Novel TNF receptor death domain ligand proteins and inhibitors of ligand binding

Abstract

domain ("TNF-R1-DD") ligand proteins are disclosed. Polynucleotides encoding the TNF-R1-DD ligand protein are also disclosed, along with vectors, host cells, and methods of making the TNF-R1-DD ligand protein. Pharmaceutical compositions containing the TNF-R1-DD ligand protein, methods of treating inflammatory conditions, and methods of inhibiting TNF-R death domain binding are also disclosed. Methods of identifying inhibitors of TNF-R death domain binding and inhibitors identified by such methods are also disclosed.

Description

[0001]This application is a continuation-in-part of application Ser. No. 08/602,228, filed Feb. 15, 1996, which was a continuation-in-part of application Ser. No. 08/533,901, filed Sep. 26, 1995, which was a continuation-in-part of application Ser. No. 08/494,440, filed Jun. 19, 1995, which was a continuation-in-part of application Ser. No. 08/327,514, filed Oct. 19, 1994.

BACKGROUND OF THE INVENTION

[0002]The present invention relates to the field of anti-inflammatory substances and other substances which act by inhibiting binding to the intracellular domain of a tumor necrosis factor receptor (hereinafter "TNF-R"), such as, for example, the P55 type (or TNF-R1) TNF receptor. More particularly, the present invention is directed to novel ligands which bind to the TNF-R intracellular domain and to inhibition or modulation of signal transduction by this receptor.

[0003]Tumor necrosis factor (herein "TNF") is a cytokine which produces a wide range of cellular activities. TNF causes an inflammatory response, which can be beneficial, such as in mounting an immune response to a pathogen, or when overexpressed can lead to other detrimental effects of inflammation.

[0004]The cellular effects of TNF are initiated by the binding of TNF to its receptors (TNF-Rs) on the surface of target cells. The isolation of polynucleotides encoding TNF-Rs and variant forms of such receptors has been described in European patent publication Nos. EP 308,378, EP 393,438, EP 433,900, EP 526,905 and EP 568,925; in PCT patent publication Nos. WO91/03553 and WO93/19777: and by Schall et al., Cell 61:361-370 (1990) (disclosing the P55 type TNF receptor). Processes for purification of TNF-Rs have also been disclosed in U.S. Pat. No. 5,296,592.

[0005]Native TNF-Rs are characterized by distinct extracellular transmembrane and intracellular domains. The primary purpose of the extracellular domain is to present a binding site for TNF on the outside of the cell. When TNF is bound to the binding site, a "signal" is transmitted to the inside of the cell through the transmembrane and intracellular domains, indicating that binding has occurred. Transmission or "transduction" of the signal to the inside of the cell occurs by a change in conformation of the transmembrane and/or intracellular domains of the receptor. This signal is "received" by the binding of proteins and other molecules to the intracellular domain of the receptor, resulting in the effects seen upon TNF stimulation. Two distinct TNF receptors of -55 kd ("TNF-R1") and -75 kd ("TNF-R2") have been identified. Numerous studies with anti-TNF receptor antibodies have demonstrated that TNF-R1 is the receptor which signals the majority of the pleiotropic activities of TNF. Recently, the domain required for signaling cytotoxicity and other TNF-mediated responses has been mapped to the -80 amino acid near the C-terminus of TNF-R1. This domain is therefore termed the "death domain" (hereinafter referred to as "TNF-R death domain" and "TNF-R1-DD") (see, Tartaglia et al., Cell 74:845-853 (1993)).

[0006]While TNF binding by TNF-Rs results in beneficial cellular effects, it is often desirable to prevent or deter TNF binding from causing other detrimental cellular effects. Although substantial effort has been expended investigating inhibition of TNF binding to the extracellular domain of TNF-R5, examination of binding of proteins and other molecules to the intracellular domain of TNF-Rs has received much less attention.

[0007]However, ligands which bind to the TNF-R intracellular domain have yet to be identified. It would be desirable to identify and isolate such ligands to examine their effects upon TNF-R signal transduction and their use as therapeutic agents for treatment of TNF-induced conditions. Furthermore, identification of such ligands would provide a means for screening for inhibitors of TNF-R/intracellular ligand binding, which will also be useful as anti-inflammatory agents.

SUMMARY OF THE INVENTION

[0008]Applicants have for the first time identified novel TNF-R1-DD ligand proteins and have isolated polynucleotides encoding such ligands. Applicants have also identified a known protein which may also bind to the death domain of TNF-R.

[0009]In one embodiment, the present invention provides a composition comprising an isolated polynucleotide encoding a protein having TNF-R1-DD ligand protein activity. In preferred embodiments, the polynucleotide is selected from the group consisting of: [0010](a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:1 from nucleotide 2 to nucleotide 1231; [0011](b) a polynucleotide comprising a fragment of the nucleotide sequence of SEQ ID NO:1; [0012](c) a polynucleotide encoding an TNF-R1-DD ligand protein comprising the amino acid sequence of SEQ ID NO:2; [0013](d) a polynucleotide encoding an TNF-R1-DD ligand protein comprising a fragment of the amino acid sequence of SEQ ID NO:2; [0014](e) a polynucleotide comprising the nucleotide sequence of SEQ ED NO:3 from nucleotide 2 to nucleotide 415; [0015](f) a polynucleotide comprising a fragment of the nucleotide sequence of SEQ ID NO:3; [0016](g) a polynucleotide encoding an TNF-R1-DD ligand protein comprising the amino acid sequence of SEQ ID NO:4; [0017](h) a polynucleotide encoding an TNF-R1-DD ligand protein comprising a fragment of the amino acid sequence of SEQ ED NO:4; [0018](i) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:9 from nucleotide 2 to nucleotide 931; [0019](j) a polynucleotide comprising a fragment of the nucleotide sequence of SEQ ID NO:9; [0020](k) a polynucleotide encoding an TNF-R1-DD ligand protein comprising the amino acid sequence of SEQ ID NO:10; [0021](l) a polynucleotide encoding an TNF-R1-DD ligand protein comprising a fragment of the amino acid sequence of SEQ ID NO:10; [0022](m) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:11 from nucleotide 2 to nucleotide 1822; [0023](n) a polynucleotide comprising a fragment of the nucleotide sequence of SEQ ID NO:1; [0024](o) a polynucleotide encoding an TNF-R1-DD ligand protein comprising the amino acid sequence of SEQ ID NO:12; [0025](p) a polynucleotide encoding an TNF-R1-DD ligand protein comprising a fragment of the amino acid sequence of SEQ ID NO:12; [0026](q) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:13 from nucleotide 3 to nucleotide 2846; [0027](r) a polynucleotide comprising a fragment of the nucleotide sequence of SEQ ID NO:13, which encodes a protein having TNF-R1-DD ligand protein activity; [0028](s) a polynucleotide encoding an TNF-R1-DD ligand protein comprising the amino acid sequence of SEQ ID NO:14; [0029](t) a polynucleotide encoding an TNF-R1-DD Ligand protein comprising a fragment of the amino acid sequence of SEQ ID NO:14 and having TNF-R1-DD ligand protein activity; [0030](u) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:15 from nucleotide 326 to nucleotide 5092; [0031](v) a polynucleotide comprising a fragment of the nucleotide sequence of SEQ ID NO:15; [0032](w) a polynucleotide encoding an TNF-R1-DD ligand protein comprising the amino acid sequence of SEQ ID NO:16; [0033](x) a polynucleotide encoding an TNF-R1-DD ligand protein comprising a fragment of the amino acid sequence of SEQ ID NO:16; [0034](y) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:17 from nucleotide 14 to nucleotide 2404; [0035](z) a polynucleotide comprising a fragment of the nucleotide sequence of SEQ ID NO:17; [0036](aa) a polynucleotide encoding an TNF-R1-DD ligand protein comprising the amino acid sequence of SEQ ID NO:18; [0037](bb) a polynucleotide encoding an TNF-R1-DD ligand protein comprising a fragment of the amino acid sequence of SEQ ID NO: 18; and [0038](cc) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotide specified in (a)-(cc).In certain preferred embodiments, the polynucleotide is operably linked to an expression control sequence. The invention also provides a host cell, including bacterial, yeast, insect and mammalian cells, transformed with such polynucleotide compositions:

[0039]Processes are also provided for producing an TNF-R1-DD ligand protein, which comprises: [0040](a) growing a culture of the host cell transformed with such polynucleotide compositions in a suitable culture medium; and [0041](b) purifying the TNF-R1-DD ligand protein from the culture.The ligand protein produced according to such methods is also provided by the present invention.

[0042]Compositions comprising a protein having TNF-R1-DD ligand protein activity are also disclosed. In preferred embodiments the protein comprises an amino acid sequence selected from the group consisting of: [0043](a) the amino acid sequence of SEQ ID NO:2; [0044](b) fragments of the amino acid sequence of SEQ ID NO:2; [0045](c) the amino acid sequence of SEQ ID NO:4; [0046](d) fragments of the amino acid sequence of SEQ ID NO:4; [0047](e) the amino acid sequence of SEQ ID NO:6; [0048](f) fragments of the amino acid sequence of SEQ ID NO:6; [0049](g) the amino acid sequence of SEQ ID NO:10; [0050](h) fragments of the amino acid sequence of SEQ ID NO:10; [0051](i) the amino acid sequence of SEQ ID NO:12; [0052](j) fragments of the amino acid sequence of SEQ ID NO:12; [0053](k) the amino acid sequence of SEQ ID NO:14; [0054](l) fragments of the amino acid sequence of SEQ ID NO:14; [0055](m) the amino acid sequence of SEQ ID NO:16; [0056](n) fragments of the amino acid sequence of SEQ ID NO:16; [0057](O) the amino acid sequence of SEQ ID NO:18; and [0058](p) fragments of the amino acid sequence of SEQ ID NO:18:the protein being substantially free from other mammalian proteins. Such compositions may further comprise a pharmaceutically acceptable carrier.

[0059]Compositions comprising an antibody which specifically reacts with such TNF-R1-DD ligand protein are also provided by the present invention.

[0060]Methods are also provided for identifying an inhibitor of TNF-R death domain binding which comprise: [0061](a) combining an TNF-R death domain protein with an TNF-R1-DD ligand protein, said combination forming a first binding mixture; [0062](b) measuring the amount of binding between the TNF-R death domain protein and the TNF-R1-DD ligand protein in the first binding mixture; [0063](c) combining a compound with the TNF-R death domain protein and an TNF-R1-DD ligand protein to form a second binding mixture; [0064](d) measuring the amount of binding in the second binding mixture; and [0065](e) comparing the amount of binding in the first binding mixture with the amount of binding in the second binding mixture;wherein the compound is capable of inhibiting TNF-R death domain binding when a decrease in the amount of binding of the second binding mixture occurs. In certain preferred embodiments the TNF-R1-DD ligand protein used in such method comprises an amino acid sequence selected from the group consisting of: [0066](a) the amino acid sequence of SEQ ID NO:2; [0067](b) fragments of the amino acid sequence of SEQ ID NO:2; [0068](c) the amino acid sequence of SEQ ID NO:4; [0069](d) fragments of the amino acid sequence of SEQ ID NO:4; [0070](e) the amino acid sequence of SEQ ID NO:6; [0071](f) fragments of the amino acid sequence of SEQ ID NO: 6; [0072](g) the amino acid sequence of SEQ ID NO:8; [0073](h) fragments of the amino acid sequence of SEQ ID NO:8; [0074](i) the amino acid sequence of SEQ ID NO:10; [0075](j) fragments of the amino acid sequence of SEQ ID NO:10; [0076](k) the amino acid sequence of SEQ ID NO:12; [0077](i) fragments of the amino acid sequence of SEQ ID NO:12; [0078](m) the amino acid sequence of SEQ ID NO:14; [0079](n) fragments of the amino acid sequence of SEQ ID NO:14; [0080](o) the amino acid sequence of SEQ ID NO: 16; [0081](p) fragments of the amino acid sequence of SEQ ID NO: 16; [0082](q) the amino acid sequence of SEQ ID NO: 18; [0083](r) fragments of the amino acid sequence of SEQ ID NO: 18.Compositions comprising inhibitors identified according to such method are also provided. Such compositions may include pharmaceutically acceptable carriers.

[0084]Methods are also provided for preventing or ameliorating an inflammatory condition which comprises administering a therapeutically effective amount of a composition comprising a protein having TNF-R1-DD ligand protein activity and a pharmaceutically acceptable carrier.

[0085]Other embodiments provide methods of inhibiting TNF-R death domain binding comprising administering a therapeutically effective amount of a composition comprising a protein having TNF-R1-DD ligand protein activity and a pharmaceutically acceptable carrier.

[0086]Methods are also provided for preventing or ameliorating an inflammatory condition which comprises administering to a mammalian subject a therapeutically effective amount of a composition comprising a pharmaceutically acceptable carrier and a protein selected from the group consisting of insulin-like growth factor binding protein-5 ("IGFBP-5"), and fragments thereof having TNF-R1-DD ligand protein activity. Such proteins may also be administered for inhibiting TNF-R death domain binding.

[0087]Methods of preventing or ameliorating an inflammatory condition or of inhibiting TNF-R death domain binding are provided, which comprise administering to a mammalian subject a therapeutically effective amount of inhibitors of TNF-R death domain binding, are also provided.

[0088]Methods of identifying an inhibitor of TNF-R death domain binding are also provided by the present invention which comprise: [0089](a) transforming a cell with a first polynucleotide encoding an TNF-R death domain protein, a second polynucleotide encoding an TNF-R1-DD ligand protein and at least one reporter gene, wherein the expression of the reporter gene is regulated by the binding of the TNF-R1-DD ligand protein encoded by the second polynucleotide to the TNF-R death domain protein encoded by the first polynucleotide; [0090](b) growing the cell in the presence of and in the absence of a compound; and [0091](c) comparing the degree of expression of the reporter gene in the presence of and in the absence of the compound;wherein the compound is capable of inhibiting TNF-R death domain binding when a decrease in the degree of expression of the reporter gene occurs. In preferred embodiments, the cell is a yeast cell and the second polynucleotide is selected from the group consisting of: [0092](a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:1 from nucleotide 2 to nucleotide 1231; [0093](b) a polynucleotide comprising a fragment of the nucleotide sequence of SEQ ID NO:1, which encodes a protein having TNF-R1-DD ligand protein activity; [0094](c) a polynucleotide encoding an TNF-R1-DD ligand protein comprising the amino acid sequence of SEQ ID NO:2; [0095](d) a polynucleotide encoding an TNF-R1-DD ligand protein comprising a fragment of the amino acid sequence of SEQ ID NO:2 and having TNF-R1-DD ligand protein activity; [0096](e) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:3 from nucleotide 2 to nucleotide 415; [0097](f) a polynucleotide comprising a fragment of the nucleotide sequence of SEQ ID NO:3, which encodes a protein having TNF-R1-DD ligand protein activity; [0098](g) a polynucleotide encoding an TNF-R1-DD ligand protein comprising the amino acid sequence of SEQ ID NO:4; [0099](h) a polynucleotide encoding an TNF-R1-DD ligand protein comprising a fragment of the amino acid sequence of SEQ ID NO:4 and having TNF-R1-DD ligand protein activity; [0100](i) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:5 from nucleotide 2 to nucleotide 559; [0101](j) a polynucleotide comprising a fragment of the nucleotide sequence of SEQ ID NO:5, which encodes a protein having TNF-R1-DD ligand protein activity; [0102](k) a polynucleotide encoding an TNF-R1-DD ligand protein comprising the amino acid sequence of SEQ ID NO:6; [0103](l) a polynucleotide encoding an TNF-R1-DD ligand protein comprising a fragment of the amino acid sequence of SEQ ID NO:6 and having TNF-R1-DD ligand protein activity; [0104](m) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:7 from nucleotide 57 to nucleotide 875; [0105](n) a polynucleotide comprising a fragment of the nucleotide sequence of SEQ ID NO:7, which encodes a protein having TNF-R1-DD ligand protein activity; [0106](o) a polynucleotide encoding an TNF-R1-DD ligand protein comprising the amino acid sequence of SEQ ID NO:8; [0107](p) a polynucleotide encoding an TNF-R1-DD ligand protein comprising a fragment of the amino acid sequence of SEQ ID NO:8 and having TNF-R1-DD ligand protein activity; [0108](q) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:9 from nucleotide 2 to nucleotide 931; [0109](r) a polynucleotide comprising a fragment of the nucleotide sequence of SEQ ID NO:9; [0110](s) a polynucleotide encoding an TNF-R1-DD ligand protein comprising the amino acid sequence of SEQ ID NO:10; [0111](t) a polynucleotide encoding an TNF-R1-DD ligand protein comprising a fragment of the amino acid sequence of SEQ ID NO:10; [0112](u) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:111 from nucleotide 2 to nucleotide 1822; [0113](v) a polynucleotide comprising a fragment of the nucleotide sequence of SEQ ID NO:11; [0114](w) a polynucleotide encoding an TNF-R1-DD ligand protein comprising the amino acid sequence of SEQ ID NO:12; [0115](x) a polynucleotide encoding an TNF-R1-DD ligand protein comprising a fragment of the amino acid sequence of SEQ ID NO:12; [0116](y) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:13 from nucleotide 3 to nucleotide 2846; [0117](z) a polynucleotide comprising a fragment of the nucleotide sequence of SEQ ID NO:13, which encodes a protein having TNF-R1-DD ligand protein activity; [0118](aa) a polynucleotide encoding an TNF-R1-DD ligand protein comprising the amino acid sequence of SEQ ID NO:14; [0119](bb) a polynucleotide encoding an TNF-R1-DD ligand protein comprising a fragment of the amino acid sequence of SEQ ID NO:14 and having TNF-R1-DD ligand protein activity; [0120](cc) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:15 from nucleotide 326 to nucleotide 5092; [0121](dd) a polynucleotide comprising a fragment of the nucleotide sequence of SEQ ID NO:15, which encodes a protein having TNF-R1-DD ligand protein activity; [0122](ee) a polynucleotide encoding an TNF-R1-DD ligand protein comprising the amino acid sequence of SEQ ID NO:16; [0123](ff) a polynucleotide encoding an TNF-R1-DD ligand protein comprising a fragment of the amino acid sequence of SEQ ID NO:16 and having TNF-R1-DD ligand protein activity; [0124](gg) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 17 from nucleotide 14 to nucleotide 2404; [0125](hh) a polynucleotide comprising a fragment of the nucleotide sequence of SEQ ID NO:17, which encodes a protein having TNF-R1-DD ligand protein activity; [0126](ii) a polynucleotide encoding an TNF-R1-DD ligand protein comprising the amino acid sequence of SEQ ID NO:18; [0127](jj) a polynucleotide encoding an TNF-R1-DD ligand protein comprising a fragment of the amino acid sequence of SEQ ID NO:18 and having TNF-R1-DD ligand and protein activity; and [0128](kk) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(jj), which encodes a protein having TNF-R1-DD ligand protein activity.

BRIEF DESCRIPTION OF THE FIGURES

[0129]FIGS. 1 and 2 depict autoradiographs demonstrating the expression of TNF-R1-DD ligand proteins of the present invention.

[0130]FIG. 3 depicts an autoradiograph demonstrating the expression of clones 1TU, 15TU and 27TU.

[0131]FIG. 4 demonstrates the binding of 1TU and 27TU to TNF-R1-DD. MBP, MBP-1TU or MBP-27TU (3 μg) was incubated with glutathione beads containing 3 μg of either GST or GST-TNF-R1-DD in 100 μl of binding buffer (0.2% Triton, 20 mM Tris pH 7.5, 140 mM NaCl, 0.1 mM EDTA, 10 mM DTT and 5% glycerol). The reaction ws performed at 4° C. for 2 hours and centrifuged to remove unbound fraction (Unbound). The beads were then washed with 500 μl binding buffer four times and resuspended into SDS-sample buffer (Bound). These samples were analyzed by Western blot using anti-MBP antibody (New England Biolab).

[0132]FIG. 5 demonstrates the ability of 15TU and 27TU to activate the JNK pathway. COS cells were contransfected with HA-tagged JNK1 and clones 15 tu or 27TU. Cells were left untreated or treated for 15 min with 50 ng/ml TNF, and HA-JNK1 was immunoprecipitated with anti-HA antibody. JNK activity was measured in an in vitro kinase assay using GST-c-jun (amino acids 1-79) as substrate, and reactions were electrophoresed on SDS-PAGE.

[0133]FIG. 6 is an autoradiograph of an SDS-PAGE gel of conditioned media from COS cells transfected with clone 3TW.

[0134]FIG. 7 is an autoradiograph which demonstrates that an antisense oligonucleotide derived from the sequence of clone 3TW inhibits TNF-induced cPLA, phosphorylation.

DETAILED DESCRIPTION OF THE INVENTION

[0135]The present inventors have for the first time identified and isolated novel polynucleotides which encode proteins which bind to the TNF-R death domain. As used herein "TNF-R" includes all receptors for tumor necrosis factor. The P55 type TNF-R is the preferred receptor for practicing the present invention.

[0136]The sequence of a polynucleotide encoding one such protein is set forth in SEQ ID NO:1 from nucleotides 2 to 1231. This polynucleotide has been identified as "clone 2DD" The amino acid sequence of the TNF-R1-DD ligand protein encoded by clone 2DD is set forth in SEQ ID NO:2. It is believed that clone 2DD is a partial cDNA clone of a longer full length coding sequence. However, as demonstrated herein the protein encoded by clone 2DD does bind the death domain of TNF-R (i.e., has "TNF-R1-DD ligand protein activity" as defined herein). Clone 2DD was deposited with the American Type Culture Collection on Oct. 13, 1994 and given the accession number ATCC 69706.

[0137]The protein encoded by clone 2DD is 410 amino acids in length. No identical or closely related sequences were found using BLASTN/BLASTX or FASTA searches. Therefore, clone 2DD encodes a novel protein.

[0138]The sequence of a polynucleotide encoding one such protein is set forth in SEQ ID NO:3 from nucleotides 2 to 415. This polynucleotide has been identified as "clone 3DD". The amino acid sequence of the TNF-R1-DD ligand protein encoded by clone 3DD is set forth in SEQ ID NO:4. It is believed that clone 3DD is a partial cDNA clone of a longer full length coding sequence. However, as demonstrated herein the protein encoded by clone 3DD does bind the death domain of TNF-R (i.e., has "TNF-R1-DD ligand protein activity" as defined herein). Clone 3DD was deposited with the American Type Culture Collection on Oct. 13, 1994 and given the accession number ATCC 69705.

[0139]The protein encoded by clone 3DD is 138 amino acids. No identical or closely related sequences were found using BLASTN/BLASTX or FASTA searches. Therefore, clone 3DD encodes a novel protein.

[0140]A full-length clone corresponding to clone 3DD was also isolated and identified as "clone 3TW". The nucleotide sequence of clone 3TW is reported as SEQ ID NO:13. Nucleotides 3 to 2846 of SEQ ID NO:13 encode a TNF-R1-DD ligand protein, the amino acid sequence of which is reported as SEQ ID NO:14. Amino acids 811 to 948 of SEQ ID NO:14 correspond to amino acids 1 to 138 of SEQ ID NO:4 (clone 3DD). Clone 3TW was deposited with the American Type Culture Collection on Sep. 26, 1995 and given the accession number ATCC 69904.

[0141]The sequence of a polynucleotide encoding another such protein is set forth in SEQ ID NO:5 from nucleotides 2 to 559. This polynucleotide has been identified as "clone 20DD." The amino acid sequence of the TNF-R1-DD ligand protein encoded by clone 20DD is set forth in SEQ ID NO:6. It is believed that clone 20DD is a partial cDNA clone of a longer full length coding sequence. However, as demonstrated herein the protein encoded by clone 20DD does bind the death domain of TNF-R (i.e., has "TNF-R1-DD ligand protein activity" as defined herein). Clone 20DD was deposited with the American Type Culture Collection on Oct. 13, 1994 and given the accession number ATCC 69704.

[0142]The protein encoded by clone 20DD is identical to amino acids 87 to 272 of insulin-like growth factor binding protein-5 ("IGFBP-5"), a sequence for which was disclosed in J. Biol. Chem. 266:10646-10653 (1991) by Shimasaki et al., which is incorporated herein by reference. The polynucleotide and amino acid sequences of IGFBP-5 are set forth in SEQ ID NO:7 and SEQ ID NO:8, respectively. Based upon the sequence identity between clone 20DD and IGFBP-5, IGFBP-5 and certain fragments thereof will exhibit TNF-R1-DD ligand binding activity (as defined herein).

[0143]The sequence of a polynucleotide encoding another such protein is set forth in SEQ ID NO:9 from nucleotides 2 to 931. This polynucleotide has been identified as "clone 1TU" The amino acid sequence of the TNF-R1-DD ligand protein encoded by clone 1TU is set forth in SEQ ID NO:10. It is believed that clone 1TU is a partial cDNA clone of a longer full length coding sequence. However, as demonstrated herein the protein encoded by clone 1TU does bind the death domain of TNF-R (i.e., has "TNF-R1-DD ligand protein activity" as defined herein). Clone 1TU was deposited with the American Type Culture Collection on Jun. 7, 1995 and given the accession number ATCC 69848.

[0144]The protein encoded by clone 1TU is 310 amino acids in length. No identical or closely related sequences were found using BLASTN/BLASTX or FASTA searches. Therefore, clone 1TU encodes a novel protein.

[0145]The sequence of a polynucleotide encoding another such protein is set forth in SEQ ID NO:11 from nucleotides 2 to 18822. This polynucleotide has been identified as "clone 27TU" The amino acid sequence of the TNF-R1-DD ligand protein encoded by clone 27TU is set forth in SEQ ID NO:12. It is believed that clone 27TU is a partial cDNA clone of a longer full length coding sequence. However, as demonstrated herein the protein encoded by clone 27TU does bind the death domain of TNF-R (i.e., has "TNF-R1-DD ligand protein activity" as defined herein). Clone 27TU was deposited with the American Type Culture Collection on Jun. 7, 1995 and given the accession number ATCC 69846.

[0146]The protein encoded by clone 27TU is 607 amino acids in length. No identical or closely related sequences were found using BLASTN/BLASTX or FASTA searches. Therefore, clone 27TU encodes a novel protein. 27TU may be a longer version of clone 2DD. 2DD encodes the same amino acid sequence (SEQ ID NO:2) as amino acids 198-607 encoded by 27TU (SEQ ID NO:12). The nucleotide sequences of 2DD and 27TU are also identical within this region of identity.

[0147]An additional "clone 15TU" was isolated which encoded a portion of the 27TU sequence (approximately amino acids 289-607 of SEQ ID NO:12). Clone 15TU was deposited with the American Type Culture Collection on Jun. 7, 1995 and given the accession number ATCC 69847. 15TU comprises the same nucleotide sequence as 27TU over this region of amino acids.

[0148]A full-length clone corresponding to clone 27TU was also isolated and identified as "clone 57TU4A". The nucleotide sequence of clone 57TU4A is reported as SEQ ID NO:15. Nucleotides 336 to 5092 of SEQ ID NO:15 encode a TNF-R1-DD ligand protein, the amino acid sequence of which is reported as SEQ ID NO:146 Amino acids 982 to 1588 of SEQ ID NO:16 correspond to amino acids 1 to 607 of SEQ. ID NO:12 (clone 27TU). Clone 57TU4A was deposited with the American Type Culture Collection on Feb. 13, 1996 and given the accession number ATCC 69988.

[0149]A full-length clone corresponding to clone 1TU was also isolated and identified as "clone 33-1B". The nucleotide sequence of clone 33-1B is reported as SEQ ID NO: 17. Nucleotides 14 to 2404 of SEQ ID NO:17 encode a TNF-R1-DD ligand protein, the amino acid sequence of which is reported as SEQ ID NO:18. Amino acids 488 to 797 of SEQ ID NO:18 correspond to amino acids 1 to 310 of SEQ ID NO:10 (clone 1TU). Clone 33-1B was deposited with the American Type Culture Collection on Aug. 13, 1996 and given the accession number ATCC

[0150]Polynucleotides hybridizing to the polynucleotides of the present invention under stringent conditions and highly stringent conditions are also part of the present invention. As used herein, "highly stringent conditions" include, for example, 0.2×SSC at 65° C.; and "stringent conditions" include, for example, 4×SSC at 65° C. or 50% formamide and 4×SSC at 42° C.

[0151]For the purposes of the present application, "TNF-R1-DD ligand protein" includes proteins which exhibit TNF-R1-DD ligand protein activity. For the purposes of the present application, a protein is defined as having "TNF-R1-DD ligand protein activity" when it binds to a protein derived from the TNF-R death domain. Activity can be measured by using any assay which will detect binding to an TNF-R death domain protein. Examples of such assays include without limitation the interaction trap assays and assays in which TNF-R death domain protein which is affixed to a surface in a manner conducive to observing binding, including without limitation those described in Examples 1 and 3. As used herein an "TNF-R death domain protein" includes the entire death domain or fragments thereof.

[0152]Fragments of the TNF-R1-DD ligand protein which are capable of interacting with the TNF-R death domain or which are capable of inhibiting TNF-R death domain binding (i.e., exhibit TNF-R1-DD ligand protein activity) are also encompassed by the present invention. Fragments of the TNF-R1-DD ligand protein may be in linear form or they may be cyclized using known methods, for example, as described in H. U. Saragovi, et al., Bio/Technology 10, 773-778 (1992) and in R. S. McDowell, et al., J. Amer. Chem. Soc. 114, 9245-9253 (1992), both of which are incorporated herein by reference. Such fragments may be fused to carrier molecules such as immunoglobulins for many purposes, including increasing the valency of TNF-R1-DD ligand protein binding sites. For example, fragments of the TNF-R1-DD ligand protein may be fused through "linker" sequences to the Fc portion of an immunoglobulin. For a bivalent form of the TNF-R1-DD ligand protein, such a fusion could be to the Fc portion of an IgG molecule. Other immunoglobulin isotypes may also be used to generate such fusions. For example, an TNF-R1-DD ligand protein--IgM fusion would generate a decavalent form of the TNF-R1-DD ligand protein of the invention.

[0153]The isolated polynucleotide of the invention may be operably linked to an expression control sequence such as the pMT2 or pED expression vectors disclosed in Kaufman et al., Nucleic Acids Res. 19, 44854490 (1991), in order to produce the TNF-R1-DD ligand protein recombinantly. Many suitable expression control sequences are known in the art. General methods of expressing recombinant proteins are also known and are exemplified in R. Kaufman, Methods in Enzymology 185, 537-566 (1990). As defined herein "operably linked" means that the isolated polynucleotide of the invention and the expression control sequence are situated within a vector or cell in such a way that the TNF-R1-DD ligand protein is expressed by a host cell which has been transformed (transfected) with the ligated polynucleotide/expression control sequence.

[0154]A number of types of cells may act as suitable host cells for expression of the TNF-R1-DD ligand protein. Host cells include, for example, monkey COS cells, Chinese Hamster Ovary (CHO) cells, human kidney 293 cells, human epidermal A431 cells, human Colo205 cells, 3T3 cells, CV-1 cells, other transformed primate cell lines, normal diploid cells, cell strains derived from in vitro culture of primary tissue, primary explants, HeLa cells, mouse L cells, BHK, HL-60, U937, HaK or Jurkat cells.

[0155]The TNF-R1-DD ligand protein may also be produced by operably linking the isolated polynucleotide of the invention to suitable control sequences in one or more insect expression vectors, and employing an insect expression system. Materials and Methods for baculovirus/insect cell expression systems are commercially available in kit form from, e.g., Invitrogen, San Diego, Calif., U.S.A. (the MaxBac® kit), and such methods are well known in the art, as described in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987), incorporated herein by reference.

[0156]Alternatively, it may be possible to produce the TNF-R1-DD ligand protein in lower eukaryotes such as yeast or in prokaryotes such as bacteria. Potentially suitable yeast strains include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains, Candida, or any yeast strain capable of expressing heterologous proteins. Potentially suitable bacterial strains include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, or any bacterial strain capable of expressing heterologous proteins. If the TNF-R1-DD ligand protein is made in yeast or bacteria. It may be necessary to modify the protein produced therein, for example by phosphorylation or glycosylation of the appropriate sites, in order to obtain the functional TNF-R1-DD ligand protein. Such covalent attachments may be accomplished using known chemical or enzymatic methods.

[0157]The TNF-R1-DD ligand protein of the invention may also be expressed as a product of transgenic animals, e.g., as a component of the milk of transgenic cows, goats, pigs, or sheep which are characterized by somatic or germ cells containing a nucleotide sequence encoding the TNF-R1-DD ligand protein.

[0158]The TNF-R1-DD ligand protein of the invention may be prepared by culturing transformed host cells under culture conditions suitable to express the recombinant protein. The resulting expressed protein may then be purified from such culture (i.e., from culture medium or cell extracts) using known purification processes, such as gel filtration and ion exchange chromatography. The purification of the TNF-R1-DD ligand protein may also include an affinity column containing the TNF-R death domain or other TNF-R death domain protein; one or more column steps over such affinity resins as concanavalin A-agarose, Heparin-Toyopearl® or Cibacrom blue 3GA Sepharose®; one or more steps involving hydrophobic interaction chromatography using such resins as phenyl ether, butyl ether, or propyl ether; or immunoaffinity chromatography.

[0159]Alternatively, the TNF-R1-DD ligand protein of the invention may also be expressed in a form which will facilitate purification. For example, it may be expressed as a fusion protein, such as those of maltose binding protein (MBP) or glutathione-S-transferase (GST). Kits for expression and purification of such fusion proteins are commercially available from New England BioLab (Beverly, Mass.) and Pharmacia (Piscataway, N.J.), respectively. The TNF-R ligand protein can also be tagged with an epitope and subsequently purified by using a specific antibody directed to such epitope. One such epitope ("Flag") is commercially available from Kodak (New Haven. CT).

[0160]Finally, one or more reverse-phase high performance liquid chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media. e.g. silica gel having pendant methyl or other aliphatic groups, can be employed to further purify the TNF-R1-DD ligand protein. Some or all of the foregoing purification steps in various combinations, can also be employed to provide a substantially homogeneous isolated recombinant protein. The TNF-R1-DD ligand protein thus purified is substantially free of other mammalian proteins and is defined in accordance with the present invention as an "isolated TNF-R1-DD ligand protein."

[0161]TNF-R1-DD ligand proteins may also be produced by known conventional chemical synthesis. Methods for constructing the proteins of the present invention by synthetic means are known to those skilled in the art. The synthetically-constructed protein sequences, by virtue of sharing primary, secondary or tertiary structural and/or conformational characteristics with TNF-R1-DD ligand proteins may possess biological properties in common therewith, including TNF-R1-DD ligand protein activity. Thus, they may be employed as biologically active or immunological substitutes for natural, purified TNF-R1-DD ligand proteins in screening of therapeutic compounds and in immunological processes for the development of antibodies.

[0162]The TNF-R1-DD ligand proteins provided herein also include proteins characterized by amino acid sequences similar to those of purified TNF-R1-DD ligand proteins but into which modification are naturally provided or deliberately engineered. For example, modifications in the peptide or DNA sequences can be made by those skilled in the art using known techniques. Modifications of interest in the TNF-R1-DD ligand protein sequences may include the replacement, insertion or deletion of a selected amino acid residue in the coding sequence. For example, one or more of the cysteine residues may be deleted or replaced with another amino acid to alter the conformation of the molecule. Mutagenic techniques for such replacement, insertion or deletion are well known to those skilled in the art (see, e.g., U.S. Pat. No. 4,518,584).

[0163]Other fragments and derivatives of the sequences of TNF-R1-DD ligand proteins which would be expected to retain TNF-R1-DD ligand protein activity in whole or in part and may thus be useful for screening or other immunological methodologies may also be easily made by those skilled in the art given the disclosures herein. Such modifications are believed to be encompassed by the present invention.

[0164]TNF-R1-DD ligand protein of the invention may also be used to screen for agents which are capable of inhibiting or blocking binding of an TNF-R1-DD ligand protein to the death domain of TNF-R, and thus may act as inhibitors of TNF-R death domain binding and/or TNF activity. Binding assays using a desired binding protein, immobilized or not, are well known in the art and may be used for this purpose using the TNF-R1-DD ligand protein of the invention. Examples 1 and 3 describe examples of such assays. Appropriate screening assays may be cell-based or cell-free. Alternatively, purified protein based screening assays may be used to identify such agents. For example, TNF-R1-DD ligand protein may be immobilized in purified form on a carrier and binding to purified TNF-R death domain may be measured in the presence and in the absence of potential inhibiting agents. A suitable binding assay may alternatively employ purified TNF-R death domain immobilized on a carrier, with a soluble form of a TNF-R1-DD ligand protein of the invention. Any TNF-R1-DD ligand protein may be used in the screening assays described above.

[0165]In such a screening assay, a first binding mixture is formed by combining TNF-R death domain protein and TNF-R1-DD ligand protein, and the amount of binding in the first binding mixture (B_o) is measured. A second binding mixture is also formed by combining TNF-R death domain protein, TNF-R1-DD ligand protein, and the compound or agent to be screened, and the amount of binding in the second binding mixture (B) is measured. The amounts of binding in the first and second binding mixtures are compared, for example, by performing a B/B_o calculation. A compound or agent is considered to be capable of inhibiting TNF-R death domain binding if a decrease in binding in the second binding mixture as compared to the first binding mixture is observed. The formulation and optimization of binding mixtures is within the level of skill in the art. Such binding mixtures may also contain buffers and salts necessary to enhance or to optimize binding, and additional control assays may be included in the screening assay of the invention.

[0166]Alternatively, appropriate screening assays may be cell based. For example, the binding or interaction between an TNF-R ligand protein and the TNF-R death domain can be measured in yeast as described below in Examples 1 and 3.

[0167]Compounds found to reduce, preferably by at least about 10%, more preferably greater than about 50% or more, the binding activity of TNF-R1-DD ligand protein to TNF-R death domain may thus be identified and then secondarily screened in other binding assays, including in vivo assays. By these means compounds having inhibitory activity for TNF-R death domain binding which may be suitable as anti-inflammatory agents may be identified.

[0168]Isolated TNF-R1-DD ligand protein may be useful in treating, preventing or ameliorating inflammatory conditions and other conditions, such as cachexia, autoimmune disease, graft versus host reaction, osteoporosis, colitis, myelogenous leukemia, diabetes, wasting, and atherosclerosis. Isolated TNF-R1-DD ligand protein may be used itself as an inhibitor of TNF-R death domain binding or to design inhibitors of TNF-R death domain binding. Inhibitors of binding of TNF-R1-DD ligand protein to the TNF-R death domain ("TNF-R intracellular binding inhibitors") are also useful for treating such conditions.

[0169]The present invention encompasses both pharmaceutical compositions and therapeutic methods of treatment or use which employ isolated TNF-R1-DD ligand protein and/or binding inhibitors of TNF-R intracellular binding.

[0170]Isolated TNF-R1-DD ligand protein or binding inhibitors (from whatever source derived, including without limitation from recombinant and non-recombinant cell lines) may be used in a pharmaceutical composition when combined with a pharmaceutically acceptable carrier. Such a composition may also contain (in addition to TNF-R1-DD ligand protein or binding inhibitor and a carrier) diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art. The term "pharmaceutically acceptable" means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient(s). The characteristics of the carrier will depend on the route of administration. The pharmaceutical composition of the invention may also contain cytokines, lymphokines, or other hematopoietic factors such as M-CSF, GM-CSF, TNF, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, L-8, IL-9, G-CSF, Meg-CSF, stem cell factor, and erythropoietin. The pharmaceutical composition may further contain other anti-inflammatory agents. Such additional factors and/or agents may be included in the pharmaceutical composition to produce a synergistic effect with isolated TNF-R1-DD ligand protein or binding inhibitor, or to minimize side effects caused by the isolated TNF-R1-DD ligand protein or binding inhibitor. Conversely, isolated TNF-R1-DD ligand protein or binding inhibitor may be included in formulations of the particular cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent to minimize side effects of the cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent.

[0171]The pharmaceutical composition of the invention may be in the form of a liposome in which isolated TNF-R1-DD ligand protein or binding inhibitor is combined, in addition to other pharmaceutically acceptable carriers, with amphipathic agents such as lipids which exist in aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers in aqueous solution. Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like. Preparation of such liposomal formulations is within the level of skill in the art, as disclosed, for example, in U.S. Pat. No. 4,235,871; U.S. Pat. No. 4,501,728; U.S. Pat. No. 4,837,028; and U.S. Pat. No. 4,737,323, all of which are incorporated herein by reference.

[0172]As used herein, the term "therapeutically effective amount" means the total amount of each active component of the pharmaceutical composition or method that is sufficient to show a meaningful patient benefit, i.e., treatment, healing, prevention or amelioration of an inflammatory response or condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions. When applied to an individual active ingredient, administered alone, the term refers to that ingredient alone. When applied to a combination, the term refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.

[0173]In practicing the method of treatment or use of the present invention, a therapeutically effective amount of isolated TNF-R1-DD ligand protein or binding inhibitor is administered to a mammal having a condition to be treated. Isolated TNF-R1-DD ligand protein or binding inhibitor may be administered in accordance with the method of the invention either alone or in combination with other therapies such as treatments employing cytokines, lymphokines or other hematopoietic factors. When co-administered with one or more cytokines, lymphokines or other hematopoietic factors, isolated TNF-R1-DD ligand protein or binding inhibitor may be administered either simultaneously with the cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors, or sequentially. If administered sequentially, the attending physician will decide on the appropriate sequence of administering isolated TNF-R1-DD ligand protein or binding inhibitor in combination with cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors.

[0174]Administration of isolated TNF-R1-DD ligand protein or binding inhibitor used in the pharmaceutical composition or to practice the method of the present invention can be carried out in a variety of conventional ways, such as oral ingestion, inhalation, or cutaneous, subcutaneous, or intravenous injection. Intravenous administration to the patient is preferred.

[0175]When a therapeutically effective amount of isolated TNF-R1-DD ligand protein or binding inhibitor is administered orally, isolated TNF-R1-DD ligand protein or binding inhibitor will be in the form of a tablet, capsule, powder, solution or elixir. When administered in tablet form, the pharmaceutical composition of the invention may additionally contain a solid carrier such as a gelatin or an adjuvant. The tablet, capsule, and powder contain from about 5 to 95% isolated TNF-R1-DD ligand protein or binding inhibitor, and preferably from about 25 to 90% isolated TNF-R1-DD ligand protein or binding inhibitor. When administered in liquid form, a liquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils may be added. The liquid form of the pharmaceutical composition may further contain physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol. When administered in liquid form, the pharmaceutical composition contains from about 0.5 to 90% by weight of isolated TNF-R1-DD ligand protein or binding inhibitor, and preferably from about 1 to 50% isolated TNF-R1-DD ligand protein or binding inhibitor.

[0176]When a therapeutically effective amount of isolated TNF-R1-DD ligand protein or binding inhibitor is administered by intravenous, cutaneous or subcutaneous injection, isolated TNF-R1-DD ligand protein or binding inhibitor will be in the form of a pyrogen-free, parenterally acceptable aqueous solution. The preparation of such parenterally acceptable protein solutions, having due regard to pH, isotonicity, stability, and the like, is within the skill in the art. A preferred pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection should contain, in addition to isolated TNF-R1-DD ligand protein or binding inhibitor, an isotonic vehicle such as Sodium Chloride Injection, Ringer's Injection, Dextrose. Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection, or other vehicle as known in the art. The pharmaceutical composition of the present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those of skill in the art.

[0177]The amount of isolated TNF-R1-DD ligand protein or binding inhibitor in the pharmaceutical composition of the present invention will depend upon the nature and severity of the condition being treated, and on the nature of prior treatments which the patient has undergone. Ultimately, the attending physician will decide the amount of isolated TNF-R1-DD ligand protein or binding inhibitor with which to treat each individual patient. Initially, the attending physician will administer low doses of isolated TNF-R1-DD ligand protein or binding inhibitor and observe the patient's response. Larger doses of isolated TNF-R1-DD ligand protein or binding inhibitor may be administered until the optimal therapeutic effect is obtained for the patient, and at that point the dosage is not increased further. It is contemplated that the various pharmaceutical compositions used to practice the method of the present invention should contain about 0.1 μg to about 100 mg of isolated TNF-R1-DD ligand protein or binding inhibitor per kg body weight.

[0178]The duration of intravenous therapy using the pharmaceutical composition of the present invention will vary, depending on the severity of the disease being treated and the condition and potential idiosyncratic response of each individual patient. It is contemplated that the duration of each application of the isolated TNF-R1-DD ligand protein or binding inhibitor will be in the range of 12 to 24 hours of continuous intravenous administration. Ultimately the attending physician will decide on the appropriate duration of intravenous therapy using the pharmaceutical composition of the present invention.

[0179]Isolated TNF-R1-DD ligand protein of the invention may also be used to immunize animals to obtain polyclonal and monoclonal antibodies which specifically react with the TNF-R1-DD ligand protein and which may inhibit TNF-R death domain binding. Such antibodies may be obtained using either the entire TNF-R1-DD ligand protein or fragments of TNF-R1-DD ligand protein as an immunogen. The peptide immunogens additionally may contain a cysteine residue at the carboxyl terminus, and are conjugated to a hapten such as keyhole limpet hemocyanin (KLH). Methods for synthesizing such peptides are known in the art, for example, as in R. P. Merrifield, J. Amer. Chem. Soc. 85, 2149-2154 (1963); J. L. Krstenansky, et al., FEBS Lett. 211, 10 (1987).

[0180]Monoclonal antibodies binding to TNF-R1-DD ligand protein or to complex carbohydrate moieties characteristic of the TNF-R1-DD ligand glycoprotein may be useful diagnostic agents for the immunodetection of TNF-R ligand protein.

[0181]Neutralizing monoclonal antibodies binding to TNF-R1-DD ligand protein or to complex carbohydrates characteristic of TNF-R1-DD ligand glycoprotein may also be useful therapeutics for both inflammatory conditions and also in the treatment of some forms of cancer where abnormal expression of TNF-R1-DD ligand protein is involved. These neutralizing monoclonal antibodies are capable of blocking the signaling function of the TNF-R1-DD ligand protein. By blocking the binding of TNF-R1-DD ligand protein, certain biological responses to TNF are either abolished or markedly reduced. In the case of cancerous cells or leukemic cells, neutralizing monoclonal antibodies against TNF-R1-DD ligand protein may be useful in detecting and preventing the metastatic spread of the cancerous cells, which may be mediated by the TNF-R1-DD ligand protein.

[0182]Due to the similarity of their sequences to the insulin growth factor binding protein ("IGFBP-5") and fragments thereof which bind to the TNF-R death domain are proteins having TNF-R1-DD ligand protein activity as defined herein. As a result, they are also useful in pharmaceutical compositions, for treating inflammatory conditions and for inhibiting TNF-R death domain binding as described above for TNF-R1-DD ligand proteins generally.

EXAMPLE 1

Cloning of TNF-R Death Domain Ligand Protein Encoding Polynucleotide

[0183]A yeast genetic selection method, the "interaction trap" [Gyuris et al. Cell 75:791-803, 1993, which is incorporated herein by reference], was used to screen W138 cell cDNA libraries (preparation, see below) for proteins that interact with the death domain of the P55 type 1 TNF receptor (TNF-R1-DD). A polynucleotide encoding amino acids 326 to 413 of the P55 type TNF receptor, TNF-R1-DD, was obtained via the polymerase chain reaction (PCR) using a grafting method. This TNF-R1-DD DNA was then cloned into pEG202 by BamHI and SalI sites, generating the bait plasmid, pEG202-TNF-R1-DD. This plasmid contains the HIS3 selectable marker, and expression of the bait, the LexA-TNF-R1-DD fusion protein, is from the strong constitutive ADH1 promoter. To create the reporter strain carrying the bait protein, yeast strain EGY48, containing the reporter sequence LexAop-Leu2 in place of the chromosomal LEU2, was transformed with pEG202-TNF-R1-DD and pSH18-34 (Ura+), which carries another reporter sequence, LexAop-lacZ. For screening cDNAs encoding proteins that interact with TNF-R1-DD, the expression vector pJG4-5 (TRP1), containing the WI38 cell cDNA library (see below for the cDNA library construction), was transformed into the above strain (EGY48/pEG202-TNF-R1-DD/pSH18-34) according to the method described by Gietz et al., Nucleic Acids Res., 20:1425 (1992).

[0184]cDNA Library Construction:

[0185]WI38 cell cDNA library: Double stranded cDNA was prepared from 3 ug of WI38 mRNA using reagents provided by the Superscript Choice System (Gibco/BRL, Gaithersberg, Md.) with the following substitutions: the first strand synthesis was primed using an oligo dT/XhoI primer/linker, and the dNTP mix was substituted with a mix containing methyl dCTP (Stratagene, LaJolla, Calif.). The cDNA was modified at both ends by addition of an EcoRI/NotI/SalI adapter linker and subsequently digested with XhoI. This produced cDNA molecules possessing an EcoRI/NotI/SalI overhang at the 5' end of the gene and an XhoI overhang at the 3' end. These fragments were then ligated into the yeast expression/fusion vector pJG4-5 (Gyuris et al., Cell, 75, 791-803, 1993), which contains at its amino terminus, the influenza virus HA1 epitope tag, the B42 acidic transcription activation domain, and the SV40 nuclear localization signal, all under the control of the galactose-dependent GAL1 promoter. The resulting plasmids were then electroporated into DH10B cells (Gibco/BRL). A total of 7.1×10⁶ colonies were plated on LB plates containing 100 ug/ml of ampicillin. These E. coli were scraped, pooled, and a large scale plasmid prep was performed using the Wizard Maxi Prep kit (Promega, Madison, Wis.), yielding 3.2 mg of supercoiled plasmid DNA.

[0186]WI38 Cell cDNA Screening Results:

[0187]1×10⁶ transformants were obtained on glucose Ura His Trp plates. These transformants were pooled and resuspended in a solution of 65% glycerol, 1 mM Tris-HCl (pH 7.5), 10 mM MgCl₂ and stored at -80° C. in 1 mL aliquots. For screening purposes, aliquots of these were diluted 10-fold into Ura^-His^-Trp^- CM dropout gal/raff medium (containing 2% galactose, 1% raffinose), which induces the expression of the library encoded proteins, and incubated at 30° C. for 4 hours. 12×10⁶ colony forming units (CFUs) were then plated on standard 10 cm galactose X-Gal Ura^-His^-Trp^-Leu^- plates at a density of 2×10⁵ CFU/plate. After three days at 30° C., about 1,000 colonies were formed (Leu.sup.+) and of those, sixty-four colonies were LacZ.sup.+. In order to test if the Leu.sup.+/LacZ.sup.+ phenotype was due to the library-encoded protein, the galactose dependency of the phenotype was tested. Expression of the library-encoded proteins was turned off by growth on glucose Ura^-His Trp master plates and then retested for galactose-dependency on glucose Ura^-His^-Trp^-Leu^-, galactose Ura^-His^-Trp^-Leu^-, glucose X-Gal Ura^-His^-Trp^-, and galactose X-Gal Ura^-His^-Trp^- plates. Of these, 32 colonies showed galactose-dependent growth on Leu^- plates and galactose-dependent blue color on X-Gal-containing medium (LacZ.sup.+ phenotype). Total yeast DNA was prepared from these colonies according to the method described previously (Hoffman and Winston, 1987). In order to analyze the cDNA sequences, PCR reactions were performed using the above yeast DNA as a template and oligo primers specific for the vector pJG4-5, flanking the cDNA insertion point. PCR products were purified (Qiagen PCR purification kit), subjected to restriction digest with the enzyme HaeIII, run on 1.8% agarose gels, and the restriction patterns compared. Similar and identical restriction patterns were grouped and representatives of each group were sequenced and compared to Genbank and other databases to identify any sequence homologies.

[0188]One clone of unique sequence ("2DD") and three clones with identical sequence ("3DD") were isolated and showed no significant sequence homologies compared to Genbank and other databases. Additionally, four other clones ("20DD") with identical sequence to a portion of human insulin-like growth factor binding protein-5 (Shunichi Shimasaki et al., J. Biol. Chem. 266:10646-10653 (1991)) were isolated. The clones "2DD," "3DD" and "20DD" were chosen for further analysis. Library vector pJG4-5 containing these clones sequences were rescued from yeast by transforming the total yeast DNAs into the E. coli strain KC8 and selecting for growth on Trp-ampicillin plates. These putative TNFR1 interacting proteins were then tested further for specificity of interaction with the TNF-R1-DD by the reintroduction of JG4-5 clone into EGY48 derivatives containing a panel of different baits, including bicoid, the cytoplasmic domain of the IL-1 receptor, and TNF-R1-DD. The above clones were found to interact only with the TNF-R1-DD. The interaction between these clones and TNF-R1-DD was thus judged to be specific.

[0189]U937 cDNA Screening Results:

[0190]A U937 cDNA library was also constructed and screened as described above. 1,020 Leu+ colonies were found and of those, 326 colonies were also LacZ+. 62 colonies of these Leu+/LacZ+ colonies showed a galactose-dependent phenotype. One of these clones, 1TU, encodes a novel sequence. Interestingly, two clones, 15TU and 27TU, encode related or identical sequences, except that 27TU contains about 864 additional nucleotides (or about 288 amino acids) at the 5' end. 15/27TU also encode a novel sequence.

EXAMPLE 2

Expression of the TNF-R1-DD Ligand Protein

[0191]cDNAs encoding TNF-R intracellular ligand proteins were released from the pJG4-5 vector with the appropriate restriction enzymes. For example, EcoRI and XhoI or NotI and XhoI were used to release cDNA from clone 2DD and clone 20DD. Where the restriction sites were also present in the internal sequence of the cDNA, PCR was performed to obtain the cDNA. For example, the cDNA fragment encoding "clone 3DD" was obtained through PCR due to the presence of an internal XhoI site. These cDNAs were then cloned into various expression vectors. These included pGEX (Pharmacia) or pMAL (New England Biolabs) for expression as a GST (Glutathione-S-transferase) or MBP (maltose binding protein) fusion protein in E. coli, a pED-based vector for mammalian expression, and pVL or pBlueBacHis (Invitrogen) for baculovirus/insect expression. For the immunodetection of TNF-R intracellular ligand expression in mammalian cells, an epitope sequence, "Flag," was inserted into the translational start site of the pED vector, generating the pED-Flag vector. cDNAs were then inserted into the pED-Flag vector. Thus, the expression of cDNA from pED-Flag yields a protein with an amino terminal Met, followed by the "Flag" sequence, Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys. Standard DEAF-Dextran or lipofectamine methods were used to transfect COS or CHO dukx cells. Immunodetection of Flag-tagged proteins was achieved using the M2 antibody (Kodak). Moreover, an immunoaffinity column using the M2 antibody, followed by elution with the "Flag" peptide, can be used for the rapid purification of the flag-tagged protein. Similarly, affinity purification of GST-, MBP- or His-tagged fusion proteins can be performed using glutathione, amylose, or nickel columns. Detailed purification protocols are provided by the manufacturers. For many fusion proteins, the TNF-R intracellular ligand can be released by the action of thrombin, factor Xa, or enterokinase cleavage. In the case where highly purified material is required, standard purification procedures, such as ion-exchange, hydrophobic, and gel filtration chromatography will be applied in addition to the affinity purification step.

[0192]FIGS. 1 and 2 depict autoradiographs demonstrating the expression of TNF-R1-DD ligand proteins in yeast and mammalian cells. FIG. 1 shows the results of expression of isolated clones of the present invention in yeast. EGY48 was transformed with pJG4-5 containing clone 2DD, 3DD or 20DD. Cells were then grown overnight in the galactose/raffinose medium. Cell lysates were prepared and subject to 4-20% SDS gel electrophoresis, followed by Western blot analysis using anti-HA antibody (12CA5, Boehringer Mannheim, Indianapolis, Ind.). FIG. 2 shows the results of expression of Flag-2DD and Flag-20DD in COS cells. COS cells were transfected with either pED-Flag (Vector control), Flag-2DD or Flag-20DD plasmid by the lipofectamine method. Thirty μg of each cell lysate were prepared and subjected to 4-20% SDS gel electrophoresis, followed by Western blot analysis using M2 antibody (Kodak). The bands in the Flag-2DD and Flag-20DD lanes indicate significant expression of the respective TNF-R1-DD ligand proteins.

EXAMPLE 3

Assays of TNF-R Death Domain Binding

[0193]Two different methods were used to assay for TNF-R1-DD ligand protein activity. The first assay measures binding in the yeast strain in "interaction trap," the system used here to screen for TNF-R1-DD interacting proteins. In this system, the expression of reporter genes from both LexAop-Leu2 and LexAop-LacZ relies on the interaction between the bait protein, in this case TNF-R1DD, and the prey, the TNF-R intracellular ligand. Thus, one can measure the strength of the interaction by the level of Leu2 or LacZ expression. The most simple method is to measure the activity of the LacZ encoded protein, β-galactosidase. This activity can be judged by the degree of blueness on the X-Gal containing medium or filter. For the quantitative measurement of β-galactosidase activity, standard assays can be found in "Methods in Yeast Genetics" Cold Spring Harbor, N.Y., 1990 (by Rose, M.D., Winston, F., and Hieter, P.).

[0194]The second assay for measuring binding is a cell-free system. An example of a typical assay is described below. Purified GST-TNF-R1-DD fusion protein (2 ug) was mixed with amylose resins bound with a GST-TNF-R1-DD intracellular ligand for 2 hour at 4° C. The mixture was then centrifuged to separate bound (remained with the beads) and unbound (remained in the supernatant) GST-TNF-R1-DD. After extensive washing, the bound GST-TNF-R1-DD was eluted with maltose and detected by Western blot analysis using a GST antibody. The TNF-R1-DD or the intracellular ligand can also be immobilized on other solid supports, such as on plates or fluorobeads. The binding can then be measured using ELISA or SPA (scintillation proximity assay).

EXAMPLE 4

Characterization of TNF-R Death Domain Ligand Protein

Mapping the Interaction Site in TNF-R1.

[0195]Many of the key amino acids for TNF-R signaling have been determined by site-directed mutagenesis (Tataglia et at., Cell 74:845-853 (1993). These amino acids are conserved between TNF-R and the Fas antigen, which is required for mediating cytotoxicity and other cellular responses. In order to test if the TNF-R intracellular proteins interact with these residues, the following mutations were constructed: F345A (substitution of phe at amino acid 345 to Ala), R347A, L351A, F345A/R347A/L351A, E369A, W378A and I408A. The ability of the mutant protein to interact with the intracellular ligand in the "interaction trap" system was tested.

Effect on the TNF-Mediated Response

[0196]The effect of the TNF-R intracellular ligands on the TNF-mediated response can be evaluated in cells overexpressing the ligands. A number of TNF-mediated responses, including transient or prolonged responses, can be measured. For example, TNF-induced kinase activity toward either MBP (myelin basic protein) or the N-terminus (amino acids 1-79) of c-jun can be measured in COS cells or CHO cells either transiently or stably overexpressing clone 2DD, 3DD or clone 20DD. The significance of these ligand proteins in TNF-mediated cytotoxicity and other cellular responses can be measured in L929 or U937 overexpressing cells. Alternatively, other functional assays, such as the induction of gene expression or PGE₂ production after prolonged incubation with TNF, can also be used to measure the TNF mediated response. Conversely, the significance of the TNF-R1-DD ligand proteins in TNF signaling can be established by lowering or eliminating the expression of the ligands. These experiments can be performed using antisense expression or transgenic mice.

Enzymatic or Functional Assays

[0197]The signal transduction events initiated by TNF binding to its receptor are still largely unknown. However, one major result of TNF binding is the stimulation of cellular serine/threonine kinase activity. In addition, TNF has been shown to stimulate the activity of PC-PLC, PLA₂, and sphingomyelinase. Therefore, some of the TNF-R1-DD ligand proteins may possess intrinsic enzymatic activity that is responsible for these activities. Therefore, enzymatic assays can be performed to test this possibility, particularly with those clones that encode proteins with sequence homology to known enzymes. In addition to enzymatic activity, based on the sequence homology to proteins with known function, other functional assays can also be measured.

EXAMPLE 5

Isolation of Full Length Clones

[0198]In many cases, cDNAs obtained from the interaction trap method each encode only a portion of the full length protein. For example, based on identity and sequence and the lack of the initiating methionine codon, clones 2DD, 3DD and 20DD apparently do not encode full length proteins. Therefore, it is desirable to isolate full length clones. The cDNAs obtained from the screening, such as clone 2DD, are used as probes, and the cDNA libraries described herein, or alternatively phage cDNA libraries, are screened to obtain full length clones in accordance with known methods (see for example, "Molecular Cloning, A Laboratory Manual", by Sambrook et al., 1989 Cold Spring Harbor).

EXAMPLE 6

Antibodies Specific for TNF-R Intracellular Ligand Protein

[0199]Antibodies specific for TNF-R intracellular ligand proteins can be produced using purified recombinant protein, as described in Example 2, as antigen. Both polyclonal and monoclonal antibodies will be produced using standard techniques, such as those described in "Antibodies, a Laboratory Manual" by Ed Harlow and David Lane (1988), Cold Spring Harbor Laboratory.

EXAMPLE 7

Characterization of Clones 1TU and 15/27TU

Specificity of Interaction

[0200]The specificity of clones 1TU, 15TU and 27TU was tested using a panel of baits. The ability of these clones to bind the TNF-R death domain was compared to their binding to the intracellular domain of the second TNF-R (TNF-R P75_IC), the entire intracellular domain of TNF-R (TNF-R p55_IC), the death domain of the fas antigen (which shares 28% identity with TNF-R-DD) (Fas_DD), the Drosophila transcription factor bicoid, and a region of the IL-1 receptor known to be critical for signalling (IL-IR_477-527). As shown in Table 1, none of these clones interacted with TNF-R p75_IC or Fas_DD, and only 1TU interacted with bicoid. In contrast, both 1TU and 15TU bound the cytoplasmic domain of the p55 TNF-R, as well as residues 477-527 of the IL-1 R. 27TU interacted relatively weakly with these sequences.

TABLE-US-00001 TABLE 1 TNF-R TNF-R IL-1R clone TNF-R_DD p75_IC p55_IC Fas_DD bicoid (477-527) 1TU +++ - +++ - ++ +++ 15TU +++ ± +++ - - ++ 27TU +++ - + - - +

Interaction with Amino Acids Critical for Signalling

[0201]The ability of each clone to interact with four single-site mutations in the TNF-R death domain (each known to abolish signalling) was measured. As shown in Table 2, each of the clones interacted less strongly with the death domain mutants than with the wild type death domain, suggesting that these clones may bind critical residues in vivo.

TABLE-US-00002 TABLE 2 clone TNF-R_DD F345A L351A W378A I408A 1TU +++ + ++ ++ + 15TU +++ + + ++ ++ 27TU +++ + + ± ++

Expression of 1TU, 15TU and 27TU

[0202]FIG. 3 depicts an autoradiograph demonstrating the expression of clones 1TU, 15TU and 27TU in yeast (A) and COS cells (B).

[0203]In (A): EGY48 was transformed with pJG4-5 containing clones 1TU, 15TU or 27TU. Cells were then grown overnight in galactose/raffinose medium. Cell lysates were prepared and subjected to 4-20% SDS gel electrophoresis, followed by Western blot analysis using anti-HA antibody (12CA5, Boehringer Mannheim).

[0204]In (B): COS cells were transfected with pED-Flag containing clones 1TU, 15TU and 27TU. Cell lysates were prepared and analyzed by Western blot using anti-Flag antibody (M2, Kodak).

Specific Binding of 1TU and 27TU to TNF-R1-DD

[0205]The interaction of 1TU and 27TU with TNF-R1-DD was tested using purified bacterially expressed fusion proteins. As shown in FIG. 4, MBP fusion proteins containing 1TU or 27TU bound only to TNF-R1-DD expressed as a GST fusion protein, but not to GST protein alone. In the control experiment, MBP protein did not bind either GST or GST/TNF-R1-DD. These results indicate that 1TU and 27TU bound specifically to the TNF-R1 death domain in vitro, confirming the data obtained in the interaction trap.

15TU and 27TU Activation of JNK Activity

[0206]The jun N-terminal kinase (JNK) is normally activated within 15 min of TNF treatment in COS cells. 15TU and 27TU were cotransfected with an epitope tagged version of JNK, HA-JNK, in duplicate. After TNF treatment, JNK was immunoprecipitated with anti-HA antibody and JNK activity was measured in immunoprecipitation kinase assays, using GST-c-jun (amino acids 1-79) as substrate). Reactions were electrophoresed on SDS-PAGE. As shown in FIG. 5, transfection of 15TU and 27TU, but not vector alone, into COS cells activated JNK even in the absence of TNF, suggesting that these clones are involved in signal transduction of TNF and the pathway leading to JNK activation in vivo.

EXAMPLE 8

Isolation, Expression and Assay of Clone 3TW

[0207]Clone 3TW was isolated from the W138 cDNA library using clone 3DD as a porbe. Clone 3TW was expressed. FIG. 6 is an autoradiograph which demonstrates expression of 3TW (indicated by arrow).

[0208]An antisense oligonucleotide was derived from the sequence of clone 3TW. The antisense oligonucleotide was assayed to determine its ability to inhibit TNF-induced cPLA₂ phosphorylation. FIG. 7 depicts the results of that experiment. Activity of the anitsense oligonucleotide (3TWAS) was compared with the full-length clone (3TWFL), Flag-3TW full length (3TWFLflag) and pED-flag vector (pEDflag). The antisense oligonucleotide inhibited phosphorylation.

Sequence CWU 1

1912158DNAHomo sapiensCDS(2)..(1231) 1agc aat gca ggt gat gga cca ggt ggc gag ggc agt gtt cac ctg gca 49Ser Asn Ala Gly Asp Gly Pro Gly Gly Glu Gly Ser Val His Leu Ala 1 5 10 15agc tct cgg ggc act ttg tct gat agt gaa att gag acc aac tct gcc 97Ser Ser Arg Gly Thr Leu Ser Asp Ser Glu Ile Glu Thr Asn Ser Ala 20 25 30aca agc acc atc ttt ggt aaa gcc cac agc ttg aag cca agc ata aag 145Thr Ser Thr Ile Phe Gly Lys Ala His Ser Leu Lys Pro Ser Ile Lys 35 40 45gag aag ctg gca ggc agc ccc att cgt act tct gaa gat gtg agc cag 193Glu Lys Leu Ala Gly Ser Pro Ile Arg Thr Ser Glu Asp Val Ser Gln 50 55 60cga gtc tat ctc tat gag gga ctc cta ggc aaa gag cgt tct act tta 241Arg Val Tyr Leu Tyr Glu Gly Leu Leu Gly Lys Glu Arg Ser Thr Leu65 70 75 80tgg gac caa atg caa ttc tgg gaa gat gcc ttc tta gat gct gtg atg 289Trp Asp Gln Met Gln Phe Trp Glu Asp Ala Phe Leu Asp Ala Val Met 85 90 95ttg gag aga gaa ggg atg ggt atg gac cag ggt ccc cag gaa atg atc 337Leu Glu Arg Glu Gly Met Gly Met Asp Gln Gly Pro Gln Glu Met Ile 100 105 110gac agg tac ctg tcc ctt gga gaa cat gac cgg aag cgc ctg gaa gat 385Asp Arg Tyr Leu Ser Leu Gly Glu His Asp Arg Lys Arg Leu Glu Asp 115 120 125gat gaa gat cgc ttg ctg gcc aca ctt ctg cac aac ctc atc tcc tac 433Asp Glu Asp Arg Leu Leu Ala Thr Leu Leu His Asn Leu Ile Ser Tyr 130 135 140atg ctg ctg atg aag gta aat aag aat gac atc cgc aag aag gtg agg 481Met Leu Leu Met Lys Val Asn Lys Asn Asp Ile Arg Lys Lys Val Arg145 150 155 160cgc cta atg gga aag tcg cac att ggg ctt gtg tac agc cag caa atc 529Arg Leu Met Gly Lys Ser His Ile Gly Leu Val Tyr Ser Gln Gln Ile 165 170 175aat gag gtg ctt gat cag ctg gcg aac ctg aat gga cgc gat ctc tct 577Asn Glu Val Leu Asp Gln Leu Ala Asn Leu Asn Gly Arg Asp Leu Ser 180 185 190atc tgg tcc agt ggc agc cgg cac atg aag aag cag aca ttt gtg gta 625Ile Trp Ser Ser Gly Ser Arg His Met Lys Lys Gln Thr Phe Val Val 195 200 205cat gca ggg aca gat aca aac gga gat atc ttt ttc atg gag gtg tgc 673His Ala Gly Thr Asp Thr Asn Gly Asp Ile Phe Phe Met Glu Val Cys 210 215 220gat gac tgt gtg gtg ttg cgt agt aac atc gga aca gtg tat gag cgc 721Asp Asp Cys Val Val Leu Arg Ser Asn Ile Gly Thr Val Tyr Glu Arg225 230 235 240tgg tgg tac gag aag ctc atc aac atg acc tac tgt ccc aag acg aag 769Trp Trp Tyr Glu Lys Leu Ile Asn Met Thr Tyr Cys Pro Lys Thr Lys 245 250 255gtg ttg tgc ttg tgg cgt aga aat ggc tct gag acc cag ctc aac aag 817Val Leu Cys Leu Trp Arg Arg Asn Gly Ser Glu Thr Gln Leu Asn Lys 260 265 270ttc tat act aaa aag tgt cgg gag ctg tac tac tgt gtg aag gac agc 865Phe Tyr Thr Lys Lys Cys Arg Glu Leu Tyr Tyr Cys Val Lys Asp Ser 275 280 285atg gag cgc gct gcc gcc cga cag caa agc atc aaa ccc gga cct gaa 913Met Glu Arg Ala Ala Ala Arg Gln Gln Ser Ile Lys Pro Gly Pro Glu 290 295 300ttg ggt ggc gag ttc cct gtg cag gac ctg aag act ggt gag ggt ggc 961Leu Gly Gly Glu Phe Pro Val Gln Asp Leu Lys Thr Gly Glu Gly Gly305 310 315 320ctg ctg cag gtg acc ctg gaa ggg atc aac ctc aaa ttc atg cac aat 1009Leu Leu Gln Val Thr Leu Glu Gly Ile Asn Leu Lys Phe Met His Asn 325 330 335cag gtt ttc ata gag ctg aat cac att aaa aag tgc aat aca gtt cga 1057Gln Val Phe Ile Glu Leu Asn His Ile Lys Lys Cys Asn Thr Val Arg 340 345 350ggc gtc ttt gtc ctg gag gaa ttt gtt cct gaa att aaa gaa gtg gtg 1105Gly Val Phe Val Leu Glu Glu Phe Val Pro Glu Ile Lys Glu Val Val 355 360 365agc cac aag tac aag aca cca atg gcc cac gaa atc tgc tac tcc gta 1153Ser His Lys Tyr Lys Thr Pro Met Ala His Glu Ile Cys Tyr Ser Val 370 375 380tta tgt ctc ttc tcg tac gtg gct gca gtt cat agc agt gag gaa gat 1201Leu Cys Leu Phe Ser Tyr Val Ala Ala Val His Ser Ser Glu Glu Asp385 390 395 400ctc aga acc ccg ccc cgg cct gtc tct agc tgatggagag gggctacgca 1251Leu Arg Thr Pro Pro Arg Pro Val Ser Ser 405 410gctgccccag cccagggcac gcccctggcc ccttgctgtt cccaagtgca cgatgctgct 1311gtgactgagg agtggatgat gctcgtgtgt cctctgcaag ccccctgctg tggcttgggt 1371gggtaccggt tatgtgtccc tctgagtgtg tcttgagcgt gtccaccttc tccctctcca 1431ctcccagaag accaaactgc cttcccctca gggctcaaga atgtgtacag tctgtggggc 1491cggtgtgaac ccactatttt gtgtccttga gacatttgtg ttgtggttcc ttgtccttgt 1551ccctggcgtt aactgtccac tgcaagagtc tggctctccc ttctctgtga cccggcatga 1611ctgggcgcct ggagcagttt cactctgtga ggagtgaggg aaccctgggg ctcaccctct 1671cagaggaagg gcacagagag gaagggaaga attggggggc agccggagtg agtggcagcc 1731tccctgcttc cttctgcatt cccaagccgg cagctactgc ccagggcccg cagtgttggc 1791tgctgcctgc cacagcctct gtgactgcag tggagcggcg aattccctgt ggcctgccac 1851gccttcggca tcagaggatg gagtggtcga ggctagtgga gtcccaggga ccgctggctg 1911ctctgcctga gcatcaggga gggggcagga aagaccaagc tgggtttgca catctgtctg 1971caggctgtct ctccaggcac ggggtgtcag gagggagaga cagcctgggt atgggcaaga 2031aatgactgta aatatttcag ccccacatta tttatagaaa atgtacagtt gtgtgaatgt 2091gaaataaatg tcctcacctc ccaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2151aaaaaaa 21582410PRTHomo sapiens 2Ser Asn Ala Gly Asp Gly Pro Gly Gly Glu Gly Ser Val His Leu Ala 1 5 10 15Ser Ser Arg Gly Thr Leu Ser Asp Ser Glu Ile Glu Thr Asn Ser Ala 20 25 30Thr Ser Thr Ile Phe Gly Lys Ala His Ser Leu Lys Pro Ser Ile Lys 35 40 45Glu Lys Leu Ala Gly Ser Pro Ile Arg Thr Ser Glu Asp Val Ser Gln 50 55 60Arg Val Tyr Leu Tyr Glu Gly Leu Leu Gly Lys Glu Arg Ser Thr Leu 65 70 75 80Trp Asp Gln Met Gln Phe Trp Glu Asp Ala Phe Leu Asp Ala Val Met 85 90 95Leu Glu Arg Glu Gly Met Gly Met Asp Gln Gly Pro Gln Glu Met Ile 100 105 110Asp Arg Tyr Leu Ser Leu Gly Glu His Asp Arg Lys Arg Leu Glu Asp 115 120 125Asp Glu Asp Arg Leu Leu Ala Thr Leu Leu His Asn Leu Ile Ser Tyr 130 135 140Met Leu Leu Met Lys Val Asn Lys Asn Asp Ile Arg Lys Lys Val Arg145 150 155 160Arg Leu Met Gly Lys Ser His Ile Gly Leu Val Tyr Ser Gln Gln Ile 165 170 175Asn Glu Val Leu Asp Gln Leu Ala Asn Leu Asn Gly Arg Asp Leu Ser 180 185 190Ile Trp Ser Ser Gly Ser Arg His Met Lys Lys Gln Thr Phe Val Val 195 200 205His Ala Gly Thr Asp Thr Asn Gly Asp Ile Phe Phe Met Glu Val Cys 210 215 220Asp Asp Cys Val Val Leu Arg Ser Asn Ile Gly Thr Val Tyr Glu Arg225 230 235 240Trp Trp Tyr Glu Lys Leu Ile Asn Met Thr Tyr Cys Pro Lys Thr Lys 245 250 255Val Leu Cys Leu Trp Arg Arg Asn Gly Ser Glu Thr Gln Leu Asn Lys 260 265 270Phe Tyr Thr Lys Lys Cys Arg Glu Leu Tyr Tyr Cys Val Lys Asp Ser 275 280 285Met Glu Arg Ala Ala Ala Arg Gln Gln Ser Ile Lys Pro Gly Pro Glu 290 295 300Leu Gly Gly Glu Phe Pro Val Gln Asp Leu Lys Thr Gly Glu Gly Gly305 310 315 320Leu Leu Gln Val Thr Leu Glu Gly Ile Asn Leu Lys Phe Met His Asn 325 330 335Gln Val Phe Ile Glu Leu Asn His Ile Lys Lys Cys Asn Thr Val Arg 340 345 350Gly Val Phe Val Leu Glu Glu Phe Val Pro Glu Ile Lys Glu Val Val 355 360 365Ser His Lys Tyr Lys Thr Pro Met Ala His Glu Ile Cys Tyr Ser Val 370 375 380Leu Cys Leu Phe Ser Tyr Val Ala Ala Val His Ser Ser Glu Glu Asp385 390 395 400Leu Arg Thr Pro Pro Arg Pro Val Ser Ser 405 4103826DNAHomo sapiensCDS(2)..(415) 3g gag gtg cag gac ctc ttc gaa gcc cag ggc aat gac cga ctg aag ctg 49Glu Val Gln Asp Leu Phe Glu Ala Gln Gly Asn Asp Arg Leu Lys Leu 1 5 10 15ctg gtg ctg tac agt gga gag gat gat gag ctg cta cag cgg gca gct 97Leu Val Leu Tyr Ser Gly Glu Asp Asp Glu Leu Leu Gln Arg Ala Ala 20 25 30gcc ggg ggc ttg gcc atg ctt acc tcc atg cgg ccc acg ctc tgc agc 145Ala Gly Gly Leu Ala Met Leu Thr Ser Met Arg Pro Thr Leu Cys Ser 35 40 45cgc att ccc caa gtg acc aca cac tgg ctg gag atc ctg cag gcc ctg 193Arg Ile Pro Gln Val Thr Thr His Trp Leu Glu Ile Leu Gln Ala Leu 50 55 60ctt ctg agc tcc aac cag gag ctg cag cac cgg ggt gct gtg gtg gtg 241Leu Leu Ser Ser Asn Gln Glu Leu Gln His Arg Gly Ala Val Val Val65 70 75 80ctg aac atg gtg gag gcc tcg agg gag att gcc agc acc ctg atg gag 289Leu Asn Met Val Glu Ala Ser Arg Glu Ile Ala Ser Thr Leu Met Glu 85 90 95agt gag atg atg gag atc ttg tca gtg cta gct aag ggt gac cac agc 337Ser Glu Met Met Glu Ile Leu Ser Val Leu Ala Lys Gly Asp His Ser 100 105 110cct gtc aca agg gct gct gca gcc tgc ctg gac aaa gca gtg gaa tat 385Pro Val Thr Arg Ala Ala Ala Ala Cys Leu Asp Lys Ala Val Glu Tyr 115 120 125ggg ctt atc caa ccc aac caa gat gga gag tgagggggtt gtccctgggc 435Gly Leu Ile Gln Pro Asn Gln Asp Gly Glu 130 135ccaaggctca tgcacacgct acctattgtg gcacggagag taaggacgga agcagctttg 495gctggtggtg gctggcatgc ccaatactct tgcccatcct cgcttgctgc cctaggatgt 555cctctgttct gagtcagcgg ccacgttcag tcacacagcc ctgcttggcc agcactgcct 615gcagcctcac tcagaggggc cctttttctg tactactgta gtcagctggg aatggggaag 675gtgcatccca acacagcctg tggatcctgg ggcatttgga agggcgcaca catcagcagc 735ctcaccagct gtgagcctgc tatcaggcct gcccctccaa taaaagtgtg tagaactcca 795aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa a 8264138PRTHomo sapiens 4Glu Val Gln Asp Leu Phe Glu Ala Gln Gly Asn Asp Arg Leu Lys Leu 1 5 10 15Leu Val Leu Tyr Ser Gly Glu Asp Asp Glu Leu Leu Gln Arg Ala Ala 20 25 30Ala Gly Gly Leu Ala Met Leu Thr Ser Met Arg Pro Thr Leu Cys Ser 35 40 45Arg Ile Pro Gln Val Thr Thr His Trp Leu Glu Ile Leu Gln Ala Leu 50 55 60Leu Leu Ser Ser Asn Gln Glu Leu Gln His Arg Gly Ala Val Val Val 65 70 75 80Leu Asn Met Val Glu Ala Ser Arg Glu Ile Ala Ser Thr Leu Met Glu 85 90 95Ser Glu Met Met Glu Ile Leu Ser Val Leu Ala Lys Gly Asp His Ser 100 105 110Pro Val Thr Arg Ala Ala Ala Ala Cys Leu Asp Lys Ala Val Glu Tyr 115 120 125Gly Leu Ile Gln Pro Asn Gln Asp Gly Glu 130 1355722DNAHomo sapiensCDS(2)..(559) 5g gag aag ccg ctg cac gcc ctg ctg cac ggc cgc ggg gtt tgc ctc aac 49Glu Lys Pro Leu His Ala Leu Leu His Gly Arg Gly Val Cys Leu Asn 1 5 10 15gaa aag agc tac cgc gag caa gtc aag atc gag aga gac tcc cgt gag 97Glu Lys Ser Tyr Arg Glu Gln Val Lys Ile Glu Arg Asp Ser Arg Glu 20 25 30cac gag gag ccc acc acc tct gag atg gcc gag gag acc tac tcc ccc 145His Glu Glu Pro Thr Thr Ser Glu Met Ala Glu Glu Thr Tyr Ser Pro 35 40 45aag atc ttc cgg ccc aaa cac acc cgc atc tcc gag ctg aag gct gaa 193Lys Ile Phe Arg Pro Lys His Thr Arg Ile Ser Glu Leu Lys Ala Glu 50 55 60gca gtg aag aag gac cgc aga aag aag ctg acc cag tcc aag ttt gtc 241Ala Val Lys Lys Asp Arg Arg Lys Lys Leu Thr Gln Ser Lys Phe Val65 70 75 80ggg gga gcc gag aac act gcc cac ccc cgg atc atc tct gaa cct gag 289Gly Gly Ala Glu Asn Thr Ala His Pro Arg Ile Ile Ser Glu Pro Glu 85 90 95atg aga cag gag tct gag cag ggc ccc tgc cgc aga cac atg gag gct 337Met Arg Gln Glu Ser Glu Gln Gly Pro Cys Arg Arg His Met Glu Ala 100 105 110tcc ctg cag gag ctc aaa gcc agc cca cgc atg gtg ccc cgt gct gtg 385Ser Leu Gln Glu Leu Lys Ala Ser Pro Arg Met Val Pro Arg Ala Val 115 120 125tac ctg ccc aat tgt gac cgc aaa gga ttc tac aag aga aag cag tgc 433Tyr Leu Pro Asn Cys Asp Arg Lys Gly Phe Tyr Lys Arg Lys Gln Cys 130 135 140aaa cct tcc cgt ggc cgc aag cgt ggc atc tgc tgg tgc gtg gac aag 481Lys Pro Ser Arg Gly Arg Lys Arg Gly Ile Cys Trp Cys Val Asp Lys145 150 155 160tac ggg atg aag ctg cca ggc atg gag tac gtt gac ggg gac ttt cag 529Tyr Gly Met Lys Leu Pro Gly Met Glu Tyr Val Asp Gly Asp Phe Gln 165 170 175tgc cac acc ttc gac agc agc aac gtt gag tgatgcgtcc ccccccaacc 579Cys His Thr Phe Asp Ser Ser Asn Val Glu 180 185tttccctcac ccccttccac ccccagcccc gactccagcc agcgcctccc tccaccccag 639gacgccactc atttcatctc atttaaggga aaaatatata tctatctatt tgaggaaaaa 699aaaaaaaaaa aaaaaaaaaa aaa 7226186PRTHomo sapiens 6Glu Lys Pro Leu His Ala Leu Leu His Gly Arg Gly Val Cys Leu Asn 1 5 10 15Glu Lys Ser Tyr Arg Glu Gln Val Lys Ile Glu Arg Asp Ser Arg Glu 20 25 30His Glu Glu Pro Thr Thr Ser Glu Met Ala Glu Glu Thr Tyr Ser Pro 35 40 45Lys Ile Phe Arg Pro Lys His Thr Arg Ile Ser Glu Leu Lys Ala Glu 50 55 60Ala Val Lys Lys Asp Arg Arg Lys Lys Leu Thr Gln Ser Lys Phe Val 65 70 75 80Gly Gly Ala Glu Asn Thr Ala His Pro Arg Ile Ile Ser Glu Pro Glu 85 90 95Met Arg Gln Glu Ser Glu Gln Gly Pro Cys Arg Arg His Met Glu Ala 100 105 110Ser Leu Gln Glu Leu Lys Ala Ser Pro Arg Met Val Pro Arg Ala Val 115 120 125Tyr Leu Pro Asn Cys Asp Arg Lys Gly Phe Tyr Lys Arg Lys Gln Cys 130 135 140Lys Pro Ser Arg Gly Arg Lys Arg Gly Ile Cys Trp Cys Val Asp Lys145 150 155 160Tyr Gly Met Lys Leu Pro Gly Met Glu Tyr Val Asp Gly Asp Phe Gln 165 170 175Cys His Thr Phe Asp Ser Ser Asn Val Glu 180 18571023DNAHomo sapiensCDS(57)..(872) 7ccctgcactc tcgctctcct gccccacccc gaggtaaagg gggcgactaa gagaag atg 59Met 1gtg ttg ctc acc gcg gtc ctc ctg ctg ctg gcc gcc tat gcg ggg ccg 107Val Leu Leu Thr Ala Val Leu Leu Leu Leu Ala Ala Tyr Ala Gly Pro 5 10 15gcc cag agc ctg ggc tcc ttc gtg cac tgc gag ccc tgc gac gag aaa 155Ala Gln Ser Leu Gly Ser Phe Val His Cys Glu Pro Cys Asp Glu Lys 20 25 30gcc ctc tcc atg tgc ccc ccc agc ccc ctg ggc tgc gag ctg gtc aag 203Ala Leu Ser Met Cys Pro Pro Ser Pro Leu Gly Cys Glu Leu Val Lys 35 40 45gag ccg ggc tgc ggc tgc tgc atg acc tgc gcc ctg gcc gag ggg cag 251Glu Pro Gly Cys Gly Cys Cys Met Thr Cys Ala Leu Ala Glu Gly Gln 50 55 60 65tcg tgc ggc gtc tac acc gag cgc tgc gcc cag ggg ctg cgc tgc ctc 299Ser Cys Gly Val Tyr Thr Glu Arg Cys Ala Gln Gly Leu Arg Cys Leu 70 75 80ccc cgg cag gac gag gag aag ccg ctg cac gcc ctg ctg cac ggc cgc 347Pro Arg Gln Asp Glu Glu Lys Pro Leu His Ala Leu Leu His Gly Arg 85 90 95ggg gtt tgc ctc aac gaa aag agc tac cgc gag caa gtc aag atc gag 395Gly Val Cys Leu Asn Glu Lys Ser Tyr Arg Glu Gln Val Lys Ile Glu 100 105 110aga gac tcc cgt gag cac gag gag ccc acc acc tct gag atg gcc gag 443Arg Asp Ser Arg Glu His Glu Glu Pro Thr Thr Ser Glu Met Ala Glu 115 120 125gag acc tac tcc ccc aag atc ttc cgg ccc aaa cac acc cgc atc tcc 491Glu Thr Tyr Ser Pro Lys Ile Phe Arg Pro Lys His Thr Arg Ile Ser130 135 140 145gag ctg aag gct gaa gca gtg aag aag gac cgc aga aag aag ctg acc 539Glu Leu Lys Ala Glu Ala Val Lys Lys Asp Arg Arg Lys Lys Leu Thr 150 155 160cag tcc aag ttt gtc ggg gga gcc gag aac

act gcc cac ccc cgg atc 587Gln Ser Lys Phe Val Gly Gly Ala Glu Asn Thr Ala His Pro Arg Ile 165 170 175atc tct gca cct gag atg aga cag gag tct gag cag ggc ccc tgc cgc 635Ile Ser Ala Pro Glu Met Arg Gln Glu Ser Glu Gln Gly Pro Cys Arg 180 185 190aga cac atg gag gct tcc ctg cag gag ctc aaa gcc agc cca cgc atg 683Arg His Met Glu Ala Ser Leu Gln Glu Leu Lys Ala Ser Pro Arg Met 195 200 205gtg ccc cgt gct gtg tac ctg ccc aat tgt gac cgc aaa gga ttc tac 731Val Pro Arg Ala Val Tyr Leu Pro Asn Cys Asp Arg Lys Gly Phe Tyr210 215 220 225aag aga aag cag tgc aaa cct tcc cgt ggc cgc aag cgt ggc atc tgc 779Lys Arg Lys Gln Cys Lys Pro Ser Arg Gly Arg Lys Arg Gly Ile Cys 230 235 240tgg tgc gtg gac aag tac ggg atg aag ctg cca ggc atg gag tac gtt 827Trp Cys Val Asp Lys Tyr Gly Met Lys Leu Pro Gly Met Glu Tyr Val 245 250 255gac ggg gac ttt cag tgc cac acc ttc gac agc agc aac gtt gag 872Asp Gly Asp Phe Gln Cys His Thr Phe Asp Ser Ser Asn Val Glu 260 265 270tgatgcgtcc ccccccaacc tttccctcac cccctcccac ccccagcccc gactccagcc 932agcgcctccc tccaccccag gacgccactc atttcatctc atttaaggga aaaatatata 992tctatctatt tgaaaaaaaa aaaaaaaacc c 10238272PRTHomo sapiens 8Met Val Leu Leu Thr Ala Val Leu Leu Leu Leu Ala Ala Tyr Ala Gly 1 5 10 15Pro Ala Gln Ser Leu Gly Ser Phe Val His Cys Glu Pro Cys Asp Glu 20 25 30Lys Ala Leu Ser Met Cys Pro Pro Ser Pro Leu Gly Cys Glu Leu Val 35 40 45Lys Glu Pro Gly Cys Gly Cys Cys Met Thr Cys Ala Leu Ala Glu Gly 50 55 60Gln Ser Cys Gly Val Tyr Thr Glu Arg Cys Ala Gln Gly Leu Arg Cys 65 70 75 80Leu Pro Arg Gln Asp Glu Glu Lys Pro Leu His Ala Leu Leu His Gly 85 90 95Arg Gly Val Cys Leu Asn Glu Lys Ser Tyr Arg Glu Gln Val Lys Ile 100 105 110Glu Arg Asp Ser Arg Glu His Glu Glu Pro Thr Thr Ser Glu Met Ala 115 120 125Glu Glu Thr Tyr Ser Pro Lys Ile Phe Arg Pro Lys His Thr Arg Ile 130 135 140Ser Glu Leu Lys Ala Glu Ala Val Lys Lys Asp Arg Arg Lys Lys Leu145 150 155 160Thr Gln Ser Lys Phe Val Gly Gly Ala Glu Asn Thr Ala His Pro Arg 165 170 175Ile Ile Ser Ala Pro Glu Met Arg Gln Glu Ser Glu Gln Gly Pro Cys 180 185 190Arg Arg His Met Glu Ala Ser Leu Gln Glu Leu Lys Ala Ser Pro Arg 195 200 205Met Val Pro Arg Ala Val Tyr Leu Pro Asn Cys Asp Arg Lys Gly Phe 210 215 220Tyr Lys Arg Lys Gln Cys Lys Pro Ser Arg Gly Arg Lys Arg Gly Ile225 230 235 240Cys Trp Cys Val Asp Lys Tyr Gly Met Lys Leu Pro Gly Met Glu Tyr 245 250 255Val Asp Gly Asp Phe Gln Cys His Thr Phe Asp Ser Ser Asn Val Glu 260 265 27091694DNAHomo sapiensCDS(2)..(931) 9c tct ctc aag gcc aac atc cct gag gtg gaa gct gtc ctc aac acc gac 49Ser Leu Lys Ala Asn Ile Pro Glu Val Glu Ala Val Leu Asn Thr Asp 1 5 10 15agg agt ttg gtg tgt gat ggg aag agg ggc tta tta act cgt ctg ctg 97Arg Ser Leu Val Cys Asp Gly Lys Arg Gly Leu Leu Thr Arg Leu Leu 20 25 30cag gtc atg aag aag gag cca gca gag tcg tct ttc agg ttt tgg caa 145Gln Val Met Lys Lys Glu Pro Ala Glu Ser Ser Phe Arg Phe Trp Gln 35 40 45gct cgg gct gtg gag agt ttc ctc cga ggg acc acc tcc tat gca gac 193Ala Arg Ala Val Glu Ser Phe Leu Arg Gly Thr Thr Ser Tyr Ala Asp 50 55 60cag atg ttc ctg ctg aag cga ggc ctc ttg gag cac atc ctt tac tgc 241Gln Met Phe Leu Leu Lys Arg Gly Leu Leu Glu His Ile Leu Tyr Cys65 70 75 80att gtg gac agc gag tgt aag tca agg gat gtg ctc cag agt tac ttt 289Ile Val Asp Ser Glu Cys Lys Ser Arg Asp Val Leu Gln Ser Tyr Phe 85 90 95gac ctc ctg ggg gag ctg atg aag ttc aac gtt gat gca ttc aag aga 337Asp Leu Leu Gly Glu Leu Met Lys Phe Asn Val Asp Ala Phe Lys Arg 100 105 110ttc aat aaa tat atc aac acc gat gca aag ttc cag gta ttc ctg aag 385Phe Asn Lys Tyr Ile Asn Thr Asp Ala Lys Phe Gln Val Phe Leu Lys 115 120 125cag atc aac agc tcc ctg gtg gac tcc aac atg ctg gtg cgc tgt gtc 433Gln Ile Asn Ser Ser Leu Val Asp Ser Asn Met Leu Val Arg Cys Val 130 135 140act ctg tcc ctg gac cga ttt gaa aac cag gtg gat atg aaa gtt gcc 481Thr Leu Ser Leu Asp Arg Phe Glu Asn Gln Val Asp Met Lys Val Ala145 150 155 160gag gta ctg tct gaa tgc cgc ctg ctc gcc tac ata tcc cag gtg ccc 529Glu Val Leu Ser Glu Cys Arg Leu Leu Ala Tyr Ile Ser Gln Val Pro 165 170 175acg cag atg tcc ttc ctc ttc cgc ctc atc aac atc atc cac gtg cag 577Thr Gln Met Ser Phe Leu Phe Arg Leu Ile Asn Ile Ile His Val Gln 180 185 190acg ctg acc cag gag aac gtc agc tgc ctc aac acc agc ctg gtg atc 625Thr Leu Thr Gln Glu Asn Val Ser Cys Leu Asn Thr Ser Leu Val Ile 195 200 205ctg atg ctg gcc cga cgg aaa gag cgg ctg ccc ctg tac ctg cgg ctg 673Leu Met Leu Ala Arg Arg Lys Glu Arg Leu Pro Leu Tyr Leu Arg Leu 210 215 220ctg cag cgg atg gag cac agc aag aag tac ccc ggc ttc ctg ctc aac 721Leu Gln Arg Met Glu His Ser Lys Lys Tyr Pro Gly Phe Leu Leu Asn225 230 235 240aac ttc cac aac ctg ctg cgc ttc tgg cag cag cac tac ctg cac aag 769Asn Phe His Asn Leu Leu Arg Phe Trp Gln Gln His Tyr Leu His Lys 245 250 255gac aag gac agc acc tgc cta gag aac agc tcc tgc atc agc ttc tca 817Asp Lys Asp Ser Thr Cys Leu Glu Asn Ser Ser Cys Ile Ser Phe Ser 260 265 270tac tgg aag gag aca gtg tcc atc ctg ttg aac ccg gac cgg cag tca 865Tyr Trp Lys Glu Thr Val Ser Ile Leu Leu Asn Pro Asp Arg Gln Ser 275 280 285ccc tct gct ctc gtt agc tac att gag gag ccc tac atg gac ata gac 913Pro Ser Ala Leu Val Ser Tyr Ile Glu Glu Pro Tyr Met Asp Ile Asp 290 295 300agg gac ttc act gag gag tgaccttggg ccaggcctcg ggaggctgct 961Arg Asp Phe Thr Glu Glu305 310gggccagtgt gggtgagcgt gggtacgatg ccacacgccc tgccctgttc ccgttcctcc 1021ctgctgctct ctgcctgccc caggtctttg ggtacaggct tggtgggagg gaagtcctag 1081aagcccttgg tccccctggg tctgagggcc ctaggtcatg gagagcctca gtccccataa 1141tgaggacagg gtaccatgcc cacctttcct tcagaaccct ggggcccagg gccacccaga 1201ggtaagagga catttagcat tagctctgtg tgagctcctg ccggtttctt ggctgtcagt 1261cagtcccaga gtggggagga agatatgggt gacccccacc ccccatctgt gagccaagcc 1321tcccttgtcc ctggcctttg gacccaggca aaggcttctg agccctgggc aggggtggtg 1381ggtaccagag aatgctgcct tcccccaagc ctgcccctct gcctcatttt cctgtagctc 1441ctctggttct gtttgctcat tggccgctgt gttcatccaa gggggttctc ccagaagtga 1501ggggcctttc cctccatccc ttggggcacg gggcagctgt gcctgccctg cctctgcctg 1561aggcagccgc tcctgcctga gcctggacat ggggcccttc cttgtgttgc caatttatta 1621acagcaaata aaccaattaa atggagacta ttaaataact ttattttaaa aatgaaaaaa 1681aaaaaaaaaa aaa 169410310PRTHomo sapiens 10Ser Leu Lys Ala Asn Ile Pro Glu Val Glu Ala Val Leu Asn Thr Asp 1 5 10 15Arg Ser Leu Val Cys Asp Gly Lys Arg Gly Leu Leu Thr Arg Leu Leu 20 25 30Gln Val Met Lys Lys Glu Pro Ala Glu Ser Ser Phe Arg Phe Trp Gln 35 40 45Ala Arg Ala Val Glu Ser Phe Leu Arg Gly Thr Thr Ser Tyr Ala Asp 50 55 60Gln Met Phe Leu Leu Lys Arg Gly Leu Leu Glu His Ile Leu Tyr Cys 65 70 75 80Ile Val Asp Ser Glu Cys Lys Ser Arg Asp Val Leu Gln Ser Tyr Phe 85 90 95Asp Leu Leu Gly Glu Leu Met Lys Phe Asn Val Asp Ala Phe Lys Arg 100 105 110Phe Asn Lys Tyr Ile Asn Thr Asp Ala Lys Phe Gln Val Phe Leu Lys 115 120 125Gln Ile Asn Ser Ser Leu Val Asp Ser Asn Met Leu Val Arg Cys Val 130 135 140Thr Leu Ser Leu Asp Arg Phe Glu Asn Gln Val Asp Met Lys Val Ala145 150 155 160Glu Val Leu Ser Glu Cys Arg Leu Leu Ala Tyr Ile Ser Gln Val Pro 165 170 175Thr Gln Met Ser Phe Leu Phe Arg Leu Ile Asn Ile Ile His Val Gln 180 185 190Thr Leu Thr Gln Glu Asn Val Ser Cys Leu Asn Thr Ser Leu Val Ile 195 200 205Leu Met Leu Ala Arg Arg Lys Glu Arg Leu Pro Leu Tyr Leu Arg Leu 210 215 220Leu Gln Arg Met Glu His Ser Lys Lys Tyr Pro Gly Phe Leu Leu Asn225 230 235 240Asn Phe His Asn Leu Leu Arg Phe Trp Gln Gln His Tyr Leu His Lys 245 250 255Asp Lys Asp Ser Thr Cys Leu Glu Asn Ser Ser Cys Ile Ser Phe Ser 260 265 270Tyr Trp Lys Glu Thr Val Ser Ile Leu Leu Asn Pro Asp Arg Gln Ser 275 280 285Pro Ser Ala Leu Val Ser Tyr Ile Glu Glu Pro Tyr Met Asp Ile Asp 290 295 300Arg Asp Phe Thr Glu Glu305 310112735DNAHomo sapiensCDS(2)..(1822) 11g gag atc agt cgg aag gtg tac aag gga atg tta gac ctc ctc aag tgt 49Glu Ile Ser Arg Lys Val Tyr Lys Gly Met Leu Asp Leu Leu Lys Cys 1 5 10 15aca gtc ctc agc ttg gag cag tcc tat gcc cac gcg ggt ctg ggt ggc 97Thr Val Leu Ser Leu Glu Gln Ser Tyr Ala His Ala Gly Leu Gly Gly 20 25 30atg gcc agc atc ttt ggg ctt ttg gag att gcc cag acc cac tac tat 145Met Ala Ser Ile Phe Gly Leu Leu Glu Ile Ala Gln Thr His Tyr Tyr 35 40 45agt aaa gaa cca gac aag cgg aag aga agt cca aca gaa agt gta aat 193Ser Lys Glu Pro Asp Lys Arg Lys Arg Ser Pro Thr Glu Ser Val Asn 50 55 60acc cca gtt ggc aag gat cct ggc cta gct ggg cgg ggg gac cca aag 241Thr Pro Val Gly Lys Asp Pro Gly Leu Ala Gly Arg Gly Asp Pro Lys65 70 75 80gct atg gca caa ctg aga gtt cca caa ctg gga cct cgg gca cca agt 289Ala Met Ala Gln Leu Arg Val Pro Gln Leu Gly Pro Arg Ala Pro Ser 85 90 95gcc aca gga aag ggt cct aag gaa ctg gac acc aga agt tta aag gaa 337Ala Thr Gly Lys Gly Pro Lys Glu Leu Asp Thr Arg Ser Leu Lys Glu 100 105 110gaa aat ttt ata gca tct att ggg cct gaa gta atc aaa cct gtc ttt 385Glu Asn Phe Ile Ala Ser Ile Gly Pro Glu Val Ile Lys Pro Val Phe 115 120 125gac ctt ggt gag aca gag gag aaa aag tcc cag atc agc gca gac agt 433Asp Leu Gly Glu Thr Glu Glu Lys Lys Ser Gln Ile Ser Ala Asp Ser 130 135 140ggt gtg agc ctg acg tct agt tcc cag agg act gat caa gac tct gtc 481Gly Val Ser Leu Thr Ser Ser Ser Gln Arg Thr Asp Gln Asp Ser Val145 150 155 160atc ggc gtg agt cca gct gtt atg atc cgc agc tca agt cag gat tct 529Ile Gly Val Ser Pro Ala Val Met Ile Arg Ser Ser Ser Gln Asp Ser 165 170 175gaa gtt agc acc gtg gtg agt aat agc tct gga gag acc ctt gga gct 577Glu Val Ser Thr Val Val Ser Asn Ser Ser Gly Glu Thr Leu Gly Ala 180 185 190gac agt gac ttg agc agc aat gca ggt gat gga cca ggt ggc gag ggc 625Asp Ser Asp Leu Ser Ser Asn Ala Gly Asp Gly Pro Gly Gly Glu Gly 195 200 205agt gtt cac ctg gca agc tct cgg ggc act ttg tct gat agt gaa att 673Ser Val His Leu Ala Ser Ser Arg Gly Thr Leu Ser Asp Ser Glu Ile 210 215 220gag acc aac tct gcc aca agc acc atc ttt ggt aaa gcc cac agc ttg 721Glu Thr Asn Ser Ala Thr Ser Thr Ile Phe Gly Lys Ala His Ser Leu225 230 235 240aag cca agc ata aag gag aag ctg gca ggc agc ccc att cgt act tct 769Lys Pro Ser Ile Lys Glu Lys Leu Ala Gly Ser Pro Ile Arg Thr Ser 245 250 255gaa gat gtg agc cag cga gtc tat ctc tat gag gga ctc cta ggc aaa 817Glu Asp Val Ser Gln Arg Val Tyr Leu Tyr Glu Gly Leu Leu Gly Lys 260 265 270gag cgt tct act tta tgg gac caa atg caa ttc tgg gaa gat gcc ttc 865Glu Arg Ser Thr Leu Trp Asp Gln Met Gln Phe Trp Glu Asp Ala Phe 275 280 285tta gat gct gtg atg ttg gag aga gaa ggg atg ggt atg gac cag ggt 913Leu Asp Ala Val Met Leu Glu Arg Glu Gly Met Gly Met Asp Gln Gly 290 295 300ccc cag gaa atg atc gac agg tac ctg tcc ctt gga gaa cat gac cgg 961Pro Gln Glu Met Ile Asp Arg Tyr Leu Ser Leu Gly Glu His Asp Arg305 310 315 320aag cgc ctg gaa gat gat gaa gat cgc ttg ctg gcc aca ctt ctg cac 1009Lys Arg Leu Glu Asp Asp Glu Asp Arg Leu Leu Ala Thr Leu Leu His 325 330 335aac ctc atc tcc tac atg ctg ctg atg aag gta aat aag aat gac atc 1057Asn Leu Ile Ser Tyr Met Leu Leu Met Lys Val Asn Lys Asn Asp Ile 340 345 350cgc aag aag gtg agg cgc cta atg gga aag tcg cac att ggg ctt gtg 1105Arg Lys Lys Val Arg Arg Leu Met Gly Lys Ser His Ile Gly Leu Val 355 360 365tac agc cag caa atc aat gag gtg ctt gat cag ctg gcg aac ctg aat 1153Tyr Ser Gln Gln Ile Asn Glu Val Leu Asp Gln Leu Ala Asn Leu Asn 370 375 380gga cgc gat ctc tct atc tgg tcc agt ggc agc cgg cac atg aag aag 1201Gly Arg Asp Leu Ser Ile Trp Ser Ser Gly Ser Arg His Met Lys Lys385 390 395 400cag aca ttt gtg gta cat gca ggg aca gat aca aac gga gat atc ttt 1249Gln Thr Phe Val Val His Ala Gly Thr Asp Thr Asn Gly Asp Ile Phe 405 410 415ttc atg gag gtg tgc gat gac tgt gtg gtg ttg cgt agt aac atc gga 1297Phe Met Glu Val Cys Asp Asp Cys Val Val Leu Arg Ser Asn Ile Gly 420 425 430aca gtg tat gag cgc tgg tgg tac gag aag ctc atc aac atg acc tac 1345Thr Val Tyr Glu Arg Trp Trp Tyr Glu Lys Leu Ile Asn Met Thr Tyr 435 440 445tgt ccc aag acg aag gtg ttg tgc ttg tgg cgt aga aat ggc tct gag 1393Cys Pro Lys Thr Lys Val Leu Cys Leu Trp Arg Arg Asn Gly Ser Glu 450 455 460acc cag ctc aac aag ttc tat act aaa aag tgt cgg gag ctg tac tac 1441Thr Gln Leu Asn Lys Phe Tyr Thr Lys Lys Cys Arg Glu Leu Tyr Tyr465 470 475 480tgt gtg aag gac agc atg gag cgc gct gcc gcc cga cag caa agc atc 1489Cys Val Lys Asp Ser Met Glu Arg Ala Ala Ala Arg Gln Gln Ser Ile 485 490 495aaa ccc gga cct gaa ttg ggt ggc gag ttc cct gtg cag gac ctg aag 1537Lys Pro Gly Pro Glu Leu Gly Gly Glu Phe Pro Val Gln Asp Leu Lys 500 505 510act ggt gag ggt ggc ctg ctg cag gtg acc ctg gaa ggg atc aac ctc 1585Thr Gly Glu Gly Gly Leu Leu Gln Val Thr Leu Glu Gly Ile Asn Leu 515 520 525aaa ttc atg cac aat cag gtt ttc ata gag ctg aat cac att aaa aag 1633Lys Phe Met His Asn Gln Val Phe Ile Glu Leu Asn His Ile Lys Lys 530 535 540tgc aat aca gtt cga ggc gtc ttt gtc ctg gag gaa ttt gtt cct gaa 1681Cys Asn Thr Val Arg Gly Val Phe Val Leu Glu Glu Phe Val Pro Glu545 550 555 560att aaa gaa gtg gtg agc cac aag tac aag aca cca atg gcc cac gaa 1729Ile Lys Glu Val Val Ser His Lys Tyr Lys Thr Pro Met Ala His Glu 565 570 575atc tgc tac tcc gta tta tgt ctc ttc tcg tac gtg gct gca gtt cat 1777Ile Cys Tyr Ser Val Leu Cys Leu Phe Ser Tyr Val Ala Ala Val His 580 585 590agc agt gag gaa gat ctc aga acc ccg ccc cgg cct gtc tct agc 1822Ser Ser Glu Glu Asp Leu Arg Thr Pro Pro Arg Pro Val Ser Ser 595 600 605tgatggagag gggctacgca gctgccccag cccagggcac gcccctggcc ccttgctgtt 1882cccaagtgca cgatgctgct gtgactgagg agtggatgat gctcgtgtgt cctctgcaag 1942ccccctgctg tggcttggtt ggttaccggt tatgtgtccc tctgagtgtg tcttgagcgt 2002gtccaccttc tccctctcca ctcccagaag accaaactgc cttcccctca gggctcaaga 2062atgtgtacag tctgtggggc cggtgtgaac ccactatttt gtgtccttga gacatttgtg 2122ttgtggttcc ttgtccttgt ccctggcgtt ataactgtcc actgcaagag tctggctctc 2182ccttctctgt gacccggcat gactgggcgc ctggagcagt ttcactctgt gaggagtgag 2242ggaaccctgg ggctcaccct ctcagaggaa gggcacagag

aggaagggaa gaattggggg 2302gcagccggag tgagtggcag cctccctgct tccttctgca ttcccaagcc ggcagctact 2362gcccagggcc cgcagtgttg gctgctgcct gccacagcct ctgtgactgc agtggagcgg 2422cgaattccct gtggcctgcc acgccttcgg catcagagga tggagtggtc gaggctagtg 2482gagtcccagg gaccgctggc tgctctgcct gagcatcagg gagggggcag gaaagaccaa 2542gctgggtttg cacatctgtc tgcaggctgt ctctccaggc acggggtgtc aggagggaga 2602gacagcctgg gtatgggcaa gaaatgactg taaatatttc agccccacat tatttataga 2662aaatgtacag ttgtgtgaat gtgaaataaa tgtcctcaac tcccaaaaaa aaaaaaaaaa 2722aaaaaaaaaa aaa 273512607PRTHomo sapiens 12Glu Ile Ser Arg Lys Val Tyr Lys Gly Met Leu Asp Leu Leu Lys Cys 1 5 10 15Thr Val Leu Ser Leu Glu Gln Ser Tyr Ala His Ala Gly Leu Gly Gly 20 25 30Met Ala Ser Ile Phe Gly Leu Leu Glu Ile Ala Gln Thr His Tyr Tyr 35 40 45Ser Lys Glu Pro Asp Lys Arg Lys Arg Ser Pro Thr Glu Ser Val Asn 50 55 60Thr Pro Val Gly Lys Asp Pro Gly Leu Ala Gly Arg Gly Asp Pro Lys 65 70 75 80Ala Met Ala Gln Leu Arg Val Pro Gln Leu Gly Pro Arg Ala Pro Ser 85 90 95Ala Thr Gly Lys Gly Pro Lys Glu Leu Asp Thr Arg Ser Leu Lys Glu 100 105 110Glu Asn Phe Ile Ala Ser Ile Gly Pro Glu Val Ile Lys Pro Val Phe 115 120 125Asp Leu Gly Glu Thr Glu Glu Lys Lys Ser Gln Ile Ser Ala Asp Ser 130 135 140Gly Val Ser Leu Thr Ser Ser Ser Gln Arg Thr Asp Gln Asp Ser Val145 150 155 160Ile Gly Val Ser Pro Ala Val Met Ile Arg Ser Ser Ser Gln Asp Ser 165 170 175Glu Val Ser Thr Val Val Ser Asn Ser Ser Gly Glu Thr Leu Gly Ala 180 185 190Asp Ser Asp Leu Ser Ser Asn Ala Gly Asp Gly Pro Gly Gly Glu Gly 195 200 205Ser Val His Leu Ala Ser Ser Arg Gly Thr Leu Ser Asp Ser Glu Ile 210 215 220Glu Thr Asn Ser Ala Thr Ser Thr Ile Phe Gly Lys Ala His Ser Leu225 230 235 240Lys Pro Ser Ile Lys Glu Lys Leu Ala Gly Ser Pro Ile Arg Thr Ser 245 250 255Glu Asp Val Ser Gln Arg Val Tyr Leu Tyr Glu Gly Leu Leu Gly Lys 260 265 270Glu Arg Ser Thr Leu Trp Asp Gln Met Gln Phe Trp Glu Asp Ala Phe 275 280 285Leu Asp Ala Val Met Leu Glu Arg Glu Gly Met Gly Met Asp Gln Gly 290 295 300Pro Gln Glu Met Ile Asp Arg Tyr Leu Ser Leu Gly Glu His Asp Arg305 310 315 320Lys Arg Leu Glu Asp Asp Glu Asp Arg Leu Leu Ala Thr Leu Leu His 325 330 335Asn Leu Ile Ser Tyr Met Leu Leu Met Lys Val Asn Lys Asn Asp Ile 340 345 350Arg Lys Lys Val Arg Arg Leu Met Gly Lys Ser His Ile Gly Leu Val 355 360 365Tyr Ser Gln Gln Ile Asn Glu Val Leu Asp Gln Leu Ala Asn Leu Asn 370 375 380Gly Arg Asp Leu Ser Ile Trp Ser Ser Gly Ser Arg His Met Lys Lys385 390 395 400Gln Thr Phe Val Val His Ala Gly Thr Asp Thr Asn Gly Asp Ile Phe 405 410 415Phe Met Glu Val Cys Asp Asp Cys Val Val Leu Arg Ser Asn Ile Gly 420 425 430Thr Val Tyr Glu Arg Trp Trp Tyr Glu Lys Leu Ile Asn Met Thr Tyr 435 440 445Cys Pro Lys Thr Lys Val Leu Cys Leu Trp Arg Arg Asn Gly Ser Glu 450 455 460Thr Gln Leu Asn Lys Phe Tyr Thr Lys Lys Cys Arg Glu Leu Tyr Tyr465 470 475 480Cys Val Lys Asp Ser Met Glu Arg Ala Ala Ala Arg Gln Gln Ser Ile 485 490 495Lys Pro Gly Pro Glu Leu Gly Gly Glu Phe Pro Val Gln Asp Leu Lys 500 505 510Thr Gly Glu Gly Gly Leu Leu Gln Val Thr Leu Glu Gly Ile Asn Leu 515 520 525Lys Phe Met His Asn Gln Val Phe Ile Glu Leu Asn His Ile Lys Lys 530 535 540Cys Asn Thr Val Arg Gly Val Phe Val Leu Glu Glu Phe Val Pro Glu545 550 555 560Ile Lys Glu Val Val Ser His Lys Tyr Lys Thr Pro Met Ala His Glu 565 570 575Ile Cys Tyr Ser Val Leu Cys Leu Phe Ser Tyr Val Ala Ala Val His 580 585 590Ser Ser Glu Glu Asp Leu Arg Thr Pro Pro Arg Pro Val Ser Ser 595 600 605133225DNAHomo sapiensCDS(3)..(2846) 13cc cag act cgc ccc gcc cca gag act gcg cct gcg cgg gca cga gac 47Gln Thr Arg Pro Ala Pro Glu Thr Ala Pro Ala Arg Ala Arg Asp 1 5 10 15acc ctc tcc gcg atg act gcc agc tca gtg gag cag ctg cgg aag gag 95Thr Leu Ser Ala Met Thr Ala Ser Ser Val Glu Gln Leu Arg Lys Glu 20 25 30ggc aat gag ctg ttc aaa tgt gga gac tac ggg ggc gcc ctg gcg gcc 143Gly Asn Glu Leu Phe Lys Cys Gly Asp Tyr Gly Gly Ala Leu Ala Ala 35 40 45tac act cag gcc ctg ggt ctg gac gcg acg ccc cag gac cag gcc gtt 191Tyr Thr Gln Ala Leu Gly Leu Asp Ala Thr Pro Gln Asp Gln Ala Val 50 55 60ctg cac cgg aac cgg gcc gcc tgc cac ctc aag ctg gaa gat tac gac 239Leu His Arg Asn Arg Ala Ala Cys His Leu Lys Leu Glu Asp Tyr Asp 65 70 75aaa gca gaa aca gag gca tcc aaa gcc att gaa aag gat ggt ggg gat 287Lys Ala Glu Thr Glu Ala Ser Lys Ala Ile Glu Lys Asp Gly Gly Asp80 85 90 95gtc aaa gca ctc tac cgg cgg agc caa gcc cta gag aag ctg ggc cgc 335Val Lys Ala Leu Tyr Arg Arg Ser Gln Ala Leu Glu Lys Leu Gly Arg 100 105 110ctg gac cag gct gtc ctt gac ctg cag aga tgt gtg agc ttg gag ccc 383Leu Asp Gln Ala Val Leu Asp Leu Gln Arg Cys Val Ser Leu Glu Pro 115 120 125aag aac aaa gtt ttc cag gag gcc ttg cgg aac atc ggg ggc cag att 431Lys Asn Lys Val Phe Gln Glu Ala Leu Arg Asn Ile Gly Gly Gln Ile 130 135 140cag gag aag gtg cga tac atg tcc tcg acg gat gcc aaa gtg gaa cag 479Gln Glu Lys Val Arg Tyr Met Ser Ser Thr Asp Ala Lys Val Glu Gln 145 150 155atg ttt cag ata ctg ttg gac cca gaa gag aag ggc act gag aaa aag 527Met Phe Gln Ile Leu Leu Asp Pro Glu Glu Lys Gly Thr Glu Lys Lys160 165 170 175caa aag gct tct cag aac ctg gtg gtg ctg gcc agg gag gat gct gga 575Gln Lys Ala Ser Gln Asn Leu Val Val Leu Ala Arg Glu Asp Ala Gly 180 185 190gcg gag aag atc ttc cgg agt aat ggg gtt cag ctc ttg caa cgt tta 623Ala Glu Lys Ile Phe Arg Ser Asn Gly Val Gln Leu Leu Gln Arg Leu 195 200 205ctg gac atg gga gag act gac ctc atg ctg gcg gct ctg cgt acg ctg 671Leu Asp Met Gly Glu Thr Asp Leu Met Leu Ala Ala Leu Arg Thr Leu 210 215 220gtt ggc att tgc tct gag cat cag tca cgg aca gtg gca acc ctg agc 719Val Gly Ile Cys Ser Glu His Gln Ser Arg Thr Val Ala Thr Leu Ser 225 230 235ata ctg gga act cgg cga gta gtc tcc atc ctg ggc gtg gaa agc cag 767Ile Leu Gly Thr Arg Arg Val Val Ser Ile Leu Gly Val Glu Ser Gln240 245 250 255gct gtg tcc ctg gct gcc tgc cac ctg ctg cag gtt atg ttt gat gcc 815Ala Val Ser Leu Ala Ala Cys His Leu Leu Gln Val Met Phe Asp Ala 260 265 270ctc aag gaa ggt gtc aaa aaa ggc ttc cga ggc aaa gaa ggt gcc atc 863Leu Lys Glu Gly Val Lys Lys Gly Phe Arg Gly Lys Glu Gly Ala Ile 275 280 285att gtg gat cct gcc cgg gag ctg aag gtc ctc atc agt aac ctc tta 911Ile Val Asp Pro Ala Arg Glu Leu Lys Val Leu Ile Ser Asn Leu Leu 290 295 300gat ctg ctg aca gag gtg ggg gtc tct ggc caa ggc cga gac aat gcc 959Asp Leu Leu Thr Glu Val Gly Val Ser Gly Gln Gly Arg Asp Asn Ala 305 310 315ctg acc ctc ctg att aaa gcg gtg ccc cgg aag tct ctc aag gac ccc 1007Leu Thr Leu Leu Ile Lys Ala Val Pro Arg Lys Ser Leu Lys Asp Pro320 325 330 335aac aac agc ctc acc ctc tgg gtc atc gac caa ggt ctg aaa aag att 1055Asn Asn Ser Leu Thr Leu Trp Val Ile Asp Gln Gly Leu Lys Lys Ile 340 345 350ttg gaa gtg ggg ggc tct cta cag gac cct cct ggg gag ctc gca gtg 1103Leu Glu Val Gly Gly Ser Leu Gln Asp Pro Pro Gly Glu Leu Ala Val 355 360 365acc gca aac agc cgc atg agc gcc tct att ctc ctc agc aag ctc ttt 1151Thr Ala Asn Ser Arg Met Ser Ala Ser Ile Leu Leu Ser Lys Leu Phe 370 375 380gat gac ctc aag tgt gat gcg gag agg gag aat ttc cac aga ctt tgt 1199Asp Asp Leu Lys Cys Asp Ala Glu Arg Glu Asn Phe His Arg Leu Cys 385 390 395gaa aac tac atc aag agc tgg ttt gag ggc caa ggg ctg gcc ggg aag 1247Glu Asn Tyr Ile Lys Ser Trp Phe Glu Gly Gln Gly Leu Ala Gly Lys400 405 410 415cta cgg gcc atc cag acg gtg tcc tgc ctc ctg cag ggc cca tgt gac 1295Leu Arg Ala Ile Gln Thr Val Ser Cys Leu Leu Gln Gly Pro Cys Asp 420 425 430gct ggc aac cgg gcc ttg gag ctg agc ggt gtc atg gag agt gtg att 1343Ala Gly Asn Arg Ala Leu Glu Leu Ser Gly Val Met Glu Ser Val Ile 435 440 445gct ctg tgt gcc tct gag cag gag gag gag cag ctg gtg gcc gtg gag 1391Ala Leu Cys Ala Ser Glu Gln Glu Glu Glu Gln Leu Val Ala Val Glu 450 455 460gct ctg atc cat gca gcc ggc aag gct aag cgg gcc tca ttc atc act 1439Ala Leu Ile His Ala Ala Gly Lys Ala Lys Arg Ala Ser Phe Ile Thr 465 470 475gcc aat ggt gtc tcg ctg ctg aag gac cta tat aag tgc agc gag aag 1487Ala Asn Gly Val Ser Leu Leu Lys Asp Leu Tyr Lys Cys Ser Glu Lys480 485 490 495gac agc atc cgc atc cgg gcg cta gtg gga ctc tgt aag ctc ggt tcg 1535Asp Ser Ile Arg Ile Arg Ala Leu Val Gly Leu Cys Lys Leu Gly Ser 500 505 510gct gga ggg act gac ttc agc atg aag cag ttt gct gaa ggc tcc act 1583Ala Gly Gly Thr Asp Phe Ser Met Lys Gln Phe Ala Glu Gly Ser Thr 515 520 525ctc aaa ctg gct aag cag tgt cga aag tgg ctg tgc aat gac cag atc 1631Leu Lys Leu Ala Lys Gln Cys Arg Lys Trp Leu Cys Asn Asp Gln Ile 530 535 540gac gca ggc act cgg cgc tgg gca gtg gag ggc ctg gct tac ctg acc 1679Asp Ala Gly Thr Arg Arg Trp Ala Val Glu Gly Leu Ala Tyr Leu Thr 545 550 555ttt gat gcc gac gtg aag gaa gag ttt gtg gag gat gcg gct gct ctg 1727Phe Asp Ala Asp Val Lys Glu Glu Phe Val Glu Asp Ala Ala Ala Leu560 565 570 575aaa gct ctg ttc cag ctc agc agg ttg gag gag agg tca gtg ctc ttt 1775Lys Ala Leu Phe Gln Leu Ser Arg Leu Glu Glu Arg Ser Val Leu Phe 580 585 590gcg gtg gcc tca gcg ctg gtg aac tgc acc aac agc tat gac tac gag 1823Ala Val Ala Ser Ala Leu Val Asn Cys Thr Asn Ser Tyr Asp Tyr Glu 595 600 605gag ccc gac ccc aag atg gtg gag ctg gcc aag tat gcc aag cag cat 1871Glu Pro Asp Pro Lys Met Val Glu Leu Ala Lys Tyr Ala Lys Gln His 610 615 620gtg ccc gag cag cac ccc aag gac aag cca agc ttc gtg cgg gct cgg 1919Val Pro Glu Gln His Pro Lys Asp Lys Pro Ser Phe Val Arg Ala Arg 625 630 635gtg aag aag ctg ctg gca gcg ggt gtg gtg tcg gcc atg gtg tgc atg 1967Val Lys Lys Leu Leu Ala Ala Gly Val Val Ser Ala Met Val Cys Met640 645 650 655gtg aag acg gag agc cct gtg ctg acc agt tcc tgc aga gag ctg ctc 2015Val Lys Thr Glu Ser Pro Val Leu Thr Ser Ser Cys Arg Glu Leu Leu 660 665 670tcc agg gtc ttc ttg gct tta gtg gaa gag gta gag gac cga ggc act 2063Ser Arg Val Phe Leu Ala Leu Val Glu Glu Val Glu Asp Arg Gly Thr 675 680 685gtg gtt gcc cag gga ggc ggc agg gcg ctg atc ccg ctg gcc ctg gaa 2111Val Val Ala Gln Gly Gly Gly Arg Ala Leu Ile Pro Leu Ala Leu Glu 690 695 700ggc acg gac gtg ggg cag aca aag gca gcc cag gcc ctt gcc aag ctc 2159Gly Thr Asp Val Gly Gln Thr Lys Ala Ala Gln Ala Leu Ala Lys Leu 705 710 715acc atc acc tcc aac ccg gag atg acc ttc cct ggc gag cgg atc tat 2207Thr Ile Thr Ser Asn Pro Glu Met Thr Phe Pro Gly Glu Arg Ile Tyr720 725 730 735gag gtg gtc cgg ccc ctc gtc tcc ctg ttg cac ctc aac tgc tca ggc 2255Glu Val Val Arg Pro Leu Val Ser Leu Leu His Leu Asn Cys Ser Gly 740 745 750ctg cag aac ttc gag gcg ctc atg gcc cta aca aac ctg gct ggg atc 2303Leu Gln Asn Phe Glu Ala Leu Met Ala Leu Thr Asn Leu Ala Gly Ile 755 760 765agc gag agg ctc cgg cag aag atc ctg aag gag aag gct gtg ccc atg 2351Ser Glu Arg Leu Arg Gln Lys Ile Leu Lys Glu Lys Ala Val Pro Met 770 775 780ata gaa ggc tac atg ttt gag gag cat gag atg atc cgc cgg gca gcc 2399Ile Glu Gly Tyr Met Phe Glu Glu His Glu Met Ile Arg Arg Ala Ala 785 790 795acg gag tgc atg tgt aac ttg gcc atg agc aag gag gtg cag gac ctc 2447Thr Glu Cys Met Cys Asn Leu Ala Met Ser Lys Glu Val Gln Asp Leu800 805 810 815ttc gaa gcc cag ggc aat gac cga ctg aag ctg ctg gtg ctg tac agt 2495Phe Glu Ala Gln Gly Asn Asp Arg Leu Lys Leu Leu Val Leu Tyr Ser 820 825 830gga gag gat gat gag ctg cta cag cgg gca gct gcc ggg ggc ttg gcc 2543Gly Glu Asp Asp Glu Leu Leu Gln Arg Ala Ala Ala Gly Gly Leu Ala 835 840 845atg ctt acc tcc atg cgg ccc acg ctc tgc agc cgc att ccc caa gtg 2591Met Leu Thr Ser Met Arg Pro Thr Leu Cys Ser Arg Ile Pro Gln Val 850 855 860acc aca cac tgg ctg gag atc ctg cag gcc ctg ctt ctg agc tcc aac 2639Thr Thr His Trp Leu Glu Ile Leu Gln Ala Leu Leu Leu Ser Ser Asn 865 870 875cag gag ctg cag cac cgg ggt gct gtg gtg gtg ctg aac atg gtg gag 2687Gln Glu Leu Gln His Arg Gly Ala Val Val Val Leu Asn Met Val Glu880 885 890 895gcc tcg agg gag att gcc agc acc ctg atg gag agt gag atg atg gag 2735Ala Ser Arg Glu Ile Ala Ser Thr Leu Met Glu Ser Glu Met Met Glu 900 905 910atc ttg tca gtg cta gct aag ggt gac cac agc cct gtc aca agg gct 2783Ile Leu Ser Val Leu Ala Lys Gly Asp His Ser Pro Val Thr Arg Ala 915 920 925gct gca gcc tgc ctg gac aaa gca gtg gaa tat ggg ctt atc caa ccc 2831Ala Ala Ala Cys Leu Asp Lys Ala Val Glu Tyr Gly Leu Ile Gln Pro 930 935 940aac caa gat gga gag tgagggggtt gtccctgggc ccaaggctca tgcacacgct 2886Asn Gln Asp Gly Glu 945acctattgtg gcacggagag taaggacgga agcagctttg gctggtggtg gctggcatgc 2946ccaatactct tgcccatcct cgcttgctgc cctaggatgt cctctgttct gagtcagcgg 3006ccacgttcag tcacacagcc ctgcttggcc agcactgcct gcagcctcac tcagaggggc 3066cctttttctg tactactgta gtcagctggg aatggggaag gtgcatccca acacagcctg 3126tggatcctgg ggcatttgga agggcgcaca catcagcagc ctcaccagct gtgagcctgc 3186tatcaggcct gcccctccaa taaaagtgtg tagaactcc 322514948PRTHomo sapiens 14Gln Thr Arg Pro Ala Pro Glu Thr Ala Pro Ala Arg Ala Arg Asp Thr 1 5 10 15Leu Ser Ala Met Thr Ala Ser Ser Val Glu Gln Leu Arg Lys Glu Gly 20 25 30Asn Glu Leu Phe Lys Cys Gly Asp Tyr Gly Gly Ala Leu Ala Ala Tyr 35 40 45Thr Gln Ala Leu Gly Leu Asp Ala Thr Pro Gln Asp Gln Ala Val Leu 50 55 60His Arg Asn Arg Ala Ala Cys His Leu Lys Leu Glu Asp Tyr Asp Lys 65 70 75 80Ala Glu Thr Glu Ala Ser Lys Ala Ile Glu Lys Asp Gly Gly Asp Val 85 90 95Lys Ala Leu Tyr Arg Arg Ser Gln Ala Leu Glu Lys Leu Gly Arg Leu 100 105 110Asp Gln Ala Val Leu Asp Leu Gln Arg Cys Val Ser Leu Glu Pro Lys 115 120 125Asn Lys Val Phe Gln Glu Ala Leu Arg Asn Ile Gly Gly Gln Ile Gln 130 135 140Glu Lys Val Arg Tyr Met Ser Ser Thr Asp Ala Lys Val Glu Gln Met145 150 155 160Phe Gln Ile Leu Leu Asp Pro Glu Glu Lys Gly Thr Glu Lys Lys Gln 165 170 175Lys Ala Ser Gln Asn Leu Val Val Leu Ala Arg Glu Asp Ala Gly Ala 180 185

190Glu Lys Ile Phe Arg Ser Asn Gly Val Gln Leu Leu Gln Arg Leu Leu 195 200 205Asp Met Gly Glu Thr Asp Leu Met Leu Ala Ala Leu Arg Thr Leu Val 210 215 220Gly Ile Cys Ser Glu His Gln Ser Arg Thr Val Ala Thr Leu Ser Ile225 230 235 240Leu Gly Thr Arg Arg Val Val Ser Ile Leu Gly Val Glu Ser Gln Ala 245 250 255Val Ser Leu Ala Ala Cys His Leu Leu Gln Val Met Phe Asp Ala Leu 260 265 270Lys Glu Gly Val Lys Lys Gly Phe Arg Gly Lys Glu Gly Ala Ile Ile 275 280 285Val Asp Pro Ala Arg Glu Leu Lys Val Leu Ile Ser Asn Leu Leu Asp 290 295 300Leu Leu Thr Glu Val Gly Val Ser Gly Gln Gly Arg Asp Asn Ala Leu305 310 315 320Thr Leu Leu Ile Lys Ala Val Pro Arg Lys Ser Leu Lys Asp Pro Asn 325 330 335Asn Ser Leu Thr Leu Trp Val Ile Asp Gln Gly Leu Lys Lys Ile Leu 340 345 350Glu Val Gly Gly Ser Leu Gln Asp Pro Pro Gly Glu Leu Ala Val Thr 355 360 365Ala Asn Ser Arg Met Ser Ala Ser Ile Leu Leu Ser Lys Leu Phe Asp 370 375 380Asp Leu Lys Cys Asp Ala Glu Arg Glu Asn Phe His Arg Leu Cys Glu385 390 395 400Asn Tyr Ile Lys Ser Trp Phe Glu Gly Gln Gly Leu Ala Gly Lys Leu 405 410 415Arg Ala Ile Gln Thr Val Ser Cys Leu Leu Gln Gly Pro Cys Asp Ala 420 425 430Gly Asn Arg Ala Leu Glu Leu Ser Gly Val Met Glu Ser Val Ile Ala 435 440 445Leu Cys Ala Ser Glu Gln Glu Glu Glu Gln Leu Val Ala Val Glu Ala 450 455 460Leu Ile His Ala Ala Gly Lys Ala Lys Arg Ala Ser Phe Ile Thr Ala465 470 475 480Asn Gly Val Ser Leu Leu Lys Asp Leu Tyr Lys Cys Ser Glu Lys Asp 485 490 495Ser Ile Arg Ile Arg Ala Leu Val Gly Leu Cys Lys Leu Gly Ser Ala 500 505 510Gly Gly Thr Asp Phe Ser Met Lys Gln Phe Ala Glu Gly Ser Thr Leu 515 520 525Lys Leu Ala Lys Gln Cys Arg Lys Trp Leu Cys Asn Asp Gln Ile Asp 530 535 540Ala Gly Thr Arg Arg Trp Ala Val Glu Gly Leu Ala Tyr Leu Thr Phe545 550 555 560Asp Ala Asp Val Lys Glu Glu Phe Val Glu Asp Ala Ala Ala Leu Lys 565 570 575Ala Leu Phe Gln Leu Ser Arg Leu Glu Glu Arg Ser Val Leu Phe Ala 580 585 590Val Ala Ser Ala Leu Val Asn Cys Thr Asn Ser Tyr Asp Tyr Glu Glu 595 600 605Pro Asp Pro Lys Met Val Glu Leu Ala Lys Tyr Ala Lys Gln His Val 610 615 620Pro Glu Gln His Pro Lys Asp Lys Pro Ser Phe Val Arg Ala Arg Val625 630 635 640Lys Lys Leu Leu Ala Ala Gly Val Val Ser Ala Met Val Cys Met Val 645 650 655Lys Thr Glu Ser Pro Val Leu Thr Ser Ser Cys Arg Glu Leu Leu Ser 660 665 670Arg Val Phe Leu Ala Leu Val Glu Glu Val Glu Asp Arg Gly Thr Val 675 680 685Val Ala Gln Gly Gly Gly Arg Ala Leu Ile Pro Leu Ala Leu Glu Gly 690 695 700Thr Asp Val Gly Gln Thr Lys Ala Ala Gln Ala Leu Ala Lys Leu Thr705 710 715 720Ile Thr Ser Asn Pro Glu Met Thr Phe Pro Gly Glu Arg Ile Tyr Glu 725 730 735Val Val Arg Pro Leu Val Ser Leu Leu His Leu Asn Cys Ser Gly Leu 740 745 750Gln Asn Phe Glu Ala Leu Met Ala Leu Thr Asn Leu Ala Gly Ile Ser 755 760 765Glu Arg Leu Arg Gln Lys Ile Leu Lys Glu Lys Ala Val Pro Met Ile 770 775 780Glu Gly Tyr Met Phe Glu Glu His Glu Met Ile Arg Arg Ala Ala Thr785 790 795 800Glu Cys Met Cys Asn Leu Ala Met Ser Lys Glu Val Gln Asp Leu Phe 805 810 815Glu Ala Gln Gly Asn Asp Arg Leu Lys Leu Leu Val Leu Tyr Ser Gly 820 825 830Glu Asp Asp Glu Leu Leu Gln Arg Ala Ala Ala Gly Gly Leu Ala Met 835 840 845Leu Thr Ser Met Arg Pro Thr Leu Cys Ser Arg Ile Pro Gln Val Thr 850 855 860Thr His Trp Leu Glu Ile Leu Gln Ala Leu Leu Leu Ser Ser Asn Gln865 870 875 880Glu Leu Gln His Arg Gly Ala Val Val Val Leu Asn Met Val Glu Ala 885 890 895Ser Arg Glu Ile Ala Ser Thr Leu Met Glu Ser Glu Met Met Glu Ile 900 905 910Leu Ser Val Leu Ala Lys Gly Asp His Ser Pro Val Thr Arg Ala Ala 915 920 925Ala Ala Cys Leu Asp Lys Ala Val Glu Tyr Gly Leu Ile Gln Pro Asn 930 935 940Gln Asp Gly Glu945156002DNAHomo sapiensCDS(326)..(5089) 15cacgtgcatg tgtagcatgc cttggttttt cctttggcat ctgaaaaagg cacaacctga 60aagacctaga acccagtgtc ggtccccagg ccctttggga caggaagaga agagccgtgt 120ggccgcgggg aggatgtcct gcggcggggc tgtcctcgcg gactgactgg actccatctc 180ccagcgggcg ccgcggcgcg gccacgcccc cccactcccc gcgcgcgccc ggtggagact 240tcgattttca gaattcctcc tgggaatgct gactccttgc ttggtgccct gatgcttctc 300tgagataaac tgatgaattg gaacc atg gtg caa aag aag aag ttc tgt cct 352 Met Val Gln Lys Lys Lys Phe Cys Pro 1 5cgg tta ctt gac tat cta gtg atc gta ggg gcc agg cac ccg agc agt 400Arg Leu Leu Asp Tyr Leu Val Ile Val Gly Ala Arg His Pro Ser Ser 10 15 20 25gat agc gtg gcc cag act cct gaa ttg cta cgg cga tac ccc ttg gag 448Asp Ser Val Ala Gln Thr Pro Glu Leu Leu Arg Arg Tyr Pro Leu Glu 30 35 40gat cac act gag ttt ccc ctg ccc cca gat gta gtg ttc ttc tgc cag 496Asp His Thr Glu Phe Pro Leu Pro Pro Asp Val Val Phe Phe Cys Gln 45 50 55ccc gag ggc tgc ctg agc gtg cgg cag cgg cgc atg agc ctt cgg gat 544Pro Glu Gly Cys Leu Ser Val Arg Gln Arg Arg Met Ser Leu Arg Asp 60 65 70gat acc tct ttt gtc ttc acc ctc act gac aag gac act gga gtc acg 592Asp Thr Ser Phe Val Phe Thr Leu Thr Asp Lys Asp Thr Gly Val Thr 75 80 85cga tat ggc atc tgt gtt aac ttc tac cgc tcc ttc caa aag cga atc 640Arg Tyr Gly Ile Cys Val Asn Phe Tyr Arg Ser Phe Gln Lys Arg Ile 90 95 100 105tct aag gag aag ggg gaa ggt ggg gca ggg tcc cgt ggg aag gaa gga 688Ser Lys Glu Lys Gly Glu Gly Gly Ala Gly Ser Arg Gly Lys Glu Gly 110 115 120acc cat gcc acc tgt gcc tca gaa gag ggt ggc act gag agc tca gag 736Thr His Ala Thr Cys Ala Ser Glu Glu Gly Gly Thr Glu Ser Ser Glu 125 130 135agt ggc tca tcc ctg cag cct ctc agt gct gac tct acc cct gat gtg 784Ser Gly Ser Ser Leu Gln Pro Leu Ser Ala Asp Ser Thr Pro Asp Val 140 145 150aac cag tct cct cgg ggc aaa cgc cgg gcc aag gcg ggg agc cgc tcc 832Asn Gln Ser Pro Arg Gly Lys Arg Arg Ala Lys Ala Gly Ser Arg Ser 155 160 165cgc aac agt act ctc acg tcc ctg tgc gtg ctc agc cac tac cct ttc 880Arg Asn Ser Thr Leu Thr Ser Leu Cys Val Leu Ser His Tyr Pro Phe170 175 180 185ttc tcc acc ttc cga gag tgt ttg tat act ctc aag cgc ctg gtg gac 928Phe Ser Thr Phe Arg Glu Cys Leu Tyr Thr Leu Lys Arg Leu Val Asp 190 195 200tgc tgt agt gag cgc ctt ctg ggc aag aaa ctg ggc atc cct cga ggc 976Cys Cys Ser Glu Arg Leu Leu Gly Lys Lys Leu Gly Ile Pro Arg Gly 205 210 215gta caa agg gac acc atg tgg cgg atc ttt act gga tcg ctg ctg gta 1024Val Gln Arg Asp Thr Met Trp Arg Ile Phe Thr Gly Ser Leu Leu Val 220 225 230gag gag aag tca agt gcc ctt ctg cat gac ctt cga gag att gag gcc 1072Glu Glu Lys Ser Ser Ala Leu Leu His Asp Leu Arg Glu Ile Glu Ala 235 240 245tgg atc tat cga ttg ctg cgc tcc cca gta ccc gtc tct ggg cag aag 1120Trp Ile Tyr Arg Leu Leu Arg Ser Pro Val Pro Val Ser Gly Gln Lys250 255 260 265cga gta gac atc gag gtc cta ccc caa gag ctc cag cca gct ctg acc 1168Arg Val Asp Ile Glu Val Leu Pro Gln Glu Leu Gln Pro Ala Leu Thr 270 275 280ttt gct ctt cca gac cca tct cga ttc acc cta gtg gat ttc cca ctg 1216Phe Ala Leu Pro Asp Pro Ser Arg Phe Thr Leu Val Asp Phe Pro Leu 285 290 295cac ctt ccc ttg gaa ctt cta ggt gtg gac gcc tgt ctc cag gtg cta 1264His Leu Pro Leu Glu Leu Leu Gly Val Asp Ala Cys Leu Gln Val Leu 300 305 310acc tgc att ctg tta gag cac aag gtg gtg cta cag tcc cga gac tac 1312Thr Cys Ile Leu Leu Glu His Lys Val Val Leu Gln Ser Arg Asp Tyr 315 320 325aat gca ctc tcc atg tct gtg atg gca ttc gtg gca atg atc tac cca 1360Asn Ala Leu Ser Met Ser Val Met Ala Phe Val Ala Met Ile Tyr Pro330 335 340 345ctg gaa tat atg ttt cct gtc atc ccg ctg cta ccc acc tgc atg gca 1408Leu Glu Tyr Met Phe Pro Val Ile Pro Leu Leu Pro Thr Cys Met Ala 350 355 360tca gca gag cag ctg ctg ttg gct cca acc ccg tac atc att ggg gtt 1456Ser Ala Glu Gln Leu Leu Leu Ala Pro Thr Pro Tyr Ile Ile Gly Val 365 370 375cct gcc agc ttc ttc ctc tac aaa ctg gac ttc aaa atg cct gat gat 1504Pro Ala Ser Phe Phe Leu Tyr Lys Leu Asp Phe Lys Met Pro Asp Asp 380 385 390gta tgg cta gtg gat ctg gac agc aat agg gtg att gcc ccc acc aat 1552Val Trp Leu Val Asp Leu Asp Ser Asn Arg Val Ile Ala Pro Thr Asn 395 400 405gca gaa gtg ctg cct atc ctg cca gaa cca gaa tca cta gag ctg aaa 1600Ala Glu Val Leu Pro Ile Leu Pro Glu Pro Glu Ser Leu Glu Leu Lys410 415 420 425aag cat tta aag cag gcc ttg gcc agc atg agt ctc aac acc cag ccc 1648Lys His Leu Lys Gln Ala Leu Ala Ser Met Ser Leu Asn Thr Gln Pro 430 435 440atc ctc aat ctg gag aaa ttt cat gag ggc cag gag atc ccc ctt ctc 1696Ile Leu Asn Leu Glu Lys Phe His Glu Gly Gln Glu Ile Pro Leu Leu 445 450 455ttg gga agg cct tct aat gac ctg cag tcc aca ccg tcc act gaa ttc 1744Leu Gly Arg Pro Ser Asn Asp Leu Gln Ser Thr Pro Ser Thr Glu Phe 460 465 470aac cca ctc atc tat ggc aat gat gtg gat tct gtg gat gtt gca acc 1792Asn Pro Leu Ile Tyr Gly Asn Asp Val Asp Ser Val Asp Val Ala Thr 475 480 485agg gtt gcc atg gta cgg ttc ttc aat tcc gcc aac gtg ctg cag gga 1840Arg Val Ala Met Val Arg Phe Phe Asn Ser Ala Asn Val Leu Gln Gly490 495 500 505ttt cag atg cac acg cgt acc ctg cgc ctc ttt cct cgg cct gtg gta 1888Phe Gln Met His Thr Arg Thr Leu Arg Leu Phe Pro Arg Pro Val Val 510 515 520gct ttt caa gct ggc tcc ttt cta gcc tca cgt ccc cgg cag act cct 1936Ala Phe Gln Ala Gly Ser Phe Leu Ala Ser Arg Pro Arg Gln Thr Pro 525 530 535ttt gcc gag aaa ttg gcc agg act cag gct gtg gag tac ttt ggg gaa 1984Phe Ala Glu Lys Leu Ala Arg Thr Gln Ala Val Glu Tyr Phe Gly Glu 540 545 550tgg atc ctt aac ccc acc aac tat gcc ttt cag cga att cac aac aat 2032Trp Ile Leu Asn Pro Thr Asn Tyr Ala Phe Gln Arg Ile His Asn Asn 555 560 565atg ttt gat cca gcc ctg att ggt gac aag cca aag tgg tat gct cat 2080Met Phe Asp Pro Ala Leu Ile Gly Asp Lys Pro Lys Trp Tyr Ala His570 575 580 585cag ctg cag cct atc cac tat cgc gtc tat gac agc aat tcc cag ctg 2128Gln Leu Gln Pro Ile His Tyr Arg Val Tyr Asp Ser Asn Ser Gln Leu 590 595 600gct gag gcc ctg agt gta cca cca gag cgg gac tct gac tcc gaa cct 2176Ala Glu Ala Leu Ser Val Pro Pro Glu Arg Asp Ser Asp Ser Glu Pro 605 610 615act gat gat agt ggc agt gat agt atg gat tat gac gat tca agc tct 2224Thr Asp Asp Ser Gly Ser Asp Ser Met Asp Tyr Asp Asp Ser Ser Ser 620 625 630tct tac tcc tcc ctt ggt gac ttt gtc agt gaa atg atg aaa tgt gac 2272Ser Tyr Ser Ser Leu Gly Asp Phe Val Ser Glu Met Met Lys Cys Asp 635 640 645att aat ggt gat act ccc aat gtg gac cct ctg aca cat gca gca ctg 2320Ile Asn Gly Asp Thr Pro Asn Val Asp Pro Leu Thr His Ala Ala Leu650 655 660 665ggg gat gcc agc gag gtg gag att gac gag ctg cag aat cag aag gaa 2368Gly Asp Ala Ser Glu Val Glu Ile Asp Glu Leu Gln Asn Gln Lys Glu 670 675 680gca gaa gag cct ggc cca gac agt gag aac tct cag gaa aac ccc cca 2416Ala Glu Glu Pro Gly Pro Asp Ser Glu Asn Ser Gln Glu Asn Pro Pro 685 690 695ctg cgc tcc agc tct agc acc aca gcc agc agc agc ccc agc act gtc 2464Leu Arg Ser Ser Ser Ser Thr Thr Ala Ser Ser Ser Pro Ser Thr Val 700 705 710atc cac gga gcc aac tct gaa cct gct gac tct acg gag atg gat gat 2512Ile His Gly Ala Asn Ser Glu Pro Ala Asp Ser Thr Glu Met Asp Asp 715 720 725aag gca gca gta ggc gtc tcc aag ccc ctc cct tcc gtg cct ccc agc 2560Lys Ala Ala Val Gly Val Ser Lys Pro Leu Pro Ser Val Pro Pro Ser730 735 740 745att ggc aaa tcg aac atg gac aga cgt cag gca gaa att gga gag ggg 2608Ile Gly Lys Ser Asn Met Asp Arg Arg Gln Ala Glu Ile Gly Glu Gly 750 755 760tca gtg cgc cgg cga atc tat gac aat cca tac ttc gag ccc caa tat 2656Ser Val Arg Arg Arg Ile Tyr Asp Asn Pro Tyr Phe Glu Pro Gln Tyr 765 770 775ggc ttt ccc cct gag gaa gat gag gat gag cag ggg gaa agt tac act 2704Gly Phe Pro Pro Glu Glu Asp Glu Asp Glu Gln Gly Glu Ser Tyr Thr 780 785 790ccc cga ttc agc caa cat gtc agt ggc aat cgg gct caa aag ctg ctg 2752Pro Arg Phe Ser Gln His Val Ser Gly Asn Arg Ala Gln Lys Leu Leu 795 800 805cgg ccc aac agc ttg aga ctg gca agt gac tca gat gca gag tca gac 2800Arg Pro Asn Ser Leu Arg Leu Ala Ser Asp Ser Asp Ala Glu Ser Asp810 815 820 825tct cgg gca agc tct ccc aac tcc acc gtc tcc aac acc agc acc gag 2848Ser Arg Ala Ser Ser Pro Asn Ser Thr Val Ser Asn Thr Ser Thr Glu 830 835 840ggc ttc ggg ggc atc atg tct ttt gcc agc agc ctc tat cgg aac cac 2896Gly Phe Gly Gly Ile Met Ser Phe Ala Ser Ser Leu Tyr Arg Asn His 845 850 855agt acc agc ttc agt ctt tca aac ctc aca ctg ccc acc aaa ggt gcc 2944Ser Thr Ser Phe Ser Leu Ser Asn Leu Thr Leu Pro Thr Lys Gly Ala 860 865 870cga gag aag gcc acg ccc ttc ccc agt ctg aaa gga aac agg agg gcg 2992Arg Glu Lys Ala Thr Pro Phe Pro Ser Leu Lys Gly Asn Arg Arg Ala 875 880 885tta gtg gat cag aag tca tct gtc att aaa cac agc cca aca gtg aaa 3040Leu Val Asp Gln Lys Ser Ser Val Ile Lys His Ser Pro Thr Val Lys890 895 900 905aga gaa cct cca tca ccc cag ggt cga tcc agc aat tct agt gag aac 3088Arg Glu Pro Pro Ser Pro Gln Gly Arg Ser Ser Asn Ser Ser Glu Asn 910 915 920cag cag ttc ctg aag gag gtg gtg cac agc gtg ctg gac ggc cag gga 3136Gln Gln Phe Leu Lys Glu Val Val His Ser Val Leu Asp Gly Gln Gly 925 930 935gtt ggc tgg ctc aac atg aaa aag gtg cgc cgg ctg ctg gag agc gag 3184Val Gly Trp Leu Asn Met Lys Lys Val Arg Arg Leu Leu Glu Ser Glu 940 945 950cag ctg cga gtc ttt gtc ctg agc aag ctg aac cgc atg gtg cag tca 3232Gln Leu Arg Val Phe Val Leu Ser Lys Leu Asn Arg Met Val Gln Ser 955 960 965gag gac gat gcc cgg cag gac atc atc ccg gat gtg gag atc agt cgg 3280Glu Asp Asp Ala Arg Gln Asp Ile Ile Pro Asp Val Glu Ile Ser Arg970 975 980 985aag gtg tac aag gga atg tta gac ctc ctc aag tgt aca gtc ctc agc 3328Lys Val Tyr Lys Gly Met Leu Asp Leu Leu Lys Cys Thr Val Leu Ser 990 995 1000ttg gag cag tcc tat gcc cac gcg ggt ctg ggt ggc atg gcc agc atc 3376Leu Glu Gln Ser Tyr Ala His Ala Gly Leu Gly Gly Met Ala Ser Ile 1005 1010 1015ttt ggg ctt ttg gag att gcc cag acc cac tac tat agt aaa gaa cca 3424Phe Gly Leu Leu Glu Ile Ala Gln Thr His Tyr Tyr Ser Lys Glu Pro

1020 1025 1030gac aag cgg aag aga agt cca aca gaa agt gta aat acc cca gtt ggc 3472Asp Lys Arg Lys Arg Ser Pro Thr Glu Ser Val Asn Thr Pro Val Gly 1035 1040 1045aag gat cct ggc cta gct ggg cgg ggg gac cca aag gct atg gca caa 3520Lys Asp Pro Gly Leu Ala Gly Arg Gly Asp Pro Lys Ala Met Ala Gln1050 1055 1060 1065ctg aga gtt cca caa ctg gga cct cgg gca cca agt gcc aca gga aag 3568Leu Arg Val Pro Gln Leu Gly Pro Arg Ala Pro Ser Ala Thr Gly Lys 1070 1075 1080ggt cct aag gaa ctg gac acc aga agt tta aag gaa gaa aat ttt ata 3616Gly Pro Lys Glu Leu Asp Thr Arg Ser Leu Lys Glu Glu Asn Phe Ile 1085 1090 1095gca tct att ggg cct gaa gta atc aaa cct gtc ttt gac ctt ggt gag 3664Ala Ser Ile Gly Pro Glu Val Ile Lys Pro Val Phe Asp Leu Gly Glu 1100 1105 1110aca gag gag aaa aag tcc cag atc agc gca gac agt ggt gtg agc ctg 3712Thr Glu Glu Lys Lys Ser Gln Ile Ser Ala Asp Ser Gly Val Ser Leu 1115 1120 1125acg tct agt tcc cag agg act gat caa gac tct gtc atc ggc gtg agt 3760Thr Ser Ser Ser Gln Arg Thr Asp Gln Asp Ser Val Ile Gly Val Ser1130 1135 1140 1145cca gct gtt atg atc cgc agc tca agt cag gat tct gaa gtt agc acc 3808Pro Ala Val Met Ile Arg Ser Ser Ser Gln Asp Ser Glu Val Ser Thr 1150 1155 1160gtg gtg agt aat agc tct gga gag acc ctt gga gct gac agt gac ttg 3856Val Val Ser Asn Ser Ser Gly Glu Thr Leu Gly Ala Asp Ser Asp Leu 1165 1170 1175agc agc aat gca ggt gat gga cca ggt ggc gag ggc agt gtt cac ctg 3904Ser Ser Asn Ala Gly Asp Gly Pro Gly Gly Glu Gly Ser Val His Leu 1180 1185 1190gca agc tct cgg ggc act ttg tct gat agt gaa att gag acc aac tct 3952Ala Ser Ser Arg Gly Thr Leu Ser Asp Ser Glu Ile Glu Thr Asn Ser 1195 1200 1205gcc aca agc acc atc ttt ggt aaa gcc cac agc ttg aag cca agc ata 4000Ala Thr Ser Thr Ile Phe Gly Lys Ala His Ser Leu Lys Pro Ser Ile1210 1215 1220 1225aag gag aag ctg gca ggc agc ccc att cgt act tct gaa gat gtg agc 4048Lys Glu Lys Leu Ala Gly Ser Pro Ile Arg Thr Ser Glu Asp Val Ser 1230 1235 1240cag cga gtc tat ctc tat gag gga ctc cta ggc aaa gag cgt tct act 4096Gln Arg Val Tyr Leu Tyr Glu Gly Leu Leu Gly Lys Glu Arg Ser Thr 1245 1250 1255tta tgg gac caa atg caa ttc tgg gaa gat gcc ttc tta gat gct gtg 4144Leu Trp Asp Gln Met Gln Phe Trp Glu Asp Ala Phe Leu Asp Ala Val 1260 1265 1270atg ttg gag aga gaa ggg atg ggt atg gac cag ggt ccc cag gaa atg 4192Met Leu Glu Arg Glu Gly Met Gly Met Asp Gln Gly Pro Gln Glu Met 1275 1280 1285atc gac agg tac ctg tcc ctt gga gaa cat gac cgg aag cgc ctg gaa 4240Ile Asp Arg Tyr Leu Ser Leu Gly Glu His Asp Arg Lys Arg Leu Glu1290 1295 1300 1305gat gat gaa gat cgc ttg ctg gcc aca ctt ctg cac aac ctc atc tcc 4288Asp Asp Glu Asp Arg Leu Leu Ala Thr Leu Leu His Asn Leu Ile Ser 1310 1315 1320tac atg ctg ctg atg aag gta aat aag aat gac atc cgc aag aag gtg 4336Tyr Met Leu Leu Met Lys Val Asn Lys Asn Asp Ile Arg Lys Lys Val 1325 1330 1335agg cgc cta atg gga aag tcg cac att ggg ctt gtg tac agc cag caa 4384Arg Arg Leu Met Gly Lys Ser His Ile Gly Leu Val Tyr Ser Gln Gln 1340 1345 1350atc aat gag gtg ctt gat cag ctg gcg aac ctg aat gga cgc gat ctc 4432Ile Asn Glu Val Leu Asp Gln Leu Ala Asn Leu Asn Gly Arg Asp Leu 1355 1360 1365tct atc tgg tcc agt ggc agc cgg cac atg aag aag cag aca ttt gtg 4480Ser Ile Trp Ser Ser Gly Ser Arg His Met Lys Lys Gln Thr Phe Val1370 1375 1380 1385gta cat gca ggg aca gat aca aac gga gat atc ttt ttc atg gag gtg 4528Val His Ala Gly Thr Asp Thr Asn Gly Asp Ile Phe Phe Met Glu Val 1390 1395 1400tgc gat gac tgt gtg gtg ttg cgt agt aac atc gga aca gtg tat gag 4576Cys Asp Asp Cys Val Val Leu Arg Ser Asn Ile Gly Thr Val Tyr Glu 1405 1410 1415cgc tgg tgg tac gag aag ctc atc aac atg acc tac tgt ccc aag acg 4624Arg Trp Trp Tyr Glu Lys Leu Ile Asn Met Thr Tyr Cys Pro Lys Thr 1420 1425 1430aag gtg ttg tgc ttg tgg cgt aga aat ggc tct gag acc cag ctc aac 4672Lys Val Leu Cys Leu Trp Arg Arg Asn Gly Ser Glu Thr Gln Leu Asn 1435 1440 1445aag ttc tat act aaa aag tgt cgg gag ctg tac tac tgt gtg aag gac 4720Lys Phe Tyr Thr Lys Lys Cys Arg Glu Leu Tyr Tyr Cys Val Lys Asp1450 1455 1460 1465agc atg gag cgc gct gcc gcc cga cag caa agc atc aaa ccc gga cct 4768Ser Met Glu Arg Ala Ala Ala Arg Gln Gln Ser Ile Lys Pro Gly Pro 1470 1475 1480gaa ttg ggt ggc gag ttc cct gtg cag gac ctg aag act ggt gag ggt 4816Glu Leu Gly Gly Glu Phe Pro Val Gln Asp Leu Lys Thr Gly Glu Gly 1485 1490 1495ggc ctg ctg cag gtg acc ctg gaa ggg atc aac ctc aaa ttc atg cac 4864Gly Leu Leu Gln Val Thr Leu Glu Gly Ile Asn Leu Lys Phe Met His 1500 1505 1510aat cag gtt ttc ata gag ctg aat cac att aaa aag tgc aat aca gtt 4912Asn Gln Val Phe Ile Glu Leu Asn His Ile Lys Lys Cys Asn Thr Val 1515 1520 1525cga ggc gtc ttt gtc ctg gag gaa ttt gtt cct gaa att aaa gaa gtg 4960Arg Gly Val Phe Val Leu Glu Glu Phe Val Pro Glu Ile Lys Glu Val1530 1535 1540 1545gtg agc cac aag tac aag aca cca atg gcc cac gaa atc tgc tac tcc 5008Val Ser His Lys Tyr Lys Thr Pro Met Ala His Glu Ile Cys Tyr Ser 1550 1555 1560gta tta tgt ctc ttc tcg tac gtg gct gca gtt cat agc agt gag gaa 5056Val Leu Cys Leu Phe Ser Tyr Val Ala Ala Val His Ser Ser Glu Glu 1565 1570 1575gat ctc aga acc ccg ccc cgg cct gtc tct agc tgatggagag gggctacgca 5109Asp Leu Arg Thr Pro Pro Arg Pro Val Ser Ser 1580 1585gctgccccag cccagggcac gcccctggcc ccttgctgtt cccaagtgca cgatgctgct 5169gtgactgagg agtggatgat gctcgtgtgt cctctgcaag ccccctgctg tggcttggtt 5229ggttaccggt tatgtgtccc tctgagtgtg tcttgagcgt gtccaccttc tccctctcca 5289ctcccagaag accaaactgc cttcccctca gggctcaaga atgtgtacag tctgtggggc 5349cggtgtgaac ccactatttt gtgtccttga gacatttgtg ttgtggttcc ttgtccttgt 5409ccctggcgtt ataactgtcc actgcaagag tctggctctc ccttctctgt gacccggcat 5469gactgggcgc ctggagcagt ttcactctgt gaggagtgag ggaaccctgg ggctcaccct 5529ctcagaggaa gggcacagag aggaagggaa gaattggggg gcagccggag tgagtggcag 5589cctccctgct tccttctgca ttcccaagcc ggcagctact gcccagggcc cgcagtgttg 5649gctgctgcct gccacagcct ctgtgactgc agtggagcgg cgaattccct gtggcctgcc 5709acgccttcgg catcagagga tggagtggtc gaggctagtg gagtcccagg gaccgctggc 5769tgctctgcct gagcatcagg gagggggcag gaaagaccaa gctgggtttg cacatctgtc 5829tgcaggctgt ctctccaggc acggggtgtc aggagggaga gacagcctgg gtatgggcaa 5889gaaatgactg taaatatttc agccccacat tatttataga aaatgtacag ttgtgtgaat 5949gtgaaataaa tgtcctcaac tcccaaaaaa aaaaaaaaaa aaaaaaaaaa aaa 6002161588PRTHomo sapiens 16Met Val Gln Lys Lys Lys Phe Cys Pro Arg Leu Leu Asp Tyr Leu Val 1 5 10 15Ile Val Gly Ala Arg His Pro Ser Ser Asp Ser Val Ala Gln Thr Pro 20 25 30Glu Leu Leu Arg Arg Tyr Pro Leu Glu Asp His Thr Glu Phe Pro Leu 35 40 45Pro Pro Asp Val Val Phe Phe Cys Gln Pro Glu Gly Cys Leu Ser Val 50 55 60Arg Gln Arg Arg Met Ser Leu Arg Asp Asp Thr Ser Phe Val Phe Thr 65 70 75 80Leu Thr Asp Lys Asp Thr Gly Val Thr Arg Tyr Gly Ile Cys Val Asn 85 90 95Phe Tyr Arg Ser Phe Gln Lys Arg Ile Ser Lys Glu Lys Gly Glu Gly 100 105 110Gly Ala Gly Ser Arg Gly Lys Glu Gly Thr His Ala Thr Cys Ala Ser 115 120 125Glu Glu Gly Gly Thr Glu Ser Ser Glu Ser Gly Ser Ser Leu Gln Pro 130 135 140Leu Ser Ala Asp Ser Thr Pro Asp Val Asn Gln Ser Pro Arg Gly Lys145 150 155 160Arg Arg Ala Lys Ala Gly Ser Arg Ser Arg Asn Ser Thr Leu Thr Ser 165 170 175Leu Cys Val Leu Ser His Tyr Pro Phe Phe Ser Thr Phe Arg Glu Cys 180 185 190Leu Tyr Thr Leu Lys Arg Leu Val Asp Cys Cys Ser Glu Arg Leu Leu 195 200 205Gly Lys Lys Leu Gly Ile Pro Arg Gly Val Gln Arg Asp Thr Met Trp 210 215 220Arg Ile Phe Thr Gly Ser Leu Leu Val Glu Glu Lys Ser Ser Ala Leu225 230 235 240Leu His Asp Leu Arg Glu Ile Glu Ala Trp Ile Tyr Arg Leu Leu Arg 245 250 255Ser Pro Val Pro Val Ser Gly Gln Lys Arg Val Asp Ile Glu Val Leu 260 265 270Pro Gln Glu Leu Gln Pro Ala Leu Thr Phe Ala Leu Pro Asp Pro Ser 275 280 285Arg Phe Thr Leu Val Asp Phe Pro Leu His Leu Pro Leu Glu Leu Leu 290 295 300Gly Val Asp Ala Cys Leu Gln Val Leu Thr Cys Ile Leu Leu Glu His305 310 315 320Lys Val Val Leu Gln Ser Arg Asp Tyr Asn Ala Leu Ser Met Ser Val 325 330 335Met Ala Phe Val Ala Met Ile Tyr Pro Leu Glu Tyr Met Phe Pro Val 340 345 350Ile Pro Leu Leu Pro Thr Cys Met Ala Ser Ala Glu Gln Leu Leu Leu 355 360 365Ala Pro Thr Pro Tyr Ile Ile Gly Val Pro Ala Ser Phe Phe Leu Tyr 370 375 380Lys Leu Asp Phe Lys Met Pro Asp Asp Val Trp Leu Val Asp Leu Asp385 390 395 400Ser Asn Arg Val Ile Ala Pro Thr Asn Ala Glu Val Leu Pro Ile Leu 405 410 415Pro Glu Pro Glu Ser Leu Glu Leu Lys Lys His Leu Lys Gln Ala Leu 420 425 430Ala Ser Met Ser Leu Asn Thr Gln Pro Ile Leu Asn Leu Glu Lys Phe 435 440 445His Glu Gly Gln Glu Ile Pro Leu Leu Leu Gly Arg Pro Ser Asn Asp 450 455 460Leu Gln Ser Thr Pro Ser Thr Glu Phe Asn Pro Leu Ile Tyr Gly Asn465 470 475 480Asp Val Asp Ser Val Asp Val Ala Thr Arg Val Ala Met Val Arg Phe 485 490 495Phe Asn Ser Ala Asn Val Leu Gln Gly Phe Gln Met His Thr Arg Thr 500 505 510Leu Arg Leu Phe Pro Arg Pro Val Val Ala Phe Gln Ala Gly Ser Phe 515 520 525Leu Ala Ser Arg Pro Arg Gln Thr Pro Phe Ala Glu Lys Leu Ala Arg 530 535 540Thr Gln Ala Val Glu Tyr Phe Gly Glu Trp Ile Leu Asn Pro Thr Asn545 550 555 560Tyr Ala Phe Gln Arg Ile His Asn Asn Met Phe Asp Pro Ala Leu Ile 565 570 575Gly Asp Lys Pro Lys Trp Tyr Ala His Gln Leu Gln Pro Ile His Tyr 580 585 590Arg Val Tyr Asp Ser Asn Ser Gln Leu Ala Glu Ala Leu Ser Val Pro 595 600 605Pro Glu Arg Asp Ser Asp Ser Glu Pro Thr Asp Asp Ser Gly Ser Asp 610 615 620Ser Met Asp Tyr Asp Asp Ser Ser Ser Ser Tyr Ser Ser Leu Gly Asp625 630 635 640Phe Val Ser Glu Met Met Lys Cys Asp Ile Asn Gly Asp Thr Pro Asn 645 650 655Val Asp Pro Leu Thr His Ala Ala Leu Gly Asp Ala Ser Glu Val Glu 660 665 670Ile Asp Glu Leu Gln Asn Gln Lys Glu Ala Glu Glu Pro Gly Pro Asp 675 680 685Ser Glu Asn Ser Gln Glu Asn Pro Pro Leu Arg Ser Ser Ser Ser Thr 690 695 700Thr Ala Ser Ser Ser Pro Ser Thr Val Ile His Gly Ala Asn Ser Glu705 710 715 720Pro Ala Asp Ser Thr Glu Met Asp Asp Lys Ala Ala Val Gly Val Ser 725 730 735Lys Pro Leu Pro Ser Val Pro Pro Ser Ile Gly Lys Ser Asn Met Asp 740 745 750Arg Arg Gln Ala Glu Ile Gly Glu Gly Ser Val Arg Arg Arg Ile Tyr 755 760 765Asp Asn Pro Tyr Phe Glu Pro Gln Tyr Gly Phe Pro Pro Glu Glu Asp 770 775 780Glu Asp Glu Gln Gly Glu Ser Tyr Thr Pro Arg Phe Ser Gln His Val785 790 795 800Ser Gly Asn Arg Ala Gln Lys Leu Leu Arg Pro Asn Ser Leu Arg Leu 805 810 815Ala Ser Asp Ser Asp Ala Glu Ser Asp Ser Arg Ala Ser Ser Pro Asn 820 825 830Ser Thr Val Ser Asn Thr Ser Thr Glu Gly Phe Gly Gly Ile Met Ser 835 840 845Phe Ala Ser Ser Leu Tyr Arg Asn His Ser Thr Ser Phe Ser Leu Ser 850 855 860Asn Leu Thr Leu Pro Thr Lys Gly Ala Arg Glu Lys Ala Thr Pro Phe865 870 875 880Pro Ser Leu Lys Gly Asn Arg Arg Ala Leu Val Asp Gln Lys Ser Ser 885 890 895Val Ile Lys His Ser Pro Thr Val Lys Arg Glu Pro Pro Ser Pro Gln 900 905 910Gly Arg Ser Ser Asn Ser Ser Glu Asn Gln Gln Phe Leu Lys Glu Val 915 920 925Val His Ser Val Leu Asp Gly Gln Gly Val Gly Trp Leu Asn Met Lys 930 935 940Lys Val Arg Arg Leu Leu Glu Ser Glu Gln Leu Arg Val Phe Val Leu945 950 955 960Ser Lys Leu Asn Arg Met Val Gln Ser Glu Asp Asp Ala Arg Gln Asp 965 970 975Ile Ile Pro Asp Val Glu Ile Ser Arg Lys Val Tyr Lys Gly Met Leu 980 985 990Asp Leu Leu Lys Cys Thr Val Leu Ser Leu Glu Gln Ser Tyr Ala His 995 1000 1005Ala Gly Leu Gly Gly Met Ala Ser Ile Phe Gly Leu Leu Glu Ile Ala 1010 1015 1020Gln Thr His Tyr Tyr Ser Lys Glu Pro Asp Lys Arg Lys Arg Ser Pro1025 1030 1035 1040Thr Glu Ser Val Asn Thr Pro Val Gly Lys Asp Pro Gly Leu Ala Gly 1045 1050 1055Arg Gly Asp Pro Lys Ala Met Ala Gln Leu Arg Val Pro Gln Leu Gly 1060 1065 1070Pro Arg Ala Pro Ser Ala Thr Gly Lys Gly Pro Lys Glu Leu Asp Thr 1075 1080 1085Arg Ser Leu Lys Glu Glu Asn Phe Ile Ala Ser Ile Gly Pro Glu Val 1090 1095 1100Ile Lys Pro Val Phe Asp Leu Gly Glu Thr Glu Glu Lys Lys Ser Gln1105 1110 1115 1120Ile Ser Ala Asp Ser Gly Val Ser Leu Thr Ser Ser Ser Gln Arg Thr 1125 1130 1135Asp Gln Asp Ser Val Ile Gly Val Ser Pro Ala Val Met Ile Arg Ser 1140 1145 1150Ser Ser Gln Asp Ser Glu Val Ser Thr Val Val Ser Asn Ser Ser Gly 1155 1160 1165Glu Thr Leu Gly Ala Asp Ser Asp Leu Ser Ser Asn Ala Gly Asp Gly 1170 1175 1180Pro Gly Gly Glu Gly Ser Val His Leu Ala Ser Ser Arg Gly Thr Leu1185 1190 1195 1200Ser Asp Ser Glu Ile Glu Thr Asn Ser Ala Thr Ser Thr Ile Phe Gly 1205 1210 1215Lys Ala His Ser Leu Lys Pro Ser Ile Lys Glu Lys Leu Ala Gly Ser 1220 1225 1230Pro Ile Arg Thr Ser Glu Asp Val Ser Gln Arg Val Tyr Leu Tyr Glu 1235 1240 1245Gly Leu Leu Gly Lys Glu Arg Ser Thr Leu Trp Asp Gln Met Gln Phe 1250 1255 1260Trp Glu Asp Ala Phe Leu Asp Ala Val Met Leu Glu Arg Glu Gly Met1265 1270 1275 1280Gly Met Asp Gln Gly Pro Gln Glu Met Ile Asp Arg Tyr Leu Ser Leu 1285 1290 1295Gly Glu His Asp Arg Lys Arg Leu Glu Asp Asp Glu Asp Arg Leu Leu 1300 1305 1310Ala Thr Leu Leu His Asn Leu Ile Ser Tyr Met Leu Leu Met Lys Val 1315 1320 1325Asn Lys Asn Asp Ile Arg Lys Lys Val Arg Arg Leu Met Gly Lys Ser 1330 1335 1340His Ile Gly Leu Val Tyr Ser Gln Gln Ile Asn Glu Val Leu Asp Gln1345 1350 1355 1360Leu Ala Asn Leu Asn Gly Arg Asp Leu Ser Ile Trp Ser Ser Gly Ser 1365 1370 1375Arg His Met Lys Lys Gln Thr Phe Val Val His Ala Gly Thr Asp Thr 1380 1385 1390Asn Gly Asp Ile Phe Phe Met Glu Val Cys Asp Asp Cys Val Val Leu 1395 1400 1405Arg Ser Asn Ile Gly Thr Val Tyr Glu Arg Trp Trp Tyr Glu Lys Leu 1410 1415 1420Ile Asn Met

Thr Tyr Cys Pro Lys Thr Lys Val Leu Cys Leu Trp Arg1425 1430 1435 1440Arg Asn Gly Ser Glu Thr Gln Leu Asn Lys Phe Tyr Thr Lys Lys Cys 1445 1450 1455Arg Glu Leu Tyr Tyr Cys Val Lys Asp Ser Met Glu Arg Ala Ala Ala 1460 1465 1470Arg Gln Gln Ser Ile Lys Pro Gly Pro Glu Leu Gly Gly Glu Phe Pro 1475 1480 1485Val Gln Asp Leu Lys Thr Gly Glu Gly Gly Leu Leu Gln Val Thr Leu 1490 1495 1500Glu Gly Ile Asn Leu Lys Phe Met His Asn Gln Val Phe Ile Glu Leu1505 1510 1515 1520Asn His Ile Lys Lys Cys Asn Thr Val Arg Gly Val Phe Val Leu Glu 1525 1530 1535Glu Phe Val Pro Glu Ile Lys Glu Val Val Ser His Lys Tyr Lys Thr 1540 1545 1550Pro Met Ala His Glu Ile Cys Tyr Ser Val Leu Cys Leu Phe Ser Tyr 1555 1560 1565Val Ala Ala Val His Ser Ser Glu Glu Asp Leu Arg Thr Pro Pro Arg 1570 1575 1580Pro Val Ser Ser1585172473DNAHomo sapiensCDS(14)..(2404) 17ccgacgagga gac atg gcg gcg gcg ccg gta gcg gct ggg tct gga gcc 49 Met Ala Ala Ala Pro Val Ala Ala Gly Ser Gly Ala 1 5 10ggc cga ggg aga cgg tcg gca gcc aca gtg gcg gct tgg ggc gga tgg 97Gly Arg Gly Arg Arg Ser Ala Ala Thr Val Ala Ala Trp Gly Gly Trp 15 20 25ggc ggc cgg ccg cgg cct ggt aac att ctg ctg cag ctg cgg cag ggc 145Gly Gly Arg Pro Arg Pro Gly Asn Ile Leu Leu Gln Leu Arg Gln Gly 30 35 40cag ctg acc ggc cgg ggc ctg gtc cgg gcg gtg cag ttc act gag act 193Gln Leu Thr Gly Arg Gly Leu Val Arg Ala Val Gln Phe Thr Glu Thr 45 50 55 60ttt ttg acg gag agg gac aaa caa tcc aag tgg agt gga att cct cag 241Phe Leu Thr Glu Arg Asp Lys Gln Ser Lys Trp Ser Gly Ile Pro Gln 65 70 75ctg ctc ctc aag ctg cac acc acc agc cac ctc cac agt gac ttt gtt 289Leu Leu Leu Lys Leu His Thr Thr Ser His Leu His Ser Asp Phe Val 80 85 90gag tgt caa aac atc ctc aag gaa att tct cct ctt ctc tcc atg gag 337Glu Cys Gln Asn Ile Leu Lys Glu Ile Ser Pro Leu Leu Ser Met Glu 95 100 105gct atg gca ttt gtt act gaa gag agg aaa ctt acc caa gaa acc act 385Ala Met Ala Phe Val Thr Glu Glu Arg Lys Leu Thr Gln Glu Thr Thr 110 115 120tat cca aat act tac att ttt gac ttg ttt gga ggt gtt gat ctt ctt 433Tyr Pro Asn Thr Tyr Ile Phe Asp Leu Phe Gly Gly Val Asp Leu Leu125 130 135 140gta gaa att ctt atg agg cct acg atc tct atc cgg gga cag aaa ctg 481Val Glu Ile Leu Met Arg Pro Thr Ile Ser Ile Arg Gly Gln Lys Leu 145 150 155aaa ata agt gat gaa atg tcc aag gac tgc ttg agt atc ctg tat aat 529Lys Ile Ser Asp Glu Met Ser Lys Asp Cys Leu Ser Ile Leu Tyr Asn 160 165 170acc tgt gtc tgt aca gag gga gtt aca aag cgt ttg gca gaa aag aat 577Thr Cys Val Cys Thr Glu Gly Val Thr Lys Arg Leu Ala Glu Lys Asn 175 180 185gac ttt gtg atc ttc ctg ttt aca ttg atg aca agt aag aag aca ttc 625Asp Phe Val Ile Phe Leu Phe Thr Leu Met Thr Ser Lys Lys Thr Phe 190 195 200tta caa aca gca acc ctc att gaa gat att tta ggt gtt aaa aag gaa 673Leu Gln Thr Ala Thr Leu Ile Glu Asp Ile Leu Gly Val Lys Lys Glu205 210 215 220atg atc cga cta gat gaa gtc ccc aat ctg agt tcc tta gta tcc aat 721Met Ile Arg Leu Asp Glu Val Pro Asn Leu Ser Ser Leu Val Ser Asn 225 230 235ttc gat cag cag cag ctc gct aat ttc tgc cgg att ctg gct gtc acc 769Phe Asp Gln Gln Gln Leu Ala Asn Phe Cys Arg Ile Leu Ala Val Thr 240 245 250att tca gag atg gat aca ggg aat gat gac aag cac acg ctt ctt gcc 817Ile Ser Glu Met Asp Thr Gly Asn Asp Asp Lys His Thr Leu Leu Ala 255 260 265aaa aat gct caa cag aag aag agc ttg agt ttg ggg cct tct gca gct 865Lys Asn Ala Gln Gln Lys Lys Ser Leu Ser Leu Gly Pro Ser Ala Ala 270 275 280gaa atc aat caa gcg gcc ctt ctc agc att cct ggc ttt gtt gag cgg 913Glu Ile Asn Gln Ala Ala Leu Leu Ser Ile Pro Gly Phe Val Glu Arg285 290 295 300ctt tgc aaa ctg gcg act cga aag gtg tca gag tca acg ggc aca gcc 961Leu Cys Lys Leu Ala Thr Arg Lys Val Ser Glu Ser Thr Gly Thr Ala 305 310 315agc ttc ctt cag gag ttg gaa gag tgg tac aca tgg cta gac aat gct 1009Ser Phe Leu Gln Glu Leu Glu Glu Trp Tyr Thr Trp Leu Asp Asn Ala 320 325 330ttg gtg cta gat gcc ctg atg cga gtg gcc aat gag gag tca gag cac 1057Leu Val Leu Asp Ala Leu Met Arg Val Ala Asn Glu Glu Ser Glu His 335 340 345aat caa gcc tcc att gtg ttc cct cct cca ggg gct tct gag gag aat 1105Asn Gln Ala Ser Ile Val Phe Pro Pro Pro Gly Ala Ser Glu Glu Asn 350 355 360ggc ctg cct cac acg tca gcc aga acc cag ctg ccc cag tca atg aag 1153Gly Leu Pro His Thr Ser Ala Arg Thr Gln Leu Pro Gln Ser Met Lys365 370 375 380att atg cat gag atc atg tac aaa ctg gaa gtg ctc tat gtc ctc tgc 1201Ile Met His Glu Ile Met Tyr Lys Leu Glu Val Leu Tyr Val Leu Cys 385 390 395gtg ctg ctg atg ggg cgt cag cga aac cag gtt cac aga atg att gca 1249Val Leu Leu Met Gly Arg Gln Arg Asn Gln Val His Arg Met Ile Ala 400 405 410gag ttc aag ctg atc cct gga ctt aat aat ttg ttt gac aaa ctg att 1297Glu Phe Lys Leu Ile Pro Gly Leu Asn Asn Leu Phe Asp Lys Leu Ile 415 420 425tgg agg aag cat tca gca tct gcc ctt gtc ctc cat ggt cac aac cag 1345Trp Arg Lys His Ser Ala Ser Ala Leu Val Leu His Gly His Asn Gln 430 435 440aac tgt gac tgt agc ccg gac atc acc ttg aag ata cag ttt ttg agg 1393Asn Cys Asp Cys Ser Pro Asp Ile Thr Leu Lys Ile Gln Phe Leu Arg445 450 455 460ctt ctt cag agc ttc agt gac cac cac gag aac aag tac ttg tta ctc 1441Leu Leu Gln Ser Phe Ser Asp His His Glu Asn Lys Tyr Leu Leu Leu 465 470 475aac aac cag gag ctg aat gaa ctc agt gcc atc tct ctc aag gcc aac 1489Asn Asn Gln Glu Leu Asn Glu Leu Ser Ala Ile Ser Leu Lys Ala Asn 480 485 490atc cct gag gtg gaa gct gtc ctc aac acc gac agg agt ttg gtg tgt 1537Ile Pro Glu Val Glu Ala Val Leu Asn Thr Asp Arg Ser Leu Val Cys 495 500 505gat ggg aag agg ggc tta tta act cgt ctg ctg cag gtc atg aag aag 1585Asp Gly Lys Arg Gly Leu Leu Thr Arg Leu Leu Gln Val Met Lys Lys 510 515 520gag cca gca gag tcg tct ttc agg ttt tgg caa gct cgg gct gtg gag 1633Glu Pro Ala Glu Ser Ser Phe Arg Phe Trp Gln Ala Arg Ala Val Glu525 530 535 540agt ttc ctc cga ggg acc acc tcc tat gca gac cag atg ttc ctg ctg 1681Ser Phe Leu Arg Gly Thr Thr Ser Tyr Ala Asp Gln Met Phe Leu Leu 545 550 555aag cga ggc ctc ttg gag cac atc ctt tac tgc att gtg gac agc gag 1729Lys Arg Gly Leu Leu Glu His Ile Leu Tyr Cys Ile Val Asp Ser Glu 560 565 570tgt aag tca agg gat gtg ctc cag agt tac ttt gac ctc ctg ggg gag 1777Cys Lys Ser Arg Asp Val Leu Gln Ser Tyr Phe Asp Leu Leu Gly Glu 575 580 585ctg atg aag ttc aac gtt gat gca ttc aag aga ttc aat aaa aat atc 1825Leu Met Lys Phe Asn Val Asp Ala Phe Lys Arg Phe Asn Lys Asn Ile 590 595 600aac acc gat gca aag ttc cag gta ttc ctg aag cag atc aac agc tcc 1873Asn Thr Asp Ala Lys Phe Gln Val Phe Leu Lys Gln Ile Asn Ser Ser605 610 615 620ctg gtg gac tcc aac atg ctg gtg cgc tgt gtc act ctg tcc ctg gac 1921Leu Val Asp Ser Asn Met Leu Val Arg Cys Val Thr Leu Ser Leu Asp 625 630 635cga ttt gaa aac cag gtg gat atg aaa gtt gcc gag gta ctg tct gaa 1969Arg Phe Glu Asn Gln Val Asp Met Lys Val Ala Glu Val Leu Ser Glu 640 645 650tgc cgc ctg ctc gcc tac ata tcc cag gtg ccc acg cag atg tcc ttc 2017Cys Arg Leu Leu Ala Tyr Ile Ser Gln Val Pro Thr Gln Met Ser Phe 655 660 665ctc ttc cgc ctc atc aac atc atc cac gtg cag acg ctg acc cag gag 2065Leu Phe Arg Leu Ile Asn Ile Ile His Val Gln Thr Leu Thr Gln Glu 670 675 680aac gtc agc tgc ctc aac acc agc ctg gtg atc ctg atg ctg gcc cga 2113Asn Val Ser Cys Leu Asn Thr Ser Leu Val Ile Leu Met Leu Ala Arg685 690 695 700cgg aaa gag cgg ctg ccc ctg tac ctg cgg ctg ctg cag cgg atg gag 2161Arg Lys Glu Arg Leu Pro Leu Tyr Leu Arg Leu Leu Gln Arg Met Glu 705 710 715cac agc aag aag tac ccc ggc ttc ctg ctc aac aac ttc cac aac ctg 2209His Ser Lys Lys Tyr Pro Gly Phe Leu Leu Asn Asn Phe His Asn Leu 720 725 730ctg cgc ttc tgg cag cag cac tac ctg cac aag gac aag gac agc acc 2257Leu Arg Phe Trp Gln Gln His Tyr Leu His Lys Asp Lys Asp Ser Thr 735 740 745tgc cta gag aac agc tcc tgc atc agc ttc tca tac tgg aag gag aca 2305Cys Leu Glu Asn Ser Ser Cys Ile Ser Phe Ser Tyr Trp Lys Glu Thr 750 755 760gtg tcc atc ctg ttg aac ccg gac cgg cag tca ccc tct gct ctc gtt 2353Val Ser Ile Leu Leu Asn Pro Asp Arg Gln Ser Pro Ser Ala Leu Val765 770 775 780agc tac att gag gag ccc tac atg gac ata gac agg gac ttc act gag 2401Ser Tyr Ile Glu Glu Pro Tyr Met Asp Ile Asp Arg Asp Phe Thr Glu 785 790 795gag tgaccttggg ccaggcctcg ggaggctgct gggccagtgt gggtgagcgt 2454Glugggtacgatg ccacacgcc 247318797PRTHomo sapiens 18Met Ala Ala Ala Pro Val Ala Ala Gly Ser Gly Ala Gly Arg Gly Arg 1 5 10 15Arg Ser Ala Ala Thr Val Ala Ala Trp Gly Gly Trp Gly Gly Arg Pro 20 25 30Arg Pro Gly Asn Ile Leu Leu Gln Leu Arg Gln Gly Gln Leu Thr Gly 35 40 45Arg Gly Leu Val Arg Ala Val Gln Phe Thr Glu Thr Phe Leu Thr Glu 50 55 60Arg Asp Lys Gln Ser Lys Trp Ser Gly Ile Pro Gln Leu Leu Leu Lys 65 70 75 80Leu His Thr Thr Ser His Leu His Ser Asp Phe Val Glu Cys Gln Asn 85 90 95Ile Leu Lys Glu Ile Ser Pro Leu Leu Ser Met Glu Ala Met Ala Phe 100 105 110Val Thr Glu Glu Arg Lys Leu Thr Gln Glu Thr Thr Tyr Pro Asn Thr 115 120 125Tyr Ile Phe Asp Leu Phe Gly Gly Val Asp Leu Leu Val Glu Ile Leu 130 135 140Met Arg Pro Thr Ile Ser Ile Arg Gly Gln Lys Leu Lys Ile Ser Asp145 150 155 160Glu Met Ser Lys Asp Cys Leu Ser Ile Leu Tyr Asn Thr Cys Val Cys 165 170 175Thr Glu Gly Val Thr Lys Arg Leu Ala Glu Lys Asn Asp Phe Val Ile 180 185 190Phe Leu Phe Thr Leu Met Thr Ser Lys Lys Thr Phe Leu Gln Thr Ala 195 200 205Thr Leu Ile Glu Asp Ile Leu Gly Val Lys Lys Glu Met Ile Arg Leu 210 215 220Asp Glu Val Pro Asn Leu Ser Ser Leu Val Ser Asn Phe Asp Gln Gln225 230 235 240Gln Leu Ala Asn Phe Cys Arg Ile Leu Ala Val Thr Ile Ser Glu Met 245 250 255Asp Thr Gly Asn Asp Asp Lys His Thr Leu Leu Ala Lys Asn Ala Gln 260 265 270Gln Lys Lys Ser Leu Ser Leu Gly Pro Ser Ala Ala Glu Ile Asn Gln 275 280 285Ala Ala Leu Leu Ser Ile Pro Gly Phe Val Glu Arg Leu Cys Lys Leu 290 295 300Ala Thr Arg Lys Val Ser Glu Ser Thr Gly Thr Ala Ser Phe Leu Gln305 310 315 320Glu Leu Glu Glu Trp Tyr Thr Trp Leu Asp Asn Ala Leu Val Leu Asp 325 330 335Ala Leu Met Arg Val Ala Asn Glu Glu Ser Glu His Asn Gln Ala Ser 340 345 350Ile Val Phe Pro Pro Pro Gly Ala Ser Glu Glu Asn Gly Leu Pro His 355 360 365Thr Ser Ala Arg Thr Gln Leu Pro Gln Ser Met Lys Ile Met His Glu 370 375 380Ile Met Tyr Lys Leu Glu Val Leu Tyr Val Leu Cys Val Leu Leu Met385 390 395 400Gly Arg Gln Arg Asn Gln Val His Arg Met Ile Ala Glu Phe Lys Leu 405 410 415Ile Pro Gly Leu Asn Asn Leu Phe Asp Lys Leu Ile Trp Arg Lys His 420 425 430Ser Ala Ser Ala Leu Val Leu His Gly His Asn Gln Asn Cys Asp Cys 435 440 445Ser Pro Asp Ile Thr Leu Lys Ile Gln Phe Leu Arg Leu Leu Gln Ser 450 455 460Phe Ser Asp His His Glu Asn Lys Tyr Leu Leu Leu Asn Asn Gln Glu465 470 475 480Leu Asn Glu Leu Ser Ala Ile Ser Leu Lys Ala Asn Ile Pro Glu Val 485 490 495Glu Ala Val Leu Asn Thr Asp Arg Ser Leu Val Cys Asp Gly Lys Arg 500 505 510Gly Leu Leu Thr Arg Leu Leu Gln Val Met Lys Lys Glu Pro Ala Glu 515 520 525Ser Ser Phe Arg Phe Trp Gln Ala Arg Ala Val Glu Ser Phe Leu Arg 530 535 540Gly Thr Thr Ser Tyr Ala Asp Gln Met Phe Leu Leu Lys Arg Gly Leu545 550 555 560Leu Glu His Ile Leu Tyr Cys Ile Val Asp Ser Glu Cys Lys Ser Arg 565 570 575Asp Val Leu Gln Ser Tyr Phe Asp Leu Leu Gly Glu Leu Met Lys Phe 580 585 590Asn Val Asp Ala Phe Lys Arg Phe Asn Lys Asn Ile Asn Thr Asp Ala 595 600 605Lys Phe Gln Val Phe Leu Lys Gln Ile Asn Ser Ser Leu Val Asp Ser 610 615 620Asn Met Leu Val Arg Cys Val Thr Leu Ser Leu Asp Arg Phe Glu Asn625 630 635 640Gln Val Asp Met Lys Val Ala Glu Val Leu Ser Glu Cys Arg Leu Leu 645 650 655Ala Tyr Ile Ser Gln Val Pro Thr Gln Met Ser Phe Leu Phe Arg Leu 660 665 670Ile Asn Ile Ile His Val Gln Thr Leu Thr Gln Glu Asn Val Ser Cys 675 680 685Leu Asn Thr Ser Leu Val Ile Leu Met Leu Ala Arg Arg Lys Glu Arg 690 695 700Leu Pro Leu Tyr Leu Arg Leu Leu Gln Arg Met Glu His Ser Lys Lys705 710 715 720Tyr Pro Gly Phe Leu Leu Asn Asn Phe His Asn Leu Leu Arg Phe Trp 725 730 735Gln Gln His Tyr Leu His Lys Asp Lys Asp Ser Thr Cys Leu Glu Asn 740 745 750Ser Ser Cys Ile Ser Phe Ser Tyr Trp Lys Glu Thr Val Ser Ile Leu 755 760 765Leu Asn Pro Asp Arg Gln Ser Pro Ser Ala Leu Val Ser Tyr Ile Glu 770 775 780Glu Pro Tyr Met Asp Ile Asp Arg Asp Phe Thr Glu Glu785 790 795198PRTArtificial SequenceDescription of Artificial Sequence Flag-tag 19Asp Tyr Lys Asp Asp Asp Asp Lys 1 5

Claims

1. A composition comprising an isolated polynucleotide encoding a protein having TNF-R1-DD ligand protein activity.

2. The composition of claim 1 wherein said polynucleotide is selected from the group consisting of:(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 from nucleotide 2 to nucleotide 1231;(b) a polynucleotide comprising a fragment of the nucleotide sequence of SEQ ID NO:1;(c) a polynucleotide encoding an TNF-R1-DD ligand protein comprising the amino acid sequence of SEQ ID NO:2;(d) a polynucleotide encoding an TNF-R1-DD ligand protein comprising a fragment of the amino acid sequence of SEQ ID NO:2; and(e) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(d).

3. The composition of claim 1 wherein said polynucleotide sequence is selected from the group consisting of:(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:3 from nucleotide 2 to nucleotide 415;(b) a polynucleotide comprising a fragment of the nucleotide sequence of SEQ ID NO:3;(c) a polynucleotide encoding an TNF-R1-DD ligand protein comprising the amino acid sequence of SEQ ID NO:4;(d) a polynucleotide encoding an TNF-R1-DD ligand protein comprising a fragment of the amino acid sequence of SEQ ID NO:4; and(e) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(d).

4. A composition of claim 1 wherein said polynucleotide is operably linked to an expression control sequence.

5. A host cell transformed with a composition of claim 4.

6. The host cell of claim 5, wherein said cell is a mammalian cell.

7. A process for producing an TNF-R1-DD ligand protein, which comprises:(a) growing a culture of the host cell of claim 5 in a suitable culture medium; and(b) purifying the TNF-R1-DD ligand protein from the culture.

8. A composition comprising a protein having TNF-R1-DD ligand protein activity.

9. The composition of claim 8 wherein said protein comprises an amino acid sequence selected from the group consisting of:(a) the amino acid sequence of SEQ ID NO:2; and(b) fragments of the amino acid sequence of SEQ ID NO:2;said protein being substantially free from other mammalian proteins.

10. The composition of claim 8 wherein said protein comprises an amino acid sequence selected from the group consisting of:(a) the amino acid sequence of SEQ ID NO:4; and(b) fragments of the amino acid sequence of SEQ ID NO:4;said protein being substantially free from other mammalian proteins.

11. The composition of claim 8 wherein said protein comprises an amino acid sequence selected from the group consisting of:(a) the amino acid sequence of SEQ ID NO:6; and(b) fragments of the amino acid sequence of SEQ ID NO:6;said protein being substantially free from other mammalian proteins.

12. The composition of claim 8, further comprising a pharmaceutically acceptable carrier.

13. A composition comprising an antibody which specifically reacts with the TNF-R1-DD ligand protein of claim 8.

14. A method of identifying an inhibitor of TNF-R death domain binding which comprises:(a) combining an TNF-R death domain protein with a composition of claim 8, said combination forming a first binding mixture;(b) measuring the amount of binding between the TNF-R death domain protein and the TNF-R1-DD ligand protein in the first binding mixture;(c) combining a compound with the TNF-R death domain protein and an TNF-R1-DD ligand protein to form a second binding mixture;(d) measuring the amount of binding in the second binding mixture; and(e) comparing the amount of binding in the first binding mixture with the amount of binding in the second binding mixture;wherein the compound is capable of inhibiting TNF-R death domain binding when a decrease in the amount of binding of the second binding mixture occurs.

15. The method of claim 14 wherein said TNF-R1-DD ligand protein comprises an amino acid sequence selected from the group consisting of:(a) the amino acid sequence of SEQ ID NO:2;(b) fragments of the amino acid sequence of SEQ ID NO:2;(c) the amino acid sequence of SEQ ID NO:4;(d) fragments of the amino acid sequence of SEQ ID NO:4;(e) the amino acid sequence of SEQ ID NO:6;(f) fragments of the amino acid sequence of SEQ ID NO:6;(g) the amino acid sequence of SEQ ID NO:8; and(h) fragments of the amino acid sequence of SEQ ID NO:8.

16. A method of preventing or ameliorating an inflammatory condition which comprises administering a therapeutically effective amount of a composition of claim 12.

17. TNF-R1-DD ligand protein produced according to the method of claim 7.

18. A method of inhibiting TNF-R death domain binding comprising administering a therapeutically effective amount of a composition of claim 12.

19. A method of preventing or ameliorating an inflammatory condition which comprises administering to a mammalian subject a therapeutically effective amount of a composition comprising a pharmaceutically acceptable carrier and a protein selected from the group consisting of IGFBP-5 and fragments thereof having TNF-R1-DD ligand protein activity.

20. A method of inhibiting TNF-R death domain binding comprising administering to a mammalian subject a therapeutically effective amount of a composition comprising a pharmaceutically acceptable carrier and a protein selected from the group consisting of IGFBP-5 and fragments thereof having TNF-R1-DD ligand protein activity.

21. A composition comprising an inhibitor identified according to the method of claim 14.

22. The composition of claim 21 further comprising a pharmaceutically acceptable carrier.

23. A method of preventing or ameliorating an inflammatory condition comprising administering to a mammalian subject a therapeutically effective amount of the composition of claim 22.

24. A method of inhibiting TNF-R death domain binding comprising administering to a mammalian subject a therapeutically effective amount of the composition of claim 22.

25. A composition comprising a pharmaceutically acceptable carrier and a protein selected from the group consisting of IGFBP-5 and fragments thereof having TNF-R1-DD ligand protein activity.

26. A method of identifying an inhibitor of TNF-R death domain binding which comprises:(a) transforming a cell with a first polynucleotide encoding an TNF-R death domain protein, a second polynucleotide encoding an TNF-R1-DD ligand protein, and at least one reporter gene, wherein the expression of the reporter gene is regulated by the binding of the TNF-R1-DD ligand protein encoded by the second polynucleotide to the TNF-R death domain protein encoded by the first polynucleotide;(b) growing the cell in the presence of and in the absence of a compound; and(c) comparing the degree of expression of the reporter gene in the presence of and in the absence of the compound;wherein the compound is capable of inhibiting TNF-R death domain binding when a decrease in the degree of expression of the reporter gene occurs.

27. The method of claim 26 wherein the second polynucleotide is selected from the group consisting of:(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:1 from nucleotide 2 to nucleotide 1231;(b) a polynucleotide comprising a fragment of the nucleotide sequence of SEQ ID NO:1, which encodes a protein having TNF-R1-DD ligand protein activity;(c) a polynucleotide encoding an TNF-R1-DD ligand protein comprising the amino acid sequence of SEQ ID NO:2;(d) a polynucleotide encoding an TNF-R1-DD ligand protein comprising a fragment of the amino acid sequence of SEQ ID NO:2 and having TNF-R1-DD ligand protein activity;(e) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:3 from nucleotide 2 to nucleotide 415;(f) a polynucleotide comprising a fragment of the nucleotide sequence of SEQ ID NO:3, which encodes a protein having TNF-R1-DD ligand protein activity;(g) a polynucleotide encoding an TNF-R1-DD ligand protein comprising the amino acid sequence of SEQ ID NO:4;(h) a polynucleotide encoding an TNF-R1-DD ligand protein comprising a fragment of the amino acid sequence of SEQ ID NO:4 and having TNF-R1-DD ligand protein activity;(i) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:5 from nucleotide 2 to nucleotide 559;(j) a polynucleotide comprising a fragment of the nucleotide sequence of SEQ ID NO:5, which encodes a protein having TNF-R1-DD ligand protein activity;(k) a polynucleotide encoding an TNF-R1-DD ligand protein comprising the amino acid sequence of SEQ ID NO:6;(l) a polynucleotide encoding an TNF-R1-DD ligand protein comprising a fragment of the amino acid sequence of SEQ ID NO:6 and having TNF-R1-DD ligand protein activity;(m) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:7 from nucleotide 57 to nucleotide 875;(n) a polynucleotide comprising a fragment of the nucleotide sequence of SEQ ID NO:7, which encodes a protein having TNF-R1-DD ligand protein activity;(o) a polynucleotide encoding an TNF-R1-DD ligand protein comprising the amino acid sequence of SEQ ID NO:8;(p) a polynucleotide encoding an TNF-R1-DD ligand protein comprising a fragment of the amino acid sequence of SEQ ID NO:8 and having TNF-R1-DD ligand protein activity; and(q) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(p), which encodes a protein having TNF-R1-DD ligand protein activity.

28. The method of claim 26 wherein the cell is a yeast cell.

29. The composition of claim 1 wherein said polynucleotide sequence is selected from the group consisting of:(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:9 from nucleotide 2 to nucleotide 931;(b) a polynucleotide comprising a fragment of the nucleotide sequence of SEQ ID NO:9;(c) a polynucleotide encoding an TNF-R1-DD ligand protein comprising the amino acid sequence of SEQ ID NO:10;(d) a polynucleotide encoding an TNF-R1-DD ligand protein comprising a fragment of the amino acid sequence of SEQ ED NO: 10; and(e) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(d).

30. The composition of claim 1 wherein said polynucleotide sequence is selected from the group consisting of:(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:11 from nucleotide 2 to nucleotide 1822;(b) a polynucleotide comprising a fragment of the nucleotide sequence of SEQ ID NO:11;(c) a polynucleotide encoding an TNF-R1-DD ligand protein comprising the amino acid sequence of SEQ ID NO:12;(d) a polynucleotide encoding an TNF-R1-DD ligand protein comprising a fragment of the amino acid sequence of SEQ ID NO: 12; and(e) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(d).

31. The composition of claim 8 wherein said protein comprises an amino acid sequence selected from the group consisting of:(a) the amino acid sequence of SEQ ID NO:10; and(b) fragments of the amino acid sequence of SEQ ID NO:10;said protein being substantially free from other mammalian proteins.

32. The composition of claim 8 wherein said protein comprises an amino acid sequence selected from the group consisting of:(a) the amino acid sequence of SEQ ID NO:12; and(b) fragments of the amino acid sequence of SEQ ID NO:12;said protein being substantially free from other mammalian proteins.

33. The method of claim 14 wherein said TNF-R1-DD ligand protein comprises an amino acid sequence selected from the group consisting of:(a) the amino acid sequence of SEQ ID NO:10;(b) fragments of the amino acid sequence of SEQ ID NO:10;(c) the amino acid sequence of SEQ ID NO:12; and(d) fragments of the amino acid sequence of SEQ ID NO:12.

34. The method of claim 26 wherein the second polynucleotide is selected from the group consisting of:(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:9 from nucleotide 2 to nucleotide 931;(b) a polynucleotide comprising a fragment of the nucleotide sequence of SEQ ID NO:9, which encodes a protein having TNF-R1-DD ligand protein activity;(c) a polynucleotide encoding an TNF-R1-DD ligand protein comprising the amino acid sequence of SEQ ID NO: 10;(d) a polynucleotide encoding an TNF-R1-DD ligand protein comprising a fragment of the amino acid sequence of SEQ ID NO:10 and having TNF-R1-DD ligand protein activity;(e) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:11 from nucleotide 2 to nucleotide 1822;(f) a polynucleotide comprising a fragment of the nucleotide sequence of SEQ ID NO:11, which encodes a protein having TNF-R1-DD ligand protein activity;(g) a polynucleotide encoding an TNF-R1-DD ligand protein comprising the amino acid sequence of SEQ ID NO:12; and(h) a polynucleotide encoding an TNF-R1-DD ligand protein comprising a fragment of the amino acid sequence of SEQ ID NO:12 and having TNF-R1-DD ligand protein activity; and(i) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(h), which encodes a protein having TNF-R1-DD ligand protein activity.

35. The composition of claim 1 wherein said polynucleotide sequence is selected from the group consisting of:(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:13 from nucleotide 3 to nucleotide 2846;(b) a polynucleotide comprising a fragment of the nucleotide sequence of SEQ ID NO:13;(c) a polynucleotide encoding an TNF-R1-DD ligand protein comprising the amino acid sequence of SEQ ID NO:14;(d) a polynucleotide encoding an TNF-R1-DD ligand protein comprising a fragment of the amino acid sequence of SEQ ID NO:14; and(e) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(d).

36. The composition of claim 8 wherein said protein comprises an amino acid sequence selected from the group consisting of:(a) the amino acid sequence of SEQ ID NO:14; and(b) fragments of the amino acid sequence of SEQ ID NO:14;said protein being substantially free from other mammalian proteins.

37. The method of claim 14 wherein said TNF-R1-DD ligand protein comprises an amino acid sequence selected from the group consisting of:(a) the amino acid sequence of SEQ ID NO:14; and(b) fragments of the amino acid sequence of SEQ ID NO:14.

38. The method of claim 26 wherein the second polynucleotide is selected from the group consisting of:(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:13 from nucleotide 3 to nucleotide 2846;(b) a polynucleotide comprising a fragment of the nucleotide sequence of SEQ ID NO:13, which encodes a protein having TNF-R1-DD ligand protein activity;(c) a polynucleotide encoding an TNF-R1-DD ligand protein comprising the amino acid sequence of SEQ ID NO:14;(d) a polynucleotide encoding an TNF-R1-DD ligand protein comprising a fragment of the amino acid sequence of SEQ ID NO:14 and having TNF-R1-DD ligand protein activity; and(e) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(d), which encodes a protein having TNF-R1-DD ligand protein activity.

39. The composition of claim 1 wherein said polynucleotide sequence is selected from the group consisting of:(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:15 from nucleotide 326 to nucleotide 5092;(b) a polynucleotide comprising a fragment of the nucleotide sequence of SEQ ID NO:15;(c) a polynucleotide encoding an TNF-R1-DD ligand protein comprising the amino acid sequence of SEQ ID NO:16;(d) a polynucleotide encoding an TNF-R1-DD ligand protein comprising a fragment of the amino acid sequence of SEQ ID NO:16; and(e) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(d).

40. The composition of claim 8 wherein said protein comprises an amino acid sequence selected from the group consisting of:(a) the amino acid sequence of SEQ ID NO:16; and(b) fragments of the amino acid sequence of SEQ ID NO:16;said protein being substantially free from other mammalian proteins.

41. The method of claim 14 wherein said TNF-R1-DD ligand protein comprises an amino acid sequence selected from the group consisting of:(a) the amino acid sequence of SEQ ID NO:16; and(b) fragments of the amino acid sequence of SEQ ID NO:16.

42. The method of claim 26 wherein the second polynucleotide is selected from the group consisting of:(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:15 from nucleotide 326 to nucleotide 5092;(b) a polynucleotide comprising a fragment of the nucleotide sequence of SEQ ID NO:15, which encodes a protein having TNF-R1-DD ligand protein activity;(c) a polynucleotide encoding an TNF-R1-DD ligand protein comprising the amino acid sequence of SEQ ID NO:16;(d) a polynucleotide encoding an TNF-R1-DD ligand protein comprising a fragment of the amino acid sequence of SEQ ID NO:16 and having TNF-R1-DD ligand protein activity; and(e) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(d), which encodes a protein having TNF-R1-DD ligand protein activity.

43. The composition of claim 1 wherein said polynucleotide sequence is selected from the group consisting of:(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:17 from nucleotide 14 to nucleotide 2404;(b) a polynucleotide comprising a fragment of the nucleotide sequence of SEQ ID NO:17;(c) a polynucleotide encoding an TNF-R1-DD ligand protein comprising the amino acid sequence of SEQ ID NO:18;(d) a polynucleotide encoding an TNF-R1-DD ligand protein comprising a fragment of the amino acid sequence of SEQ ID NO:18; and(e) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(d).

44. The composition of claim 8 wherein said protein comprises an amino acid sequence selected from the group consisting of:(a) the amino acid sequence of SEQ ID NO:18; and(b) fragments of the amino acid sequence of SEQ ID NO: 18;said protein being substantially free from other mammalian proteins.

45. The method of claim 14 wherein said TNF-R1-DD ligand protein comprises an amino acid sequence selected from the group consisting of:(a) the amino acid sequence of SEQ ID NO:18; and(b) fragments of the amino acid sequence of SEQ ID NO:18.

46. The method of claim 26 wherein the second polynucleotide is selected from the group consisting of:(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:17 from nucleotide 14 to nucleotide 2404;(b) a polynucleotide comprising a fragment of the nucleotide sequence of SEQ ID NO:17, which encodes a protein having TNF-R1-DD ligand protein activity;(c) a polynucleotide encoding an TNF-R1-DD ligand protein comprising the amino acid sequence of SEQ ID NO: 18;(d) a polynucleotide encoding an TNF-R1-DD ligand protein comprising a fragment of the amino acid sequence of SEQ ID NO:18 and having TNF-R1-DD ligand protein activity; and(e) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(d), which encodes a protein having TNF-R1-DD ligand protein activity.

Images