Patent application title: STABILIZED LOW AFFINITY CONFORMATION OF INTEGRINS FOR DRUG DISCOVERY
Inventors:
Timothy A. Springer (Chestnut Hill, MA, US)
Bing Hao Luo (Baton Rouge, LA, US)
IPC8 Class: AG01N3368FI
USPC Class:
436501
Class name: Chemistry: analytical and immunological testing biospecific ligand binding assay
Publication date: 2015-02-12
Patent application number: 20150044779
Abstract:
The methods and compositions described herein are based, in part, on the
discovery that the introduction of a disulfide bond into an integrin
polypeptide by the substitution of at least one cysteine residue in the
polypeptide permits stabilization of the integrin in a "closed/inactive"
state. This stabilizing disulfide bond permits integrins to be screened
for a candidate molecule that can bind to the closed state. In
particular, this approach can be used to screen for agents that bind to
the closed state of an integrin polypeptide, and are useful as
therapeutic treatments to prevent integrin activation.Claims:
1. A method for identifying a candidate modulator of integrin activity,
the method comprising (a) contacting an integrin polypeptide with a
candidate agent, wherein the integrin polypeptide is locked into a
desired conformation; and (b) detecting binding of the candidate agent to
the integrin polypeptide, wherein binding of the candidate agent to the
integrin polypeptide is indicative that the candidate agent is a
candidate modulator of integrin activity.Description:
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of U.S. Ser. No. 12/645,958, filed Dec. 23, 2009, which claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 61/141,145 filed on Dec. 29, 2008, the contents of which are incorporated herein by reference in its entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which has been submitted via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jan. 12, 2010, is named 03339306.txt, and is 170,131 bytes in size.
FIELD OF THE INVENTION
[0003] The field of the invention relates to stabilized integrins and uses thereof.
BACKGROUND
[0004] Integrins are cell adhesion receptors that transmit bidirectional signals across the plasma membrane and link the extracellular environment of a cell to the actin cytoskeleton. The conformation of the integrin extracellular domain and its affinity for ligand are dynamically regulated by a process termed "inside-out signaling." Rapid upregulation of adhesiveness of integrins on platelets and white blood cells mediates hemostasis and leukocyte trafficking to sites of inflammation. By coupling to the actin cytoskeleton, integrins promote firm adhesion and provide traction for lamellipodium protrusion and locomotion. In migrating cells the adhesiveness of integrins is spatially and temporally regulated so that integrins are activated near the leading edge to support lamellipod extension and deactivated near the trailing edge to support uropod retraction and internalization (Alon and Dustin, 2007; Arnaout et al., 2005; Broussard et al., 2008; Calderwood, 2004; Evans and Calderwood, 2007; Luo et al., 2007).
[0005] Integrin αIIbβ3, the most abundant receptor on platelets, binds to fibrinogen and von Willebrand factor, and mediates platelet aggregation and association with injured vessel walls. Inherited mutations in its αIIb or β3 subunits result in the bleeding disorder Glanzmann's thrombasthenia. RGD-mimetic small molecules and an antibody to αIIbβ3 are prescribed for the prevention of thrombosis ((Springer et al., 2008; Xiao et al., 2004).
[0006] The integrin α and β subunits have large N-terminal extracellular domains, single-pass transmembrane domains, and usually short C-terminal cytoplasmic domains. The first crystal structure of an integrin ectodomain, of αVβ3, represented a huge advance (Xiong et al., 2001; Xiong et al., 2002). Together with subsequent work, ten of twelve domains in the ectofragment were revealed in a bent conformation (Xiao et al., 2004; Xiong et al., 2004). A ligand-binding head formed by both subunits is followed by legs in each subunit that connect to the transmembrane domains. There is an extreme bend at knees between the upper and lower legs. Integrin epidermal growth factor-like (I-EGF) domains 1 and 2 at the β-knee were disordered in the previous αVβ3 structure. Crystals of β2 leg fragments containing I-EGF domains 1 and 2 have been solved in two different orientations (Shi et al., 2007), but the conformation of these domains in the bent integrin conformation remains unknown.
[0007] Subsequent to the αVβ3 crystal structure, mutational studies on cell surface integrins and EM studies on αVβ3, αLβ2, and αXβ2 integrins demonstrated that the bent conformation is the physiologically relevant, low affinity integrin conformation (Nishida et al., 2006; Takagi et al., 2002). Nonetheless, a cryo EM study on αIIbβ3 revealed a different, less compact conformation with a different arrangement of leg domains (Adair and Yeager, 2002). Furthermore, two recent studies have revealed extended conformations of αIIbβ3 but failed to find a bent conformation (Rocco et al., 2008; Ye et al., 2008). Crystal structure studies on αIIbβ3 are important to resolve these controversies. Revealing the structure within a complete ectodomain of the bent β-knee is important to understanding the mechanism of integrin extension. Moreover, no integrin crystal structure to date has described the bent structure in the light of current knowledge that it is physiologically relevant, is in a low-affinity state, and with the aim of understanding how the bent conformation is stabilized and how it transitions to extended conformations. The previous αVβ3 bent conformation was described as "not expected to occur in the membrane-bound receptor," and being in "its active (ligand competent) state" (Xiong et al., 2001).
[0008] Most studies find that upon activation, integrins extend. Upon extension, the headpiece can remain in the closed conformation, as when bent, or transition to an open conformation with high affinity for ligand (Xiao et al., 2004). In contrast, a "deadbolt model" posits that activation can occur in the absence of extension (Arnaout et al., 2005). Binding of cytoskeletal proteins such as talin and kindlins to the integrin β cytoplasmic domain appears to interfere with α/β cytoplasmic domain association, and induce integrin extension (Wegener and Campbell, 2008). However, there is currently no known feature of integrin structure that would enable cytoskeleton binding to couple to the extended, open conformation with high affinity for ligand. This would appear to be important to fulfill the key role of integrins in integrating the extracellular and intracellular environments.
[0009] Three closely linked metal ion binding sites in the β I domain are especially important in ligand binding. Mg2+ at the central, metal ion-dependent adhesion site (MIDAS) site directly coordinates the acidic sidechain shared by all integrin ligands. However, in previous unliganded, bent αVβ3 structures, the MIDAS and one adjacent site were unoccupied, and it was proposed that metal binding was either caused by integrin activation or induced by ligand binding (Xiong et al., 2002) However, crystals have not been reported with a combination of the two metal ions important for integrin ligand binding, Mg2+ and Ca2+. Therefore, in current comparisons between low and high affinity β I domain conformations, the changes associated with ligand binding and metal binding cannot be deconvoluted.
SUMMARY OF THE CLAIMS
[0010] The methods described herein are based in part on the discovery that a disulfide bond can be introduced to an integrin polypeptide by the substitution of at least one cysteine residue in the polypeptide. The disulfide bond(s) formed in the integrin polypeptide stabilize the integrin in a "closed/inactive" state, which permits the integrins to be screened for a candidate molecule that can bind to the closed state. In particular, this approach can be used to screen for agents that bind to the closed state of an integrin polypeptide, and would be useful as therapeutic treatments to prevent integrin activation.
[0011] In one aspect, the methods described herein relate to a method of identifying a candidate modulator of integrin activity, comprising (a) contacting an integrin polypeptide with a candidate agent, wherein the integrin polypeptide is locked into a desired conformation; and (b) detecting binding of the candidate agent to the integrin polypeptide, wherein binding of the candidate agent to the integrin polypeptide is indicative that the candidate agent is a candidate modulator of integrin activity.
[0012] In one embodiment of this aspect and all other aspects described herein, The method of the candidate agent is selected from the group consisting of an antibody, a small molecule, a chemical, a peptide, and a peptidomimetic.
[0013] In another embodiment of this aspect and all other aspects described herein, the candidate modulator stabilizes the integrin polypeptide into a closed conformation. Alternatively, the candidate modulator can induce a conformational shift from the open conformation to the closed conformation.
[0014] In another embodiment of this aspect and all other aspects described herein, the candidate modulator inhibits binding of an integrin ligand to the integrin polypeptide. Alternatively, the candidate modulator may act at a site distant from the integrin ligand site to prevent integrin-mediated activity in response to ligand binding--that is, ligand binding may occur but activation of the integrin activity is blocked (e.g., non-competitive inhibition).
[0015] In another embodiment of this aspect and all other aspects described herein, the integrin polypeptide is selected from the group consisting of αVβ3, αIIbβ3, αVb6, αVβ1, αVβ5, αMβ2, αXβ2, αLβ2, and αVβ8.
[0016] In another embodiment of this aspect and all other aspects described herein, wherein locking the integrin polypeptide into the desired conformation comprises introducing a stabilizing disulfide bond into the integrin polypeptide.
[0017] In another embodiment of this aspect and all other aspects described herein, the disulfide bond is formed by a cysteine residue substitution of at least one amino acid residue of said integrin polypeptide.
[0018] In another embodiment of this aspect and all other aspects described herein, the substitution comprises a mutation selected from the group consisting of: L959C (human αIIb), E960C (human αIIb), I955C (human αV), Q956C (human αV), V664C (human β3), P688C (human β3), L662C (human β6), P686C (human β6), A619C (human β8), and F636C (human β3).
[0019] In another embodiment of this aspect and all other aspects described herein, the candidate agent is further assayed for activation or inhibition of integrin activity. In one embodiment, a cell-based assay is used to determine integrin activity.
[0020] Another aspect described herein is an integrin polypeptide composition stabilized in the "closed" conformation. In one embodiment, the integrin polypeptide is stabilized by substitution of at least one amino acid residue for a cysteine residue, wherein a disulfide bond is formed.
[0021] In another embodiment of this aspect and all other aspects described herein, the substitution comprises a mutation selected from the group consisting of: L959C (human αIIb), E960C (human αIIb), I955C (human αV), Q956C (human αV), V664C (human β3), P688C (human β3), L662C (human β6), P686C (human β6), A619C (human β8), and F636C (human β3).
[0022] In another embodiment of this aspect, the composition further comprises a solid support such as a bead, a dish, a well, a plate, etc.
DEFINITIONS
[0023] As used herein, a "modified integrin I-domain polypeptide" or "modified integrin polypeptide" includes an integrin I-domain polypeptide that has been altered with respect to the wild-type sequence or the native state such that at least one disulfide bond has been introduced into the polypeptide thereby stabilizing the integrin in a desired conformation. An integrin polypeptide is considered "locked into a desired conformation" if the disulfide bond prevents a conformational shift in the integrin polypeptide from occurring under non-denaturing conditions (i.e., denaturing conditions can be induced by e.g., high temperatures, the presence of reducing agents (such as β-mercaptoethanol, dithiothreitol), the presence of strong denaturing reagents (such as 6M guanidinium hydrochloride, 8M urea, or 1% sodium dodecyl sulfate), or any combination thereof).
[0024] As used herein, the term "stabilizing disulfide bond" is used to describe substitution of at least one cysteine residue that permits the formation of a disulfide bond, which in turn prevents a conformational shift in the integrin polypeptide even in the presence of an activating ligand. The "stabilizing disulfide bond" is introduced to the polypeptide by one of skill in the art and does not reflect a natural or native disulfide bond of the polypeptide. However, it is contemplated that an integrin polypeptide with such a stabilizing disulfide bond can be found in nature due to a mutation in amino acid sequence.
[0025] As used herein, the term "binding of the candidate agent" refers to an interaction of a candidate agent with an integrin polypeptide stabilized in a closed conformation. Since the conformation of the integrin polypeptide is held in place by a disulfide bond, the term "binding" reflects an interaction and is insufficient to indicate the inhibitory or activating activity of the compound. Further screening assays for integrin activity, as described herein, should be used to determine the action of a candidate agent.
[0026] As used herein, the term "candidate agent" includes a compound or other agent that is capable of at least binding to an integrin polypeptide or modified polypeptide as described herein. In an alternative embodiment, the compound or agent is "a modulator of integrin activity," which is capable of modulating or regulating at least one integrin activity, as described herein. Modulators of integrin activity may include, but are not limited to, small organic or inorganic molecules, nucleic acid molecules, peptides, antibodies, and the like. A modulator of integrin activity can be an inducer or inhibitor of integrin-mediated activities such as cell adhesion or ligand binding. As used herein, an "inducer of integrin activity" stimulates, enhances, and/or mimics an integrin activity. As used herein, an "inhibitor of integrin activity" reduces, blocks or antagonizes an integrin activity.
[0027] As used interchangeably herein, the terms "integrin activity", or "integrin-mediated activity" refer to an activity exerted by an integrin polypeptide or nucleic acid molecule on an integrin responsive cell, or on integrin ligand or receptor, as determined in vitro and in vivo, according to standard techniques. In one embodiment, an integrin activity is the ability to mediate cell adhesion events, e.g, cell to cell, or cell to extracellular matrix adhesion. In another embodiment, an integrin activity can be measured as the ability to transduce cellular signaling events. In yet another embodiment, an integrin activity is the ability to bind a ligand, e.g., ICAM.
[0028] As used herein, the term "inhibition of integrin activity" refers to a decrease in ligand activated integrin activity of at least 10% as assessed using a cell-based integrin assay; preferably the activity of the integrin is decreased by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or even 100% (i.e., no activity) integrin activity in the same cell-based integrin assay.
[0029] As used herein the term "comprising" or "comprises" is used in reference to compositions, methods, and respective component(s) thereof, that are essential to the invention, yet open to the inclusion of unspecified elements, whether essential or not.
[0030] As used herein the term "consisting essentially of" refers to those elements required for a given embodiment. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.
[0031] The term "consisting of" refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.
BRIEF DESCRIPTION OF THE FIGURES
[0032] FIGS. 1A-1B show disulfide bond formation in mutant receptors and effect on function. 293T cells were co-transfected with full-length, wild-type or mutant integrin subunits to express the indicated α/β pairs on the cell surface. FIG. A. Integrin heterodimers were immunoprecipitated from 35S-labeled cell detergent lysates with anti-β3 mAb AP3 and subjected to non-reducing SDS-PAGE and fluorography. Molecular size markers are shown on the left. FIG. B. Soluble fibrinogen binding to 293T transfectants in the presence of 1 mM Ca2+/1 mM Mg2+ (white) or 1 mM Mn2+ plus 10 mg/ml PT25-2 antibody (black). Binding was measured as the mean fluorescence intensity of FITC-conjugated fibrinogen staining as a percentage of mean fluorescence intensity of staining with Cy3-conjugated AP3 mAb. Methods were as described previously (Luo, et al. 2004; Zhu, et al. 2007).
[0033] FIG. 2 shows disulfide formation efficiency of the αLβ2 cys mutants on cell surface. The lane numbers in the left panel correspond to the combination of αL and β2 subunits connected by lines shown in the right panel. #1 was not well expressed in this particular experiment. Figure discloses SEQ ID NOS 104 and 81, respectively, in order of appearance.
[0034] FIG. 3 shows expression level of disulfide locked αLβ2 mutants on 293T cell surface.
[0035] FIG. 4 shows exemplary LFA-1 disulfide mutants on 293T cell.
[0036] FIGS. 5A-5C show LFA-1 crosslinking on cell surface. FIG. 5A shows sequences around the cysteine mutations are shown (SEQ ID NOS 85-94, respectively, in order of appearance). Cysteine mutations were introduced and are shown, and the names of each exemplary construct are labeled at the right. FIG. 5B shows expression of the tested α/β combinations was tested using a FACS assay to identify cell surface expression. Disulfide bond formation was detected by Western blotting of lysed cell after transfection. FIG. 5C shows a schematic representation of the α/β combinations tested and shown in FIG. 2B (SEQ ID NOS 104 and 81, respectively, in order of appearance).
[0037] FIGS. 6A-6C show soluble integrin αLβ2 (LFA-1) and αXβ2(CR4) crosslinking and activation. FIG. 6A shows C-terminal sequence of constructs used for soluble integrin expression (SEQ ID NOS 95-103, respectively, in order of appearance). The cysteine mutations were introduced and are shown. The name of each construct is depicted at the right of each sequence. Tags and other features are labeled above the sequences. His6 tag disclosed as SEQ ID NO: 105. FIG. 6B shows exemplary α/β combinations were tested in transfection and protein purification experiments. Crosslinking was confirmed by SDS-PAGE after protein purification and TEV cleavage. FIG. 6C shows activation status of purified integrin proteins was monitored by KIM127 exposure in 1 mM Ca2+/Mg2+.
[0038] FIGS. 7A-7C show optimal sequence alignments of exemplary integrin alpha subunits and include a sequence alignment of AV_HU--4504763 (SEQ ID NO: 1), AV_MO--6680486 (SEQ ID NO: 2), A5_HU--124946 (SEQ ID NO: 3), A5_MO--6754378 (SEQ ID NO:4), AB_HU--124951 (SEQ ID NO: 5), AB_MO--12643835 (SEQ ID NO: 6), A8_HU--1708570 (SEQ ID NO: 7), A6_HU--4557675 (SEQ ID NO: 8), A6_MO--7110659 (SEQ ID NO: 9), A7_HU--4504753 (SEQ ID NO: 10), A7_MO1--3378244 (SEQ ID NO: 11), A7_MO2--3378244 (SEQ ID NO: 12), A3_HU-11467963 (SEQ ID NO: 13), A3_MO--7305189 (SEQ ID NO: 14), A4_HU--4504749 (SEQ ID NO: 15), A4_MO--7110657 (SEQ ID NO: 16), A9_HU--2833247 (SEQ ID NO: 17), A1_HU--2829468 (SEQ ID NO: 18), A2_HU--4504743 (SEQ ID NO: 19), A2_MO--6680478 (SEQ ID NO: 20), A10_HU--6650628 (SEQ ID NO: 21), A11_HU--12643894 (SEQ ID NO: 22), AE--1HU--6007851 (SEQ ID NO: 23), AE_MO--6680482 (SEQ ID NO: 24), AD_HU--12643717 (SEQ ID NO: 25), AX_HU--4504765 (SEQ ID NO: 26), AX_MO--10946646 (SEQ ID NO: 27), AM_HU--1708572 (SEQ ID NO: 28), AM_MO--124956 (SEQ ID NO: 29), AL_HU--1170591 (SEQ ID NO: 30), and AL_MO--124953 (SEQ ID NO: 31).
[0039] FIGS. 8A-8I show optimal sequence alignments of exemplary integrin beta subunits and include a sequence alignment of sequences B1_HUMAN--4504767 (SEQ ID NO: 32), B1_MOUSE--124964 (SEQ ID NO: 33), B2_HUMAN--14780741 0332-H (SEQ ID NO: 34), B2_MOUSE--3183523 (SEQ ID NO: 35), B3_HUMAN--2119640 (SEQ ID NO: 36), B3_MOUSE--7949057 (SEQ ID NO: 37), B4_HUMAN--14768997 (SEQ ID NO: 38), B4_MOUSE--484472 (SEQ ID NO: 39), B5_HUMAN--106776 (SEQ ID NO: 40), B5_MOUSE--3478697 y236-C (SEQ ID NO: 41), B6_HUMAN--9625002 (SEQ ID NO: 42), B6_MOUSE--10946686 (SEQ ID NO: 43), B7_HUMAN--4504777 (SEQ ID NO: 44), B7_MOUSE--7305193 (SEQ ID NO: 45), B8_HUMAN--4504779 (SEQ ID NO: 46), and PACTOLU_MOUSE--3287491 (SEQ ID NO: 47). "SNTT" disclosed as residues 646-649 of SEQ ID NO: 47.
DETAILED DESCRIPTION
[0040] The methods described herein are based in part on the discovery that the introduction of a disulfide bond into an integrin polypeptide by the substitution of at least one cysteine residue in the polypeptide permits stabilization of the integrin in a "closed/inactive" state. This stabilizing disulfide bond permits integrins to be screened for a candidate molecule that can bind to the closed state. In particular, this approach can be used to screen for agents that bind to the closed state of an integrin polypeptide, and would be useful as therapeutic treatments to prevent integrin activation.
[0041] The αIIbβ3 Crystal Structure.
[0042] A crystal structure for molecule I in αIIbβ3 crystals was determined and ribbon diagrams were prepared (data not shown). The following characteristics are noted. In one model, the αIIbβ3 is extended by torsion at the α and β-knees. Upon superstition of molecules 1 and 2 of αIIbβ3 and αVβ3 (Xiong et al., 2004) the structures indicate "breathing". It is determined that there is a variation in the distance of the lower α-leg from the lower β-leg, opening its cleft, and variation in the lower β-leg: αIIbβ3 molecule 1 and αVβ3. A view of the α-subunit only rotated about 90° indicates variation in the distance of the lower α-leg from the upper α-headpiece: αIIbβ3 molecule 1 and molecule 2; αVβ3. The headpieces of αIIbβ3 molecule 1 and αVβ3 show breathing at the β I/hybrid domain interface. Further information regarding the solved crystal structure for the αIIbβ3 integrin can be found in a published paper by the inventors (Zhu, et al., Molecular Cell (2008) 32(6): 849-862).
Disulfide Bonds
[0043] Disulfide bond formation occurs between two cysteine residues that are appropriately positioned within the three-dimensional structure of an integrin polypeptide. In one embodiment of the invention, a polypeptide is stabilized in the closed conformation by introducing at least one cysteine substitution into the amino acid sequence such that a disulfide bond is formed. The introduction of a single cysteine substitution is performed in circumstances in which an additional cysteine residue is present in the native amino acid sequence of the polypeptide at an appropriate position such that a disulfide bond is formed. Alternatively, in another embodiment, two cysteine substitutions are introduced into the amino acid sequence of the polypeptide at positions that allow a disulfide bond to form, thereby stabilizing the polypeptide in a desired conformation.
[0044] In one embodiment of the invention, cysteine substitutions are introduced such that the formation of a disulfide bond is favored only in one protein conformation (i.e., a closed conformation), such that the protein is stabilized in that particular conformation.
[0045] Preparation of a modified polypeptide of the invention by introducing cysteine substitutions can be achieved by mutagenesis of DNA encoding the integrin polypeptide of interest. For example, an isolated nucleic acid molecule encoding a modified integrin I-domain polypeptide can be created by introducing one or more nucleotide substitutions into the nucleotide sequence of an integrin gene such that one or more amino acid substitutions, e.g., cysteine substitutions, are introduced into the encoded protein. Mutations can be introduced into a nucleic acid sequence by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis.
[0046] Some non-limiting examples of substituted cysteine residues in α/β subunits include the following and are denoted with a bold, underlined C:
TABLE-US-00001 Subunit Human αIIb 946 RGEAQVWTQLLRACEERA- 963; Human αIIb 946 RGEAQVWTQLLRALCERA- 963; Human αv 942 TNSTLVTTNVTWGCQPAPM 960; Human αv 942 TNSTLVTTNVTWGICPAPM 960; Human β3 663 CCVRFQYYEDSS--GKSILYVVEEPECPKG 690; Human β3 663 CVVRFQYYEDSS--GKSILYVVEEPECCKG 690; Human β6 661 CCITFLITTDNE--GKTIIHSINEKDCPKP 688; Human β6 661 CLITFLITTDNE--GKTIIHSINEKDCCKP 688; Human β8 619 CCLMEQQ-----------HYVDQTSECFSS 637; and Human β8 619 CALMEQQ-----------HYVDQTSECCSS 637.
[0047] In another embodiment, the method of the invention can be used to stabilize a protein in a biologically inactive conformation, e.g., a conformation that is enzymatically inactive or does not have ligand binding capacity and/or effector functions, e.g., a "closed" conformation.
[0048] Proteins that are stabilized in a particular conformation find use in, for example, in proteomic screening technologies. In proteomic screens of tissues and disease states, antibodies, polypeptide, and/or small molecules that are specific for, e.g., an inactive protein conformer, can be used to assess the activity of different cellular signaling, metabolic, and adhesive pathways. Thus, associations can be made between specific diseases and the activation of specific biochemical and signaling pathways. Furthermore, the methods described herein relate to polypeptides, antibodies, and small molecules identified using the methods described herein and uses for same, e.g., to treat, for example, inflammatory disorders. Conformer-specific reagents can also be placed on chips and used to screen tissue extracts, or used to stain tissue sections. Furthermore, drugs or antibodies, e.g., anti-integrin antibodies which specifically recognize a modified integrin I-domain polypeptide, e.g., an anti-LFA-1 antibody which specifically recognizes a modified LFA-1 I-domain polypeptide, that are selective for a particular conformer, e.g., an open conformer or a closed conformer, may provide differential therapeutic effects. Therefore, selective screening assays using a protein stabilized in a particular conformer can be used to rationally obtain compounds with a desired activity.
Integrins
[0049] Integrins exist on cell surfaces in an inactive conformation that does not bind ligand. Upon cell activation, integrins change shape (conformation) and can bind ligand. Over 20 different integrin heterodimers (different α and β subunit combinations) exist that are expressed in a selective fashion on all cells in the body. After activation, integrins bind in a specific manner to protein ligands on the surface of other cells, in the extracellular matrix, or that are assembled in the clotting or complement cascades. Integrins on leukocytes are of central importance in leukocyte emigration and in inflammatory and immune responses. Ligands for the leukocyte integrin Mac-1 (αMβ2) include the inflammation-associated cell surface molecule ICAM-1, the complement component iC3b, and the clotting component fibrinogen. Ligands for the leukocyte integrin LFA-1 (αLβ2) include ICAM-1, ICAM-2, and ICAM-3. Antibodies to leukocyte integrins can block many types of inflammatory and auto-immune diseases, by, e.g., modulating, or inhibiting, for example, cell to cell interactions, or cell to extracellular matrix interactions. Integrins on platelets are important in clotting and in heart disease and approved drugs that interact with platelet integrin function include the antibody abciximab (Reopro®) and the peptide-like antagonist eptifibatide (Integrilin®). Integrins on connective tissue cells, epithelium, and endothelium are important in disease states affecting these cells. They regulate cell growth, differentiation, wound healing, fibrosis, apoptosis, and angiogenesis. Integrins on cancerous cells regulate invasion and metastasis.
[0050] It is contemplated herein that an agent can be used to bind to integrins in the "closed conformation" in order to stabilize integrins in their off state and modify or prevent integrin activation.
[0051] One embodiment of the methods described herein provides a modified integrin I-domain polypeptide comprising at least one disulfide bond, such that the modified I-domain polypeptide is stabilized in a desired conformation. A modified integrin I-domain polypeptide of the invention may be derived from an I-domain of an integrin a subunit including α1, α2, α10, α11, αD, αV, αX, αM, αE, αL (CD11a), αM (CD11b) and αX (CD11c).
[0052] Also contemplated herein are integrins with conservative substitutions. Conservative substitutions (substituents) typically include the substitution of one amino acid for another with similar characteristics (e.g., charge, size, shape, and other biological properties) such as substitutions within the following groups: valine, glycine; glycine, alanine; valine, isoleucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. The non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. It is also contemplated herein that a substitution can occur among amino acid groups with varying characteristics. Some non-limiting examples of substituted cysteine residues in α/β subunits include the following and are denoted with a bold, underlined C:
TABLE-US-00002 Subunit Human αIIb 946 RGEAQVWTQLLRACEERA-; 963 (SEQ ID NO: 48) Human αIIb 946 RGEAQVWTQLLRALCERA-; 963 (SEQ ID NO: 49) Human αv 942 TNSTLVTTNVTWGCQPAPM; 960 (SEQ ID NO: 50) Human αv 942 TNSTLVTTNVTWGICPAPM; 960 (SEQ ID NO: 51) Human β3 663 CCVRFQYYEDSS--GKSILYVVEEPECPKG; 690 (SEQ ID NO: 52) Human β3 663 CVVRFQYYEDSS--GKSILYVVEEPECCKG; 690 (SEQ ID NO: 53) Human β6 661 CCITFLITTDNE--GKTIIHSINEKDCPKP; 688 (SEQ ID NO: 54) Human β6 661 CLITFLITTDNE--GKTIIHSINEKDCCKP; 688 (SEQ ID NO: 55) Human β8 619 CCLMEQQ-----------HYVDQTSECFSS; 637 (SEQ ID NO: 56) and Human β8 619 CALMEQQ-----------HYVDQTSECCSS. 637 (SEQ ID NO: 57)
[0053] In a preferred embodiment, a cysteine residue is substituted into the integrin polypeptide to permit the formation of a disulfide bond. In one embodiment, a modified integrin I-domain polypeptide of the invention is encoded by an amino acid sequence containing at least one cysteine substitution, or alternatively two cysteine substitutions, as compared to the wild-type sequence.
[0054] The introduction of cysteine residues at appropriate positions within the amino acid sequence of the I-domain polypeptide allows for the formation of a disulfide bond that stabilizes the domain in a particular conformation, e.g., an inactive "closed" conformation. For example, the αL L289C/K294C mutant and the αM Q163C/R313C mutants are stabilized in an inactive or "closed" conformation that does not bind ligand.
[0055] In one embodiment, described herein is a modified integrin I-domain which is comprised within an integrin subunit, and which may be further associated with an integrin β subunit. In another embodiment, a modified integrin I-domain polypeptide of the invention is a soluble polypeptide. Furthermore, the invention provides a modified integrin I-domain polypeptide which is operatively linked to a heterologous polypeptide. Modified integrin polypeptides of the invention include modified integrin I-domain and I-like domain polypeptides that are comprised within an integrin α or β subunit polypeptide, respectively; soluble modified integrin I-domain and I-like domain polypeptides; and modified integrin I-domain and I-like domain polypeptides that are operatively linked to a heterologous polypeptide, e.g., fusion proteins.
[0056] Some non-limiting examples of substituted cysteine residues in α/β subunits include the following and are denoted with a bold, underlined C:
TABLE-US-00003 Subunit Human αIIb 946 RGEAQVWTQLLRACEERA- 963; Human αIIb 946 RGEAQVWTQLLRALCERA- 963; Human αv 942 TNSTLVTTNVTWGCQPAPM 960; Human αv 942 TNSTLVTTNVTWGICPAPM 960; Human β3 663 CCVRFQYYEDSS--GKSILYVVEEPECPKG 690; Human β3 663 CVVRFQYYEDSS--GKSILYVVEEPECCKG 690; Human β6 661 CCITFLITTDNE--GKTIIHSINEKDCPKP 688; Human β6 661 CLITFLITTDNE--GKTIIHSINEKDCCKP 688; Human β8 619 CCLMEQQ-----------HYVDQTSECFSS 637; and Human β8 619 CALMEQQ-----------HYVDQTSECCSS 637.
[0057] The cDNAs for multiple human integrin α and β subunit polypeptides have been cloned and sequenced, and the polypeptide sequences have been determined (see, for example, GenBank Accession Numbers: NM--002203 (α2), AF112345 (α10), NM--012211 (α1), NM--005353 (αD), NM--002208 (αE), NM--000887 (αX), NM--000632 (αM), NM--002209 (αL), X68742 and P56199 (α1), NM--000211 (β2), NM--000212 (β3), NM--002214 (β8)). In addition, the sequences encoding integrin α and β subunit polypeptides from other species are available in the art. Furthermore, as described previously, three dimensional structure of the αM, αL, α1 and α2 I-domains has been solved (Lee, J-O, et al. (1995) Structure 3:1333-1340; Lee, J-O, et al. (199S) Cell 80:631-638; Qu, A and Leahy, D J (1995) Proc Natl Acad Sci USA 92:10277-10281; Qu, A and Leahy, D J (1996) Structure 4:931-942; Emsley, J et al. (1997) J Biol Chem 272:28512-28517; Baldwin, E T et al. (1998) Structure 6:923-935; Kallen, J et al. (1999) J Mol Biol 292:1-9).
[0058] Isolated modified integrin polypeptides as described herein preferably have an amino acid sequence that is sufficiently identical to the amino acid sequence of a native integrin polypeptide, yet which comprise at least one, and alternatively two cysteine substitutions, such that a disulfide bond is formed that stabilizes the polypeptide in a desired conformation. As used herein, the term "sufficiently identical" refers to an amino acid (or nucleotide) sequence which contains a sufficient or minimum number of identical or equivalent (e.g., an amino acid residue that has a similar side chain) amino acid residues (or nucleotides) to an integrin amino acid (or nucleotide) sequence such that the polypeptide shares common structural domains or motifs, and/or a common functional activity with a native integrin polypeptide. For example, amino acid or nucleotide sequences which share at least 30%, 40%, or 50%, preferably 60%, more preferably 70%, 75%, 80%, 85% or 90%, 91%, 92%, 93%, 94%, 95% or greater identity and share a common functional activity (e.g., an activity of a modified integrin I-domain or I-like domain as described herein) are defined herein as sufficiently identical. An integrin I-domain polypeptide may differ in amino acid sequence from the integrin polypeptides disclosed herein due to natural allelic variation or mutagenesis.
[0059] To determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In a preferred embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 80%, or 90% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology"). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
[0060] The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In one embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available on the world wide web at gcg.com), using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available on the world wide web at gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, the percent identity between two amino acid or nucleotide sequences is determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
[0061] Alternatively, sequences aligned for optimal comparison purposes can be used to find residues that are homologous among a variety of α or β integrin subunits, such that substitution of a cysteine residue known to stabilize an integrin in a closed conformation in one isoform can be extended to another isoform. One of skill in the art can readily align sequences in an optimal manner to determine a preferred site for cysteine substitution. Provided herein are sequences aligned in an optimal manner for the purpose of determining a preferred site for cysteine substitution. For example, mutation sites in αVβ1, and αVβ5 can be determined from alignment with αVβ6 and αVβ8 sequences. Alternatively, mutation sites in αMβ2 can be determined from alignment with the αXβ2 sequence.
[0062] In one embodiment, modified integrin polypeptides are produced by recombinant DNA techniques. For example, a modified integrin polypeptide can be isolated from a host cell transfected with a polynucleotide sequence encoding a modified integrin polypeptide (e.g., a I-domain polypeptide or a soluble I-domain fusion protein) using an appropriate purification scheme using standard protein purification techniques. Alternative to recombinant expression, a modified integrin polypeptide can be synthesized chemically using standard peptide synthesis techniques.
Integrins and Disease
[0063] Integrins are key targets in many diseases. Accordingly, isolated high affinity I-30 domains of the invention, as well as antibodies, or small molecule antagonists selective for activated leukocyte integrins can be used to modulate, e.g., inhibit or prevent, autoimmune and inflammatory disease, transplant rejection, ischemia/reperfusion injury as in hypovolemic shock, myocardial infarct, and cerebral shock. Furthermore, co-crystals of high affinity I domains bound to natural ligands and/or small molecule antagonists can readily be made, which will enable computational drug design, and advance modification and improvement of drug development candidates.
[0064] Accordingly, in one aspect the methods described herein provide a method for identifying a modulator of integrin activity comprising assaying the ability of a test compound to bind to a modified integrin I-domain polypeptide which is stabilized in the closed conformation. In another embodiment, the invention provides a method for identifying a compound capable of modulating the interaction of an integrin and a cognate ligand wherein binding of a ligand to a modified integrin I-domain polypeptide, which is stabilized in the closed conformation, is assayed in the presence and absence of a test compound.
[0065] As used herein, an integrin mediated disorder includes, for example, an inflammatory or immune system disorder, and/or a cellular proliferative disorder. Examples of integrin-mediated disorders include myocardial infarction, stroke, restenosis, transplant rejection, graft versus host disease or host versus graft disease, and reperfusion injury. An inflammatory or immune system disorder includes, but is not limited to adult respiratory distress syndrome (ARDS), multiple organ injury syndromes secondary to septicemia or trauma, viral infection, inflammatory bowel disease, ulcerative colitis, Crohn's disease, leukocyte adhesion deficiency II syndrome, thermal injury, hemodialysis, leukapheresis, peritonitis, chronic obstructive pulmonary disease, lung inflammation, asthma, acute appendicitis, dermatoses with acute inflammatory components, wound healing, septic shock, acute glomerulonephritis, nephritis, amyloidosis, reactive arthritis, rheumatoid arthritis, chronic bronchitis, Sjorgen's syndrome, sarcoidosis, scleroderma, lupus, polymyositis, Reiter's syndrome, psoriasis, dermatitis, pelvic inflammatory disease, inflammatory breast disease, orbital inflammatory disease, immune deficiency disorders (e.g., HIV, common variable immunodeficiency, congenital X-linked infantile hypogammaglobulinemia, transient hypogammaglobulinemia, selective IgA deficiency, necrotizing enterocolitis, granulocyte transfusion associated syndromes, cytokine-induced toxicity, chronic mucocutaneous candidiasis, severe combined immunodeficiency), autoimmune disorders, and acute purulent meningitis or other central nervous system inflammatory disorders.
Screening Assays
[0066] The methods described herein (also referred to herein as a "screening assay") can be used to identify modulators, i.e., candidate or test compounds or agents (e.g., peptides, antibodies, peptidomimetics, small molecules (organic or inorganic) or other drugs) which modulate integrin activity. These assays are designed to identify compounds, for example, that bind to an integrin I-domain polypeptide, e.g., an integrin I-domain polypeptide in an active conformation, binds to other proteins that interact with an integrin I-domain polypeptide, induce binding, and modulate the interaction of an integrin I-domain polypeptide with other proteins, e.g., an integrin ligand, e.g., ICAM, and thus modulate integrin activity.
[0067] In the case of an integrin stabilized in the closed conformation, a lack of integrin activity indicates that the integrin is stabilized in the "off" position. In order to screen candidate modulators that bind to this particular conformation, it is necessary to measure binding of the candidate agent to the integrin, rather than assessing integrin activity. Binding assays are known in the art and can be achieved using e.g., radioligand binding assays or fluorescence-detected binding. Candidate modulators that are capable of binding an integrin stabilized in a desired conformation will need to be confirmed as an inhibitor or stimulator of integrin activity using an integrin that is not stabilized in a particular confirmation. Integrin activity assays for such purposes are well known in the art and/or are described herein.
[0068] In an alternate embodiment, a soluble, recombinant high affinity integrin I-domain can be used to screen for small molecule antagonists that interfere with integrin ligand binding. Furthermore, antagonists, e.g., antibodies, with direct/competitive and indirect/noncompetitive modes of inhibition can be discriminated, based on comparison with effects on wild-type integrin I-domains which show minimal ligand binding activity. For example, an indirect inhibitor should inhibit ligand binding by an activated, wild-type integrin I-domain, but not by a disulfide-locked high affinity I-domain.
[0069] In another embodiment, an assay is a cell-based assay comprising contacting a cell expressing a modified integrin polypeptide on the cell surface with a test compound and determining the ability of the test compound to modulate (e.g., induce or inhibit) an integrin activity. For example, a cell expressing a modified integrin I-domain polypeptide stabilized in an open conformation on the cell surface is contacted with a test compound, and the ability of the test compound to modulate adhesion to an integrin ligand is determined, as described herein.
[0070] In another embodiment, the ability of a test compound to modulate integrin ligand binding can also be determined, for example, by coupling a modified integrin I-domain polypeptide that is stabilized in e.g., an open conformation with a detectable label such that the binding of the modified integrin polypeptide can be determined by detecting the amount of labeled integrin I-domain binding to an immobilized integrin ligand.
[0071] Animal-based model systems, such as an animal model of inflammation, may be used, for example, as part of screening strategies designed to identify compounds which are modulators of integrin activity. Thus, the animal-based models may be used to identify drugs, pharmaceuticals, therapies and interventions which may be effective in modulating inflammation and treating integrin-mediated disorders. For example, animal models may be exposed to a compound suspected of exhibiting an ability to modulate integrin activity, and the response of the animals to the exposure may be monitored by assessing inflammatory activity before and after treatment. Transgenic animals, e.g., transgenic mice, which express modified integrin I-domain polypeptides as described herein can also be used to identify drugs, pharmaceuticals, therapies and interventions which may be effective in modulating inflammation and treating integrin-mediated disorders
[0072] In another aspect, the methods described herein pertain to a combination of two or more of the assays described herein. For example, a modulator of integrin activity can be identified using a cell-based assay, and the ability of the agent to modulate integrin activity can be confirmed in vivo, e.g., in an animal such as an animal model for inflammation.
[0073] Moreover, screening assays can be used to identify inducers of integrin activity, for example, that mimic the activity of a integrin polypeptide, e.g., the binding of an integrin to a ligand or receptor, or the activity of an integrin towards an integrin responsive cell. Such compounds may include, but are not limited to, peptides, antibodies, or small organic or inorganic compounds. An anti-integrin antibody, e.g., an anti-LFA-1 antibody, which selectively binds to an open, activated conformer can be used to assess the ability of a test compound to activate, inactivate, or prevent activation of an integrin.
[0074] The test compounds can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the `one-bead one-compound` library method; and synthetic library methods using affinity chromatography selection. The biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, K. S. (1997) Anticancer Drug Des. 12:145).
[0075] Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al. (1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061; and in Gallop et al. (1994) J. Med. Chem. 37:1233.
[0076] Libraries of compounds may be presented in solution (e.g., Houghten (1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (Ladner U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. '409), plasmids (Cull et al. (1992) Proc Natl Acad Sci USA 89:1865-1869) or on phage (Scott and Smith (1990) Science 249:386-390); (Devlin (1990) Science 249:404-406); (Cwirla et al. (1990) Proc. Natl. Acad. Sci. 87:6378-6382); (Felici (1991) J. Mol. Biol. 222:301-310); (Ladner supra.).
[0077] The methods described herein further pertain to novel agents identified by the above-described screening assays. With regard to intervention, any treatments which modulate integrin activity and/or inflammatory activity should be considered as candidates for human therapeutic intervention.
Other Embodiments
[0078] Reported herein are disulfide bonds between integrin α and β subunits in their C-terminal domains or in the linkers between these C-terminal domains and the transmembrane domain. These disulfides may be used in either intact integrins on the cell surface or truncated extracellular domain fragments. The results with disulfide bonds introduced into cell surface integrins are predictive of those that can be successfully introduced into extracellular domain fragments.
[0079] Also shown herein are disulfide bonds that stabilize integrins in the bent, low affinity conformation. The stabilized integrins bind ligands and ligand-mimetic Fab less well. The stabilized integrins have utility for screening for conformation-dependent antibodies and drug molecules. Antibodies and drugs may be identified that are selective for the active, non-bent, or inactive, bent integrins, using either cell surface integrins or extracellular domain fragments.
[0080] In addition, stabilized integrins on cells, in comparison with wild-type integrins, can be used to select for antibodies selective for the bent, inactive conformation or extended, active conformation. Alternatively, stabilized and wild-type ectodomain integrin fragments can be used to screen for drugs selective for the bent inactive conformation. Such drugs would bind to and stabilize the bent, inactive conformation, but not the extended, active conformation. Thus they would represent a novel class of non-competitive integrin antagonists.
[0081] The present invention may be as described in any one of the following numbered paragraphs.
[0082] 1. A method for identifying a candidate modulator of integrin activity, the method comprising (a) contacting an integrin polypeptide with a candidate agent, wherein the integrin polypeptide is locked into a desired conformation; and (b) detecting binding of the candidate agent to the integrin polypeptide, wherein binding of the candidate agent to the integrin polypeptide is indicative that the candidate agent is a candidate modulator of integrin activity.
[0083] 2. The method of paragraph 1, wherein the candidate agent is selected from the group consisting of an antibody, a small molecule, a chemical, a peptide, and a peptidomimetic.
[0084] 3. The method of paragraph 1 or 2, wherein the candidate modulator stabilizes the integrin polypeptide into a closed conformation.
[0085] 4. The method of paragraphs 1, 2, or 3 wherein the candidate modulator inhibits binding of an integrin ligand to the integrin polypeptide.
[0086] 5. The method of any one of paragraphs 1-4, wherein the integrin polypeptide is selected from the group consisting of αVβ3, αIIbβ3, αVb6, αVβ1, αVβ5, αMβ2, αXβ2, αLβ2, and αVβ8.
[0087] 6. The method of any one of paragraphs 1-5, wherein locking the integrin polypeptide into the desired conformation comprises introducing a stabilizing disulfide bond into the integrin polypeptide.
[0088] 7. The method of any one of paragraphs 1-6, wherein the disulfide bond is formed by a cysteine residue substitution of at least one amino acid residue of the integrin polypeptide.
[0089] 8. The method of any one of paragraphs 1-7, wherein the substitution comprises a mutation selected from the group consisting of: L959C (human αIIb), E960C (human αIIb), I955C (human αV), Q956C (human αV), V664C (human β3), P688C (human β3), L662C (human β6), P686C (human β6), A619C (human β8), and F636C (human β3).
[0090] 9. The method of any one of paragraphs 1-8, wherein an optimal sequence alignment is used to identify homologous residues for a cysteine substitution in an integrin polypeptide selected from the group consisting of αVβ3, αIIbβ3, αVb6, αVβ1, αVβ5, αMβ2, αXβ2, αLβ2, and αVβ8.
[0091] 10. The method of any one of paragraphs 1-9, wherein the candidate agent is assayed for activation or inhibition of integrin activity.
[0092] 11. The method of any one of paragraphs 1-10, wherein a cell-based assay is used to determine integrin activity.
[0093] 12. An integrin polypeptide composition comprising: a modified integrin polypeptide, wherein the integrin polypeptide is locked in a closed conformation.
[0094] 13. The composition of paragraph 12, wherein the integrin polypeptide is modified by substitution of at least one amino acid residue for a cysteine residue, whereby a disulfide bond is formed.
[0095] 14. The composition of paragraph 12 or 13, wherein the substitution comprises a mutation selected from the group consisting of: L959C (human αIIb), E960C (human αIIb), I955C (human αV), Q956C (human αV), V664C (human β3), P688C (human β3), L662C (human β6), P686C (human β6), A619C (human β8), and F636C (human β3).
EXAMPLES
Example 1
Structure of a Complete Integrin Ectodomain
[0096] Herein the inventors describe a crystal structure of platelet integrin αIIbβ3 in the bent conformation useful for screening for agents that bind to the integrin in its bent conformation. Crystals in Ca2+ and Mg2+ show that physiologically in the low affinity state the metal binding sites in the β I domain are fully occupied. Of two different αIIbβ3 molecules in the asymmetric unit, one has density for all integrin domains. Thus, the conformation in the bent state is revealed of I-EGF domains 1 and 2 at the β-knee, at the epicenter of conformational change. The overall structure, the linkages between domains, the arrangement of the legs within the bent structure, and the effect of hybrid domain swing-out on affinity for ligand, have profound implications for the mechanism of integrin activation. Use of this information in models of extended integrins experiencing forces at sites of cell adhesion reveals how integrin affinity is regulated by force exerted parallel to the membrane by a motile actin cytoskeleton. Integrin structure and mechanochemistry provides a natural mechanism for increasing integrin affinity upon cytoskeleton attachment and decreasing it upon cytoskeleton disassembly.
αIIbβ3 Crystal Structure and Negative Stain EM
[0097] A 2.55 Å resolution crystal structure of the complete αIIbβ3 ectodomain in Ca2+ and Mg2+ has been refined to an Rfree of 26.8% (FIG. 1A, Table 1). In comparisons to αVβ3 below, differences in resolution and refinement should be kept in mind. The 3.1 Å αVβ3 structure is refined to an Rfree of 36.7% (Xiong et al., 2004). αIIbβ3 has 95% and 0.4% residues in favored and outlier Ramachandran regions, respectively, and geometry in the 98th percentile (where 100 is the best); whereas αVβ3 has 76% and 6.7% residues in favored and outlier regions, respectively, and geometry in the 21st percentile; all values are as reported by MOLPROBITY (Davis et al., 2007). Water molecules, which have important roles in protein structures such as in forming hydrogen bonds and metal coordinations, have been added to the αIIbβ3 but not to the αVβ3 structure, as appropriate for their respective resolutions. No cis-prolines are present in the αVβ3 structure, whereas 6 are present in the αIIbβ3 structure. Two of the cis-prolines, Pro-163 and Pro-169, are in the ligand-binding β3 I domain. The region around cis-Pro-169 has an electron density typical for the αIIbβ3 structure. There is a shift in the sequence-to-structure register between αVβ3 and αIIbβ3 at β3 167-176, in the specificity-determining loop that forms the outer rim of the ligand-binding pocket in the β3 I domain. Thus, with its higher resolution and better refinement, the αIIbβ3 structure provides details about backbone conformation, hydrogen bonding, and side-chain packing that are important for understanding ligand and metal binding; and for accurate molecular dynamics simulations and structure-guided mutagenesis. Furthermore, for the first time, the structure factors for an integrin ectodomain have been deposited, opening access to the experimental electron density upon which the atomic models are based.
[0098] Overall Bent Structure.
[0099] The overall arrangement of domains in the two independent αIIbβ3 molecules in the crystal asymmetric unit is similar to that seen in αVβ3 crystals (FIG. 1C), except for differences in angles between domains described below that give insights into breathing. A similar bent conformation in solution in physiologic divalent cations is seen for three distinct αIIbβ3 constructs in negative stain EM with class averaging (FIG. 1F-H). The bent integrins from the three types of constructs are indistinguishable from one another (FIG. 1F panels 1-3, G panels 1-2, H panels 1-4) and show excellent cross-correlation with the αIIbβ3 crystal structure (FIG. 1F panels 1 and 5, G panels 1 and 5, and H, panels 1, 5 and 6). One construct was clasped by appending to the α and β ectodomain C-termini 15-residue linkers containing TEV protease sites, followed by ACID and BASE peptides that associate in an α-helical coiled-coil (Nishida et al., 2006). Association near the C-termini of the α and β subunit ectodomains that is provided in vivo by the association between the αIIb, and β3 transmembrane domains (Luo et al., 2004) is mimicked by the clasp (Takagi et al., 2002). The clasped αIIbβ3 particles were 64% bent and 32% extended (with 4% unclassified) (FIG. 1F). Unclasped particles, in which the clasp was removed with TEV protease, were 44% bent and 52% extended (FIG. 1G). A third construct, which was identical to that used in crystallization, contained cysteines introduced in C-terminal portions of the αIIb and β3 subunits in positions that resulted in efficient disulfide bond formation in cell surface integrins (FIG. 2). These mutations, αIIb-L959C and β3-P688C, stabilized the integrin in a bent, closed conformation that closely mimics the bent, closed conformation seen in the clasped and unclasped constructs that lack this disulfide. The disulfide-bonded construct was 100% bent (FIG. 1H).
[0100] The differing proportion of bent particles in the three preparations shows that tighter association near the C-termini correlated with maintenance of the bent conformation, and also, with resistance to activation on the cell surface (FIG. 2). This is in agreement with work on other soluble integrin preparations, and a large body of work on cell surface integrins, which has shown that association of the α and β subunit transmembrane and cytoplasmic domains stabilizes integrins in the low-affinity state and in the bent conformation (Luo et al., 2007).
[0101] The differing proportion of bent particles in the three preparations shows that tighter association near the C-termini correlated with maintenance of the bent conformation, and also, with resistance to activation on the cell surface (FIG. 2). This is in agreement with work on other soluble integrin preparations, and a large body of work on cell surface integrins, which has shown that association of the α and β subunit transmembrane and cytoplasmic domains stabilizes integrins in the low-affinity state and in the bent conformation (Luo et al., 2007).
[0102] Similar bent conformations have previously been described in EM studies of the resting states of αVβ3, αXβ2, and αLβ2 (Nishida et al., 2006; Takagi et al., 2002). Furthermore, extensive studies using mutations and antibodies to ligand-induced binding sites show that αIIbβ3 is compact on the cell surface when resting, and extended when activated (Honda et al., 1995; Luo et al., 2007). The similarity in packing of two independent examples of αIIbβ3 and of αVβ3 in crystal lattices and similar appearance of multiple soluble integrins in EM, together with the work cited above, strongly suggests that the bent crystal structure determined here is representative of the resting state of most, if not all, integrins. However, three cryo EM, EM, and hydrodynamic studies of detergent soluble αIIbβ3 from platelets have reached conclusions that are incompatible with one another, and with the domain arrangement seen here (Adair and Yeager, 2002; Rocco et al., 2008; Ye et al., 2008). The difficulty in obtaining a consensus view on αIIbβ3 structure may reflect the delicate equilibrium between bent and extended structures (FIG. 1G-H), averaging over ensembles of bent and extended conformations, the poor association of the αIIb and β3 transmembrane domains in detergent (Wegener and Campbell, 2008), and the ease with which the αIIb and β3 subunits dissociate, even on the platelet surface (Luo et al., 2003).
[0103] Briefly, αIIb and β 3 ectodomains were fused to C-terminal segments containing a tobacco etch protease (TEV) site, ACID or BASE coiled-coils, and strep II or His6 (SEQ ID NO: 105) tags, with or without αIIb-L959C and β3-P688C mutations to introduce a disulfide bond. Proteins were purified from CHO Lec 3.2.8.1 cell supernatants. αIIbβ3 with the extra disulfide bond and the C-terminal tag removed by TEV protease in buffer containing 1 mM CaCl2 was crystallized in 10% PEG 3350, 50 mM magnesium acetate, and 0.1 M imidazole, pH 7.0. Diffraction data collected at 19-ID of APS was solved using molecular replacement in space group P41. Final refinement with REFMAC5 utilized TLS and NCS. Crystals of the αIIbβ3 ectodomain contain two molecules per asymmetric unit. Density is present for all ectodomain residues (αIIb 1-959 and β3 1-690) except for five loops, and in one molecule, the C-terminal portion of the β-tail domain. Thirteen or 18 N-linked carbohydrate residues are visualized in each molecule. I-EGF1 from the complete αIIbβ3 ectodomain was used to model density for this domain in re-refined αIIbβ3 headpiece structures with (Springer et al., 2008) or without Fab (Table 1).
[0104] Conceptual advances since the previously described αVβ3 crystal structures allow us to describe the bent αIIbβ3 crystal structure in light of its physiological relevance as the low affinity integrin state, and as the starting point for integrin extension. Furthermore, the αIIbβ3 structure reveals I-EGF domains 1 and 2, and a highly acute bend between them in the bent conformation (FIG. 1A). In contrast, I-EGF domains 2, 3, and 4 extend in an almost straight orientation, with an approximate 90° left-handed twist between successive domains, to cover most of the length of the lower β-leg (FIG. 1A). The β-knee, at the junction between I-EGF1 and I-EGF2, is flanked on one side by the PSI domain and on the other by a knob-like projection in the thigh domain (FIG. 1A).
[0105] DNA constructs of the extracellular domains of soluble αIIbβ3 were made and expressed as described previously (Takagi, et al), or with modifications as described below to introduce an additional disulfide. αIIb extracellular domain residues 1-963 were fused with a tobacco etch virus (TEV) protease site, acidic coiled coil and StrepII tag to give the C-terminal sequence QLLRALEERA/TGGLENLYFQGGENAQCEKELQALEKENAQLEWELQALEKELAQWSHPQFEK (SEQ ID NO: 58), where the slash marks the fusion position, and then inserted into the pcDNA3.1 vector with hygromycin resistance gene. β3 extracellular domain residues 1-690 were fused with a TEV protease site, basic coiled coil and His6 (SEQ ID NO: 105) tag to give the C-terminal sequence VVEEPECPKG/TSGLENLYFQGGKNAQCKKKLQALKKKNAQLKWKLQALKKKLAQGGHHHITH H (SEQ ID NO: 59), where the slash marks the fusion position, and then inserted into the pEF1 vector with the puromycin resistance gene. Cysteine mutations αIIb-L959C and β3-P688C were introduced at the underlined positions in the above sequences using a site-directed mutagenesis kit. Plasmid DNA of the αIIb and β3 constructs was co-transfected into CHO Lec 3.2.8.1 cells using electroporation. Cells were cultured in selection medium containing puromycin and hygromycin for about 10 days until single colonies were obtained. ELISA was used with mAb 7E3 as the capturing antibody and biotinylated mAb AP3 as the detecting antibody to screen for clones with high expression cell lines. Three rounds of screening of approximately 150 colonies yielded one clone (clone #11) with an expression level of about 5 mg/L for the disulfide-bonded construct. The clone was expanded and cultured in roller bottles.
[0106] Overall Extended Structure.
[0107] In the extended conformation of αIIbβ3, the α and β-legs straighten at the knees, and extend away from rather than fold up against the headpiece (FIGS. 1F and G, panel 4). The headpiece fragment excised from the crystal structure cross-correlates excellently with the headpiece seen in EM (FIGS. 1F and G, panels 6-8). Furthermore, cross-correlation demonstrated that in Ca2+ and Mg2+, extended αIIbβ3 predominantly assumes the closed headpiece conformation with low affinity for ligand, as seen in the bent crystal structure, rather than the open conformation with high affinity. Most extended class averages, whether with clasped or unclasped αIIbβ3, show the α-leg crossing over or under the β-leg (FIG. 1F, G, panel 4). Leg crossing appears to be a consequence of upper leg configuration in the bent conformation with the long axis of I-EGF 1 pointing toward the α-knee (FIG. 1A). When the bent crystal structure is extended at the α and β-knees, leg crossing results. However, the legs are highly flexible, and for clarity are shown side-by-side in FIG. 1B. Extended integrins with crossed and uncrossed legs have also been seen for activated αVβ3, αXβ2, and detergent soluble αIIbβ3 integrins (Iwasaki et al., 2005; Nishida et al., 2006; Takagi et al., 2002).
[0108] After physiological activation of αIIbβ3 on platelets or treatment with high concentrations of ligands, multiple ligand-induced binding site (LIBS) epitopes are exposed. These epitopes map to the lower β-leg, and to the PSI domain (Honda et al., 1995). The lower β-leg is buried in a cleft in the bent conformation (FIG. 1A), but will be exposed in the extended conformation (FIG. 1B). Similarly, the LIBS epitope in the PSI domain, mapped to residues 1-6 (Honda et al., 1995), is masked by I-EGF2 in the bent conformation (FIG. 1A). By contrast, this epitope is exposed after extension at the I-EGF1/I-EGF2 interface in the β-knee brings I-EGF2 away from the PSI domain (FIG. 1B). The previous functional studies, together with the location of these epitopes within the αIIbβ3 structure, demonstrate that bent and extended αIIbβ3 represent latent and activated integrins, respectively, contradict suggestions that αIIbβ3 is extended in the resting state (Rocco et al., 2008; Ye et al., 2008), and agree with election tomography of active, detergent soluble αIIbβ3 showing that it is extended (Iwasaki et al., 2005).
Methods
Crystallography
[0109] Briefly, αIIb and β 3 ectodomains were fused to C-terminal segments containing a tobacco etch protease (TEV) site, ACID or BASE coiled-coils, and strep II or His6 tags, with or without αIIb-L959C and β3-P688C mutations to introduce a disulfide bond. Proteins were purified from CHO Lec 3.2.8.1 cell supernatants. αIIb β3 with the extra disulfide bond and the C-terminal tag removed by TEV protease in buffer containing 1 mM CaCl2 was crystallized in 10% PEG 3350, 50 mM magnesium acetate, and 0.1 M imidazole, pH 7.0. Diffraction data collected at 19-ID of APS was solved using molecular replacement in space group P41. Final refinement with REFMAC5 utilized TLS and NCS. Crystals of the αIIb β3 ectodomain contain two molecules per asymmetric unit. Density is present for all ectodomain residues (αIIb 1-959 and β3 1-690) except for five loops, and in one molecule, the C-terminal portion of the β-tail domain. Thirteen or 18 N-linked carbohydrate residues are visualized in each molecule. I-EGF1 from the complete αIIb β3 ectodomain was used to model density for this domain in re-refined αIIb β3 headpiece structures with (Springer et al., 2008) or without Fab (Table 1).
Negative Stain EM
[0110] The clasped and unclasped αIIb β3 was purified on a Superdex 200 HR column equilibrated with TBS plus 1 mM Ca2+ and 1 mM Mg2+. The peak fraction was adsorbed to glow discharged carbon-coated copper grids, stained with uranyl formate, and inspected with an FEI Tecnai 12 electron microscope operated at 120 kV. Images were acquired at a nominal magnification of 67,000×. Imaging plates were scanned and digitized with a Ditabis micron imaging plate scanner (DITABIS Digital Biomedical Imaging System, AG, Pforzheim, Germany) using a step size of 15 μm and 2×2 pixels were averaged to yield a final pixel size of 4.46 Å at the specimen level. 2,000-5,000 particles were interactively collected, windowed into 75×75-pixel individual images, and subjected to ten cycles of multi-reference alignment and classification. Image processing and cross-correlation using the SPIDER image processing package (Frank et al., 1996) was as described previously (Nishida et al., 2006).
Example 2
Disulfide-Stabilized Integrins for Antibody, Ligand-Binding and Drug Screening Methods
Production of Soluble αIIbβ3
[0111] DNA constructs of the extracellular domains of soluble αIIbβ3 were made and expressed as described previously (Takagi, et al), or with modifications as described below to introduce an additional disulfide. αIIb extracellular domain residues 1-963 were fused with a tobacco etch virus (TEV) protease site, acidic coiled coil and StrepII tag to give the C-terminal sequence QLLRALEERA/TGGLENLYFQGGENAQCEKELQALEKENAQLEWELQALEKELAQWSH PQFEK, where the slash marks the fusion position, and then inserted into the pcDNA3.1 vector with hygromycin resistance gene. β3 extracellular domain residues 1-690 were fused with a TEV protease site, basic coiled coil and His6 tag to give the C-terminal sequence VVEEPECPKG/TSGLENLYFQGGKNAQCKKKLQALKKKNAQLKWKLQALKKKLAQGG HHHHTIH, where the slash marks the fusion position, and then inserted into the pEF1 vector with the puromycin resistance gene. Cysteine mutations αIIb-L959C and β3-P688C were introduced at the underlined positions in the above sequences using a site-directed mutagenesis kit. Plasmid DNA of the αIIb and β3 constructs was co-transfected into CHO Lec 3.2.8.1 cells using electroporation. Cells were cultured in selection medium containing puromycin and hygromycin for about 10 days until single colonies were obtained. ELISA was used with mAb 7E3 as the capturing antibody and biotinylated mAb AP3 as the detecting antibody to screen for clones with high expression cell lines. Three rounds of screening of approximately 150 colonies yielded one clone (clone #11) with an expression level of about 5 mg/L for the disulfide-bonded construct. The clone was expanded and cultured in roller bottles.
[0112] The culture supernatant was concentrated by ultra-filtration and exchanged into 25 mM TrisHCl (pH 8.0) and 300 mM NaCl, plus 5 mM CaCl2 and 10 mM imidazole (loading buffer). The solution was loaded onto a Ni-NTA matrix (QIAGEN®) column (5 ml of resin per 1 liter of culture supernatant) pre-equilibrated with loading buffer. The column was then washed with ten bed-volumes of loading buffer plus 20 mM imidazole and the bound proteins were eluted with five bed-volumes of the loading buffer plus 250 mM imidazole. Eluted proteins were concentrated with an Amicon YM-30 filter (Millipore, Bedford, Mass.) into 20 mM TrisHCl (pH 7.5) and 150 mM NaCl (TBS), plus 5 mM CaCl2, and loaded on a Strep-Tactin column (IBA, St. Louis, Mo.), which was washed with ten bed-volumes of the same buffer. Protein was eluted with the same buffer plus 5 mM desthiobiotin. Purified αIIbβ3 was concentrated with an Amicon YM-30 centrifugal filter to about 1 mg/ml and treated with TEV protease (2.5 units of enzyme per μg αIIbβ3) at 25° C. for 16 hr in TBS plus 5 mM CaCl2. The unclasped αIIbβ3 protein was collected in the flow-through of a second Ni-NTA chromatography step. Purified αIIbβ3 was subjected to Superdex 200 chromatography (Amersham, Piscataway, N.J.) in 20 mM TrisHCl (pH 8), 150 mM NaCl, 1 mM CaCl2.
Negative Stain EM.
[0113] The clasped and unclasped αIIbβ3 was purified on a Superdex 200 HR column in Tris saline, 1 mM Ca2+, 1 mM Mg2+. The peak fraction was adsorbed to glow discharged carbon-coated copper grids, stained with uranylformate, and inspected with an FEI Tecnai 12 electron microscope operated at 120 kV. Images were acquired at a nominal magnification of 67,000×. Imaging plates were scanned and digitized with a Ditabis micron imaging plate scanner (DITABIS Digital Biomedical Imaging System, AG, Pforzheim, Germany) using a step size of 15 μm and 2×2 pixels were averaged to yield a final pixel size of 4.46 Å at the specimen level. 2,000-5,000 particles were interactively collected, windowed into 75×75-pixel individual images, and subjected to ten cycles of multi-reference alignment and classification. Images were processed and cross-correlated using SPIDER (Frank®, et al) as described (Nishida, et al).
Disulfide Crosslinking and Immunoprecipitation
[0114] Twenty-four hours after transfection, 293T cells in 12-well plates with 1.5 ml DMEM medium containing 10% FCS were pre-treated with 15 μg/ml of 2-BP for 1 hour, the medium was replaced with 0.75 ml Met, Cys-free RPMI 1640 (Sigma R-7513), supplemented with 10% dialyzed FCS, 10 μl [35S] cysteine/methionine (10mCi/ml, PerkinElmer Life Science), 15 μg/ml 2-BP. After 1.5 h at 37° C., 0.75 ml of RPMI 1640 containing 10% FCS, 500 μg/ml cysteine, 100 μg/ml methionine, and 15 μg/ml 2-BP was added, and cells chased for at least 17 hours. Cells were detached by vigorous pipetting, washed, and suspended (106 cells in 100 μl) in Tris-buffered saline (TBS, 20 mM Tris-HCl, pH 7.5, 150 mM NaCl) containing 1 mM Ca2+/1 mM Mg2+ and proteinase inhibitors (1 μg/ml each aprotinin, leupeptin, and pepstatin). The cells were kept intact or broken by 3 cycles of freezing on dry ice and thawing. Saponin (40 μg/ml) gave results identical to freeze-thawing, but freeze-thawing was adapted as the least membrane-perturbing. After chilling on ice for 5 minutes, 200 μM CuSO4/1000 μM o-phenanthroline was added by 10 fold dilution from stock solution, and cells were incubated on ice for another 10 minutes. N-ethylmaleimide (10 mM) was added and after 10 minutes on ice, cells were lysed with an equal volume of TBS containing 2% Triton X-100 and 0.1% NP-40 for 10 minutes on ice. Cell lysates were cleared by centrifugation at 14,000 RPM for 10 minutes and immunoprecipitated with anti-β3 mAb AP3 and protein G agarose at 4° C. for 1 hour. The precipitated proteins were subjected to non-reducing 7.5% SDS-PAGE. The SDS-PAGE gel was dried and exposed for 3 h to storage phosphor screens which were measured with a Storm PhosphorImager (Molecular Dynamics, Sunnyvale, Calif., United States). Disulfide bond formation was quantitated as the intensity of the disulfide-bonded heterodimer band divided by the sum of the intensity of αIIb, β3, and heterodimer bands. Specific intensity of each band was determined by subtraction of background intensity.
[0115] For constitutively cross-linked extracellular and exofacial residues, crosslinking was also measured in redox buffer and after DTT treatment followed by Cu-phenanthroline. For redox buffer treatment, cells were suspended in pH 8.2 TBS containing 1 mM Ca2+/1 mM Mg2+ and 5 mM cysteamine/1 mM cystamine, and incubated at 37° C. for 1 hour. Following addition of 10 mM N-ethylmaleimide, cells were lysed and immunoprecipitated as described above.
Results
Disulfide Bonds Near the Ectodomain-Transmembrane Domain Junction of αIIbβ3
[0116] The interface between the αIIb and β3 TM domains has been defined by scanning the TM domains with cysteine and determining the propensity for disulfide bond formation (Luo, et al). Similarly disulfide-bond formation between residues just outside the plasma membrane in intact integrins expressed on the surface of 293 cells was examined (FIG. 2A). A cysteine introduced at residue 688 in the β3 tail domain efficiently formed an inter-subunit disulfide bond with a cysteine introduced at either residue 959 or 960 in the αIIb calf-2 domain (FIG. 2A).
[0117] αXβ2 was clasped at its C-terminal residues shown herein in the following table. Following the protein sequence, generic coiled coil and hexameric histidine (SEQ ID NO: 105) tag were added to the C terminal of construct. Soluble expression of ectodomain was performed via transfecting 293S cells with PEI (Polyethylenimine) method. Following five days of incubation in 37° C., DMEM media containing 10% FCS and 10% CO2, Western blotting with anti-His antibody was performed to investigate formation disulfide formation. Table below discloses SEQ ID NOS 60-69, respectively, in order of appearance.
##STR00001##
[0118] Disulfide cross-links between αIIb and β3 transmembrane residues prevent transmission of activation signals across the membrane both in the inside-out and outside-in directions; however, they do not prevent activation of extracellular ligand binding by extracellular signals, such as Mn2+ and activating antibody (Luo, et al; Zhu, et al). Similarly, the α965C/β693C and α965C/β691C mutants with inter-subunit disulfide bonds in the linker regions could be activated by extracellular stimuli to bind the ligand fibrinogen as efficiently as wild-type (FIG. 2B). However, the α959C/β688C and α960C/β688C mutants with inter-subunit disulfide bonds between C-terminal β tail domain and calf-2 residues were partially resistant to activation by Mn2+ and PT25-2 antibody. These results indicate that the tighter association between the α and β subunits enforced by the more ectodomain-proximal disulfide between the β-tail and calf-2 domains makes them more resistant to activation.
[0119] The greater stability (higher frequency) of bent particles in αIIbβ3 preparations with than without the αIIb959C/β3688C disulfide correlates with the greater resistance to activation of cell-surface αIIb,959C/β3688C than wild-type αIIbβ3 (FIG. 2B). This finding is consistent with conclusions from EM and functional studies on αVβ3 and αXβ2 integrins, that the bent conformation represents the resting state and integrin activation requires extension (Takagi, et al; Nishida, et al).
EM Studies on Disulfide-Mutant Integrins
[0120] A similar bent conformation in solution with physiologic divalent cations is seen for three distinct αIIbβ3 constructs in negative stain EM with class averaging (FIG. 1F-H). The bent integrins from the three types of constructs are indistinguishable from one another (FIG. 1F panels 1-3, G panels 1-2, H panels 1-4) and show excellent cross-correlation with the αIIbβ3 crystal structure (FIG. 1F panels 1 and 5, G panels 1 and 5, and H, panels 1, 5 and 6). One construct was clasped by appending to the α and β ectodomain C-termini 15-residue linkers containing TEV protease sites, followed by an α-helical coiled-coil (Nishida, et al). Association near the C-termini of the α and β subunit ectodomains provided in vivo by association between the αIIb and β3 transmembrane domains (Luo, et al) is mimicked by the clasp (Takagi, et al). The clasped αIIbβ3 particles were 64% bent and 32% extended (with 4% unclassified) (FIG. 1F). Unclasped particles, in which the clasp was removed with TEV protease, were 44% bent and 52% extended (FIG. 1G). A third construct, which was identical to that used in crystallization, contained cysteines introduced in C-terminal portions of the αIIb and β3 subunits in positions that resulted in efficient disulfide bond formation in cell surface integrins. The disulfide-bonded construct was 100% bent (FIG. 1H).
[0121] The differing proportion of bent particles in the three preparations shows that tighter association near the C-termini correlates with maintenance of the bent conformation, and also, with resistance to activation on the cell surface (FIG. 2). This is in agreement with work on other soluble integrin preparations, and a large body of work on cell surface integrins, which has shown that association of the α and β subunit transmembrane and cytoplasmic domains stabilizes integrins in the low-affinity state and in the bent conformation (reviewed in Luo, et al).
Example 3
[0122] αXβ2 was clasped at its C-terminal residues shown herein in the following table. Following the protein sequence, generic coiled coil and hexameric histidine tag were added to the C terminal of construct. Soluble expression of ectodomain was performed via transfecting 293S cells with PEI (Polyethylenimine) method. Following five days of incubation in 37° C., DMEM media containing 10% FCS and 10% CO2, Western blotting with anti-His antibody was performed to investigate formation disulfide formation.
##STR00002##
[0123] 5 constructs for both αX and β2 were transfected, and 25 (5×5) combinations of heterodimeric formation were tested by Western blotting. 5 constructs of αX with P677C of β2 constructs resulted in partial formation of disulfide linkage, whereas rest of heterodimeric combinations displayed completely formation dilsulfide. On the other hand, expression level of P677C of β2 construct was comparable to wild type expression based on ELISA, and considerable lessening of expression level was observed for other 20 combinations.
TABLE-US-00004 TABLE 1 X-ray diffraction data and refinement Protein αIIbβ3 ectodomain αIIbβ3 headpiece Spacegroup P41 P62 Unit cell (a, b, c) (Å) 81.3, 81.3, 654.6 332.1, 332.1, 88.3 (α, β, γ) (°) 90, 90, 90 90, 90, 120 Wavelength (Å) 0.97934 0.9760 Resolution (Å) 50-2.55 45-2.90 Number of reflections 614,293/135,066 1,251,268/122,126 (total/unique) Completeness (%) 98.6/93.9* 98.3/93.9* I/σ(I) 12.2/2.1* 17.4/3.0* Rmerge (%).sup. 7.1/56.6* 9.7/60.2* Rwork.sup. /Rfree.sup..dagger-dbl..dagger-dbl. 0.233/0.268 0.174/0.196 RMSD: Bond (Å) 0.003 0.006 Angle (°) 0.736 0.659 Ramachandran plot** 95.0%/4.6%/0.4% 96.9%/2.9%/0.2% PDB code (prev. 1TYE) *Asterisked numbers correspond to the last resolution shell. .sup. Rmerge = ΣhΣi|Ii(h) - <I(h)>|/ΣhΣiIi(h), where Ii(h) and <I(h)> are the ith and mean measurement of the intensity of reflection h. .sup. Rwork = Σh||Fobs(h)| - |Fcalc (h)||/Σh|Fobs (h)|, where Fobs (h) and Fcalc (h) are the observed and calculated structure factors, respectively. No I/σ cutoff was applied. .sup..dagger-dbl..dagger-dbl.Rfree is the R value obtained for a test set of reflections consisting of a randomly selected 1.3% subset of the data set excluded from refinement. **Residues in favorable, allowed, and outlier regions of the Ramachandran plot as reported by MOLPROBITY (Davis et al., 2007).
TABLE-US-00005 TABLE 2 Variation in inter-domain angles in integrinsa. bent αIIbβ3b bent αIIbβ3c bent αIIbβ3d open αIIbβ3e bent β3f frag β2g Domain interface bent αIIbβ3 bent αVβ3 open αIIbβ3 open αIIbβ3 bent β2 frag β2 α β-propeller - α thigh 0.5° 9.7-9.9° -- -- -- -- α thigh - α calf1 1.3° 19-20° -- -- -- -- α calf1 - α calf2 3.4° 14-17° -- -- -- -- β I - β hybrid 0.2° 6.7-6.7° 58-70° 1.2-12° -- -- β hybrid - β PSI 0.4° 7.2-7.8° 2.0-11° 1.8-9.2° 18-27° 5.0-8.3° β PSI - β I-EGF1 0.8° -- 3.4-13° 5.7-18° 5.5-40° 5.5-41° β hybrid - β I-EGF1 0.5° -- 6.8-23° 11-26° 29-51° 3.7-46° β I-EGF1 - β I-EGF2 0.4° -- -- -- 140-170° 67° β I-EGF2 - β I-EGF3 1.2° -- -- -- 8.4-8.5° -- β I-EGF3 - β I-EGF4 2.3° 5.4-7.2° -- -- -- -- β I-EGF4 - β ankle 1.2° 10-11° -- -- -- -- β I-ankle - β TD .sup. 46°h 18°i -- -- -- -- α β-propeller - β I 0.2° 3.0-3.3° 1.7-2.8° 0.6-1.1° -- -- aEach pair of domains from two molecules were superposed using the first domain, and the change in angle upon superimposing the second domain was calculated. Dashes indicate where no comparison is possible, because only one or no domain pairs are available. bTwo molecules in current structure (1 × 1). cTwo molecules in current structure versus PDB code IU8C (2 × 1). dTwo molecules in current structure versus PDB 2VDR and three molecules in PDB ITYE (2 × 4). eComparisons among 2VDR and three molecules in ITYE (3 × 4/2). fTwo molecules in current structure, and PDB 1U8C versus PDB 1YUK, PDB 2P26, and PDB 2P28 (3 × 3 to 3 × 1, depending on fragment length). gcomparisons among PDB 1YUK, 2P26, and 2P28 (3 to 1 comparisons depending on fragment length). hResidues common to molecules 1 and 2 in βTD are used, 606-612 iαIIbβ3 molecule 1 compared to αVβ3
TABLE-US-00006 TABLE 3 αXβ2-GCG constructs prepared to investigate the clasping of C-terminal region of ectodomain. Table discloses SEQ ID NOS 70-81, respectively, in order of appearance. ##STR00003## Residues changed to Cys are always both preceded and followed by Gly to introduce local flexibility to Cys. *Clasping sequence of crystallized αXβB2-GCG -TCSH construct are shown on wild type sequence as red and asterisked.
TABLE-US-00007 TABLE 4 Cu--Ph oxidation/ αIIbβ3 Constitutive disulfideb Redox buffer treat. intact cell alpha beta Exp1 Exp2 Average Exp1 Exp2 Average Exp1 Average R957 E686 28 28 0 0 R957 C687 2 2 R957 P688 95 82 88.5 R957 G690 15 15 R957 P691 10 10 R957 D692 12 12 A958 E686 100 100 54 54 A958 C687 2 2 A958 P688 96 96 A958 G690 12 12 A958 P691 15 15 A958 D692 8 8 L959 E686 94 94 L959 C687 2 2 L959 P688 98 98 100 100 L959 G690 30 30 L959 P691 12 12 L959 D692 2 2 E960 E686 92 92 59 59 E960 C687 3 3 E960 P688 91 91 98 98 E960 G690 77 77 E960 P691 64 64 E960 D692 15 15 R962 P691 100 100 56 56 100 100 R962 D692 67 67 5 5 72 72 R962 I693 77 77 3 3 82 82 R962 L694 8 8 0 0 6 6 R962 V695 0 0 0 0 0 0 R962 V696 0 0 0 0 0 0 R962 L697 0 0 0 0 0 0 A963 P691 100 100 30 38 34 100 100 A963 D692 41 41 2 5 3.5 44 44 A963 I693 100 100 19 24 21.5 100 100 A963 L694 5 5 0 0 0 13 13 A963 V695 1 1 1 3 3 A963 V696 0 2 1 13 13 A963 L697 1 0 0.5 6 6 I964 P691 60 60 61 54 57.5 59 59 I964 D692 77 77 21 19 20 80 80 I964 I693 74 74 90 89 89.5 92 92 I964 L694 33 33 5 8 6.5 35 35 I964 V695 18 18 4 6 5 16 16 I964 V696 31 31 30 54 42 39 39 I964 L697 6 6 5 6 5.5 6 6 I964 L698 0 0 0 0 0 0 0 Values are % disulfide bond formation
TABLE-US-00008 TABLE 5 5A 052504 FACS (LFA-1 disulfide mutants on 293T cell) Mean Sample No Sample description X63 MHM24 m24 A03 KIM127(1) KIM127(2) mock -- -- 6.34 6.17 6.27 6.79 5.54 #2 E1061C G676C 97.38 35.81 59.26 8.27 9.59 #3 K1062C G676C 117.13 57.43 67.16 11.14 15.81 #4 Q1063C G676C 95.90 47.81 61.04 12.12 12.15 #5 K1062C P677C 102.18 43.50 58.30 10.26 9.36 #6 Q1063C P677C 116.87 52.29 72.99 10.89 14.43 #7 M1064C P677C 125.86 52.95 77.00 10.77 11.85 #8 Q1063C N678C 88.46 38.63 52.54 8.54 12.25 #9 M1064C N678C 4.06 147.12 57.10 88.01 10.03 12.09 WT WT WT 5.96 158.96 66.32 105.85 12.15 13.91 5B Normalization Mean value normalized with expression level (MHM24) Sample No Sample description X63 MHM24 m24 A03 KIM127(1) KIM127(2) mock -- -- 6.34 -- -- -- -- #2 E1061C G676C 97.38 0.33 0.58 0.016 0.044 #3 K1062C G676C 117.13 0.46 0.55 0.039 0.093 #4 Q1063C G676C 95.90 0.46 0.61 0.060 0.074 #5 K1062C P677C 102.18 0.39 0.54 0.036 0.040 #6 Q1063C P677C 116.87 0.42 0.60 0.037 0.080 #7 M1064C P677C 125.86 0.39 0.59 0.033 0.053 #8 Q1063C N678C 88.46 0.40 0.56 0.021 0.082 #9 M1064C N678C 4.06 147.12 0.36 0.58 0.023 0.047 WT WT WT 5.96 158.96 0.39 0.65 0.035 0.055 5C Normalization (WT is set to 1.0) Relative to wild-type Sample No Sample description X63 MHM24 m24 A03 KIM127(1) KIM127(2) mock -- -- 6.34 -- -- -- -- #2 E1061C G676C 97.38 0.83 0.89 0.46 0.81 #3 K1062C G676C 117.13 1.17 0.84 1.12 1.69 #4 Q1063C G676C 95.90 1.18 0.94 1.69 1.35 #5 K1062C P677C 102.18 0.99 0.83 1.03 0.73 #6 Q1063C P677C 116.87 1.06 0.93 1.06 1.47 #7 M1064C P677C 125.86 0.99 0.91 0.95 0.96 #8 Q1063C N678C 88.46 1.00 0.86 0.61 1.49 #9 M1064C N678C 4.06 147.12 0.92 0.89 0.66 0.85 WT WT WT 5.96 158.96 1.00 1.00 1.00 1.00 5D Summary of WB and FACS result Relative to wild-type di- Expres- Sample No Sample description sulfide sio m24 A03 KIM127(1) KIM127(2) mock -- -- - - -- -- -- -- #2 E1061C G676C ++ ++ 0.83 0.89 0.46 0.81 #3 K1062C G676C ++ ++ 1.17 0.84 1.12 1.69 #4 Q1063C G676C + ++ 1.18 0.94 1.69 1.35 #5 K1062C P677C ++ ++ 0.99 0.83 1.03 0.73 #6 Q1063C P677C ++ +++ 1.06 0.93 1.06 1.47 #7 M1064C P677C ++ +++ 0.99 0.91 0.95 0.96 #8 Q1063C N678C - ++ 1.00 0.86 0.61 1.49 #9 M1064C N678C + +++ 0.92 0.89 0.66 0.85 WT WT WT - +++ 1.00 1.00 1.00 1.00 5E Summary of WB result ve to wild-type di- Expres- Sample No Sample description sulfide sion mock -- -- - - #2 E1061C G676C ++ ++ #3 K1062C G676C ++ ++ indicates data missing or illegible when filed
Sequence CWU
1
1
1051159PRTHomo sapiens 1Arg Asp Leu Ala Leu Ser Glu Gly Asp Ile His Thr
Leu Gly Cys Gly 1 5 10
15 Val Ala Gln Cys Leu Lys Ile Val Cys Gln Val Gly Arg Leu Asp Arg
20 25 30 Gly Lys Ser
Ala Ile Leu Tyr Val Lys Ser Leu Leu Trp Thr Glu Thr 35
40 45 Phe Met Asn Lys Glu Asn Gln Asn
His Ser Tyr Ser Leu Lys Ser Ser 50 55
60 Ala Ser Phe Asn Val Ile Glu Phe Pro Tyr Lys Asn Leu
Pro Ile Glu 65 70 75
80 Asp Ile Thr Asn Ser Thr Leu Val Thr Thr Asn Val Thr Trp Gly Ile
85 90 95 Gln Pro Ala Pro
Met Pro Val Pro Val Trp Val Ile Ile Leu Ala Val 100
105 110 Leu Ala Gly Leu Leu Leu Leu Ala Val
Leu Val Phe Val Met Tyr Arg 115 120
125 Met Gly Phe Phe Lys Arg Val Arg Pro Pro Gln Glu Glu Gln
Glu Arg 130 135 140
Glu Gln Leu Gln Pro His Glu Asn Gly Glu Gly Asn Ser Glu Thr 145
150 155 2159PRTMus musculus 2Arg
Gly Leu Thr Leu Arg Glu Gly Asp Val His Thr Leu Gly Cys Gly 1
5 10 15 Ile Ala Lys Cys Leu Gln
Ile Thr Cys Gln Val Gly Arg Leu Asp Arg 20
25 30 Gly Lys Ser Ala Ile Leu Tyr Val Lys Ser
Leu Leu Trp Thr Glu Thr 35 40
45 Phe Met Asn Lys Glu Asn Gln Asn His Ser Tyr Ser Leu Lys
Ser Ser 50 55 60
Ala Ser Phe Asn Ile Ile Glu Phe Pro Tyr Lys Asn Leu Pro Ile Glu 65
70 75 80 Asp Leu Phe Asn Ser
Thr Leu Val Thr Thr Asn Ile Thr Trp Gly Ile 85
90 95 Gln Pro Ala Pro Met Pro Val Pro Val Trp
Val Ile Ile Leu Ala Val 100 105
110 Leu Ala Gly Leu Leu Leu Leu Ala Val Leu Val Phe Val Met Tyr
Arg 115 120 125 Met
Gly Phe Phe Lys Arg Val Arg Pro Pro Gln Glu Glu Gln Glu Arg 130
135 140 Glu Gln Leu Gln Pro His
Glu Asn Gly Glu Gly Asn Ser Glu Thr 145 150
155 3153PRTHomo sapiens 3Pro Ser Arg Ser Ser Ala Ser
Ser Gly Pro Gln Ile Leu Lys Cys Pro 1 5
10 15 Glu Ala Glu Cys Phe Arg Leu Arg Cys Glu Leu
Gly Pro Leu His Gln 20 25
30 Gln Glu Ser Gln Ser Leu Gln Leu His Phe Arg Val Trp Ala Lys
Thr 35 40 45 Phe
Leu Gln Arg Glu His Gln Pro Phe Ser Leu Gln Cys Glu Ala Val 50
55 60 Tyr Lys Ala Leu Lys Met
Pro Tyr Arg Ile Leu Pro Arg Gln Leu Pro 65 70
75 80 Gln Lys Glu Arg Gln Val Ala Thr Ala Val Gln
Trp Thr Lys Ala Glu 85 90
95 Gly Ser Tyr Gly Val Pro Leu Trp Ile Ile Ile Leu Ala Ile Leu Phe
100 105 110 Gly Leu
Leu Leu Leu Gly Leu Leu Ile Tyr Ile Leu Tyr Lys Leu Gly 115
120 125 Phe Phe Lys Arg Ser Leu Pro
Tyr Gly Thr Ala Met Glu Lys Ala Gln 130 135
140 Leu Lys Pro Pro Ala Thr Ser Asp Ala 145
150 4153PRTMus musculus 4Pro Gly Arg Ser Ser Thr Ala
Ser Gly Thr Gln Val Leu Lys Cys Pro 1 5
10 15 Glu Ala Lys Cys Phe Arg Leu Arg Cys Glu Phe
Gly Pro Leu His Arg 20 25
30 Gln Glu Ser Arg Ser Leu Gln Leu His Phe Arg Val Trp Ala Lys
Thr 35 40 45 Phe
Leu Gln Arg Glu Tyr Gln Pro Phe Ser Leu Gln Cys Glu Ala Val 50
55 60 Tyr Glu Ala Leu Lys Met
Pro Tyr Gln Ile Leu Pro Arg Gln Leu Pro 65 70
75 80 Gln Lys Lys Leu Gln Val Ala Thr Ala Val Gln
Trp Thr Lys Ala Glu 85 90
95 Gly Ser Asn Gly Val Pro Leu Trp Ile Ile Ile Leu Ala Ile Leu Phe
100 105 110 Gly Leu
Leu Leu Leu Gly Leu Leu Ile Tyr Val Leu Tyr Lys Leu Gly 115
120 125 Phe Phe Lys Arg Ser Leu Pro
Tyr Gly Thr Ala Met Glu Lys Ala Gln 130 135
140 Leu Lys Pro Pro Ala Thr Ser Asp Ala 145
150 5143PRTHomo sapiens 5Pro Glu Gln Pro Ser Arg Leu
Gln Asp Pro Val Leu Val Ser Cys Asp 1 5
10 15 Ser Ala Pro Cys Thr Val Val Gln Cys Asp Leu
Gln Glu Met Ala Arg 20 25
30 Gly Gln Arg Ala Met Val Thr Val Leu Ala Phe Leu Trp Leu Pro
Ser 35 40 45 Leu
Tyr Gln Arg Pro Leu Asp Gln Phe Val Leu Gln Ser His Ala Trp 50
55 60 Phe Asn Val Ser Ser Leu
Pro Tyr Ala Val Pro Pro Leu Ser Leu Pro 65 70
75 80 Arg Gly Glu Ala Gln Val Trp Thr Gln Leu Leu
Arg Ala Leu Glu Glu 85 90
95 Arg Ala Ile Pro Ile Trp Trp Val Leu Val Gly Val Leu Gly Gly Leu
100 105 110 Leu Leu
Leu Thr Ile Leu Val Leu Ala Met Trp Lys Val Gly Phe Phe 115
120 125 Lys Arg Asn Arg Pro Pro Leu
Glu Glu Asp Asp Glu Glu Gly Glu 130 135
140 6143PRTMus musculus 6Gln Gly Pro Lys Pro Gly Gln Gln
Asp Pro Val Leu Val Ser Cys Asp 1 5 10
15 Gly Ser Ala Ser Cys Thr Val Val Glu Cys Glu Leu Arg
Glu Met Val 20 25 30
Arg Gly Gln Arg Ala Met Val Thr Val Gln Ala Met Leu Gly Leu Ser
35 40 45 Ser Leu Arg Gln
Arg Pro Gln Glu Gln Phe Val Leu Gln Ser His Ala 50
55 60 Trp Phe Asn Val Ser Ser Leu Pro
Tyr Ser Val Pro Val Val Ser Leu 65 70
75 80 Pro Ser Gly Gln Ala Arg Val Gln Thr Gln Leu Leu
Arg Ala Leu Glu 85 90
95 Glu Arg Ala Ile Pro Val Trp Trp Val Leu Val Gly Val Leu Gly Gly
100 105 110 Leu Leu Leu
Leu Thr Leu Leu Val Leu Ala Met Trp Lys Ala Gly Phe 115
120 125 Phe Lys Arg Asn Arg Pro Pro Leu
Glu Glu Asp Glu Glu Glu Glu 130 135
140 7159PRTHomo sapiens 7Lys Arg Asp Val His Val Val Glu Phe
His Arg Gln Ser Pro Ala Lys 1 5 10
15 Ile Leu Asn Cys Thr Asn Ile Glu Cys Leu Gln Ile Ser Cys
Ala Val 20 25 30
Gly Arg Leu Glu Gly Gly Glu Ser Ala Val Leu Lys Val Arg Ser Arg
35 40 45 Leu Trp Ala His
Thr Phe Leu Gln Arg Lys Asn Asp Pro Tyr Ala Leu 50
55 60 Ala Ser Leu Val Ser Phe Glu Val
Lys Lys Met Pro Tyr Thr Asp Gln 65 70
75 80 Pro Ala Lys Leu Pro Glu Gly Ser Ile Ala Ile Lys
Thr Ser Val Ile 85 90
95 Trp Ala Thr Pro Asn Val Ser Phe Ser Ile Pro Leu Trp Val Ile Ile
100 105 110 Leu Ala Ile
Leu Leu Gly Leu Leu Val Leu Ala Ile Leu Thr Leu Ala 115
120 125 Leu Trp Lys Cys Gly Phe Phe Asp
Arg Ala Arg Pro Pro Gln Glu Asp 130 135
140 Met Thr Asp Arg Glu Gln Leu Thr Asn Asp Lys Thr Pro
Glu Ala 145 150 155
8156PRTHomo sapiens 8Phe Ala Glu Arg Lys Tyr Gln Thr Leu Asn Cys Ser Val
Asn Val Asn 1 5 10 15
Cys Val Asn Ile Arg Cys Pro Leu Arg Gly Leu Asp Ser Lys Ala Ser
20 25 30 Leu Ile Leu Arg
Ser Arg Leu Trp Asn Ser Thr Phe Leu Glu Glu Tyr 35
40 45 Ser Lys Leu Asn Tyr Leu Asp Ile Leu
Met Arg Ala Phe Ile Asp Val 50 55
60 Thr Ala Ala Ala Glu Asn Ile Arg Leu Pro Asn Ala Gly
Thr Gln Val 65 70 75
80 Arg Val Thr Val Phe Pro Ser Lys Thr Val Ala Gln Tyr Ser Gly Val
85 90 95 Pro Trp Trp Ile
Ile Leu Val Ala Ile Leu Ala Gly Ile Leu Met Leu 100
105 110 Ala Leu Leu Val Phe Ile Leu Trp Lys
Cys Gly Phe Phe Lys Arg Asn 115 120
125 Lys Lys Asp His Tyr Asp Ala Thr Tyr His Lys Ala Glu Ile
His Ala 130 135 140
Gln Pro Ser Asp Lys Glu Arg Leu Thr Ser Asp Ala 145 150
155 9156PRTMus musculus 9Phe Pro Glu Arg Lys Tyr Gln
Thr Leu Asn Cys Ser Val Asn Val Arg 1 5
10 15 Cys Val Asn Ile Arg Cys Pro Leu Arg Gly Leu
Asp Thr Lys Ala Ser 20 25
30 Leu Val Leu Cys Ser Arg Leu Trp Asn Ser Thr Phe Leu Glu Glu
Tyr 35 40 45 Ser
Lys Leu Asn Tyr Leu Asp Ile Leu Val Arg Ala Ser Ile Asp Val 50
55 60 Thr Ala Ala Ala Gln Asn
Ile Lys Leu Pro His Ala Gly Thr Gln Val 65 70
75 80 Arg Val Thr Val Phe Pro Ser Lys Thr Val Ala
Gln Tyr Ser Gly Val 85 90
95 Ala Trp Trp Ile Ile Leu Leu Ala Val Leu Ala Gly Ile Leu Met Leu
100 105 110 Ala Leu
Leu Val Phe Leu Leu Trp Lys Cys Gly Phe Phe Lys Arg Asn 115
120 125 Lys Lys Asp His Tyr Asp Ala
Thr Tyr His Lys Ala Glu Ile His Thr 130 135
140 Gln Pro Ser Asp Lys Glu Arg Leu Thr Ser Asp Ala
145 150 155 10203PRTHomo sapiens
10Trp Pro Val Ser Ser Ala Glu Lys Lys Lys Asn Ile Thr Leu Asp Cys 1
5 10 15 Ala Arg Gly Thr
Ala Asn Cys Val Val Phe Ser Cys Pro Leu Tyr Ser 20
25 30 Phe Asp Arg Ala Ala Val Leu His Val
Trp Gly Arg Leu Trp Asn Ser 35 40
45 Thr Phe Leu Glu Glu Tyr Ser Ala Val Lys Ser Leu Glu Val
Ile Val 50 55 60
Arg Ala Asn Ile Thr Val Lys Ser Ser Ile Lys Asn Leu Met Leu Arg 65
70 75 80 Asp Ala Ser Thr Val
Ile Pro Val Met Val Tyr Leu Asp Pro Met Ala 85
90 95 Val Val Ala Glu Gly Val Pro Trp Trp Val
Ile Leu Leu Ala Val Leu 100 105
110 Ala Gly Leu Leu Val Leu Ala Leu Leu Val Leu Leu Leu Trp Lys
Met 115 120 125 Gly
Phe Phe Lys Arg Ala Lys His Pro Glu Ala Thr Val Pro Gln Tyr 130
135 140 His Ala Val Lys Ile Pro
Arg Glu Asp Arg Gln Gln Phe Lys Glu Glu 145 150
155 160 Lys Thr Gly Thr Ile Leu Arg Asn Asn Trp Gly
Ser Pro Arg Arg Glu 165 170
175 Gly Pro Asp Ala His Pro Ile Leu Ala Ala Asp Gly His Pro Glu Leu
180 185 190 Gly Pro
Asp Gly His Pro Gly Pro Gly Thr Ala 195 200
11202PRTMus musculus 11Trp Pro Val Ser Ser Ala Glu Lys Arg Asn Val
Thr Leu Asp Cys Ala 1 5 10
15 Gln Gly Thr Ala Lys Cys Val Val Phe Ser Cys Pro Leu Tyr Ser Phe
20 25 30 Asp Arg
Ala Ala Val Leu His Val Trp Gly Arg Leu Trp Asn Ser Thr 35
40 45 Phe Leu Glu Glu Tyr Met Ala
Val Lys Ser Leu Glu Val Ile Val Arg 50 55
60 Ala Asn Ile Thr Val Lys Ser Ser Ile Lys Asn Leu
Leu Leu Arg Asp 65 70 75
80 Ala Ser Thr Val Ile Pro Val Met Val Tyr Leu Asp Pro Met Ala Val
85 90 95 Val Val Glu
Gly Val Pro Trp Trp Val Ile Leu Leu Ala Val Leu Ala 100
105 110 Gly Leu Leu Val Leu Ala Leu Leu
Val Leu Leu Leu Trp Lys Leu Gly 115 120
125 Phe Phe Lys Arg Ala Lys His Pro Glu Ala Thr Val Pro
Gln Tyr His 130 135 140
Ala Val Lys Ile Pro Arg Glu Asp Arg Gln Gln Phe Lys Glu Glu Lys 145
150 155 160 Thr Gly Thr Ile
Gln Arg Ser Asn Trp Gly Asn Ser Gln Trp Glu Gly 165
170 175 Ser Asp Ala His Pro Ile Leu Ala Ala
Asp Trp His Pro Glu Leu Gly 180 185
190 Pro Asp Gly His Pro Val Pro Ala Thr Ala 195
200 12202PRTMus musculus 12Trp Pro Val Ser Ser Ala
Glu Lys Arg Asn Val Thr Leu Asp Cys Ala 1 5
10 15 Gln Gly Thr Ala Lys Cys Val Val Phe Ser Cys
Pro Leu Tyr Ser Phe 20 25
30 Asp Arg Ala Ala Val Leu His Val Trp Gly Arg Leu Trp Asn Ser
Thr 35 40 45 Phe
Leu Glu Glu Tyr Met Ala Val Lys Ser Leu Glu Val Ile Val Arg 50
55 60 Ala Asn Ile Thr Val Lys
Ser Ser Ile Lys Asn Leu Leu Leu Arg Asp 65 70
75 80 Ala Ser Thr Val Ile Pro Val Met Val Tyr Leu
Asp Pro Met Ala Val 85 90
95 Val Val Glu Gly Val Pro Trp Trp Val Ile Leu Leu Ala Val Leu Ala
100 105 110 Gly Leu
Leu Val Leu Ala Leu Leu Val Leu Leu Leu Trp Lys Leu Gly 115
120 125 Phe Phe Lys Arg Ala Lys His
Pro Glu Ala Thr Val Pro Gln Tyr His 130 135
140 Ala Val Lys Ile Pro Arg Glu Asp Arg Gln Gln Phe
Lys Glu Glu Lys 145 150 155
160 Thr Gly Thr Ile Gln Arg Ser Asn Trp Gly Asn Ser Gln Trp Glu Gly
165 170 175 Ser Asp Ala
His Pro Ile Leu Ala Ala Asp Trp His Pro Glu Leu Gly 180
185 190 Pro Asp Gly His Pro Val Pro Ala
Thr Ala 195 200 13162PRTHomo sapiens
13Leu Ala Ala Ala Lys Lys Ala Lys Ser Glu Thr Val Leu Thr Cys Ala 1
5 10 15 Thr Gly Arg Ala
His Cys Val Trp Leu Glu Cys Pro Ile Pro Asp Ala 20
25 30 Pro Val Val Thr Asn Val Thr Val Lys
Ala Arg Val Trp Asn Ser Thr 35 40
45 Phe Ile Glu Asp Tyr Arg Asp Phe Asp Arg Val Arg Val Asn
Gly Trp 50 55 60
Ala Thr Leu Phe Leu Arg Thr Ser Ile Pro Thr Ile Asn Met Glu Asn 65
70 75 80 Lys Thr Thr Trp Phe
Ser Val Asp Ile Asp Ser Glu Leu Val Glu Glu 85
90 95 Leu Pro Ala Glu Ile Glu Leu Trp Leu Val
Leu Val Ala Val Gly Ala 100 105
110 Gly Leu Leu Leu Leu Gly Leu Ile Ile Leu Leu Leu Trp Lys Cys
Gly 115 120 125 Phe
Phe Lys Arg Ala Arg Thr Arg Ala Leu Tyr Glu Ala Lys Arg Gln 130
135 140 Lys Ala Glu Met Lys Ser
Gln Pro Ser Glu Thr Glu Arg Leu Thr Asp 145 150
155 160 Asp Tyr 14162PRTMus musculus 14Leu Ala Ala
Ala Lys Lys Ala Lys Ser Glu Thr Val Leu Thr Cys Ser 1 5
10 15 Asn Gly Arg Ala Arg Cys Val Trp
Leu Glu Cys Pro Leu Pro Asp Thr 20 25
30 Ser Asn Ile Thr Asn Val Thr Val Lys Ala Arg Val Trp
Asn Ser Thr 35 40 45
Phe Ile Glu Asp Tyr Lys Asp Phe Asp Arg Val Arg Val Asp Gly Trp 50
55 60 Ala Thr Leu Phe
Leu Arg Thr Ser Ile Pro Thr Ile Asn Met Glu Asn 65 70
75 80 Lys Thr Thr Trp Phe Ser Val Asp Ile
Asp Ser Glu Leu Val Glu Glu 85 90
95 Leu Pro Ala Glu Ile Glu Leu Trp Leu Val Leu Val Ala Val
Gly Ala 100 105 110
Gly Leu Leu Leu Leu Gly Leu Ile Ile Leu Leu Leu Trp Lys Cys Gly
115 120 125 Phe Phe Lys Arg
Ala Arg Thr Arg Ala Leu Tyr Glu Ala Lys Arg Gln 130
135 140 Lys Ala Glu Met Lys Ser Gln Pro
Ser Glu Thr Glu Arg Leu Thr Asp 145 150
155 160 Asp Tyr 15154PRTHomo sapiens 15Phe Leu Ser Lys
Thr Asp Lys Arg Leu Leu Tyr Cys Ile Lys Ala Asp 1 5
10 15 Pro His Cys Leu Asn Phe Leu Cys Asn
Phe Gly Lys Met Glu Ser Gly 20 25
30 Lys Glu Ala Ser Val His Ile Gln Leu Glu Gly Arg Pro Ser
Ile Leu 35 40 45
Glu Met Asp Glu Thr Ser Ala Leu Lys Phe Glu Ile Arg Ala Thr Gly 50
55 60 Phe Pro Glu Pro Asn
Pro Arg Val Ile Glu Leu Asn Lys Asp Glu Asn 65 70
75 80 Val Ala His Val Leu Leu Glu Gly Leu His
His Gln Arg Pro Lys Arg 85 90
95 Tyr Phe Thr Ile Val Ile Ile Ser Ser Ser Leu Leu Leu Gly Leu
Ile 100 105 110 Val
Leu Leu Leu Ile Ser Tyr Val Met Trp Lys Ala Gly Phe Phe Lys 115
120 125 Arg Gln Tyr Lys Ser Ile
Leu Gln Glu Glu Asn Arg Arg Asp Ser Trp 130 135
140 Ser Tyr Ile Asn Ser Lys Ser Asn Asp Asp 145
150 16154PRTMus musculus 16Phe Leu Ser
Lys Thr Asp Lys Arg Leu Leu Tyr Cys Met Lys Ala Asp 1 5
10 15 Gln His Cys Leu Asp Phe Leu Cys
Asn Phe Gly Lys Met Glu Ser Gly 20 25
30 Lys Glu Ala Ser Val His Ile Gln Leu Glu Gly Arg Pro
Ser Ile Leu 35 40 45
Glu Met Asp Glu Thr Ser Ser Leu Lys Phe Glu Ile Lys Ala Thr Ala 50
55 60 Phe Pro Glu Pro
His Pro Lys Val Ile Glu Leu Asn Lys Asp Glu Asn 65 70
75 80 Val Ala His Val Phe Leu Glu Gly Leu
His His Gln Arg Pro Lys Arg 85 90
95 His Phe Thr Ile Ile Ile Ile Thr Ile Ser Leu Leu Leu Gly
Leu Ile 100 105 110
Val Leu Leu Leu Ile Ser Cys Val Met Trp Lys Ala Gly Phe Phe Lys
115 120 125 Arg Gln Tyr Lys
Ser Ile Leu Gln Glu Glu Asn Arg Arg Asp Ser Trp 130
135 140 Ser Tyr Val Asn Ser Lys Ser Asn
Asp Asp 145 150 17156PRTHomo sapiens
17Phe Phe Thr Lys Ser Gly Arg Lys Val Leu Asp Cys Glu Lys Pro Gly 1
5 10 15 Ile Ser Cys Leu
Thr Ala His Cys Asn Phe Ser Ala Leu Ala Lys Glu 20
25 30 Glu Ser Arg Thr Ile Asp Ile Tyr Met
Leu Leu Asn Thr Glu Ile Leu 35 40
45 Lys Lys Asp Ser Ser Ser Val Ile Gln Phe Met Ser Arg Ala
Lys Val 50 55 60
Lys Val Asp Pro Ala Leu Arg Val Val Glu Ile Ala His Gly Asn Pro 65
70 75 80 Glu Glu Val Thr Val
Val Phe Glu Ala Leu His Asn Leu Glu Pro Arg 85
90 95 Gly Tyr Val Val Gly Trp Ile Ile Ala Ile
Ser Leu Leu Val Gly Ile 100 105
110 Leu Ile Phe Leu Leu Leu Ala Val Leu Leu Trp Lys Met Gly Phe
Phe 115 120 125 Arg
Arg Arg Tyr Lys Glu Ile Ile Glu Ala Glu Lys Asn Arg Lys Glu 130
135 140 Asn Glu Asp Ser Trp Asp
Trp Val Gln Lys Asn Gln 145 150 155
18125PRTHomo sapiens 18Lys Arg Gly Thr Ile Leu Asp Cys Asn Thr Cys Lys
Phe Ala Thr Ile 1 5 10
15 Thr Cys Asn Leu Thr Ser Ser Asp Ile Ser Gln Val Asn Val Ser Leu
20 25 30 Ile Leu Trp
Lys Pro Thr Phe Ile Lys Ser Tyr Phe Ser Ser Leu Asn 35
40 45 Leu Thr Ile Arg Gly Glu Leu Arg
Ser Glu Asn Ala Ser Leu Val Leu 50 55
60 Ser Ser Ser Asn Gln Lys Arg Glu Leu Val Ile Gln Ile
Ser Lys Asp 65 70 75
80 Gly Leu Pro Gly Arg Val Pro Leu Trp Val Ile Leu Leu Ser Ala Phe
85 90 95 Ala Gly Leu Leu
Leu Leu Met Leu Leu Ile Leu Ala Leu Trp Lys Ile 100
105 110 Gly Phe Phe Lys Arg Pro Leu Lys Lys
Lys Met Glu Lys 115 120 125
19139PRTHomo sapiens 19Arg His Thr Lys Glu Leu Asn Cys Arg Thr Ala Ser
Cys Ser Asn Val 1 5 10
15 Thr Cys Trp Leu Lys Asp Val His Met Lys Gly Glu Tyr Phe Val Asn
20 25 30 Val Thr Thr
Arg Ile Trp Asn Gly Thr Phe Ala Ser Ser Thr Phe Gln 35
40 45 Thr Val Gln Leu Thr Ala Ala Ala
Glu Ile Asn Thr Tyr Asn Pro Glu 50 55
60 Ile Tyr Val Ile Glu Asp Asn Thr Val Thr Ile Pro Leu
Val Ile Met 65 70 75
80 Lys Pro Asp Glu Lys Ala Glu Val Pro Thr Gly Val Ile Ile Gly Ser
85 90 95 Ile Ile Ala Gly
Ile Leu Leu Leu Leu Ala Leu Val Ala Ile Leu Trp 100
105 110 Lys Leu Gly Phe Phe Lys Arg Lys Tyr
Glu Lys Met Thr Lys Asn Pro 115 120
125 Asp Glu Ile Asp Glu Thr Thr Glu Leu Ser Ser 130
135 20139PRTMus musculus 20Arg His Thr Lys
Glu Leu Asp Cys Arg Thr Thr Ser Cys Ser Asn Ile 1 5
10 15 Thr Cys Trp Leu Lys Asp Leu His Met
Lys Ala Glu Tyr Phe Ile Asn 20 25
30 Val Thr Thr Arg Val Trp Asn Arg Thr Phe Ala Ala Ser Thr
Phe Gln 35 40 45
Thr Val Gln Leu Thr Ala Ala Ala Glu Ile Asp Thr His Asn Pro Gln 50
55 60 Leu Phe Val Ile Glu
Glu Asn Ala Val Thr Ile Pro Leu Val Ile Met 65 70
75 80 Lys Pro Thr Glu Lys Ala Glu Val Pro Thr
Gly Val Ile Ile Gly Ser 85 90
95 Ile Ile Ala Gly Ile Leu Leu Leu Leu Ala Met Thr Ala Gly Leu
Trp 100 105 110 Lys
Leu Gly Phe Phe Lys Arg Gln Tyr Lys Lys Met Gly Gln Asn Pro 115
120 125 Asp Glu Met Asp Glu Thr
Thr Glu Leu Asn Ser 130 135
21135PRTHomo sapiens 21Gln His Thr Asn Arg Leu Asn Gly Ser Asn Thr Gln
Cys Gln Val Val 1 5 10
15 Arg Cys His Leu Gly Gln Leu Ala Lys Gly Thr Glu Val Ser Val Gly
20 25 30 Leu Leu Arg
Leu Val His Asn Glu Phe Phe Arg Arg Ala Lys Phe Lys 35
40 45 Ser Leu Thr Val Val Ser Thr Phe
Glu Leu Gly Thr Glu Glu Gly Ser 50 55
60 Val Leu Gln Leu Thr Glu Ala Ser Arg Trp Ser Glu Ser
Val Leu Glu 65 70 75
80 Val Val Gln Thr Arg Pro Ile Leu Ile Ser Leu Trp Ile Leu Ile Gly
85 90 95 Ser Val Leu Gly
Gly Leu Leu Leu Leu Ala Leu Leu Val Phe Cys Leu 100
105 110 Trp Lys Leu Gly Phe Phe Ala His Lys
Lys Ile Pro Glu Glu Glu Lys 115 120
125 Arg Glu Glu Lys Leu Glu Gln 130 135
22136PRTHomo sapiens 22Arg Arg Ala Pro Gln Leu Asn His Ser Asn Ser Asp
Val Val Ser Ile 1 5 10
15 Asn Cys Asn Ile Arg Leu Val Pro Asn Gln Glu Ile Asn Phe His Leu
20 25 30 Leu Gly Asn
Leu Trp Leu Arg Ser Leu Lys Ala Leu Lys Tyr Lys Ser 35
40 45 Met Lys Ile Met Val Asn Ala Ala
Leu Gln Arg Gln Phe His Ser Pro 50 55
60 Phe Ile Phe Arg Glu Glu Asp Pro Ser Arg Gln Ile Val
Phe Glu Ile 65 70 75
80 Ser Lys Gln Glu Asp Trp Gln Val Pro Ile Trp Ile Ile Val Gly Ser
85 90 95 Thr Leu Gly Gly
Leu Leu Leu Leu Ala Leu Leu Val Leu Ala Leu Trp 100
105 110 Lys Leu Gly Phe Phe Arg Ser Ala Arg
Arg Arg Arg Glu Pro Gly Leu 115 120
125 Asp Pro Thr Pro Lys Val Leu Glu 130
135 23136PRTHomo sapiens 23Ser Ser Val Gln His Val Glu Glu Trp His
Ser Val Ser Cys Val Ile 1 5 10
15 Ala Ser Asp Lys Glu Asn Val Thr Val Ala Ala Glu Ile Ser Trp
Asp 20 25 30 His
Ser Glu Glu Leu Leu Lys Asp Val Thr Glu Leu Gln Ile Leu Gly 35
40 45 Glu Ile Ser Phe Asn Lys
Ser Leu Tyr Glu Gly Leu Asn Ala Glu Asn 50 55
60 His Arg Thr Lys Ile Val Val Val Phe Leu Lys
Asp Glu Lys Tyr His 65 70 75
80 Ser Leu Pro Ile Ile Ile Lys Gly Ser Val Gly Gly Leu Leu Val Leu
85 90 95 Ile Val
Ile Leu Val Ile Leu Phe Lys Cys Gly Phe Phe Lys Arg Lys 100
105 110 Tyr Gln Gln Leu Asn Leu Glu
Ser Ile Arg Lys Ala Gln Leu Lys Ser 115 120
125 Glu Asn Leu Leu Glu Glu Glu Asn 130
135 24134PRTMus musculus 24Asp Pro Val Gln His Val Lys Glu
Trp His Ser Val Val Cys Ala Ile 1 5 10
15 Thr Ser Asn Lys Glu Asn Val Thr Val Ala Ala Glu Ile
Ser Val Gly 20 25 30
His Thr Lys Gln Leu Leu Arg Asp Val Ser Glu Leu Pro Ile Leu Gly
35 40 45 Glu Ile Ser Phe
Asn Lys Ser Leu Tyr Glu Gly Leu Asn Ala Glu Asn 50
55 60 His Arg Thr Lys Ile Val Val Ile
Phe Leu Lys Glu Glu Glu Thr Arg 65 70
75 80 Ser Leu Pro Leu Ile Ile Gly Ser Ser Ile Gly Gly
Leu Leu Val Leu 85 90
95 Val Val Ile Ile Ala Ile Leu Phe Lys Cys Gly Phe Phe Lys Arg Lys
100 105 110 Tyr Gln Gln
Leu Asn Leu Glu Ser Thr Arg Arg Ala Gln Leu Lys Ala 115
120 125 Asp Ser Leu Leu Gln Asp 130
25152PRTHomo sapiens 25Ser Arg Ser Pro Met Leu Asp Cys
Ser Ile Ala Asp Cys Leu Gln Phe 1 5 10
15 Arg Cys Asp Val Pro Ser Phe Ser Val Gln Glu Glu Leu
Asp Phe Thr 20 25 30
Leu Lys Gly Asn Leu Ser Phe Gly Trp Val Arg Glu Thr Leu Gln Lys
35 40 45 Lys Val Leu Val
Val Ser Val Ala Glu Ile Thr Phe Asp Thr Ser Val 50
55 60 Tyr Ser Gln Leu Pro Gly Gln Glu
Ala Phe Met Arg Ala Val Met Glu 65 70
75 80 Met Val Leu Glu Glu Asp Glu Val Tyr Asn Ala Ile
Pro Ile Ile Met 85 90
95 Gly Ser Ser Val Gly Ala Leu Leu Leu Leu Ala Leu Ile Thr Ala Thr
100 105 110 Leu Tyr Lys
Leu Gly Phe Phe Lys Arg His Tyr Lys Glu Met Leu Glu 115
120 125 Asp Lys Pro Glu Asp Thr Ala Thr
Phe Ser Gly Asp Asp Phe Ser Cys 130 135
140 Val Ala Pro Asn Val Pro Leu Ser 145
150 26149PRTHomo sapiens 26Gln Lys Asn Pro Val Leu Asp Cys Ser
Ile Ala Gly Cys Leu Arg Phe 1 5 10
15 Arg Cys Asp Val Pro Ser Phe Ser Val Gln Glu Glu Leu Asp
Phe Thr 20 25 30
Leu Lys Gly Asn Leu Ser Phe Gly Trp Val Arg Gln Ile Leu Gln Lys
35 40 45 Lys Val Ser Val
Val Ser Val Ala Glu Ile Thr Phe Asp Thr Ser Val 50
55 60 Tyr Ser Gln Leu Pro Gly Gln Glu
Ala Phe Met Arg Ala Val Thr Thr 65 70
75 80 Thr Val Leu Glu Lys Tyr Lys Val His Asn Pro Thr
Pro Leu Ile Val 85 90
95 Gly Ser Ser Ile Gly Gly Leu Leu Leu Leu Ala Leu Ile Thr Ala Val
100 105 110 Leu Tyr Lys
Val Gly Phe Phe Lys Arg Gln Tyr Lys Glu Met Met Glu 115
120 125 Glu Ala Asn Gly Gln Ile Ala Pro
Glu Asn Gly Thr Gln Thr Pro Ser 130 135
140 Pro Pro Ser Glu Lys 145 27146PRTMus
musculus 27Gln Lys Ser Pro Val Leu Asp Cys Ser Ile Ala Asp Cys Leu His
Leu 1 5 10 15 Arg
Cys Asp Ile Pro Ser Leu Gly Ile Leu Asp Glu Leu Tyr Phe Ile
20 25 30 Leu Lys Gly Asn Leu
Ser Phe Gly Trp Ile Ser Gln Thr Leu Gln Lys 35
40 45 Lys Val Leu Leu Leu Ser Glu Ala Glu
Ile Thr Phe Asn Thr Ser Val 50 55
60 Tyr Ser Gln Leu Pro Gly Gln Glu Ala Phe Leu Arg Ala
Val Thr Lys 65 70 75
80 Thr Val Leu Glu Met Tyr Lys Val His Asn Pro Val Pro Leu Ile Val
85 90 95 Gly Ser Ser Val
Gly Gly Leu Leu Leu Leu Ala Ile Ile Thr Ala Ile 100
105 110 Leu Tyr Lys Ala Gly Phe Phe Lys Arg
Gln Tyr Lys Glu Met Leu Glu 115 120
125 Glu Ala Asn Gly Gln Phe Val Ser Asp Gly Thr Pro Thr Pro
Gln Val 130 135 140
Ala Gln 145 28138PRTHomo sapiens 28Arg Lys Ala Pro Val Val Asn Cys
Ser Ile Ala Val Cys Gln Arg Ile 1 5 10
15 Gln Cys Asp Ile Pro Phe Phe Gly Ile Gln Glu Glu Phe
Asn Ala Thr 20 25 30
Leu Lys Gly Asn Leu Ser Phe Asp Trp Tyr Ile Lys Thr Ser His Asn
35 40 45 His Leu Leu Ile
Val Ser Thr Ala Glu Ile Leu Phe Asn Asp Ser Val 50
55 60 Phe Thr Leu Leu Pro Gly Gln Gly
Ala Phe Val Arg Ser Val Thr Glu 65 70
75 80 Thr Lys Val Glu Pro Phe Glu Val Pro Asn Pro Leu
Pro Leu Ile Val 85 90
95 Gly Ser Ser Val Gly Gly Leu Leu Leu Leu Ala Leu Ile Thr Ala Ala
100 105 110 Leu Tyr Lys
Leu Gly Phe Phe Lys Arg Gln Tyr Lys Asp Met Met Ser 115
120 125 Glu Gly Gly Pro Pro Gly Ala Glu
Pro Gln 130 135 29138PRTMus musculus
29Glu Arg Thr Pro Val Leu Asn Cys Ser Val Ala Val Cys Lys Arg Ile 1
5 10 15 Gln Cys Asp Leu
Pro Ser Phe Asn Thr Gln Glu Ile Phe Asn Val Thr 20
25 30 Leu Lys Gly Asn Leu Ser Phe Asp Trp
Tyr Ile Lys Thr Ser His Gly 35 40
45 His Leu Leu Leu Val Ser Ser Thr Glu Ile Leu Phe Asn Asp
Ser Ala 50 55 60
Phe Ala Leu Leu Pro Gly Gln Glu Ser Tyr Val Arg Ser Val Thr Glu 65
70 75 80 Thr Lys Val Glu Pro
Tyr Glu Val His Asn Pro Val Pro Leu Ile Val 85
90 95 Gly Ser Ser Ile Gly Gly Leu Val Leu Leu
Ala Leu Ile Thr Ala Gly 100 105
110 Leu Tyr Lys Leu Gly Phe Phe Lys Arg Gln Tyr Lys Asp Met Met
Asn 115 120 125 Glu
Ala Ala Pro Gln Asp Ala Pro Pro Gln 130 135
30163PRTHomo sapiens 30Asp Ala Ala Glu Pro Cys Leu Pro Gly Ala Leu Phe
Arg Cys Pro Val 1 5 10
15 Val Phe Arg Gln Glu Ile Leu Val Gln Val Ile Gly Thr Leu Glu Leu
20 25 30 Val Gly Glu
Ile Glu Ala Ser Ser Met Phe Ser Leu Cys Ser Ser Leu 35
40 45 Ser Ile Ser Phe Asn Ser Ser Lys
His Phe His Leu Tyr Gly Ser Asn 50 55
60 Ala Ser Leu Ala Gln Val Val Met Lys Val Asp Val Val
Tyr Glu Lys 65 70 75
80 Gln Met Leu Tyr Leu Tyr Val Leu Ser Gly Ile Gly Gly Leu Leu Leu
85 90 95 Leu Leu Leu Ile
Phe Ile Val Leu Tyr Lys Val Gly Phe Phe Lys Arg 100
105 110 Asn Leu Lys Glu Lys Met Glu Ala Gly
Arg Gly Val Pro Asn Gly Ile 115 120
125 Pro Ala Glu Asp Ser Glu Gln Leu Ala Ser Gly Gln Glu Ala
Gly Asp 130 135 140
Pro Gly Cys Leu Lys Pro Leu His Glu Lys Asp Ser Glu Ser Gly Gly 145
150 155 160 Gly Lys Asp
31159PRTMus musculus 31Ser Glu Ala Glu Gln Pro Cys Leu Pro Gly Val Gln
Phe Arg Cys Pro 1 5 10
15 Ile Val Phe Arg Trp Glu Ile Leu Ile Gln Val Thr Gly Thr Val Glu
20 25 30 Leu Ser Lys
Glu Ile Lys Ala Ser Ser Thr Leu Ser Leu Cys Ser Ser 35
40 45 Leu Ser Val Ser Phe Asn Ser Ser
Lys His Phe His Leu Tyr Gly Ser 50 55
60 Lys Ala Ser Glu Ala Gln Val Leu Val Lys Val Asp Leu
Ile His Glu 65 70 75
80 Lys Glu Met Leu His Val Tyr Val Leu Ser Gly Ile Gly Gly Leu Val
85 90 95 Leu Leu Phe Leu
Ile Phe Leu Ala Leu Tyr Lys Val Gly Phe Phe Lys 100
105 110 Arg Asn Leu Lys Glu Lys Met Glu Ala
Asp Gly Gly Val Pro Asn Gly 115 120
125 Ser Pro Pro Glu Asp Thr Asp Pro Leu Ala Val Pro Gly Glu
Glu Thr 130 135 140
Lys Asp Met Gly Cys Leu Glu Pro Leu Arg Glu Ser Asp Lys Asp 145
150 155 32778PRTHomo sapiens 32Gln
Thr Asp Glu Asn Arg Cys Leu Lys Ala Asn Ala Lys Ser Cys Gly 1
5 10 15 Glu Cys Ile Gln Ala Gly
Pro Asn Cys Gly Trp Cys Thr Asn Ser Thr 20
25 30 Phe Leu Gln Glu Gly Met Pro Thr Ser Ala
Arg Cys Asp Asp Leu Glu 35 40
45 Ala Leu Lys Lys Lys Gly Cys Pro Pro Asp Asp Ile Glu Asn
Pro Arg 50 55 60
Gly Ser Lys Asp Ile Lys Lys Asn Lys Asn Val Thr Asn Arg Ser Lys 65
70 75 80 Gly Thr Ala Glu Lys
Leu Lys Pro Glu Asp Ile His Gln Ile Gln Pro 85
90 95 Gln Gln Leu Val Leu Arg Leu Arg Ser Gly
Glu Pro Gln Thr Phe Thr 100 105
110 Leu Lys Phe Lys Arg Ala Glu Asp Tyr Pro Ile Asp Leu Tyr Tyr
Leu 115 120 125 Met
Asp Leu Ser Tyr Ser Met Lys Asp Asp Leu Glu Asn Val Lys Ser 130
135 140 Leu Gly Thr Asp Leu Met
Asn Glu Met Arg Arg Ile Thr Ser Asp Phe 145 150
155 160 Arg Ile Gly Phe Gly Ser Phe Val Glu Lys Thr
Val Met Pro Tyr Ile 165 170
175 Ser Thr Thr Pro Ala Lys Leu Arg Asn Pro Cys Thr Ser Glu Gln Asn
180 185 190 Cys Thr
Thr Pro Phe Ser Tyr Lys Asn Val Leu Ser Leu Thr Asn Lys 195
200 205 Gly Glu Val Phe Asn Glu Leu
Val Gly Lys Gln Arg Ile Ser Gly Asn 210 215
220 Leu Asp Ser Pro Glu Gly Gly Phe Asp Ala Ile Met
Gln Val Ala Val 225 230 235
240 Cys Gly Ser Leu Ile Gly Trp Arg Asn Val Thr Arg Leu Leu Val Phe
245 250 255 Ser Thr Asp
Ala Gly Phe His Phe Ala Gly Asp Gly Lys Leu Gly Gly 260
265 270 Ile Val Leu Pro Asn Asp Gly Gln
Cys His Leu Glu Asn Asn Met Tyr 275 280
285 Thr Met Ser His Tyr Tyr Asp Tyr Pro Ser Ile Ala His
Leu Val Gln 290 295 300
Lys Leu Ser Glu Asn Asn Ile Gln Thr Ile Phe Ala Val Thr Glu Glu 305
310 315 320 Phe Gln Pro Val
Tyr Lys Glu Leu Lys Asn Leu Ile Pro Lys Ser Ala 325
330 335 Val Gly Thr Leu Ser Ala Asn Ser Ser
Asn Val Ile Gln Leu Ile Ile 340 345
350 Asp Ala Tyr Asn Ser Leu Ser Ser Glu Val Ile Leu Glu Asn
Gly Lys 355 360 365
Leu Ser Glu Gly Val Thr Ile Ser Tyr Lys Ser Tyr Cys Lys Asn Gly 370
375 380 Val Asn Gly Thr Gly
Glu Asn Gly Arg Lys Cys Ser Asn Ile Ser Ile 385 390
395 400 Gly Asp Glu Val Gln Phe Glu Ile Ser Ile
Thr Ser Asn Lys Cys Pro 405 410
415 Lys Lys Asp Ser Asp Ser Phe Lys Ile Arg Pro Leu Gly Phe Thr
Glu 420 425 430 Glu
Val Glu Val Ile Leu Gln Tyr Ile Cys Glu Cys Glu Cys Gln Ser 435
440 445 Glu Gly Ile Pro Glu Ser
Pro Lys Cys His Glu Gly Asn Gly Thr Phe 450 455
460 Glu Cys Gly Ala Cys Arg Cys Asn Glu Gly Arg
Val Gly Arg His Cys 465 470 475
480 Glu Cys Ser Thr Asp Glu Val Asn Ser Glu Asp Met Asp Ala Tyr Cys
485 490 495 Arg Lys
Glu Asn Ser Ser Glu Ile Cys Ser Asn Asn Gly Glu Cys Val 500
505 510 Cys Gly Gln Cys Val Cys Arg
Lys Arg Asp Asn Thr Asn Glu Ile Tyr 515 520
525 Ser Gly Lys Phe Cys Glu Cys Asp Asn Phe Asn Cys
Asp Arg Ser Asn 530 535 540
Gly Leu Ile Cys Gly Gly Asn Gly Val Cys Lys Cys Arg Val Cys Glu 545
550 555 560 Cys Asn Pro
Asn Tyr Thr Gly Ser Ala Cys Asp Cys Ser Leu Asp Thr 565
570 575 Ser Thr Cys Glu Ala Ser Asn Gly
Gln Ile Cys Asn Gly Arg Gly Ile 580 585
590 Cys Glu Cys Gly Val Cys Lys Cys Thr Asp Pro Lys Phe
Gln Gly Gln 595 600 605
Thr Cys Glu Met Cys Gln Thr Cys Leu Gly Val Cys Ala Glu His Lys 610
615 620 Glu Cys Val Gln
Cys Arg Ala Phe Asn Lys Gly Glu Lys Lys Asp Thr 625 630
635 640 Cys Thr Gln Glu Cys Ser Tyr Phe Asn
Ile Thr Lys Val Glu Ser Arg 645 650
655 Asp Lys Leu Pro Gln Pro Val Gln Pro Asp Pro Val Ser His
Cys Lys 660 665 670
Glu Lys Asp Val Asp Asp Cys Trp Phe Tyr Phe Thr Tyr Ser Val Asn
675 680 685 Gly Asn Asn Glu
Val Met Val His Val Val Glu Asn Pro Glu Cys Pro 690
695 700 Thr Gly Pro Asp Ile Ile Pro Ile
Val Ala Gly Val Val Ala Gly Ile 705 710
715 720 Val Leu Ile Gly Leu Ala Leu Leu Leu Ile Trp Lys
Leu Leu Met Ile 725 730
735 Ile His Asp Arg Arg Glu Phe Ala Lys Phe Glu Lys Glu Lys Met Asn
740 745 750 Ala Lys Trp
Asp Thr Gly Glu Asn Pro Ile Tyr Lys Ser Ala Val Thr 755
760 765 Thr Val Val Asn Pro Lys Tyr Glu
Gly Lys 770 775 33778PRTMus musculus
33Gln Thr Asp Lys Asn Arg Cys Leu Lys Ala Asn Ala Lys Ser Cys Gly 1
5 10 15 Glu Cys Ile Gln
Ala Gly Pro Asn Cys Gly Trp Cys Thr Asn Thr Thr 20
25 30 Phe Leu Gln Glu Gly Met Pro Thr Ser
Ala Arg Cys Asp Asp Leu Glu 35 40
45 Ala Leu Lys Lys Lys Gly Cys Gln Pro Ser Asp Ile Glu Asn
Pro Arg 50 55 60
Gly Ser Gln Thr Ile Lys Lys Asn Lys Asn Val Thr Asn Arg Ser Lys 65
70 75 80 Gly Met Ala Glu Lys
Leu Arg Pro Glu Asp Ile Thr Gln Ile Gln Pro 85
90 95 Gln Gln Leu Leu Leu Lys Leu Arg Ser Gly
Glu Pro Gln Lys Phe Thr 100 105
110 Leu Lys Phe Lys Arg Ala Glu Asp Tyr Pro Ile Asp Leu Tyr Tyr
Leu 115 120 125 Met
Asp Leu Ser Tyr Ser Met Lys Asp Asp Leu Glu Asn Val Lys Ser 130
135 140 Leu Gly Thr Asp Leu Met
Asn Glu Met Arg Arg Ile Thr Ser Asp Phe 145 150
155 160 Arg Ile Gly Phe Gly Ser Phe Val Glu Lys Thr
Val Met Pro Tyr Ile 165 170
175 Ser Thr Thr Pro Ala Lys Leu Arg Asn Pro Cys Thr Ser Glu Gln Asn
180 185 190 Cys Thr
Ser Pro Phe Ser Tyr Lys Asn Val Leu Ser Leu Thr Asp Arg 195
200 205 Gly Glu Phe Phe Asn Glu Leu
Val Gly Gln Gln Arg Ile Ser Gly Asn 210 215
220 Leu Asp Ser Pro Glu Gly Gly Phe Asp Ala Ile Met
Gln Val Ala Val 225 230 235
240 Cys Gly Ser Leu Ile Gly Trp Arg Asn Val Thr Arg Leu Leu Val Phe
245 250 255 Ser Thr Asp
Ala Gly Phe His Phe Ala Gly Asp Gly Lys Leu Gly Gly 260
265 270 Ile Val Leu Pro Asn Asp Gly Gln
Cys His Leu Glu Asn Asn Val Tyr 275 280
285 Thr Met Ser His Tyr Tyr Asp Tyr Pro Ser Ile Ala His
Leu Val Gln 290 295 300
Lys Leu Ser Glu Asn Asn Ile Gln Thr Ile Phe Ala Val Thr Glu Glu 305
310 315 320 Phe Gln Pro Val
Tyr Lys Glu Leu Lys Asn Leu Ile Pro Lys Ser Ala 325
330 335 Val Gly Thr Leu Ser Gly Asn Ser Ser
Asn Val Ile Gln Leu Ile Ile 340 345
350 Asp Ala Tyr Asn Ser Leu Ser Ser Glu Val Ile Leu Glu Asn
Ser Lys 355 360 365
Leu Pro Asp Gly Val Thr Ile Asn Tyr Lys Ser Tyr Cys Lys Asn Gly 370
375 380 Val Asn Gly Thr Gly
Glu Asn Gly Arg Lys Cys Ser Asn Ile Ser Ile 385 390
395 400 Gly Asp Glu Val Gln Phe Glu Ile Ser Ile
Thr Ala Asn Lys Cys Pro 405 410
415 Asn Lys Glu Ser Glu Thr Ile Lys Ile Lys Pro Leu Gly Phe Thr
Glu 420 425 430 Glu
Val Glu Val Val Leu Gln Phe Ile Cys Lys Cys Asn Cys Gln Ser 435
440 445 His Gly Ile Pro Ala Ser
Pro Lys Cys His Glu Gly Asn Gly Thr Phe 450 455
460 Glu Cys Gly Ala Cys Arg Cys Asn Glu Gly Arg
Val Gly Arg His Cys 465 470 475
480 Glu Cys Ser Thr Asp Glu Val Asn Ser Glu Asp Met Asp Ala Tyr Cys
485 490 495 Arg Lys
Glu Asn Ser Ser Glu Ile Cys Ser Asn Asn Gly Glu Cys Val 500
505 510 Cys Gly Gln Cys Val Cys Arg
Lys Arg Asp Asn Thr Asn Glu Ile Tyr 515 520
525 Ser Gly Lys Phe Cys Glu Cys Asp Asn Phe Asn Cys
Asp Arg Ser Asn 530 535 540
Gly Leu Ile Cys Gly Gly Asn Gly Val Cys Arg Cys Arg Val Cys Glu 545
550 555 560 Cys Tyr Pro
Asn Tyr Thr Gly Ser Ala Cys Asp Cys Ser Leu Asp Thr 565
570 575 Gly Pro Cys Leu Ala Ser Asn Gly
Gln Ile Cys Asn Gly Arg Gly Ile 580 585
590 Cys Glu Cys Gly Ala Cys Lys Cys Thr Asp Pro Lys Phe
Gln Gly Pro 595 600 605
Thr Cys Glu Thr Cys Gln Thr Cys Leu Gly Val Cys Ala Glu His Lys 610
615 620 Glu Cys Val Gln
Cys Arg Ala Phe Asn Lys Gly Glu Lys Lys Asp Thr 625 630
635 640 Cys Ala Gln Glu Cys Ser His Phe Asn
Leu Thr Lys Val Glu Ser Arg 645 650
655 Glu Lys Leu Pro Gln Pro Val Gln Val Asp Pro Val Thr His
Cys Lys 660 665 670
Glu Lys Asp Ile Asp Asp Cys Trp Phe Tyr Phe Thr Tyr Ser Val Asn
675 680 685 Gly Asn Asn Glu
Ala Ile Val His Val Val Glu Thr Pro Asp Cys Pro 690
695 700 Thr Gly Pro Asp Ile Ile Pro Ile
Val Ala Gly Val Val Ala Gly Ile 705 710
715 720 Val Leu Ile Gly Leu Ala Leu Leu Leu Ile Trp Lys
Leu Leu Met Ile 725 730
735 Ile His Asp Arg Arg Glu Phe Ala Lys Phe Glu Lys Glu Lys Met Asn
740 745 750 Ala Lys Trp
Asp Thr Gly Glu Asn Pro Ile Tyr Lys Ser Ala Val Thr 755
760 765 Thr Val Val Asn Pro Lys Tyr Glu
Gly Lys 770 775 34747PRTHomo sapiens
34Gln Glu Cys Thr Lys Phe Lys Val Ser Ser Cys Arg Glu Cys Ile Glu 1
5 10 15 Ser Gly Pro Gly
Cys Thr Trp Cys Gln Lys Leu Asn Phe Thr Gly Pro 20
25 30 Gly Asp Pro Asp Ser Ile Arg Cys Asp
Thr Arg Pro Gln Leu Leu Met 35 40
45 Arg Gly Cys Ala Ala Asp Asp Ile Met Asp Pro Thr Ser Leu
Ala Glu 50 55 60
Thr Gln Glu Asp His Asn Gly Gly Gln Lys Gln Leu Ser Pro Gln Lys 65
70 75 80 Val Thr Leu Tyr Leu
Arg Pro Gly Gln Ala Ala Ala Phe Asn Val Thr 85
90 95 Phe Arg Arg Ala Lys Gly Tyr Pro Ile Asp
Leu Tyr Tyr Leu Met Asp 100 105
110 Leu Ser Tyr Ser Met Leu Asp Asp Leu Arg Asn Val Lys Lys Leu
Gly 115 120 125 Gly
Asp Leu Leu Arg Ala Leu Asn Glu Ile Thr Glu Ser Gly Arg Ile 130
135 140 Gly Phe Gly Ser Phe Val
Asp Lys Thr Val Leu Pro Phe Val Asn Thr 145 150
155 160 His Pro Asp Lys Leu Arg Asn Pro Cys Pro Asn
Lys Glu Lys Glu Cys 165 170
175 Gln Pro Pro Phe Ala Phe Arg His Val Leu Lys Leu Thr Asn Asn Ser
180 185 190 Asn Gln
Phe Gln Thr Glu Val Gly Lys Gln Leu Ile Ser Gly Asn Leu 195
200 205 Asp Ala Pro Glu Gly Gly Leu
Asp Ala Met Met Gln Val Ala Ala Cys 210 215
220 Pro Glu Glu Ile Gly Trp Arg Asn Val Thr Arg Leu
Leu Val Phe Ala 225 230 235
240 Thr Asp Asp Gly Phe His Phe Ala Gly Asp Gly Lys Leu Gly Ala Ile
245 250 255 Leu Thr Pro
Asn Asp Gly Arg Cys His Leu Glu Asp Asn Leu Tyr Lys 260
265 270 Arg Ser Asn Glu Phe Asp Tyr Pro
Ser Val Gly Gln Leu Ala His Lys 275 280
285 Leu Ala Glu Asn Asn Ile Gln Pro Ile Phe Ala Val Thr
Ser Arg Met 290 295 300
Val Lys Thr Tyr Glu Lys Leu Thr Glu Ile Ile Pro Lys Ser Ala Val 305
310 315 320 Gly Glu Leu Ser
Glu Asp Ser Ser Asn Val Val His Leu Ile Lys Asn 325
330 335 Ala Tyr Asn Lys Leu Ser Ser Arg Val
Phe Leu Asp His Asn Ala Leu 340 345
350 Pro Asp Thr Leu Lys Val Thr Tyr Asp Ser Phe Cys Ser Asn
Gly Val 355 360 365
Thr His Arg Asn Gln Pro Arg Gly Asp Cys Asp Gly Val Gln Ile Asn 370
375 380 Val Pro Ile Thr Phe
Gln Val Lys Val Thr Ala Thr Glu Cys Ile Gln 385 390
395 400 Glu Gln Ser Phe Val Ile Arg Ala Leu Gly
Phe Thr Asp Ile Val Thr 405 410
415 Val Gln Val Leu Pro Gln Cys Glu Cys Arg Cys Arg Asp Gln Ser
Arg 420 425 430 Asp
Arg Ser Leu Cys His Gly Lys Gly Phe Leu Glu Cys Gly Ile Cys 435
440 445 Arg Cys Asp Thr Gly Tyr
Ile Gly Lys Asn Cys Glu Cys Gln Thr Gln 450 455
460 Gly Arg Ser Ser Gln Glu Leu Glu Gly Ser Cys
Arg Lys Asp Asn Asn 465 470 475
480 Ser Ile Ile Cys Ser Gly Leu Gly Asp Cys Val Cys Gly Gln Cys Leu
485 490 495 Cys His
Thr Ser Asp Val Pro Gly Lys Leu Ile Tyr Gly Gln Tyr Cys 500
505 510 Glu Cys Asp Thr Ile Asn Cys
Glu Arg Tyr Asn Gly Gln Val Cys Gly 515 520
525 Gly Pro Gly Arg Gly Leu Cys Phe Cys Gly Lys Cys
Arg Cys His Pro 530 535 540
Gly Phe Glu Gly Ser Ala Cys Gln Cys Glu Arg Thr Thr Glu Gly Cys 545
550 555 560 Leu Asn Pro
Arg Arg Val Glu Cys Ser Gly Arg Gly Arg Cys Arg Cys 565
570 575 Asn Val Cys Glu Cys His Ser Gly
Tyr Gln Leu Pro Leu Cys Gln Glu 580 585
590 Cys Pro Gly Cys Pro Ser Pro Cys Gly Lys Tyr Ile Ser
Cys Ala Glu 595 600 605
Cys Leu Lys Phe Glu Lys Gly Pro Phe Gly Lys Asn Cys Ser Ala Ala 610
615 620 Cys Pro Gly Leu
Gln Leu Ser Asn Asn Pro Val Lys Gly Arg Thr Cys 625 630
635 640 Lys Glu Arg Asp Ser Glu Gly Cys Trp
Val Ala Tyr Thr Leu Glu Gln 645 650
655 Gln Asp Gly Met Asp Arg Tyr Leu Ile Tyr Val Asp Glu Ser
Arg Glu 660 665 670
Cys Val Ala Gly Pro Asn Ile Ala Ala Ile Val Gly Gly Thr Val Ala
675 680 685 Gly Ile Val Leu
Ile Gly Ile Leu Leu Leu Val Ile Trp Lys Ala Leu 690
695 700 Ile His Leu Ser Asp Leu Arg Glu
Tyr Arg Arg Phe Glu Lys Glu Lys 705 710
715 720 Leu Lys Ser Gln Trp Asn Asn Asp Asn Pro Leu Phe
Lys Ser Ala Thr 725 730
735 Thr Thr Val Met Asn Pro Lys Phe Ala Glu Ser 740
745 35748PRTMus musculus 35Gln Glu Cys Thr Lys Tyr Lys
Val Ser Ser Cys Arg Asp Cys Ile Gln 1 5
10 15 Ser Gly Pro Gly Cys Ser Trp Cys Gln Lys Leu
Asn Phe Thr Gly Pro 20 25
30 Gly Glu Pro Asp Ser Leu Arg Cys Asp Thr Arg Ala Gln Leu Leu
Leu 35 40 45 Lys
Gly Cys Pro Ala Asp Asp Ile Met Asp Pro Arg Ser Ile Ala Asn 50
55 60 Pro Glu Phe Asp Gln Arg
Gly Gln Arg Lys Gln Leu Ser Pro Gln Lys 65 70
75 80 Val Thr Leu Tyr Leu Arg Pro Gly Gln Ala Ala
Ala Phe Asn Val Thr 85 90
95 Phe Arg Arg Ala Lys Gly Tyr Pro Ile Asp Leu Tyr Tyr Leu Met Asp
100 105 110 Leu Ser
Tyr Ser Met Leu Asp Asp Leu Asn Asn Val Lys Lys Leu Gly 115
120 125 Gly Asp Leu Leu Gln Ala Leu
Asn Glu Ile Thr Glu Ser Gly Arg Ile 130 135
140 Gly Phe Gly Ser Phe Val Asp Lys Thr Val Leu Pro
Phe Val Asn Thr 145 150 155
160 His Pro Glu Lys Leu Arg Asn Pro Cys Pro Asn Lys Glu Lys Ala Cys
165 170 175 Gln Pro Pro
Phe Ala Phe Arg His Val Leu Lys Leu Thr Asp Asn Ser 180
185 190 Asn Gln Phe Gln Thr Glu Val Gly
Lys Gln Leu Ile Ser Gly Asn Leu 195 200
205 Asp Ala Pro Glu Gly Gly Leu Asp Ala Ile Met Gln Val
Ala Ala Cys 210 215 220
Pro Glu Glu Ile Gly Trp Arg Asn Val Thr Arg Leu Leu Val Phe Ala 225
230 235 240 Thr Asp Asp Gly
Phe His Phe Ala Gly Asp Gly Lys Leu Gly Ala Ile 245
250 255 Leu Thr Pro Asn Asp Gly Arg Cys His
Leu Glu Asp Asn Met Tyr Lys 260 265
270 Arg Ser Asn Glu Phe Asp Tyr Pro Ser Val Gly Gln Leu Ala
His Lys 275 280 285
Leu Ser Glu Ser Asn Ile Gln Pro Ile Phe Ala Val Thr Lys Lys Met 290
295 300 Val Lys Thr Tyr Glu
Lys Leu Thr Glu Ile Ile Pro Lys Ser Ala Val 305 310
315 320 Gly Glu Leu Ser Asp Asp Ser Ser Asn Val
Val Gln Leu Ile Lys Asn 325 330
335 Ala Tyr Tyr Lys Leu Ser Ser Arg Val Phe Leu Asp His Ser Thr
Leu 340 345 350 Pro
Asp Thr Leu Lys Val Thr Tyr Asp Ser Phe Cys Ser Asn Gly Ala 355
360 365 Ser Ser Ile Gly Lys Ser
Arg Gly Asp Cys Asp Gly Val Gln Ile Asn 370 375
380 Asn Pro Val Thr Phe Gln Val Lys Val Met Ala
Ser Glu Cys Ile Gln 385 390 395
400 Glu Gln Ser Phe Val Ile Arg Ala Leu Gly Phe Thr Asp Thr Val Thr
405 410 415 Val Gln
Val Arg Pro Gln Cys Glu Cys Gln Cys Arg Asp Gln Ser Arg 420
425 430 Glu Gln Ser Leu Cys Gly Gly
Lys Gly Val Met Glu Cys Gly Ile Cys 435 440
445 Arg Cys Glu Ser Gly Tyr Ile Gly Lys Asn Cys Glu
Cys Gln Thr Gln 450 455 460
Gly Arg Ser Ser Gln Glu Leu Glu Arg Asn Cys Arg Lys Asp Asn Ser 465
470 475 480 Ser Ile Val
Cys Ser Gly Leu Gly Asp Cys Ile Cys Gly Gln Cys Val 485
490 495 Cys His Thr Ser Asp Val Pro Asn
Lys Glu Ile Phe Gly Gln Tyr Cys 500 505
510 Glu Cys Asp Asn Val Asn Cys Glu Arg Tyr Asn Ser Gln
Val Cys Gly 515 520 525
Gly Ser Asp Arg Gly Ser Cys Asn Cys Gly Lys Cys Ser Cys Lys Pro 530
535 540 Gly Tyr Glu Gly
Ser Ala Cys Gln Cys Gln Arg Ser Thr Thr Gly Cys 545 550
555 560 Leu Asn Ala Arg Leu Val Glu Cys Ser
Gly Arg Gly His Cys Gln Cys 565 570
575 Asn Arg Cys Ile Cys Asp Glu Gly Tyr Gln Pro Pro Met Cys
Glu Asp 580 585 590
Cys Pro Ser Cys Gly Ser His Cys Arg Asp Asn His Thr Ser Cys Ala
595 600 605 Glu Cys Leu Lys
Phe Asp Lys Gly Pro Phe Glu Lys Asn Cys Ser Val 610
615 620 Gln Cys Ala Gly Met Thr Leu Gln
Thr Ile Pro Leu Lys Lys Lys Pro 625 630
635 640 Cys Lys Glu Arg Asp Ser Glu Gly Cys Trp Ile Thr
Tyr Thr Leu Gln 645 650
655 Gln Lys Asp Gly Arg Asn Ile Tyr Asn Ile His Val Glu Asp Ser Leu
660 665 670 Glu Cys Val
Lys Gly Pro Asn Val Ala Ala Ile Val Gly Gly Thr Val 675
680 685 Val Gly Val Val Leu Ile Gly Val
Leu Leu Leu Val Ile Trp Lys Ala 690 695
700 Leu Thr His Leu Thr Asp Leu Arg Glu Tyr Arg Arg Phe
Glu Lys Glu 705 710 715
720 Lys Leu Lys Ser Gln Trp Asn Asn Asp Asn Pro Leu Phe Lys Ser Ala
725 730 735 Thr Thr Thr Val
Met Asn Pro Lys Phe Ala Glu Ser 740 745
36762PRTHomo sapiens 36Gly Pro Asn Ile Cys Thr Thr Arg Gly Val Ser
Ser Cys Gln Gln Cys 1 5 10
15 Leu Ala Val Ser Pro Met Cys Ala Trp Cys Ser Asp Glu Ala Leu Pro
20 25 30 Leu Gly
Ser Pro Arg Cys Asp Leu Lys Glu Asn Leu Leu Lys Asp Asn 35
40 45 Cys Ala Pro Glu Ser Ile Glu
Phe Pro Val Ser Glu Ala Arg Val Leu 50 55
60 Glu Asp Arg Pro Leu Ser Asp Lys Gly Ser Gly Asp
Ser Ser Gln Val 65 70 75
80 Thr Gln Val Ser Pro Gln Arg Ile Ala Leu Arg Leu Arg Pro Asp Asp
85 90 95 Ser Lys Asn
Phe Ser Ile Gln Val Arg Gln Val Glu Asp Tyr Pro Val 100
105 110 Asp Ile Tyr Tyr Leu Met Asp Leu
Ser Tyr Ser Met Lys Asp Asp Leu 115 120
125 Trp Ser Ile Gln Asn Leu Gly Thr Lys Leu Ala Thr Gln
Met Arg Lys 130 135 140
Leu Thr Ser Asn Leu Arg Ile Gly Phe Gly Ala Phe Val Asp Lys Pro 145
150 155 160 Val Ser Pro Tyr
Met Tyr Ile Ser Pro Pro Glu Ala Leu Glu Asn Pro 165
170 175 Cys Tyr Asp Met Lys Thr Thr Cys Leu
Pro Met Phe Gly Tyr Lys His 180 185
190 Val Leu Thr Leu Thr Asp Gln Val Thr Arg Phe Asn Glu Glu
Val Lys 195 200 205
Lys Gln Ser Val Ser Arg Asn Arg Asp Ala Pro Glu Gly Gly Phe Asp 210
215 220 Ala Ile Met Gln Ala
Thr Val Cys Asp Glu Lys Ile Gly Trp Arg Asn 225 230
235 240 Asp Ala Ser His Leu Leu Val Phe Thr Thr
Asp Ala Lys Thr His Ile 245 250
255 Ala Leu Asp Gly Arg Leu Ala Gly Ile Val Gln Pro Asn Asp Gly
Gln 260 265 270 Cys
His Val Gly Ser Asp Asn His Tyr Ser Ala Ser Thr Thr Met Asp 275
280 285 Tyr Pro Ser Leu Gly Leu
Met Thr Glu Lys Leu Ser Gln Lys Asn Ile 290 295
300 Asn Leu Ile Phe Ala Val Thr Glu Asn Val Val
Asn Leu Tyr Gln Asn 305 310 315
320 Tyr Ser Glu Leu Ile Pro Gly Thr Thr Val Gly Val Leu Ser Met Asp
325 330 335 Ser Ser
Asn Val Leu Gln Leu Ile Val Asp Ala Tyr Gly Lys Ile Arg 340
345 350 Ser Lys Val Glu Leu Glu Val
Arg Asp Leu Pro Glu Glu Leu Ser Leu 355 360
365 Ser Phe Asn Ala Thr Cys Leu Asn Asn Glu Val Ile
Pro Gly Leu Lys 370 375 380
Ser Cys Met Gly Leu Lys Ile Gly Asp Thr Val Ser Phe Ser Ile Glu 385
390 395 400 Ala Lys Val
Arg Gly Cys Pro Gln Glu Lys Glu Lys Ser Phe Thr Ile 405
410 415 Lys Pro Val Gly Phe Lys Asp Ser
Leu Ile Val Gln Val Thr Phe Asp 420 425
430 Cys Asp Cys Ala Cys Gln Ala Gln Ala Glu Pro Asn Ser
His Arg Cys 435 440 445
Asn Asn Gly Asn Gly Thr Phe Glu Cys Gly Val Cys Arg Cys Gly Pro 450
455 460 Gly Trp Leu Gly
Ser Gln Cys Glu Cys Ser Glu Glu Asp Tyr Arg Pro 465 470
475 480 Ser Gln Gln Asp Glu Cys Ser Pro Arg
Glu Gly Gln Pro Val Cys Ser 485 490
495 Gln Arg Gly Glu Cys Leu Cys Gly Gln Cys Val Cys His Ser
Ser Asp 500 505 510
Phe Gly Lys Ile Thr Gly Lys Tyr Cys Glu Cys Asp Asp Phe Ser Cys
515 520 525 Val Arg Tyr Lys
Gly Glu Met Cys Ser Gly His Gly Gln Cys Ser Cys 530
535 540 Gly Asp Cys Leu Cys Asp Ser Asp
Trp Thr Gly Tyr Tyr Cys Asn Cys 545 550
555 560 Thr Thr Arg Thr Asp Thr Cys Met Ser Ser Asn Gly
Leu Leu Cys Ser 565 570
575 Gly Arg Gly Lys Cys Glu Cys Gly Ser Cys Val Cys Ile Gln Pro Gly
580 585 590 Ser Tyr Gly
Asp Thr Cys Glu Lys Cys Pro Thr Cys Pro Asp Ala Cys 595
600 605 Thr Phe Lys Lys Glu Cys Val Glu
Cys Lys Lys Phe Asp Arg Gly Ala 610 615
620 Leu His Asp Glu Asn Thr Cys Asn Arg Tyr Cys Arg Asp
Glu Ile Glu 625 630 635
640 Ser Val Lys Glu Leu Lys Asp Thr Gly Lys Asp Ala Val Asn Cys Thr
645 650 655 Tyr Lys Asn Glu
Asp Asp Cys Val Val Arg Phe Gln Tyr Tyr Glu Asp 660
665 670 Ser Ser Gly Lys Ser Ile Leu Tyr Val
Val Glu Glu Pro Glu Cys Pro 675 680
685 Lys Gly Pro Asp Ile Leu Val Val Leu Leu Ser Val Met Gly
Ala Ile 690 695 700
Leu Leu Ile Gly Leu Ala Ala Leu Leu Ile Trp Lys Leu Leu Ile Thr 705
710 715 720 Ile His Asp Arg Lys
Glu Phe Ala Lys Phe Glu Glu Glu Arg Ala Arg 725
730 735 Ala Lys Trp Asp Thr Ala Asn Asn Pro Leu
Tyr Lys Glu Ala Thr Ser 740 745
750 Thr Phe Thr Asn Ile Thr Tyr Arg Gly Thr 755
760 37762PRTMus musculus 37Glu Ser Asn Ile Cys Thr Thr
Arg Gly Val Asn Ser Cys Gln Gln Cys 1 5
10 15 Leu Ala Val Ser Pro Val Cys Ala Trp Cys Ser
Asp Glu Thr Leu Ser 20 25
30 Gln Gly Ser Pro Arg Cys Asn Leu Lys Glu Asn Leu Leu Lys Asp
Asn 35 40 45 Cys
Ala Pro Glu Ser Ile Glu Phe Pro Val Ser Glu Ala Gln Ile Leu 50
55 60 Glu Ala Arg Pro Leu Ser
Ser Lys Gly Ser Gly Ser Ser Ala Gln Ile 65 70
75 80 Thr Gln Val Ser Pro Gln Arg Ile Val Leu Arg
Leu Arg Pro Asp Asp 85 90
95 Ser Lys Ile Phe Ser Leu Gln Val Arg Gln Val Glu Asp Tyr Pro Val
100 105 110 Asp Ile
Tyr Tyr Leu Met Asp Leu Ser Phe Ser Met Lys Asp Asp Leu 115
120 125 Ser Ser Ile Gln Thr Leu Gly
Thr Lys Leu Ala Ser Gln Met Arg Lys 130 135
140 Leu Thr Ser Asn Leu Arg Ile Gly Phe Gly Ala Phe
Val Asp Lys Pro 145 150 155
160 Val Ser Pro Tyr Met Tyr Ile Ser Pro Pro Gln Ala Ile Lys Asn Pro
165 170 175 Cys Tyr Asn
Met Lys Asn Ala Cys Leu Pro Met Phe Gly Tyr Lys His 180
185 190 Val Leu Thr Leu Thr Asp Gln Val
Ser Arg Phe Asn Glu Glu Val Lys 195 200
205 Lys Gln Ser Val Ser Arg Asn Arg Asp Ala Pro Glu Gly
Gly Phe Asp 210 215 220
Ala Ile Met Gln Ala Thr Val Cys Asp Glu Lys Ile Gly Trp Arg Asn 225
230 235 240 Asp Ala Ser His
Leu Leu Val Phe Thr Thr Asp Ala Lys Thr His Ile 245
250 255 Ala Leu Asp Gly Arg Leu Ala Gly Ile
Val Leu Pro Asn Asp Gly His 260 265
270 Cys His Ile Gly Thr Asp Asn His Tyr Ser Ala Ser Thr Thr
Met Asp 275 280 285
Tyr Pro Ser Leu Gly Leu Met Thr Glu Lys Leu Ser Gln Lys Asn Ile 290
295 300 Asn Leu Ile Phe Ala
Val Thr Glu Asn Val Val Ser Leu Tyr Gln Asn 305 310
315 320 Tyr Ser Glu Leu Ile Pro Gly Thr Thr Val
Gly Val Leu Ser Asp Asp 325 330
335 Ser Ser Asn Val Leu Gln Leu Ile Val Asp Ala Tyr Gly Lys Ile
Arg 340 345 350 Ser
Lys Val Glu Leu Glu Val Arg Asp Leu Pro Gly Glu Leu Ser Leu 355
360 365 Ser Phe Asn Ala Thr Cys
Leu Asn Asn Glu Val Ile Pro Gly Leu Lys 370 375
380 Ser Cys Val Gly Leu Lys Ile Gly Asp Thr Val
Ser Phe Ser Ile Glu 385 390 395
400 Ala Lys Val Arg Gly Cys Pro Gln Glu Lys Glu Gln Ser Phe Thr Ile
405 410 415 Lys Pro
Val Gly Phe Lys Asp Ser Leu Thr Val Gln Val Thr Phe Asp 420
425 430 Cys Asp Cys Ala Cys Gln Ala
Phe Ala Gln Pro Ser Ser Pro Arg Cys 435 440
445 Asn Asn Gly Asn Gly Thr Phe Glu Cys Gly Val Cys
Arg Cys Asp Gln 450 455 460
Gly Trp Leu Gly Ser Met Cys Glu Cys Ser Glu Glu Asp Tyr Arg Pro 465
470 475 480 Ser Gln Gln
Glu Glu Cys Ser Pro Lys Glu Gly Gln Pro Ile Cys Ser 485
490 495 Gln Arg Gly Glu Cys Leu Cys Gly
Gln Cys Val Cys His Ser Ser Asp 500 505
510 Phe Gly Lys Ile Thr Gly Lys Tyr Cys Glu Cys Asp Asp
Phe Ser Cys 515 520 525
Val Arg Tyr Lys Gly Glu Met Cys Ser Gly His Gly Gln Cys Asn Cys 530
535 540 Gly Asp Cys Val
Cys Asp Ser Asp Trp Thr Gly Tyr Tyr Cys Asn Cys 545 550
555 560 Thr Thr Arg Thr Asp Thr Cys Met Ser
Thr Asn Gly Leu Leu Cys Ser 565 570
575 Gly Arg Gly Asn Cys Glu Cys Gly Ser Cys Val Cys Val Gln
Pro Gly 580 585 590
Ser Tyr Gly Asp Thr Cys Glu Lys Cys Pro Thr Cys Pro Asp Ala Cys
595 600 605 Ser Phe Lys Lys
Glu Cys Val Glu Cys Lys Lys Phe Asn Arg Gly Thr 610
615 620 Leu His Glu Glu Asn Thr Cys Ser
Arg Tyr Cys Arg Asp Asp Ile Glu 625 630
635 640 Gln Val Lys Glu Leu Thr Asp Thr Gly Lys Asn Ala
Val Asn Cys Thr 645 650
655 Tyr Lys Asn Glu Asp Asp Cys Val Val Arg Phe Gln Tyr Tyr Glu Asp
660 665 670 Thr Ser Gly
Arg Ala Val Leu Tyr Val Val Glu Glu Pro Glu Cys Pro 675
680 685 Lys Gly Pro Asp Ile Leu Val Val
Leu Leu Ser Val Met Gly Ala Ile 690 695
700 Leu Leu Ile Gly Leu Ala Thr Leu Leu Ile Trp Lys Leu
Leu Ile Thr 705 710 715
720 Ile His Asp Arg Lys Glu Phe Ala Lys Phe Glu Glu Glu Arg Ala Arg
725 730 735 Ala Lys Trp Asp
Thr Ala Asn Asn Pro Leu Tyr Lys Glu Ala Thr Ser 740
745 750 Thr Phe Thr Asn Ile Thr Tyr Arg Gly
Thr 755 760 38772PRTHomo sapiens 38Asn
Arg Cys Lys Lys Ala Pro Val Lys Ser Cys Thr Glu Cys Val Arg 1
5 10 15 Val Asp Lys Asp Cys Ala
Tyr Cys Thr Asp Glu Met Phe Arg Asp Arg 20
25 30 Arg Cys Asn Thr Gln Ala Glu Leu Leu Ala
Ala Gly Cys Gln Arg Glu 35 40
45 Ser Ile Val Val Met Glu Ser Ser Phe Gln Ile Thr Glu Glu
Thr Gln 50 55 60
Ile Asp Thr Thr Leu Arg Arg Ser Gln Met Ser Pro Gln Gly Leu Arg 65
70 75 80 Val Arg Leu Arg Pro
Gly Glu Glu Arg His Phe Glu Leu Glu Val Phe 85
90 95 Glu Pro Leu Glu Ser Pro Val Asp Leu Tyr
Ile Leu Met Asp Phe Ser 100 105
110 Asn Ser Met Ser Asp Asp Leu Asp Asn Leu Lys Lys Met Gly Gln
Asn 115 120 125 Leu
Ala Arg Val Leu Ser Gln Leu Thr Ser Asp Tyr Thr Ile Gly Phe 130
135 140 Gly Lys Phe Val Asp Lys
Val Ser Val Pro Gln Thr Asp Met Arg Pro 145 150
155 160 Glu Lys Leu Lys Glu Pro Trp Pro Asn Ser Asp
Pro Pro Phe Ser Phe 165 170
175 Lys Asn Val Ile Ser Leu Thr Glu Asp Val Asp Glu Phe Arg Asn Lys
180 185 190 Leu Gln
Gly Glu Arg Ile Ser Gly Asn Leu Asp Ala Pro Glu Gly Gly 195
200 205 Phe Asp Ala Ile Leu Gln Thr
Ala Val Cys Thr Arg Asp Ile Gly Trp 210 215
220 Arg Pro Asp Ser Thr His Leu Leu Val Phe Ser Thr
Glu Ser Ala Phe 225 230 235
240 His Tyr Glu Ala Asp Gly Ala Asn Val Leu Ala Gly Ile Met Ser Arg
245 250 255 Asn Asp Glu
Arg Cys His Leu Asp Thr Thr Gly Thr Tyr Thr Gln Tyr 260
265 270 Arg Thr Gln Asp Tyr Pro Ser Val
Pro Thr Leu Val Arg Leu Leu Ala 275 280
285 Lys His Asn Ile Ile Pro Ile Phe Ala Val Thr Asn Tyr
Ser Tyr Ser 290 295 300
Tyr Tyr Glu Lys Leu His Thr Tyr Phe Pro Val Ser Ser Leu Gly Val 305
310 315 320 Leu Gln Glu Asp
Ser Ser Asn Ile Val Glu Leu Leu Glu Glu Ala Phe 325
330 335 Asn Arg Ile Arg Ser Asn Leu Asp Ile
Arg Ala Leu Asp Ser Pro Arg 340 345
350 Gly Leu Arg Thr Glu Val Thr Ser Lys Met Phe Gln Lys Thr
Arg Thr 355 360 365
Gly Ser Phe His Ile Arg Arg Gly Glu Val Gly Ile Tyr Gln Val Gln 370
375 380 Leu Arg Ala Leu Glu
His Val Asp Gly Thr His Val Cys Gln Leu Pro 385 390
395 400 Glu Asp Gln Lys Gly Asn Ile His Leu Lys
Pro Ser Phe Ser Asp Gly 405 410
415 Leu Lys Met Asp Ala Gly Ile Ile Cys Asp Val Cys Thr Cys Glu
Leu 420 425 430 Gln
Lys Glu Val Arg Ser Ala Arg Cys Ser Phe Asn Gly Asp Phe Val 435
440 445 Cys Gly Gln Cys Val Cys
Ser Glu Gly Trp Ser Gly Gln Thr Cys Asn 450 455
460 Cys Ser Thr Gly Ser Leu Ser Asp Ile Gln Pro
Cys Leu Arg Glu Gly 465 470 475
480 Glu Asp Lys Pro Cys Ser Gly Arg Gly Glu Cys Gln Cys Gly His Cys
485 490 495 Val Cys
Tyr Gly Glu Gly Arg Tyr Glu Gly Gln Phe Cys Glu Tyr Asp 500
505 510 Asn Phe Gln Cys Pro Arg Thr
Ser Gly Phe Leu Cys Asn Asp Arg Gly 515 520
525 Arg Cys Ser Met Gly Gln Cys Val Cys Glu Pro Gly
Trp Thr Gly Pro 530 535 540
Ser Cys Asp Cys Pro Leu Ser Asn Ala Thr Cys Ile Asp Ser Asn Gly 545
550 555 560 Gly Ile Cys
Asn Gly Arg Gly His Cys Glu Cys Gly Arg Cys His Cys 565
570 575 His Gln Gln Ser Leu Tyr Thr Asp
Thr Ile Cys Glu Ile Asn Tyr Ser 580 585
590 Ala Ile His Pro Gly Leu Cys Glu Asp Leu Arg Ser Cys
Val Gln Cys 595 600 605
Gln Ala Trp Gly Thr Gly Glu Lys Lys Gly Arg Thr Cys Glu Glu Cys 610
615 620 Asn Phe Lys Val
Lys Met Val Asp Glu Leu Lys Arg Ala Glu Glu Val 625 630
635 640 Val Val Arg Cys Ser Phe Arg Asp Glu
Asp Asp Asp Cys Thr Tyr Ser 645 650
655 Tyr Thr Met Glu Gly Asp Gly Ala Pro Gly Pro Asn Ser Thr
Val Leu 660 665 670
Val His Lys Lys Lys Asp Cys Pro Pro Gly Ser Phe Trp Trp Leu Ile
675 680 685 Pro Leu Leu Leu
Leu Leu Leu Pro Leu Leu Ala Leu Leu Leu Leu Leu 690
695 700 Cys Trp Lys Tyr Cys Ala Cys Cys
Lys Ala Cys Leu Ala Leu Leu Pro 705 710
715 720 Cys Cys Asn Arg Gly His Met Val Gly Phe Lys Glu
Asp His Tyr Met 725 730
735 Leu Arg Glu Asn Leu Met Ala Ser Asp His Leu Asp Thr Pro Met Leu
740 745 750 Arg Ser Gly
Asn Leu Lys Gly Arg Asp Val Val Arg Trp Lys Val Thr 755
760 765 Asn Asn Met Gln 770
39771PRTMus musculus 39Asn Arg Cys Lys Lys Ala Gln Val Lys Ser Cys Thr
Glu Cys Ile Arg 1 5 10
15 Val Asp Lys Ser Cys Ala Tyr Cys Thr Asp Glu Leu Phe Lys Glu Arg
20 25 30 Arg Cys Asn
Thr Gln Ala Asp Val Leu Ala Ala Gly Cys Arg Gly Glu 35
40 45 Ser Ile Leu Val Met Glu Ser Ser
Leu Glu Ile Thr Glu Asn Thr Gln 50 55
60 Ile Val Thr Ser Leu His Arg Ser Gln Val Ser Pro Gln
Gly Leu Gln 65 70 75
80 Val Arg Leu Arg Arg Gly Glu Glu Arg Thr Phe Val Phe Gln Val Phe
85 90 95 Glu Pro Leu Glu
Ser Pro Val Asp Leu Tyr Ile Leu Met Asp Phe Ser 100
105 110 Asn Ser Met Ser Asp Asp Leu Asp Asn
Leu Lys Gln Met Gly Gln Asn 115 120
125 Leu Ala Lys Ile Leu Arg Gln Leu Thr Ser Asp Tyr Thr Ile
Gly Phe 130 135 140
Gly Lys Phe Val Asp Lys Val Ser Val Pro Gln Thr Asp Met Arg Pro 145
150 155 160 Glu Lys Leu Lys Glu
Pro Trp Pro Asn Ser Asp Pro Pro Phe Ser Phe 165
170 175 Lys Asn Val Ile Ser Leu Thr Glu Asn Val
Glu Glu Phe Trp Asn Lys 180 185
190 Leu Gln Gly Glu Arg Ile Ser Gly Asn Leu Asp Ala Pro Glu Gly
Gly 195 200 205 Phe
Asp Ala Ile Leu Gln Thr Ala Val Cys Thr Arg Asp Ile Gly Trp 210
215 220 Arg Ala Asp Ser Thr His
Leu Leu Val Phe Ser Thr Glu Ser Ala Phe 225 230
235 240 His Tyr Glu Ala Asp Gly Ala Asn Val Leu Ala
Gly Ile Met Asn Arg 245 250
255 Asn Asp Glu Lys Cys His Leu Asp Ala Ser Gly Ala Tyr Thr Gln Tyr
260 265 270 Lys Thr
Gln Asp Tyr Pro Ser Val Pro Thr Leu Val Arg Leu Leu Ala 275
280 285 Lys His Asn Ile Ile Pro Ile
Phe Ala Val Thr Asn Tyr Ser Tyr Ser 290 295
300 Tyr Tyr Glu Lys Leu His Lys Tyr Phe Pro Val Ser
Ser Leu Gly Val 305 310 315
320 Leu Gln Glu Asp Ser Ser Asn Ile Val Glu Leu Leu Glu Glu Ala Phe
325 330 335 Tyr Arg Ile
Arg Ser Asn Leu Asp Ile Arg Ala Leu Asp Ser Pro Arg 340
345 350 Gly Leu Arg Thr Glu Val Thr Ser
Asp Thr Leu Gln Lys Thr Glu Thr 355 360
365 Gly Ser Phe His Ile Lys Arg Gly Glu Val Gly Thr Tyr
Asn Val His 370 375 380
Leu Arg Ala Val Glu Asp Ile Asp Gly Thr His Val Cys Gln Leu Ala 385
390 395 400 Lys Glu Asp Gln
Gly Gly Asn Ile His Leu Lys Pro Ser Phe Ser Asp 405
410 415 Gly Leu Arg Met Asp Ala Ser Val Ile
Cys Asp Val Cys Pro Cys Glu 420 425
430 Leu Gln Lys Glu Val Arg Ser Ala Arg Cys His Phe Arg Gly
Asp Phe 435 440 445
Met Cys Gly His Cys Val Cys Asn Glu Gly Trp Ser Gly Lys Thr Cys 450
455 460 Asn Cys Ser Thr Gly
Ser Leu Ser Asp Thr Gln Pro Cys Leu Arg Glu 465 470
475 480 Gly Glu Asp Lys Pro Cys Ser Gly His Gly
Glu Cys Gln Cys Gly Arg 485 490
495 Cys Val Cys Tyr Gly Glu Gly Arg Tyr Glu Gly His Phe Cys Glu
Tyr 500 505 510 Asp
Asn Phe Gln Cys Pro Arg Thr Ser Gly Phe Leu Cys Asn Asp Arg 515
520 525 Gly Arg Cys Ser Met Gly
Glu Cys Val Cys Glu Pro Gly Trp Thr Gly 530 535
540 Arg Ser Cys Asp Cys Pro Leu Ser Asn Ala Thr
Cys Ile Asp Ser Asn 545 550 555
560 Gly Gly Ile Cys Asn Gly Arg Gly Tyr Cys Glu Cys Gly Arg Cys His
565 570 575 Cys Asn
Gln Gln Ser Leu Tyr Thr Asp Thr Thr Cys Glu Ile Asn Tyr 580
585 590 Ser Ala Ile Leu Gly Leu Cys
Glu Asp Leu Arg Ser Cys Val Gln Cys 595 600
605 Gln Ala Trp Gly Thr Gly Glu Lys Lys Gly Arg Ala
Cys Asp Asp Cys 610 615 620
Pro Phe Lys Val Lys Met Val Asp Glu Leu Lys Lys Glu Glu Val Val 625
630 635 640 Glu Tyr Cys
Ser Phe Arg Asp Glu Asp Asp Asp Cys Thr Tyr Ser Tyr 645
650 655 Asn Val Glu Gly Asp Gly Ser Pro
Gly Pro Asn Ser Thr Val Leu Val 660 665
670 His Lys Lys Lys Asp Cys Leu Pro Ala Pro Ser Trp Trp
Leu Ile Pro 675 680 685
Leu Leu Ile Phe Leu Leu Leu Leu Leu Ala Leu Leu Leu Leu Leu Cys 690
695 700 Trp Lys Tyr Cys
Ala Cys Cys Lys Ala Cys Leu Gly Leu Leu Pro Cys 705 710
715 720 Cys Asn Arg Gly His Met Val Gly Phe
Lys Glu Asp His Tyr Met Leu 725 730
735 Arg Glu Asn Leu Met Ala Ser Asp His Leu Asp Thr Pro Met
Leu Arg 740 745 750
Ser Gly Asn Leu Lys Gly Arg Asp Thr Val Arg Trp Lys Ile Thr Asn
755 760 765 Asn Val Gln
770 40775PRTHomo sapiens 40Gly Leu Asn Ile Cys Thr Ser Gly Ser Ala
Thr Ser Cys Glu Glu Cys 1 5 10
15 Leu Leu Ile His Pro Lys Cys Ala Trp Cys Ser Lys Glu Asp Phe
Gly 20 25 30 Ser
Pro Arg Ser Ile Thr Ser Arg Cys Asp Leu Arg Ala Asn Leu Val 35
40 45 Lys Asn Gly Cys Gly Gly
Glu Ile Glu Ser Pro Ala Ser Ser Phe His 50 55
60 Val Leu Arg Ser Leu Pro Leu Ser Ser Lys Gly
Ser Gly Ser Ala Gly 65 70 75
80 Trp Asp Val Ile Gln Met Thr Pro Gln Glu Ile Ala Val Asn Leu Arg
85 90 95 Pro Gly
Asp Lys Thr Thr Phe Gln Leu Gln Val Arg Gln Val Glu Tyr 100
105 110 Pro Val Asp Leu Tyr Tyr Leu
Met Asp Leu Ser Leu Ser Met Lys Asp 115 120
125 Asp Leu Asp Asn Ile Arg Ser Leu Gly Thr Lys Leu
Ala Glu Glu Met 130 135 140
Arg Lys Leu Thr Ser Asn Phe Arg Leu Gly Phe Gly Ser Phe Val Asp 145
150 155 160 Lys Asp Ile
Ser Pro Phe Ser Tyr Thr Ala Pro Arg Tyr Gln Thr Asn 165
170 175 Pro Cys Ile Gly Tyr Lys Leu Phe
Pro Asn Cys Val Pro Ser Phe Gly 180 185
190 Phe Arg His Leu Leu Pro Leu Thr Asp Arg Val Asp Ser
Phe Asn Glu 195 200 205
Glu Val Arg Lys Gln Arg Val Ser Arg Asn Arg Asp Ala Pro Glu Gly 210
215 220 Gly Phe Asp Ala
Val Leu Gln Ala Ala Val Cys Lys Glu Lys Ile Gly 225 230
235 240 Trp Arg Lys Asp Ala Leu His Leu Leu
Val Phe Thr Thr Asp Asp Val 245 250
255 Pro His Ile Ala Leu Asp Gly Lys Leu Gly Gly Leu Val Gln
Pro His 260 265 270
Asp Gly Gln Cys His Leu Asn Glu Ala Asn Glu Tyr Thr Ala Ser Asn
275 280 285 Gln Met Asp Tyr
Pro Ser Leu Ala Leu Leu Gly Glu Lys Leu Ala Glu 290
295 300 Asn Asn Ile Asn Leu Ile Phe Ala
Val Thr Lys Asn His Tyr Met Leu 305 310
315 320 Tyr Lys Asn Phe Thr Ala Leu Ile Pro Gly Thr Thr
Val Glu Ile Leu 325 330
335 Asp Gly Asp Ser Lys Asn Ile Ile Gln Leu Ile Ile Asn Ala Tyr Asn
340 345 350 Ser Ile Arg
Ser Lys Val Glu Leu Ser Val Trp Asp Gln Pro Glu Asp 355
360 365 Leu Asn Leu Phe Phe Thr Ala Thr
Cys Gln Asp Gly Val Ser Tyr Pro 370 375
380 Gly Gln Arg Lys Cys Glu Gly Leu Lys Ile Gly Asp Thr
Ala Ser Phe 385 390 395
400 Glu Val Ser Leu Glu Ala Arg Ser Cys Pro Ser Arg His Thr Glu His
405 410 415 Val Phe Ala Leu
Arg Pro Val Gly Phe Arg Asp Ser Leu Glu Val Gly 420
425 430 Val Thr Tyr Asn Cys Thr Cys Gly Cys
Ser Val Gly Leu Glu Pro Asn 435 440
445 Ser Ala Arg Cys Asn Gly Ser Gly Thr Tyr Val Cys Gly Leu
Cys Glu 450 455 460
Cys Ser Pro Gly Tyr Leu Gly Thr Arg Cys Glu Cys Gln Asp Gly Glu 465
470 475 480 Asn Gln Ser Val Tyr
Gln Asn Leu Cys Arg Glu Ala Glu Gly Lys Pro 485
490 495 Leu Cys Ser Gly Arg Gly Asp Cys Ser Cys
Asn Gln Cys Ser Cys Phe 500 505
510 Glu Ser Glu Phe Gly Lys Ile Tyr Gly Pro Phe Cys Glu Cys Asp
Asn 515 520 525 Phe
Ser Cys Ala Arg Asn Lys Gly Val Leu Cys Ser Gly His Gly Glu 530
535 540 Cys His Cys Gly Glu Cys
Lys Cys His Ala Gly Tyr Ile Gly Asp Asn 545 550
555 560 Cys Asn Cys Ser Thr Asp Ile Ser Thr Cys Arg
Gly Arg Asp Gly Gln 565 570
575 Ile Cys Ser Glu Arg Gly His Cys Leu Cys Gly Gln Cys Gln Cys Thr
580 585 590 Glu Pro
Gly Ala Phe Gly Glu Met Cys Glu Lys Cys Pro Thr Cys Pro 595
600 605 Asp Ala Cys Ser Thr Lys Arg
Asp Cys Val Glu Cys Pro Leu Leu His 610 615
620 Ser Gly Lys Pro Asp Asn Gln Thr Cys His Ser Leu
Cys Arg Asp Glu 625 630 635
640 Val Ile Thr Trp Val Asp Thr Ile Val Lys Asp Asp Gln Glu Ala Val
645 650 655 Leu Cys Phe
Tyr Lys Thr Ala Lys Asp Cys Val Met Met Phe Thr Tyr 660
665 670 Val Glu Leu Pro Ser Gly Lys Ser
Asn Leu Thr Val Leu Arg Glu Pro 675 680
685 Glu Cys Gly Asn Thr Pro Asn Ala Met Thr Ile Leu Leu
Ala Val Val 690 695 700
Gly Ser Ile Leu Leu Val Gly Leu Ala Leu Leu Ala Ile Trp Lys Leu 705
710 715 720 Leu Val Thr Ile
His Asp Arg Arg Glu Phe Ala Lys Phe Gln Ser Glu 725
730 735 Arg Ser Arg Ala Arg Tyr Glu Met Ala
Ser Asn Pro Leu Tyr Arg Lys 740 745
750 Pro Ile Ser Thr His Thr Val Asp Phe Thr Phe Asn Lys Phe
Asn Lys 755 760 765
Ser Tyr Asn Gly Thr Val Asp 770 775 41776PRTMus
musculus 41Gly Leu Asn Ile Cys Thr Ser Gly Ser Ala Thr Ser Cys Glu Glu
Cys 1 5 10 15 Leu
Leu Ile His Pro Lys Cys Ala Trp Cys Ser Lys Glu Tyr Phe Gly
20 25 30 Asn Pro Arg Ser Ile
Thr Ser Arg Cys Asp Leu Lys Ala Asn Leu Ile 35
40 45 Arg Asn Gly Cys Glu Gly Glu Ile Glu
Ser Pro Ala Ser Ser Thr His 50 55
60 Val Leu Arg Asn Leu Pro Leu Ser Cys Lys Gly Ser Ser
Ala Thr Gly 65 70 75
80 Ser Asp Val Ile Gln Met Thr Pro Gln Glu Ile Ala Val Ser Leu Arg
85 90 95 Pro Gly Glu Gln
Thr Thr Phe Gln Leu Gln Val Arg Gln Val Glu Asp 100
105 110 Tyr Pro Val Asp Leu Tyr Tyr Leu Met
Asp Leu Ser Leu Ser Met Lys 115 120
125 Asp Asp Leu Glu Asn Ile Arg Ser Leu Gly Thr Lys Leu Ala
Glu Glu 130 135 140
Met Arg Lys Leu Thr Ser Asn Phe Arg Leu Gly Phe Gly Ser Phe Val 145
150 155 160 Asp Lys Asp Ile Ser
Pro Phe Ser Tyr Thr Ala Pro Arg Tyr Gln Thr 165
170 175 Asn Pro Cys Ile Gly Tyr Lys Leu Phe Pro
Asn Cys Val Pro Ser Phe 180 185
190 Gly Phe Arg His Leu Leu Pro Leu Thr Asp Arg Val Asp Ser Phe
Asn 195 200 205 Glu
Glu Val Arg Lys Gln Arg Val Ser Arg Asn Arg Asp Ala Pro Glu 210
215 220 Gly Gly Phe Asp Ala Val
Leu Gln Ala Ala Val Cys Lys Glu Lys Ile 225 230
235 240 Gly Trp Arg Lys Asp Ala Leu His Leu Leu Val
Phe Thr Thr Asp Asp 245 250
255 Val Pro His Ile Ala Leu Asp Gly Lys Leu Gly Gly Leu Val Gln Pro
260 265 270 His Asp
Gly Gln Cys His Leu Asn Glu Ala Asn Glu Tyr Thr Ala Ser 275
280 285 Asn Gln Met Asp Tyr Pro Ser
Leu Ala Leu Leu Gly Glu Lys Leu Ala 290 295
300 Glu Asn Asn Ile Asn Leu Ile Phe Ala Val Thr Lys
Asn His Tyr Met 305 310 315
320 Leu Tyr Lys Asn Phe Thr Ala Leu Ile Pro Gly Thr Thr Val Glu Ile
325 330 335 Leu His Gly
Asp Ser Lys Asn Ile Ile Gln Leu Ile Ile Asn Ala Tyr 340
345 350 Ser Ser Ile Arg Ala Lys Val Glu
Leu Ser Val Trp Asp Gln Pro Glu 355 360
365 Asp Leu Asn Leu Phe Phe Thr Ala Thr Cys Gln Asp Gly
Ile Ser Tyr 370 375 380
Pro Gly Gln Arg Lys Cys Glu Gly Leu Lys Ile Gly Asp Thr Ala Ser 385
390 395 400 Phe Glu Val Ser
Val Glu Ala Arg Ser Cys Pro Gly Arg Gln Ala Ala 405
410 415 Gln Ser Phe Thr Leu Arg Pro Val Gly
Phe Arg Asp Ser Leu Gln Val 420 425
430 Glu Val Ala Tyr Asn Cys Thr Cys Gly Cys Ser Thr Gly Leu
Glu Pro 435 440 445
Asn Ser Ala Arg Cys Ser Gly Asn Gly Thr Tyr Thr Cys Gly Leu Cys 450
455 460 Glu Cys Asp Pro Gly
Tyr Leu Gly Thr Arg Cys Glu Cys Gln Glu Gly 465 470
475 480 Glu Asn Gln Ser Gly Tyr Gln Asn Leu Cys
Arg Glu Ala Glu Gly Lys 485 490
495 Pro Leu Cys Ser Gly Arg Gly Glu Cys Ser Cys Asn Gln Cys Ser
Cys 500 505 510 Phe
Glu Ser Glu Phe Gly Arg Ile Tyr Gly Pro Phe Cys Glu Cys Asp 515
520 525 Ser Phe Ser Cys Ala Arg
Asn Lys Gly Val Leu Cys Ser Gly His Gly 530 535
540 Glu Cys His Cys Gly Glu Cys Lys Cys His Ala
Gly Tyr Ile Gly Asp 545 550 555
560 Asn Cys Asn Cys Ser Thr Asp Val Ser Thr Cys Arg Ala Lys Asp Gly
565 570 575 Gln Ile
Cys Ser Asp Arg Gly Arg Cys Val Cys Gly Gln Cys Gln Cys 580
585 590 Thr Glu Pro Gly Ala Phe Gly
Glu Thr Cys Glu Lys Cys Pro Thr Cys 595 600
605 Pro Asp Ala Cys Ser Ser Lys Arg Asp Cys Val Glu
Cys Leu Leu Leu 610 615 620
His Gln Gly Lys Pro Asp Asn Gln Thr Cys His His Gln Cys Lys Asp 625
630 635 640 Glu Val Ile
Thr Trp Val Asp Thr Ile Val Lys Asp Asp Gln Glu Ala 645
650 655 Val Leu Cys Phe Tyr Lys Thr Ala
Lys Asp Cys Val Met Met Phe Ser 660 665
670 Tyr Thr Glu Leu Pro Asn Gly Arg Ser Asn Leu Thr Val
Leu Arg Glu 675 680 685
Pro Glu Cys Gly Ser Ala Pro Asn Ala Met Thr Ile Leu Leu Ala Val 690
695 700 Val Gly Ser Ile
Leu Leu Ile Gly Met Ala Leu Leu Ala Ile Trp Lys 705 710
715 720 Leu Leu Val Thr Ile His Asp Arg Arg
Glu Phe Ala Lys Phe Gln Ser 725 730
735 Glu Arg Ser Arg Ala Arg Tyr Glu Met Ala Ser Asn Pro Leu
Tyr Arg 740 745 750
Lys Pro Ile Ser Thr His Thr Val Asp Phe Ala Phe Asn Lys Phe Asn
755 760 765 Lys Ser Tyr Asn
Gly Ser Val Asp 770 775 42771PRTHomo sapiens
42His Val Gln Gly Gly Cys Ala Leu Gly Gly Ala Glu Thr Cys Glu Asp 1
5 10 15 Cys Leu Leu Ile
Gly Pro Gln Cys Ala Trp Cys Ala Gln Glu Asn Phe 20
25 30 Thr His Pro Ser Gly Val Gly Glu Arg
Cys Asp Thr Pro Ala Asn Leu 35 40
45 Leu Ala Lys Gly Cys Gln Leu Asn Phe Ile Glu Asn Pro Val
Ser Gln 50 55 60
Val Glu Ile Leu Lys Asn Lys Pro Leu Ser Val Gly Arg Gln Lys Asn 65
70 75 80 Ser Ser Asp Ile Val
Gln Ile Ala Pro Gln Ser Leu Ile Leu Lys Leu 85
90 95 Arg Pro Gly Gly Ala Gln Thr Leu Gln Val
His Val Arg Gln Thr Glu 100 105
110 Asp Tyr Pro Val Asp Leu Tyr Tyr Leu Met Asp Leu Ser Ala Ser
Met 115 120 125 Asp
Asp Asp Leu Asn Thr Ile Lys Glu Leu Gly Ser Arg Leu Ser Lys 130
135 140 Glu Met Ser Lys Leu Thr
Ser Asn Phe Arg Leu Gly Phe Gly Ser Phe 145 150
155 160 Val Glu Lys Pro Val Ser Pro Phe Val Lys Thr
Thr Pro Glu Glu Ile 165 170
175 Ala Asn Pro Cys Ser Ser Ile Pro Tyr Phe Cys Leu Pro Thr Phe Gly
180 185 190 Phe Lys
His Ile Leu Pro Leu Thr Asn Asp Ala Glu Arg Phe Asn Glu 195
200 205 Ile Val Lys Asn Gln Lys Ile
Ser Ala Asn Ile Asp Thr Pro Glu Gly 210 215
220 Gly Phe Asp Ala Ile Met Gln Ala Ala Val Cys Lys
Glu Lys Ile Gly 225 230 235
240 Trp Arg Asn Asp Ser Leu His Leu Leu Val Phe Val Ser Asp Ala Asp
245 250 255 Ser His Phe
Gly Met Asp Ser Lys Leu Ala Gly Ile Val Ile Pro Asn 260
265 270 Asp Gly Leu Cys His Leu Asp Ser
Lys Asn Glu Tyr Ser Met Ser Thr 275 280
285 Val Leu Glu Tyr Pro Thr Ile Gly Gln Leu Ile Asp Lys
Leu Val Gln 290 295 300
Asn Asn Val Leu Leu Ile Phe Ala Val Thr Gln Glu Gln Val His Leu 305
310 315 320 Tyr Glu Asn Tyr
Ala Lys Leu Ile Pro Gly Ala Thr Val Gly Leu Leu 325
330 335 Gln Lys Asp Ser Gly Asn Ile Leu Gln
Leu Ile Ile Ser Ala Tyr Glu 340 345
350 Glu Leu Arg Ser Glu Val Glu Leu Glu Val Leu Gly Asp Thr
Glu Gly 355 360 365
Leu Asn Leu Ser Phe Thr Ala Ile Cys Asn Asn Gly Thr Leu Phe Gln 370
375 380 His Gln Lys Lys Cys
Ser His Met Lys Val Gly Asp Thr Ala Ser Phe 385 390
395 400 Ser Val Thr Val Asn Ile Pro His Cys Glu
Arg Arg Ser Arg His Ile 405 410
415 Ile Ile Lys Pro Val Gly Leu Gly Asp Ala Leu Glu Leu Leu Val
Ser 420 425 430 Pro
Glu Cys Asn Cys Asp Cys Gln Lys Glu Val Glu Val Asn Ser Ser 435
440 445 Lys Cys His His Gly Asn
Gly Ser Phe Gln Cys Gly Val Cys Ala Cys 450 455
460 His Pro Gly His Met Gly Pro Arg Cys Glu Cys
Gly Glu Asp Met Leu 465 470 475
480 Ser Thr Asp Ser Cys Lys Glu Ala Pro Asp His Pro Ser Cys Ser Gly
485 490 495 Arg Gly
Asp Cys Tyr Cys Gly Gln Cys Ile Cys His Leu Ser Pro Tyr 500
505 510 Gly Asn Ile Tyr Gly Pro Tyr
Cys Gln Cys Asp Asn Phe Ser Cys Val 515 520
525 Arg His Lys Gly Leu Leu Cys Gly Gly Asn Gly Asp
Cys Asp Cys Gly 530 535 540
Glu Cys Val Cys Arg Ser Gly Trp Thr Gly Glu Tyr Cys Asn Cys Thr 545
550 555 560 Thr Ser Thr
Asp Ser Cys Val Ser Glu Asp Gly Val Leu Cys Ser Gly 565
570 575 Arg Gly Asp Cys Val Cys Gly Lys
Cys Val Cys Thr Asn Pro Gly Ala 580 585
590 Ser Gly Pro Thr Cys Glu Arg Cys Pro Thr Cys Gly Asp
Pro Cys Asn 595 600 605
Ser Lys Arg Ser Cys Ile Glu Cys His Leu Ser Ala Ala Gly Gln Ala 610
615 620 Arg Glu Glu Cys
Val Asp Lys Cys Lys Leu Ala Gly Ala Thr Ile Ser 625 630
635 640 Glu Glu Glu Asp Phe Ser Lys Asp Gly
Ser Val Ser Cys Ser Leu Gln 645 650
655 Gly Glu Asn Glu Cys Leu Ile Thr Phe Leu Ile Thr Thr Asp
Asn Glu 660 665 670
Gly Lys Thr Ile Ile His Ser Ile Asn Glu Lys Asp Cys Pro Lys Pro
675 680 685 Pro Asn Ile Pro
Met Ile Met Leu Gly Val Ser Leu Ala Ile Leu Leu 690
695 700 Ile Gly Val Val Leu Leu Cys Ile
Trp Lys Leu Leu Val Ser Phe His 705 710
715 720 Asp Arg Lys Glu Val Ala Lys Phe Glu Ala Glu Arg
Ser Lys Ala Lys 725 730
735 Trp Gln Thr Gly Thr Asn Pro Leu Tyr Arg Gly Ser Thr Ser Thr Phe
740 745 750 Lys Asn Val
Thr Tyr Lys His Arg Glu Lys Gln Lys Val Asp Leu Ser 755
760 765 Thr Asp Cys 770
43770PRTMus musculus 43His Val Gln Gly Gly Cys Ala Trp Gly Gly Ala Glu
Ser Cys Ser Asp 1 5 10
15 Cys Leu Leu Thr Gly Pro His Cys Ala Trp Cys Ser Gln Glu Asn Phe
20 25 30 Thr His Leu
Ser Gly Ala Gly Glu Arg Cys Asp Thr Pro Ala Asn Leu 35
40 45 Leu Ala Lys Gly Cys Gln Leu Pro
Phe Ile Glu Asn Pro Val Ser Arg 50 55
60 Ile Glu Val Leu Gln Asn Lys Pro Leu Ser Val Gly Arg
Gln Lys Asn 65 70 75
80 Ser Ser Asp Ile Val Gln Ile Ala Pro Gln Ser Leu Val Leu Lys Leu
85 90 95 Arg Pro Gly Arg
Glu Gln Thr Leu Gln Val Gln Val Arg Gln Thr Glu 100
105 110 Asp Tyr Pro Val Asp Leu Tyr Tyr Leu
Met Asp Leu Ser Ala Ser Met 115 120
125 Asp Asp Asp Leu Asn Thr Ile Lys Glu Leu Gly Ser Arg Leu
Ala Lys 130 135 140
Glu Met Ser Lys Leu Thr Ser Asn Phe Arg Leu Gly Phe Gly Ser Phe 145
150 155 160 Val Glu Lys Pro Val
Ser Pro Phe Met Lys Thr Thr Pro Glu Glu Ile 165
170 175 Thr Asn Pro Cys Ser Ser Ile Pro Tyr Phe
Cys Leu Pro Thr Phe Gly 180 185
190 Phe Lys His Ile Leu Pro Leu Thr Asp Asp Ala Glu Arg Phe Asn
Glu 195 200 205 Ile
Val Arg Lys Gln Lys Ile Ser Ala Asn Ile Asp Thr Pro Glu Gly 210
215 220 Gly Phe Asp Ala Ile Met
Gln Ala Ala Val Cys Lys Glu Lys Ile Gly 225 230
235 240 Trp Arg Asn Asp Ser Leu His Leu Leu Val Phe
Val Ser Asp Ala Asp 245 250
255 Ser His Phe Gly Met Asp Ser Lys Leu Ala Gly Ile Val Ile Pro Asn
260 265 270 Asp Gly
Leu Cys His Leu Asp His Arg Asn Glu Tyr Ser Met Ser Thr 275
280 285 Val Leu Glu Tyr Pro Thr Ile
Gly Gln Leu Ile Asp Lys Leu Val Gln 290 295
300 Asn Asn Val Leu Leu Ile Phe Ala Val Thr Gln Glu
Gln Val His Leu 305 310 315
320 Tyr Glu Asn Tyr Ala Lys Leu Ile Pro Gly Ala Thr Val Gly Leu Leu
325 330 335 Gln Lys Asp
Ser Gly Asn Ile Leu Gln Leu Ile Ile Ser Ala Tyr Glu 340
345 350 Glu Leu Arg Ser Glu Val Glu Leu
Glu Val Leu Gly Asp Thr Glu Gly 355 360
365 Leu Asn Leu Ser Phe Thr Ala Leu Cys Asn Asn Gly Val
Leu Phe Pro 370 375 380
His Gln Lys Lys Cys Ser His Met Lys Val Gly Asp Thr Ala Ser Phe 385
390 395 400 Asn Val Thr Val
Ser Val Ser Asn Cys Glu Lys Arg Ser Arg Asn Leu 405
410 415 Ile Ile Lys Pro Val Gly Leu Gly Asp
Thr Leu Glu Ile Leu Val Ser 420 425
430 Ala Glu Cys Asp Cys Asp Cys Gln Arg Glu Ile Glu Thr Asn
Ser Ser 435 440 445
Lys Cys His Asn Gly Asn Gly Ser Phe Gln Cys Gly Val Cys Thr Cys 450
455 460 Asn Pro Gly His Met
Gly Pro His Cys Glu Cys Gly Glu Asp Met Val 465 470
475 480 Ser Thr Asp Ser Cys Lys Glu Ser Pro Gly
His Pro Ser Cys Ser Gly 485 490
495 Arg Gly Asp Cys Tyr Cys Gly Gln Cys Ile Cys His Leu Ser Pro
Tyr 500 505 510 Gly
Ser Ile Tyr Gly Pro Tyr Cys Gln Cys Asp Asn Phe Ser Cys Leu 515
520 525 Arg His Lys Gly Leu Leu
Cys Gly Asp Asn Gly Asp Cys Asp Cys Gly 530 535
540 Glu Cys Val Cys Arg Asp Gly Trp Thr Gly Glu
Tyr Cys Asn Cys Thr 545 550 555
560 Thr Asn Arg Asp Ser Cys Thr Ser Glu Asp Gly Val Leu Cys Ser Gly
565 570 575 Arg Gly
Asp Cys Val Cys Gly Lys Cys Val Cys Arg Asn Pro Gly Ala 580
585 590 Ser Gly Pro Thr Cys Glu Arg
Cys Pro Thr Cys Gly Asp Pro Cys Asn 595 600
605 Ser Lys Arg Ser Cys Ile Glu Cys Tyr Leu Ser Ala
Asp Gly Gln Ala 610 615 620
Gln Glu Glu Cys Ala Asp Lys Cys Lys Ala Ile Gly Ala Thr Ile Ser 625
630 635 640 Glu Glu Asp
Phe Ser Lys Asp Thr Ser Val Ser Cys Ser Leu Gln Gly 645
650 655 Glu Asn Glu Cys Leu Ile Thr Phe
Leu Ile Thr Thr Asp Asn Glu Gly 660 665
670 Lys Thr Ile Ile His Asn Ile Asn Glu Lys Asp Cys Pro
Lys Pro Pro 675 680 685
Asn Ile Pro Met Ile Met Leu Gly Val Ser Leu Ala Ile Leu Leu Ile 690
695 700 Gly Val Val Leu
Leu Cys Ile Trp Lys Leu Leu Val Ser Phe His Asp 705 710
715 720 Arg Lys Glu Val Ala Lys Phe Glu Ala
Glu Arg Ser Lys Ala Lys Trp 725 730
735 Gln Thr Gly Thr Asn Pro Leu Tyr Arg Gly Ser Thr Ser Thr
Phe Lys 740 745 750
Asn Val Thr Tyr Lys His Arg Glu Lys His Lys Ala Gly Leu Ser Ser
755 760 765 Asp Gly 770
44779PRTHomo sapiens 44Glu Leu Asp Ala Lys Ile Pro Ser Thr Gly Asp Ala
Thr Glu Trp Arg 1 5 10
15 Asn Pro His Leu Ser Met Leu Gly Ser Cys Gln Pro Ala Pro Ser Cys
20 25 30 Gln Lys Cys
Ile Leu Ser His Pro Ser Cys Ala Trp Cys Lys Gln Leu 35
40 45 Asn Phe Thr Ala Ser Gly Glu Ala
Glu Ala Arg Arg Cys Ala Arg Arg 50 55
60 Glu Glu Leu Leu Ala Arg Gly Cys Pro Leu Glu Glu Leu
Glu Glu Pro 65 70 75
80 Arg Gly Gln Gln Glu Val Leu Gln Asp Gln Pro Leu Ser Gln Gly Ala
85 90 95 Arg Gly Glu Gly
Ala Thr Gln Leu Ala Pro Gln Arg Val Arg Val Thr 100
105 110 Leu Arg Pro Gly Glu Pro Gln Gln Leu
Gln Val Arg Phe Leu Arg Ala 115 120
125 Glu Gly Tyr Pro Val Asp Leu Tyr Tyr Leu Met Asp Leu Ser
Tyr Ser 130 135 140
Met Lys Asp Asp Leu Glu Arg Val Arg Gln Leu Gly His Ala Leu Leu 145
150 155 160 Val Arg Leu Gln Glu
Val Thr His Ser Val Arg Ile Gly Phe Gly Ser 165
170 175 Phe Val Asp Lys Thr Val Leu Pro Phe Val
Ser Thr Val Pro Ser Lys 180 185
190 Leu Arg His Pro Cys Pro Thr Arg Leu Glu Arg Cys Gln Ser Pro
Phe 195 200 205 Ser
Phe His His Val Leu Ser Leu Thr Gly Asp Ala Gln Ala Phe Glu 210
215 220 Arg Glu Val Gly Arg Gln
Ser Val Ser Gly Asn Leu Asp Ser Pro Glu 225 230
235 240 Gly Gly Phe Asp Ala Ile Leu Gln Ala Ala Leu
Cys Gln Glu Gln Ile 245 250
255 Gly Trp Arg Asn Val Ser Arg Leu Leu Val Phe Thr Ser Asp Asp Thr
260 265 270 Phe His
Thr Ala Gly Asp Gly Lys Leu Gly Gly Ile Phe Met Pro Ser 275
280 285 Asp Gly His Cys His Leu Asp
Ser Asn Gly Leu Tyr Ser Arg Ser Thr 290 295
300 Glu Phe Asp Tyr Pro Ser Val Gly Gln Val Ala Gln
Ala Leu Ser Ala 305 310 315
320 Ala Asn Ile Gln Pro Ile Phe Ala Val Thr Ser Ala Ala Leu Pro Val
325 330 335 Tyr Gln Glu
Leu Ser Lys Leu Ile Pro Lys Ser Ala Val Gly Glu Leu 340
345 350 Ser Glu Asp Ser Ser Asn Val Val
Gln Leu Ile Met Asp Ala Tyr Asn 355 360
365 Ser Leu Ser Ser Thr Val Thr Leu Glu His Ser Ser Leu
Pro Pro Gly 370 375 380
Val His Ile Ser Tyr Glu Ser Gln Cys Glu Gly Pro Glu Lys Arg Glu 385
390 395 400 Gly Lys Ala Glu
Asp Arg Gly Gln Cys Asn His Val Arg Ile Asn Gln 405
410 415 Thr Val Thr Phe Trp Val Ser Leu Gln
Ala Thr His Cys Leu Pro Glu 420 425
430 Pro His Leu Leu Arg Leu Arg Ala Leu Gly Phe Ser Glu Glu
Leu Ile 435 440 445
Val Glu Leu His Thr Leu Cys Asp Cys Asn Cys Ser Asp Thr Gln Pro 450
455 460 Gln Ala Pro His Cys
Ser Asp Gly Gln Gly His Leu Gln Cys Gly Val 465 470
475 480 Cys Ser Cys Ala Pro Gly Arg Leu Gly Arg
Leu Cys Glu Cys Ser Val 485 490
495 Ala Glu Leu Ser Ser Pro Asp Leu Glu Ser Gly Cys Arg Ala Pro
Asn 500 505 510 Gly
Thr Gly Pro Leu Cys Ser Gly Lys Gly His Cys Gln Cys Gly Arg 515
520 525 Cys Ser Cys Ser Gly Gln
Ser Ser Gly His Leu Cys Glu Cys Asp Asp 530 535
540 Ala Ser Cys Glu Arg His Glu Gly Ile Leu Cys
Gly Gly Phe Gly Arg 545 550 555
560 Cys Gln Cys Gly Val Cys His Cys His Ala Asn Arg Thr Gly Arg Ala
565 570 575 Cys Glu
Cys Ser Gly Asp Met Asp Ser Cys Ile Ser Pro Glu Gly Gly 580
585 590 Leu Cys Ser Gly His Gly Arg
Cys Lys Cys Asn Arg Cys Gln Cys Leu 595 600
605 Asp Gly Tyr Tyr Gly Ala Leu Cys Asp Gln Cys Pro
Gly Cys Lys Thr 610 615 620
Pro Cys Glu Arg His Arg Asp Cys Ala Glu Cys Gly Ala Phe Arg Thr 625
630 635 640 Gly Pro Leu
Ala Thr Asn Cys Ser Thr Ala Cys Ala His Thr Asn Val 645
650 655 Thr Leu Ala Leu Ala Pro Ile Leu
Asp Asp Gly Trp Cys Lys Glu Arg 660 665
670 Thr Leu Asp Asn Gln Leu Phe Phe Phe Leu Val Glu Asp
Asp Ala Arg 675 680 685
Gly Thr Val Val Leu Arg Val Arg Pro Gln Glu Lys Gly Ala Asp His 690
695 700 Thr Gln Ala Ile
Val Leu Gly Cys Val Gly Gly Ile Val Ala Val Gly 705 710
715 720 Leu Gly Leu Val Leu Ala Tyr Arg Leu
Ser Val Glu Ile Tyr Asp Arg 725 730
735 Arg Glu Tyr Ser Arg Phe Glu Lys Glu Gln Gln Gln Leu Asn
Trp Lys 740 745 750
Gln Asp Ser Asn Pro Leu Tyr Lys Ser Ala Ile Thr Thr Thr Ile Asn
755 760 765 Pro Arg Phe Gln
Glu Ala Asp Ser Pro Thr Leu 770 775
45786PRTMus musculus 45Glu Leu Asp Thr Lys Ile Thr Ser Ser Gly Glu Ala
Ala Glu Trp Glu 1 5 10
15 Asp Pro Asp Leu Ser Leu Gln Gly Ser Cys Gln Pro Val Pro Ser Cys
20 25 30 Gln Lys Cys
Ile Leu Ser His Pro Ser Cys Ala Trp Cys Lys Gln Leu 35
40 45 Asn Phe Thr Ala Ser Gly Glu Ala
Glu Ala Arg Arg Cys Ala Arg Arg 50 55
60 Glu Glu Leu Leu Ala Arg Gly Cys Pro Ala Gln Glu Leu
Glu Glu Pro 65 70 75
80 Arg Gly Arg Gln Glu Val Leu Gln Asp Lys Pro Leu Ser Gln Gly Asp
85 90 95 Arg Gly Glu Gly
Ala Thr Gln Leu Pro Gln Arg Ile Arg Val Thr Leu 100
105 110 Arg Pro Gly Glu Pro Gln Lys Phe Arg
Val Arg Phe Leu Arg Ala Ala 115 120
125 Gly Tyr Pro Val Asp Leu Tyr Tyr Leu Met Asp Leu Ser Tyr
Ser Met 130 135 140
Lys Asp Asp Leu Glu Arg Val Arg Gln Leu Gly His Ala Leu Leu Val 145
150 155 160 Arg Leu Gln Glu Val
Thr His Ser Val Arg Ile Gly Phe Gly Ser Phe 165
170 175 Val Asp Lys Thr Val Leu Pro Phe Val Ser
Thr Val Pro Ser Lys Leu 180 185
190 His His Pro Cys Pro Ser Arg Leu Glu Arg Cys Gln Pro Pro Phe
Ser 195 200 205 Phe
His His Val Leu Ser Leu Thr Gly Asp Ala Gln Ala Phe Glu Arg 210
215 220 Glu Val Gly Arg Gln Asn
Val Ser Gly Asn Leu Asp Ser Pro Glu Gly 225 230
235 240 Gly Phe Asp Ala Ile Leu Gln Ala Ala Leu Cys
Gln Glu Gln Ile Gly 245 250
255 Trp Arg Asn Val Ser Arg Leu Leu Val Phe Thr Ser Asp Asp Thr Phe
260 265 270 His Thr
Ala Gly Asp Gly Lys Leu Gly Gly Ile Phe Met Pro Ser Asp 275
280 285 Gly Arg Cys His Leu Asp Ser
Asn Gly Val Tyr Thr Asn Ser Ala Glu 290 295
300 Phe Asp Tyr Pro Ser Val Gly Gln Val Ala Gln Ala
Leu Thr Ala Ala 305 310 315
320 Asn Ile Gln Pro Ile Phe Ala Val Thr Gly Ala Thr Leu Pro Val Tyr
325 330 335 Gln Glu Leu
Arg Gln Leu Ile Pro Lys Ser Ala Val Gly Glu Leu Ser 340
345 350 Glu Asp Ser Ser Asn Val Val Gln
Leu Ile Met Asp Ala Tyr Asp Ser 355 360
365 Leu Ser Ser Thr Val Thr Leu Glu His Ser Pro Leu Pro
Pro Gly Val 370 375 380
Ser Ile Ser Phe Glu Ser His Cys Lys Gly Pro Glu Lys Thr Glu Gly 385
390 395 400 Glu Ala Gly Asp
Arg Gly Gln Cys Asn Asp Val Arg Val Asn Gln Thr 405
410 415 Val Asp Phe Trp Val Thr Leu Gln Ala
Thr His Cys Leu Pro Glu Ala 420 425
430 His Val Leu Arg Leu Trp Ala Leu Gly Phe Ser Glu Glu Leu
Thr Val 435 440 445
Glu Leu His Thr Val Cys Asp Cys Asn Cys Gly Asp Ala Gln Pro His 450
455 460 Ala Pro Tyr Cys Ser
Asp Gly Gln Gly Asp Leu Gln Cys Gly Ile Cys 465 470
475 480 Ser Cys Ala Pro Gly Arg Leu Gly Gln Leu
Cys Glu Cys Ser Glu Ala 485 490
495 Asp Leu Ser Ser Pro Asp Leu Glu Ser Gly Cys Arg Ala Pro Asn
Gly 500 505 510 Thr
Gly Pro Leu Cys Ser Gly Lys Gly Arg Cys Gln Cys Gly Arg Cys 515
520 525 Ser Cys Ser Gly Gln Ser
Ser Gly His Leu Cys Glu Cys Asp Asp Ala 530 535
540 Ser Cys Glu Arg His Glu Gly Ile Leu Cys Gly
Gly Phe Gly His Cys 545 550 555
560 Gln Cys Gly Val Cys His Cys His Ala Asn His Thr Gly Arg Ala Cys
565 570 575 Glu Cys
Ser Lys Ser Val Asp Ser Cys Val Ser Pro Glu Gly Gly Leu 580
585 590 Cys Ser Gly His Gly Tyr Cys
Lys Cys Asn Arg Cys Gln Cys Leu Asp 595 600
605 Gly Tyr Tyr Gly Ala Leu Cys Asp Gln Cys Leu Gly
Cys Lys Ser Pro 610 615 620
Cys Glu Gln Tyr Arg Asp Cys Ala Glu Cys Gly Ala Phe Gly Thr Gly 625
630 635 640 Pro Leu Ala
Ala Asn Cys Ser Val Val Cys Ala Asp Val Asn Val Thr 645
650 655 Leu Thr Leu Ala Pro Asn Leu Asp
Asp Gly Trp Cys Lys Glu Arg Thr 660 665
670 Ile Asp Asn Gln Leu Phe Phe Phe Leu Val Glu His Ala
Ala Ser Gly 675 680 685
Ile Val Leu Arg Val Arg Pro Gln Glu Lys Gly Val Asp His Thr Arg 690
695 700 Ala Ile Ile Leu
Gly Cys Thr Gly Gly Ile Val Ala Val Gly Leu Gly 705 710
715 720 Leu Val Leu Ala Tyr Arg Leu Ser Val
Glu Ile Tyr Asp Arg Arg Glu 725 730
735 Tyr Arg Arg Phe Glu Lys Glu Gln Gln Gln Leu Asn Trp Lys
Gln Asp 740 745 750
Asn Asn Pro Leu Tyr Lys Ser Ala Ile Thr Thr Thr Val Asn Pro Arg
755 760 765 Phe Gln Gly Thr
Asn Gly Arg Ser Pro Ser Leu Ser Leu Thr Arg Glu 770
775 780 Ala Asp 785 46727PRTHomo
sapiens 46Glu Asp Asn Arg Cys Ala Ser Ser Asn Ala Ala Ser Cys Ala Arg Cys
1 5 10 15 Leu Ala
Leu Gly Pro Glu Cys Gly Trp Cys Val Gln Glu Asp Phe Ile 20
25 30 Ser Gly Gly Ser Arg Ser Glu
Arg Cys Asp Ile Val Ser Asn Leu Ile 35 40
45 Ser Lys Gly Cys Ser Val Asp Ser Ile Glu Tyr Pro
Ser Val His Val 50 55 60
Ile Ile Pro Thr Glu Asn Glu Ile Asn Thr Gln Val Thr Pro Gly Glu 65
70 75 80 Val Ser Ile
Gln Leu Arg Pro Gly Ala Glu Ala Asn Phe Met Leu Lys 85
90 95 Val His Pro Leu Lys Lys Tyr Pro
Val Asp Leu Tyr Tyr Leu Val Asp 100 105
110 Val Ser Ala Ser Met His Asn Asn Ile Glu Lys Leu Asn
Ser Val Gly 115 120 125
Asn Asp Leu Ser Arg Lys Met Ala Phe Phe Ser Arg Asp Phe Arg Leu 130
135 140 Gly Phe Gly Ser
Tyr Val Asp Lys Thr Val Ser Pro Tyr Ile Ser Ile 145 150
155 160 His Pro Glu Arg Ile His Asn Gln Cys
Ser Asp Tyr Asn Leu Asp Cys 165 170
175 Met Pro Pro His Gly Tyr Ile His Val Leu Ser Leu Thr Glu
Asn Ile 180 185 190
Thr Glu Phe Glu Lys Ala Val His Arg Gln Lys Ile Ser Gly Asn Ile
195 200 205 Asp Thr Pro Glu
Gly Gly Phe Asp Ala Met Leu Gln Ala Ala Val Cys 210
215 220 Glu Ser His Ile Gly Trp Arg Lys
Glu Ala Lys Arg Leu Leu Leu Val 225 230
235 240 Met Thr Asp Gln Thr Ser His Leu Ala Leu Asp Ser
Lys Leu Ala Gly 245 250
255 Ile Val Val Pro Asn Asp Gly Asn Cys His Leu Lys Asn Asn Val Tyr
260 265 270 Val Lys Ser
Thr Thr Met Glu His Pro Ser Leu Gly Gln Leu Ser Glu 275
280 285 Lys Leu Ile Asp Asn Asn Ile Asn
Val Ile Phe Ala Val Gln Gly Lys 290 295
300 Gln Phe His Trp Tyr Lys Asp Leu Leu Pro Leu Leu Pro
Gly Thr Ile 305 310 315
320 Ala Gly Glu Ile Glu Ser Lys Ala Ala Asn Leu Asn Asn Leu Val Val
325 330 335 Glu Ala Tyr Gln
Lys Leu Ile Ser Glu Val Lys Val Gln Val Glu Asn 340
345 350 Gln Val Gln Gly Ile Tyr Phe Asn Ile
Thr Ala Ile Cys Pro Asp Gly 355 360
365 Ser Arg Lys Pro Gly Met Glu Gly Cys Arg Asn Val Thr Ser
Asn Asp 370 375 380
Glu Val Leu Phe Asn Val Thr Val Thr Met Lys Lys Cys Asp Val Thr 385
390 395 400 Gly Gly Lys Asn Tyr
Ala Ile Ile Lys Pro Ile Gly Phe Asn Glu Thr 405
410 415 Ala Lys Ile His Ile His Arg Asn Cys Ser
Cys Gln Cys Glu Asp Asn 420 425
430 Arg Gly Pro Lys Gly Lys Cys Val Asp Glu Thr Phe Leu Asp Ser
Lys 435 440 445 Cys
Phe Gln Cys Asp Glu Asn Lys Cys His Phe Asp Glu Asp Gln Phe 450
455 460 Ser Ser Glu Ser Cys Lys
Ser His Lys Asp Gln Pro Val Cys Ser Gly 465 470
475 480 Arg Gly Val Cys Val Cys Gly Lys Cys Ser Cys
His Lys Ile Lys Leu 485 490
495 Gly Lys Val Tyr Gly Lys Tyr Cys Glu Lys Asp Asp Phe Ser Cys Pro
500 505 510 Tyr His
His Gly Asn Leu Cys Ala Gly His Gly Glu Cys Glu Ala Gly 515
520 525 Arg Cys Gln Cys Phe Ser Gly
Trp Glu Gly Asp Arg Cys Gln Cys Pro 530 535
540 Ser Ala Ala Ala Gln His Cys Val Asn Ser Lys Gly
Gln Val Cys Ser 545 550 555
560 Gly Arg Gly Thr Cys Val Cys Gly Arg Cys Glu Cys Thr Asp Pro Arg
565 570 575 Ser Ile Gly
Arg Phe Cys Glu His Cys Pro Thr Cys Tyr Thr Ala Cys 580
585 590 Lys Glu Asn Trp Asn Cys Met Gln
Cys Leu His Pro His Asn Leu Ser 595 600
605 Gln Ala Ile Leu Asp Gln Cys Lys Thr Ser Cys Ala Leu
Met Glu Gln 610 615 620
Gln His Tyr Val Asp Gln Thr Ser Glu Cys Phe Ser Ser Pro Ser Tyr 625
630 635 640 Leu Arg Ile Phe
Phe Ile Ile Phe Ile Val Thr Phe Leu Ile Gly Leu 645
650 655 Leu Lys Val Leu Ile Ile Arg Gln Val
Ile Leu Gln Trp Asn Ser Asn 660 665
670 Lys Ile Lys Ser Ser Ser Asp Tyr Arg Val Ser Ala Ser Lys
Lys Asp 675 680 685
Lys Leu Ile Leu Gln Ser Val Cys Thr Arg Ala Val Thr Tyr Arg Arg 690
695 700 Glu Lys Pro Glu Glu
Ile Lys Met Asp Ile Ser Lys Leu Asn Ala His 705 710
715 720 Glu Thr Phe Arg Cys Asn Phe
725 47649PRTMus musculus 47Gln Leu Cys Thr Lys Asp Asn Val
Ser Thr Cys Gln Asp Cys Ile Arg 1 5 10
15 Ser Gly Pro Ser Cys Ala Trp Cys Gln Lys Leu Asn Phe
Thr Gly Arg 20 25 30
Gly Glu Pro Asp Ser Val Arg Cys Asp Thr Pro Glu Gln Leu Leu Leu
35 40 45 Lys Gly Cys Thr
Ser Glu Tyr Leu Val Asp Pro Lys Ser Leu Ala Glu 50
55 60 Ser Gln Glu Asp Lys Glu Arg Asp
Gln Arg Gln Leu Ser Pro Arg Asn 65 70
75 80 Val Thr Val Phe Leu Arg Pro Gly Gln Ala Ala Thr
Phe Lys Val Asp 85 90
95 Phe Gln Arg Thr Gln Asp Asn Ser Val Asp Leu Tyr Phe Leu Met Gly
100 105 110 Leu Ser Gly
Ser Ala Gln Gly His Leu Ser Asn Val Gln Thr Leu Gly 115
120 125 Ser Asp Leu Leu Lys Ala Leu Asn
Glu Ile Ser Arg Ser Gly Arg Ile 130 135
140 Gly Phe Gly Ser Ile Val Asn Met Thr Phe Gln His Ile
Leu Lys Leu 145 150 155
160 Thr Ala Asp Ser Ser Gln Phe Gln Arg Glu Leu Arg Lys Gln Leu Val
165 170 175 Ser Gly Lys Leu
Ala Thr Pro Lys Gly Gln Leu Asp Ala Val Val Gln 180
185 190 Val Ala Ile Cys Leu Gly Glu Ile Gly
Trp Arg Asn Gly Thr Arg Phe 195 200
205 Leu Val Leu Val Thr Asp Asn Asp Phe His Leu Ala Lys Asp
Lys Thr 210 215 220
Leu Gly Thr Arg Gln Asn Thr Ser Asp Gly Arg Cys His Leu Asp Asp 225
230 235 240 Gly Met Tyr Arg Ser
Arg Gly Glu Pro Asp Tyr Gln Ser Val Val Gln 245
250 255 Leu Ala Ser Lys Leu Ala Glu Asn Asn Ile
Gln Pro Ile Phe Val Val 260 265
270 Pro Ser Arg Met Val Lys Thr Tyr Glu Lys Leu Thr Thr Phe Ile
Pro 275 280 285 Lys
Leu Thr Ile Gly Glu Leu Ser Asp Asp Ser Ser Asn Val Ala Gln 290
295 300 Leu Ile Arg Asn Ala Tyr
Ser Lys Leu Ser Ser Ile Val Val Leu Asn 305 310
315 320 His Ser Thr Ile Pro Ser Ile Leu Lys Val Thr
Tyr Asp Ser Tyr Cys 325 330
335 Ser Asn Gly Thr Ser Asn Pro Gly Lys Pro Ser Gly Asp Cys Ser Gly
340 345 350 Val Gln
Ile Asn Asp Gln Val Thr Phe Gln Val Asn Ile Thr Ala Ser 355
360 365 Glu Cys Phe Arg Glu Gln Phe
Phe Phe Ile Gln Ala Leu Gly Phe Met 370 375
380 Asp Ser Val Thr Val Arg Val Leu Pro Leu Cys Glu
Cys Gln Cys Gln 385 390 395
400 Glu Gln Ser Gln His His Ser Leu Cys Gly Gly Lys Gly Ala Met Glu
405 410 415 Cys Gly Ile
Cys Arg Cys Asn Ser Gly Tyr Ala Gly Lys Asn Cys Glu 420
425 430 Cys Gln Thr Gln Gly Pro Ser Ser
Gln Asp Leu Glu Gly Ser Cys Arg 435 440
445 Lys Asp Asn Ser Ser Ile Met Cys Ser Gly Leu Gly Asp
Cys Ile Cys 450 455 460
Gly Gln Cys Glu Cys His Thr Ser Asp Ile Pro Asn Lys Glu Ile Tyr 465
470 475 480 Gly Gln Tyr Cys
Glu Cys Asp Asn Val Asn Cys Glu Arg Tyr Asp Gly 485
490 495 Gln Val Cys Gly Gly Pro Glu Arg Gly
His Cys Ser Cys Gly Arg Cys 500 505
510 Phe Cys Arg Tyr Ser Phe Val Gly Ser Ala Cys Gln Cys Arg
Met Ser 515 520 525
Thr Ser Gly Cys Leu Asn Asn Arg Met Val Glu Cys Ser Gly His Gly 530
535 540 Arg Cys Tyr Cys Asn
Arg Cys Leu Cys Asp Pro Gly Tyr Gln Pro Pro 545 550
555 560 Leu Cys Glu Lys Arg Pro Gly Tyr Phe His
Arg Cys Ser Glu Tyr Tyr 565 570
575 Ser Cys Ala Arg Cys Leu Lys Asp Asn Ser Ala Ile Lys Cys Arg
Glu 580 585 590 Cys
Trp Asn Leu Leu Phe Ser Asn Thr Pro Phe Ser Asn Lys Thr Cys 595
600 605 Met Thr Glu Arg Asp Ser
Glu Gly Cys Trp Thr Thr Tyr Thr Leu Tyr 610 615
620 Gln Pro Asp Gln Ser Asp Ile Asn Ser Ile Tyr
Ile Lys Glu Ser Leu 625 630 635
640 Val Cys Ala Glu Ile Ser Asn Thr Thr 645
4818PRTHomo sapiens 48Arg Gly Glu Ala Gln Val Trp Thr Gln Leu Leu
Arg Ala Cys Glu Glu 1 5 10
15 Arg Ala 4918PRTHomo sapiens 49Arg Gly Glu Ala Gln Val Trp Thr
Gln Leu Leu Arg Ala Leu Cys Glu 1 5 10
15 Arg Ala 5019PRTHomo sapiens 50Thr Asn Ser Thr Leu
Val Thr Thr Asn Val Thr Trp Gly Cys Gln Pro 1 5
10 15 Ala Pro Met 5119PRTHomo sapiens 51Thr
Asn Ser Thr Leu Val Thr Thr Asn Val Thr Trp Gly Ile Cys Pro 1
5 10 15 Ala Pro Met 5228PRTHomo
sapiens 52Cys Cys Val Arg Phe Gln Tyr Tyr Glu Asp Ser Ser Gly Lys Ser Ile
1 5 10 15 Leu Tyr
Val Val Glu Glu Pro Glu Cys Pro Lys Gly 20
25 5328PRTHomo sapiens 53Cys Val Val Arg Phe Gln Tyr Tyr Glu
Asp Ser Ser Gly Lys Ser Ile 1 5 10
15 Leu Tyr Val Val Glu Glu Pro Glu Cys Cys Lys Gly
20 25 5426PRTHomo sapiens 54Cys Cys Ile
Thr Phe Leu Ile Thr Thr Asp Asn Glu Gly Lys Thr His 1 5
10 15 Ser Ile Asn Glu Lys Asp Cys Pro
Lys Pro 20 25 5526PRTHomo sapiens 55Cys
Leu Ile Thr Phe Leu Ile Thr Thr Asp Asn Glu Gly Lys Thr His 1
5 10 15 Ser Ile Asn Glu Lys Asp
Cys Cys Lys Pro 20 25 5619PRTHomo
sapiens 56Cys Cys Leu Met Glu Gln Gln His Tyr Val Asp Gln Thr Ser Glu Cys
1 5 10 15 Phe Ser
Ser 5719PRTHomo sapiens 57Cys Ala Leu Met Glu Gln Gln His Tyr Val Asp Gln
Thr Ser Glu Cys 1 5 10
15 Cys Ser Ser 5862PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 58Gln Leu Leu Arg Ala Leu Glu Glu Arg
Ala Thr Gly Gly Leu Glu Asn 1 5 10
15 Leu Tyr Phe Gln Gly Gly Glu Asn Ala Gln Cys Glu Lys Glu
Leu Gln 20 25 30
Ala Leu Glu Lys Glu Asn Ala Gln Leu Glu Trp Glu Leu Gln Ala Leu
35 40 45 Glu Lys Glu Leu
Ala Gln Trp Ser His Pro Gln Phe Glu Lys 50 55
60 5962PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 59Val Val Glu Glu Pro Glu Cys Pro Lys
Gly Thr Ser Gly Leu Glu Asn 1 5 10
15 Leu Tyr Phe Gln Gly Gly Lys Asn Ala Gln Cys Lys Lys Lys
Leu Gln 20 25 30
Ala Leu Lys Lys Lys Asn Ala Gln Leu Lys Trp Lys Leu Gln Ala Leu
35 40 45 Lys Lys Lys Leu
Ala Gln Gly Gly His His His His His His 50 55
60 6051PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 60Glu Lys Gly Cys Gly Leu Gln Thr Leu
Phe Gln Gly Pro Leu Gly Ala 1 5 10
15 Gln Gly Glu Lys Glu Leu Gln Ala Leu Glu Lys Glu Asn Ala
Gln Leu 20 25 30
Glu Trp Glu Leu Gln Ala Leu Glu Lys Glu Leu Ala Gln His His His
35 40 45 His His His
50 6152PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 61Glu Lys Tyr Gly Cys Gly Leu Gln Thr Leu Phe
Gln Gly Pro Leu Gly 1 5 10
15 Ala Gln Gly Glu Lys Glu Leu Gln Ala Leu Glu Lys Glu Asn Ala Gln
20 25 30 Leu Glu
Trp Glu Leu Gln Ala Leu Glu Lys Glu Leu Ala Gln His His 35
40 45 His His His His 50
6253PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 62Glu Lys Tyr Lys Gly Cys Gly Leu Gln Thr Leu
Phe Gln Gly Pro Leu 1 5 10
15 Gly Ala Gln Gly Glu Lys Glu Leu Gln Ala Leu Glu Lys Glu Asn Ala
20 25 30 Gln Leu
Glu Trp Glu Leu Gln Ala Leu Glu Lys Glu Leu Ala Gln His 35
40 45 His His His His His 50
6354PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 63Glu Lys Tyr Lys Val Gly Cys Gly Leu Gln Thr
Leu Phe Gln Gly Pro 1 5 10
15 Leu Gly Ala Gln Gly Glu Lys Glu Leu Gln Ala Leu Glu Lys Glu Asn
20 25 30 Ala Gln
Leu Glu Trp Glu Leu Gln Ala Leu Glu Lys Glu Leu Ala Gln 35
40 45 His His His His His His
50 6455PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 64Glu Lys Tyr Lys Val His Gly Cys Gly
Leu Gln Thr Leu Phe Gln Gly 1 5 10
15 Pro Leu Gly Ala Gln Gly Glu Lys Glu Leu Gln Ala Leu Glu
Lys Glu 20 25 30
Asn Ala Gln Leu Glu Trp Glu Leu Gln Ala Leu Glu Lys Glu Leu Ala
35 40 45 Gln His His His
His His His 50 55 6551PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
65Glu Cys Gly Cys Gly Leu Gln Thr Leu Phe Gln Gly Pro Leu Gly Ala 1
5 10 15 Gln Gly Lys Lys
Lys Leu Gln Ala Leu Lys Lys Lys Asn Ala Gln Leu 20
25 30 Lys Trp Lys Leu Gln Ala Leu Lys Lys
Lys Leu Ala Gln His His His 35 40
45 His His His 50 6652PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
66Glu Cys Val Gly Cys Gly Leu Gln Thr Leu Phe Gln Gly Pro Leu Gly 1
5 10 15 Ala Gln Gly Lys
Lys Lys Leu Gln Ala Leu Lys Lys Lys Asn Ala Gln 20
25 30 Leu Lys Trp Lys Leu Gln Ala Leu Lys
Lys Lys Leu Ala Gln His His 35 40
45 His His His His 50 6753PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
67Glu Cys Val Ala Gly Cys Gly Leu Gln Thr Leu Phe Gln Gly Pro Leu 1
5 10 15 Gly Ala Gln Gly
Lys Lys Lys Leu Gln Ala Leu Lys Lys Lys Asn Ala 20
25 30 Gln Leu Lys Trp Lys Leu Gln Ala Leu
Lys Lys Lys Leu Ala Gln His 35 40
45 His His His His His 50
6854PRTArtificial SequenceDescription of Artificial Sequence Synthetic
polypeptide 68Glu Cys Val Ala Gly Gly Cys Gly Leu Gln Thr Leu Phe Gln
Gly Pro 1 5 10 15
Leu Gly Ala Gln Gly Lys Lys Lys Leu Gln Ala Leu Lys Lys Lys Asn
20 25 30 Ala Gln Leu Lys Trp
Lys Leu Gln Ala Leu Lys Lys Lys Leu Ala Gln 35
40 45 His His His His His His 50
6955PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 69Glu Cys Val Ala Gly Pro Gly Cys Gly Leu Gln
Thr Leu Phe Gln Gly 1 5 10
15 Pro Leu Gly Ala Gln Gly Lys Lys Lys Leu Gln Ala Leu Lys Lys Lys
20 25 30 Asn Ala
Gln Leu Lys Trp Lys Leu Gln Ala Leu Lys Lys Lys Leu Ala 35
40 45 Gln His His His His His His
50 55 7012PRTHomo sapiens 70Glu Lys Tyr Lys Val His
Asn Pro Thr Pro Leu Ile 1 5 10
716PRTArtificial SequenceDescription of Artificial Sequence Synthetic
peptide 71Glu Lys Tyr Gly Cys Gly 1 5
727PRTArtificial SequenceDescription of Artificial Sequence Synthetic
peptide 72Glu Lys Tyr Lys Gly Cys Gly 1 5
738PRTArtificial SequenceDescription of Artificial Sequence Synthetic
peptide 73Glu Lys Tyr Lys Val Gly Cys Gly 1 5
749PRTArtificial SequenceDescription of Artificial Sequence Synthetic
peptide 74Glu Lys Tyr Lys Val His Gly Cys Gly 1 5
7510PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 75Glu Lys Tyr Lys Val His Asn Gly Cys Gly 1
5 10 765PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 76Glu Cys Gly Cys Gly 1
5 776PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 77Glu Cys Val Gly Cys Gly 1 5
787PRTArtificial SequenceDescription of Artificial Sequence Synthetic
peptide 78Glu Cys Val Ala Gly Cys Gly 1 5
798PRTArtificial SequenceDescription of Artificial Sequence Synthetic
peptide 79Glu Cys Val Ala Gly Gly Cys Gly 1 5
809PRTArtificial SequenceDescription of Artificial Sequence Synthetic
peptide 80Glu Cys Val Ala Gly Pro Gly Cys Gly 1 5
8110PRTHomo sapiens 81Glu Cys Val Ala Gly Pro Asn Ile Ala Ala 1
5 10 824PRTUnknownDescription of Unknown
Integrin alpha sequence 82Arg Ala Leu Glu 1
837PRTUnknownDescription of Unknown Integrin beta sequence 83Pro Asp Ile
Leu Val Val Leu 1 5 848PRTUnknownDescription of
Unknown Integrin beta sequence 84Pro Asp Ile Leu Val Val Leu Leu 1
5 8529PRTHomo sapiens 85Leu Tyr Gly Ser Asn Ala Ser
Leu Ala Gln Val Val Met Lys Val Asp 1 5
10 15 Val Val Tyr Glu Lys Gln Met Leu Tyr Leu Tyr
Val Leu 20 25
8629PRTArtificial SequenceDescription of Artificial Sequence Synthetic
peptide 86Leu Tyr Gly Ser Asn Ala Ser Leu Ala Gln Val Val Met Lys Val
Asp 1 5 10 15 Val
Val Tyr Cys Lys Gln Met Leu Tyr Leu Tyr Val Leu 20
25 8729PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 87Leu Tyr Gly Ser Asn Ala Ser
Leu Ala Gln Val Val Met Lys Val Asp 1 5
10 15 Val Val Tyr Glu Cys Gln Met Leu Tyr Leu Tyr
Val Leu 20 25
8829PRTArtificial SequenceDescription of Artificial Sequence Synthetic
peptide 88Leu Tyr Gly Ser Asn Ala Ser Leu Ala Gln Val Val Met Lys Val
Asp 1 5 10 15 Val
Val Tyr Glu Lys Cys Met Leu Tyr Leu Tyr Val Leu 20
25 8929PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 89Leu Tyr Gly Ser Asn Ala Ser
Leu Ala Gln Val Val Met Lys Val Asp 1 5
10 15 Val Val Tyr Glu Lys Gln Cys Leu Tyr Leu Tyr
Val Leu 20 25 9029PRTHomo
sapiens 90Met Asp Arg Tyr Leu Ile Tyr Val Asp Glu Ser Arg Glu Cys Val Ala
1 5 10 15 Gly Pro
Asn Ile Ala Ala Ile Val Gly Gly Thr Val Ala 20
25 9129PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 91Met Asp Arg Tyr Leu Ile Tyr Val Asp Glu
Ser Arg Cys Cys Val Ala 1 5 10
15 Gly Pro Asn Ile Ala Ala Ile Val Gly Gly Thr Val Ala
20 25 9229PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 92Met
Asp Arg Tyr Leu Ile Tyr Val Asp Glu Ser Arg Glu Cys Val Ala 1
5 10 15 Cys Pro Asn Ile Ala Ala
Ile Val Gly Gly Thr Val Ala 20 25
9329PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 93Met Asp Arg Tyr Leu Ile Tyr Val Asp Glu Ser Arg
Glu Cys Val Ala 1 5 10
15 Gly Cys Asn Ile Ala Ala Ile Val Gly Gly Thr Val Ala
20 25 9429PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 94Met
Asp Arg Tyr Leu Ile Tyr Val Asp Glu Ser Arg Glu Cys Val Ala 1
5 10 15 Gly Pro Cys Ile Ala Ala
Ile Val Gly Gly Thr Val Ala 20 25
9556PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 95Tyr Glu Lys Gln Thr Gly Gly Leu Glu Asn Leu
Tyr Phe Gln Gly Gly 1 5 10
15 Glu Asn Ala Gln Cys Glu Lys Glu Leu Gln Ala Leu Glu Lys Glu Asn
20 25 30 Ala Gln
Leu Glu Trp Glu Leu Gln Ala Leu Glu Lys Glu Leu Ala Gln 35
40 45 Trp Ser His Pro Gln Phe Glu
Lys 50 55 9656PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 96Tyr Cys Lys Gln Thr
Gly Gly Leu Glu Asn Leu Tyr Phe Gln Gly Gly 1 5
10 15 Glu Asn Ala Gln Cys Glu Lys Glu Leu Gln
Ala Leu Glu Lys Glu Asn 20 25
30 Ala Gln Leu Glu Trp Glu Leu Gln Ala Leu Glu Lys Glu Leu Ala
Gln 35 40 45 Trp
Ser His Pro Gln Phe Glu Lys 50 55
9756PRTArtificial SequenceDescription of Artificial Sequence Synthetic
polypeptide 97Tyr Glu Lys Gly Cys Gly Gly Leu Glu Asn Leu Tyr Phe Gln
Gly Gly 1 5 10 15
Glu Asn Ala Gln Cys Glu Lys Glu Leu Gln Ala Leu Glu Lys Glu Asn
20 25 30 Ala Gln Leu Glu Trp
Glu Leu Gln Ala Leu Glu Lys Glu Leu Ala Gln 35
40 45 Trp Ser His Pro Gln Phe Glu Lys
50 55 9857PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 98Tyr Lys Val His Gly Thr
Gly Gly Leu Glu Asn Leu Tyr Phe Gln Gly 1 5
10 15 Gly Glu Asn Ala Gln Cys Glu Lys Glu Leu Gln
Ala Leu Glu Lys Glu 20 25
30 Asn Ala Gln Leu Glu Trp Glu Leu Gln Ala Leu Glu Lys Glu Leu
Ala 35 40 45 Gln
Trp Ser His Pro Gln Phe Glu Lys 50 55
9957PRTArtificial SequenceDescription of Artificial Sequence Synthetic
polypeptide 99Tyr Lys Val His Gly Cys Gly Gly Leu Glu Asn Leu Tyr Phe
Gln Gly 1 5 10 15
Gly Glu Asn Ala Gln Cys Glu Lys Glu Leu Gln Ala Leu Glu Lys Glu
20 25 30 Asn Ala Gln Leu Glu
Trp Glu Leu Gln Ala Leu Glu Lys Glu Leu Ala 35
40 45 Gln Trp Ser His Pro Gln Phe Glu Lys
50 55 10057PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 100Val Ala Gly Pro Asp
Thr Ser Gly Leu Glu Asn Leu Tyr Phe Gln Gly 1 5
10 15 Gly Lys Asn Ala Gln Cys Lys Lys Lys Leu
Gln Ala Leu Lys Lys Lys 20 25
30 Asn Ala Gln Leu Lys Trp Lys Leu Gln Ala Leu Lys Lys Lys Leu
Ala 35 40 45 Gln
Gly Gly His His His His His His 50 55
10157PRTArtificial SequenceDescription of Artificial Sequence Synthetic
polypeptide 101Cys Ala Gly Pro Asp Thr Ser Gly Leu Glu Asn Leu Tyr Phe
Gln Gly 1 5 10 15
Gly Lys Asn Ala Gln Cys Lys Lys Lys Leu Gln Ala Leu Lys Lys Lys
20 25 30 Asn Ala Gln Leu Lys
Trp Lys Leu Gln Ala Leu Lys Lys Lys Leu Ala 35
40 45 Gln Gly Gly His His His His His His
50 55 10257PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 102Val Ala Cys Pro Asp
Thr Ser Gly Leu Glu Asn Leu Tyr Phe Gln Gly 1 5
10 15 Gly Lys Asn Ala Gln Cys Lys Lys Lys Leu
Gln Ala Leu Lys Lys Lys 20 25
30 Asn Ala Gln Leu Lys Trp Lys Leu Gln Ala Leu Lys Lys Lys Leu
Ala 35 40 45 Gln
Gly Gly His His His His His His 50 55
10357PRTArtificial SequenceDescription of Artificial Sequence Synthetic
polypeptide 103Val Ala Gly Pro Asp Gly Cys Gly Leu Glu Asn Leu Tyr Phe
Gln Gly 1 5 10 15
Gly Lys Asn Ala Gln Cys Lys Lys Lys Leu Gln Ala Leu Lys Lys Lys
20 25 30 Asn Ala Gln Leu Lys
Trp Lys Leu Gln Ala Leu Lys Lys Lys Leu Ala 35
40 45 Gln Gly Gly His His His His His His
50 55 10410PRTHomo sapiens 104Val Val Tyr Glu
Lys Gln Met Leu Tyr Leu 1 5 10
1056PRTArtificial SequenceDescription of Artificial Sequence Synthetic
6xHis tag 105His His His His His His 1 5
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