PILR ALPHA INTERACTIONS AND METHODS OF MODIFYING SAME

Abstract

Described herein is a novel receptor-ligand interaction and agents that may modify and/or block the interaction. Methods, uses, reagents and kits for the modulation of ligand activities related to its interaction with the novel receptor are disclosed. Also disclosed are therapeutic uses of reagents in treating inflammation-related disorders.

Description

RELATED APPLICATIONS

[0001] This application is a divisional of U.S. patent application Ser. No. 15/397,399, filed on Jan. 3, 2017, now U.S. Pat. No. 10,160,807, which is a divisional of U.S. patent application Ser. No. 14/598,870, filed on Jan. 16, 2015, now U.S. Pat. No. 9,556,269, which is a continuation of U.S. patent application Ser. No. 13/921,557, filed on June. 19, 2013, now abandoned, which is a continuation of International Application No. PCT/US2011/066753, having an international filing date of Dec. 22, 2011, which claims benefit of priority applications U.S. Provisional Application No. 61/510,453, filed Jul. 21, 2011, and U.S. Provisional Application No. 61/426,945, filed Dec. 23, 2010, the entire contents of each of which are incorporated herein by reference.

SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on May 22, 2019, is named 50474-095004_Sequence_Listing_05.22.2019_ST25 and is 172,536 bytes in size.

TECHNICAL FIELD

[0003] Described herein is a novel receptor-ligand interaction and agents that may modify and/or block the interaction. Methods, uses, reagents and kits for the modulation of ligand activities related to its interaction with the novel receptor are disclosed. Also disclosed are therapeutic uses of reagents in treating inflammation-related disorders. Specifically, this disclosure relates to PILR.alpha., its ligands and methods of modulating their interaction. Also disclosed herein are methods of treating PILR.alpha.-mediated inflammation and/or diseases, e.g., pathogenic infection, autoimmune arthritis, etc.

BACKGROUND

[0004] Immune function is regulated by the balance of activating and inhibitory signals mediated by immune cell-surface receptors (Lanier, Nature Immunol. (2001) 2:23-27; Smith et al., Immunol. Rev. (2001) 181:115-125; Moretta et al., Annu. Rev. Immunol. (2001) 19:197-223; Diefenbach & Raulet, Curr. Opin. Immunol. (2003) 15:37-44.). Among these receptors, there are families that harbor extracellular regions that have a highly conserved amino-acid sequence but have different intracellular domains responsible for activation and inhibitory signaling. Thus, activating and inhibitory members of these families can generally recognize the same or very similar ligands with slightly different specificities, presumably resulting in fine-tuning of immune regulation. Since they are involved in both the activation and inhibition of immune function, they are generally referred to as paired receptor families. Paired immunoglobulin-like (Ig-like) type 2 receptors (PILRs) are one of the paired receptor families. Inhibitory receptors possess immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in their cytoplasmic domains and deliver inhibitory signals via tyrosine phosphatases, such as SHP-1. The ITIM-bearing receptor PILR-alpha recruits SHP-1 via its amino-terminal SH2 domain and is likely to have cellular inhibitory potential. The lack of a cytoplasmic tail and the presence of the transmembrane lysine residue in the second receptor, PILR-beta, suggest its potential activating function. See Fournier et al., J. Immunol. (2000) 165:1197-1209; Mousseau et al., J. Biol. Chem. (2000) 275:4467-4474.; Shiratori et al., J. Exp. Med. (2004) 199:525-533. Although the ligand for mouse PILR-alpha and PILR-beta was identified as CD99 (Shiratori et al., J. Exp. Med. (2004) 199:525-533), to date no human ligand has been identified for human PILRs.

[0005] Herpes simplex virus type 1 (HSV-1) is a member of the alphaherpesvirus subfamily and can cause recurrent mucocutaneous lesions on the mouth, face, or genitalia and potentially meningitis or encephalitis. Membrane glycoprotein B (gB) of HSV-1 is a second ligand for PILR-alpha (Satoh et al., Cell (2008)132:935-944). The interaction of HSV-1 gB with PILRa mediates viral entry and cell-cell fusion. Interestingly, expression of PILRa on cells enhances HSV-1 entry, whereas expression of PILRb does not (Fan and Longnecker, J. Virol. (2010) 84(17):8664-8672). This suggests HSV-1 gB is not a ligand of PILRb, and subtle amino acid differences between a and b play a role in ligand selectivity. Interestingly, binding of PILRa to HSV-1 gB also requires sialylated O-glycans (T53, T480) (Fan et al., J. Virol. (2009) 83(15):7384-7390). PILRa specifically associates with HSV-1 gB, but not with other HSV-1 glycoproteins, although some other envelope proteins are known to be O-glycosylated (Fan et al., J. Virol. (2009) 83(15):7384-7390).

[0006] Although PILR-alpha and PILR-beta are abundantly expressed on myeloid cells, very little is known about their role in host defense against extracellular bacterial infection or in immune function. Thus, elucidating the role for PILR-alpha and PILR-beta is a necessary prerequisite for the prevention/treatment of diseases/conditions associated with PILR-alpha and/or PILR-beta dysfunction. The present disclosure identifies sialidated glycans as necessary and sufficient for hPILRa binding. The present disclosure also provides a method and compositions, such as selective binding agents, to modulate the interactions of the PILR and its ligands. Specifically, described herein are novel reagents and methods based on the interaction of infective microorganisms and/or endogenous ligands for the prevention/treatment of diseases/conditions associated with PILR-alpha and/or PILR-beta activity.

BRIEF SUMMARY OF THE INVENTION

[0007] The present disclosure relates to various methods, uses, reagents, and kits based on modulation of the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA.

[0008] One embodiment of the invention relates to an agent that may block the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FccRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA.

[0009] Another embodiment of the present invention relates to a composition that may comprise (1) an agent that may block the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA, and (2) a pharmaceutically acceptable carrier.

[0010] Another embodiment of present invention further relates to a method for blocking the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA; the method may comprise the step of administering an effective amount of an agent that may block the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA.

[0011] Another embodiment of the present invention provides an interaction occurring at the cell surface and a method for blocking the cell surface interaction between a PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA may comprise contacting cells (a cell expressing PILR-alpha and/or a cell expressing a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA) with an effective amount of an agent that may block the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA.

[0012] Another embodiment of the present invention provides a method for inhibiting production of an inflammatory mediator by a cell, the method may comprise blocking the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA.

[0013] Another embodiment of the present invention relates to the use of an agent that may block the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA for the preparation of a medicament that may block the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA.

[0014] Another embodiment of the present invention relates to the use of an agent for treating an inflammatory disease/condition in a subject and/or for the preparation of a medicament for treating an inflammatory disease in a subject, wherein the agent may block the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA.

[0015] Another embodiment of the present invention relates to a method for identifying a compound capable of blocking the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA; the method may comprise measuring the binding of PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA in the presence versus the absence of an agent, wherein a lower binding of PILR-alpha to said ligand in the presence of the agent (in comparison with the absence of the agent) may be indicative that the agent is capable of blocking the interaction between PILR-alpha and said ligand.

[0016] Another embodiment of the present invention relates to a method for identifying a compound capable of inhibiting and/or decreasing inflammation; the method may comprise measuring the binding of PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA in the presence versus the absence of the agent, wherein a lower binding of PILR-alpha to the ligand in the presence of the agent may be indicative that the agent is capable of inhibiting or decreasing inflammation.

[0017] Another embodiment of the present invention provides a method of treating an inflammatory disease or condition in a subject; the method may comprise blocking the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA in the subject.

[0018] Another embodiment of the present invention relates to a use of an agent capable of blocking the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA for treating an inflammatory disease or condition in a subject.

[0019] Another embodiment of the present invention relates to a use of an agent capable of blocking the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA for the preparation of a medicament for treating an inflammatory disease or condition in a subject.

[0020] Another embodiment of the present invention relates to a composition for treating an inflammatory disease or condition in a subject comprising (1) an agent capable of blocking the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA and (2) a pharmaceutically acceptable carrier.

[0021] Another embodiment of the present invention relates to an agent that may stimulate the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA.

[0022] Another embodiment of the present invention relates to a composition that may comprise (1) an agent that may stimulate the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA and (2) a pharmaceutically acceptable carrier.

[0023] Another embodiment of the present invention relates to a method for stimulating the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA; the method may comprise the step of administering an effective amount of an agent that may stimulate the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA.

[0024] In an embodiment of the present invention, the interaction may occur at the cell surface and a method for stimulating the cell surface interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA may comprise contacting cells (a cell expressing PILR-alpha and/or a cell expressing a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA) with an effective amount of an agent that may stimulate the interaction between PILR-alpha and said ligand.

[0025] An additional embodiment of the present invention provides a method for inhibiting production of an inflammatory mediator by a cell, the method may comprise stimulating the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA.

[0026] A further embodiment of the present invention relates to the use of an agent that may stimulate the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA for the preparation of a medicament that may stimulate the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA.

[0027] An embodiment of the present invention relates to the use of an agent for treating an inflammatory disease in a subject and/or for the preparation of a medicament for treating an inflammatory disease in a subject wherein the agent may stimulate the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA.

[0028] Another embodiment of the present invention relates to a method for identifying a compound capable of stimulating the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA; the method may comprise measuring the binding of PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA in the presence versus the absence of an agent, wherein an elevated binding of PILR-alpha and said ligand in the presence of the agent (in comparison with the absence of the agent) may be indicative that the agent is capable of stimulating the interaction between PILR-alpha and said ligand.

[0029] Another further embodiment of the present invention relates to a method for identifying a compound capable of stimulating the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA; the method may comprise measuring PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA activity in the presence or absence of the agent, wherein an elevated PILR-alpha activity in the presence of the agent may be indicative that the agent is stimulating the interaction between PILR-alpha and said ligand.

[0030] Another embodiment of the present invention relates to a method for identifying a compound capable of inhibiting and/or decreasing inflammation; the method may comprise measuring the binding of PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA in the presence versus the absence of the agent, wherein an elevated binding of PILR-alpha and said ligand in the presence of the agent may be indicative that the agent is capable of inhibiting or decreasing inflammation.

[0031] Yet another embodiment of the present invention provides a method of identifying a compound capable of inhibiting or decreasing inflammation; the method may comprise measuring PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA activity in the presence versus the absence of the agent, wherein an elevated PILR-alpha activity in the presence of the agent may be indicative that the agent is capable of inhibiting or decreasing inflammation.

[0032] A further embodiment of the present invention provides a method of treating an inflammatory disease or condition in a subject; the method may comprise stimulating the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA in the subject.

[0033] Another embodiment of the present invention relates to a use of an agent capable of stimulating the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA for treating an inflammatory disease or condition in a subject.

[0034] Another embodiment of the present invention relates to a use of an agent capable of stimulating the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA for the preparation of a medicament for treating an inflammatory disease or condition in a subject.

[0035] Another embodiment of the present invention relates to a composition for treating an inflammatory disease or condition in a subject comprising an agent capable of stimulating the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA and a pharmaceutically acceptable carrier.

[0036] In some aspects of any of the foregoing embodiments the ligand may be any one of NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA.

[0037] One more embodiment of the present invention relates to variants of PILR-alpha. In one aspect, the variant comprises an alteration at a residue corresponding to R133 in SEQ ID NO:1. The alteration may be the addition, deletion or substitution of the residue. The variant may comprise a mutation corresponding R133A. In another aspect, the variant comprises an alteration at a residue corresponding to R126 in SEQ ID NO:3. The alteration may be the addition, deletion or substitution of the residue. The variant may comprise a mutation corresponding R126A. In a further aspect, the variant comprises an alteration that abrogates binding of PILRa to a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA. In a further aspect, the variant comprises an alteration that inhibits or decreases binding of PILRa to a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA.

[0038] An additional embodiment of the present invention relates to a binding motif for ligands of PILR-alpha. In one aspect, the ligands comprise a sialidated glycan domain. In a second aspect, the human PILR-alpha binds to a ligand is via a sialoadhesin homology domain.

[0039] In a different embodiment, at least one ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA interacts with PILR-beta. In an aspect, the ligand binds to PILR-beta but with lower affinity than to PILR-alpha.

[0040] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the scope and spirit of the invention will become apparent to one skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0041] FIGS. 1A-1E illustrate that PILRa binds to mouse but not human CD99 and that transfer of mouse CD99 PKAPT motif to human CD99 restores PILR.alpha. binding. FIG. 1A: 293T cells were transfected with mouse and human CD99 expression vectors and the transfectants were stained with hPILR.alpha.-Fc (black line), mouse PILR.alpha.-Fc (black line) or control Ig (grey area). CD99 expressing cells were gated and their binding to PILR.alpha.-Fc was shown. FIG. 1B: Comparison of profiles of O-glycans from human and mouse CD99-Fc fusion proteins. O-glycans were released by reductive .beta.-elimination and permethylated then analyzed by MALDI-TOF MS. FIG. 1C: shows the glycosylation motifs in mouse CD99 and the variant human CD99 where the mouse PKAPT motif was inserted (GB_HSV1/476-484: SEQ ID NO: 67, GB_HSV1/48-57: SEQ ID NO: 68, CD99_MOUSE/41-49: SEQ ID NO: 69, CD99 MOUSE/46-54: SEQ ID NO: 70, CD99_HUMAN/46-51: SEQ ID NO: 71, CD99_HUMAN/46-54: SEQ ID NO: 72). See FIG. 5A in Shiratori et al., J. Exp. Med. (2004) 199(4):525-533 for the mouse CD99 sequence. FIG. 1D: illustrates the recovery of binding of human CD99 with the PKAPT motif inserted (i.e., the PKAPT motif from mouse CD99 was inserted into the human CD99) to both mouse and human PILRa. Both human and mouse PILRa bind mouse CD99 (second row) but fail to bind human CD99 (third row). FIG. 1E: 293T cells were transfected with mouse, human CD99 or human CD99 with mouse PKAPT motif inserted after T41 and the transfectants were stained with hPILR.alpha.-Fc (black line), mouse PILR.alpha.-Fc (black line) or control Ig (grey area).

[0042] FIGS. 2A-2E illustrate novel PILR.alpha. ligands NPDC1 and COLEC12. FIG. 2A: COS7 cells were transfected with hNPDC1 and hCOLEC12 expression vectors, stained with hPILR.alpha.-AP, mPILR.alpha.-AP and control supernatant followed with AP substrate. FIG. 2B: 293T cells were transfected with mCD99, hNPDC1 and hCOLEC12, and the transfectants were stained with isotype control, hPILR.alpha.-Fc or mouse PILR.alpha.-Fc (black line). Binding to mock transfectants (grey area) represents background binding. Transfected ligand expressing cells were gated and their binding to PILR.alpha.-Fc was shown. FIG. 2C: 293T cells were transfected with human and mouse PILR.alpha., and the transfectants were stained with hNPDC1-Fc or hCOLEC12-his (black line). Binding to mock transfectants (grey area) represents background binding. PILR.alpha. positive cells were gated and ligand fusion staining was shown. FIG. 2D: Radioligand assay was used to determine the equilibrium binding affinity of hPILR.alpha.-Fc to hNPDC1 transiently expressed on 293T cells. .sup.125I labeled hPILRa.alpha.-Fc was allowed to bind to cells in the presence of increasing amounts of unlabeled hPRILa-Fc. The average equilibrium K.sub.D from two replicate assays was 49 nM. FIG. 2E: SPR equilibrium binding analysis of hPILR.alpha.-Fc binding to immobilized hCOLEC12 is shown. The equilibrium K.sub.D for hPILR.alpha./hCOLEC12 was 1.1 uM.

[0043] FIG. 3 shows that human NPDC1 and COLEC12 are glycosylated with sialylated glycans. Comparison of MALDI-TOF MS spectra of permethylated O-glycans, released by reductive .beta.-elimination, from human NPDC1-Fc and human COLEC12-his. Molecular ions of permethylated glycans (glycan alditols) were detected in positive ion mode, as sodium adducts (M+Na).sup.+.

[0044] FIG. 4 shows that sialylated glycans on NPDC1 or COLEC12 are required for their binding to PILR.alpha.. The binding of selected proteins to hPILR.alpha. was determined by surface plasmon resonance. Human PILR.alpha.-Fc (25 ug/ml) was immobilized to a CMS sensor chip. Fusion proteins with and without Sialidase A treatment were used as analytes (1 .mu.M). The difference between the responses of negative control protein and the testing proteins was shown.

[0045] FIGS. 5A-5G illustrate that a conserved Arginine site in PILR.alpha. is required for its binding to ligand. FIG. 5A: Amino acid sequence alignment of PILR.alpha. from human (SEQ ID NO: 73), chimp (SEQ ID NO: 74), cow (SEQ ID NO: 75), dog (SEQ ID NO: 76), rat (SEQ ID NO: 77), and mouse (SEQ ID NO: 78); PIL.beta. from human (SEQ ID NO: 79) and mouse (SEQ ID NO: 80); and N-terminal of SIGLEC1 from human (SEQ ID NO: 81) and mouse (SEQ ID NO: 82). The positions of Ig fold residues are designated by an arrow above the sequence (.dwnarw.) based on comparison with Igk/l and TCRb V set Ig domains. Conserved non-Ig PILR.alpha. residues are designated by an .sctn. above the sequence. SIGLEC1 residues involved in the sialic acid-binding are boxed (dashed lines). Conserved Siglec residues across the family are boxed (solid lines). Asterisks represent PILR.alpha. amino acids that are important for sialic acid interaction. Solid arrows beneath the sequence denote the positions corresponding to active sites of SIGLEC1 crystal structure (and PILR.alpha. homology model). The underlined segments designate beta strands in PILR. Black circles represent the mutational binding analysis that have been done on PILR.alpha.. The pairwise-percentage-residue identity between PILR.alpha. and SIGLEC1 was 23%. FIG. 5B: 293T cells were transfected with wild type human and mouse PILR.alpha. (grey line), or human PILR.alpha.R126A and mouse PILR.alpha.133A (black line) expression constructs, and the transfectants were stained with mCD99-Fc, hNPDC1-Fc or hCOLEC12-his, their binding to mock transfectants (grey area) was shown as background binding. PILR.alpha. positive cells were gated and ligand fusion staining was shown. FIG. 5C: 293T cells were transfected with mouse CD99, human NPDC1, human COLEC12, or HSV1 gB expression vectors, and the transfectants were stained with hPILR.alpha.-Fc or mPILR.alpha.-Fc (grey line), hPILR.alpha.R126A-Fc or mPILR.alpha.R133A-Fc (black line), their binding to mock transfectants (grey area) was shown as background binding. Transfected ligand expressing cells were gated and PILR.alpha.-Fc staining was shown. FIG. 5D: 293T cells were infected with HSV1. Twenty-four hours later (FIG. 5D-1) glycoprotein B expression in HSV1 (black line) or mock (grey area) infected cells was shown; FIG. 5D-2, hPILR.alpha.-Fc or mPILR.alpha.-Fc (grey line), hPILR.alpha.R126A-Fc or mPILR.alpha.R133A-Fc (black line) binding to HSV1 infected cells was shown, their binding to mock transfectants (grey area) was shown as background binding. FIG. 5E: WT hPILRa-Fc and hPILRa R126-Fc were immobilized on a chip and their binding to hNPDC1, hCOLEC12, and mCD99 was compared. All three proteins showed strong binding to WT hPILRa (upper three dashed lines on the graph). Little or no binding was observed with mutant hPILRa, suggesting this conserved Arg site is necessary for binding of PILRa to its ligands (lower three lines on the graph with the Mut designation). FIG. 5F: The binding of ligand fusion proteins to wild type and Arginine mutated human PILR.alpha.. The binding of selected proteins to hPILR.alpha. was determined by SPR. Human PILR.alpha.-Fc and PILR.alpha.R126A-Fc was immobilized to a sensor chip. Fusion proteins were used as analytes (1 .mu.M). F. The binding of hPILR.alpha.-Fc or mPILR.alpha.-Fc (grey line), hPILR.alpha.R126A-Fc or mPILR.alpha.R133A-Fc (black line) to human PBMC, T cells and monocytes (FIG. 5F-1), mouse thymocyte, CD8+ T and B cells (FIG. 5F-2) was shown, the binding of isotype control to these cells (grey area) was shown as background binding. FIG. 5G: alignment of four human PILRa isoforms, NP_038467 (SEQ ID NO: 83), NP_840056 (SEQ ID NO: 84), AAH17812 (SEQ ID NO: 85), and NP_840057 (SEQ ID NO: 86).

[0046] FIG. 6 illustrates the binding of PILRa-Fc to human NPDC1 and human COLEC12 expressing cells. HEK293T cells were transfected with human NPDC1, human COLEC12, human CD99 or vector. Transfected 293T cells were incubated with control immunoglobulins or the indicated the PILR constructs indicated on the top followed by an incubation with a second antibody and analyzed by flow cytometry.

[0047] FIG. 7 illustrates the PILRa-Fc binding to ligand transfectants.

[0048] FIGS. 8A-8C illustrate the binding of (A) mouse and (B) human PILRa and the variant PILRa fusion proteins (i.e. R126A for human and R133A for mouse) to 3 different human ligands--COLEC12, CLEC4G and FceRII. FIG. 8C also shows the binding of mPILRb to the same ligands. Note that the variant mPILRa does not bind and that mPILRb binds to a lesser degree than mPILRa for CLEC4G and FceRII.

[0049] FIGS. 9A-9C illustrate the binding of (A) mouse and (B) human PILRa and the variant PILRa fusion proteins (i.e. R126A for human and R133A for mouse) to 3 different human ligands--MAG, FceRII and LRRTM4. FIG. 9C also shows the binding of mPILRb to the same ligands. Note that the variant mPILRa does not bind and that mPILRb binds to a lesser degree than mPILRa for FcRII.

[0050] FIGS. 10A-10C illustrate the binding of (A) mouse and (B) human PILRa and the variant PILRa fusion proteins (i.e. R126A for human and R133A for mouse) to 3 different human ligands--BR3, IL2Ra and PSS8. FIG. 10C also shows the binding of mPILRb to the same ligands. Note that the variant mPILRa does not bind and that mPILRb binds to a lesser degree than mPILRa for BR3.

[0051] FIG. 11 is an extension of FIGS. 6-10C and illustrates the binding of anti-ragweed (control), hPILRa, and the variant hPILRa (R126A) fusion proteins to 2 different human ligands--hNPDC1 and mCD99. Neo indicates cells transformed with a neomycin containing vector used as a negative control. Note that the variant hPILRa does not bind any ligands tested or to the control.

[0052] FIG. 12 is a table showing the various tissue and/or cell types that express each of the listed ligands.

DETAILED DESCRIPTION

[0053] The invention will now be described in detail by way of reference only using the following definitions and examples. All patents and publications, including all sequences disclosed within such patents and publications, referred to herein are expressly incorporated by reference.

[0054] Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D ED., John Wiley and Sons, New York (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide one of skill with a general dictionary of many of the terms used in this invention. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. Practitioners are particularly directed to Sambrook et al., 1989, and Ausubel F M et al., 1993, for definitions and terms of the art. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary.

[0055] Numeric ranges are inclusive of the numbers defining the range.

[0056] Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.

[0057] The headings provided herein are not limitations of the various aspects or embodiments of the invention which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole.

Definitions

PILR Family Members

[0058] As used herein, "PILR" refers to paired immunoglobulin-like receptors (PILR) alpha and beta. They are related type I transmembrane receptors bearing a highly similar (83% identity) extracellular Ig-like variable (V)-type domain but divergent intracellular signaling domains. When only one of the members is being referenced, it will be designated as either PILR.alpha. or PILR.beta..

[0059] Mouse PILR-alpha (mPILR.alpha.) as used herein refers to the protein having the amino acid sequence of SEQ ID NO: 1, which includes a potential signal sequence. Human PILR-alpha (hPILR.alpha.) as used herein refers to the protein having the amino acid sequence of SEQ ID NO: 3, which includes a potential signal sequence. Paired immunoglobulin-like receptor alpha, ITIM-containing inhibitory receptor of the PILR family that associates with SHP-1 (PTPN6) and SHP-2 (PTPN11), involved in signaling and inhibits activation-dependent calcium mobilization in monocytic cells. PILR-alpha may also be referred to as PILRa or PILR.alpha. herein.

[0060] Mouse PILR-beta (mPILRb) as used herein refers to the protein having the amino acid sequence of SEQ ID NO:33 Human PILR-beta (hPILRb) as used herein refers to the protein having the amino acid sequence of SEQ ID NO:35, which includes a potential signal sequence. Paired immunoglobulin like type 2 receptor beta, may play a role in transmembrane receptor protein tyrosine kinase signaling pathway. Increased expression of the gene encoding the protein is associated with acute lymphoblastic leukemias. PILR-beta may also be referred to as PILRb or PILR.beta. herein.

PILR Ligands

[0061] NPDC1 as used herein refers to the protein having the amino acid sequence of SEQ ID NO: 5, which includes a potential signal sequence. Neural proliferation, differentiation and control, I (NPDC1) is specifically expressed in neural cells when they stop to divide and begin to differentiate. It may also regulate transcription, cell proliferation, neuron differentiation, and organ morphogenesis. Its expression is developmentally regulated and persists in the adult; it increases in the embryonic brain, in distinct, defined regions, and is correlated with growth arrest and terminal differentiation. NPDC1 has long hydrophobic stretch of amino acids (residues 13-29), a coiled-coil region (amino acids 93-120), a transmembrane domain (amino acids 191-207), an acidic domain (amino acids 277-307), and MAP-kinases consensus sites (amino acids 234-244) (Evrard and Rouget, (2005) J. Neuro. Res. 79:747-755). It may be clipped and exist in a soluble form.

[0062] COLEC12 as used herein refers to the protein having the amino acid sequence of SEQ ID NO: 7. Collectin subfamily member 12 (COLEC12) is a type II transmembrane glycoprotein that binds bacteria through its lectin domain and may play a role in host defense.

[0063] ETBR as used herein refers to the protein having the amino acid sequence of SEQ ID NO: 8, which includes a potential signal sequence. Endothelin receptor type B (ETBR) regulates angiogenesis, smooth muscle contraction, and cell migration; gene mutation correlates with melanoma, Waardenburg syndrome, and Hirschsprung disease, aberrantly expressed in asthma, atherosclerosis, and several cancers.

[0064] CLEC4G as used herein refers to the protein having the amino acid sequence of SEQ ID NO: 10, which includes a potential signal sequence. C type lectin superfamily 4 member G (CLEC4G) is a homodimerizing protein that functions as a pathogen associated molecular pattern receptor, may play a role in cell-cell adhesion, antigen processing, and presentation. See also, Liu et al., (2004) J. Biol. Chem. 279(18):18748-58.

[0065] BR3 as used herein refers to the protein having the amino acid sequence of SEQ ID NO: 12. BR3 is a 184-residue type III transmembrane protein expressed on the surface of B cells (Thompson, et al., (2001) Science 293:2108-2111; Yan, et al., (2001) Curr. Biol. 11:1547-1552). BR3 is expressed in a variety of disease tissue including multiple myeloma and non-Hodgkin Lymphoma (Novak, A J (2004) Blood 104:2247-2253; Novak, A J (2004) Blood 103:689-694). BR3 mediates NF-kappaB activation and IL10 production, elevated mRNA levels are associated with systemic lupus erythematosus and multiple sclerosis.

[0066] MAG as used herein refers to the protein having the amino acid sequence of SEQ ID NO: 13, which includes a potential signal sequence. Myelin associated glycoprotein (MAG, also called SIGLEC4A) may play a role in cell adhesion. An increase in autoimmune antibody correlates with autism, demyelinating diseases, and polyneuropathies associated with paraproteinemias. Gene polymorphism is associated with schizophrenia.

[0067] IL-2Ra as used herein refers to the protein having the amino acid sequence of SEQ ID NO: 15, which includes a potential signal sequence. Interleukin 2 receptor alpha, plays a role in regulation of T cell mediated immune response, expression is altered in several neoplasms, immune system and inflammatory diseases, Parkinson disease, asthma, and type I diabetes mellitus.

[0068] FceRII as used herein refers to the protein having the amino acid sequence of SEQ ID NO: 17. Fc fragment of IgE low affinity II receptor, acts in thymocyte maturation, histamine secretion, and TNF production, regulates NO production in monocytes, upregulated in hypogammaglobulinaemia, Kawasaki disease, Graves thyrotoxicosis, and chronic uremia.

[0069] LRRTM4 as used herein refers to the protein having the amino acid sequence of SEQ ID NO: 18, which includes a potential signal sequence. Leucine rich repeat transmembrane neuronal 4 may stimulate beta-secretase mediated processing of beta-amyloid-precursor protein, may play a role in brain development and is associated with Alzheimer disease. LRRTM4 contains nine leucine rich repeats.

[0070] DAG1 as used herein refers to the protein having the amino acid sequence of SEQ ID NO: 20, which includes a potential signal sequence. Dystroglycan 1 or dystrophin-associated glycoprotein 1 (DAG1) is an extracellular matrix glycoprotein that acts in muscle contraction, may be involved in synaptic transmission and establishment of cell polarity, aberrant protein expression correlates with muscular dystrophies and several neoplasms.

[0071] APLP1 as used herein refers to the protein having the amino acid sequence of SEQ ID NO: 22, which includes a potential signal sequence. Amyloid beta precursor like protein 1 (APLP1) is an alpha 2A adrenergic receptor binding protein that regulates proteolysis of amyloid precursor proteins, negatively regulates endocytosis; map position of corresponding gene correlates with Alzheimer disease.

[0072] PTPRN as used herein refers to the protein having the amino acid sequence of SEQ ID NO: 24, which includes a potential signal sequence. Protein tyrosine phosphatase receptor-type N (PTPRN) is a putative transmembrane receptor protein tyrosine phosphatase that acts as an autoantigen in type 1 diabetes mellitus.

[0073] WDR31 as used herein refers to the protein having the amino acid sequence of SEQ ID NO: 26, which includes a potential signal sequence. WD repeat domain 31 (WDR31) contains five WD domain G-beta repeats, has low similarity to S. pombe Cpc2p, which is required for normal mating, sporulation, and protein translation and is a putative receptor for S. pombe Pck2p during cell wall synthesis and morphogenesis.

[0074] PSS8 as used herein refers to the protein having the amino acid sequence of SEQ ID NO: 27, which includes a potential signal sequence. Protease serine 8 or prostasin (PSS8) is a serine protease that plays a role in regulation of the amiloride-sensitive epithelial sodium channel, overexpressed in ovarian cancer cells; corresponding gene expression is downregulated in prostate cancers.

[0075] SIGLEC7 as used herein refers to the protein having the amino acid sequence of SEQ ID NO: 29, which includes a potential signal sequence. Sialic acid binding Ig-like lectin 7 (SIGLEC7) is a disialoganglioside-binding inhibitory receptor in natural killer cells that may act in myelopoiesis. It interacts with the lipooligosaccharide (LOS) on the surface of Campylobacter jejuni.

[0076] IL15-RA as used herein refers to the protein having the amino acid sequence of SEQ ID NO: 31, which includes a potential signal sequence. Interleukin 15 receptor alpha (IL15-RA), the interleukin-15 (IL15)-binding subunit of the receptor, functions in signal transduction, cell proliferation, and natural killer cell survival, may be involved in the cellular defense response to various pathogens.

[0077] The term "antibody" herein is used in the broadest sense and refers to any immunoglobulin (Ig) molecule comprising two heavy chains and two light chains, and any fragment, mutant, variant or derivation thereof which so long as they exhibit the desired biological activity (e.g., epitope binding activity). Examples of antibodies include monoclonal antibodies, polyclonal antibodies, multispecific antibodies and antibody fragments.

[0078] The Kabat numbering system is generally used when referring to a residue in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g, Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The "EU numbering system" or "EU index" is generally used when referring to a residue in an immunoglobulin heavy chain constant region (e.g., the EU index reported in Kabat et al., supra). The "EU index as in Kabat" refers to the residue numbering of the human IgG1 EU antibody. Unless stated otherwise herein, references to residue numbers in the variable domain of antibodies means residue numbering by the Kabat numbering system. Unless stated otherwise herein, references to residue numbers in the constant domain of antibodies means residue numbering by the EU numbering system (e.g., see U.S. Provisional Application No. 60/640,323, Figures for EU numbering).

[0079] The term "multispecific antibody" is used in the broadest sense and specifically covers an antibody that has polyepitopic specificity. Such multispecific antibodies include, but are not limited to, an antibody comprising a heavy chain variable domain (V.sub.H) and a light chain variable domain (V.sub.L), where the V.sub.HV.sub.L unit has polyepitopic specificity, antibodies having two or more V.sub.L and V.sub.H domains with each V.sub.HV.sub.L unit binding to a different epitope, antibodies having two or more single variable domains with each single variable domain binding to a different epitope, full length antibodies, antibody fragments such as Fab, Fv, dsFv, scFv, diabodies, bispecific diabodies and triabodies, antibody fragments that have been linked covalently or non-covalently. "Polyepitopic specificity" refers to the ability to specifically bind to two or more different epitopes on the same or different target(s). "Monospecific" refers to the ability to bind only one epitope. According to one embodiment the multispecific antibody is an IgG antibody that binds to each epitope with an affinity of 5 .mu.M to 0.001 pM, 3 .mu.M to 0.001 pM, 1 .mu.M to 0.001 pM, 0.5 .mu.M to 0.001 pM, or 0.1 .mu.M to 0.001 pM.

[0080] In the studies described herein, Applicants have demonstrated that PILR is a functional receptor for sialidated glycan ligands. Specifically, the ligand is selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA.

Other Definitions

[0081] The terms "PILR gene" or "PILR nucleic acid molecule" or "polynucleotide" refers to a nucleic acid molecule comprising or consisting of a nucleotide sequence encoding a specific PILR polypeptide. Exemplary nucleotide sequences are set forth in FIG. 1A of Fournier et al., J. Immunol. (2000) 165:1197-1209 and NM_013439 for human PILRa; multiple cDNAs have been identified for PILR.beta. (Wilson et al., (2006) Physiol. Genomics 27:201-18.) and annotated by NCBI, e.g., NM_178238.1, NM_178238.2, for human PILRb.

[0082] The term "polypeptide allelic variant" refers to the polypeptide encoded by one of several possible naturally occurring alternate forms of a gene occupying a given locus on a chromosome of an organism or a population of organisms.

[0083] The term "polypeptide derivatives" refers to a polypeptide that has been chemically modified.

[0084] The term "polypeptide fragment" refers to a polypeptide that comprises a truncation at the amino terminus (with or without a leader sequence) and/or a truncation at the carboxy terminus of the polypeptide whose sequence is as defined herein. Polypeptide fragments may result from alternative RNA splicing or from in vivo protease activity. For transmembrane or membrane-bound forms of the polypeptides, preferred fragments include soluble forms such as those lacking a transmembrane or membrane-binding domain.

[0085] In preferred embodiments, truncations comprise about 10 amino acids, or about 20 amino acids, or about 50 amino acids, or about 75 amino acids, or about 100 amino acids, or more than about 100 amino acids. The polypeptide fragments so produced will comprise about 25 contiguous amino acids, or about 50 amino acids, or about 75 amino acids, or about 100 amino acids, or about 150 amino acids, or about 200 amino acids. Such polypeptide fragments may optionally comprise an amino terminal methionine residue. It will be appreciated that such fragments can be used, for example, to generate antibodies to the polypeptides.

[0086] The term "PILR fusion polypeptide" refers to a fusion of one or more amino acids (such as a heterologous peptide or polypeptide) at the amino or carboxy terminus of a PILR polypeptide. In an aspect the heterologous polypeptide is an Fc region of on IgG.

[0087] The term "polypeptide ortholog" refers to a polypeptide from another species that corresponds to an polypeptide as defined herein. For example, mouse and human PILRa polypeptides are considered orthologs of each other. For ease of reference, the human and mouse PILRa polypeptide sequences are aligned and shown in FIG. 1C of Fournier et al., (2000) supra.

[0088] The term "polypeptide variants" refers to, for example, PILRa polypeptides comprising amino acid sequences having one or more amino acid sequence substitutions, deletions (such as internal deletions and/or PILRa polypeptide fragments), and/or additions (such as internal additions and/or PILRa fusion polypeptides) as compared to the PILRa polypeptide as defined above. Variants may be naturally occurring (e.g., PILRa polypeptide allelic variants, PILRa polypeptide orthologs and PILRa polypeptide splice variants) or may be artificially constructed. Such PILRa polypeptide variants may be prepared from the corresponding nucleic acid molecules having a DNA sequence that varies accordingly from the DNA sequence as defined above for the PILRa gene. In preferred embodiments, the variants have from 1 to 3, or from 1 to 5, or from 1 to 10, or from 1 to 15, or from 1 to 20, or from 1 to 25, or from 1 to 50, or from 1 to 75, or from 1 to 100, or more than 100 amino acid substitutions, insertions, additions and/or deletions, wherein the substitutions may be conservative, or non-conservative, or any combination thereof.

[0089] In general, a polypeptide "variant" (i.e. a variant of any polypeptide disclosed herein) means a biologically active polypeptide having at least about 80% amino acid sequence identity with the corresponding native sequence polypeptide. Such variants include, for instance, polypeptides wherein one or more amino acid (naturally occurring amino acid and/or a non-naturally occurring amino acid) residues are added, or deleted, at the N- and/or C-terminus of the polypeptide. Ordinarily, a variant will have at least about 80% amino acid sequence identity, or at least about 90% amino acid sequence identity, or at least about 95% or more amino acid sequence identity with the native sequence polypeptide. Variants also include polypeptide fragments (e.g., subsequences, truncations, etc.), typically biologically active, of the native sequence.

[0090] "Percent (%) amino acid sequence identity" herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in a selected sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are obtained as described below by using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc. has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087, and is publicly available through Genentech, Inc., South San Francisco, Calif. The ALIGN-2 program should be compiled for use on a UNIX operating system, e.g., digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

[0091] For purposes herein, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows:

[0092] 100 times the fraction X/Y where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A.

[0093] The term "heterologous" when used with reference to portions of a nucleic acid indicates that the nucleic acid comprises two or more subsequences that are not normally found in the same relationship to each other in nature. For instance, the nucleic acid is typically recombinantly produced, having two or more sequences, e.g., from unrelated genes arranged to make a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source. Similarly, a heterologous protein will often refer to two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).

[0094] "Essentially pure" protein means a composition comprising at least about 90% by weight of the protein, based on total weight of the composition, preferably at least about 95% by weight, more preferably at least about 90% by weight, even more preferably at least about 95% by weight. "Essentially homogeneous" protein means a composition comprising at least about 99% by weight of protein, based on total weight of the composition.

[0095] The term "antagonist" when used herein refers to a molecule capable of neutralizing, blocking, inhibiting, abrogating, reducing or interfering with the activities of a protein of the invention including its binding to one or more receptors in the case of a ligand or binding to one or more ligands in case of a receptor. Antagonists include antibodies and antigen-binding fragments thereof, proteins, peptides, glycoproteins, glycopeptides, glycolipids, polysaccharides, oligosaccharides, nucleic acids, bioorganic molecules, peptidomimetics, pharmacological agents and their metabolites, transcriptional and translation control sequences, and the like. Antagonists also include small molecule inhibitors of a protein of the invention, and fusions proteins, receptor molecules and derivatives which bind specifically to protein thereby sequestering its binding to its target, antagonist variants of the protein, antisense molecules directed to a protein of the invention, RNA aptamers, and ribozymes against a protein of the invention.

[0096] A "blocking" antibody or an "antagonist" antibody is one which inhibits or reduces biological activity of the antigen it binds. Certain blocking antibodies or antagonist antibodies substantially or completely inhibit the biological activity of the antigen.

[0097] The term "antigen" refers to a molecule or a portion of a molecule capable of being bound by a selective binding agent, such as an antibody, and additionally capable of being used in an animal to produce antibodies capable of binding to an epitope of each antigen. An antigen may have one or more epitopes.

[0098] The terms "active" and "biologically active" polypeptides refer to polypeptides having at least one activity characteristic of the reference polypeptide. For example, the enzymatic activity associated with a protease is proteolysis and, thus, an active protease has proteolytic activity. It follows that the biological activity of a given protein refers to any biological activity typically attributed to that protein by those of skill in the art. as defined above.

[0099] As used herein, the term "immunoadhesin" designates antibody-like molecules that combine the binding specificity of a heterologous protein (an "adhesin") with the effector functions of immunoglobulin constant domains. Structurally, the immunoadhesins comprise a fusion of an amino acid sequence with the desired binding specificity that is other than the antigen recognition and binding site of an antibody (i.e., is "heterologous"), and an immunoglobulin constant domain sequence. The adhesin part of an immunoadhesin molecule typically is a contiguous amino acid sequence comprising at least the binding site of a receptor or a ligand--such as a PILRa, for example. The immunoglobulin constant domain sequence in the immunoadhesin can be obtained from any immunoglobulin, such as IgG-1, IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE, IgD, or IgM.

[0100] In one aspect thereof, the present invention relates to an agent that may block the interaction between a ligand (as defined herein) and a PILR family member. In another aspect, the present invention relates to an agent that may modulate the interaction between a ligand (as defined herein) and a PILR family member.

[0101] As used herein, an "agent" that may block the interaction between a ligand (as defined herein) and PILRa may be a protein. For example, such protein may be an (isolated) antibody, or antigen-binding fragment (portion) thereof, that may specifically bind to a ligand (as defined herein) and/or PILRa. The antibody may be, for example, a monoclonal antibody and/or a polyclonal antibody. Monoclonal antibodies (MAbs) may be made by one of several procedures available to one of skill in the art, for example, by fusing antibody producing cells with immortalized cells and thereby making a hybridoma. The general methodology for fusion of antibody producing B cells to an immortal cell line is well within the province of one skilled in the art. Another example is the generation of MAbs from mRNA extracted from bone marrow and spleen cells of immunized animals using combinatorial antibody library technology. One drawback of MAbs derived from animals or from derived cell lines is that although they may be administered to a patient for diagnostic or therapeutic purposes, they are often recognized as foreign antigens by the immune system and are unsuitable for continued use. Antibodies that are not recognized as foreign antigens by the human immune system have greater potential for both diagnosis and treatment. Methods for generating human and humanized antibodies are now well known in the art.

[0102] Polyclonal antibodies may be obtained by immunizing a selected animal with a protein or polypeptide (for example without limitation a ligand or PILRa or PILRb). Serum from the animal may be collected and treated according to known procedures. Polyclonal antibodies to the protein or polypeptide of interest may then be purified by affinity chromatography. Techniques for producing polyclonal antisera are well known in the art.

[0103] Antibodies may originate for example, from a mouse, rat or any other mammal. The antibody may also be a human antibody which may be obtained, for example, from a transgenic non-human mammal capable of expressing human immunoglobulin genes. The antibody may also be a humanized antibody which may comprise, for example, one or more complementarity determining regions of non-human origin. It may also comprise a surface residue of a human antibody and/or framework regions of a human antibody. The antibody may also be a chimeric antibody which may comprise, for example, variable domains of a non-human antibody and constant domains of a human antibody. Suitable antibodies may also include, for example, an antigen-binding fragment, a Fab fragment; a F(ab')2 fragment, and Fv fragment; or a single-chain antibody comprising an antigen-binding fragment (e.g., a single chain Fv). An antibody encompassed in the present invention may be an antibody binding specifically to PILRa. In an embodiment, an antibody encompassed in the present invention may be an antibody binding specifically to a ligand (as described herein).

[0104] Anti-PILR agents (e.g. antibodies) may be experimentally tested and validated using in vivo and in vitro assays. Suitable assays include, but are not limited to, activity assays and binding assays.

[0105] The activity of a ligand interaction with PILR may, among other means, be measured by the following methods:

[0106] Suitable assays for thymocyte or splenocyte cytotoxicity include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3. 1-3.19; Chapter 7, Immunologic studies in Humans); Herrmann, et al., Proc. Natl. Acad. Sci. USA 78:2488-2492 (1981); Herrmann, et al., J. Immunol. 128:1968-1974 (1982); Handa, et al., J. Immunol. 135:1564-1572 (1985); Takai, et al., I. Immunol. 137:3494-3500 (1986); Takai, et al., J. Immunol. 140:508-512 (1988); Bowman, et al., J. Virology 61:1992-1998; Bertagnolli, et al., Cellular Immunology 133:327-341 (1991); Brown, et al., J. Immunol. 153:3079-3092 (1994).

[0107] Assays for T-cell-dependent immunoglobulin responses and isotype switching (which will identify, among others, proteins that modulate T-cell dependent antibody responses and that affect Th1/Th2 profiles) include, without limitation, those described in: Maliszewski, J. Immunol. 144:3028-3033 (1990); and Assays for B cell function: In vitro antibody production, Mond, J. J. and Brunswick, M. In Current Protocols in Immunology. J. E. e.a. Coligan eds. Vol 1 pp. 3.8.1-3.8.16, John Wiley and Sons, Toronto. 1994.

[0108] Mixed lymphocyte reaction (MLR) assays (which will identify, among others, proteins that generate predominantly Th1 and CTL responses) include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai, et al., J. Immunol. 137:3494-3500 (1986); Takai, et al., J. Immunol. 140:508-512 (1988); Bertagnolli, et al., J. Immunol. 149:3778-3783 (1992).

[0109] Dendritic cell-dependent assays (which will identify, among others, proteins expressed by dendritic cells that activate naive T-cells) include, without limitation, those described in: Guery et al., J. Immunol. 134:536-544 (1995); Inaba et al., J. Exp. Med. 173:549-559 (1991); Macatonia, et al., J. Immunol. 154:5071-5079 (1995); Porgador, et al., J. Exp. Med. 182:255-260 (1995); Nair, et al., J. Virology 67:4062-4069 (1993); Huang, et al., Science 264:961-965 (1994); Macatonia, et al., J. Exp. Med. 169:1255-1264 (1989); Bhardwaj, et al., J. Clin. Invest. 94:797-807 (1994); and Inaba, et al., J. Exp. Med. 172:631-640 (1990).

[0110] Assays for lymphocyte survival/apoptosis (which will identify, among others, proteins that prevent apoptosis after superantigen induction and proteins that regulate lymphocyte homeostasis) include, without limitation, those described in: Darzynkiewicz et al., Cytometry 13:795-808 (1992); Gorczyca, et al., Leukemia 7:659-670 (1993); Gorczyca, et al., Cancer Res. 53:1945-1951 (1993); Itoh, et al., Cell 66:233-243 (1991); Zacharchuk, J. Immunol. 145:4037-4045 (1990); Zamai, et al., Cytometty 14:891-897 (1993); Gorczyca, et al., Int. J. Oncol. 1:639-648 (1992).

[0111] Assays for proteins that influence early steps of T-cell commitment and development include, without limitation, those described in: Antica, et al., Blood 84:111-117 (1994); Fine, etal., Cell. Immunol. 155:111-122, (1994); Galy, et al., Blood 85:2770-2778 (1995); Toki, et al., Proc. Nat. Acad. Sci. USA 88:7548-7551 (1991).

[0112] According to the present invention, a (protein) agent may also be a "soluble protein". Soluble proteins (purified) of the invention may be obtained from any techniques well known in the art. For example, a soluble protein may be obtained by transfecting a recombinant DNA molecule expressing solely the extracellular region of a molecule and/or portion thereof followed by purification. In another example, a protein and/or a portion of a protein (for example an extracellular region exempt of its transmembrane and cytoplasmic domains) may be fused to a constant domain (Fc portion) of an immunoglobulin. A (purified) soluble protein of the present invention may be soluble PILR and/or portion thereof. By "portion" (of soluble protein for example) it is meant a portion that exhibits similar (biological) activity yet is smaller in size. An agent of the present invention may be soluble PILRa. An agent of the present invention may be portions of soluble PILRa. Human PILRa (SEQ ID NO: 3) is a 303 amino acid protein. Its extracellular domain is approximately 170 amino acids in length. A (purified) soluble human PILRa may have a sequence that may consist from about residue 20 to residue 207 of SEQ ID NO:3. The present invention relates to and explicitly incorporates herein each and every specific member and combination of sub-ranges therein whatsoever. Thus, any specified range or group is to be understood as a shorthand way of referring to each and every member of a range or group individually as well as each and every possible sub-ranges or sub-groups encompassed therein; and similarly with respect to any sub-ranges or sub-groups therein.

[0113] As used herein, the term "block" or "inhibit" refers to a decrease in one or more given measurable activity by at least 10% relative to a reference and/or control. Where inhibition is desired, such inhibition is preferably at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more, up to and including 100%, i.e., complete inhibition or absence of the given activity. As used herein, the term "substantially inhibits/blocks" refers to a decrease in a given measurable activity by at least 50% relative to a reference. For example, "substantially inhibits" refers to a decrease in a given measurable activity of at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% and up to and including 100% relative to a reference. As used herein, "blocks/prevents/inhibits/impairs/lowers the interaction", with reference to the binding of a ligand that binds to a receptor refers to a decrease in binding by at least 10% relative to a reference. An agent may block the binding of a ligand to a receptor-expressing cells. "Inhibits the interaction" and/or "block the binding" preferably refers to a decrease in binding of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more, up to and including 100%. A "receptor" as provided for herein means PILRa or PILRb. A "ligand" as provided for herein is selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA. A general feature of a ligand is glycan modification, e.g., sialidated glycans.

[0114] A "composition" of the invention including an agent may be manufactured in a conventional manner. In particular, it is formulated with a pharmaceutically acceptable diluent or carrier, e.g., water or a saline solution such as phosphate buffer saline. In general, a diluent or carrier is selected on the basis of the mode and route of administration, as well as standard pharmaceutical practice. Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, it may be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of compositions may be brought about by including in the composition an agent which delays absorption, for example, monostearate salts and gelatin. Moreover, an agent of the invention may be administered in a time release formulation, for example in a composition which includes a slow release polymer. The active agents may be prepared with carriers that will protect the agent against rapid release, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers may be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid and polylactic, polyglycolic copolymers (PLG). Many methods for the preparation of such formulations are patented or generally known to those skilled in the relevant art. The present invention relates to compositions that may comprise an agent capable of modulating PILR (e.g., PILRa or PILRb) activity and a pharmacologically acceptable carrier. In one embodiment, such compositions include an agent that may block the interaction between a ligand and PILRa to treat an PILRa-related disease (for example an immune-related disease and/or inflammatory disease and/or microbial infection).

[0115] As used herein "pharmaceutically acceptable carrier" or excipient includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. In one embodiment, the carrier is suitable for parenteral administration. Alternatively, the carrier may be suitable for intravenous, intraperitoneal, intramuscular, sublingual or oral administration. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media is incompatible with the active agent, use thereof in the pharmaceutical compositions of the invention is contemplated. Supplementary active compounds may also be incorporated into the compositions.

[0116] "Administration" of a composition may be performed by any suitable routes. Such routes may include parenteral, pulmonary, nasal and/or oral routes. In one embodiment, the pharmaceutical composition may be intra-muscular (IM), subcutaneous (SC), intra-dermal (ID), intra-venous (IV) and/or intra-peritoneal (IP) routes using any suitable means.

[0117] The term "effective amount" is intended to mean an amount of an agent sufficient to substantially block the interaction between a ligand (as defined herein) and PILR (e.g., PILRa or PILRb). An effective amount may also encompass either "therapeutically effective amount" and/or "prophylactically effective amount". A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result, such as a reduction in disease progression and/or alleviation of the symptoms associated with a disease. A therapeutically effective amount of modulators of a PILR activity may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the agent to elicit a desired response in the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response. A therapeutically effective amount is also one in which any toxic or detrimental effects of the agent are outweighed by the therapeutically beneficial effects. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result, such as preventing and/or inhibiting (reducing) the rate of disease onset or progression. A prophylactically effective amount may be determined as described above for the therapeutically effective amount. For any particular subject, specific dosage regimens may be adjusted over time according to the individual need and the professional judgment of the person administering of the compositions.

[0118] During inflammation, various molecules may be secreted by cells. Such molecules may be referred to as "inflammatory mediators". As will be appreciated by one skilled in the art, these inflammatory mediators may be, for example and without limitation, amines, eicosanoids, growth factors, reactive oxygen species, enzymes (for example a proteinase), chemokines, cytokines, etc.

[0119] Measuring the binding of a ligand (as defined herein) to PILRa may be performed using (without limitation) such suitable assays as quantitative comparisons comparing kinetic and equilibrium binding constants. The kinetic association rate (k.sub.on) and dissociation rate (k.sub.off), and the equilibrium binding constants (K.sub.d) may be determined using surface plasmon resonance on a BIAcore.TM. instrument following the standard procedure in the literature. Binding properties of these interactions may also be assessed by flow cytometry and/or by solid phase binding assay.

[0120] The present invention also relates to a method of identifying a compound capable of blocking the interaction between a ligand and PILRa; the method may comprise measuring a ligand-mediated PILRa activity in the presence or absence of the agent, wherein a lower PILRa activity in the presence of the agent may be indicative that the agent is blocking the interaction between a ligand and PILRa.

[0121] As used herein, "an activity mediated by a ligand" or "a ligand-mediated PILRa activity" is an activity involving or resulting from the binding of a ligand to PILRa, and includes, but is not limited to, binding to PILRa, the induction of T cells to produce and secrete cytokines (for example IL-2, IL-10, IFN-.gamma. and TNF-.alpha.), the synthesis of inflammatory molecules (inflammatory mediators) such as IL-6, IL-8 and metalloproteinases and T-cell proliferation (or inhibition thereof), etc. It will be understood that the ligand-mediated activity may depend on the specific ligand, e.g., NPCD1, BR3, etc., being evaluated.

[0122] In an embodiment the use may be for the treatment or prevention of inflammatory-related diseases or condition, or a microbial infection in the subject.

[0123] One of skill in the art will recognize that while the some definitions have been written with reference to a specific polypeptide they are equally applicable to other polypeptides described herein.

[0124] Although various embodiments of the invention are disclosed herein, many adaptations and modifications may be made within the scope of the invention in accordance with the common general knowledge of those skilled in the relevant art. Such modifications include the substitution of known equivalents for any aspect of the invention in order to achieve the same result in substantially the same way. Furthermore, numeric ranges are inclusive of the numbers defining the range. In the claims, the word "comprising" is used as an open-ended term, substantially equivalent to the phrase "including, but not limited to". The following examples are illustrative of various aspects of the invention, and do not limit the broad aspects of the invention as disclosed herein.

[0125] In the experimental disclosure which follows, the following abbreviations apply: eq (equivalents); M (Molar); .mu.M (micromolar); N (Normal); mol (moles); mmol (millimoles); .mu.mol (micromoles); nmol (nanomoles); g (grams); mg (milligrams); kg (kilograms); .mu.g (micrograms); L (liters); ml (milliliters); .mu.l (microliters); cm (centimeters); mm (millimeters); .mu.m (micrometers); nm (nanometers); .degree. C. (degrees Centigrade); h (hours); min (minutes); sec (seconds); msec (milliseconds); Ci (Curies) mCi (milliCuries); .mu.Ci (microCuries); TLC (thin layer achromatography);. CIA (Collagen Induced Arthritis); EAE (Experimental allergy encephalitis); AIA (Antibody induced arthritis);DTH (Delayed Type Hypersensitivity)

EXAMPLES

[0126] The present invention is described in further detain in the following examples which are not in any way intended to limit the scope of the invention as claimed. The attached Figures are meant to be considered as integral parts of the specification and description of the invention. All references cited are herein specifically incorporated by reference for all that is described therein. The following examples are offered to illustrate, but not to limit the claimed invention.

[0127] Cells and Reagents used in the Examples were as follows: All transfections were performed with Fugene 6 (Roche) or Lipofectamine 2000 (Invitrogen) according to the manufacturers protocol. Mouse total RNA adult tissue panel was purchased from Zyagen (San Diego, Calif.). Recombinant human cytokines were from Peprotech (Rocky Hill, N.J.). The following anti-mouse antibodies were used for flow cytometry: anti-mPILRa, anti-mCD99 and anti-mCOLEC12 are all from R&D Systems (Minneapolis, Minn.), anti-FLAG (M2) monoclonal antibody was purchased from Sigma-Aldrich and labeled with Alexa Flour-647 monoclonal antibody labeling kit from Invitrogen, and anti-mouse IgG2a-FITC was from BD Biosciences (San Jose, Calif.).

Example 1

Recombinant Production of PILR, Ligands and Antibodies Thereof

[0128] This example illustrates preparation of potentially glycosylated forms of the desired ligands or PILR proteins (either of which is referred to in this example as a desired protein) by recombinant expression in mammalian cells. Preparations of PILR antibodies are also described.

[0129] The vector, pRK5 (see EP 307,247, published Mar. 15, 1989), was employed as the expression vector in all instances. Optionally, DNA encoding the desired protein was ligated into pRK5 with selected restriction enzymes to allow insertion of such DNA using ligation methods such as described in Sambrook et al., supra. Epitope-tagged variants of the desired protein may also be expressed in cells. The DNA encoding the desired protein was ligated into pRK5 containing the desired epitope tag (poly-His, FLAG, human IgG.sub.1 Fc) in frame with the desired epitope tag.

[0130] The predicted extracellular domains of human and mouse PILR were cloned without the transmembrane domain into the pRK5 vector containing a C-terminal human IgG.sub.1-Fc or 8.times.-His tag, or with the transmembrane domain into the pRK5 vector containing a C-terminal GFP tag.

[0131] Soluble forms of these proteins were produced in a CHO cell transient transfection and purified by affinity chromatography using anti-FLAG (M2) agarose affinity gel (Sigma-Aldrich) for FLAG-tagged proteins, Ni-NTA agarose (Qiagen) for 8.times.-His-tagged proteins, or protein-A Sepharose (Amersham Pharmacia) for IgG1-Fc fusion proteins. Proteins were further separated from aggregates and contaminants with a Superdex 200 gel-filtration column and/or MonoQ/S ion exchange columns (Amersham). Protein purity was assessed by SDS-PAGE followed by SimplyBlue Safe Stain (Invitrogen) and purified proteins were aliqouted and frozen at -80.degree. C. until needed.

[0132] In one embodiment, the selected host cells may be HEK293T cells. Human 293 cells (ATCC.RTM. CCL 1573) were grown to 50-80% confluence in tissue culture plates in medium such as DMEM supplemented with fetal calf serum and optionally, nutrient components and/or antibiotics. 1-10 .mu.g of DNA encoding the desired protein ligated into pRK5 was introduced into HEK293T cells using commercially available transfection reagents SUPERFECT.RTM. (QIAGEN), LIPOFECTAMINE.RTM. (INVITROGEN.RTM.) or FUGENE.RTM. (ROCHE.RTM.) according to manufacturer's instructions. 18-24 hours after the transfections, the culture medium was removed and tested in selected bioassays or cells were harvested using 10 mM EDTA in 20 mM Na phosphate buffer, pH7.4, and tested in selected bioassays.

[0133] Stable expression of the desired protein was achieved in HEK293T cells by cloning DNA encoding the desired protein into pRK5 vector with a selection marker that confers resistance to the antibiotic GENETICIN.RTM.. For stable expression of desired proteins, cells were transfected as described and allowed to grow in DMEM with a concentration of GENETICIN.RTM. that would permit growth of cells in which the desired vector had integrated into the genome (1-0.5 .mu.g/ml).

[0134] In another embodiment, the epitope tagged versions of the desired protein can be expressed in host CHO cells. Twelve micrograms of the desired plasmid DNA was introduced into approximately 10 million CHO cells using commercially available transfection reagents SUPERFECT.RTM. (QIAGEN.RTM.), DOSPER.RTM., LIPOFECTAMINE.RTM. (INVITROGEN.RTM.) or FUGENE.RTM. (Boehringer Mannheim) according to manufacturer's instructions. The cells are grown as described in Lucas et al. (Nucl. Acids Res. (1996) 24:9 1774-1779). Approximately 3.times.10.sup.-7 cells are frozen in an ampule for further growth and production as described below.

[0135] The ampules containing the plasmid DNA are thawed by placement into a water bath and mixed by vortexing. The contents are pipetted into a centrifuge tube containing 10 mLs of media and centrifuged at 1000 rpm for 5 minutes. The supernatant was aspirated and the cells were resuspended in 10 mL of selective media (0.2 .mu.m filtered PS20 with 5% 0.2 .mu.m diafiltered fetal bovine serum). The cells are then aliquoted into a 100 mL spinner containing 90 mL of selective media. After 1-2 days, the cells are transferred into a 250 mL spinner filled with 150 mL selective growth medium and incubated at 37.degree. C. After another 2-3 days, 250 mL, 500 mL and 2000 mL spinners are seeded with 3.times.10.sup.5 cells/mL. The cell media was exchanged with fresh media by centrifugation and resuspension in production medium. Although any suitable CHO media may be employed, a production medium described in U.S. Pat. No. 5,122,469, issued Jun. 16, 1992 may actually be used. A 3 L production spinner was seeded at 1.2.times.10.sup.6 cells/mL. On day 0, the cell number and pH was determined. On day 1, the spinner was sampled and sparging with filtered air was commenced. On day 2, the spinner was sampled, the temperature shifted to 33.degree. C., and 30 mL of 500 g/L glucose and 0.6 mL of 10% antifoam (e.g., 35% polydimethylsiloxane emulsion, Dow Corning 365 Medical Grade Emulsion) taken. Throughout the production, the pH was adjusted as necessary to keep it at around 7.2. After 10 days, or until the viability dropped below 70%, the cell culture was harvested by centrifugation and filtering through a 0.22 .mu.m filter. The filtrate was either stored at 4.degree. C. or immediately loaded onto columns for purification.

[0136] For the poly-His tagged constructs, the proteins are purified using a Ni-NTA column (Qiagen). Before purification, imidazole was added to the conditioned media to a concentration of 5 mM. The conditioned media was pumped onto a 6 ml Ni-NTA column equilibrated at 4.degree. C., in 20 mM Hepes, pH 7.4, buffer containing 0.3 M NaCl and 5 mM imidazole at a flow rate of 4-5 ml/min. After loading, the column was washed with additional equilibration buffer and the protein eluted with equilibration buffer containing 0.25 M imidazole. The purified protein was then run over a Superdex 5200 gel filtration column and/or a MonoQ/S ion exchange column (Applied Biosystems) to remove aggregated or proteolysed protein or any contaminants and subsequently concentrated and dialyzed into PBS. The homogeneity was assessed by SDS polyacrylamide gels and by N-terminal amino acid sequencing by Edman degradation. Proteins were stored at -80.degree. C. until used in bioassays.

[0137] For the FLAG-epitope tagged constructs, the proteins are purified using an anti-FLAG (M2) agarose column (Sigma). The conditioned media was pumped onto a 6 ml anti-FLAG column equilibrated at 4.degree. C. with 20 mM Na phosphate buffer, pH 7.4. After loading, the column was washed extensively with equilibration buffer before elution with 100 mM citric acid, pH 3.5. The eluted protein was immediately neutralized by collecting 1 ml fractions into tubes containing 275 .mu.L of 1 M Tris buffer, pH 9. The highly purified protein was subsequently run over size exclusion chromatography, dialyzed, analyzed, and stored as above for the poly-His tagged proteins.

[0138] Immunoadhesin (Fc-containing) constructs are purified from the conditioned media as follows. The conditioned medium was pumped onto a 5 ml Protein A column (Pharmacia) which had been equilibrated in 20 mM Na phosphate buffer, pH 7.4. After loading, the column was washed extensively with equilibration buffer before elution with 100 mM citric acid, pH 3.5. The eluted protein was immediately neutralized by collecting 1 ml fractions into tubes containing 275 .mu.L of 1 M Tris buffer, pH 9. The highly purified protein was subsequently run over size exclusion chromatography, dialyzed, analyzed, and stored as above for the poly-His tagged proteins.

[0139] 4 to 6-week-old Armenian hamsters (Cytogen) or mice were immunized with 2 .mu.g/injection each murine and human recombinant PILR.alpha. proteins. The immunogens are resuspended in monophosphoryl lipid A/trehalose dicorynomycolate adjuvant and injected via footpad or I.P. at 3 to 4 day intervals for a total of 10 boosts. Three days after the final boost, lymphocytes from immunized hamster spleens and lymph nodes were harvested for fusion with SP2/0 myeloma cells (American Type Culture Collection) by using the Cyto Pulse CEEF-50 apparatus (Cyto Pulse Sciences). Briefly, after washing twice with Cytofusion Medium C (Cyto Pulse Sciences), the isolated lymphocytes and SP2/0 cells were mixed at a 1:1 ratio and then resuspended at 10 million cells/ml in Cytofusion Medium C, electrofusion was performed according to manufacturer's guidance. Fused cells were cultured in ClonaCell-HY Medium C (StemCell Technologies) overnight at 37.degree. C. in a 7% CO.sub.2 incubator. The next day, fused cells were centrifuged and resuspended in 10 ml ClonaCell-HY Medium C and then gently mixed with 90 ml Methylcellulose-based ClonaCell-HY Medium D (StemCell Technologies) containing HAT components. The fused cells were plated into 100 mm Petri dishes (Becton Dickinson) and allowed to grow in 37.degree. C. in a 7% CO.sub.2 incubator. After 7-10 days incubation, the single hybridoma clones were picked by ClonePix (Genetix, United Kingdom) and transferred into 96-well cell culture plates (Becton Dickinson) with 200 .mu.L/well ClonaCell-HY Medium E (StemCell Technologies). Hybridoma culture media was changed prior to ELISA screening. All ELISA positive clones were further screened by FACS. After at least 2 rounds of single cell subcloning by limiting dilution, final clones were scaled up and the supernatants were collected for antibody purification. The hybridoma supernatants were purified by Protein A affinity chromatography, then sterile filtered (0.2 .mu.m pore size, Nalgene Nunc International, NY, USA) and stored at 4.degree. C. in PBS. The purified mAbs were confirmed by ELISA and FACS before testing on functional assays. The isotypes of purified mAbs were determined by the mouse monoclonal antibody isotyping kit from Roche Diagnostics Corporation. The isotypes of purified hamster mAbs were determined by ELISA.

Example 2

Sialidation of PILRa Ligands

[0140] This example uses one PILRa ligand, mCD99, to illustrate the importance of sialidation on the ligand for PILRa binding. Mouse PILR.alpha. binds to mCD99 with relatively low affinity, however, it is unclear whether PILR.alpha. can bind human CD99. Shiratori et al. (2004) J Exp Med 199, 525-533; Tabata et al. (2008) J Biol Chem 283, 8893-8901. To test this, we expressed mouse or human CD99 in 293T cells to see whether they can bind to PILR.alpha.-mIgG2a (PILR.alpha.-Fc) fusion proteins. We found both mouse and human PILR.alpha.-Fc fusions bound mCD99 transfectants. However, neither protein bound to hCD99 transfectants (FIG. 1A). This suggests that the CD99 interaction with PILR.alpha. is not conserved at least across human and mouse species. However a conserved PILR.alpha. interaction domain between mouse and human PILR.alpha. could mediate its binding to mCD99.

[0141] The differential binding of mouse and human CD99 to PILR.alpha. may be the result of the low sequence identity between CD99 homologs (about 41% in the extracellular domain) or the differences in glycosylation (type and/or a number of glycans). It has been shown that sialylated O-linked glycans in mCD99 play an essential role in PILR.alpha. binding to mCD99. Wang et al. (2008) J Immunol 180, 1686-1693. hCD99 has been reported to be O-glycosylated, and similarly to mCD99, does not have any potential N-glycosylation sites. Gelin et al. (1989) EMBO J 8, 3253-3259. Lack of binding of PILR.alpha. to hCD99 may therefore be related to the differences in its O-glycosylation pattern. To examine this possibility, we first compared profiles of O-glycans from human and mouse CD99-Fc fusion proteins. O-glycans were released by reductive .beta.-elimination and permethylated prior to analysis by MALDI-TOF MS. Comparison of MALDI-TOF spectra (FIG. 1B), demonstrated qualitatively similar O-glycosylation profiles of human and mouse CD99. Two major mono- and di-sialylated O-glycans, having the following compositions: NeuAc.sub.1Hex.sub.1HexNAc.sub.1 and NeuAc.sub.1Hex.sub.1HexNAc.sub.1, were detected in both proteins. The composition of observed O-glycans is consistent with the presence of sialylated Gal.beta.1-3GalNAc core 1 structures. North et al. (2010) J Biol Chem 285, 5759-5775; Olson et al. (2005) Glycobiology 15, 177-191.

[0142] Since O-glycans of human and mouse CD99 did not differ qualitatively, we next examined whether there might be a difference in the number of O-glycans present on each of the proteins. LC-MS analysis of reduced fusion proteins (with and without PNGase F and Sialidase digestion) showed that hCD99-Fc carried two O-glycans and mCD99-Fc carried three O-glycans with the following composition: NeuAc.sub.1-2Hex.sub.1HexNAc.sub.1. In the case of mCD99, two adjacent O-glycosylation sites, Thr-45 and Thr-50 (NMKPT.sup.45PKAPT.sup.50PKKPS; SEQ ID NO: 64) are relevant for PILR.alpha. recognition. Wang et al. (2008). Sequence alignment of human and mouse CD99 showed that Thr-41 of hCD99 corresponds to the Thr-45 O-glycosylation site of mCD99 and that hCD99 lacks the second potential O-glycosylation site corresponding to Thr-50 of mCD99. Therefore, we performed LC-MS tryptic peptide mapping of hCD99-Fc to determine the localization of O-glycans and to examine whether the peptide containing Thr-41 is indeed O-glycosylated. Two sialylated glycopeptides with following sequence were detected: APDGGFLDLSDALPDNENKKPTAIPK (hCD99-derived; SEQ ID NO: 65) and GPTIKPCPPCK (mIgG2a Fc-derived; SEQ ID NO: 66). Each glycopeptide was glycosylated with a single NeuAc.sub.1-2Hex.sub.1HexNAc.sub.1 O-glycan. The presence of two sialylated glycopeptides was consistent with the LC-MS analysis of reduced hCD99-Fc. The detected sialoglycopeptide APDGGFLDLSDALPDNENKKPT.sup.41AIPK (SEQ ID NO: 65) contained Thr-41 corresponding to Thr-45 O-glycosylation site of mCD99 and a Ser residue which potentially could be O-glycosylated. However, the exact site of O-glycosylation could not have been sequenced through the performed type of LC-MS experiment. The presence of an O-glycan on the GPTIKPCPPCK (SEQ ID NO: 66) peptide is consistent with the previous report of it being O-glycosylated in the truncated version of mouse IgG2a. Masuda et al. (1999) Mol Immunol 36, 993-1003.

[0143] Our results indicated that human and mouse CD99 have similar O-glycans but that the presence of a common core of 1 NeuAc.sub.1-2Hex.sub.1HexNAc.sub.1 O-glycan per se on hCD99 is not sufficient for PILR.alpha. binding. A major difference between the mouse and human CD99 O-glycan modifications is that mCD99 has two O-glycosylation sites (Thr-45 and Thr-50) versus one in the human counterpart. Wang et al. (2008). We asked whether the introduction of the mCD99 region P.sup.46KAPT.sup.50 (PKAPT) into hCD99 can confer the binding of PILR.alpha. to hCD99. We expressed full-length mCD99, hCD99 and hCD99PKAPT in 293T cells, and again tested the binding of hPILR.alpha.-Fc or mPILR.alpha.-Fc by FACS. We found that the insertion of mCD99 PKAPT motif into hCD99, directly following the Thr-41 O-glycosylation site, was able to induce binding of human or mouse PILR.alpha. to hCD99PKAPT expressing cells. Surprisingly, this interaction was similar in magnitude to human or mouse PILR.alpha. binding to mCD99. This suggests the sialylated O-glycans are required for CD99 binding to PILR.alpha. (FIG. 1C).

Example 3

Identification of Novel PILRa Ligands

[0144] This example demonstrates the identification of novel ligands for PILRa.

[0145] It has been shown that mCD99, HSV1 glycoprotein B and PANP are PILR.alpha. ligands. CD99 is expressed on T-cells, B-cells, NK cells, monocytes and neutrophils. However, hPILRa is expressed predominately in cells of the myelomonocytic lineage, including monocytes/macrophages, granulocytes, and dendritic cells (DC). Discordant PILRa-Fc binding and CD99 expression as well as a low affinity interaction with CD99 (2.2 mM) suggested that there may be additional ligands for PILRa. To identify novel PILRa ligands and to further investigate the nature of PILR.alpha. interactions with its ligands as well as biological functions thereof, a cDNA expression library was screened with an Alkaline phosphatase (AP)-tagged PILR.alpha. construct.

[0146] The extracellular domains of hPILR.alpha. (Met1-196Thr) and mPILR.alpha. (Met1-197Val) were cloned into the expression vector pRK5 as fusions to C-terminal alkaline phosphatase (AP) tag. 293T cells were transfected with PILR.alpha.-AP-pRK5 constructs and Fugene 6 Transfection Reagent (Roche) according to the manufacturer's instructions. Three days after transfection, supernatants were collected for screening. COST cells were plated in a 24-well format and transfected with 10 ul of Origene library DNA (Origene, human DNA library containing 20 k genes) per well with Fugene 6 Reagent. Two days later, cells were incubated with human or mouse PILR.alpha.-Ap supernatants for 45 min at room temperature (RT). Then cells were fixed with 4% PFA for 15 min at RT. After removing fixation buffer, cells were blocked with 100 mM Glycine in HBS (20 mM Hepes, PH7.2, 150 mM NaCl) for 15 min at RT. Cells were then rinsed and incubated in HBS for 90 min at 65.degree. C. HBS was removed and Western Blue Substrate (Promega) was added and color was developed for 30 min to 1 hr.

[0147] The resulting positive clones were identified and further analyzed using FACS. The ligands that bound hPILRa are NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA. See FIGS. 6-11. The expression of these newly identified PILRa ligands on various cell types are summarized in FIG. 12.

[0148] The hPILR.alpha.-AP binding to human NPDC1 and human COLEC12 expressing cells are subject to further analysis (FIG. 2A). Similar to mCD99, both mouse or human PILR.alpha.-AP were able to bind to hNPDC1 and hCOLEC12 expressing cells suggesting that a conserved interaction domain mediates PILR.alpha. binding to these ligands. NPDC1 is a type I transmembrane protein and has been identified as a neural-specific gene involved in the control of cell proliferation and differentiation. Galiana et al. (1995) Proc Natl Acad Sci USA 92, 1560-1564. COLEC12 is a type II transmembrane collectin family member as also known as collectin placenta 1 (CL-P1) and Scavenger Receptor with C-type Lectin (SRCL) type I. Ohtani et al. (2001) J Biol Chem 276, 44222-44228; Nakamura et al. (2001) Biochem Biophy Res Commun 280, 1028-1035.

[0149] To confirm the binding of PILR.alpha. to hNPDC1 and hCOLEC12 on the cell surface, N-terminal flag-tagged hNPDC1 or C-terminal his tagged hCOLEC12 were expressed in 293T cells, and then tested for mPILR.alpha.-Fc and hPILR.alpha.-Fc binding. Mock transfected cells were used as a negative control and mCD99 transfected cells were used as positive control. Both mPILR.alpha.-Fc and hPILR.alpha.-Fc bound to mCD99, hNPDC1 and hCOLEC12 expressing cells (FIG. 2B). Similar results were also obtained by hNPDC1-Fc or hCOLEC12-his in PILR.alpha. transfectants by FACS analysis. Again, both hNPDC1-Fc and hCOLEC12-his bound to hPILR.alpha. as well as mPILR.alpha. tranfectants, (FIG. 2C). Using an equilibrium competition radioligand assay, the affinity (K.sub.D) of hPILR.alpha.-Fc binding to cell-surface expressed hNPCD1 was determined to be 49 nM (FIG. 2D). Because the sensitivity of the radioligand competition assay was too low to measure the lower affinity interaction between hPILR.alpha.-Fc and hCOLEC12, we used SPR instead (FIG. 2E). Using this method, the equilibrium K.sub.D of this interaction was found to be 1.1 uM. Taken together these studies demonstrate specific binding of PILR.alpha. to human NPDC1 and COLEC12.

[0150] The presence of sialylated O-glycans is required for PILR.alpha. binding to its known ligands including mCD99, HSV1-gB and the recently identified PANP. Kogure et al. (2011) Biochem Biophys Res Commun 405, 428-533; Wang et al. (2008); Wang et al. (2009) J Virol 83, 13042-13045. Human NPDC1 and COLEC12 have multiple potential O-glycosylation sites when analyzed using the NetOGlyc 3.1 prediction server. Julenius et al. (2005) Glycobiology 15, 153-164. hNPDC1 does not have any potential N-glycosylation sites, while hCOLEC12 has multiple sites. To determine whether both proteins are indeed O-glycosylated, they were analyzed in similar manner to mouse and human CD99. MALDI-TOF MS analysis of permethylated O-glycans released by reductive .beta.-elimination from hNPDC1-Fc and hCOLEC12-his confirmed the presence of O-glycans, qualitatively similar to those determined in mouse and human CD99 (FIG. 3). In addition, analysis of hCOLEC12 by a combination of HPLC charge profiling of fluorescent 2-aminobenzoic acid labeled glycans and MALDI-TOF of permethylated unlabeled glycans also demonstrated its complex N-glycosylation profile. Neutral glycans were mostly of the high-mannose type (predominantly Man-5) and complex afucosylated or core-fucosylated bi-, tri- and tetra-antennary glycans, galactosylated to various degrees. Negatively charged glycans were sialylated (0-4 NANA), complex type bi-, tri- and tetra-antennary N-glycans, core-fucosylated and mostly fully galactosylated.

[0151] In order to test whether sialylated glycans on NPDC1 and COLEC12 are required for their binding to PILR.alpha., we performed Surface Plasmon Resonance analysis using Biacore with hNPDC1 and hCOLEC12 fusion proteins with and without Sialidase A treatment. Before Sialidase A treatment, the proteins showed good binding affinity to hPILR.alpha. (FIG. 4). However, after Sialidase A treatment the proteins showed little or no binding to hPILR.alpha. (FIG. 4). mCD99 was used as a control and showed similar results to both hNPDC1 and hCOLEC12 (FIG. 4). These studies suggest that the sialyated glycans on hNPDC1 and hCOLEC12 are required for their binding to hPILR.alpha..

[0152] Our binding analysis indicates that hNPDC1 binds with higher affinity (K.sub.D=49 nM) than hCOLEC12 (K.sub.D=1 .mu.M). Although PILR.alpha. binds to NPDC1 with relative higher affinity, both hPILR.alpha. and mPILR.alpha. do not bind to mNPDC1. Similar to CD99, differences in NPDC1 glycosylation may be responsible for this observation. NPDC1 has been identified as a neuronal-specific gene involved in the control of cell proliferation and differentiation. Galiana et al. (1995) Proc Natl Acad Sci USA 92, 1560-1564. Our results suggest that PILR.alpha. recognizes several ligands, some of which are not conserved between mouse and human. The identification of multiple PILR.alpha. ligands in the neuronal system (PANP and NPDC1) suggests that PILR.alpha. might play a role in the CNS.

[0153] COLEC12 is the only member of the Collectin scavenger receptor family that is expressed as a cell surface transmembrane protein and its ECD contains coiled-coil, collagen-like, and C-type lectin/carbohydrate domains. Ohtani et al. (2001) J Biol Chem 276, 44222-44228. COLEC12 is expressed in vascular endothelia cells and monocytes to mediate the uptake of oxidized low density lipoprotein and microbes. Ohtani et al. (2001) J Biol Chem 276, 44222-44228; Nakamura et al. (2001) Biochem Biophys Res Commun 280, 1028-1035. Interestingly, Collectins have been shown to interact with other inhibitory receptors such as SIRP.alpha. to modulate lung pathophysiology. Janssen et al. (2008) Am J Respir Crit Care Med 178, 158-167. PILR.alpha. is emerging as a receptor that recognizes a specific group of ligands in cellular or pathogenic sources with unique sialic acid pattern. Correspondingly, we found that human and mouse PILR.alpha.-Fc fusions bind to mouse thymocytes, peripheral CD8+ T and activated CD4+ T, B, NK, NKT cells, granulocytes and monocytes, as well as a majority of human PBMC. These data suggest that PILR.alpha. ligands are broadly expressed in immune cells. Since the presence of sialylated glycans is a common feature of all known PILR.alpha. ligands, it is apparent that PILR.alpha. has additional cellular ligands.

Example 4

Conserved PILRa Domain for its Interaction with Diverse Sialylated Ligands

[0154] This example demonstrates a conserved domain on PILRa critical for its binding to sialylated ligands.

[0155] Sialylated glycan modifications appear to be a general feature of all identified PILR.alpha. ligands and coincidently few other receptors such as Siglecs. Kogure et al., (2011) Biochem Biophys Res Commun 405, 428-433; Wang et al. (2008); Crocker et al. (2007) Nat Rev Immunol 7, 255-266; Wang et al. (2009). A conserved Arg site in the ECD of Siglecs plays a critical role in their binding to sialic acid. May et al. (1998) Mol Cell 1, 719-728; Ikehara et al. (2004) J Biol Chem 279, 43117-43125; Vinson et al. (1996) J Biol Chem 271, 9267-9272. The PILR.alpha. ECD has two, similar Arg sites (human Arg 96, mouse arginine 103; human R126, mouse R133) that are highly conserved across species (FIG. 5A). The second Arg site (mouse R133, human R126) corresponds to the critical Arg site that is required for the binding of Siglecs to sialic acid in the ligands. May et al. (1998) Mol Cell 1, 719-728. The hPILR.alpha. R126 is not an Ig fold stabilizing residue (FIG. 5A). We examined whether mutation of this Arg site in mouse or human PILR.alpha. affects their binding to ligands. First, we tested the binding of ligand fusion proteins including mCD99-Fc, hNPDC1-Fc and hCOLEC12-his to cell surface-expressed WT or Arg mutated hPILR.alpha.R126A and mPILR.alpha.R131A. The Arg mutation did not affect cell surface expression of human and mouse PILR.alpha.. We found that all fusion proteins bound to wild type human and mouse transfectants, but none of them bound to hPILR.alpha.R126A and mPILR.alpha.R133A transfectants (FIG. 5B), suggesting Arg126 in hPILRa and Arg133 in mouse PILR.alpha. are required for PILR.alpha. binding to ligands. We also generated WT and Arg mutant human and mouse PILR.alpha.-Fc fusion proteins, and examined their binding to cell surface expressed ligands. The result showed that WT human and mouse PILR.alpha.-Fc bound to mCD99, hNPDC1, hCOLEC12 and HSV-1 gB transfectants, while hPILR.alpha.R126A-Fc and mPILR.alpha.R133A-Fc did not bind to these ligand transfectants (FIG. 5C). Glycoprotein B expression could be detected on HSV1 infected cells 24 hrs after infection (FIG. 5D-1), we therefore tested wild type and Arg mutant human and mouse PILR.alpha.-Fc binding to HSV1 infected cells. Again, we observed the binding of only WT hPILR.alpha.-Fc and mPILR.alpha.-Fc but not hPILR.alpha.R126A and mPILR.alpha.R133A to HSV1 infected 293T cells (FIG. 5D-2). These studies further support that this conserved Arg site is necessary for PILR.alpha. binding to its ligands.

[0156] To better quantify the binding of WT versus Arg mutant PILR.alpha. binding to various ligands, we performed SPR analysis. The WT hPILR.alpha.-Fc and hPILR.alpha. R126-Fc were immobilized on a chip and their binding to hNPDC1, hCOLEC12, and mCD99 was compared (FIG. 5E). All three proteins showed strong binding with the WT hPILR.alpha. (FIG. 5E). However, little or no binding was observed with the mutant hPILR.alpha., suggesting this conserved Arg site is necessary for binding of PILR.alpha. to its ligands (FIG. 5E).

[0157] Our data suggests that PILR.alpha.-ligand interactions require a conserved Arg motif on PILR.alpha. and specific sialylated decorations on the ligands. Since sialylation occur on many cell surface proteins, we predict PILR.alpha. ligands should be broadly expressed in various cells and hPILR.alpha.R126A/mPILR.alpha.133A should not bind to the natural ligands expressed on the surface of primary cells. To test this, we stained mouse and human hematopoietic cells with human or mouse PILR.alpha.-Fc. We found that both human and mouse PILR.alpha.-Fc bound to the majority of human PBMC (FIG. 5F-1). PILR.alpha.-Fc highly bound to human T cells and monocyte subsets (FIG. 5F-1). For murine cells, PILR.alpha.-Fc fusions highly bound to CD8+ peripheral T cells and thymocytes, as well as peripheral B cells (FIG. 5F-2). However neither hPILR.alpha.R126A-Fc nor mPILR.alpha.133A-Fc can bind to human PBMCs (FIG. 5F-1), mouse thymocytes, or peripheral T and B cells (FIG. 5F-2). These results suggest that R126 in human PILR.alpha. and R133 in murine PILR.alpha. are required for their binding to cellular ligands expressed in primary cells.

[0158] Our study identifies the ARG126 (mouse ARG133) as a critical contact residue in hPILR.alpha. (FIG. 5A). Others have shown that TRP139 is also important in mediating hPILR.alpha. interaction with its ligand gB. Fan et al. (2010) J Virol 84, 8664-8672. Given the similarities of the binding residues between PILR.alpha. and Siglec family, we built a homology model of PILR.alpha. to gain insights into its contact residues.

[0159] The PILR.alpha. Sequence was aligned with the N terminal of the mouse Sialoadhesion from 1QFO.PDB using MOE2010.10 Protein Align application. Blosum62 was used as the alignment matrix, with tree-based build-up, Gap Start penalty of 7 and Gap Extend penalty of 1, Iteration limit of 100 and Failure limit of 10. The aligned structures were used to build a homology model using the Homology Model application in MOE2010.10. The crystal structure of the first chain in the SIGLEC1 was used as the template, while the ligand atoms were used as the `environment` during model building. May et al. (1998) Mol Cell 1, 719-728; Munday and Crocker (1999) J Leukoc Biol 66, 705-711. The C, N-terminal outgaps were not built. A total of 25 models were built with fine minimization, and the final model was put through the Protonate3D procedure to detect the correct protonation states and was finely minimized at the end. The Merck force field (MMFF94x) with Born solvation method was used to reproduce the small molecule interactions of the active site. The active site-ligand interactions of SIGLEC1 and those of the PILR.alpha. model were rendered using the Ligand Interaction Diagram application of MOE2010.10. Clark and Labute (2007) J Chem Inf Model 47, 1933-1944. The active site-ligand contacts are coded according to Table 1.

TABLE-US-00001 TABLE 1 Ligand contacts of Siglec-1 and the PILRa model. Siglec-1 PILRa Contact Active Site Ligand Active Site Ligand Code Residue/Atom Residue/Atom Residue/Atom Residue/Atom Comments C01 TRP2.Ring SIA201.C11 TYR33.Ring SIA201.C11 3.6 .ANG. vs 5.4 .ANG. C02 TYR44.OH GAL202.O6 ARG74.NH1/NH2 GAL202.O6 [1] C03 ARG97.NH1 SIA201.O1A ARG126.NH1 SIA201.O1A identical C04 ARG97.NH2 SIA201.O1B ARG126.NH2 SIA201.O1B identical C05 SER103.O SIA201.O4 THR131 SIA201.O4 2.8 .ANG. vs 4.8 .ANG. C06 ARG132 SIA201.O4/O10 Unique to PILRa C07 ASN104.CA SIA201.O4 GLN137.OE1 SIA201.N5 5 .ANG. vs 4.4 .ANG. C08 ARG105.O SIA201.N5 GLN138.O SIA201.N5 Identical C09 ARG105.NE SIA201.O1A GLN138 SIA201.O1A Identical C10 TRP106.Ring SIA201.C9 TRP139.Ring SIA201.C9 Identical C11 LEU107.O SIA201.O9 GLN140.O SIA201.O9 Identical C12 LEU107.N SIA201.O8 GLN140.N SIA201.O8 Identical C13 LEU107.CD2 GAL202.O6 GLN140.NE2 GAL202.O6 3.8 .ANG. vs 2.8 .ANG. C14 ASP108 SER141 [2] Ligand atoms according to crystal structure (1QFO.pdb). Some corresponding atomic distances are shown under comments for Siglec-1 and PILRa respectively. [1] Contact possible via altered conformation; [2] Both residues about 5 .ANG. away from SIA201.O9.

[0160] Despite the low sequence identity between SIGLEC1 and PILR.alpha. (FIG. 5A), it is clear that most of the contacts are strikingly similar, even when the active site residues are not identical. First, the C03, C04 contacts (SIGLEC1.ARG97 versus PILR.alpha..ARG126) and the C10 contact (SIGLEC1.TRP106 versus PILR.alpha..TRP139) are identical. SIGLEC1.SER103 and PILR.alpha..THR131 both accept a hydrogen bond via their backbone carboxylate oxygens from the ligand Sialic acid O4 (contact C05). The backbone carboxylate oxygen of SIGLEC1.ARG105 and that of PILR.alpha..GLN138 accept a hydrogen bond from N5 of the ligand sialic acid (contact C08). For contact C09, the guanidine nitrogen of this Arg in SIGLEC1 interacts with the carboxylate of ligand sialic acid O1A (4.1 .ANG.), while the side chain amino in the corresponding residue (PILR.alpha..GLN138) has the same contact, only slightly weaker (4.7 .ANG.). The peptidic backbone of SIGLEC1.LEU107 and the corresponding PILR.alpha..GLN140 have identical contacts with the ligand; their amino group contacts the ligand sialic acid O8 (coded as C11) and their carboxylate oxygen can accept a hydrogen bond from the ligand Sialic acid O9 (coded as C12). In comparison, the side chain amide group of PILR.alpha..GLN140 is able to contact ligand galactose O6, which in SIGLEC1, the non-polar side chain of LEU108 has a non-favorable interaction with the ligand. This contact is coded as C13. The hydroxyl group of Tyrosine 44 of SIGLEC1 donates a hydrogen bond to O6 of the ligand galactose (contact code C02). The corresponding residue in PILR.alpha. (Arg73) cannot make this contact in the bent conformation observed in the model, where it seems to prefer to hydrogen bond with PILR.alpha..PHE124.O, but manual rotation of the side chain along CG-CD can restore not one but two hydrogen bonds with the same oxygen in the ligand (galactose O6) with slight repulsion from PILR.alpha..GLN140.NE2. Overall, it is likely that this contact (C02) can be present in PILR.alpha., even stronger than SIGLEC1. Similarly, while SIGLEC1.TRP2 has proton-Pi interaction with the ligand Sialic acid C11 with the distance of 3.6 A, the base conformation of the corresponding residue (PILR.alpha..TYR33) is slightly further (5.4 A), but a 30 degree rotation of the tyrosine side chain along CA-CB makes perfect overlay of the two aromatic systems, suggesting that the two receptors might have nearly identical contacts at this site too (C01). The PILR.alpha..ARG132 which has no corresponding residue in SIGLEC1 crystal structure and shows no interaction with the ligand in the base conformation. However, after rotating the side chain along C.alpha.-C.beta. or C.beta.-C.gamma., the side chain can make hydrogen bonds with the ligand sialic acid O4, O10. These interactions (C06) are unique to PILR.alpha. suggesting that distinctive features exist between SIGLEC1 and PILRa receptors. It is interesting to note that the conserved interaction residues are predominantly located in carboxyl end of PILR.alpha. between 123 to 142 amino acids (FIG. 5A, arrows). In summary, PILR.alpha. active site is very similar to the SIGLEC1, with at least two additional hydrogen bonding contacts (Arg132, Gln140). These residues are exposed to solvent, even in the complex, suggesting possible contacts with the protein domains of other ligands.

[0161] Despite only 41% amino acid identity between the human and mouse PILR.alpha. ECDs, we find that both proteins can still interact with similar ligands and primary cells types. This suggests that a conserved interacting domain has been selected during evolution. Correspondingly, the alignment of PILR.alpha. sequences points to high degree of conservation among potential contact residues (FIG. 5A) some of which are shared with Siglecs family of receptors) (Arginine 126, Tryptophane 139). Tabata et al. (2008) J Biol Chem 283, 8893-8901. Our studies demonstrate that one common binding mechanism involves the recognition of one or several sialic acid modifications in all ligands by the conserved arginine site in mouse and human PILR.alpha.. We have come to this conclusion by showing that ligand fusion proteins including mCD99-Fc, hNPDC1-Fc and hCOLEC12-his do not bind to hPILR.alpha.R126A and mPILR.alpha.R133A expressed on cell surface while they all bind to the wild type human or mouse PILR.alpha. (FIG. 5B). Second, we find that mutated fusion proteins hPILR.alpha.R126A-Fc and mPILR.alpha.R133A-Fc do not bind to cell surface expressed ligands including mCD99, hNPDC1, hCOLEC12 and HSV1 gB (FIGS. 5C-5D). Third, our surface plasmon resonance analysis clearly shows that only wild type hPILR.alpha.-Fc but not mutated hPILR.alpha.R126A-Fc can bind to various ligands. Finally, hPILR.alpha.R126A-Fc and mPILR.alpha.R133A-Fc fail to interact with natural ligand(s) expressed in primary hematopoietic cells (FIG. 5F), suggesting this conserved Arg site is required for PILR.alpha. binding to all nature ligands. PILR.alpha.'s uniform binding interaction mode by sialic acid recognition might have evolved to trigger a conserved signaling pathway and functional outcome depending on which ligand(s) are binding to PILR.alpha.. The convergence of PILR.alpha. sequences across different species may be indicative of receptor genes that are responding to evolutionary pressure provided by pathogens or unknown ligands.

[0162] A highly conserved and essential arginine residue (Arg97 in SIGLEC1) contacts the carboxylate group of sialic acid and two tryptophans that interact with the N-acetyl and glycerol moieties of N-acetyl neuraminic acid. May et al. (1998) Mol Cell 1, 719-728; Munday et al. (1999) J Leukoc Biol 66, 705-711. Our functional data with arginine-mutated PILR.alpha. is consistent with a model in which Siglecs and PILR.alpha. have some similarity in their ligand interaction domains. Our analysis identifies arginine 126 as a key contact residue in hPILR.alpha. (Arg 133 in mPILR.alpha.) and its location appears to be critical to mediate PILR.alpha. interaction with sialic acid on gB as well as other ligands. Further support of this model is provided by recent data that the Tryptophan-139 residue is also critical for human PILR.alpha. binding to HSV1 gB. Fan et al. (2010) J Virol 84, 8664-8672. This Tryptophan-139 is not present in PILR.beta. and the converse mutation of L139W in PILR.beta. does not confer the ability to mediate binding to HSV1 gB, suggesting that there are other amino acids that play a role in the binding of PILR.alpha. with gB. Fan et al. (2010) J Virol 84, 8664-8672. Based on known structure of SIGLEC1:sialic acid complex, it is possible to speculate on how PILR.alpha. might recognize sialic acid containing ligands and rationalize the results of previously reported PILR.alpha. mutations. May et al. (1998) Mol Cell 1, 719-728. The homology model of the structure of PILR.alpha. identifies active site residues that are almost identical between the Siglecs and PILR.alpha.. In the absence of a crystal structure for PILR.alpha., high similarities among the active site-ligand contacts between PILR.alpha. and SIGLEC1, even in case of non-identical residues allows us to confide in the homology model, and provides us with an expanded hypothesis for ligand binding of PILR.alpha. which can further analyzed via additional mutation studies. Structural resolution of PILR.alpha. in the context of sialylated proteins will be necessary to define the interaction domains. SIGLEC1 and multiple CD33-related Siglecs can interact with sialic acids on pathogens such as N. meningitidis, C. jejuni, group B Streptococcus and Trypanosoma cruzi. Crocker et al. (2007) Nat Rev Immunol 7, 255-266. It will be interesting to know whether PILR.alpha. could also directly interact with similar pathogens, which are known to carry sialylated sugar modifications in their surface structures.

[0163] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Sequence CWU 1

1

631302PRTMus sp. 1Met Ala Leu Leu Ile Ser Leu Pro Gly Gly Thr Pro Ala Met Ala Gln1 5 10 15Ile Leu Leu Leu Leu Ser Ser Ala Cys Leu His Ala Gly Asn Ser Glu 20 25 30Arg Ser Asn Arg Lys Asn Gly Phe Gly Val Asn Gln Pro Glu Ser Cys 35 40 45Ser Gly Val Gln Gly Gly Ser Ile Asp Ile Pro Phe Ser Phe Tyr Phe 50 55 60Pro Trp Lys Leu Ala Lys Asp Pro Gln Met Ser Ile Ala Trp Arg Trp65 70 75 80Lys Asp Phe His Gly Glu Phe Ile Tyr Asn Ser Ser Leu Pro Phe Ile 85 90 95His Glu His Phe Lys Gly Arg Leu Ile Leu Asn Trp Thr Gln Gly Gln 100 105 110Thr Ser Gly Val Leu Arg Ile Leu Asn Leu Lys Glu Ser Asp Gln Thr 115 120 125Arg Tyr Phe Gly Arg Val Phe Leu Gln Thr Thr Glu Gly Ile Gln Phe 130 135 140Trp Gln Ser Ile Pro Gly Thr Gln Leu Asn Val Thr Asn Ala Thr Cys145 150 155 160Thr Pro Thr Thr Leu Pro Ser Thr Thr Ala Ala Thr Ser Ala His Thr 165 170 175Gln Asn Asp Ile Thr Glu Val Lys Ser Ala Asn Ile Gly Gly Leu Asp 180 185 190Leu Gln Thr Thr Val Gly Leu Ala Thr Ala Ala Ala Val Phe Leu Val 195 200 205Gly Val Leu Gly Leu Ile Val Phe Leu Trp Trp Lys Arg Arg Arg Gln 210 215 220Gly Gln Lys Thr Lys Ala Glu Ile Pro Ala Arg Glu Pro Leu Glu Thr225 230 235 240Ser Glu Lys His Glu Ser Val Gly His Glu Gly Gln Cys Met Asp Pro 245 250 255Lys Glu Asn Pro Lys Asp Asn Asn Ile Val Tyr Ala Ser Ile Ser Leu 260 265 270Ser Ser Pro Thr Ser Pro Gly Thr Ala Pro Asn Leu Pro Val His Gly 275 280 285Asn Pro Gln Glu Glu Thr Val Tyr Ser Ile Val Lys Ala Lys 290 295 3002274PRTMus sp. 2Gly Asn Ser Glu Arg Ser Asn Arg Lys Asn Gly Phe Gly Val Asn Gln1 5 10 15Pro Glu Ser Cys Ser Gly Val Gln Gly Gly Ser Ile Asp Ile Pro Phe 20 25 30Ser Phe Tyr Phe Pro Trp Lys Leu Ala Lys Asp Pro Gln Met Ser Ile 35 40 45Ala Trp Arg Trp Lys Asp Phe His Gly Glu Phe Ile Tyr Asn Ser Ser 50 55 60Leu Pro Phe Ile His Glu His Phe Lys Gly Arg Leu Ile Leu Asn Trp65 70 75 80Thr Gln Gly Gln Thr Ser Gly Val Leu Arg Ile Leu Asn Leu Lys Glu 85 90 95Ser Asp Gln Thr Arg Tyr Phe Gly Arg Val Phe Leu Gln Thr Thr Glu 100 105 110Gly Ile Gln Phe Trp Gln Ser Ile Pro Gly Thr Gln Leu Asn Val Thr 115 120 125Asn Ala Thr Cys Thr Pro Thr Thr Leu Pro Ser Thr Thr Ala Ala Thr 130 135 140Ser Ala His Thr Gln Asn Asp Ile Thr Glu Val Lys Ser Ala Asn Ile145 150 155 160Gly Gly Leu Asp Leu Gln Thr Thr Val Gly Leu Ala Thr Ala Ala Ala 165 170 175Val Phe Leu Val Gly Val Leu Gly Leu Ile Val Phe Leu Trp Trp Lys 180 185 190Arg Arg Arg Gln Gly Gln Lys Thr Lys Ala Glu Ile Pro Ala Arg Glu 195 200 205Pro Leu Glu Thr Ser Glu Lys His Glu Ser Val Gly His Glu Gly Gln 210 215 220Cys Met Asp Pro Lys Glu Asn Pro Lys Asp Asn Asn Ile Val Tyr Ala225 230 235 240Ser Ile Ser Leu Ser Ser Pro Thr Ser Pro Gly Thr Ala Pro Asn Leu 245 250 255Pro Val His Gly Asn Pro Gln Glu Glu Thr Val Tyr Ser Ile Val Lys 260 265 270Ala Lys3303PRTHomo sapiens 3Met Gly Arg Pro Leu Leu Leu Pro Leu Leu Pro Leu Leu Leu Pro Pro1 5 10 15Ala Phe Leu Gln Pro Ser Gly Ser Thr Gly Ser Gly Pro Ser Tyr Leu 20 25 30Tyr Gly Val Thr Gln Pro Lys His Leu Ser Ala Ser Met Gly Gly Ser 35 40 45Val Glu Ile Pro Phe Ser Phe Tyr Tyr Pro Trp Glu Leu Ala Thr Ala 50 55 60Pro Asp Val Arg Ile Ser Trp Arg Arg Gly His Phe His Arg Gln Ser65 70 75 80Phe Tyr Ser Thr Arg Pro Pro Ser Ile His Lys Asp Tyr Val Asn Arg 85 90 95Leu Phe Leu Asn Trp Thr Glu Gly Gln Lys Ser Gly Phe Leu Arg Ile 100 105 110Ser Asn Leu Gln Lys Gln Asp Gln Ser Val Tyr Phe Cys Arg Val Glu 115 120 125Leu Asp Thr Arg Ser Ser Gly Arg Gln Gln Trp Gln Ser Ile Glu Gly 130 135 140Thr Lys Leu Ser Ile Thr Gln Ala Val Thr Thr Thr Thr Gln Arg Pro145 150 155 160Ser Ser Met Thr Thr Thr Trp Arg Leu Ser Ser Thr Thr Thr Thr Thr 165 170 175Gly Leu Arg Val Thr Gln Gly Lys Arg Arg Ser Asp Ser Trp His Ile 180 185 190Ser Leu Glu Thr Ala Val Gly Val Ala Val Ala Val Thr Val Leu Gly 195 200 205Ile Met Ile Leu Gly Leu Ile Cys Leu Leu Arg Trp Arg Arg Arg Lys 210 215 220Gly Gln Gln Arg Thr Lys Ala Thr Thr Pro Ala Arg Glu Pro Phe Gln225 230 235 240Asn Thr Glu Glu Pro Tyr Glu Asn Ile Arg Asn Glu Gly Gln Asn Thr 245 250 255Asp Pro Lys Leu Asn Pro Lys Asp Asp Gly Ile Val Tyr Ala Ser Leu 260 265 270Ala Leu Ser Ser Ser Thr Ser Pro Arg Ala Pro Pro Ser His Arg Pro 275 280 285Leu Lys Ser Pro Gln Asn Glu Thr Leu Tyr Ser Val Leu Lys Ala 290 295 3004284PRTHomo sapiens 4Gln Pro Ser Gly Ser Thr Gly Ser Gly Pro Ser Tyr Leu Tyr Gly Val1 5 10 15Thr Gln Pro Lys His Leu Ser Ala Ser Met Gly Gly Ser Val Glu Ile 20 25 30Pro Phe Ser Phe Tyr Tyr Pro Trp Glu Leu Ala Thr Ala Pro Asp Val 35 40 45Arg Ile Ser Trp Arg Arg Gly His Phe His Arg Gln Ser Phe Tyr Ser 50 55 60Thr Arg Pro Pro Ser Ile His Lys Asp Tyr Val Asn Arg Leu Phe Leu65 70 75 80Asn Trp Thr Glu Gly Gln Lys Ser Gly Phe Leu Arg Ile Ser Asn Leu 85 90 95Gln Lys Gln Asp Gln Ser Val Tyr Phe Cys Arg Val Glu Leu Asp Thr 100 105 110Arg Ser Ser Gly Arg Gln Gln Trp Gln Ser Ile Glu Gly Thr Lys Leu 115 120 125Ser Ile Thr Gln Ala Val Thr Thr Thr Thr Gln Arg Pro Ser Ser Met 130 135 140Thr Thr Thr Trp Arg Leu Ser Ser Thr Thr Thr Thr Thr Gly Leu Arg145 150 155 160Val Thr Gln Gly Lys Arg Arg Ser Asp Ser Trp His Ile Ser Leu Glu 165 170 175Thr Ala Val Gly Val Ala Val Ala Val Thr Val Leu Gly Ile Met Ile 180 185 190Leu Gly Leu Ile Cys Leu Leu Arg Trp Arg Arg Arg Lys Gly Gln Gln 195 200 205Arg Thr Lys Ala Thr Thr Pro Ala Arg Glu Pro Phe Gln Asn Thr Glu 210 215 220Glu Pro Tyr Glu Asn Ile Arg Asn Glu Gly Gln Asn Thr Asp Pro Lys225 230 235 240Leu Asn Pro Lys Asp Asp Gly Ile Val Tyr Ala Ser Leu Ala Leu Ser 245 250 255Ser Ser Thr Ser Pro Arg Ala Pro Pro Ser His Arg Pro Leu Lys Ser 260 265 270Pro Gln Asn Glu Thr Leu Tyr Ser Val Leu Lys Ala 275 2805325PRTHomo sapiens 5Met Ala Thr Pro Leu Pro Pro Pro Ser Pro Arg His Leu Arg Leu Leu1 5 10 15Arg Leu Leu Leu Ser Gly Leu Val Leu Gly Ala Ala Leu Arg Gly Ala 20 25 30Ala Ala Gly His Pro Asp Val Ala Ala Cys Pro Gly Ser Leu Asp Cys 35 40 45Ala Leu Lys Arg Arg Ala Arg Cys Pro Pro Gly Ala His Ala Cys Gly 50 55 60Pro Cys Leu Gln Pro Phe Gln Glu Asp Gln Gln Gly Leu Cys Val Pro65 70 75 80Arg Met Arg Arg Pro Pro Gly Gly Gly Arg Pro Gln Pro Arg Leu Glu 85 90 95Asp Glu Ile Asp Phe Leu Ala Gln Glu Leu Ala Arg Lys Glu Ser Gly 100 105 110Gln Ser Thr Pro Pro Leu Pro Lys Asp Arg Gln Arg Leu Pro Glu Pro 115 120 125Ala Thr Leu Gly Phe Ser Ala Arg Gly Gln Gly Leu Glu Leu Gly Leu 130 135 140Pro Ser Thr Pro Gly Thr Pro Thr Pro Thr Pro His Thr Ser Leu Gly145 150 155 160Ser Pro Val Ser Ser Asp Pro Val His Met Ser Pro Leu Glu Pro Arg 165 170 175Gly Gly Gln Gly Asp Gly Leu Ala Leu Val Leu Ile Leu Ala Phe Cys 180 185 190Val Ala Gly Ala Ala Ala Leu Ser Val Ala Ser Leu Cys Trp Cys Arg 195 200 205Leu Gln Arg Glu Ile Arg Leu Thr Gln Lys Ala Asp Tyr Ala Thr Ala 210 215 220Lys Ala Pro Gly Ser Pro Ala Ala Pro Arg Ile Ser Pro Gly Asp Gln225 230 235 240Arg Leu Ala Gln Ser Ala Glu Met Tyr His Tyr Gln His Gln Arg Gln 245 250 255Gln Met Leu Cys Leu Glu Arg His Lys Glu Pro Pro Lys Glu Leu Asp 260 265 270Thr Ala Ser Ser Asp Glu Glu Asn Glu Asp Gly Asp Phe Thr Val Tyr 275 280 285Glu Cys Pro Gly Leu Ala Pro Thr Gly Glu Met Glu Val Arg Asn Pro 290 295 300Leu Phe Asp His Ala Ala Leu Ser Ala Pro Leu Pro Ala Pro Ser Ser305 310 315 320Pro Pro Ala Leu Pro 3256291PRTHomo sapiens 6Gly His Pro Asp Val Ala Ala Cys Pro Gly Ser Leu Asp Cys Ala Leu1 5 10 15Lys Arg Arg Ala Arg Cys Pro Pro Gly Ala His Ala Cys Gly Pro Cys 20 25 30Leu Gln Pro Phe Gln Glu Asp Gln Gln Gly Leu Cys Val Pro Arg Met 35 40 45Arg Arg Pro Pro Gly Gly Gly Arg Pro Gln Pro Arg Leu Glu Asp Glu 50 55 60Ile Asp Phe Leu Ala Gln Glu Leu Ala Arg Lys Glu Ser Gly Gln Ser65 70 75 80Thr Pro Pro Leu Pro Lys Asp Arg Gln Arg Leu Pro Glu Pro Ala Thr 85 90 95Leu Gly Phe Ser Ala Arg Gly Gln Gly Leu Glu Leu Gly Leu Pro Ser 100 105 110Thr Pro Gly Thr Pro Thr Pro Thr Pro His Thr Ser Leu Gly Ser Pro 115 120 125Val Ser Ser Asp Pro Val His Met Ser Pro Leu Glu Pro Arg Gly Gly 130 135 140Gln Gly Asp Gly Leu Ala Leu Val Leu Ile Leu Ala Phe Cys Val Ala145 150 155 160Gly Ala Ala Ala Leu Ser Val Ala Ser Leu Cys Trp Cys Arg Leu Gln 165 170 175Arg Glu Ile Arg Leu Thr Gln Lys Ala Asp Tyr Ala Thr Ala Lys Ala 180 185 190Pro Gly Ser Pro Ala Ala Pro Arg Ile Ser Pro Gly Asp Gln Arg Leu 195 200 205Ala Gln Ser Ala Glu Met Tyr His Tyr Gln His Gln Arg Gln Gln Met 210 215 220Leu Cys Leu Glu Arg His Lys Glu Pro Pro Lys Glu Leu Asp Thr Ala225 230 235 240Ser Ser Asp Glu Glu Asn Glu Asp Gly Asp Phe Thr Val Tyr Glu Cys 245 250 255Pro Gly Leu Ala Pro Thr Gly Glu Met Glu Val Arg Asn Pro Leu Phe 260 265 270Asp His Ala Ala Leu Ser Ala Pro Leu Pro Ala Pro Ser Ser Pro Pro 275 280 285Ala Leu Pro 2907742PRTHomo sapiens 7Met Lys Asp Asp Phe Ala Glu Glu Glu Glu Val Gln Ser Phe Gly Tyr1 5 10 15Lys Arg Phe Gly Ile Gln Glu Gly Thr Gln Cys Thr Lys Cys Lys Asn 20 25 30Asn Trp Ala Leu Lys Phe Ser Ile Ile Leu Leu Tyr Ile Leu Cys Ala 35 40 45Leu Leu Thr Ile Thr Val Ala Ile Leu Gly Tyr Lys Val Val Glu Lys 50 55 60Met Asp Asn Val Thr Gly Gly Met Glu Thr Ser Arg Gln Thr Tyr Asp65 70 75 80Asp Lys Leu Thr Ala Val Glu Ser Asp Leu Lys Lys Leu Gly Asp Gln 85 90 95Thr Gly Lys Lys Ala Ile Ser Thr Asn Ser Glu Leu Ser Thr Phe Arg 100 105 110Ser Asp Ile Leu Asp Leu Arg Gln Gln Leu Arg Glu Ile Thr Glu Lys 115 120 125Thr Ser Lys Asn Lys Asp Thr Leu Glu Lys Leu Gln Ala Ser Gly Asp 130 135 140Ala Leu Val Asp Arg Gln Ser Gln Leu Lys Glu Thr Leu Glu Asn Asn145 150 155 160Ser Phe Leu Ile Thr Thr Val Asn Lys Thr Leu Gln Ala Tyr Asn Gly 165 170 175Tyr Val Thr Asn Leu Gln Gln Asp Thr Ser Val Leu Gln Gly Asn Leu 180 185 190Gln Asn Gln Met Tyr Ser His Asn Val Val Ile Met Asn Leu Asn Asn 195 200 205Leu Asn Leu Thr Gln Val Gln Gln Arg Asn Leu Ile Thr Asn Leu Gln 210 215 220Arg Ser Val Asp Asp Thr Ser Gln Ala Ile Gln Arg Ile Lys Asn Asp225 230 235 240Phe Gln Asn Leu Gln Gln Val Phe Leu Gln Ala Lys Lys Asp Thr Asp 245 250 255Trp Leu Lys Glu Lys Val Gln Ser Leu Gln Thr Leu Ala Ala Asn Asn 260 265 270Ser Ala Leu Ala Lys Ala Asn Asn Asp Thr Leu Glu Asp Met Asn Ser 275 280 285Gln Leu Asn Ser Phe Thr Gly Gln Met Glu Asn Ile Thr Thr Ile Ser 290 295 300Gln Ala Asn Glu Gln Asn Leu Lys Asp Leu Gln Asp Leu His Lys Asp305 310 315 320Ala Glu Asn Arg Thr Ala Ile Lys Phe Asn Gln Leu Glu Glu Arg Phe 325 330 335Gln Leu Phe Glu Thr Asp Ile Val Asn Ile Ile Ser Asn Ile Ser Tyr 340 345 350Thr Ala His His Leu Arg Thr Leu Thr Ser Asn Leu Asn Glu Val Arg 355 360 365Thr Thr Cys Thr Asp Thr Leu Thr Lys His Thr Asp Asp Leu Thr Ser 370 375 380Leu Asn Asn Thr Leu Ala Asn Ile Arg Leu Asp Ser Val Ser Leu Arg385 390 395 400Met Gln Gln Asp Leu Met Arg Ser Arg Leu Asp Thr Glu Val Ala Asn 405 410 415Leu Ser Val Ile Met Glu Glu Met Lys Leu Val Asp Ser Lys His Gly 420 425 430Gln Leu Ile Lys Asn Phe Thr Ile Leu Gln Gly Pro Pro Gly Pro Arg 435 440 445Gly Pro Arg Gly Asp Arg Gly Ser Gln Gly Pro Pro Gly Pro Thr Gly 450 455 460Asn Lys Gly Gln Lys Gly Glu Lys Gly Glu Pro Gly Pro Pro Gly Pro465 470 475 480Ala Gly Glu Arg Gly Pro Ile Gly Pro Ala Gly Pro Pro Gly Glu Arg 485 490 495Gly Gly Lys Gly Ser Lys Gly Ser Gln Gly Pro Lys Gly Ser Arg Gly 500 505 510Ser Pro Gly Lys Pro Gly Pro Gln Gly Pro Ser Gly Asp Pro Gly Pro 515 520 525Pro Gly Pro Pro Gly Lys Glu Gly Leu Pro Gly Pro Gln Gly Pro Pro 530 535 540Gly Phe Gln Gly Leu Gln Gly Thr Val Gly Glu Pro Gly Val Pro Gly545 550 555 560Pro Arg Gly Leu Pro Gly Leu Pro Gly Val Pro Gly Met Pro Gly Pro 565 570 575Lys Gly Pro Pro Gly Pro Pro Gly Pro Ser Gly Ala Val Val Pro Leu 580 585 590Ala Leu Gln Asn Glu Pro Thr Pro Ala Pro Glu Asp Asn Ser Cys Pro 595 600 605Pro His Trp Lys Asn Phe Thr Asp Lys Cys Tyr Tyr Phe Ser Val Glu 610 615 620Lys Glu Ile Phe Glu Asp Ala Lys Leu Phe Cys Glu Asp Lys Ser Ser625 630 635 640His Leu Val Phe Ile Asn Thr Arg Glu Glu Gln Gln Trp Ile Lys Lys 645 650 655Gln Met Val Gly Arg Glu Ser His Trp Ile Gly Leu Thr Asp Ser Glu 660 665 670Arg Glu Asn Glu Trp Lys Trp Leu Asp Gly Thr Ser Pro Asp Tyr Lys 675 680

685Asn Trp Lys Ala Gly Gln Pro Asp Asn Trp Gly His Gly His Gly Pro 690 695 700Gly Glu Asp Cys Ala Gly Leu Ile Tyr Ala Gly Gln Trp Asn Asp Phe705 710 715 720Gln Cys Glu Asp Val Asn Asn Phe Ile Cys Glu Lys Asp Arg Glu Thr 725 730 735Val Leu Ser Ser Ala Leu 7408442PRTHomo sapiens 8Met Gln Pro Pro Pro Ser Leu Cys Gly Arg Ala Leu Val Ala Leu Val1 5 10 15Leu Ala Cys Gly Leu Ser Arg Ile Trp Gly Glu Glu Arg Gly Phe Pro 20 25 30Pro Asp Arg Ala Thr Pro Leu Leu Gln Thr Ala Glu Ile Met Thr Pro 35 40 45Pro Thr Lys Thr Leu Trp Pro Lys Gly Ser Asn Ala Ser Leu Ala Arg 50 55 60Ser Leu Ala Pro Ala Glu Val Pro Lys Gly Asp Arg Thr Ala Gly Ser65 70 75 80Pro Pro Arg Thr Ile Ser Pro Pro Pro Cys Gln Gly Pro Ile Glu Ile 85 90 95Lys Glu Thr Phe Lys Tyr Ile Asn Thr Val Val Ser Cys Leu Val Phe 100 105 110Val Leu Gly Ile Ile Gly Asn Ser Thr Leu Leu Arg Ile Ile Tyr Lys 115 120 125Asn Lys Cys Met Arg Asn Gly Pro Asn Ile Leu Ile Ala Ser Leu Ala 130 135 140Leu Gly Asp Leu Leu His Ile Val Ile Asp Ile Pro Ile Asn Val Tyr145 150 155 160Lys Leu Leu Ala Glu Asp Trp Pro Phe Gly Ala Glu Met Cys Lys Leu 165 170 175Val Pro Phe Ile Gln Lys Ala Ser Val Gly Ile Thr Val Leu Ser Leu 180 185 190Cys Ala Leu Ser Ile Asp Arg Tyr Arg Ala Val Ala Ser Trp Ser Arg 195 200 205Ile Lys Gly Ile Gly Val Pro Lys Trp Thr Ala Val Glu Ile Val Leu 210 215 220Ile Trp Val Val Ser Val Val Leu Ala Val Pro Glu Ala Ile Gly Phe225 230 235 240Asp Ile Ile Thr Met Asp Tyr Lys Gly Ser Tyr Leu Arg Ile Cys Leu 245 250 255Leu His Pro Val Gln Lys Thr Ala Phe Met Gln Phe Tyr Lys Thr Ala 260 265 270Lys Asp Trp Trp Leu Phe Ser Phe Tyr Phe Cys Leu Pro Leu Ala Ile 275 280 285Thr Ala Phe Phe Tyr Thr Leu Met Thr Cys Glu Met Leu Arg Lys Lys 290 295 300Ser Gly Met Gln Ile Ala Leu Asn Asp His Leu Lys Gln Arg Arg Glu305 310 315 320Val Ala Lys Thr Val Phe Cys Leu Val Leu Val Phe Ala Leu Cys Trp 325 330 335Leu Pro Leu His Leu Ser Arg Ile Leu Lys Leu Thr Leu Tyr Asn Gln 340 345 350Asn Asp Pro Asn Arg Cys Glu Leu Leu Ser Phe Leu Leu Val Leu Asp 355 360 365Tyr Ile Gly Ile Asn Met Ala Ser Leu Asn Ser Cys Ile Asn Pro Ile 370 375 380Ala Leu Tyr Leu Val Ser Lys Arg Phe Lys Asn Cys Phe Lys Ser Cys385 390 395 400Leu Cys Cys Trp Cys Gln Ser Phe Glu Glu Lys Gln Ser Leu Glu Glu 405 410 415Lys Gln Ser Cys Leu Lys Phe Lys Ala Asn Asp His Gly Tyr Asp Asn 420 425 430Phe Arg Ser Ser Asn Lys Tyr Ser Ser Ser 435 4409416PRTHomo sapiens 9Glu Glu Arg Gly Phe Pro Pro Asp Arg Ala Thr Pro Leu Leu Gln Thr1 5 10 15Ala Glu Ile Met Thr Pro Pro Thr Lys Thr Leu Trp Pro Lys Gly Ser 20 25 30Asn Ala Ser Leu Ala Arg Ser Leu Ala Pro Ala Glu Val Pro Lys Gly 35 40 45Asp Arg Thr Ala Gly Ser Pro Pro Arg Thr Ile Ser Pro Pro Pro Cys 50 55 60Gln Gly Pro Ile Glu Ile Lys Glu Thr Phe Lys Tyr Ile Asn Thr Val65 70 75 80Val Ser Cys Leu Val Phe Val Leu Gly Ile Ile Gly Asn Ser Thr Leu 85 90 95Leu Arg Ile Ile Tyr Lys Asn Lys Cys Met Arg Asn Gly Pro Asn Ile 100 105 110Leu Ile Ala Ser Leu Ala Leu Gly Asp Leu Leu His Ile Val Ile Asp 115 120 125Ile Pro Ile Asn Val Tyr Lys Leu Leu Ala Glu Asp Trp Pro Phe Gly 130 135 140Ala Glu Met Cys Lys Leu Val Pro Phe Ile Gln Lys Ala Ser Val Gly145 150 155 160Ile Thr Val Leu Ser Leu Cys Ala Leu Ser Ile Asp Arg Tyr Arg Ala 165 170 175Val Ala Ser Trp Ser Arg Ile Lys Gly Ile Gly Val Pro Lys Trp Thr 180 185 190Ala Val Glu Ile Val Leu Ile Trp Val Val Ser Val Val Leu Ala Val 195 200 205Pro Glu Ala Ile Gly Phe Asp Ile Ile Thr Met Asp Tyr Lys Gly Ser 210 215 220Tyr Leu Arg Ile Cys Leu Leu His Pro Val Gln Lys Thr Ala Phe Met225 230 235 240Gln Phe Tyr Lys Thr Ala Lys Asp Trp Trp Leu Phe Ser Phe Tyr Phe 245 250 255Cys Leu Pro Leu Ala Ile Thr Ala Phe Phe Tyr Thr Leu Met Thr Cys 260 265 270Glu Met Leu Arg Lys Lys Ser Gly Met Gln Ile Ala Leu Asn Asp His 275 280 285Leu Lys Gln Arg Arg Glu Val Ala Lys Thr Val Phe Cys Leu Val Leu 290 295 300Val Phe Ala Leu Cys Trp Leu Pro Leu His Leu Ser Arg Ile Leu Lys305 310 315 320Leu Thr Leu Tyr Asn Gln Asn Asp Pro Asn Arg Cys Glu Leu Leu Ser 325 330 335Phe Leu Leu Val Leu Asp Tyr Ile Gly Ile Asn Met Ala Ser Leu Asn 340 345 350Ser Cys Ile Asn Pro Ile Ala Leu Tyr Leu Val Ser Lys Arg Phe Lys 355 360 365Asn Cys Phe Lys Ser Cys Leu Cys Cys Trp Cys Gln Ser Phe Glu Glu 370 375 380Lys Gln Ser Leu Glu Glu Lys Gln Ser Cys Leu Lys Phe Lys Ala Asn385 390 395 400Asp His Gly Tyr Asp Asn Phe Arg Ser Ser Asn Lys Tyr Ser Ser Ser 405 410 41510293PRTHomo sapiens 10Met Asp Thr Thr Arg Tyr Ser Lys Trp Gly Gly Ser Ser Glu Glu Val1 5 10 15Pro Gly Gly Pro Trp Gly Arg Trp Val His Trp Ser Arg Arg Pro Leu 20 25 30Phe Leu Ala Leu Ala Val Leu Val Thr Thr Val Leu Trp Ala Val Ile 35 40 45Leu Ser Ile Leu Leu Ser Lys Ala Ser Thr Glu Arg Ala Ala Leu Leu 50 55 60Asp Gly His Asp Leu Leu Arg Thr Asn Ala Ser Lys Gln Thr Ala Ala65 70 75 80Leu Gly Ala Leu Lys Glu Glu Val Gly Asp Cys His Ser Cys Cys Ser 85 90 95Gly Thr Gln Ala Gln Leu Gln Thr Thr Arg Ala Glu Leu Gly Glu Ala 100 105 110Gln Ala Lys Leu Met Glu Gln Glu Ser Ala Leu Arg Glu Leu Arg Glu 115 120 125Arg Val Thr Gln Gly Leu Ala Glu Ala Gly Arg Gly Arg Glu Asp Val 130 135 140Arg Thr Glu Leu Phe Arg Ala Leu Glu Ala Val Arg Leu Gln Asn Asn145 150 155 160Ser Cys Glu Pro Cys Pro Thr Ser Trp Leu Ser Phe Glu Gly Ser Cys 165 170 175Tyr Phe Phe Ser Val Pro Lys Thr Thr Trp Ala Ala Ala Gln Asp His 180 185 190Cys Ala Asp Ala Ser Ala His Leu Val Ile Val Gly Gly Leu Asp Glu 195 200 205Gln Gly Phe Leu Thr Arg Asn Thr Arg Gly Arg Gly Tyr Trp Leu Gly 210 215 220Leu Arg Ala Val Arg His Leu Gly Lys Val Gln Gly Tyr Gln Trp Val225 230 235 240Asp Gly Val Ser Leu Ser Phe Ser His Trp Asn Gln Gly Glu Pro Asn 245 250 255Asp Ala Trp Gly Arg Glu Asn Cys Val Met Met Leu His Thr Gly Leu 260 265 270Trp Asn Asp Ala Pro Cys Asp Ser Glu Lys Asp Gly Trp Ile Cys Glu 275 280 285Lys Arg His Asn Cys 29011247PRTHomo sapiens 11Val Ile Leu Ser Ile Leu Leu Ser Lys Ala Ser Thr Glu Arg Ala Ala1 5 10 15Leu Leu Asp Gly His Asp Leu Leu Arg Thr Asn Ala Ser Lys Gln Thr 20 25 30Ala Ala Leu Gly Ala Leu Lys Glu Glu Val Gly Asp Cys His Ser Cys 35 40 45Cys Ser Gly Thr Gln Ala Gln Leu Gln Thr Thr Arg Ala Glu Leu Gly 50 55 60Glu Ala Gln Ala Lys Leu Met Glu Gln Glu Ser Ala Leu Arg Glu Leu65 70 75 80Arg Glu Arg Val Thr Gln Gly Leu Ala Glu Ala Gly Arg Gly Arg Glu 85 90 95Asp Val Arg Thr Glu Leu Phe Arg Ala Leu Glu Ala Val Arg Leu Gln 100 105 110Asn Asn Ser Cys Glu Pro Cys Pro Thr Ser Trp Leu Ser Phe Glu Gly 115 120 125Ser Cys Tyr Phe Phe Ser Val Pro Lys Thr Thr Trp Ala Ala Ala Gln 130 135 140Asp His Cys Ala Asp Ala Ser Ala His Leu Val Ile Val Gly Gly Leu145 150 155 160Asp Glu Gln Gly Phe Leu Thr Arg Asn Thr Arg Gly Arg Gly Tyr Trp 165 170 175Leu Gly Leu Arg Ala Val Arg His Leu Gly Lys Val Gln Gly Tyr Gln 180 185 190Trp Val Asp Gly Val Ser Leu Ser Phe Ser His Trp Asn Gln Gly Glu 195 200 205Pro Asn Asp Ala Trp Gly Arg Glu Asn Cys Val Met Met Leu His Thr 210 215 220Gly Leu Trp Asn Asp Ala Pro Cys Asp Ser Glu Lys Asp Gly Trp Ile225 230 235 240Cys Glu Lys Arg His Asn Cys 24512184PRTHomo sapiens 12Met Arg Arg Gly Pro Arg Ser Leu Arg Gly Arg Asp Ala Pro Ala Pro1 5 10 15Thr Pro Cys Val Pro Ala Glu Cys Phe Asp Leu Leu Val Arg His Cys 20 25 30Val Ala Cys Gly Leu Leu Arg Thr Pro Arg Pro Lys Pro Ala Gly Ala 35 40 45Ser Ser Pro Ala Pro Arg Thr Ala Leu Gln Pro Gln Glu Ser Val Gly 50 55 60Ala Gly Ala Gly Glu Ala Ala Leu Pro Leu Pro Gly Leu Leu Phe Gly65 70 75 80Ala Pro Ala Leu Leu Gly Leu Ala Leu Val Leu Ala Leu Val Leu Val 85 90 95Gly Leu Val Ser Trp Arg Arg Arg Gln Arg Arg Leu Arg Gly Ala Ser 100 105 110Ser Ala Glu Ala Pro Asp Gly Asp Lys Asp Ala Pro Glu Pro Leu Asp 115 120 125Lys Val Ile Ile Leu Ser Pro Gly Ile Ser Asp Ala Thr Ala Pro Ala 130 135 140Trp Pro Pro Pro Gly Glu Asp Pro Gly Thr Thr Pro Pro Gly His Ser145 150 155 160Val Pro Val Pro Ala Thr Glu Leu Gly Ser Thr Glu Leu Val Thr Thr 165 170 175Lys Thr Ala Gly Pro Glu Gln Gln 18013626PRTHomo sapiens 13Met Ile Phe Leu Thr Ala Leu Pro Leu Phe Trp Ile Met Ile Ser Ala1 5 10 15Ser Arg Gly Gly His Trp Gly Ala Trp Met Pro Ser Ser Ile Ser Ala 20 25 30Phe Glu Gly Thr Cys Val Ser Ile Pro Cys Arg Phe Asp Phe Pro Asp 35 40 45Glu Leu Arg Pro Ala Val Val His Gly Val Trp Tyr Phe Asn Ser Pro 50 55 60Tyr Pro Lys Asn Tyr Pro Pro Val Val Phe Lys Ser Arg Thr Gln Val65 70 75 80Val His Glu Ser Phe Gln Gly Arg Ser Arg Leu Leu Gly Asp Leu Gly 85 90 95Leu Arg Asn Cys Thr Leu Leu Leu Ser Asn Val Ser Pro Glu Leu Gly 100 105 110Gly Lys Tyr Tyr Phe Arg Gly Asp Leu Gly Gly Tyr Asn Gln Tyr Thr 115 120 125Phe Ser Glu His Ser Val Leu Asp Ile Val Asn Thr Pro Asn Ile Val 130 135 140Val Pro Pro Glu Val Val Ala Gly Thr Glu Val Glu Val Ser Cys Met145 150 155 160Val Pro Asp Asn Cys Pro Glu Leu Arg Pro Glu Leu Ser Trp Leu Gly 165 170 175His Glu Gly Leu Gly Glu Pro Ala Val Leu Gly Arg Leu Arg Glu Asp 180 185 190Glu Gly Thr Trp Val Gln Val Ser Leu Leu His Phe Val Pro Thr Arg 195 200 205Glu Ala Asn Gly His Arg Leu Gly Cys Gln Ala Ser Phe Pro Asn Thr 210 215 220Thr Leu Gln Phe Glu Gly Tyr Ala Ser Met Asp Val Lys Tyr Pro Pro225 230 235 240Val Ile Val Glu Met Asn Ser Ser Val Glu Ala Ile Glu Gly Ser His 245 250 255Val Ser Leu Leu Cys Gly Ala Asp Ser Asn Pro Pro Pro Leu Leu Thr 260 265 270Trp Met Arg Asp Gly Thr Val Leu Arg Glu Ala Val Ala Glu Ser Leu 275 280 285Leu Leu Glu Leu Glu Glu Val Thr Pro Ala Glu Asp Gly Val Tyr Ala 290 295 300Cys Leu Ala Glu Asn Ala Tyr Gly Gln Asp Asn Arg Thr Val Gly Leu305 310 315 320Ser Val Met Tyr Ala Pro Trp Lys Pro Thr Val Asn Gly Thr Met Val 325 330 335Ala Val Glu Gly Glu Thr Val Ser Ile Leu Cys Ser Thr Gln Ser Asn 340 345 350Pro Asp Pro Ile Leu Thr Ile Phe Lys Glu Lys Gln Ile Leu Ser Thr 355 360 365Val Ile Tyr Glu Ser Glu Leu Gln Leu Glu Leu Pro Ala Val Ser Pro 370 375 380Glu Asp Asp Gly Glu Tyr Trp Cys Val Ala Glu Asn Gln Tyr Gly Gln385 390 395 400Arg Ala Thr Ala Phe Asn Leu Ser Val Glu Phe Ala Pro Val Leu Leu 405 410 415Leu Glu Ser His Cys Ala Ala Ala Arg Asp Thr Val Gln Cys Leu Cys 420 425 430Val Val Lys Ser Asn Pro Glu Pro Ser Val Ala Phe Glu Leu Pro Ser 435 440 445Arg Asn Val Thr Val Asn Glu Ser Glu Arg Glu Phe Val Tyr Ser Glu 450 455 460Arg Ser Gly Leu Val Leu Thr Ser Ile Leu Thr Leu Arg Gly Gln Ala465 470 475 480Gln Ala Pro Pro Arg Val Ile Cys Thr Ala Arg Asn Leu Tyr Gly Ala 485 490 495Lys Ser Leu Glu Leu Pro Phe Gln Gly Ala His Arg Leu Met Trp Ala 500 505 510Lys Ile Gly Pro Val Gly Ala Val Val Ala Phe Ala Ile Leu Ile Ala 515 520 525Ile Val Cys Tyr Ile Thr Gln Thr Arg Arg Lys Lys Asn Val Thr Glu 530 535 540Ser Pro Ser Phe Ser Ala Gly Asp Asn Pro Pro Val Leu Phe Ser Ser545 550 555 560Asp Phe Arg Ile Ser Gly Ala Pro Glu Lys Tyr Glu Ser Glu Arg Arg 565 570 575Leu Gly Ser Glu Arg Arg Leu Leu Gly Leu Arg Gly Glu Pro Pro Glu 580 585 590Leu Asp Leu Ser Tyr Ser His Ser Asp Leu Gly Lys Arg Pro Thr Lys 595 600 605Asp Ser Tyr Thr Leu Thr Glu Glu Leu Ala Glu Tyr Ala Glu Ile Arg 610 615 620Val Lys62514607PRTHomo sapiens 14Gly His Trp Gly Ala Trp Met Pro Ser Ser Ile Ser Ala Phe Glu Gly1 5 10 15Thr Cys Val Ser Ile Pro Cys Arg Phe Asp Phe Pro Asp Glu Leu Arg 20 25 30Pro Ala Val Val His Gly Val Trp Tyr Phe Asn Ser Pro Tyr Pro Lys 35 40 45Asn Tyr Pro Pro Val Val Phe Lys Ser Arg Thr Gln Val Val His Glu 50 55 60Ser Phe Gln Gly Arg Ser Arg Leu Leu Gly Asp Leu Gly Leu Arg Asn65 70 75 80Cys Thr Leu Leu Leu Ser Asn Val Ser Pro Glu Leu Gly Gly Lys Tyr 85 90 95Tyr Phe Arg Gly Asp Leu Gly Gly Tyr Asn Gln Tyr Thr Phe Ser Glu 100 105 110His Ser Val Leu Asp Ile Val Asn Thr Pro Asn Ile Val Val Pro Pro 115 120 125Glu Val Val Ala Gly Thr Glu Val Glu Val Ser Cys Met Val Pro Asp 130 135 140Asn Cys Pro Glu Leu Arg Pro Glu Leu Ser Trp Leu Gly His Glu Gly145 150 155 160Leu Gly Glu Pro Ala Val Leu Gly Arg Leu Arg Glu Asp Glu Gly Thr 165 170 175Trp Val Gln Val Ser Leu Leu His Phe Val Pro Thr Arg Glu Ala Asn 180 185 190Gly His Arg Leu Gly Cys Gln Ala Ser Phe Pro Asn

Thr Thr Leu Gln 195 200 205Phe Glu Gly Tyr Ala Ser Met Asp Val Lys Tyr Pro Pro Val Ile Val 210 215 220Glu Met Asn Ser Ser Val Glu Ala Ile Glu Gly Ser His Val Ser Leu225 230 235 240Leu Cys Gly Ala Asp Ser Asn Pro Pro Pro Leu Leu Thr Trp Met Arg 245 250 255Asp Gly Thr Val Leu Arg Glu Ala Val Ala Glu Ser Leu Leu Leu Glu 260 265 270Leu Glu Glu Val Thr Pro Ala Glu Asp Gly Val Tyr Ala Cys Leu Ala 275 280 285Glu Asn Ala Tyr Gly Gln Asp Asn Arg Thr Val Gly Leu Ser Val Met 290 295 300Tyr Ala Pro Trp Lys Pro Thr Val Asn Gly Thr Met Val Ala Val Glu305 310 315 320Gly Glu Thr Val Ser Ile Leu Cys Ser Thr Gln Ser Asn Pro Asp Pro 325 330 335Ile Leu Thr Ile Phe Lys Glu Lys Gln Ile Leu Ser Thr Val Ile Tyr 340 345 350Glu Ser Glu Leu Gln Leu Glu Leu Pro Ala Val Ser Pro Glu Asp Asp 355 360 365Gly Glu Tyr Trp Cys Val Ala Glu Asn Gln Tyr Gly Gln Arg Ala Thr 370 375 380Ala Phe Asn Leu Ser Val Glu Phe Ala Pro Val Leu Leu Leu Glu Ser385 390 395 400His Cys Ala Ala Ala Arg Asp Thr Val Gln Cys Leu Cys Val Val Lys 405 410 415Ser Asn Pro Glu Pro Ser Val Ala Phe Glu Leu Pro Ser Arg Asn Val 420 425 430Thr Val Asn Glu Ser Glu Arg Glu Phe Val Tyr Ser Glu Arg Ser Gly 435 440 445Leu Val Leu Thr Ser Ile Leu Thr Leu Arg Gly Gln Ala Gln Ala Pro 450 455 460Pro Arg Val Ile Cys Thr Ala Arg Asn Leu Tyr Gly Ala Lys Ser Leu465 470 475 480Glu Leu Pro Phe Gln Gly Ala His Arg Leu Met Trp Ala Lys Ile Gly 485 490 495Pro Val Gly Ala Val Val Ala Phe Ala Ile Leu Ile Ala Ile Val Cys 500 505 510Tyr Ile Thr Gln Thr Arg Arg Lys Lys Asn Val Thr Glu Ser Pro Ser 515 520 525Phe Ser Ala Gly Asp Asn Pro Pro Val Leu Phe Ser Ser Asp Phe Arg 530 535 540Ile Ser Gly Ala Pro Glu Lys Tyr Glu Ser Glu Arg Arg Leu Gly Ser545 550 555 560Glu Arg Arg Leu Leu Gly Leu Arg Gly Glu Pro Pro Glu Leu Asp Leu 565 570 575Ser Tyr Ser His Ser Asp Leu Gly Lys Arg Pro Thr Lys Asp Ser Tyr 580 585 590Thr Leu Thr Glu Glu Leu Ala Glu Tyr Ala Glu Ile Arg Val Lys 595 600 60515272PRTHomo sapiens 15Met Asp Ser Tyr Leu Leu Met Trp Gly Leu Leu Thr Phe Ile Met Val1 5 10 15Pro Gly Cys Gln Ala Glu Leu Cys Asp Asp Asp Pro Pro Glu Ile Pro 20 25 30His Ala Thr Phe Lys Ala Met Ala Tyr Lys Glu Gly Thr Met Leu Asn 35 40 45Cys Glu Cys Lys Arg Gly Phe Arg Arg Ile Lys Ser Gly Ser Leu Tyr 50 55 60Met Leu Cys Thr Gly Asn Ser Ser His Ser Ser Trp Asp Asn Gln Cys65 70 75 80Gln Cys Thr Ser Ser Ala Thr Arg Asn Thr Thr Lys Gln Val Thr Pro 85 90 95Gln Pro Glu Glu Gln Lys Glu Arg Lys Thr Thr Glu Met Gln Ser Pro 100 105 110Met Gln Pro Val Asp Gln Ala Ser Leu Pro Gly His Cys Arg Glu Pro 115 120 125Pro Pro Trp Glu Asn Glu Ala Thr Glu Arg Ile Tyr His Phe Val Val 130 135 140Gly Gln Met Val Tyr Tyr Gln Cys Val Gln Gly Tyr Arg Ala Leu His145 150 155 160Arg Gly Pro Ala Glu Ser Val Cys Lys Met Thr His Gly Lys Thr Arg 165 170 175Trp Thr Gln Pro Gln Leu Ile Cys Thr Gly Glu Met Glu Thr Ser Gln 180 185 190Phe Pro Gly Glu Glu Lys Pro Gln Ala Ser Pro Glu Gly Arg Pro Glu 195 200 205Ser Glu Thr Ser Cys Leu Val Thr Thr Thr Asp Phe Gln Ile Gln Thr 210 215 220Glu Met Ala Ala Thr Met Glu Thr Ser Ile Phe Thr Thr Glu Tyr Gln225 230 235 240Val Ala Val Ala Gly Cys Val Phe Leu Leu Ile Ser Val Leu Leu Leu 245 250 255Ser Gly Leu Thr Trp Gln Arg Arg Gln Arg Lys Ser Arg Arg Thr Ile 260 265 27016251PRTHomo sapiens 16Glu Leu Cys Asp Asp Asp Pro Pro Glu Ile Pro His Ala Thr Phe Lys1 5 10 15Ala Met Ala Tyr Lys Glu Gly Thr Met Leu Asn Cys Glu Cys Lys Arg 20 25 30Gly Phe Arg Arg Ile Lys Ser Gly Ser Leu Tyr Met Leu Cys Thr Gly 35 40 45Asn Ser Ser His Ser Ser Trp Asp Asn Gln Cys Gln Cys Thr Ser Ser 50 55 60Ala Thr Arg Asn Thr Thr Lys Gln Val Thr Pro Gln Pro Glu Glu Gln65 70 75 80Lys Glu Arg Lys Thr Thr Glu Met Gln Ser Pro Met Gln Pro Val Asp 85 90 95Gln Ala Ser Leu Pro Gly His Cys Arg Glu Pro Pro Pro Trp Glu Asn 100 105 110Glu Ala Thr Glu Arg Ile Tyr His Phe Val Val Gly Gln Met Val Tyr 115 120 125Tyr Gln Cys Val Gln Gly Tyr Arg Ala Leu His Arg Gly Pro Ala Glu 130 135 140Ser Val Cys Lys Met Thr His Gly Lys Thr Arg Trp Thr Gln Pro Gln145 150 155 160Leu Ile Cys Thr Gly Glu Met Glu Thr Ser Gln Phe Pro Gly Glu Glu 165 170 175Lys Pro Gln Ala Ser Pro Glu Gly Arg Pro Glu Ser Glu Thr Ser Cys 180 185 190Leu Val Thr Thr Thr Asp Phe Gln Ile Gln Thr Glu Met Ala Ala Thr 195 200 205Met Glu Thr Ser Ile Phe Thr Thr Glu Tyr Gln Val Ala Val Ala Gly 210 215 220Cys Val Phe Leu Leu Ile Ser Val Leu Leu Leu Ser Gly Leu Thr Trp225 230 235 240Gln Arg Arg Gln Arg Lys Ser Arg Arg Thr Ile 245 25017321PRTHomo sapiens 17Met Glu Glu Gly Gln Tyr Ser Glu Ile Glu Glu Leu Pro Arg Arg Arg1 5 10 15Cys Cys Arg Arg Gly Thr Gln Ile Val Leu Leu Gly Leu Val Thr Ala 20 25 30Ala Leu Trp Ala Gly Leu Leu Thr Leu Leu Leu Leu Trp His Trp Asp 35 40 45Thr Thr Gln Ser Leu Lys Gln Leu Glu Glu Arg Ala Ala Arg Asn Val 50 55 60Ser Gln Val Ser Lys Asn Leu Glu Ser His His Gly Asp Gln Met Ala65 70 75 80Gln Lys Ser Gln Ser Thr Gln Ile Ser Gln Glu Leu Glu Glu Leu Arg 85 90 95Ala Glu Gln Gln Arg Leu Lys Ser Gln Asp Leu Glu Leu Ser Trp Asn 100 105 110Leu Asn Gly Leu Gln Ala Asp Leu Ser Ser Phe Lys Ser Gln Glu Leu 115 120 125Asn Glu Arg Asn Glu Ala Ser Asp Leu Leu Glu Arg Leu Arg Glu Glu 130 135 140Val Thr Lys Leu Arg Met Glu Leu Gln Val Ser Ser Gly Phe Val Cys145 150 155 160Asn Thr Cys Pro Glu Lys Trp Ile Asn Phe Gln Arg Lys Cys Tyr Tyr 165 170 175Phe Gly Lys Gly Thr Lys Gln Trp Val His Ala Arg Tyr Ala Cys Asp 180 185 190Asp Met Glu Gly Gln Leu Val Ser Ile His Ser Pro Glu Glu Gln Asp 195 200 205Phe Leu Thr Lys His Ala Ser His Thr Gly Ser Trp Ile Gly Leu Arg 210 215 220Asn Leu Asp Leu Lys Gly Glu Phe Ile Trp Val Asp Gly Ser His Val225 230 235 240Asp Tyr Ser Asn Trp Ala Pro Gly Glu Pro Thr Ser Arg Ser Gln Gly 245 250 255Glu Asp Cys Val Met Met Arg Gly Ser Gly Arg Trp Thr Asp Ala Phe 260 265 270Cys Asp Arg Lys Leu Gly Ala Trp Val Cys Asp Arg Leu Ala Thr Cys 275 280 285Thr Pro Pro Ala Ser Glu Gly Ser Ala Glu Ser Met Gly Pro Asp Ser 290 295 300Arg Pro Asp Pro Asp Gly Arg Leu Pro Thr Pro Ser Ala Pro Leu His305 310 315 320Ser18590PRTHomo sapiens 18Met Gly Phe His Leu Ile Thr Gln Leu Lys Gly Met Ser Val Val Leu1 5 10 15Val Leu Leu Pro Thr Leu Leu Leu Val Met Leu Thr Gly Ala Gln Arg 20 25 30Ala Cys Pro Lys Asn Cys Arg Cys Asp Gly Lys Ile Val Tyr Cys Glu 35 40 45Ser His Ala Phe Ala Asp Ile Pro Glu Asn Ile Ser Gly Gly Ser Gln 50 55 60Gly Leu Ser Leu Arg Phe Asn Ser Ile Gln Lys Leu Lys Ser Asn Gln65 70 75 80Phe Ala Gly Leu Asn Gln Leu Ile Trp Leu Tyr Leu Asp His Asn Tyr 85 90 95Ile Ser Ser Val Asp Glu Asp Ala Phe Gln Gly Ile Arg Arg Leu Lys 100 105 110Glu Leu Ile Leu Ser Ser Asn Lys Ile Thr Tyr Leu His Asn Lys Thr 115 120 125Phe His Pro Val Pro Asn Leu Arg Asn Leu Asp Leu Ser Tyr Asn Lys 130 135 140Leu Gln Thr Leu Gln Ser Glu Gln Phe Lys Gly Leu Arg Lys Leu Ile145 150 155 160Ile Leu His Leu Arg Ser Asn Ser Leu Lys Thr Val Pro Ile Arg Val 165 170 175Phe Gln Asp Cys Arg Asn Leu Asp Phe Leu Asp Leu Gly Tyr Asn Arg 180 185 190Leu Arg Ser Leu Ser Arg Asn Ala Phe Ala Gly Leu Leu Lys Leu Lys 195 200 205Glu Leu His Leu Glu His Asn Gln Phe Ser Lys Ile Asn Phe Ala His 210 215 220Phe Pro Arg Leu Phe Asn Leu Arg Ser Ile Tyr Leu Gln Trp Asn Arg225 230 235 240Ile Arg Ser Ile Ser Gln Gly Leu Thr Trp Thr Trp Ser Ser Leu His 245 250 255Asn Leu Asp Leu Ser Gly Asn Asp Ile Gln Gly Ile Glu Pro Gly Thr 260 265 270Phe Lys Cys Leu Pro Asn Leu Gln Lys Leu Asn Leu Asp Ser Asn Lys 275 280 285Leu Thr Asn Ile Ser Gln Glu Thr Val Asn Ala Trp Ile Ser Leu Ile 290 295 300Ser Ile Thr Leu Ser Gly Asn Met Trp Glu Cys Ser Arg Ser Ile Cys305 310 315 320Pro Leu Phe Tyr Trp Leu Lys Asn Phe Lys Gly Asn Lys Glu Ser Thr 325 330 335Met Ile Cys Ala Gly Pro Lys His Ile Gln Gly Glu Lys Val Ser Asp 340 345 350Ala Val Glu Thr Tyr Asn Ile Cys Ser Glu Val Gln Val Val Asn Thr 355 360 365Glu Arg Ser His Leu Val Pro Gln Thr Pro Gln Lys Pro Leu Ile Ile 370 375 380Pro Arg Pro Thr Ile Phe Lys Pro Asp Val Thr Gln Ser Thr Phe Glu385 390 395 400Thr Pro Ser Pro Ser Pro Gly Phe Gln Ile Pro Gly Ala Glu Gln Glu 405 410 415Tyr Glu His Val Ser Phe His Lys Ile Ile Ala Gly Ser Val Ala Leu 420 425 430Phe Leu Ser Val Ala Met Ile Leu Leu Val Ile Tyr Val Ser Trp Lys 435 440 445Arg Tyr Pro Ala Ser Met Lys Gln Leu Gln Gln His Ser Leu Met Lys 450 455 460Arg Arg Arg Lys Lys Ala Arg Glu Ser Glu Arg Gln Met Asn Ser Pro465 470 475 480Leu Gln Glu Tyr Tyr Val Asp Tyr Lys Pro Thr Asn Ser Glu Thr Met 485 490 495Asp Ile Ser Val Asn Gly Ser Gly Pro Cys Thr Tyr Thr Ile Ser Gly 500 505 510Ser Arg Glu Cys Glu Met Pro His His Met Lys Pro Leu Pro Tyr Tyr 515 520 525Ser Tyr Asp Gln Pro Val Ile Gly Tyr Cys Gln Ala His Gln Pro Leu 530 535 540His Val Thr Lys Gly Tyr Glu Thr Val Ser Pro Glu Gln Asp Glu Ser545 550 555 560Pro Gly Leu Glu Leu Gly Arg Asp His Ser Phe Ile Ala Thr Ile Ala 565 570 575Arg Ser Ala Ala Pro Ala Ile Tyr Leu Glu Arg Ile Ala Asn 580 585 59019560PRTHomo sapiens 19Gln Arg Ala Cys Pro Lys Asn Cys Arg Cys Asp Gly Lys Ile Val Tyr1 5 10 15Cys Glu Ser His Ala Phe Ala Asp Ile Pro Glu Asn Ile Ser Gly Gly 20 25 30Ser Gln Gly Leu Ser Leu Arg Phe Asn Ser Ile Gln Lys Leu Lys Ser 35 40 45Asn Gln Phe Ala Gly Leu Asn Gln Leu Ile Trp Leu Tyr Leu Asp His 50 55 60Asn Tyr Ile Ser Ser Val Asp Glu Asp Ala Phe Gln Gly Ile Arg Arg65 70 75 80Leu Lys Glu Leu Ile Leu Ser Ser Asn Lys Ile Thr Tyr Leu His Asn 85 90 95Lys Thr Phe His Pro Val Pro Asn Leu Arg Asn Leu Asp Leu Ser Tyr 100 105 110Asn Lys Leu Gln Thr Leu Gln Ser Glu Gln Phe Lys Gly Leu Arg Lys 115 120 125Leu Ile Ile Leu His Leu Arg Ser Asn Ser Leu Lys Thr Val Pro Ile 130 135 140Arg Val Phe Gln Asp Cys Arg Asn Leu Asp Phe Leu Asp Leu Gly Tyr145 150 155 160Asn Arg Leu Arg Ser Leu Ser Arg Asn Ala Phe Ala Gly Leu Leu Lys 165 170 175Leu Lys Glu Leu His Leu Glu His Asn Gln Phe Ser Lys Ile Asn Phe 180 185 190Ala His Phe Pro Arg Leu Phe Asn Leu Arg Ser Ile Tyr Leu Gln Trp 195 200 205Asn Arg Ile Arg Ser Ile Ser Gln Gly Leu Thr Trp Thr Trp Ser Ser 210 215 220Leu His Asn Leu Asp Leu Ser Gly Asn Asp Ile Gln Gly Ile Glu Pro225 230 235 240Gly Thr Phe Lys Cys Leu Pro Asn Leu Gln Lys Leu Asn Leu Asp Ser 245 250 255Asn Lys Leu Thr Asn Ile Ser Gln Glu Thr Val Asn Ala Trp Ile Ser 260 265 270Leu Ile Ser Ile Thr Leu Ser Gly Asn Met Trp Glu Cys Ser Arg Ser 275 280 285Ile Cys Pro Leu Phe Tyr Trp Leu Lys Asn Phe Lys Gly Asn Lys Glu 290 295 300Ser Thr Met Ile Cys Ala Gly Pro Lys His Ile Gln Gly Glu Lys Val305 310 315 320Ser Asp Ala Val Glu Thr Tyr Asn Ile Cys Ser Glu Val Gln Val Val 325 330 335Asn Thr Glu Arg Ser His Leu Val Pro Gln Thr Pro Gln Lys Pro Leu 340 345 350Ile Ile Pro Arg Pro Thr Ile Phe Lys Pro Asp Val Thr Gln Ser Thr 355 360 365Phe Glu Thr Pro Ser Pro Ser Pro Gly Phe Gln Ile Pro Gly Ala Glu 370 375 380Gln Glu Tyr Glu His Val Ser Phe His Lys Ile Ile Ala Gly Ser Val385 390 395 400Ala Leu Phe Leu Ser Val Ala Met Ile Leu Leu Val Ile Tyr Val Ser 405 410 415Trp Lys Arg Tyr Pro Ala Ser Met Lys Gln Leu Gln Gln His Ser Leu 420 425 430Met Lys Arg Arg Arg Lys Lys Ala Arg Glu Ser Glu Arg Gln Met Asn 435 440 445Ser Pro Leu Gln Glu Tyr Tyr Val Asp Tyr Lys Pro Thr Asn Ser Glu 450 455 460Thr Met Asp Ile Ser Val Asn Gly Ser Gly Pro Cys Thr Tyr Thr Ile465 470 475 480Ser Gly Ser Arg Glu Cys Glu Met Pro His His Met Lys Pro Leu Pro 485 490 495Tyr Tyr Ser Tyr Asp Gln Pro Val Ile Gly Tyr Cys Gln Ala His Gln 500 505 510Pro Leu His Val Thr Lys Gly Tyr Glu Thr Val Ser Pro Glu Gln Asp 515 520 525Glu Ser Pro Gly Leu Glu Leu Gly Arg Asp His Ser Phe Ile Ala Thr 530 535 540Ile Ala Arg Ser Ala Ala Pro Ala Ile Tyr Leu Glu Arg Ile Ala Asn545 550 555 56020895PRTHomo sapiens 20Met Arg Met Ser Val Gly Leu Ser Leu Leu Leu Pro Leu Trp Gly Arg1 5 10 15Thr Phe Leu Leu Leu Leu Ser Val Val Met Ala Gln Ser His Trp Pro 20 25 30Ser Glu Pro Ser Glu Ala Val Arg Asp Trp Glu Asn Gln Leu Glu Ala 35 40 45Ser Met His Ser Val Leu Ser Asp Leu His

Glu Ala Val Pro Thr Val 50 55 60Val Gly Ile Pro Asp Gly Thr Ala Val Val Gly Arg Ser Phe Arg Val65 70 75 80Thr Ile Pro Thr Asp Leu Ile Ala Ser Ser Gly Asp Ile Ile Lys Val 85 90 95Ser Ala Ala Gly Lys Glu Ala Leu Pro Ser Trp Leu His Trp Asp Ser 100 105 110Gln Ser His Thr Leu Glu Gly Leu Pro Leu Asp Thr Asp Lys Gly Val 115 120 125His Tyr Ile Ser Val Ser Ala Thr Arg Leu Gly Ala Asn Gly Ser His 130 135 140Ile Pro Gln Thr Ser Ser Val Phe Ser Ile Glu Val Tyr Pro Glu Asp145 150 155 160His Ser Asp Leu Gln Ser Val Arg Thr Ala Ser Pro Asp Pro Gly Glu 165 170 175Val Val Ser Ser Ala Cys Ala Ala Asp Glu Pro Val Thr Val Leu Thr 180 185 190Val Ile Leu Asp Ala Asp Leu Thr Lys Met Thr Pro Lys Gln Arg Ile 195 200 205Asp Leu Leu His Arg Met Arg Ser Phe Ser Glu Val Glu Leu His Asn 210 215 220Met Lys Leu Val Pro Val Val Asn Asn Arg Leu Phe Asp Met Ser Ala225 230 235 240Phe Met Ala Gly Pro Gly Asn Pro Lys Lys Val Val Glu Asn Gly Ala 245 250 255Leu Leu Ser Trp Lys Leu Gly Cys Ser Leu Asn Gln Asn Ser Val Pro 260 265 270Asp Ile His Gly Val Glu Ala Pro Ala Arg Glu Gly Ala Met Ser Ala 275 280 285Gln Leu Gly Tyr Pro Val Val Gly Trp His Ile Ala Asn Lys Lys Pro 290 295 300Pro Leu Pro Lys Arg Val Arg Arg Gln Ile His Ala Thr Pro Thr Pro305 310 315 320Val Thr Ala Ile Gly Pro Pro Thr Thr Ala Ile Gln Glu Pro Pro Ser 325 330 335Arg Ile Val Pro Thr Pro Thr Ser Pro Ala Ile Ala Pro Pro Thr Glu 340 345 350Thr Met Ala Pro Pro Val Arg Asp Pro Val Pro Gly Lys Pro Thr Val 355 360 365Thr Ile Arg Thr Arg Gly Ala Ile Ile Gln Thr Pro Thr Leu Gly Pro 370 375 380Ile Gln Pro Thr Arg Val Ser Glu Ala Gly Thr Thr Val Pro Gly Gln385 390 395 400Ile Arg Pro Thr Met Thr Ile Pro Gly Tyr Val Glu Pro Thr Ala Val 405 410 415Ala Thr Pro Pro Thr Thr Thr Thr Lys Lys Pro Arg Val Ser Thr Pro 420 425 430Lys Pro Ala Thr Pro Ser Thr Asp Ser Thr Thr Thr Thr Thr Arg Arg 435 440 445Pro Thr Lys Lys Pro Arg Thr Pro Arg Pro Val Pro Arg Val Thr Thr 450 455 460Lys Val Ser Ile Thr Arg Leu Glu Thr Ala Ser Pro Pro Thr Arg Ile465 470 475 480Arg Thr Thr Thr Ser Gly Val Pro Arg Gly Gly Glu Pro Asn Gln Arg 485 490 495Pro Glu Leu Lys Asn His Ile Asp Arg Val Asp Ala Trp Val Gly Thr 500 505 510Tyr Phe Glu Val Lys Ile Pro Ser Asp Thr Phe Tyr Asp His Glu Asp 515 520 525Thr Thr Thr Asp Lys Leu Lys Leu Thr Leu Lys Leu Arg Glu Gln Gln 530 535 540Leu Val Gly Glu Lys Ser Trp Val Gln Phe Asn Ser Asn Ser Gln Leu545 550 555 560Met Tyr Gly Leu Pro Asp Ser Ser His Val Gly Lys His Glu Tyr Phe 565 570 575Met His Ala Thr Asp Lys Gly Gly Leu Ser Ala Val Asp Ala Phe Glu 580 585 590Ile His Val His Arg Arg Pro Gln Gly Asp Arg Ala Pro Ala Arg Phe 595 600 605Lys Ala Lys Phe Val Gly Asp Pro Ala Leu Val Leu Asn Asp Ile His 610 615 620Lys Lys Ile Ala Leu Val Lys Lys Leu Ala Phe Ala Phe Gly Asp Arg625 630 635 640Asn Cys Ser Thr Ile Thr Leu Gln Asn Ile Thr Arg Gly Ser Ile Val 645 650 655Val Glu Trp Thr Asn Asn Thr Leu Pro Leu Glu Pro Cys Pro Lys Glu 660 665 670Gln Ile Ala Gly Leu Ser Arg Arg Ile Ala Glu Asp Asp Gly Lys Pro 675 680 685Arg Pro Ala Phe Ser Asn Ala Leu Glu Pro Asp Phe Lys Ala Thr Ser 690 695 700Ile Thr Val Thr Gly Ser Gly Ser Cys Arg His Leu Gln Phe Ile Pro705 710 715 720Val Val Pro Pro Arg Arg Val Pro Ser Glu Ala Pro Pro Thr Glu Val 725 730 735Pro Asp Arg Asp Pro Glu Lys Ser Ser Glu Asp Asp Val Tyr Leu His 740 745 750Thr Val Ile Pro Ala Val Val Val Ala Ala Ile Leu Leu Ile Ala Gly 755 760 765Ile Ile Ala Met Ile Cys Tyr Arg Lys Lys Arg Lys Gly Lys Leu Thr 770 775 780Leu Glu Asp Gln Ala Thr Phe Ile Lys Lys Gly Val Pro Ile Ile Phe785 790 795 800Ala Asp Glu Leu Asp Asp Ser Lys Pro Pro Pro Ser Ser Ser Met Pro 805 810 815Leu Ile Leu Gln Glu Glu Lys Ala Pro Leu Pro Pro Pro Glu Tyr Pro 820 825 830Asn Gln Ser Val Pro Glu Thr Thr Pro Leu Asn Gln Asp Thr Met Gly 835 840 845Glu Tyr Thr Pro Leu Arg Asp Glu Asp Pro Asn Ala Pro Pro Tyr Gln 850 855 860Pro Pro Pro Pro Phe Thr Val Pro Met Glu Gly Lys Gly Ser Arg Pro865 870 875 880Lys Asn Met Thr Pro Tyr Arg Ser Pro Pro Pro Tyr Val Pro Pro 885 890 89521868PRTHomo sapiens 21Gln Ser His Trp Pro Ser Glu Pro Ser Glu Ala Val Arg Asp Trp Glu1 5 10 15Asn Gln Leu Glu Ala Ser Met His Ser Val Leu Ser Asp Leu His Glu 20 25 30Ala Val Pro Thr Val Val Gly Ile Pro Asp Gly Thr Ala Val Val Gly 35 40 45Arg Ser Phe Arg Val Thr Ile Pro Thr Asp Leu Ile Ala Ser Ser Gly 50 55 60Asp Ile Ile Lys Val Ser Ala Ala Gly Lys Glu Ala Leu Pro Ser Trp65 70 75 80Leu His Trp Asp Ser Gln Ser His Thr Leu Glu Gly Leu Pro Leu Asp 85 90 95Thr Asp Lys Gly Val His Tyr Ile Ser Val Ser Ala Thr Arg Leu Gly 100 105 110Ala Asn Gly Ser His Ile Pro Gln Thr Ser Ser Val Phe Ser Ile Glu 115 120 125Val Tyr Pro Glu Asp His Ser Asp Leu Gln Ser Val Arg Thr Ala Ser 130 135 140Pro Asp Pro Gly Glu Val Val Ser Ser Ala Cys Ala Ala Asp Glu Pro145 150 155 160Val Thr Val Leu Thr Val Ile Leu Asp Ala Asp Leu Thr Lys Met Thr 165 170 175Pro Lys Gln Arg Ile Asp Leu Leu His Arg Met Arg Ser Phe Ser Glu 180 185 190Val Glu Leu His Asn Met Lys Leu Val Pro Val Val Asn Asn Arg Leu 195 200 205Phe Asp Met Ser Ala Phe Met Ala Gly Pro Gly Asn Pro Lys Lys Val 210 215 220Val Glu Asn Gly Ala Leu Leu Ser Trp Lys Leu Gly Cys Ser Leu Asn225 230 235 240Gln Asn Ser Val Pro Asp Ile His Gly Val Glu Ala Pro Ala Arg Glu 245 250 255Gly Ala Met Ser Ala Gln Leu Gly Tyr Pro Val Val Gly Trp His Ile 260 265 270Ala Asn Lys Lys Pro Pro Leu Pro Lys Arg Val Arg Arg Gln Ile His 275 280 285Ala Thr Pro Thr Pro Val Thr Ala Ile Gly Pro Pro Thr Thr Ala Ile 290 295 300Gln Glu Pro Pro Ser Arg Ile Val Pro Thr Pro Thr Ser Pro Ala Ile305 310 315 320Ala Pro Pro Thr Glu Thr Met Ala Pro Pro Val Arg Asp Pro Val Pro 325 330 335Gly Lys Pro Thr Val Thr Ile Arg Thr Arg Gly Ala Ile Ile Gln Thr 340 345 350Pro Thr Leu Gly Pro Ile Gln Pro Thr Arg Val Ser Glu Ala Gly Thr 355 360 365Thr Val Pro Gly Gln Ile Arg Pro Thr Met Thr Ile Pro Gly Tyr Val 370 375 380Glu Pro Thr Ala Val Ala Thr Pro Pro Thr Thr Thr Thr Lys Lys Pro385 390 395 400Arg Val Ser Thr Pro Lys Pro Ala Thr Pro Ser Thr Asp Ser Thr Thr 405 410 415Thr Thr Thr Arg Arg Pro Thr Lys Lys Pro Arg Thr Pro Arg Pro Val 420 425 430Pro Arg Val Thr Thr Lys Val Ser Ile Thr Arg Leu Glu Thr Ala Ser 435 440 445Pro Pro Thr Arg Ile Arg Thr Thr Thr Ser Gly Val Pro Arg Gly Gly 450 455 460Glu Pro Asn Gln Arg Pro Glu Leu Lys Asn His Ile Asp Arg Val Asp465 470 475 480Ala Trp Val Gly Thr Tyr Phe Glu Val Lys Ile Pro Ser Asp Thr Phe 485 490 495Tyr Asp His Glu Asp Thr Thr Thr Asp Lys Leu Lys Leu Thr Leu Lys 500 505 510Leu Arg Glu Gln Gln Leu Val Gly Glu Lys Ser Trp Val Gln Phe Asn 515 520 525Ser Asn Ser Gln Leu Met Tyr Gly Leu Pro Asp Ser Ser His Val Gly 530 535 540Lys His Glu Tyr Phe Met His Ala Thr Asp Lys Gly Gly Leu Ser Ala545 550 555 560Val Asp Ala Phe Glu Ile His Val His Arg Arg Pro Gln Gly Asp Arg 565 570 575Ala Pro Ala Arg Phe Lys Ala Lys Phe Val Gly Asp Pro Ala Leu Val 580 585 590Leu Asn Asp Ile His Lys Lys Ile Ala Leu Val Lys Lys Leu Ala Phe 595 600 605Ala Phe Gly Asp Arg Asn Cys Ser Thr Ile Thr Leu Gln Asn Ile Thr 610 615 620Arg Gly Ser Ile Val Val Glu Trp Thr Asn Asn Thr Leu Pro Leu Glu625 630 635 640Pro Cys Pro Lys Glu Gln Ile Ala Gly Leu Ser Arg Arg Ile Ala Glu 645 650 655Asp Asp Gly Lys Pro Arg Pro Ala Phe Ser Asn Ala Leu Glu Pro Asp 660 665 670Phe Lys Ala Thr Ser Ile Thr Val Thr Gly Ser Gly Ser Cys Arg His 675 680 685Leu Gln Phe Ile Pro Val Val Pro Pro Arg Arg Val Pro Ser Glu Ala 690 695 700Pro Pro Thr Glu Val Pro Asp Arg Asp Pro Glu Lys Ser Ser Glu Asp705 710 715 720Asp Val Tyr Leu His Thr Val Ile Pro Ala Val Val Val Ala Ala Ile 725 730 735Leu Leu Ile Ala Gly Ile Ile Ala Met Ile Cys Tyr Arg Lys Lys Arg 740 745 750Lys Gly Lys Leu Thr Leu Glu Asp Gln Ala Thr Phe Ile Lys Lys Gly 755 760 765Val Pro Ile Ile Phe Ala Asp Glu Leu Asp Asp Ser Lys Pro Pro Pro 770 775 780Ser Ser Ser Met Pro Leu Ile Leu Gln Glu Glu Lys Ala Pro Leu Pro785 790 795 800Pro Pro Glu Tyr Pro Asn Gln Ser Val Pro Glu Thr Thr Pro Leu Asn 805 810 815Gln Asp Thr Met Gly Glu Tyr Thr Pro Leu Arg Asp Glu Asp Pro Asn 820 825 830Ala Pro Pro Tyr Gln Pro Pro Pro Pro Phe Thr Val Pro Met Glu Gly 835 840 845Lys Gly Ser Arg Pro Lys Asn Met Thr Pro Tyr Arg Ser Pro Pro Pro 850 855 860Tyr Val Pro Pro86522650PRTHomo sapiens 22Met Gly Pro Ala Ser Pro Ala Ala Arg Gly Leu Ser Arg Arg Pro Gly1 5 10 15Gln Pro Pro Leu Pro Leu Leu Leu Pro Leu Leu Leu Leu Leu Leu Arg 20 25 30Ala Gln Pro Ala Ile Gly Ser Leu Ala Gly Gly Ser Pro Gly Ala Ala 35 40 45Glu Ala Pro Gly Ser Ala Gln Val Ala Gly Leu Cys Gly Arg Leu Thr 50 55 60Leu His Arg Asp Leu Arg Thr Gly Arg Trp Glu Pro Asp Pro Gln Arg65 70 75 80Ser Arg Arg Cys Leu Arg Asp Pro Gln Arg Val Leu Glu Tyr Cys Arg 85 90 95Gln Met Tyr Pro Glu Leu Gln Ile Ala Arg Val Glu Gln Ala Thr Gln 100 105 110Ala Ile Pro Met Glu Arg Trp Cys Gly Gly Ser Arg Ser Gly Ser Cys 115 120 125Ala His Pro His His Gln Val Val Pro Phe Arg Cys Leu Pro Gly Glu 130 135 140Phe Val Ser Glu Ala Leu Leu Val Pro Glu Gly Cys Arg Phe Leu His145 150 155 160Gln Glu Arg Met Asp Gln Cys Glu Ser Ser Thr Arg Arg His Gln Glu 165 170 175Ala Gln Glu Ala Cys Ser Ser Gln Gly Leu Ile Leu His Gly Ser Gly 180 185 190Met Leu Leu Pro Cys Gly Ser Asp Arg Phe Arg Gly Val Glu Tyr Val 195 200 205Cys Cys Pro Pro Pro Gly Thr Pro Asp Pro Ser Gly Thr Ala Val Gly 210 215 220Asp Pro Ser Thr Arg Ser Trp Pro Pro Gly Ser Arg Val Glu Gly Ala225 230 235 240Glu Asp Glu Glu Glu Glu Glu Ser Phe Pro Gln Pro Val Asp Asp Tyr 245 250 255Phe Val Glu Pro Pro Gln Ala Glu Glu Glu Glu Glu Thr Val Pro Pro 260 265 270Pro Ser Ser His Thr Leu Ala Val Val Gly Lys Val Thr Pro Thr Pro 275 280 285Arg Pro Thr Asp Gly Val Asp Ile Tyr Phe Gly Met Pro Gly Glu Ile 290 295 300Ser Glu His Glu Gly Phe Leu Arg Ala Lys Met Asp Leu Glu Glu Arg305 310 315 320Arg Met Arg Gln Ile Asn Glu Val Met Arg Glu Trp Ala Met Ala Asp 325 330 335Asn Gln Ser Lys Asn Leu Pro Lys Ala Asp Arg Gln Ala Leu Asn Glu 340 345 350His Phe Gln Ser Ile Leu Gln Thr Leu Glu Glu Gln Val Ser Gly Glu 355 360 365Arg Gln Arg Leu Val Glu Thr His Ala Thr Arg Val Ile Ala Leu Ile 370 375 380Asn Asp Gln Arg Arg Ala Ala Leu Glu Gly Phe Leu Ala Ala Leu Gln385 390 395 400Ala Asp Pro Pro Gln Ala Glu Arg Val Leu Leu Ala Leu Arg Arg Tyr 405 410 415Leu Arg Ala Glu Gln Lys Glu Gln Arg His Thr Leu Arg His Tyr Gln 420 425 430His Val Ala Ala Val Asp Pro Glu Lys Ala Gln Gln Met Arg Phe Gln 435 440 445Val His Thr His Leu Gln Val Ile Glu Glu Arg Val Asn Gln Ser Leu 450 455 460Gly Leu Leu Asp Gln Asn Pro His Leu Ala Gln Glu Leu Arg Pro Gln465 470 475 480Ile Gln Glu Leu Leu His Ser Glu His Leu Gly Pro Ser Glu Leu Glu 485 490 495Ala Pro Ala Pro Gly Gly Ser Ser Glu Asp Lys Gly Gly Leu Gln Pro 500 505 510Pro Asp Ser Lys Asp Asp Thr Pro Met Thr Leu Pro Lys Gly Ser Thr 515 520 525Glu Gln Asp Ala Ala Ser Pro Glu Lys Glu Lys Met Asn Pro Leu Glu 530 535 540Gln Tyr Glu Arg Lys Val Asn Ala Ser Val Pro Arg Gly Phe Pro Phe545 550 555 560His Ser Ser Glu Ile Gln Arg Asp Glu Leu Ala Pro Ala Gly Thr Gly 565 570 575Val Ser Arg Glu Ala Val Ser Gly Leu Leu Ile Met Gly Ala Gly Gly 580 585 590Gly Ser Leu Ile Val Leu Ser Met Leu Leu Leu Arg Arg Lys Lys Pro 595 600 605Tyr Gly Ala Ile Ser His Gly Val Val Glu Val Asp Pro Met Leu Thr 610 615 620Leu Glu Glu Gln Gln Leu Arg Glu Leu Gln Arg His Gly Tyr Glu Asn625 630 635 640Pro Thr Tyr Arg Phe Leu Glu Glu Arg Pro 645 65023612PRTHomo sapiens 23Ser Leu Ala Gly Gly Ser Pro Gly Ala Ala Glu Ala Pro Gly Ser Ala1 5 10 15Gln Val Ala Gly Leu Cys Gly Arg Leu Thr Leu His Arg Asp Leu Arg 20 25 30Thr Gly Arg Trp Glu Pro Asp Pro Gln Arg Ser Arg Arg Cys Leu Arg 35 40 45Asp Pro Gln Arg Val Leu Glu Tyr Cys Arg Gln Met Tyr Pro Glu Leu 50 55 60Gln Ile Ala Arg Val Glu Gln Ala Thr Gln Ala Ile Pro Met Glu Arg65 70 75 80Trp Cys Gly Gly Ser Arg Ser Gly Ser Cys Ala His Pro His His Gln 85 90 95Val Val Pro Phe Arg Cys Leu Pro Gly Glu Phe Val Ser Glu Ala Leu

100 105 110Leu Val Pro Glu Gly Cys Arg Phe Leu His Gln Glu Arg Met Asp Gln 115 120 125Cys Glu Ser Ser Thr Arg Arg His Gln Glu Ala Gln Glu Ala Cys Ser 130 135 140Ser Gln Gly Leu Ile Leu His Gly Ser Gly Met Leu Leu Pro Cys Gly145 150 155 160Ser Asp Arg Phe Arg Gly Val Glu Tyr Val Cys Cys Pro Pro Pro Gly 165 170 175Thr Pro Asp Pro Ser Gly Thr Ala Val Gly Asp Pro Ser Thr Arg Ser 180 185 190Trp Pro Pro Gly Ser Arg Val Glu Gly Ala Glu Asp Glu Glu Glu Glu 195 200 205Glu Ser Phe Pro Gln Pro Val Asp Asp Tyr Phe Val Glu Pro Pro Gln 210 215 220Ala Glu Glu Glu Glu Glu Thr Val Pro Pro Pro Ser Ser His Thr Leu225 230 235 240Ala Val Val Gly Lys Val Thr Pro Thr Pro Arg Pro Thr Asp Gly Val 245 250 255Asp Ile Tyr Phe Gly Met Pro Gly Glu Ile Ser Glu His Glu Gly Phe 260 265 270Leu Arg Ala Lys Met Asp Leu Glu Glu Arg Arg Met Arg Gln Ile Asn 275 280 285Glu Val Met Arg Glu Trp Ala Met Ala Asp Asn Gln Ser Lys Asn Leu 290 295 300Pro Lys Ala Asp Arg Gln Ala Leu Asn Glu His Phe Gln Ser Ile Leu305 310 315 320Gln Thr Leu Glu Glu Gln Val Ser Gly Glu Arg Gln Arg Leu Val Glu 325 330 335Thr His Ala Thr Arg Val Ile Ala Leu Ile Asn Asp Gln Arg Arg Ala 340 345 350Ala Leu Glu Gly Phe Leu Ala Ala Leu Gln Ala Asp Pro Pro Gln Ala 355 360 365Glu Arg Val Leu Leu Ala Leu Arg Arg Tyr Leu Arg Ala Glu Gln Lys 370 375 380Glu Gln Arg His Thr Leu Arg His Tyr Gln His Val Ala Ala Val Asp385 390 395 400Pro Glu Lys Ala Gln Gln Met Arg Phe Gln Val His Thr His Leu Gln 405 410 415Val Ile Glu Glu Arg Val Asn Gln Ser Leu Gly Leu Leu Asp Gln Asn 420 425 430Pro His Leu Ala Gln Glu Leu Arg Pro Gln Ile Gln Glu Leu Leu His 435 440 445Ser Glu His Leu Gly Pro Ser Glu Leu Glu Ala Pro Ala Pro Gly Gly 450 455 460Ser Ser Glu Asp Lys Gly Gly Leu Gln Pro Pro Asp Ser Lys Asp Asp465 470 475 480Thr Pro Met Thr Leu Pro Lys Gly Ser Thr Glu Gln Asp Ala Ala Ser 485 490 495Pro Glu Lys Glu Lys Met Asn Pro Leu Glu Gln Tyr Glu Arg Lys Val 500 505 510Asn Ala Ser Val Pro Arg Gly Phe Pro Phe His Ser Ser Glu Ile Gln 515 520 525Arg Asp Glu Leu Ala Pro Ala Gly Thr Gly Val Ser Arg Glu Ala Val 530 535 540Ser Gly Leu Leu Ile Met Gly Ala Gly Gly Gly Ser Leu Ile Val Leu545 550 555 560Ser Met Leu Leu Leu Arg Arg Lys Lys Pro Tyr Gly Ala Ile Ser His 565 570 575Gly Val Val Glu Val Asp Pro Met Leu Thr Leu Glu Glu Gln Gln Leu 580 585 590Arg Glu Leu Gln Arg His Gly Tyr Glu Asn Pro Thr Tyr Arg Phe Leu 595 600 605Glu Glu Arg Pro 61024979PRTHomo sapiens 24Met Arg Arg Pro Arg Arg Pro Gly Gly Leu Gly Gly Ser Gly Gly Leu1 5 10 15Arg Leu Leu Leu Cys Leu Leu Leu Leu Ser Ser Arg Pro Gly Gly Cys 20 25 30Ser Ala Val Ser Ala His Gly Cys Leu Phe Asp Arg Arg Leu Cys Ser 35 40 45His Leu Glu Val Cys Ile Gln Asp Gly Leu Phe Gly Gln Cys Gln Val 50 55 60Gly Val Gly Gln Ala Arg Pro Leu Leu Gln Val Thr Ser Pro Val Leu65 70 75 80Gln Arg Leu Gln Gly Val Leu Arg Gln Leu Met Ser Gln Gly Leu Ser 85 90 95Trp His Asp Asp Leu Thr Gln Tyr Val Ile Ser Gln Glu Met Glu Arg 100 105 110Ile Pro Arg Leu Arg Pro Pro Glu Pro Arg Pro Arg Asp Arg Ser Gly 115 120 125Leu Ala Pro Lys Arg Pro Gly Pro Ala Gly Glu Leu Leu Leu Gln Asp 130 135 140Ile Pro Thr Gly Ser Ala Pro Ala Ala Gln His Arg Leu Pro Gln Pro145 150 155 160Pro Val Gly Lys Gly Gly Ala Gly Ala Ser Ser Ser Leu Ser Pro Leu 165 170 175Gln Ala Glu Leu Leu Pro Pro Leu Leu Glu His Leu Leu Leu Pro Pro 180 185 190Gln Pro Pro His Pro Ser Leu Ser Tyr Glu Pro Ala Leu Leu Gln Pro 195 200 205Tyr Leu Phe His Gln Phe Gly Ser Arg Asp Gly Ser Arg Val Ser Glu 210 215 220Gly Ser Pro Gly Met Val Ser Val Gly Pro Leu Pro Lys Ala Glu Ala225 230 235 240Pro Ala Leu Phe Ser Arg Thr Ala Ser Lys Gly Ile Phe Gly Asp His 245 250 255Pro Gly His Ser Tyr Gly Asp Leu Pro Gly Pro Ser Pro Ala Gln Leu 260 265 270Phe Gln Asp Ser Gly Leu Leu Tyr Leu Ala Gln Glu Leu Pro Ala Pro 275 280 285Ser Arg Ala Arg Val Pro Arg Leu Pro Glu Gln Gly Ser Ser Ser Arg 290 295 300Ala Glu Asp Ser Pro Glu Gly Tyr Glu Lys Glu Gly Leu Gly Asp Arg305 310 315 320Gly Glu Lys Pro Ala Ser Pro Ala Val Gln Pro Asp Ala Ala Leu Gln 325 330 335Arg Leu Ala Ala Val Leu Ala Gly Tyr Gly Val Glu Leu Arg Gln Leu 340 345 350Thr Pro Glu Gln Leu Ser Thr Leu Leu Thr Leu Leu Gln Leu Leu Pro 355 360 365Lys Gly Ala Gly Arg Asn Pro Gly Gly Val Val Asn Val Gly Ala Asp 370 375 380Ile Lys Lys Thr Met Glu Gly Pro Val Glu Gly Arg Asp Thr Ala Glu385 390 395 400Leu Pro Ala Arg Thr Ser Pro Met Pro Gly His Pro Thr Ala Ser Pro 405 410 415Thr Ser Ser Glu Val Gln Gln Val Pro Ser Pro Val Ser Ser Glu Pro 420 425 430Pro Lys Ala Ala Arg Pro Pro Val Thr Pro Val Leu Leu Glu Lys Lys 435 440 445Ser Pro Leu Gly Gln Ser Gln Pro Thr Val Ala Gly Gln Pro Ser Ala 450 455 460Arg Pro Ala Ala Glu Glu Tyr Gly Tyr Ile Val Thr Asp Gln Lys Pro465 470 475 480Leu Ser Leu Ala Ala Gly Val Lys Leu Leu Glu Ile Leu Ala Glu His 485 490 495Val His Met Ser Ser Gly Ser Phe Ile Asn Ile Ser Val Val Gly Pro 500 505 510Ala Leu Thr Phe Arg Ile Arg His Asn Glu Gln Asn Leu Ser Leu Ala 515 520 525Asp Val Thr Gln Gln Ala Gly Leu Val Lys Ser Glu Leu Glu Ala Gln 530 535 540Thr Gly Leu Gln Ile Leu Gln Thr Gly Val Gly Gln Arg Glu Glu Ala545 550 555 560Ala Ala Val Leu Pro Gln Thr Ala His Ser Thr Ser Pro Met Arg Ser 565 570 575Val Leu Leu Thr Leu Val Ala Leu Ala Gly Val Ala Gly Leu Leu Val 580 585 590Ala Leu Ala Val Ala Leu Cys Val Arg Gln His Ala Arg Gln Gln Asp 595 600 605Lys Glu Arg Leu Ala Ala Leu Gly Pro Glu Gly Ala His Gly Asp Thr 610 615 620Thr Phe Glu Tyr Gln Asp Leu Cys Arg Gln His Met Ala Thr Lys Ser625 630 635 640Leu Phe Asn Arg Ala Glu Gly Pro Pro Glu Pro Ser Arg Val Ser Ser 645 650 655Val Ser Ser Gln Phe Ser Asp Ala Ala Gln Ala Ser Pro Ser Ser His 660 665 670Ser Ser Thr Pro Ser Trp Cys Glu Glu Pro Ala Gln Ala Asn Met Asp 675 680 685Ile Ser Thr Gly His Met Ile Leu Ala Tyr Met Glu Asp His Leu Arg 690 695 700Asn Arg Asp Arg Leu Ala Lys Glu Trp Gln Ala Leu Cys Ala Tyr Gln705 710 715 720Ala Glu Pro Asn Thr Cys Ala Thr Ala Gln Gly Glu Gly Asn Ile Lys 725 730 735Lys Asn Arg His Pro Asp Phe Leu Pro Tyr Asp His Ala Arg Ile Lys 740 745 750Leu Lys Val Glu Ser Ser Pro Ser Arg Ser Asp Tyr Ile Asn Ala Ser 755 760 765Pro Ile Ile Glu His Asp Pro Arg Met Pro Ala Tyr Ile Ala Thr Gln 770 775 780Gly Pro Leu Ser His Thr Ile Ala Asp Phe Trp Gln Met Val Trp Glu785 790 795 800Ser Gly Cys Thr Val Ile Val Met Leu Thr Pro Leu Val Glu Asp Gly 805 810 815Val Lys Gln Cys Asp Arg Tyr Trp Pro Asp Glu Gly Ala Ser Leu Tyr 820 825 830His Val Tyr Glu Val Asn Leu Val Ser Glu His Ile Trp Cys Glu Asp 835 840 845Phe Leu Val Arg Ser Phe Tyr Leu Lys Asn Val Gln Thr Gln Glu Thr 850 855 860Arg Thr Leu Thr Gln Phe His Phe Leu Ser Trp Pro Ala Glu Gly Thr865 870 875 880Pro Ala Ser Thr Arg Pro Leu Leu Asp Phe Arg Arg Lys Val Asn Lys 885 890 895Cys Tyr Arg Gly Arg Ser Cys Pro Ile Ile Val His Cys Ser Asp Gly 900 905 910Ala Gly Arg Thr Gly Thr Tyr Ile Leu Ile Asp Met Val Leu Asn Arg 915 920 925Met Ala Lys Gly Val Lys Glu Ile Asp Ile Ala Ala Thr Leu Glu His 930 935 940Val Arg Asp Gln Arg Pro Gly Leu Val Arg Ser Lys Asp Gln Phe Glu945 950 955 960Phe Ala Leu Thr Ala Val Ala Glu Glu Val Asn Ala Ile Leu Lys Ala 965 970 975Leu Pro Gln25945PRTHomo sapiens 25Val Ser Ala His Gly Cys Leu Phe Asp Arg Arg Leu Cys Ser His Leu1 5 10 15Glu Val Cys Ile Gln Asp Gly Leu Phe Gly Gln Cys Gln Val Gly Val 20 25 30Gly Gln Ala Arg Pro Leu Leu Gln Val Thr Ser Pro Val Leu Gln Arg 35 40 45Leu Gln Gly Val Leu Arg Gln Leu Met Ser Gln Gly Leu Ser Trp His 50 55 60Asp Asp Leu Thr Gln Tyr Val Ile Ser Gln Glu Met Glu Arg Ile Pro65 70 75 80Arg Leu Arg Pro Pro Glu Pro Arg Pro Arg Asp Arg Ser Gly Leu Ala 85 90 95Pro Lys Arg Pro Gly Pro Ala Gly Glu Leu Leu Leu Gln Asp Ile Pro 100 105 110Thr Gly Ser Ala Pro Ala Ala Gln His Arg Leu Pro Gln Pro Pro Val 115 120 125Gly Lys Gly Gly Ala Gly Ala Ser Ser Ser Leu Ser Pro Leu Gln Ala 130 135 140Glu Leu Leu Pro Pro Leu Leu Glu His Leu Leu Leu Pro Pro Gln Pro145 150 155 160Pro His Pro Ser Leu Ser Tyr Glu Pro Ala Leu Leu Gln Pro Tyr Leu 165 170 175Phe His Gln Phe Gly Ser Arg Asp Gly Ser Arg Val Ser Glu Gly Ser 180 185 190Pro Gly Met Val Ser Val Gly Pro Leu Pro Lys Ala Glu Ala Pro Ala 195 200 205Leu Phe Ser Arg Thr Ala Ser Lys Gly Ile Phe Gly Asp His Pro Gly 210 215 220His Ser Tyr Gly Asp Leu Pro Gly Pro Ser Pro Ala Gln Leu Phe Gln225 230 235 240Asp Ser Gly Leu Leu Tyr Leu Ala Gln Glu Leu Pro Ala Pro Ser Arg 245 250 255Ala Arg Val Pro Arg Leu Pro Glu Gln Gly Ser Ser Ser Arg Ala Glu 260 265 270Asp Ser Pro Glu Gly Tyr Glu Lys Glu Gly Leu Gly Asp Arg Gly Glu 275 280 285Lys Pro Ala Ser Pro Ala Val Gln Pro Asp Ala Ala Leu Gln Arg Leu 290 295 300Ala Ala Val Leu Ala Gly Tyr Gly Val Glu Leu Arg Gln Leu Thr Pro305 310 315 320Glu Gln Leu Ser Thr Leu Leu Thr Leu Leu Gln Leu Leu Pro Lys Gly 325 330 335Ala Gly Arg Asn Pro Gly Gly Val Val Asn Val Gly Ala Asp Ile Lys 340 345 350Lys Thr Met Glu Gly Pro Val Glu Gly Arg Asp Thr Ala Glu Leu Pro 355 360 365Ala Arg Thr Ser Pro Met Pro Gly His Pro Thr Ala Ser Pro Thr Ser 370 375 380Ser Glu Val Gln Gln Val Pro Ser Pro Val Ser Ser Glu Pro Pro Lys385 390 395 400Ala Ala Arg Pro Pro Val Thr Pro Val Leu Leu Glu Lys Lys Ser Pro 405 410 415Leu Gly Gln Ser Gln Pro Thr Val Ala Gly Gln Pro Ser Ala Arg Pro 420 425 430Ala Ala Glu Glu Tyr Gly Tyr Ile Val Thr Asp Gln Lys Pro Leu Ser 435 440 445Leu Ala Ala Gly Val Lys Leu Leu Glu Ile Leu Ala Glu His Val His 450 455 460Met Ser Ser Gly Ser Phe Ile Asn Ile Ser Val Val Gly Pro Ala Leu465 470 475 480Thr Phe Arg Ile Arg His Asn Glu Gln Asn Leu Ser Leu Ala Asp Val 485 490 495Thr Gln Gln Ala Gly Leu Val Lys Ser Glu Leu Glu Ala Gln Thr Gly 500 505 510Leu Gln Ile Leu Gln Thr Gly Val Gly Gln Arg Glu Glu Ala Ala Ala 515 520 525Val Leu Pro Gln Thr Ala His Ser Thr Ser Pro Met Arg Ser Val Leu 530 535 540Leu Thr Leu Val Ala Leu Ala Gly Val Ala Gly Leu Leu Val Ala Leu545 550 555 560Ala Val Ala Leu Cys Val Arg Gln His Ala Arg Gln Gln Asp Lys Glu 565 570 575Arg Leu Ala Ala Leu Gly Pro Glu Gly Ala His Gly Asp Thr Thr Phe 580 585 590Glu Tyr Gln Asp Leu Cys Arg Gln His Met Ala Thr Lys Ser Leu Phe 595 600 605Asn Arg Ala Glu Gly Pro Pro Glu Pro Ser Arg Val Ser Ser Val Ser 610 615 620Ser Gln Phe Ser Asp Ala Ala Gln Ala Ser Pro Ser Ser His Ser Ser625 630 635 640Thr Pro Ser Trp Cys Glu Glu Pro Ala Gln Ala Asn Met Asp Ile Ser 645 650 655Thr Gly His Met Ile Leu Ala Tyr Met Glu Asp His Leu Arg Asn Arg 660 665 670Asp Arg Leu Ala Lys Glu Trp Gln Ala Leu Cys Ala Tyr Gln Ala Glu 675 680 685Pro Asn Thr Cys Ala Thr Ala Gln Gly Glu Gly Asn Ile Lys Lys Asn 690 695 700Arg His Pro Asp Phe Leu Pro Tyr Asp His Ala Arg Ile Lys Leu Lys705 710 715 720Val Glu Ser Ser Pro Ser Arg Ser Asp Tyr Ile Asn Ala Ser Pro Ile 725 730 735Ile Glu His Asp Pro Arg Met Pro Ala Tyr Ile Ala Thr Gln Gly Pro 740 745 750Leu Ser His Thr Ile Ala Asp Phe Trp Gln Met Val Trp Glu Ser Gly 755 760 765Cys Thr Val Ile Val Met Leu Thr Pro Leu Val Glu Asp Gly Val Lys 770 775 780Gln Cys Asp Arg Tyr Trp Pro Asp Glu Gly Ala Ser Leu Tyr His Val785 790 795 800Tyr Glu Val Asn Leu Val Ser Glu His Ile Trp Cys Glu Asp Phe Leu 805 810 815Val Arg Ser Phe Tyr Leu Lys Asn Val Gln Thr Gln Glu Thr Arg Thr 820 825 830Leu Thr Gln Phe His Phe Leu Ser Trp Pro Ala Glu Gly Thr Pro Ala 835 840 845Ser Thr Arg Pro Leu Leu Asp Phe Arg Arg Lys Val Asn Lys Cys Tyr 850 855 860Arg Gly Arg Ser Cys Pro Ile Ile Val His Cys Ser Asp Gly Ala Gly865 870 875 880Arg Thr Gly Thr Tyr Ile Leu Ile Asp Met Val Leu Asn Arg Met Ala 885 890 895Lys Gly Val Lys Glu Ile Asp Ile Ala Ala Thr Leu Glu His Val Arg 900 905 910Asp Gln Arg Pro Gly Leu Val Arg Ser Lys Asp Gln Phe Glu Phe Ala 915 920 925Leu Thr Ala Val Ala Glu Glu Val Asn Ala Ile Leu Lys Ala Leu Pro 930 935 940Gln94526366PRTHomo sapiens 26Met Leu Leu Leu Arg Cys Gln Leu Lys Gln Ala Pro Pro Gln Lys Val1 5 10 15Ser Phe Arg Phe Cys Val Val Met Gly Lys Gln Gln Ser Lys Leu Lys 20 25 30His Ser Thr Tyr Lys Tyr

Gly Pro Asp Glu Ile Ile Glu Glu Arg Ile 35 40 45Gln Thr Lys Ala Phe Gln Glu Tyr Ser Pro Ala His Met Asp Thr Val 50 55 60Ser Val Val Ala Ala Leu Asn Ser Asp Leu Cys Val Ser Gly Gly Lys65 70 75 80Asp Lys Thr Val Val Ala Tyr Asn Trp Lys Thr Gly Asn Val Val Lys 85 90 95Arg Phe Lys Gly His Glu His Glu Ile Thr Lys Val Ala Cys Ile Pro 100 105 110Lys Ser Ser Gln Phe Phe Ser Ala Ser Arg Asp Arg Met Val Met Met 115 120 125Trp Asp Leu His Gly Ser Ser Gln Pro Arg Gln Gln Leu Cys Gly His 130 135 140Ala Met Val Val Thr Gly Leu Ala Val Ser Pro Asp Ser Ser Gln Leu145 150 155 160Cys Thr Gly Ser Arg Asp Asn Thr Leu Leu Leu Trp Asp Val Val Thr 165 170 175Gly Gln Ser Val Glu Arg Ala Ser Val Ser Arg Asn Val Val Thr His 180 185 190Leu Cys Trp Val Pro Arg Glu Pro Tyr Ile Leu Gln Thr Ser Glu Asp 195 200 205Lys Thr Leu Arg Leu Trp Asp Ser Arg Gly Leu Gln Val Ala His Met 210 215 220Phe Pro Ala Lys Gln His Ile Gln Thr Tyr Cys Glu Val Ser Val Asp225 230 235 240Gly His Lys Cys Ile Ser Cys Ser Asn Gly Phe Gly Gly Glu Gly Cys 245 250 255Glu Ala Thr Leu Trp Asp Leu Arg Gln Thr Arg Asn Arg Ile Cys Glu 260 265 270Tyr Lys Gly His Phe Gln Thr Val Ala Ser Cys Val Phe Leu Pro Arg 275 280 285Ala Leu Ala Leu Met Pro Leu Ile Ala Thr Ser Ser His Asp Cys Lys 290 295 300Val Lys Ile Trp Asn Gln Asp Thr Gly Ala Cys Leu Phe Thr Leu Ser305 310 315 320Leu Asp Gly Ser Gly Pro Leu Thr Ser Leu Ala Val Gly Asp Ala Ile 325 330 335Ser Leu Leu Cys Ala Ser Phe Asn Arg Gly Ile His Leu Leu Arg Met 340 345 350Asp His Ser Gln Gly Leu Glu Leu Gln Glu Val Ala Ala Phe 355 360 36527343PRTHomo sapiens 27Met Ala Gln Lys Gly Val Leu Gly Pro Gly Gln Leu Gly Ala Val Ala1 5 10 15Ile Leu Leu Tyr Leu Gly Leu Leu Arg Ser Gly Thr Gly Ala Glu Gly 20 25 30Ala Glu Ala Pro Cys Gly Val Ala Pro Gln Ala Arg Ile Thr Gly Gly 35 40 45Ser Ser Ala Val Ala Gly Gln Trp Pro Trp Gln Val Ser Ile Thr Tyr 50 55 60Glu Gly Val His Val Cys Gly Gly Ser Leu Val Ser Glu Gln Trp Val65 70 75 80Leu Ser Ala Ala His Cys Phe Pro Ser Glu His His Lys Glu Ala Tyr 85 90 95Glu Val Lys Leu Gly Ala His Gln Leu Asp Ser Tyr Ser Glu Asp Ala 100 105 110Lys Val Ser Thr Leu Lys Asp Ile Ile Pro His Pro Ser Tyr Leu Gln 115 120 125Glu Gly Ser Gln Gly Asp Ile Ala Leu Leu Gln Leu Ser Arg Pro Ile 130 135 140Thr Phe Ser Arg Tyr Ile Arg Pro Ile Cys Leu Pro Ala Ala Asn Ala145 150 155 160Ser Phe Pro Asn Gly Leu His Cys Thr Val Thr Gly Trp Gly His Val 165 170 175Ala Pro Ser Val Ser Leu Leu Thr Pro Lys Pro Leu Gln Gln Leu Glu 180 185 190Val Pro Leu Ile Ser Arg Glu Thr Cys Asn Cys Leu Tyr Asn Ile Asp 195 200 205Ala Lys Pro Glu Glu Pro His Phe Val Gln Glu Asp Met Val Cys Ala 210 215 220Gly Tyr Val Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp Ser Gly Gly225 230 235 240Pro Leu Ser Cys Pro Val Glu Gly Leu Trp Tyr Leu Thr Gly Ile Val 245 250 255Ser Trp Gly Asp Ala Cys Gly Ala Arg Asn Arg Pro Gly Val Tyr Thr 260 265 270Leu Ala Ser Ser Tyr Ala Ser Trp Ile Gln Ser Lys Val Thr Glu Leu 275 280 285Gln Pro Arg Val Val Pro Gln Thr Gln Glu Ser Gln Pro Asp Ser Asn 290 295 300Leu Cys Gly Ser His Leu Ala Phe Ser Ser Ala Pro Ala Gln Gly Leu305 310 315 320Leu Arg Pro Ile Leu Phe Leu Pro Leu Gly Leu Ala Leu Gly Leu Leu 325 330 335Ser Pro Trp Leu Ser Glu His 34028311PRTHomo sapiens 28Ala Glu Ala Pro Cys Gly Val Ala Pro Gln Ala Arg Ile Thr Gly Gly1 5 10 15Ser Ser Ala Val Ala Gly Gln Trp Pro Trp Gln Val Ser Ile Thr Tyr 20 25 30Glu Gly Val His Val Cys Gly Gly Ser Leu Val Ser Glu Gln Trp Val 35 40 45Leu Ser Ala Ala His Cys Phe Pro Ser Glu His His Lys Glu Ala Tyr 50 55 60Glu Val Lys Leu Gly Ala His Gln Leu Asp Ser Tyr Ser Glu Asp Ala65 70 75 80Lys Val Ser Thr Leu Lys Asp Ile Ile Pro His Pro Ser Tyr Leu Gln 85 90 95Glu Gly Ser Gln Gly Asp Ile Ala Leu Leu Gln Leu Ser Arg Pro Ile 100 105 110Thr Phe Ser Arg Tyr Ile Arg Pro Ile Cys Leu Pro Ala Ala Asn Ala 115 120 125Ser Phe Pro Asn Gly Leu His Cys Thr Val Thr Gly Trp Gly His Val 130 135 140Ala Pro Ser Val Ser Leu Leu Thr Pro Lys Pro Leu Gln Gln Leu Glu145 150 155 160Val Pro Leu Ile Ser Arg Glu Thr Cys Asn Cys Leu Tyr Asn Ile Asp 165 170 175Ala Lys Pro Glu Glu Pro His Phe Val Gln Glu Asp Met Val Cys Ala 180 185 190Gly Tyr Val Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp Ser Gly Gly 195 200 205Pro Leu Ser Cys Pro Val Glu Gly Leu Trp Tyr Leu Thr Gly Ile Val 210 215 220Ser Trp Gly Asp Ala Cys Gly Ala Arg Asn Arg Pro Gly Val Tyr Thr225 230 235 240Leu Ala Ser Ser Tyr Ala Ser Trp Ile Gln Ser Lys Val Thr Glu Leu 245 250 255Gln Pro Arg Val Val Pro Gln Thr Gln Glu Ser Gln Pro Asp Ser Asn 260 265 270Leu Cys Gly Ser His Leu Ala Phe Ser Ser Ala Pro Ala Gln Gly Leu 275 280 285Leu Arg Pro Ile Leu Phe Leu Pro Leu Gly Leu Ala Leu Gly Leu Leu 290 295 300Ser Pro Trp Leu Ser Glu His305 31029316PRTHomo sapiens 29Met Leu Leu Leu Leu Leu Leu Pro Leu Leu Trp Gly Arg Glu Arg Val1 5 10 15Glu Gly Gln Lys Ser Asn Arg Lys Asp Tyr Ser Leu Thr Met Gln Ser 20 25 30Ser Val Thr Val Gln Glu Gly Met Cys Ala His Val Arg Cys Ser Phe 35 40 45Ser Tyr Pro Val Asp Ser Gln Thr Asp Ser Asp Pro Val His Gly Tyr 50 55 60Trp Phe Arg Ala Gly Asn Asp Ile Ser Trp Lys Ala Pro Val Ala Thr65 70 75 80Asn Asn Pro Ala Trp Ala Val Gln Glu Glu Thr Arg Asp Arg Phe His 85 90 95Leu Leu Gly Asp Pro Gln Thr Lys Asn Cys Thr Leu Ser Ile Arg Asp 100 105 110Ala Arg Met Ser Asp Ala Gly Arg Tyr Phe Phe Arg Met Glu Lys Gly 115 120 125Asn Ile Lys Trp Asn Tyr Lys Tyr Asp Gln Leu Ser Val Asn Val Thr 130 135 140Asp Pro Pro Gln Asn Leu Thr Val Thr Val Phe Gln Gly Glu Gly Thr145 150 155 160Ala Ser Thr Ala Leu Gly Asn Ser Ser Ser Leu Ser Val Leu Glu Gly 165 170 175Gln Ser Leu Arg Leu Val Cys Ala Val Asp Ser Asn Pro Pro Ala Arg 180 185 190Leu Ser Trp Thr Trp Arg Ser Leu Thr Leu Tyr Pro Ser Gln Pro Ser 195 200 205Asn Pro Leu Val Leu Glu Leu Gln Val His Leu Gly Asp Glu Gly Glu 210 215 220Phe Thr Cys Arg Ala Gln Asn Ser Leu Gly Ser Gln His Val Ser Leu225 230 235 240Asn Leu Ser Leu Gln Gln Glu Tyr Thr Gly Lys Met Arg Pro Val Ser 245 250 255Gly Val Leu Leu Gly Ala Val Gly Gly Ala Gly Ala Thr Ala Leu Val 260 265 270Phe Leu Ser Phe Cys Val Ile Phe Ile Val Val Arg Ser Cys Arg Lys 275 280 285Lys Ser Ala Arg Pro Ala Ala Asp Val Gly Asp Ile Gly Met Lys Asp 290 295 300Ala Asn Thr Ile Arg Gly Ser Ala Leu Arg Val Thr305 310 31530298PRTHomo sapiens 30Gln Lys Ser Asn Arg Lys Asp Tyr Ser Leu Thr Met Gln Ser Ser Val1 5 10 15Thr Val Gln Glu Gly Met Cys Ala His Val Arg Cys Ser Phe Ser Tyr 20 25 30Pro Val Asp Ser Gln Thr Asp Ser Asp Pro Val His Gly Tyr Trp Phe 35 40 45Arg Ala Gly Asn Asp Ile Ser Trp Lys Ala Pro Val Ala Thr Asn Asn 50 55 60Pro Ala Trp Ala Val Gln Glu Glu Thr Arg Asp Arg Phe His Leu Leu65 70 75 80Gly Asp Pro Gln Thr Lys Asn Cys Thr Leu Ser Ile Arg Asp Ala Arg 85 90 95Met Ser Asp Ala Gly Arg Tyr Phe Phe Arg Met Glu Lys Gly Asn Ile 100 105 110Lys Trp Asn Tyr Lys Tyr Asp Gln Leu Ser Val Asn Val Thr Asp Pro 115 120 125Pro Gln Asn Leu Thr Val Thr Val Phe Gln Gly Glu Gly Thr Ala Ser 130 135 140Thr Ala Leu Gly Asn Ser Ser Ser Leu Ser Val Leu Glu Gly Gln Ser145 150 155 160Leu Arg Leu Val Cys Ala Val Asp Ser Asn Pro Pro Ala Arg Leu Ser 165 170 175Trp Thr Trp Arg Ser Leu Thr Leu Tyr Pro Ser Gln Pro Ser Asn Pro 180 185 190Leu Val Leu Glu Leu Gln Val His Leu Gly Asp Glu Gly Glu Phe Thr 195 200 205Cys Arg Ala Gln Asn Ser Leu Gly Ser Gln His Val Ser Leu Asn Leu 210 215 220Ser Leu Gln Gln Glu Tyr Thr Gly Lys Met Arg Pro Val Ser Gly Val225 230 235 240Leu Leu Gly Ala Val Gly Gly Ala Gly Ala Thr Ala Leu Val Phe Leu 245 250 255Ser Phe Cys Val Ile Phe Ile Val Val Arg Ser Cys Arg Lys Lys Ser 260 265 270Ala Arg Pro Ala Ala Asp Val Gly Asp Ile Gly Met Lys Asp Ala Asn 275 280 285Thr Ile Arg Gly Ser Ala Leu Arg Val Thr 290 29531267PRTHomo sapiens 31Met Ala Pro Arg Arg Ala Arg Gly Cys Arg Thr Leu Gly Leu Pro Ala1 5 10 15Leu Leu Leu Leu Leu Leu Leu Arg Pro Pro Ala Thr Arg Gly Ile Thr 20 25 30Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val Lys Ser 35 40 45Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys 50 55 60Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn Lys Ala65 70 75 80Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg Asp 85 90 95Pro Ala Leu Val His Gln Arg Pro Ala Pro Pro Ser Thr Val Thr Thr 100 105 110Ala Gly Val Thr Pro Gln Pro Glu Ser Leu Ser Pro Ser Gly Lys Glu 115 120 125Pro Ala Ala Ser Ser Pro Ser Ser Asn Asn Thr Ala Ala Thr Thr Ala 130 135 140Ala Ile Val Pro Gly Ser Gln Leu Met Pro Ser Lys Ser Pro Ser Thr145 150 155 160Gly Thr Thr Glu Ile Ser Ser His Glu Ser Ser His Gly Thr Pro Ser 165 170 175Gln Thr Thr Ala Lys Asn Trp Glu Leu Thr Ala Ser Ala Ser His Gln 180 185 190Pro Pro Gly Val Tyr Pro Gln Gly His Ser Asp Thr Thr Val Ala Ile 195 200 205Ser Thr Ser Thr Val Leu Leu Cys Gly Leu Ser Ala Val Ser Leu Leu 210 215 220Ala Cys Tyr Leu Lys Ser Arg Gln Thr Pro Pro Leu Ala Ser Val Glu225 230 235 240Met Glu Ala Met Glu Ala Leu Pro Val Thr Trp Gly Thr Ser Ser Arg 245 250 255Asp Glu Asp Leu Glu Asn Cys Ser His His Leu 260 26532237PRTHomo sapiens 32Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val1 5 10 15Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly 20 25 30Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn 35 40 45Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile 50 55 60Arg Asp Pro Ala Leu Val His Gln Arg Pro Ala Pro Pro Ser Thr Val65 70 75 80Thr Thr Ala Gly Val Thr Pro Gln Pro Glu Ser Leu Ser Pro Ser Gly 85 90 95Lys Glu Pro Ala Ala Ser Ser Pro Ser Ser Asn Asn Thr Ala Ala Thr 100 105 110Thr Ala Ala Ile Val Pro Gly Ser Gln Leu Met Pro Ser Lys Ser Pro 115 120 125Ser Thr Gly Thr Thr Glu Ile Ser Ser His Glu Ser Ser His Gly Thr 130 135 140Pro Ser Gln Thr Thr Ala Lys Asn Trp Glu Leu Thr Ala Ser Ala Ser145 150 155 160His Gln Pro Pro Gly Val Tyr Pro Gln Gly His Ser Asp Thr Thr Val 165 170 175Ala Ile Ser Thr Ser Thr Val Leu Leu Cys Gly Leu Ser Ala Val Ser 180 185 190Leu Leu Ala Cys Tyr Leu Lys Ser Arg Gln Thr Pro Pro Leu Ala Ser 195 200 205Val Glu Met Glu Ala Met Glu Ala Leu Pro Val Thr Trp Gly Thr Ser 210 215 220Ser Arg Asp Glu Asp Leu Glu Asn Cys Ser His His Leu225 230 23533224PRTMus sp. 33Met Ala Leu Leu Ile Ser Leu Pro Gly Gly Thr Pro Ala Met Ala Gln1 5 10 15Val Leu Leu Leu Leu Ser Ser Gly Cys Leu His Ala Gly Asn Ser Glu 20 25 30Arg Tyr Asn Arg Lys Asn Gly Phe Gly Val Asn Gln Pro Glu Arg Cys 35 40 45Ser Gly Val Gln Gly Gly Ser Ile Asp Ile Pro Phe Ser Phe Tyr Phe 50 55 60Pro Trp Lys Leu Ala Lys Asp Pro Gln Met Ser Ile Ala Trp Lys Trp65 70 75 80Lys Asp Phe His Gly Glu Val Ile Tyr Asn Ser Ser Leu Pro Phe Ile 85 90 95His Glu His Phe Lys Gly Arg Leu Ile Leu Asn Trp Thr Gln Gly Gln 100 105 110Thr Ser Gly Val Leu Arg Ile Leu Asn Leu Lys Glu Ser Asp Gln Ala 115 120 125Gln Tyr Phe Ser Arg Val Asn Leu Gln Ser Thr Glu Gly Met Lys Leu 130 135 140Trp Gln Ser Ile Pro Gly Thr Gln Leu Asn Val Thr Gln Ala Leu Asn145 150 155 160Thr Thr Met Arg Ser Pro Phe Ile Val Thr Ser Glu Phe Thr Thr Ala 165 170 175Gly Leu Glu His Thr Ser Asp Gln Arg Asn Pro Ser Leu Met Asn Leu 180 185 190Gly Ala Met Val Thr Met Leu Leu Ala Lys Val Leu Val Ile Val Leu 195 200 205Val Tyr Gly Trp Met Ile Phe Leu Arg Trp Lys Gln Arg Pro Ala His 210 215 22034196PRTMus sp. 34Gly Asn Ser Glu Arg Tyr Asn Arg Lys Asn Gly Phe Gly Val Asn Gln1 5 10 15Pro Glu Arg Cys Ser Gly Val Gln Gly Gly Ser Ile Asp Ile Pro Phe 20 25 30Ser Phe Tyr Phe Pro Trp Lys Leu Ala Lys Asp Pro Gln Met Ser Ile 35 40 45Ala Trp Lys Trp Lys Asp Phe His Gly Glu Val Ile Tyr Asn Ser Ser 50 55 60Leu Pro Phe Ile His Glu His Phe Lys Gly Arg Leu Ile Leu Asn Trp65 70 75 80Thr Gln Gly Gln Thr Ser Gly Val Leu Arg Ile Leu Asn Leu Lys Glu 85 90 95Ser Asp Gln Ala Gln Tyr Phe Ser Arg Val Asn Leu Gln Ser Thr Glu 100 105 110Gly Met Lys Leu Trp Gln Ser Ile Pro Gly Thr Gln Leu Asn Val Thr 115 120 125Gln Ala Leu Asn Thr Thr Met Arg Ser Pro Phe Ile Val Thr Ser

Glu 130 135 140Phe Thr Thr Ala Gly Leu Glu His Thr Ser Asp Gln Arg Asn Pro Ser145 150 155 160Leu Met Asn Leu Gly Ala Met Val Thr Met Leu Leu Ala Lys Val Leu 165 170 175Val Ile Val Leu Val Tyr Gly Trp Met Ile Phe Leu Arg Trp Lys Gln 180 185 190Arg Pro Ala His 19535227PRTHomo sapiens 35Met Gly Arg Pro Leu Leu Leu Pro Leu Leu Leu Leu Leu Gln Pro Pro1 5 10 15Ala Phe Leu Gln Pro Gly Gly Ser Thr Gly Ser Gly Pro Ser Tyr Leu 20 25 30Tyr Gly Val Thr Gln Pro Lys His Leu Ser Ala Ser Met Gly Gly Ser 35 40 45Val Glu Ile Pro Phe Ser Phe Tyr Tyr Pro Trp Glu Leu Ala Ile Val 50 55 60Pro Asn Val Arg Ile Ser Trp Arg Arg Gly His Phe His Gly Gln Ser65 70 75 80Phe Tyr Ser Thr Arg Pro Pro Ser Ile His Lys Asp Tyr Val Asn Arg 85 90 95Leu Phe Leu Asn Trp Thr Glu Gly Gln Glu Ser Gly Phe Leu Arg Ile 100 105 110Ser Asn Leu Arg Lys Glu Asp Gln Ser Val Tyr Phe Cys Arg Val Glu 115 120 125Leu Asp Thr Arg Arg Ser Gly Arg Gln Gln Leu Gln Ser Ile Lys Gly 130 135 140Thr Lys Leu Thr Ile Thr Gln Ala Val Thr Thr Thr Thr Thr Trp Arg145 150 155 160Pro Ser Ser Thr Thr Thr Ile Ala Gly Leu Arg Val Thr Glu Ser Lys 165 170 175Gly His Ser Glu Ser Trp His Leu Ser Leu Asp Thr Ala Ile Arg Val 180 185 190Ala Leu Ala Val Ala Val Leu Lys Thr Val Ile Leu Gly Leu Leu Cys 195 200 205Leu Leu Leu Leu Trp Trp Arg Arg Arg Lys Gly Ser Arg Ala Pro Ser 210 215 220Ser Asp Phe22536208PRTHomo sapiens 36Gln Pro Gly Gly Ser Thr Gly Ser Gly Pro Ser Tyr Leu Tyr Gly Val1 5 10 15Thr Gln Pro Lys His Leu Ser Ala Ser Met Gly Gly Ser Val Glu Ile 20 25 30Pro Phe Ser Phe Tyr Tyr Pro Trp Glu Leu Ala Ile Val Pro Asn Val 35 40 45Arg Ile Ser Trp Arg Arg Gly His Phe His Gly Gln Ser Phe Tyr Ser 50 55 60Thr Arg Pro Pro Ser Ile His Lys Asp Tyr Val Asn Arg Leu Phe Leu65 70 75 80Asn Trp Thr Glu Gly Gln Glu Ser Gly Phe Leu Arg Ile Ser Asn Leu 85 90 95Arg Lys Glu Asp Gln Ser Val Tyr Phe Cys Arg Val Glu Leu Asp Thr 100 105 110Arg Arg Ser Gly Arg Gln Gln Leu Gln Ser Ile Lys Gly Thr Lys Leu 115 120 125Thr Ile Thr Gln Ala Val Thr Thr Thr Thr Thr Trp Arg Pro Ser Ser 130 135 140Thr Thr Thr Ile Ala Gly Leu Arg Val Thr Glu Ser Lys Gly His Ser145 150 155 160Glu Ser Trp His Leu Ser Leu Asp Thr Ala Ile Arg Val Ala Leu Ala 165 170 175Val Ala Val Leu Lys Thr Val Ile Leu Gly Leu Leu Cys Leu Leu Leu 180 185 190Leu Trp Trp Arg Arg Arg Lys Gly Ser Arg Ala Pro Ser Ser Asp Phe 195 200 205375PRTMus sp. 37Pro Lys Ala Pro Thr1 5388PRTArtificial SequenceSynthetic 8xHis tag 38His His His His His His His His1 53915PRTMus sp. 39Asn Met Lys Pro Thr Pro Lys Ala Pro Thr Pro Lys Lys Pro Ser1 5 10 154026PRTHomo sapiens 40Ala Pro Asp Gly Gly Phe Leu Asp Leu Ser Asp Ala Leu Pro Asp Asn1 5 10 15Glu Asn Lys Lys Pro Thr Ala Ile Pro Lys 20 254111PRTHomo sapiens 41Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys1 5 10429PRTHerpes simplex virus 42Pro Pro Asn Pro Thr Pro Pro Pro Pro1 5439PRTHerpes simplex virus 43Gly Gly Pro Ala Thr Pro Ala Pro Pro1 5449PRTMus sp. 44Asn Met Lys Pro Thr Pro Lys Ala Pro1 5459PRTMus sp. 45Pro Lys Ala Pro Thr Pro Lys Lys Pro1 5466PRTHomo sapiens 46Ala Ile Pro Lys Lys Pro1 5479PRTHomo sapiens 47Pro Lys Ala Pro Thr Pro Lys Lys Pro1 54814PRTMus sp. 48Asn Met Lys Pro Thr Pro Lys Ala Pro Thr Pro Lys Lys Pro1 5 104914PRTHomo sapiens 49Asn Lys Lys Pro Thr Ala Ile Pro Lys Lys Pro Ser Ala Gly1 5 105019PRTArtificial SequenceSynthetic peptide 50Asn Lys Lys Pro Thr Pro Lys Ala Pro Thr Ala Ile Pro Lys Lys Pro1 5 10 15Ser Ala Gly51120PRTHomo sapiens 51Tyr Leu Tyr Gly Val Thr Gln Pro Lys His Leu Ser Ala Ser Met Gly1 5 10 15Gly Ser Val Glu Ile Pro Phe Ser Phe Tyr Tyr Pro Trp Glu Leu Ala 20 25 30Thr Ala Pro Asp Val Arg Ile Ser Trp Arg Arg Gly His Phe His Arg 35 40 45Gln Ser Phe Tyr Ser Thr Arg Pro Pro Ser Ile His Lys Asp Tyr Val 50 55 60Asn Arg Leu Phe Leu Asn Trp Thr Glu Gly Gln Lys Ser Gly Phe Leu65 70 75 80Arg Ile Ser Asn Leu Gln Lys Gln Asp Gln Ser Val Tyr Phe Cys Arg 85 90 95Val Glu Leu Asp Thr Arg Ser Ser Gly Arg Gln Gln Trp Gln Ser Ile 100 105 110Glu Gly Thr Lys Leu Ser Ile Thr 115 12052120PRTPan sp. 52Tyr Pro Tyr Gly Val Thr Gln Pro Lys His Leu Ser Ala Ser Met Gly1 5 10 15Gly Ser Val Glu Ile Pro Phe Ser Phe Tyr Tyr Pro Trp Glu Leu Ala 20 25 30Thr Ala Pro Asp Val Arg Ile Ser Trp Arg Arg Gly His Phe His Gly 35 40 45Gln Ser Phe Tyr Ser Thr Arg Pro Pro Ser Ile His Lys Asp Tyr Val 50 55 60Asn Arg Leu Phe Leu Asn Trp Thr Glu Gly Gln Lys Ser Gly Phe Leu65 70 75 80Arg Ile Ser Asn Leu Arg Lys Gln Asp Gln Ser Val Tyr Phe Cys Arg 85 90 95Val Glu Leu Asp Thr Arg Ser Ser Gly Arg Gln Gln Trp Gln Ser Ile 100 105 110Glu Gly Thr Lys Leu Ser Ile Thr 115 12053120PRTBos sp. 53Gln His Tyr Glu Met Lys Gln Pro Arg Asp Leu Ser Ala Pro Glu Gly1 5 10 15Gly Ser Ile Leu Ile Pro Phe Ser Phe Ser His Pro Gly Glu Leu Ala 20 25 30Lys Val Pro Asn Met Arg Ile Phe Trp Arg Trp Lys His Phe His Gly 35 40 45Glu Phe Ile Tyr Asn Thr Ser Pro Leu Phe Thr His Lys Asn Phe Lys 50 55 60Asn Arg Leu Ile Leu Asn Trp Lys Glu Pro Glu Lys Asn Gly Ser Leu65 70 75 80Gln Ile Ser Asn Leu Arg Arg Glu Asp Gln Ser Met Tyr Phe Cys Arg 85 90 95Val Gln Leu Asp Thr Leu Arg Asp Gly Lys Gln Lys Trp Gln Ser Ile 100 105 110Glu Gly Thr Lys Leu Thr Ile Thr 115 12054120PRTCanis sp. 54Met Asp Phe Arg Met Asp Gln Pro Glu His Leu Ser Ala Pro Lys Gly1 5 10 15Gly Thr Val His Ile Asn Phe Thr Phe Tyr Tyr Cys Gly Ala Leu Ala 20 25 30Lys Asp Pro Arg Val Ser Ile Ala Leu Lys Arg Thr His Phe His Gly 35 40 45Glu Val Ile Tyr Asn Ser Thr Arg His Phe Val His Glu Asp Tyr Lys 50 55 60Asp Arg Ile Ile Leu Asn Leu Pro Glu Gly Gln Lys Ser Gly Phe Leu65 70 75 80Gln Ile Leu Asn Leu Arg Glu Glu Asp Glu Asn Met Tyr Phe Cys Arg 85 90 95Val Gln Leu Lys Thr Gln Arg Phe Gly Leu Gln Val Trp Gln Ser Ile 100 105 110Leu Gly Thr Lys Leu Thr Ile Asn 115 12055119PRTRattus sp. 55Tyr Asp Tyr Gly Val Asp Gln Pro Ala Val Leu Ser Gly Val Gln Gly1 5 10 15Ser Ser Ile Glu Ile Pro Phe Ser Phe Tyr Phe Pro Trp Asn Leu Thr 20 25 30Lys Asp Pro Gln Met Ser Ile Ala Trp Arg Trp Lys Asn Phe His Gly 35 40 45Glu Phe Ile Tyr Asn Ser Thr Gln Pro Phe Ile His Glu His Phe Lys 50 55 60Asp Arg Leu Ile Met Asn Trp Thr Gln Gly Gln Thr Ser Gly Val Leu65 70 75 80Arg Ile Leu Asn Phe Lys Lys Asn Asp Gln Ala Thr Tyr Phe Gly Arg 85 90 95Val Leu Leu Gln Thr Thr Glu Gly Met Lys Val Trp Gln Ser Ile Pro 100 105 110Gly Thr Asn Leu Thr Val Thr 11556119PRTMus sp. 56Asn Gly Phe Gly Val Asn Gln Pro Glu Ser Cys Ser Gly Val Gln Gly1 5 10 15Gly Ser Ile Asp Ile Pro Phe Ser Phe Tyr Phe Pro Trp Lys Leu Ala 20 25 30Lys Asp Pro Gln Met Ser Ile Ala Trp Arg Trp Lys Asp Phe His Gly 35 40 45Glu Phe Ile Tyr Asn Ser Ser Leu Pro Phe Ile His Glu His Phe Lys 50 55 60Gly Arg Leu Ile Leu Asn Trp Thr Gln Gly Gln Thr Ser Gly Val Leu65 70 75 80Arg Ile Leu Asn Leu Lys Glu Ser Asp Gln Thr Arg Tyr Phe Gly Arg 85 90 95Val Phe Leu Gln Thr Thr Glu Gly Ile Gln Phe Trp Gln Ser Ile Pro 100 105 110Gly Thr Gln Leu Asn Val Thr 11557120PRTHomo sapiens 57Tyr Leu Tyr Gly Val Thr Gln Pro Lys His Leu Ser Ala Ser Met Gly1 5 10 15Gly Ser Val Glu Ile Pro Phe Ser Phe Tyr Tyr Pro Trp Glu Leu Ala 20 25 30Ile Val Pro Asn Val Arg Ile Ser Trp Arg Arg Gly His Phe His Gly 35 40 45Gln Ser Phe Tyr Ser Thr Arg Pro Pro Ser Ile His Lys Asp Tyr Val 50 55 60Asn Arg Leu Phe Leu Asn Trp Thr Glu Gly Gln Glu Ser Gly Phe Leu65 70 75 80Arg Ile Ser Asn Leu Arg Lys Glu Asp Gln Ser Val Tyr Phe Cys Arg 85 90 95Val Glu Leu Asp Thr Arg Arg Ser Gly Arg Gln Gln Leu Gln Ser Ile 100 105 110Lys Gly Thr Lys Leu Thr Ile Thr 115 12058119PRTMus sp. 58Asn Gly Phe Gly Val Asn Gln Pro Glu Arg Cys Ser Gly Val Gln Gly1 5 10 15Gly Ser Ile Asp Ile Pro Phe Ser Phe Tyr Phe Pro Trp Lys Leu Ala 20 25 30Lys Asp Pro Gln Met Ser Ile Ala Trp Lys Trp Lys Asp Phe His Gly 35 40 45Glu Val Ile Tyr Asn Ser Ser Leu Pro Phe Ile His Glu His Phe Lys 50 55 60Gly Arg Leu Ile Leu Asn Trp Thr Gln Gly Gln Thr Ser Gly Val Leu65 70 75 80Arg Ile Leu Asn Leu Lys Glu Ser Asp Gln Ala Gln Tyr Phe Ser Arg 85 90 95Val Asn Leu Gln Ser Thr Glu Gly Met Lys Leu Trp Gln Ser Ile Pro 100 105 110Gly Thr Gln Leu Asn Val Thr 11559118PRTHomo sapiens 59Ala Ser Trp Gly Val Ser Ser Pro Gln Asp Val Gln Gly Val Lys Gly1 5 10 15Ser Cys Leu Leu Ile Pro Cys Ile Phe Ser Phe Pro Ala Asp Val Glu 20 25 30Val Pro Asp Gly Ile Thr Ala Ile Trp Tyr Tyr Asp Tyr Ser Gly Gln 35 40 45Arg Gln Val Val Ser His Ser Ala Asp Pro Lys Leu Val Glu Ala Arg 50 55 60Phe Arg Gly Arg Thr Glu Phe Met Gly Asn Pro Glu His Arg Val Cys65 70 75 80Asn Leu Leu Leu Lys Asp Leu Gln Pro Glu Asp Ser Gly Ser Tyr Asn 85 90 95Phe Arg Phe Glu Ile Ser Glu Val Asn Arg Trp Ser Asp Val Lys Gly 100 105 110Thr Leu Val Thr Val Thr 11560118PRTMus sp. 60Thr Thr Trp Gly Val Ser Ser Pro Lys Asn Val Gln Gly Leu Ser Gly1 5 10 15Ser Cys Leu Leu Ile Pro Cys Ile Phe Ser Tyr Pro Ala Asp Val Pro 20 25 30Val Ser Asn Gly Ile Thr Ala Ile Trp Tyr Tyr Asp Tyr Ser Gly Lys 35 40 45Arg Gln Val Val Ile His Ser Gly Asp Pro Lys Leu Val Asp Lys Arg 50 55 60Phe Arg Gly Arg Ala Glu Leu Met Gly Asn Met Asp His Lys Val Cys65 70 75 80Asn Leu Leu Leu Lys Asp Leu Lys Pro Glu Asp Ser Gly Thr Tyr Asn 85 90 95Phe Arg Phe Glu Ile Ser Asp Ser Asn Arg Trp Leu Asp Val Lys Gly 100 105 110Thr Thr Val Thr Val Thr 11561230PRTHomo sapiens 61Met Gly Arg Pro Leu Leu Leu Pro Leu Leu Pro Leu Leu Leu Pro Pro1 5 10 15Ala Phe Leu Gln Pro Ser Gly Ser Thr Gly Ser Gly Pro Ser Tyr Leu 20 25 30Tyr Gly Val Thr Gln Pro Lys His Leu Ser Ala Ser Met Gly Gly Ser 35 40 45Val Glu Ile Pro Phe Ser Phe Tyr Tyr Pro Trp Glu Leu Ala Thr Ala 50 55 60Pro Asp Val Arg Ile Ser Trp Arg Arg Gly His Phe His Arg Gln Ser65 70 75 80Phe Tyr Ser Thr Arg Pro Pro Ser Ile His Lys Asp Tyr Val Asn Arg 85 90 95Leu Phe Leu Asn Trp Thr Glu Gly Gln Lys Ser Gly Phe Leu Arg Ile 100 105 110Ser Asn Leu Gln Lys Gln Asp Gln Ser Val Tyr Phe Cys Arg Val Glu 115 120 125Leu Asp Thr Arg Ser Ser Gly Arg Gln Gln Trp Gln Ser Ile Glu Gly 130 135 140Thr Lys Leu Ser Ile Thr Gln Gly Gln Gln Arg Thr Lys Ala Thr Thr145 150 155 160Pro Ala Arg Glu Pro Phe Gln Asn Thr Glu Glu Pro Tyr Glu Asn Ile 165 170 175Arg Asn Glu Gly Gln Asn Thr Asp Pro Lys Leu Asn Pro Lys Asp Asp 180 185 190Gly Ile Val Tyr Ala Ser Leu Ala Leu Ser Ser Ser Thr Ser Pro Arg 195 200 205Ala Pro Pro Ser His Arg Pro Leu Lys Ser Pro Gln Asn Glu Thr Leu 210 215 220Tyr Ser Val Leu Lys Ala225 23062226PRTHomo sapiens 62Met Gly Arg Pro Leu Leu Leu Pro Leu Leu Pro Leu Leu Leu Pro Pro1 5 10 15Ala Phe Leu Gln Pro Ser Gly Ser Thr Gly Ser Gly Pro Ser Tyr Leu 20 25 30Tyr Gly Val Thr Gln Pro Lys His Leu Ser Ala Ser Met Gly Gly Ser 35 40 45Val Glu Ile Pro Phe Ser Phe Tyr Tyr Pro Trp Glu Leu Ala Thr Ala 50 55 60Pro Asp Val Arg Ile Ser Trp Arg Arg Gly His Phe His Gly Gln Ser65 70 75 80Phe Tyr Ser Thr Arg Pro Pro Ser Ile His Lys Asp Tyr Val Asn Arg 85 90 95Leu Phe Leu Asn Trp Thr Glu Gly Gln Lys Ser Gly Phe Leu Arg Ile 100 105 110Ser Asn Leu Gln Lys Gln Asp Gln Ser Val Tyr Phe Cys Arg Val Glu 115 120 125Leu Asp Thr Arg Ser Ser Gly Arg Gln Gln Trp Gln Ser Ile Glu Gly 130 135 140Thr Lys Leu Ser Ile Thr Gln Gly Gln Gln Arg Thr Lys Ala Thr Thr145 150 155 160Pro Ala Arg Glu Pro Phe Gln Asn Thr Glu Glu Pro Tyr Glu Asn Ile 165 170 175Arg Asn Glu Gly Gln Asn Thr Asp Pro Lys Leu Asn Pro Lys Leu His 180 185 190Leu Thr Gln Ser Thr Ser Gln Pro Pro Ser Pro Gln Glu Pro Pro Glu 195 200 205Arg Asp Pro Val Leu Cys Leu Lys Gly Leu Thr Asn Gly Gln Pro Ser 210 215 220Gln Asp22563175PRTHomo sapiens 63Met Gly Arg Pro Leu Leu Leu Pro Leu Leu Pro Leu Leu Leu Pro Pro1 5 10 15Ala Phe Leu Gln Pro Ser Gly Ser Thr Gly Ser Gly Pro Ser Tyr Leu 20 25 30Tyr Gly Val Thr Gln Pro Lys His Leu Ser Ala Ser Met Gly Gly Ser 35 40 45Val Glu Ile Pro Phe Ser Phe Tyr Tyr Pro Trp Glu Leu Ala Thr Ala 50 55 60Pro Asp Val Arg Ile Ser Trp Arg Arg Gly His Phe His Arg Gln Ser65 70 75 80Phe Tyr Ser Thr Arg Pro Pro Ser Ile His Lys Asp Tyr Val Asn Arg 85 90 95Leu Phe Leu Asn Trp Thr Glu Gly Gln Lys Ser Gly Phe Leu Arg Ile 100 105

110Ser Asn Leu Gln Lys Gln Asp Gln Ser Val Tyr Phe Cys Arg Val Glu 115 120 125Leu Asp Thr Arg Ser Ser Gly Arg Gln Gln Trp Gln Ser Ile Glu Gly 130 135 140Thr Lys Leu Ser Ile Thr Gln Gly Asn Pro Ser Lys Thr Gln Arg Ser145 150 155 160His Met Arg Ile Ser Gly Met Lys Asp Lys Ile Gln Ile Pro Ser 165 170 175

Claims

1. A method for modulating the cell surface interaction between a ligand and PILR-alpha comprising contacting cells with an effective amount of an agent that may modulate the interaction between said ligand and PILR-alpha, wherein the ligand is selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA.

2. The method of claim 1, wherein the cells express PILR-alpha.

3. The method of claim 1, wherein the cells express NPDC1.

4. The method of claim 1, wherein the cells express COLEC12.

5. The method of claim 1, wherein the cells express ETBR.

6. The method of claim 1, wherein the cells express CLEC4G.

7. The method of claim 1, wherein the cells express BR3.

8. The method of claim 1, wherein the cells express MAG.

9. The method of claim 1, wherein the cells express IL-2Ra.

10. The method of claim 1, wherein the cells express FceRII.

11. The method of claim 1, wherein the cells express LRRTM4.

12. The method of claim 1, wherein the cells express DAG1.

13. The method of claim 1, wherein the cells express APLP1.

14. The method of claim 1, wherein the cells express PTPRN.

15. The method of claim 1, wherein the cells express WDR31.

16. The method of claim 1, wherein the cells express PSS8.

17. The method of claim 1, wherein the cells express SIGLEC7.

18. The method of claim 1, wherein the cells express IL15-RA.

19. An agent for modulating the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA.

20. A method for enhancing pathogen clearance in a subject comprising blocking the interaction between PILRa and a surface entry protein on the pathogen.

21. The method of claim 20 wherein the pathogen is selected from Listeria, Herpes Simplex Virus-1, Neisseria meningitis, Haemophius influenzae, group B Streptococcus, Campylobacter jejuni, Staph aureus, and Escherichia coli.