a portion of SEQ ID NO:149) LTSRPLPDIPVRPSKS; (a portion of SEQ ID NO:156) NTNRPLPPTPDGLDVR; (a portion of SEQ ID NO:158) MKDRVLPPIPTVESAV; (a portion of SEQ ID NO:153) LQSRPLPLPPQSSYPI; (a portion of SEQ ID NO:159) FINRRLPALPPDNSLL; (a portion of SEQ ID NO:151) FRALPLPPTPDNPFAG; and (a portion of SEQ ID NO:147) LYSAIAPDPPPRNSSS. (a portion of SEQ ID NO:154)
116. The method of claim 97 where the second molecule is a peptide that binds to the SH3 domain of p53bp2, said peptide comprising an amino acid sequence selected from the group consisting of:
YDASSAPQRPPLPVRKSRP; (SEQ ID NO:183) EYVNASPERPPIPGRKSRP; (SEQ ID NO:184) WNGIAIPGRPEIPPRASRP; (SEQ ID NO:185) SMIFIYPERPSPPPRFSRP; (SEQ ID NO:186) GVEEWNPERPQIPLRLSRP; (SEQ ID NO:187) WVVDSRPDIPLRRSLP; (SEQ ID NO:188) VVPLGRPEIPLRKSLP; (SEQ ID NO:189) GGTVGRPPIPERKSVD; (SEQ ID NO:190) YSHAGRPEVPPRQSKP; (SEQ ID NO:191) FSAAARPDIPSRASTP; (SEQ ID NO:192) LYIPKRPEVPPRRHEA; (SEQ ID NO:193) NNISARPPLPSRQNPP; and (SEQ ID NO:194) MAGTPRPAVPQRMNPP. (SEQ ID NO:195)
117. The method of claim 97 where the second molecule is a peptide that binds to the N terminal SH3 domain of Crk, said peptide comprising an amino acid sequence selected from the group consisting of:
GQPAGDPDPPPLPAKF; (SEQ ID NO:197) FEQTGVPLLPPKSFKY; (SEQ ID NO:198) IFGDPPPPIPMKGRSL; (SEQ ID NO:199) SNQGSIPVLPIKRVQY; (SEQ ID NO:200) NYVNALPPGPPLPAKN; (SEQ ID NO:201) SSDPERPVLPPKLWSV; (SEQ ID NO:202) HFGPSKPPLPIKTRIT; (SEQ ID NO:203) DWKVPEPPVPKLPLKQ; (SEQ ID NO:204) ATSEGLPILPSKVGSY; (SEQ ID NO:205) NANVSAPRAPAFPVKT; (SEQ ID NO:206) EMVLGPPVPPKRGTVV; (SEQ ID NO:207) AGSRHPPTLPPKESGG; and (SEQ ID NO:208) SVAADPPRLPAKSRPQ. (SEQ ID NO:209)
118. The method of claim 97 where the second molecule is a peptide that binds to the SH3 domain of Yes, said peptide comprising an amino acid sequence selected from the group consisting of:
ITMRPLPALPGHGQIH; (SEQ ID NO:211) LPRRPLPDLPMAAGKG; (SEQ ID NO:212) LGSRPLPPTPRQWPEV; (SEQ ID NO:213) STIRPLPAIPRDTLLT; (SEQ ID NO:214) RSGRPLPPIPEVGHNV; (SEQ ID NO:215) IGSRPLPWTPDDLGSA; (SEQ ID NO:216) LAQRELPGLPAGAGVS; (SEQ ID NO:217) IPGRALPELPPQRALP; (SEQ ID NO:218) FVGRELPPTPRTVIPW; (SEQ ID NO:219) DPRSALPALPLTPLQT; and (SEQ ID NO:220) SPHDVLPALPDSHSKS. (SEQ ID NO:221)
119. The method of claim 97 where the second molecule is a peptide that binds to the N terminal SH3 domain of Grb2, said peptide comprising an amino acid sequence selected from the group consisting of:
KWDSLLPALPPAFTVE; (SEQ ID NO:224) RWDQVLPELPTSKGQI; (SEQ ID NO:225) RFDFPLPTHPNLQKAH; (SEQ ID NO:226) RLDSPLPALPPTVMQN; (SEQ ID NO:227) RWGAPLPPLPEYSWST; (SEQ ID NO:228) YWDMPLPRLPGEEPSL; (SEQ ID NO:229) RFDYNLPDVPLSLGTA; (SEQ ID NO:230) TKKPNAPLPPLPAYMG; (SEQ ID NO:231) KWDLDLPPEPMSLGNY; (SEQ ID NO:232) YYQRPLPPLPLSHFES; (SEQ ID NO:234) YYRKPLPNLPRGQTDD; (SEQ ID NO:235) YFDKPLPESPGALMSL; (SEQ ID NO:236) YFSRALPGLPERQEAH; (SEQ ID NO:237) SLWDPLPPIPQSKTSV; (SEQ ID NO:239) SYYDPLPKLPDPGDLG; (SEQ ID NO:240) KLYYPLPPVPFKDTKH; and (SEQ ID NO:241) DPYDALPETPSMKASQ. (SEQ ID NO:242)
120. The method of claim 97 where the second molecule is a peptide having an amino acid sequence selected from the group consisting of: SEQ ID NOs: 250-252, 254, 256-259, 261, 262, 264-266, 269-272, 275, 280, 281, 286-289, 291, 294, and 295.
121. The method of claim 97 where the second molecule is a peptide having an amino acid sequence selected from the group consisting of: SEQ ID NOs: 296-453.
122. A method of identifying a compound that affects the binding of a molecule comprising an SH3 domain and a ligand of the SH3 domain, the method comprising:
(a) contacting the SH3 domain and the ligand under conditions conducive to binding in the presence of a candidate compound and measuring the amount of binding between the SH3 domain and the ligand;
(b) comparing the amount of binding in step (a) with the amount of binding known or determined to occur between the molecule and the ligand in the absence of the candidate compound, where a difference in the amount of binding between step (a) and the amount of binding known or determined to occur between the molecule and the ligand in the absence of the candidate compound indicates that the candidate compound is a compound that affects the binding of the molecule comprising an SH3 domain and the ligand.
123. A kit comprising, in one or more containers:
(a) a purified first molecule comprising an SH3 domain;
(b) a purified second molecule that binds to the SH3 domain.
124. The kit of claim 123 wherein said second molecule comprises a peptide having an amino acid sequence selected from the group consisting of: SEQ ID NOs:107- 112, 114-126, 128-140, 142-159, 167, 168-172, 174, 176-195, 197-209, 211-221, 224-232, 234-237, 239-242, 250-252, 254, 256-259, 261, 262, 264-266, 269-272, 275, 280, 281, 286-289, 291, 294-453.
125. A purified peptide that binds to the SH3 domain of Src, said peptide comprising the amino acid sequence
LX1X2RPLPX3ψPX4X5 (SEQ ID NO:454)
where ψ represents aliphatic amino acid residues and X1, X2, X3, X4, and X5 represent any amino acid; except that if
X3=P, ψ=L, X4=P, and X5=P, then:
where X1=F, then X2 is not H or R; or
where X1=S, then X2 is not R, H, A, N, T, G, V, M, or W; or
where X1=C, then X2 is not S or G; or
where X1=R, then X2 is not T or F; or
where X1=A, then X2 is not R, Q, N, S, or L; or
where X1=Q, then X2 is not M; or
where X1=L, then X2 is not R; or
where X1=I, then X2 is not A; or
where X1=P, then X2 is not P, W, or R; or
where X1=G, then X2 is not S or R; or
where X1=T, then X2 is not T.
126. A purified peptide that binds to the SH3 domain of Yes, said peptide comprising the amino acid sequence
ψX1X2RPLPX3LPX4X5 (SEQ ID NO:455)
where ψ represents aliphatic amino acid residues and X1, X2, X3, X4, and X5 represent any amino acid; except that if
X3=P, X4=P, and X5=P, then:
when ψ=L,
where X1=F, then X2 is not H or R; or
where X1=S, then X2 is not R, H, A, N, T, G, V, M, or W; or
where X1=C, then X2 is not S or G; or
where X1=R, then X2 is not T or F; or
where X1=A, then X2 is not R, Q, N, S, or L; or
where X1=Q, then X2 is not M; or
where X1=L, then X2 is not R; or
where X1=I, then X2 is not A; or
where X1=P, then X2 is not P, W, or R; or
where X1=G, then X2 is not S or R; or
where X1=T, then X2 is not T; and
when ψ=P,
where X1=A, then X2 is not R; or
where X1=S, then X2 is not R or Y; or
where X1=M, then X2 is not S; or
where X1=V, then X2 is not G; or
where X1=R, then X2 is not S; or
where X1=I, then X2 is not R; and
when ψ=A,
where X1=A, then X2 is not K; and
when ψ=V,
where X1=A, then X2 is not C or Q; or
where X1=P, then X2 is not P; and
when ψ=I,
where X1=G, then X2 is not H; or
where X1=T, then X2 is not S; or
where X1=R, then X2 is not S.
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