Modified truncated complement system regulators

Abstract

Analogs of regulators of complement activation (RCA) proteins which have altered specificities and affinities for the targets C3b and/or C4b are described. These analogs are obtained by substituting amino acids which affect the complement inhibitory activities of these proteins, substituting, rearranging or adding SCRs (short consensus repeats) or SCR regions to the proteins, deleting amino acid sequences, and combinations thereof.

Description

RELATED APPLICATIONS

[0001] This application is a continuation of U.S. application Ser. No. 08/126,505, filed Sep. 24, 1993 (Now U.S. Pat. No. 6,897,290 B1), which is a continuation-in-part of U.S. application Ser. No. 07/695,514, filed May 3, 1991 (Now abandoned). The entire teachings of U.S. application Ser. No. 08/126,505 (Now U.S. Pat. No. 6,897,290 B1) are incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] The invention relates to modified and/or shortened forms of complement regulators derived from regulatory proteins of complement activation (RCA), especially CR1.

[0003] The complement system serves to aid in the removal of foreign substances and of immune complexes from animal hosts. This system and its regulation is reviewed by Hourcade, D., et al., Advances in Immunol (1989) 45: 381-416. Briefly, the complement system generates, either by a "classical pathway" or an "alternative pathway," C3b which binds to target immune complexes or foreign substances and marks them for destruction or clearance. C3b is generated from its precursor C3 by the proteolytic enzymes collectively designated "C3 convertase." One form of C3 convertase is generated in the classical pathway by the association of the proteins C4b and C2a. The other form is generated in the alternative pathway by association of C3b and Bb. Both C3 convertases can associate with an additional C3b subunit to form the C5 convertases, C3bBbC3b and C4bC2aC3b, both of which are active in the production of the C5-C9 membrane attack complex which can cause cell lysis, and the production of C5a, a major proinflammatory agent.

[0004] Both C3b, and less directly, C4b, are agonists in the complement system. This is shown in the diagram in FIG. 1.

[0005] The complement system is regulated via a number of interrelated mechanisms. There are two general mechanisms for inhibition of the destructive components of the complement system. The first mechanism is generally reversible, facilitating the dissociation of the C3 convertases--i.e., C3b from Bb and C4b from C2a. Facilitation of dissociation is sometimes known as decay acceleration. The dissociation may also involve reversible binding of the antagonist proteins to C3b or C4b components, thus preventing their reassociation. The other mechanism, which is an irreversible inactivation process, results from proteolytic cleavage of the C3 convertase components C3b or C4b by the serine protease factor I. This proteolytic cleavage occurs only in the presence of a cofactor. Both general regulatory mechanisms, the facilitation of dissociation of C3b and C4b and the inactivation of C3B and C4b through cleavage by factor I, also apply to the inhibition of the alternative pathway C5 convertase (C3bBbC3b) and the classical pathway C5 convertase (C4bC2aC3b).

[0006] The proteins encoded by a region of the genome which is designated the "regulators of complement activation" (RCA) gene cluster are involved in both of the foregoing mechanisms. Currently, it is known that at least six complement proteins are encoded by this region. These are summarized in Table 1.

TABLE-US-00001 TABLE 1 RCA Proteins: Functional Profile Primary Decay Acceleration Cofactor Name Ligand(s) (Dissociation) Activity CR1 C3b/C4b + + MCP C3b/C4b - + DAF C3b/C4b + - C3 Convertases C4bp C4b + + Factor H C3b + + CR2 C3dg - ND

[0007] These proteins share certain structural similarities which are further described below.

[0008] The reversible binding to C4b or C3b to dissociate the C3 convertases is effected by two plasma proteins designated C4 binding protein (C4bp) and factor H, and by two membrane proteins designated decay acceleration factor (DAF) and complement receptor 1 (CR1). Reversible binding to C4b is effected by C4bp, DAF and CR1 while reversible binding to C3b is effected by factor H, DAF and CR1.

[0009] The irreversible inactivation of the C3 convertases resulting from proteolytic cleavage of convertase components C3b or C4b by the enzyme factor I can occur by virtue of cofactor activity effected by the above-mentioned factor H and C4bp in the plasma and by CR1 and membrane cofactor protein (MCP) at the cell surface. Cofactor activity for cleavage of C3b is effected by factor H, CR1 and MCP while cofactor activity for cleavage of C4b is effected by C4bp, CR1 and MCP. It is also possible that the sixth protein, complement receptor 2 (CR2), has this cofactor activity at the cell surface.

[0010] In summary, of the six proteins encoded by the RCA gene cluster, factor H, C4bp, and CR1 have both reversible dissociation activity and irreversible cofactor activity; DAF has only reversible dissociation activity, and MCP and possibly CR2 have only irreversible cofactor activities. CR1, DAF and MCP interact with both C3b and C4b; C4bp interacts primarily with C4b, and factor H interacts primarily with C3b.

[0011] The cDNAs corresponding to CR1, CR2, DAF, MCP, C4bp, and factor H have all been obtained and sequenced. Evaluation of these comparative sequences has lead to the alignment set forth in FIG. 2A which shows the organization of the RCA proteins into short consensus repeat ("SCR") containing and non-SCR-containing regions with the N-terminal ends at the left. In this figure, TM refers to transmembrane domain, C to cytoplasmic domain, 0 to 0-linked glycosylation domain, G to glycolipid anchor, U to domain with unknown significance and D to a disulfide bridge-containing domain.

[0012] There is considerable uniformity among the RCA family of proteins. All of them are composed of 60-70 amino acid repeating units commonly designated "short consensus repeats" (SCRs). Each SCR shares a number of invariant or highly conserved amino acid residues with other SCRs in the same protein or SCRs in other family members. Those members of the family which are membrane bound also have at their C termini either transmembrane regions and intracellular regions or a glycolipid anchor.

[0013] The SCRs form the extracellular portions of those members of the family which are membrane-bound and almost all of the protein structure in the secreted members. Two covalently-crosslinked cysteine pairs establish two loops within each SCR. The smallest family members are DAF and MCP; each contains four SCRs followed by an 0-linked glycosylation region. DAF is terminated with a glycolipid anchor while MCP ends with an extracytoplasmic segment of unknown significance, a transmembrane region and an intracellular domain. Of the secreted members of the family, factor H contains twenty SCRs, while the native form of C4bp is an association of seven subunits of eight SCRs (the C4bp alpha chains) and one subunit of three SCRs (the C4bp beta chain). Both C4bp chains conclude with non-SCR domains that interconnect the chains through disulfide linkages. CR2 contains sixteen SCRs, a transmembrane region and an intracellular domain. The most common polymorphic form of CR1 contains four repeating units of seven similar SCRs (long homologous repeats or LHRS) numbered 1-28, followed by an additional two SCRs designated 29 and 30, a transmembrane region and an intracellular region.

[0014] Klickstein, L. B., et al., J. Exy. Med. (1988) 168:1699-1717, described the identification of distinct C3b and C4b recognition sites in CR1 using deletion mutagenesis. They concluded that a singled primary C4b binding site is located in SCR 1-2 (SEQ ID NOS: 1 and 3), while two major C3b binding sites are located in SCR 8-9 (SEQ ID NOS: 2 and 4), and SCR 15-16. C3b cofactor activity was localized to SCR 8-9 and SCR 15-16. More recently it has been shown the CR1 active site containing SCR 8-9 extends to SCR 10, and by analogy, the active site that contains SCR 15-16 (which is only one amino acid different than SCR 8-9) must extend to SCR 17. (Kalli, et al., J. Exp. Med. 174, 1451-1460 (1991); Makrides, et al., J. Biol. Chem. 267, 24754-24761 (1992)). The CR1 active site containing SCR 1-2 extends to SCR 3 and/or 4, as reported by Makrides, et al., (1992).

[0015] The murine C4bp binding site, and presumably the C4b cofactor and C4bC2a decay acceleration active sites, was reported to extend from SCRs 1-3 in the alpha chain by Ogata, et al., J. Immunology 150, 2273-2280 (1993).

[0016] The factor H binding site, and probably the C3b cofactor and C3bBb decay acceleration active sites, lies within the first five SCRs. The CR2 binding site for C3b proteolytic products extends through the first two SCRs, Kalli, et al., J. Immunology 147: 509-594 (1991); Carel, et al., J. Biol. Chem. 265: 12293-12299 (1990).

[0017] The MCP active sites extend through all four SCRs: SCRs 2-4 are required for C3b and C4b cofactor activity. SCR 1 appears unnecessary for C3b cofactor activity and binding but appears necessary for efficient C4b cofactor activity and binding, as reported by Adams, et al., J. Immunology 147:3005-3011 (1991). The DAF active sites extend through SCRs 2-4, as reported by Coyne, et al., J. Immunology 149: 2906-2913 (1992).

[0018] Hourcade, D., et al., J. Exp. Med. 168: 1255-1270 (1988), described a cDNA clone designated CR1-4 that encodes the first eight and one-half amino terminal SCRs of CR1. This cDNA was transfected into COS cells which resulted in the synthesis of a secreted truncated form of CR1 with a molecular weight of 78 kd (Krych, N. et al., Proc. Natl. Acad. Sci. USA 88:4353-4357 (1991). This shortened form of the protein, as shown herein below, binds mainly C4b. This shortened form has now been determined to have C4b cofactor activity, as described herein.

[0019] The multiple binding sites of CR1 can cooperate in their interactions with C3b-containing targets. In vitro, CR1 binds C3-C3b dimers much more tightly than C3b monomers because binding to dimers can occur simultaneously at two sites in the same CR1 molecule, as reported by Wong and Farrell, J. Immunol. (1991) 146:656; Ross and Medof Adv. Immunol. (1985) 37:217). Deletion of one of the two primary C3b binding sites can reduce the binding of CR1 to C3-C3b by a factor of ten, as reported by Wong and Farrell, J. Immunol. (1991) 146:656. It is likely that the primary C4b binding site also cooperates with the primary C3b binding sites in interactions with targets that contain both C3b and C4b. These effects have an important consequence in vivo: CR1 has a higher affinity for targets densely coated with C3b and with targets densely coated with C3b plus C4b.

[0020] The C5 convertases, which are important in the stimulation of inflammation and in lysis of some target cells, are composed of multiple CR1 ligands: The classical C5 convertase contains C3b and C4b (C4bC3bC2a) while the alternative pathway C5 convertase contains two C3b proteins (C3bC3bBb). Inactivation of the C5 convertases by CR1 can also involve cooperation between more than one CR1 binding site. Wong and Farrell. J. Immunol. (1991) 146:656 showed that more than one CR1 C3b binding site may be essential for effective inhibition of alternative pathway C3 and C5 convertases.

[0021] The proteins encoded by the RCA gene cluster can be prepared recombinantly and used in diagnosis and therapy for the regulation of the complement system. The problems of transplantation of xenografts are reviewed by Platt, J. L., et al., in Immunology Today (1990) 11:450-457. Evidence has accumulated that the immediate hyperacute rejection of discordant xenografts is caused by recipient complement activity. Transgenic animals expressing human complement regulators (such as DAF or MCP) on cell surfaces could be an abundant source of organs that would be protected from hyperacute rejection in human recipients. A soluble complement inhibitor could also play a role in protecting xenografts from complement-mediated rejection.

[0022] The ability of a recombinant soluble form of CR1 to inhibit inflammation in the reversed passive Arthus reaction in rats was described by Yeh, C. G., et al., J. Immunol (1991) 146:250-256. This soluble CR1 was obtained from Chinese hamster ovary (CHO) cells expressing a CR1 genetic construct which has been mutated to remove the transmembrane and cytoplasmic domains. The ability of a similar soluble CR1, produced recombinantly in CHO cells, to inhibit post-ischemic myocardial inflammation and necrosis in rats was reported by Weissman, H. F., et al., Science (1990) 249: 146-151.

[0023] Proteins related to the RCA proteins have also been shown to be produced by viruses, presumably as a mechanism whereby infection by the virus can be facilitated, as reported by Kotwaal, J., et al., Nature (1988) 335:176-178; McNearney, T. A., J Exp Med (1987) 16:1525-1535.

[0024] Complete inhibition of the complement system on a long-term basis is not likely to be desirable in most individuals. In some cases of autoimmune disease, inhibition of the classical pathway alone may be sufficient. In the case of the xenograft transplants, however, stringent inhibition of both pathways may be important. Similar stringency may be required for other applications. Accordingly, alternative modulators of the complement system with regulatable binding activities would be desirable.

[0025] It is therefore an object of the present invention to provide modified complement regulators which can be administered in soluble form for treatment of inflammatory disorders or to reduce an individuals ability to reject foreign materials.

[0026] It is a further object of the present invention to provide modified complement regulators which are shorter and more easily and economically produced than the more complex naturally occurring proteins.

[0027] It is another object of the present invention to provide complement regulators which combine the activities of different complement regulators to provide enhanced capability of inhibiting complement proteins specifically and systemically, both in the classical and alternative pathways.

SUMMARY OF THE INVENTION

[0028] Analogs of RCA-encoded proteins which are modified full-length or truncated forms of these regulatory proteins are described and demonstrated to inhibit complement. These analogs include RCA proteins having site-specific mutations, RCA protein hybrids which contain one or more SCRs from more than one RCA protein, modifications of RCA recombinants in which SCRs are arranged in different orders, and truncated versions of RCA proteins containing as few three SCRs. The modified proteins retain complement inhibitory activity, which may be altered in specificity, affinity, or mechanism.

[0029] The modified proteins are demonstrated by the .DELTA.SCR11, subst1 mutation of CR1-4 (8,9) which carries only three complete SCRs (amino acids 1-194, SCR 1-3) and has C3b activity; the 1ar, 5r, 8br mutation of CR1-4 (8,9) which has accentuated C3b and C4b activities; a three SCR form composed of the first 194 amino acids of 1ar, 5r, 8br; mutants 10, 10, 10, 11, 11c, 8a, 6b, 14, and 15, which are mutations of CR1-4 with enhanced activities; and CR1-4 stop7, CR1-4 (8,9) stop7, and 1ar, 5r, 8br stop 7, which are all shorter (7 SCR) forms of their respective parent proteins (CR1-4, CR1-4 (8,9) and 1ar, 5r, 8br, respectively).

[0030] These analogs are useful in controlling the complement system, and thus may be useful in the treatment of autoimmune diseases, the suppression of rejection of transplants, in diagnosis and the reduction in tissue damage associated with myocardial infarctions and cerebral vascular accidents. They may also play a role in the diagnosis of conditions associated with complement activation and immune complex formation.

BRIEF DESCRIPTION OF THE DRAWINGS

[0031] FIG. 1 is a schematic of the classical and alternate complement pathways.

[0032] FIGS. 2A and 2B compare the amino acid sequences of SCR-1 and SCR-8 (FIG. 2A) and SCR-2 and SCR-9 (FIG. 2B) of full-length CR1, indicating the substitutions made in SCR-1 and SCR-2 based on the amino acid residue present in corresponding SCR, as described in Examples 2 and 3 and Table 2.

[0033] FIGS. 3A and 3B compare the amino acid sequences of SCR-1 and SCR-8 (SEQ ID NOS: 1 and 2, respectively) (FIG. 3A) and SCR-2 and SCR-9 (SEQ ID NOS: 3 and 4 respectively) (FIG. 3B) of full-length CR1. The amino acid sequences of SCR 15-16 are identical to those of SCR 8-9, with the exception of a T at position 110. The enumeration is consistent with that of Hourcade et al., J. Exp. Med. (1988) 168:1255-1270, with the first amino acid (Q) of the mature receptor designated as amino acid 1. The corresponding position in CR1 SCR-8 is similarly designated.

DETAILED DESCRIPTION OF THE INVENTION

[0034] Described herein is a family of RCA protein analogs which can be used to modulate the complement system. The analogues can be used to alter the binding specificity of the members of this protein family in both membrane-bound and soluble forms.

[0035] As used herein, "RCA proteins" refers to proteins encoded by the gene cluster at the genetic region1q32, which encodes six known proteins effective in complement regulation: factor H, C4bp, CR1, CR2, DAF and MCP. In addition, apparent coding regions similar to the amino terminal coding region of the CR1 gene and the MCP gene have been located in this cluster, although it is unclear whether these sequences are expressed (Hourcade, D., et al., J. Biol. Chem. 265:974-980 (1990); Hourcade, D., et al., Genomics 12:289-300 (1992)). The term "RCA proteins" also refers to a group of proteins which include short consensus repeats (SCR) which are homologous to SCR in CR1, CR2, MCP, DAF, factor H, and C4bp.

[0036] The modified proteins described herein are collectively referred to as "modified RCA proteins". These include truncated, hybrid and recombined forms of the RCA proteins. "Truncated" proteins are shorter versions of the RCA proteins, typically modified so as to remove the C-terminal regions which effect membrane binding or secretion and sometimes modified further by deletion of one or more SCRs. "Hybrid" proteins are RCA proteins that are composed of portions, i.e., the SCRs, of one RCA protein combined with SCRs of one or more other RCA proteins. "Recombined" forms are those wherein the SCRs of an RCA protein are rearranged in a new order. Also included are proteins having site specific substitutions and deletions of one or more amino acids. "Modified RCA proteins" include proteins which result from combinations of these changes.

[0037] CR1 contains 30 SCR, followed by transmembrane and cytoplasmic regions. CR2 contains 16 SCR, followed by transmembrane and cytoplasmic regions. DAF and MCP each contain four SCR, followed by glycolipid anchor regions. The C4 binding protein is a more complex protein, having seven alpha subunits and one beta subunit. The alpha subunit consists of eight SCR and the beta subunit consists of three SCR. Factor H consists of twenty SCR.

[0038] In some embodiments, modifications are made using corresponding SCRs of the protein as sites for alteration. By "corresponding SCR" is meant the most highly homologous SCR as judged by the amino acid sequences of the protein. Exon structure can in some cases facilitate this assignment. As noted above, SCRs 1-3 of CR1 correspond to SCRs 2-4 of DAF. SCRs 1-3 of factor H, CR1, C4bp and MCP are corresponding sequences among these proteins. CR1 is organized into a series of long homologous repeats (LHRS) containing 7 SCRs so that CR1 SCRs 1-7 correspond to CR1 SCRs 8-14; 15-21; and 22-28. CR2 is organized into a series of long homologous repeats of 4 SCRs in length. SCRs 1-2 of CR1 correspond to SCRs 3-4, SCRs 7-8, SCRs 11-12 and SCRs 15-16 of CR2.

[0039] These proteins are characterized by C4b-binding activity, C3b-binding activity, C4b cofactor activity, and C3b cofactor activity. In general, it takes two to three SCRs for each activity. Activities which are biologically important include decay acceleration or dissociation, C3b cofactor activity and C4b cofactor activity. Cofactor activity requires binding but binding alone may not be sufficient for cofactor activity.

[0040] It is generally accepted that CR1 C4b binding and cofactor activity requires SCRs 1, 2 and 3, 8, 9, and 10, or 15, 16, and 17, which are corresponding regions of the protein. C3b binding and cofactor activity requires SCRs 8, 9, and 10, or 15, 16, and 17, which are corresponding regions of the protein. MCP C4b binding and cofactor activity requires SCRs 1, 2, 3, and 4. MCP C3b binding requires SCRs 3 and 4; cofactor activity requires SCRs 2, 3, and 4. C4bp C4b binding and cofactor activity requires SCRs 1, 2, and 3. DAF decay accelerating activity requires SCRs 2, 3, and 4. Factor H C3b binding activity is mediated by the first five SCRs.

[0041] Based on these discoveries, it is possible to design a more potent soluble complement inhibitor by modifying corresponding regions to increase affinity for C4b and C3b or to design soluble complement inhibitors that specifically inhibit one part of the complement system. These modifications can be in the form of specific substitutions of amino acids that alter C3b or C4b binding within corresponding SCRs of CR1 or other RCA proteins, or substitution of SCRs from one protein into another.

Substitution of SCR Regions from One Regulatory Protein into a Second Regulatory Protein

[0042] The identification of the amino acid sequences essential (or refractory) to binding to C4b and C3b and C4b and C3b cofactor activity permits transposition of similar sequences into corresponding regions of the same protein or corresponding regions of other family members or alteration of sequences which bind C3b and C4b so as to alter their affinities. Corresponding regions have been identified by degree of amino acid sequence homology.

[0043] In the case of CR1, four corresponding regions of interest are SCRs 1-3, SCRs 8-10, SCRs 15-17 and SCRs 22-24. The SCR portions 2-4 for DAF correspond to 1-3, 8-10, 15-17 and 22-24 for CR1. Substitution of portions of DAF with homologous CR1 sequences provides forms of DAF with cofactor activity and/or binding activity, such as is exhibited by CR1. Similarly, substitutions of portions of MCP with homologous sequences provides forms of MCP with increased binding affinity and cofactor activity and/or increased dissociation activity.

[0044] Specific Amino Acid Substitutions

[0045] Addition of Binding Sites

[0046] Specific amino acids are selected for substitution based on studies that elucidate their roles in complement regulation in specific active sites. Substitution can be employed in order to alter the activity of additional RCA active sites in the same or other proteins. In this manner, binding and cofactor sites can be added to SCRs not normally contributing directly to binding capacity.

[0047] The standard one letter abbreviations for amino acids are used herein.

[0048] For example, the C3b and C4b binding and f 4 cofactor sequence in CR1, N-A-A-H-W-S-T-K-P-P-I-C-Q (amino acids 49-61 of SEQ ID NO: 4) can be transferred to corresponding locations or to locations referenced to conserved amino acids in alternative SCRs to confer C3b binding. Conversely, the homologous sequence in SCR-2 of CR1, i.e., D-T-V-W-D-N-E-T-P-I-C-D (amino acids 49-61 of SEQ ID NO: 3) can be transferred by substitution to other locations in C3b-binding SCRs in order to decrease C3b and C4b binding and cofactor activity. The C4b binding regions are shown to be associated with three separate critical locations in SCR-1 and SCR-2 of CR1 in the proximity of amino acids 35, 64-65, and 92-94. Alterations in amino acid sequences of the corresponding SCRs in CR1 or in additional RCA family members or their truncated, hybrid, or recombined forms in these positions alter C4b binding and cofactor activities and in at least one case alter C3b binding and cofactor activities.

[0049] Substitution of Similar Amino Acids

[0050] Structurally similar amino acids can be substituted in such transfers for some of the specified amino acids. Structurally similar amino acids include: (I, L, V); (F, Y); (K, R); (Q, N); (D, E); and (G, A).

[0051] Deletion of Amino Acids

[0052] It also may be advantageous to delete amino acids from specific active sites in order to alter or enhance complement regulatory activity.

[0053] Construction of Truncated Forms

[0054] In some embodiments, it will be advantageous to delete a specific activity by deletion of a region known to have a particular activity. It may also be desirable to delete the region of the protein which anchors the naturally occurring protein to the cell surface, for example, the transmembrane and cytoplasmic regions or the glycolipid anchor region.

[0055] Modifications which Enhance Cofactor Activity

[0056] In general, either C3b or C4b cofactor activity can be enhanced by substitutions which increase the binding activity of the other factor, i.e., to increase C4b cofactor activity, amino acids are substituted into the modified protein which increase C3b binding and vice versa. This is demonstrated in the examples, specifically by the mutants shown in Table 2. An example of a single amino acid substitution that enhances both C4b cofactor activity and C3b cofactor activity is CR1 mutant 6b: changing the G at 79 to D increases C4b cofactor, as well as C3b binding and cofactor activity.

[0057] Preparation of the Analogs

[0058] The modified proteins described herein are most conveniently prepared using recombinant techniques, although in some cases they can be prepared by enzymatic cleavage, for example, to yield truncated or soluble forms of the naturally occurring proteins. The genes encoding the various members of the RCA protein family are of known sequence and are published.

[0059] cDNA encoding CR1 has been described by Klickstein, L. B., et al., J. Exp. Med. (1987) 165: 1095, Klickstein, L. B., et al., J. Exp. Med. (1988) 168:1699; Hourcade, D., et al., J. Exp. Med. (1988) 168: 1255, the teachings of which are incorporated by reference. SEQ ID NO: 12 is the nucleotide sequence and SEQ ID NO: 13 is the amino acid sequence for CR1. Sequence ID No 14 is the nucleotide sequence and SEQ ID NO: 15 is the amino acid sequence for CR2. SEQ ID NO: 16 is the nucleotide sequence and SEQ ID NO: 17 is the amino acid sequence for DAF. SEQ ID NO: 18 is the nucleotide sequence and SEQ ID NO: 9 is the amino acid sequence for MCP.

[0060] The cDNA encoding CR2 has been described by Moore, M. D., et al., Proc. Natl. Acad. Sci. USA (1987) 84:9194, and by Weiss, J. J., et al., J. Exp. Med. (1988) 167:1047, the teachings of which are incorporated by reference.

[0061] The cDNA encoding DAF has been described by Caras, I. W., et al., Nature (1987) 25:545, and by Medof, M. E., et al., Proc. Natl. Acad. Sci. USA (1987) 84:2007, the teachings of which are incorporated by reference.

[0062] The cDNA encoding MCP has been described by Lublin, D. M., et al., J. Exp. Med. (1988) 168:181, the teachings of which are incorporated by reference.

[0063] The cDNA encoding the C4bp alpha chain has been described by Chung, L. P., et al., Biochem. J. (1985) 230:133, and the cDNA encoding the C4bp beta chain has been described by Hillarp, A., and Dahlback, B., Proc. Natl. Acad. Sci. USA (1990) 87:1183, the teachings of which are incorporated by reference.

[0064] The cDNA encoding factor H has been described by Ripoche, J., et al., Biochem. J. (1988) 249:593, the teachings of which are incorporated by reference.

[0065] Since the cDNAs encoding these proteins are known and the amino acid sequences have been deduced, comparison of corresponding regions of the various proteins of the member families has been possible. In addition, the availability of the cDNA sequence makes possible the preparation of genetic constructs encoding truncated forms and other modified forms of the proteins using standard site-directed mutagenesis techniques, such as those described by Kunkel, T. A., et al., Methods Enzymol (1987) 154: 367-382.

[0066] Accordingly, the first step in the preparation of the analogs requires identification of the corresponding region of the target protein through sequence homology and site-directed mutagenesis in this region of the gene to alter C4b or C3b binding properties.

[0067] After the gene encoding the analog is prepared, the modified gene is expressed using standard recombinant techniques. The gene sequence is ligated into a suitable expression vector under the control of sequences known to be appropriate to the desired host. Production of recombinant proteins in microbial systems such as E. coli, B. subtilis, various strains of yeasts, and other fungi, such as Aspergillus, is well known. It may be advantageous to produce the desired analogs in cells of higher organisms as well, such as the standard BPV/C127 system, the Baculovirus/insect cell system, CHO cells, COS cells, and other mammalian cells, or in transgenic animals. Standard expression systems in various cell lines are well known and standard in the art.

[0068] Transgenic animals can be constructed for several species. The gene is placed under the control of a suitable promoter, for example, the metallothionine promoter or a tissue specific promoter, and the gene microinjected into an embryo, which is then implanted into a surrogate mother. Production in transgenic animals is important in the context of preparing transplants for use in other species.

[0069] Construction of Transgenic Animals.

[0070] Animal Sources

[0071] Animals suitable for transgenic experiments can be obtained from standard commercial sources. These include animals such as mice and rats for testing of genetic manipulation procedures, as well as larger animals such as pigs, cows, sheep, goats, and other animals that have been genetically engineered using techniques known to those skilled in the art. These techniques are briefly summarized below based principally on manipulation of mice and rats.

[0072] Microinjection Procedures

[0073] The procedures for manipulation of the embryo and for microinjection of DNA are described in detail in Hogan et al. Manipulating the mouse embryo, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1986), the teachings of which are incorporated herein. These techniques are readily applicable to embryos of other animal species, and, although the success rate is lower, it is considered to be a routine practice to those skilled in this art.

[0074] Transgenic Animals

[0075] Female animals are induced to superovulate using methodology adapted from the standard techniques used with mice, that is, with an injection of pregnant mare serum gonadotrophin (PMSG; Sigma) followed 48 hours later by an injection of human chorionic gonadotrophin (hCG; Sigma). Females are placed with males immediately after hCG injection. Approximately one day after hCG, the mated females are sacrificed and embryos are recovered from excised oviducts and placed in Dulbecco's phosphate buffered saline with 0.5% bovine serum albumin (BSA; Sigma). Surrounding cumulus cells are removed with hyaluronidase (1 mg/ml). Pronuclear embryos are then washed and placed in Earle's balanced salt solution containing 0.5% BSA (EBSS) in a 37.5.degree. C. incubator with a humidified atmosphere at 5% CO.sub.2, 95% air until the time of injection.

[0076] Randomly cycling adult females are mated with vasectomized males to induce a false pregnancy, at the same time as donor females. At the time of embryo transfer, the recipient females are anesthetized and the oviducts are exposed by an incision through the body wall directly over the oviduct. The ovarian bursa is opened and the embryos to be transferred are inserted into the infundibulum. After the transfer, the incision is closed by suturing.

[0077] Embryonic Stem (ES) Cell Methods

[0078] Introduction of cDNA into ES Cells:

[0079] Methods for the culturing of ES cells and the, subsequent production of transgenic animals, the introduction of DNA into ES cells by a variety of methods such as electroporation, calcium phosphate/DNA precipitation, and direct injection are described in detail in Teratocarcinomas and embryonic stem cells, a practical approach, ed. E. J. Robertson, (IRL Press 1987), the teachings of which are incorporated herein. Selection of the desired clone of transgene-containing ES cells is accomplished through one of several means. In cases involving sequence specific gene integration, a nucleic acid sequence for recombination with the gene of interest or sequences for controlling expression thereof is co-precipitated with a gene encoding a marker such as neomycin resistance. Transfection is carried out by one of several methods described in detail in Lovell-Badge, in Teratocarcinomas and embryonic stem cells, a practical approach, ed. E. J. Robertson, (IRL Press 1987) or in Potter et al Proc. Natl. Acad. Sci. USA 81, 7161 (1984). Calcium phosphate/DNA precipitation, direct injection, and electroporation are the preferred methods. In these procedures, a number of ES cells, for example, 0.5.times.10.sup.6, are plated into tissue culture dishes and transfected with a mixture of the linearized nucleic acid sequence and 1 mg of pSV2neo DNA (Southern and Berg, J. Mol. Appl. Gen. 1:327-341 (1982)) precipitated in the presence of 50 mg lipofectin in a final volume of 100 .mu.l. The cells are fed with selection medium containing 10% fetal bovine serum in DMEM supplemented with an antibiotic such as G418 (between 200 and 500 .mu.g/ml). Colonies of cells resistant to G418 are isolated using cloning rings and expanded. DNa is extracted from drug resistant clones and Southern blotting experiments using the nucleic acid sequence as a probe are used to identify those clones carrying the desired nucleic acid sequences. In some experiments, PCR methods are used to identify the clones of interest.

[0080] DNA molecules introduced into ES cells can also be integrated into the chromosome through the process of homologous recombination, described by Capecchi, (1989). Direct injection results in a high efficiency of integration. Desired clones are identified through PCR of DNA prepared from pools of injected ES cells. Positive cells within the pools are identified by PCR subsequent to cell cloning (Zimmer and Gruss, Nature 338, 150-153 (1989)). DNA introduction by electroporation is less efficient and requires a selection step. Methods for positive selection of the recombination event (i.e., neo resistance) and dual positive-negative selection (i.e., neo resistance and ganciclovir resistance) and the subsequent identification of the desired clones by PCR have been described by Joyner et al., Nature 338, 153-156 (1989) and Capecchi, (1989), the teachings of which are incorporated herein.

[0081] Embryo Recovery and ES Cell Injection

[0082] Naturally cycling or superovulated females mated with males are used to harvest embryos for the injection of ES cells. Embryos of the appropriate age are recovered after successful mating. Embryos are flushed from the uterine horns of mated females and placed in Dulbecco's modified essential medium plus 10% calf serum for injection with ES cells. Approximately 10-20 ES cells are injected into blastocysts using a glass microneedle with an internal diameter of approximately 20 .mu.m.

[0083] Transfer of Embryos to Pseudolpregnant Females

[0084] Randomly cycling adult females are paired with vasectomized males. Recipient females are mated such that they will be at 2.5 to 3.5 days post-mating (for mice, or later for larger animals) when required for implantation with blastocysts containing ES cells. At the time of embryo transfer, the recipient females are anesthetized. The ovaries are exposed by making an incision in the body wall directly over the oviduct and the ovary and uterus are externalized. A hole is made in the uterine horn with a needle through which the blastocysts are transferred. After the transfer, the ovary and uterus are pushed back into the body and the incision is closed by suturing. This procedure is repeated on the opposite side if additional transfers are to be made.

[0085] Identification of Transgenic Animals

[0086] Samples (1-2 cm of mouse tails) are removed from young animals. For larger animals, blood or other tissue can be used. To test for chimeras in the homologous recombination experiments, i.e., to look for contribution of the targeted ES cells to the animals, coat color has been used in mice, although blood could be examined in larger animals. DNA is prepared and analyzed by both Southern blot and PCR to detect transgenic founder (F.sub.0) animals and their progeny (F.sub.1 and F.sub.2).

[0087] Once the transgenic animals are identified, lines are established by conventional breeding and used as the donors for tissue removal and implantation using standard techniques for implantation into humans.

Purification of Analogs

[0088] The analogs recombinantly produced in culture or animals can be purified from the cell culture using standard purification techniques such as chromatography, for example, immunoaffinity or ion-exchange chromatography, and electrophoresis, generally using the same procedures as have been published for use in purifying the naturally occurring material.

[0089] While recombinant production of the analogs is the most convenient and practical method of preparing the proteins, it may also be desirable to synthesize the analogs using protein synthesis techniques, such as standard solid-phase peptide synthesis technology. This approach may be suitable in particular in the case of truncated forms of the RCA protein family having modified amino acid sequences, especially in view of the discovery that proteins containing as few as three SCRs have useful biological activity.

[0090] Assay Systems

[0091] The analogs are tested for the desired biological activities among those characteristic of the RCA family using in vitro or in vivo assays. In vitro systems such as those described by Wong and Farrell (J. Immunol. (1991) 146:656) can be used to measure effects on the complement pathways. In vivo and general biological effects can be assessed as described by Weisman, et al. (Science (1990) 249:146); or Yeh, et al. (J. Immunol. (1991) 146:250), the teachings of which are incorporated by reference.

[0092] Binding Assays.

[0093] Affinity chromatography columns were prepared as described by Krych, et al., Proc. Natl. Acad. Sci. USA 88, 4353-4357. Binding assays were performed using 100 ml of iC3-Sepharose (iC3-S) or C4b-Sepharose (C4b-S) and 1.8 ml of medium containing of the mutant protein. Media were diluted to desired concentrations of NaCl. After 1 hr on a rotator at room temperature, samples were centrifuged, media removed and bound protein eluted from the Sepharose using 400 mM NaCl with 1% NP-40. Eluted proteins were quantitated by ELISA, using two monoclonal anti-CR1 antibodies, 3D9 and E11 (Hogg, et al., Eur. J. Immunol. 14, 236-240 (1984), O'Shea, et al., J. Immunol. 134, 2580-2587 (1985)). Since neither monoclonal antibody reacts with CR1 SCRs-1, 2, 8 or 9, all of the mutants derived from either CR1-4 or CR1-4 (8, 9) could be quantitated: using this assay. For each mutant protein at least three binding assays from different transfections were performed.

[0094] Assays for Cofactor Activity

[0095] C3 and C4 were purified according to the method of Dykman, et al., Proc. Natl. Acad. Sci. USA 80, 1698-1702 (1983), Dykman., et al., J. Exp. Med. 157, 2160-2165 (1983) or purchased (Quidel, San Diego, Calif.), converted to C3b and C4b and labeled with .sup.125I using Iodogen coated beads (Pierce). Cofactor assays were performed using 200 ng of labeled C3b or C4b, 60 ng of factor I (Quidel) and media with mutant proteins. Amounts of the cofactor proteins were estimated in ELISA assay based on a standard curve of secreted CR1 (sCR1, Weisman, et al., Science 249, 146-151 (1990)). To test for cleavage of C3b, samples containing approximately 6 pg of the mutant proteins were incubated for 1 hr at 37.degree. C. To test for cleavage of C4b, samples were incubated for up to 16 hours at 37.degree. C. After incubation, samples were reduced by boiling in the buffer containing 2% SDS and 5% beta-mercaptoethanol in 0.25% TRIS, pH 6.8 and electrophoresed on a 4-20% SDS-PAG (Integrated Separations) or on a 10% self-made gel. After drying the gels were autoradiographed at -70.degree. C. using an intensifying screen.

[0096] Preparation and Administration of Pharmaceutical Compositions

[0097] The most potent analogs based on the in vitro assays are tested in vivo. In general, the in vitro assays are accepted as highly correlated with the corresponding in vivo activity. The appropriate dosages are determined by comparing the in vitro activity of the naturally occurring protein with that of the analog, comparing the in vitro activity of the naturally occurring protein with the in vivo activity of the naturally occurring protein, then calculating the expected in vivo activity of the analog, adjusting for any measured differences in half-life.

[0098] Complement activation can account for substantial tissue damage in a wide variety of autoimmune/immune complex mediated syndromes such as systemic lupus erythematosus, rheumatoid arthritis, hemolytic anemias, myasthenia gravis and others. Inhibition of the complement systems is a desirable therapeutic intervention in these cases. In some instances, specific inhibition of the classical pathway alone by RCA analogs could be preferred since long-term inhibition of the alternative pathway could lead to side effects.

[0099] Inhibition of complement activation could also be desirable in cases that involve tissue damage brought about by vascular injury such as myocardial infarction, cerebral vascular accidents or acute shock lung syndrome. In these cases, the complement system may contribute to the destruction of partially damaged tissue as in reperfusion injury. Highly stringent inhibition of complement for relatively brief periods might be preferred in these instances and soluble RCA analogs designed for higher potency may prove especially useful.

[0100] Complement inhibition may also prove important in the prevention of xenograft rejection. Organs derived from animals transgenic for human DAF or MCP may be protected at least in part from complement-mediated hyperacute rejection by the expression of transgenic DAF or MCP on the cell surfaces of the xenograft. Animals transgenic for RCA analogs designed for higher potency may provide more successful xenografts. Soluble RCA analogs may also prove useful in protecting the transplant in the recipient.

[0101] Soluble analogs having decreased activity may also be useful as competitive inhibitors of the natural inhibitors, in cases where an increased complement mediated response is desirable or where an individual has a disorder in which their immunity is compromised by overproduction of the natural inhibitors.

[0102] The analogs can be administered locally or systemically in pharmaceutically acceptable carries such as saline, phosphate buffered saline, or a controlled release formulation. The dosage level and mode of administration of the analogs depend on the nature of the analog, the nature of the condition to be treated, and the history of the individual patient. Systemic administration is generally required, which may be by injection or by transmucosal or transdermal delivery. Administration by injection may be intravenous, intramuscular, intraperitoneal or subcutaneous. Formulations for injection are generally biocompatible solutions of the active ingredient such as Hank's solution or Ringer's solution. Formulations for transdermal or transmucosal administration generally include penetrants such as fusidic acid or bile salts in combination with detergents or surface-active agents. The formulations can then be manufactured as aerosols, suppositories, or patches. Oral administration is generally not favored for protein or peptide active ingredients; however, if suitably formulated so as to be protected from the digestive enzymes, oral administration can also be employed.

[0103] Suitable formulations for a desired mode of administration can be found in Remington's Pharmaceutical Sciences, latest edition, Mack Publishing Co., Easton, Pa. The dosage levels and precise formulations are obtainable by routine optimization procedures as is generally known in the art.

[0104] Diagnostic Applications

[0105] The analogs which are capable of binding C3b and/or C4b are useful as diagnostic tools in assessing the presence, absence or amount of C3b or C4b or C3b/C4b-bearing immune complexes in biological fluids. Such assays take advantage of the ability of the analog specifically to bind C3b and/or C4b and can be conducted in a variety of formats as is generally known. Formats for specific binding partner assays include direct and competitive formats, sandwich assays, and agglutination assays. Complexation between members of the specific binding pair can be conducted in solution or on a solid phase and can be detected using a variety of labeling techniques including fluorescence, radioisotopes, chromophores, and visible particles.

[0106] Typical reagent kits useful in assays for C3b and/or C4b and/or C3b/C4b-bearing immune complexes include the analog specifically binding to the analyte, optionally coupled to a solid support and additional labeling reagents useful in the assay. For example, one of many formats for the assay might include treating the sample to be tested with a solid support to which is coupled the analog as a specific binding partner, washing the support which has been treated with sample suspected of containing analyte, and then treating the washed support with anti-C3b or anti-C4b antibody labeled with an enzyme such as horseradish peroxidase. The presence of labeled enzyme on a support then is detected by addition of a substrate solution which results in the development of a color in the presence of the enzyme.

[0107] The present invention will be further understood by reference to the following non-limiting examples.

EXAMPLE 1

Binding Specificity of Truncated CR1

[0108] The cDNA which encodes the first 543 amino acids of mature CR1 (SCR 1-8 and 1/2 of SCR-9) was transfected into COS cells to obtain a secreted protein designated CR1-4.

[0109] Two mouse monoclonal anti-CR1 antibodies were used to determine the immunoreactivity of CR1-4, and as a method to assay for this protein. Antibody E11 (Hogg, N., et al., Eur. J. Immunol. (1984) 14:236-243), and 3D9 (O'Shea, J. J., et al., J. Immunol. (1985) 134:2580-2587), recognized CR1 and bound to the recombinantly produced CR1-4.

[0110] Binding to C4b or to C3b was tested using either C4b or iC3 (C3 containing a broken thioester bond analogous in reactivity and function to C3b) bound to a Sepharose.TM. support as described by Dykman, T., et al., Proc. Natl. Acad. Sci. USA (1983) 80:1698-1702, and Cole, J. L., et al., Proc. Natl. Acad. Sci. USA (1985) 82:859-863. Binding to these solid-supported substrates was verified by testing with an ELISA for CR1.

[0111] The C4b derivatized support was able to bind CR1-4. Most efficient binding occurred at between 12.5 and 25 mM salt.

[0112] Solubilized C4b inhibited binding of the CR1-4 protein to C4b-derivatized support but soluble iC3 did not inhibit this binding. This confirms that CR1-4 binds primarily C4b but not iC3 (C3b).

EXAMPLE 2

Construction of CR1-4 Analogs

[0113] Various mutated forms of CR1-4 were constructed and tested for binding activity to C4b and iC3 as described above in Example 1. Table 2 shows the analogs constructed and the binding and cofactor activities of the analogs. In Table 2, the modifications are shown by the number of the amino acid and the conversion effected. The numbers correspond to those set forth in FIGS. 2A and 2B and FIGS. 3A and 3B which set forth the amino sequences of SCR-1 (SEQ ID NO: 1), SCR-2, (SEQ ID NO: 3) SCR-8 (SEQ ID NO: 2) and SCR-9 (SEQ ID NO: 4) in CR1.

[0114] The combined changes shown in truncated forms should be predictive of the activity of a modified full length protein. There is strong evidence available that establishes three primary binding and cofactor sites in the major polymorphic form (30 SCRs) of CR1. One site interacts almost exclusively with C4b (SCRs 1-4) while the other two sites, nearly identical in amino acid sequence, interact with both C4b and C3b (SCRs 8-11 and SCRs 15-18) [Klickstein, L. B., et al., J. Exp. Med. 168: 1699-1717 (1988); Kalli, et al., J. Exp. Med. 174: 1451-1460 (1991); Makrides, S. C., et al., J. Biol. Chem. 267:24754-24761 (1992)].

[0115] Electron microscope analysis of CR1 suggests that CR1 is a semi-rigid rod composed of individual globular domains (the SCRs). Thus, CR1 is pictured as an elongated protein bearing well-separated active sites. These sites may not work cooperatively. A comparison of several polymorphic forms, containing the first site (SCRs 1-4) and one, two and three copies of the second site, demonstrated little difference among them in cofactor and decay acceleration assays, as reported by Seya, T., et al., J. Immunology 135: 2661 (1985). Indeed, after the filing of the application to which this claims priority, it was shown that modified membrane forms of CR1 bearing a single active site alone (SCR 1-4 or SCR 15-18) could protect CHO cells from human complement-mediated lysis (Makrides, S. C., et al., J. Biol. Chem. 267: 24754-24761 (1992)). This provides evidence that a simple CR1 form with a single active site could be biologically active, and that modifications that improve the activity of each primary site in the constructs could be incorporated together in a multiple-site CR1 form, yielding an improved regulator.

TABLE-US-00002 TABLE 2 CR 1-4 Mutants C4b C3b C4b- C3b- Cofactor cofactor Designation Description.sup.a binding binding activity activity CR1-4 original peptide +++ + ++ + .DELTA.SCR-1 amino acids 1-60 deleted - - ND ND .DELTA.SCR-2 amino acids 61-122 deleted - - ND ND .DELTA.SCR-3 amino acids 123-194 deleted ND ND - - CR1-4 Stop 7 truncated after amino acid 449 ND + ++ + 1 35: G.fwdarw. E (amino acid 35 of SEQ ID NOS: 1 and 2, respectively); + + - ++ 37: S.fwdarw. Y (amino acid 37 of SEQ ID NOS: 1 and 2, respectively) 1a 35: G.fwdarw. E (amino acid 35 of SEQ ID NOS: 1 and 2, respectively) + + + + 1b 37: S.fwdarw. Y (amino acid 37 of SEQ ID NOS: 1 and 2, respectively) ++ ++ + + 2 44, 47, 49: I . . . K . . . S.fwdarw. T . . . D . . . L +++ + ++ + (amino acids 44, 47 and 49 of SEQ ID NOS: 1 and 2, respectively) 3 52.fwdarw. 54: T-G-A.fwdarw. S-S-P (amino acids 52-54 of SEQ ID NOS: 1 and 2, respectively) +++ + ++ + 4 57, 59: R . . . R.fwdarw. V . . . K +++ + ++ + (amino acids 57, 59 of SEQ ID NOS: 1 and 2, respectively) 5 64, 65: R-N.fwdarw. K-T (amino acids 4, 5 of SEQ ID NOS: 3 and 4, respectively) + + - + 5a 64: R.fwdarw. K (amino acid 4 of SEQ ID NOS: 3 and 4, respectively) +/- - ND ND 5b 65: N.fwdarw. T (amino acid 5 of SEQ ID NOS: 3 and 4, respectively) +/- - ND ND 6 78, 79: K-G.fwdarw. T-D (amino acids 18, 19 of SEQ ID NOS: 3 and 4, respectively) +++ + ++ + 6b 79: G.fwdarw. D (amino acid 19 of SEQ ID NOS: 3 and 4, respectively) +++ +++ +++ +++ 1b, 6b 37, 79: S.fwdarw. Y; G.fwdarw. D (amino acid 37 of SEQ ID NOS: 1 and 2, and amino acid 19 of +++ ++ + + SEQ ID NOS: 3 and 4, respectively) 7 85, 87: Q . . . K.fwdarw. R . . . N (amino acids 25, 27 of SEQ ID NOS: 3 and 4) +++ + ++ + 8 92, 94: K . . . Y.fwdarw. T . . . H (amino acids 32 and 34 of SEQ ID NOS: 3 and 4, respectively) + + - + 8a 92: K.fwdarw. T (amino acid 32 of SEQ ID NOS: 3 and 4, respectively) +++ + +++ ++ 8b 94: Y.fwdarw. H (amino acid 34 of SEQ ID NOS: 3 and 4, respectively) + + - + 9 99, 103: S . . . T.fwdarw. H . . . E +++ + ++ + (amino acids 38 and 43 of SEQ ID NOS: 3 and 4, respectively) 10 109-112: D-T-V-I.fwdarw. N-A-A-H ++++ ++ +++ ++ (amino acids 49-52 of SEQ ID NOS: 3 and 4, respectively) 10, 11 109-112: D-T-V-I.fwdarw. N-A-A-H; +++ +++ +++ +++ 114-117, 121: D-N-E-T . . . D.fwdarw. S-T-K-P . . . Q (amino acids 49-52 of SEQ ID NOS: 3 and 4, respectively); (amino acids 54-61 of SEQ ID NOS: 3 and 4, respectively) 11 114-117, 121: D-N-E-T . . . D.fwdarw. S-T-K-P . . . Q +++ ++ ++ + (amino acids 54-61 of SEQ ID NOS: 3 and 4, respectively) 11a 114: D.fwdarw. S (amino acid 54 of SEQ ID NOS: 3 and 4, respectively) +++ + ND ND 11b 115: N.fwdarw. T (amino acid 55 of SEQ ID NOS: 3 and 4, respectively) +++ + ND ND 11c 116: E.fwdarw. K (amino acid 56 of SEQ ID NOS: 3 and 4, respectively) ++++ ++ +++ +++ 11d 117: T-P (amino acid 57 of SEQ ID NOS: 3 and 4, respectively) +++ + ND ND 11c, d 116, 117: E-T.fwdarw. K-P (amino acids 56 and 57 of SEQ ID NOS: 3 and 4, respectively) ++++ ++ +++ +++ 11e 121: D.fwdarw. Q (amino acid 61 of SEQ ID NOS: 3 and 4, respectively) +++ + ND ND 11.DELTA.SCR1 1-60 deleted, 114-117, 121: ++ ++ ++ ++ D-N-E-T . . . D.fwdarw. S-T-K-P . . . Q (amino acids 54-61 of SEQ ID NOS: 3 and 4, respectively) 12 1, 3: Q . . . N.fwdarw. H . . . Q (amino acids 1-3 of SEQ ID NOS: 1 and 2, respectively) + + ++ + 13 6-9: E-W-L-P.fwdarw. D-H-F-L (amino acids of 6-9 of SEQ ID NOS: 1 and 2, respectively) + + ++ + 14 12-16, 18-21: R-P-T-N-L . . . D-E-F-E.fwdarw. K-L-K-T-Q . . . N-A-S-D ++++ ++ +++ +++ (amino acids 12-16 and 18-21 of SEQ ID NOS: 1 and 2, respectively) 27, 29: Y . . . N.fwdarw. S . . . K (amino acids 27 and 29 of SEQ ID NOS: 1 and 2, respectively) ++++ ++ +++ +++ ND Not determined. . . . intervening amino acids which were not changed.

[0116] As shown in Table 2, deletion of either SCR-1 or SCR-2 results in loss of C4b-binding activity; thus both regions are required for binding to C4b. Binding to C3b is conferred by insertion of the SCR-9 sequence S-T-K-P-(P-I-C)-Q (amino acids 54-61 of SEQ ID NO: 4) into SCR-2 (SEQ ID NO: 3); however, deletion of SCR-1 (SEQ ID NO: 1) partially eliminates binding to C3b in this analog. Thus, efficient binding to C3b requires not only the relevant sequence in SCR-2 (SEQ ID NO: 3), but an additional portion of SCR-1 (SEQ ID NO: 1). The ability to bind C3b conferred by altering the sequence in SCR-2 (SEQ ID NO: 3) does not affect binding to C4b.

[0117] As also shown by Table 2, it is possible to weaken or destroy binding to C4b by altering amino acids 35, 64-65 or 94. It is possible to strengthen C4b binding by altering amino acid 92.

[0118] These results indicate that by manipulation of C3b and C4b binding sites in CR1 the affinity and specificity of CR1-4 can be altered. Similar alterations may be made to corresponding regions in additional members of the RCA protein family.

EXAMPLE 3

Construction of a More Potent Soluble CR1 Analog

[0119] A stringent inhibitor of the complement system has applications where higher potency is required. In this example, the sCR1 sequence is used as starting material for a family of analogs of higher inhibitory capacity for both classical and alternative pathways. Other CR1 truncated, full-length, recombined or hybrid forms of CR1 could also be used.

[0120] As noted above, the sCR1 protein contains four corresponding regions: SCRs 1-2 (SEQ ID NO: 1 and 3 respectively), SCRs 8-9 (SEQ ID NO: 2 and 4 respectively), SCRs 15-16 and SCRs 22-23. The primary C4b-binding site is within SCRs 1-2 (SEQ ID NO: 1 and 3 respectively) and the two primary C3b-binding sites are in SCRs 8-9 (SEQ ID NO: 2 and 4 respectively) and SCRs 15-16. SCRs 22-23 has no reported binding activity although it is highly homologous in amino acid sequence to the other three corresponding regions.

[0121] One or more substitutions are introduced into the corresponding positions of soluble CR1 SCRs 1-2, 8-9, 15-16, and 22-23. Specific substitutions designed to increase C3b and C4b cofactor activity and binding activity are selected from the following:

TABLE-US-00003 positions substitution 35 G (amino acid 35 of SEQ ID NO: 1) 64-65 R-N (amino acids 4-5 of SEQ ID NO: 3) 94 Y (amino acid 34 of SEQ ID NO: 3) 109-112 N-A-A-H (amino acids 49-52 of SEQ ID NO: 4) 114-121 S-T-K-P-P-I-C-Q (amino acids 54-61 of SEQ ID NO: 4) 109-121 N-A-A-H-W-S-T-K-P-P-I-C-Q (amino acids 49-61 of SEQ ID NO: 4) 92 T (amino acid 32 of SEQ ID NO: 4) 79 D (amino acid 19 of SEQ ID NO: 4) 12-16 K-L-K-T-Q (amino acids 12-16 of SEQ ID NO: 2) 18-21 N-A-S-D (amino acids 18-21 of SEQ ID NO: 2) 12-21 K-L-K-T-Q-T-N-A-S-D (amino acids 12-21 of SEQ ID NO: 2) 27-29 S-L-K (amino acids 27-29 of SEQ ID NO: 2)

[0122] In some cases, some of these amino acids may already be present in the corresponding position. In some cases structurally similar amino acids may be substituted instead of those substituted above.

[0123] The use of these substitutions at some or all four corresponding positions will yield a full length soluble CR1 form with more potent active sites, and with an additional active site at SCR 22-24.

[0124] Substitutions described above were introduced into the four corresponding regions of interest in order to increase the affinity of sCR1 for its C3b and C4b-containing targets. These modifications were designed to increase the use of existing coenzyme and dissociation functions and potentially to establish new coenzyme and dissociation functions. The introduction of amino acid sequences significant to C3b binding into the C4b binding region presumably would not interfere substantially with C4b activities since such a substitution in CR1-4 did not detectably alter the C4b binding properties of CR1-4. The introduction of amino acids significant to C4b-binding into C3b-binding regions presumably would not interfere substantially with C3b binding activities since substantial C3b-binding occurs in CR1-4 mutant 11, in which many amino acids specific to SCRs 1-2, including those significant in C4b binding, are already present.

[0125] Specific substitutions as shown in FIGS. 2A and 2B and FIGS. 3A and 3B designed to increase affinity to C4b were described in Example 3: SCRs 1-2 (SEQ ID NO: 1 and 3 respectively), the primary C4b-binding site of CR1, modified by replacement of K (amino acid 32 of SEQ ID NO: 3) at position 92 with T (amino acid 32 of SEQ ID NO: 4); SCRs 8-9 (SEQ ID NOS: 2 and 4) and the identical SCRs 15-16 modified by replacement of E (amino acid 35 of SEQ ID NO: 2) at a position corresponding to 35 by G (amino acid 35 of SEQ ID NO: 1)(or A), H (amino acid 34 of SEQ ID NO: 4) at a position corresponding to 94 by Y (amino acid 34 of SEQ ID NO: 3) (or F), K (amino acid 4 of SEQ ID NO: 4) at a position corresponding to 64 by R (amino acid 4 of SEQ ID NO: 3) and T (amino acid 5 of SEQ ID NO: 4) at a position corresponding to 65 by N (amino acid 5 of SEQ ID NO: 3) (or Q). SCRs 22-23 were also modified by replacement of G at a position corresponding to 64 by R (amino acid 4 of SEQ ID NO: 3) (or K) (amino acid 4 of SEQ ID NO: 4), P at a position corresponding to 65 with N (amino acid 5 of SEQ ID NO: 3) (or Q), E at a position corresponding to 92 with T (amino acid 32 of SEQ ID NO: 4) at a position corresponding to 94 with Y (amino acid 34 of SEQ ID NO: 3).

[0126] Together, these modifications result in higher affinity for C4b and may also result in the establishment of new coenzyme or dissociation functions or the improvement in the capacity of the coenzyme and dissociation functions present on the sCR1 starting material.

[0127] As described in previous examples, substitution of a short stretch of amino acids into SCRs 1-2 (SEQ ID NOS: 1 and 3) of CR1-4 by corresponding amino acids of SCRs 8-9 (amino acid 34 of SEQ ID NO: 3) confers C3b binding capacity to CR1-4. Replacement of D-N-E-T-P-I-C-D (amino acid 54-61 of SEQ ID NO: 3) at position 114-121 in SCR1 SCRs 1-2 by S-T-K-P-P-I-C-Q (amino acid 54-61 of SEQ ID NO: 4) increases the affinity of sCR1 for C3b. Further, replacement of D-K-K-A-P-I-C-D (SEQ ID NO: 5) at positions 114-121 in SCRs 22-23 by S-T-K-P-P-I-C-Q (amino acids 54-61 of SEQ ID NO: 4) increases the affinity of sCR1 for C3b. Some structurally similar amino acids may also be substituted in the sequence.

[0128] A more potent complement inhibitor, in general, provides increased C4b-binding and increased C3b-binding. This is achieved by introducing all the modifications set forth above.

EXAMPLE 4

Construction Of Truncated CR1 Mutants Having Increased Activity

[0129] More than 25 mutants were assayed for cofactor activity at several different salt concentrations. The assay was done as described in Adams, E. M., Brown, M .C., Nunge, M., Krych, M., and Atkinson, J. P. (1991) J. Immunology, 147: 3005-3011.

[0130] A number of mutations of the active sites in SCRs 1-3, the site that normally interacts with C4b and not C3b, which improve on the natural activity of this site, both for C4b and C3b cofactor activity. At least one mutant in the active sites in SCR 8-11, which is apparently the more potent natural active site, that improves on the cofactor activity of this site for both C3b and C4b was also detected.

[0131] In summary, specific CR1 proteins that have both C3b and C4b cofactor activity and are in some ways an improvement on the full length soluble CR1 have now been identified. These constructs are less than one-fourth (seven SCR constructs) to a third the size of soluble CR1 (sCR1) and have both C4b binding and cofactor activity as well as C3b binding and cofactor activity. Constructs including as few as three SCRs appear to have C3b cofactor activity, indicative of C3b binding activity. In some cases the data indicates that the modified active site appears to be more potent than the natural active sites.

[0132] These forms and their activities are shown in Table 3:

TABLE-US-00004 TABLE 3 Activities of CR1-4 (8, 9) Mutants C4b C3b C4b- C3b- cofactor cofactor Designation Description binding binding activity activity CR1-4 (8, 9) * ++++ ++++ ++++ ++++ .DELTA.SGR10 Deletion of amino acids 123-194 ND ND -- -- ND ND -- -- .DELTA.SCR11, ** ND ND ND ++++ subst1 CR1-4 (8, 9) stop 7 truncated after amino acid 449 ND ++++ ++++ ++++ 1br 37: Y.fwdarw.S (amino acid 37 of SEQ ID NOS: 2 and 1, ++++ +++ ++++ ++ respectively) 2r 44, 47, 49: T . . . D . . . L.fwdarw.I . . . K . . . S ++++ +++ ++++ +++ (amino acids 44, 47 and 49 of SEQ ID NOS: 2 and 1, respectively) 3r 52-54, 57, 59: +++ ++ ++++ ++++ S-S-P . . . V . . . K.fwdarw.T-G-A . . . R . . . R (amino acids 52-54, 57, 59 of SEQ ID NOS: 2 and 1, respectively) 6r 78-79, 82: T-D . . . V.fwdarw.K-G . . . F ++ ++++ ++++ ++++ (amino acids 18-19, 22 of SEQ ID NOS: 4 and 3, respectively) 7r 85, 87: R . . . N.fwdarw.Q . . . K ++++ +++ ++++ ++++ (amino acids 25, 27 of SEQ ID NOS: 4 and 3, respectively) 8r 92, 94: T . . . H.fwdarw.K . . . Y ++++ ++++ ++++ ++++ (amino acids 32-34 of SEQ ID NOS: 4 and 3, respectively) 9r 99, 103, 106: ++++ ++++ +++ ++++ H . . . E . . . L.fwdarw.S . . . T . . . I (amino acids 39, 43 and 46 of SEQ ID NOS: 4 and 3, 4 and 3, respectively) 10r 109-112: N-A-A-H.fwdarw.D-T-V-I +++ ++ ++ ++ (amino acids 49-52 of SEQ ID NOS: 4 and 3, respectively) 10br 110: A.fwdarw.T (amino acid 50 of SEQ ID NOS: 4 and 3 ++++ ++++ ++++ ++++ respectively) 10cr 111: A.fwdarw.V (amino acid 51 of SEQ ID NOS: 4 and 3, ++++ ++++ ++++ ++++ respectively) 10dr 112: H.fwdarw.I (amino acid 52 of SEQ ID NOS: 4 and 3, ++++ ++++ ++++ ++++ respectively) 10ar 109: N.fwdarw.D (amino acid 49 of SEQ ID NOS: 4 and 3, ++ ++ + ++ respectively) 11r 114-117, 121: S-T-K-P . . . Q.fwdarw.D-N-E-T . . . D +++ ++ +++ + (amino acids 54-57, 61 of SEQ ID NOS: 4 and 3, respectively) 11ar 114: S.fwdarw.D (amino acid 54 of SEQ ID NOS: 4 and 3, ++++ ++++ ++++ ++++ respectively) 11br 115: T.fwdarw.N (amino acid 55 of SEQ ID NOS: 4 and 3, ++++ ++++ ++++ ++++ respectively) 11er 121: Q.fwdarw.D (amino acid 61 of SEQ ID NOS: 4 and 3, ++++ ++++ ++++ ++++ respectively) 11cr 116: K.fwdarw.E (amino acid 56 of SEQ ID NOS: 4 and 3, +++ +++ + + respectively) 11dr 117: P.fwdarw.T (amino acid 57 of SEQ ID NOS: 4 and 3, +++ +++ ++++ ++++ respectively) 10, 11r 109-112, 114-117, 121: + + ++ -- N-A-A-H . . . S-T-K-P . . . Q.fwdarw.D-T-V-I . . . D-N-E-T . . . D (amino acids 49-52, 54-57, 61 of SEQ ID NOS: 4 and 3, respectively) 12r 1, 3: H . . . Q.fwdarw.Q . . . N ++++ ++++ ++++ ++++ (amino acids 1 and 3 of SEQ ID NOS: 2 and 1, respectively) 13r 6-9: D-H-F-L.fwdarw.E-W-L-P ++++ ++++ ++++ ++++ (amino acids 6-9 of SEQ ID NOS: 2 and 1, respectively) 14r 12-16, 18-21: +++ +++ ++++ +++ K-L-K-T-Q- . . . N-A-S-D.fwdarw.R-P-T-N-L . . . D-E-F-E (amino acids 12-16, 18-21 of SEQ ID NOS: 2 and 1, respectively) 15r 27, 29: S . . . K.fwdarw.Y . . . N +++ ++++ ++++ +++ (amino acids 27 and 29 of SEQ ID NOS: 2 and 1, respectively) 1ar, 5r 35, 64-65, 94: E . . . K-T . . . H.fwdarw.G . . . R-N . . . Y +++++ +++++ +++++ +++++ 8br (amino acid 35 of SEQ ID NOS: 2 and 1, amino acids 4-5 and 34 of SEQ ID NOS: 4 and 3 respectively) *CR1-4 with its first 122 amino acids (SCR1-2) replaced with CR1 amino acids 497-618 (SCR 8-9), as enumerated by Klickstein, L. B., J. Exp. Med. 168: 1699-1717. **CR1-4 (8, 9) with deletion of 194-253; substitution of amino acids 271-543 with: T-R-T-T-F-H-L-G-R-K-C-S-T-A-V-S-P-A-T-T-S-E-G-L-R-L-C-A-A-H-P-R-E-T-G-A-L- -Q-P-P-H-V-K (SEQ ID NO: 11)

EXAMPLE 5

DAF Analogs

[0133] The membrane-bound complement regulator DAF facilitates the dissociation of C3b and C4b-containing convertases but does not bind C3b or C4b, nor does it serve as cofactor for factor I-mediated proteolytic inactivation of C3b or C4b. It would be desirable, under certain situations, to increase the complement regulatory activity of DAF or of truncated, recombined or hybrid forms of DAF.

[0134] Based on amino sequence homology, DAF SCRs 2-3 corresponds to the CR1 active regions. Homology between DAF SCRs 2-3 and CR1 SCRs 1-2 is 40% while homology between DAF SCRs 2-3 and CR1 SCRs 8-9 is 39%. Since there are several unmatched amino acids in these alignments, the position numbers of DAF do not precisely match those of CR1.

[0135] One or more substitutions are introduced into the DAF SCRs 2-3. Specific substitutions designed to confer C3b and C4b cofactor activity and binding activity are selected from the following:

[0136] DAF position(s) enumerated as in Lublin and Atkinson, Ann. Rev. Immunol. 9:431 (1991):

TABLE-US-00005 DAF position DAF sequence Substitution 180-187 S-D-P-L-P-E-C-R S-T-K-P-P-I-C-Q (SEQ ID NO: 6) (amino acids 54-61 of SEQ ID NO: 4) 175-178 S-S-V-Q N-A-A-H (SEQ ID NO: 7) (amino acids 49-52 of SEQ ID NO: 4) 175-187 S-D-P-L-P-E-C-R-S-S-V-Q S-T-K-P-P-I-C-Q-N-A-A-H (SEQ ID NO: 8) (SEQ ID NO: 9) 130 P R 145 G D 77-84 Q-P-Y-I-T-Q-N-Y K-L-K-T-Q-T-N-A-S-D (SEQ ID NO: 10) (amino acids 12-21 of SEQ ID NO: 2) 90-92 V-V-E S-L-K (amino acids 27-29 of SEQ ID NO: 2)

[0137] Replacement of S-D-P-L-P-E-C-R (SEQ ID NO: 6) at DAF position 180-187 (using the enumeration in Lublin and Atkinson, Ann. Rev. Immunol. (1989) 7:35-58) with S-T-K-P-P-I-C-O (amino acid 54-61 of SEQ ID NO: 4) increases the affinity of DAF for C3b. Replacement of S-D-P-L-P-E-C (amino acid 1-7 of SEQ ID NO: 6) with S-T-K-P-P-I-C-Q (amino acid 54-61 of SEQ ID NO: 4) leaves R at the end of this segment, since R is found in the corresponding positions of CR1 SCR 1-2 (SEQ ID NOS: 1 and 3)and CR1 SCR 8-9 (SEQ ID NOS: 2 and 4). Some structurally similar amino acids can be substituted in the S-T-K-P-P-I-C-Q (amino acid 54-61 of SEQ ID NO: 4) sequence.

[0138] Replacement of P at DAF position 130 with R increases the affinity of DAF for C4b. All other amino acids found important in C4b interactions in CR1 are already present in DAF SCRs 2-3.

[0139] These substitutions, by increasing the affinity of DAF for C3b and, possibly, C4b, enhance the respective 5 inhibitory effects of DAF on complement activation.

EXAMPLE 6

Analogs of Factor H

[0140] Factor H is a plasma protein consisting solely of 20 SCRs. Factor H exhibits C3b binding and C3b cofactor and dissociation capacity but no apparent ability to inactivate or bind C4b. Since factor H has already evolved as a plasma protein, it could be advantageous to use it as the starting material for soluable RCA analogs. Although the active sites of factor H are not precisely known, a proteolytic fragment composed of the first 5.5 SCRs of factor H exhibits C3b-binding and cofactor activity (Alsenz et al., Biochem. J. (1984) 389).

[0141] To increase the affinity of factor H for C3b binding, N-A-A-H-W-S-T-K-P-P-I-C-Q (amino acid 49-61 of SEQ ID NO: 4) is introduced into several of the SCRs, none bearing a close correspondence to CR1 SCR 8-9. In one embodiment, the first six SCRs are left unmodified, thus retaining the original active site(s), and the remaining fourteen SCRs are modified by substitution of the N-A-A-H-W-S-T-K-P-P-I-C-Q (amino acid 49-61 of SEQ ID NO: 4) into the position(s) homologous to CR1. Some structurally similar amino acids can substitute in the N-A-A-H-W-S-T-K-P-P-I-C-Q (amino acid 49-61 of SEQ ID NO: 4) sequence. Homologous positions are readily identified because the W that precedes this C3b binding segment is found in most SCRs: in all factor H SCRs except SCR 10 and SCR 20 while the C within the C3b binding segment is found in all the known SCRs. While not all substitutions will necessarily confer added binding activity, since these factor H SCRs are less homologous to the CR1 SCR 1-2 and CR1 SCR 8-9 regions. However, at least some modified H SCRs gain C3b-binding capacity, resulting in a factor H analog with much higher affinity for C3b. As discussed above, higher affinity would lead to the greater use of the active sites already present in the first six H SCRs.

[0142] Modifications and variations of the present invention will be obvious to those skilled in the art from the foregoing detailed description. These modifications and variations are intended to come within the scope of the following claims.

Sequence CWU 1

1

19160PRTHomo sapiensSCR-1 1Gln Cys Asn Ala Pro Glu Trp Leu Pro Phe Ala Arg Pro Thr Asn Leu 1 5 10 15Thr Asp Glu Phe Glu Phe Pro Ile Gly Thr Tyr Leu Asn Tyr Glu Cys 20 25 30Arg Pro Gly Tyr Ser Gly Arg Pro Phe Ser Ile Ile Cys Leu Lys Asn 35 40 45Ser Val Trp Thr Gly Ala Lys Asp Arg Cys Arg Arg 50 55 60260PRTHomo sapiensSCR-8 2His Cys Gln Ala Pro Asp His Phe Leu Phe Ala Lys Leu Lys Thr Gln 1 5 10 15Thr Asn Ala Ser Asp Phe Pro Ile Gly Thr Ser Leu Lys Tyr Glu Cys 20 25 30Arg Pro Glu Tyr Tyr Gly Arg Pro Phe Ser Ile Thr Cys Leu Asp Asn 35 40 45Leu Val Trp Ser Ser Pro Lys Asp Val Cys Lys Arg 50 55 60362PRTHomo sapiensSCR-2 3Lys Ser Cys Arg Asn Pro Pro Asp Pro Val Asn Gly Met Val His Val 1 5 10 15Ile Lys Gly Ile Gln Phe Gly Ser Gln Ile Lys Tyr Ser Cys Thr Lys 20 25 30Gly Tyr Arg Leu Ile Gly Ser Ser Ser Ala Thr Cys Ile Ile Ser Gly 35 40 45Asp Thr Val Ile Trp Asp Asn Glu Thr Pro Ile Cys Asp Arg 50 55 60462PRTHomo sapiensSCR-9 4Lys Ser Cys Lys Thr Pro Pro Asp Pro Val Asn Gly Met Val His Val 1 5 10 15Ile Thr Asp Ile Gln Val Gly Ser Arg Ile Asn Tyr Ser Cys Thr Thr 20 25 30Gly His Arg Leu Ile Gly His Ser Ser Ala Glu Cys Ile Leu Ser Gly 35 40 45Asn Ala Ala His Trp Ser Thr Lys Pro Pro Ile Cys Gln Arg 50 55 6058PRTHomo sapiensPositions 114-121 in SCRs 22-23 5Asp Lys Lys Ala Pro Ile Cys Asp1 568PRTHomo sapiensDAF Positions 180-187 6Ser Asp Pro Leu Pro Glu Cys Arg1 574PRTHomo sapiensDAF Positions 175-178 7Ser Ser Val Gln1812PRTHomo sapiensDAF Positions 175-187 8Ser Asp Pro Leu Pro Glu Cys Arg Ser Ser Val Gln 1 5 10912PRTArtificial SequenceSubstitution at positions 175-187 of human DAF 9Ser Thr Lys Pro Pro Ile Cys Gln Asn Ala Ala His 1 5 10108PRTHomo sapiensDAF Positions 77-84 10Gln Pro Tyr Ile Thr Gln Asn Tyr1 51143PRTArtificial SequenceSubstitution at positions 271-543 in the delta SCR11 analog of human CR1 11Thr Arg Thr Thr Phe His Leu Gly Arg Lys Cys Ser Thr Ala Val Ser 1 5 10 15Pro Ala Thr Thr Ser Glu Gly Leu Arg Leu Cys Ala Ala His Pro Arg 20 25 30Glu Thr Gly Ala Leu Gln Pro Pro His Val Lys 35 40126801DNAHomo sapiensCR-1 12caa tgc aat gcc cca gaa tgg ctt cca ttt gcc agg cct acc aac cta 48Gln Cys Asn Ala Pro Glu Trp Leu Pro Phe Ala Arg Pro Thr Asn Leu1 5 10 15act gat gag ttt gag ttt ccc att ggg aca tat ctg aac tat gaa tgc 96Thr Asp Glu Phe Glu Phe Pro Ile Gly Thr Tyr Leu Asn Tyr Glu Cys20 25 30cgc cct ggt tat tcc gga aga ccg ttt tct atc atc tgc cta aaa aac 144Arg Pro Gly Tyr Ser Gly Arg Pro Phe Ser Ile Ile Cys Leu Lys Asn35 40 45tca gtc tgg act ggt gct aag gac agg tgc aga cgt aaa tca tgt cgt 192Ser Val Trp Thr Gly Ala Lys Asp Arg Cys Arg Arg Lys Ser Cys Arg50 55 60aat cct cca gat cct gtg aat ggc atg gtg cat gtg atc aaa ggc atc 240Asn Pro Pro Asp Pro Val Asn Gly Met Val His Val Ile Lys Gly Ile65 70 75 80cag ttc gga tcc caa att aaa tat tct tgt act aaa gga tac cga ctc 288Gln Phe Gly Ser Gln Ile Lys Tyr Ser Cys Thr Lys Gly Tyr Arg Leu85 90 95att ggt tcc tcg tct gcc aca tgc atc atc tca ggt gat act gtc att 336Ile Gly Ser Ser Ser Ala Thr Cys Ile Ile Ser Gly Asp Thr Val Ile100 105 110tgg gat aat gaa aca cct att tgt gac aga att cct tgt ggg cta ccc 384Trp Asp Asn Glu Thr Pro Ile Cys Asp Arg Ile Pro Cys Gly Leu Pro115 120 125ccc acc atc acc aat gga gat ttc att agc acc aac aga gag aat ttt 432Pro Thr Ile Thr Asn Gly Asp Phe Ile Ser Thr Asn Arg Glu Asn Phe130 135 140cac tat gga tca gtg gtg acc tac cgc tgc aat cct gga agc gga ggg 480His Tyr Gly Ser Val Val Thr Tyr Arg Cys Asn Pro Gly Ser Gly Gly145 150 155 160aga aag gtg ttt gag ctt gtg ggt gag ccc tcc ata tac tgc acc agc 528Arg Lys Val Phe Glu Leu Val Gly Glu Pro Ser Ile Tyr Cys Thr Ser165 170 175aat gac gat caa gtg ggc atc tgg agc ggc ccc gcc cct cag tgc att 576Asn Asp Asp Gln Val Gly Ile Trp Ser Gly Pro Ala Pro Gln Cys Ile180 185 190ata cct aac aaa tgc acg cct cca aat gtg gaa aat gga ata ttg gta 624Ile Pro Asn Lys Cys Thr Pro Pro Asn Val Glu Asn Gly Ile Leu Val195 200 205tct gac aac aga agc tta ttt tcc tta aat gaa gtt gtg gag ttt agg 672Ser Asp Asn Arg Ser Leu Phe Ser Leu Asn Glu Val Val Glu Phe Arg210 215 220tgt cag cct ggc ttt gtc atg aaa gga ccc cgc cgt gtg aag tgc cag 720Cys Gln Pro Gly Phe Val Met Lys Gly Pro Arg Arg Val Lys Cys Gln225 230 235 240gcc ctg aac aaa tgg gag ccg gag cta cca agc tgc tcc agg gta tgt 768Ala Leu Asn Lys Trp Glu Pro Glu Leu Pro Ser Cys Ser Arg Val Cys245 250 255cag cca cct cca gat gtc ctg cat gct gag cgt acc caa agg gac aag 816Gln Pro Pro Pro Asp Val Leu His Ala Glu Arg Thr Gln Arg Asp Lys260 265 270gac aac ttt tca cct ggg cag gaa gtg ttc tac agc tgt gag ccc ggc 864Asp Asn Phe Ser Pro Gly Gln Glu Val Phe Tyr Ser Cys Glu Pro Gly275 280 285tac gac ctc aga ggg gct gcg tct atg cgc tgc aca ccc cag gga gac 912Tyr Asp Leu Arg Gly Ala Ala Ser Met Arg Cys Thr Pro Gln Gly Asp290 295 300tgg agc cct gca gcc ccc aca tgt gaa gtg aaa tcc tgt gat gac ttc 960Trp Ser Pro Ala Ala Pro Thr Cys Glu Val Lys Ser Cys Asp Asp Phe305 310 315 320atg ggc caa ctt ctt aat ggc cgt gtg cta ttt cca gta aat ctc cag 1008Met Gly Gln Leu Leu Asn Gly Arg Val Leu Phe Pro Val Asn Leu Gln325 330 335ctt gga gca aaa gtg gat ttt gtt tgt gat gaa gga ttt caa tta aaa 1056Leu Gly Ala Lys Val Asp Phe Val Cys Asp Glu Gly Phe Gln Leu Lys340 345 350ggc agc tct gct agt tac tgt gtc ttg gct gga atg gaa agc ctt tgg 1104Gly Ser Ser Ala Ser Tyr Cys Val Leu Ala Gly Met Glu Ser Leu Trp355 360 365aat agc agt gtt cca gtg tgt gaa caa atc ttt tgt cca agt cct cca 1152Asn Ser Ser Val Pro Val Cys Glu Gln Ile Phe Cys Pro Ser Pro Pro370 375 380gtt att cct aat ggg aga cac aca gga aaa cct ctg gaa gtc ttt ccc 1200Val Ile Pro Asn Gly Arg His Thr Gly Lys Pro Leu Glu Val Phe Pro385 390 395 400ttt gga aaa gca gta aat tac aca tgc gac ccc cac cca gac aga ggg 1248Phe Gly Lys Ala Val Asn Tyr Thr Cys Asp Pro His Pro Asp Arg Gly405 410 415acg agc ttc gac ctc att gga gag agc acc atc cgc tgc aca agt gac 1296Thr Ser Phe Asp Leu Ile Gly Glu Ser Thr Ile Arg Cys Thr Ser Asp420 425 430cct caa ggg aat ggg gtt tgg agc agc cct gcc cct cgc tgt gga att 1344Pro Gln Gly Asn Gly Val Trp Ser Ser Pro Ala Pro Arg Cys Gly Ile435 440 445ctg ggt cac tgt caa gcc cca gat cat ttt ctg ttt gcc aag ttg aaa 1392Leu Gly His Cys Gln Ala Pro Asp His Phe Leu Phe Ala Lys Leu Lys450 455 460acc caa acc aat gca tct gac ttt ccc att ggg aca tct tta aag tac 1440Thr Gln Thr Asn Ala Ser Asp Phe Pro Ile Gly Thr Ser Leu Lys Tyr465 470 475 480gaa tgc cgt cct gag tac tac ggg agg cca ttc tct atc aca tgt cta 1488Glu Cys Arg Pro Glu Tyr Tyr Gly Arg Pro Phe Ser Ile Thr Cys Leu485 490 495gat aac ctg gtc tgg tca agt ccc aaa gat gtc tgt aaa cgt aaa tca 1536Asp Asn Leu Val Trp Ser Ser Pro Lys Asp Val Cys Lys Arg Lys Ser500 505 510tgt aaa act cct cca gat cca gtg aat ggc atg gtg cat gtg atc aca 1584Cys Lys Thr Pro Pro Asp Pro Val Asn Gly Met Val His Val Ile Thr515 520 525gac atc cag gtt gga tcc aga atc aac tat tct tgt act aca ggg cac 1632Asp Ile Gln Val Gly Ser Arg Ile Asn Tyr Ser Cys Thr Thr Gly His530 535 540cga ctc att ggt cac tca tct gct gaa tgt atc ctc tcg ggc aat gct 1680Arg Leu Ile Gly His Ser Ser Ala Glu Cys Ile Leu Ser Gly Asn Ala545 550 555 560gcc cat tgg agc acg aag ccg cca att tgt caa cga att cct tgt ggg 1728Ala His Trp Ser Thr Lys Pro Pro Ile Cys Gln Arg Ile Pro Cys Gly565 570 575cta ccc ccc acc atc gcc aat gga gat ttc att agc acc aac aga gag 1776Leu Pro Pro Thr Ile Ala Asn Gly Asp Phe Ile Ser Thr Asn Arg Glu580 585 590aat ttt cac tat gga tca gtg gtg acc tac cgc tgc aat cct gga agc 1824Asn Phe His Tyr Gly Ser Val Val Thr Tyr Arg Cys Asn Pro Gly Ser595 600 605gga ggg aga aag gtg ttt gag ctt gtg ggt gag ccc tcc ata tac tgc 1872Gly Gly Arg Lys Val Phe Glu Leu Val Gly Glu Pro Ser Ile Tyr Cys610 615 620acc agc aat gac gat caa gtg ggc atc tgg agc ggc ccg gcc cct cag 1920Thr Ser Asn Asp Asp Gln Val Gly Ile Trp Ser Gly Pro Ala Pro Gln625 630 635 640tgc att ata cct aac aaa tgc acg cct cca aat gtg gaa aat gga ata 1968Cys Ile Ile Pro Asn Lys Cys Thr Pro Pro Asn Val Glu Asn Gly Ile645 650 655ttg gta tct gac aac aga agc tta ttt tcc tta aat gaa gtt gtg gag 2016Leu Val Ser Asp Asn Arg Ser Leu Phe Ser Leu Asn Glu Val Val Glu660 665 670ttt agg tgt cag cct ggc ttt gtc atg aaa gga ccc cgc cgt gtg aag 2064Phe Arg Cys Gln Pro Gly Phe Val Met Lys Gly Pro Arg Arg Val Lys675 680 685tgc cag gcc ctg aac aaa tgg gag ccg gag cta cca agc tgc tcc agg 2112Cys Gln Ala Leu Asn Lys Trp Glu Pro Glu Leu Pro Ser Cys Ser Arg690 695 700gta tgt cag cca cct cca gat gtc ctg cat gct gag cgt acc caa agg 2160Val Cys Gln Pro Pro Pro Asp Val Leu His Ala Glu Arg Thr Gln Arg705 710 715 720gac aag gac aac ttt tca ccc ggg cag gaa gtg ttc tac agc tgt gag 2208Asp Lys Asp Asn Phe Ser Pro Gly Gln Glu Val Phe Tyr Ser Cys Glu725 730 735ccc ggc tat gac ctc aga ggg gct gcg tct atg cgc tgc aca ccc cag 2256Pro Gly Tyr Asp Leu Arg Gly Ala Ala Ser Met Arg Cys Thr Pro Gln740 745 750gga gac tgg agc cct gca gcc ccc aca tgt gaa gtg aaa tcc tgt gat 2304Gly Asp Trp Ser Pro Ala Ala Pro Thr Cys Glu Val Lys Ser Cys Asp755 760 765gac ttc atg ggc caa ctt ctt aat ggc cgt gtg cta ttt cca gta aat 2352Asp Phe Met Gly Gln Leu Leu Asn Gly Arg Val Leu Phe Pro Val Asn770 775 780ctc cag ctt gga gca aaa gtg gat ttt gtt tgt gat gaa gga ttt caa 2400Leu Gln Leu Gly Ala Lys Val Asp Phe Val Cys Asp Glu Gly Phe Gln785 790 795 800tta aaa ggc agc tct gct agt tat tgt gtc ttg gct gga atg gaa agc 2448Leu Lys Gly Ser Ser Ala Ser Tyr Cys Val Leu Ala Gly Met Glu Ser805 810 815ctt tgg aat agc agt gtt cca gtg tgt gaa caa atc ttt tgt cca agt 2496Leu Trp Asn Ser Ser Val Pro Val Cys Glu Gln Ile Phe Cys Pro Ser820 825 830cct cca gtt att cct aat ggg aga cac aca gga aaa cct ctg gaa gtc 2544Pro Pro Val Ile Pro Asn Gly Arg His Thr Gly Lys Pro Leu Glu Val835 840 845ttt ccc ttt gga aaa gca gta aat tac aca tgc gac ccc cac cca gac 2592Phe Pro Phe Gly Lys Ala Val Asn Tyr Thr Cys Asp Pro His Pro Asp850 855 860aga ggg acg agc ttc gac ctc att gga gag agc acc atc cgc tgc aca 2640Arg Gly Thr Ser Phe Asp Leu Ile Gly Glu Ser Thr Ile Arg Cys Thr865 870 875 880agt gac cct caa ggg aat ggg gtt tgg agc agc cct gcc cct cgc tgt 2688Ser Asp Pro Gln Gly Asn Gly Val Trp Ser Ser Pro Ala Pro Arg Cys885 890 895gga att ctg ggt cac tgt caa gcc cca gat cat ttt ctg ttt gcc aag 2736Gly Ile Leu Gly His Cys Gln Ala Pro Asp His Phe Leu Phe Ala Lys900 905 910ttg aaa acc caa acc aat gca tct gac ttt ccc att ggg aca tct tta 2784Leu Lys Thr Gln Thr Asn Ala Ser Asp Phe Pro Ile Gly Thr Ser Leu915 920 925aag tac gaa tgc cgt cct gag tac tac ggg agg cca ttc tct atc aca 2832Lys Tyr Glu Cys Arg Pro Glu Tyr Tyr Gly Arg Pro Phe Ser Ile Thr930 935 940tgt cta gat aac ctg gtc tgg tca agt ccc aaa gat gtc tgt aaa cgt 2880Cys Leu Asp Asn Leu Val Trp Ser Ser Pro Lys Asp Val Cys Lys Arg945 950 955 960aaa tca tgt aaa act cct cca gat cca gtg aat ggc atg gtg cat gtg 2928Lys Ser Cys Lys Thr Pro Pro Asp Pro Val Asn Gly Met Val His Val965 970 975atc aca gac atc cag gtt gga tcc aga atc aac tat tct tgt act aca 2976Ile Thr Asp Ile Gln Val Gly Ser Arg Ile Asn Tyr Ser Cys Thr Thr980 985 990ggg cac cga ctc att ggt cac tca tct gct gaa tgt atc ctc tca ggc 3024Gly His Arg Leu Ile Gly His Ser Ser Ala Glu Cys Ile Leu Ser Gly995 1000 1005aat act gcc cat tgg agc acg aag ccg cca att tgt caa cga att cct 3072Asn Thr Ala His Trp Ser Thr Lys Pro Pro Ile Cys Gln Arg Ile Pro1010 1015 1020tgt ggg cta ccc cca acc atc gcc aat gga gat ttc att agc acc aac 3120Cys Gly Leu Pro Pro Thr Ile Ala Asn Gly Asp Phe Ile Ser Thr Asn1025 1030 1035 1040aga gag aat ttt cac tat gga tca gtg gtg acc tac cgc tgc aat ctt 3168Arg Glu Asn Phe His Tyr Gly Ser Val Val Thr Tyr Arg Cys Asn Leu1045 1050 1055gga agc aga ggg aga aag gtg ttt gag ctt gtg ggt gag ccc tcc ata 3216Gly Ser Arg Gly Arg Lys Val Phe Glu Leu Val Gly Glu Pro Ser Ile1060 1065 1070tac tgc acc agc aat gac gat caa gtg ggc atc tgg agc ggc ccc gcc 3264Tyr Cys Thr Ser Asn Asp Asp Gln Val Gly Ile Trp Ser Gly Pro Ala1075 1080 1085cct cag tgc att ata cct aac aaa tgc acg cct cca aat gtg gaa aat 3312Pro Gln Cys Ile Ile Pro Asn Lys Cys Thr Pro Pro Asn Val Glu Asn1090 1095 1100gga ata ttg gta tct gac aac aga agc tta ttt tcc tta aat gaa gtt 3360Gly Ile Leu Val Ser Asp Asn Arg Ser Leu Phe Ser Leu Asn Glu Val1105 1110 1115 1120gtg gag ttt agg tgt cag cct ggc ttt gtc atg aaa gga ccc cgc cgt 3408Val Glu Phe Arg Cys Gln Pro Gly Phe Val Met Lys Gly Pro Arg Arg1125 1130 1135gtg aag tgc cag gcc ctg aac aaa tgg gag cca gag tta cca agc tgc 3456Val Lys Cys Gln Ala Leu Asn Lys Trp Glu Pro Glu Leu Pro Ser Cys1140 1145 1150tcc agg gtg tgt cag ccg cct cca gaa atc ctg cat ggt gag cat acc 3504Ser Arg Val Cys Gln Pro Pro Pro Glu Ile Leu His Gly Glu His Thr1155 1160 1165cca agc cat cag gac aac ttt tca cct ggg cag gaa gtg ttc tac agc 3552Pro Ser His Gln Asp Asn Phe Ser Pro Gly Gln Glu Val Phe Tyr Ser1170 1175 1180tgt gag cct ggc tat gac ctc aga ggg gct gcg tct ctg cac tgc aca 3600Cys Glu Pro Gly Tyr Asp Leu Arg Gly Ala Ala Ser Leu His Cys Thr1185 1190 1195 1200ccc cag gga gac tgg agc cct gaa gcc ccg aga tgt gca gtg aaa tcc 3648Pro Gln Gly Asp Trp Ser Pro Glu Ala Pro Arg Cys Ala Val Lys Ser1205 1210 1215tgt gat gac ttc ttg ggt caa ctc cct cat ggc cgt gtg cta ttt cca 3696Cys Asp Asp Phe Leu Gly Gln Leu Pro His Gly Arg Val Leu Phe Pro1220 1225 1230ctt aat ctc cag ctt ggg gca aag gtg tcc ttt gtc tgt gat gaa ggg 3744Leu Asn Leu Gln Leu Gly Ala Lys Val Ser Phe Val Cys Asp Glu Gly1235 1240 1245ttt cgc tta aag ggc agt tcc gtt agt cat tgt gtc ttg gtt gga atg 3792Phe Arg Leu Lys Gly Ser Ser Val Ser His Cys Val Leu Val Gly Met1250 1255 1260aga agc ctt tgg aat aac agt gtt cct gtg tgt gaa cat atc ttt tgt 3840Arg Ser Leu Trp Asn Asn Ser Val Pro Val Cys Glu His Ile Phe Cys1265 1270 1275 1280cca aat cct cca gct atc ctt aat ggg aga cac aca gga act ccc tct 3888Pro Asn Pro Pro Ala Ile Leu Asn Gly Arg His Thr Gly Thr Pro Ser1285 1290 1295gga gat att ccc tat gga aaa gaa ata tct tac aca tgt gac ccc cac 3936Gly Asp Ile Pro Tyr Gly Lys Glu Ile Ser Tyr Thr Cys Asp Pro His1300 1305 1310cca gac aga ggg atg acc ttc aac ctc att ggg gag agc acc atc cgc 3984Pro Asp Arg Gly Met Thr Phe Asn Leu Ile Gly Glu Ser Thr Ile Arg1315 1320 1325tgc aca agt gac cct cat ggg aat ggg gtt tgg agc agc cct gcc cct 4032Cys Thr Ser Asp Pro His Gly Asn Gly Val Trp Ser Ser Pro Ala Pro1330

1335 1340cgc tgt gaa ctt tct gtt cgt gct ggt cac tgt aaa acc cca gag cag 4080Arg Cys Glu Leu Ser Val Arg Ala Gly His Cys Lys Thr Pro Glu Gln1345 1350 1355 1360ttt cca ttt gcc agt cct acg atc cca att aat gac ttt gag ttt cca 4128Phe Pro Phe Ala Ser Pro Thr Ile Pro Ile Asn Asp Phe Glu Phe Pro1365 1370 1375gtc ggg aca tct ttg aat tat gaa tgc cgt cct ggg tat ttt ggg aaa 4176Val Gly Thr Ser Leu Asn Tyr Glu Cys Arg Pro Gly Tyr Phe Gly Lys1380 1385 1390atg ttc tct atc tcc tgc cta gaa aac ttg gtc tgg tca agt gtt gaa 4224Met Phe Ser Ile Ser Cys Leu Glu Asn Leu Val Trp Ser Ser Val Glu1395 1400 1405gac aac tgt aga cga aaa tca tgt gga cct cca cca gaa ccc ttc aat 4272Asp Asn Cys Arg Arg Lys Ser Cys Gly Pro Pro Pro Glu Pro Phe Asn1410 1415 1420gga atg gtg cat ata aac aca gat aca cag ttt gga tca aca gtt aat 4320Gly Met Val His Ile Asn Thr Asp Thr Gln Phe Gly Ser Thr Val Asn1425 1430 1435 1440tat tct tgt aat gaa ggg ttt cga ctc att ggt tcc cca tct act act 4368Tyr Ser Cys Asn Glu Gly Phe Arg Leu Ile Gly Ser Pro Ser Thr Thr1445 1450 1455tgt ctc gtc tca ggc aat aat gtc aca tgg gat aag aag gca cct att 4416Cys Leu Val Ser Gly Asn Asn Val Thr Trp Asp Lys Lys Ala Pro Ile1460 1465 1470tgt gag atc ata tct tgt gag cca cct cca acc ata tcc aat gga gac 4464Cys Glu Ile Ile Ser Cys Glu Pro Pro Pro Thr Ile Ser Asn Gly Asp1475 1480 1485ttc tac agc aac aat aga aca tct ttt cac aat gga acg gtg gta act 4512Phe Tyr Ser Asn Asn Arg Thr Ser Phe His Asn Gly Thr Val Val Thr1490 1495 1500tac cag tgc cac act gga cca gat gga gaa cag ctg ttt gag ctt gtg 4560Tyr Gln Cys His Thr Gly Pro Asp Gly Glu Gln Leu Phe Glu Leu Val1505 1510 1515 1520gga gaa cgg tca ata tat tgc acc agc aaa gat gat caa gtt ggt gtt 4608Gly Glu Arg Ser Ile Tyr Cys Thr Ser Lys Asp Asp Gln Val Gly Val1525 1530 1535tgg agc agc cct ccc cct cgg tgt att tct act aat aaa tgc aca gct 4656Trp Ser Ser Pro Pro Pro Arg Cys Ile Ser Thr Asn Lys Cys Thr Ala1540 1545 1550cca gaa gtt gaa aat gca att aga gta cca gga aac agg agt ttc ttt 4704Pro Glu Val Glu Asn Ala Ile Arg Val Pro Gly Asn Arg Ser Phe Phe1555 1560 1565tcc ctc act gag atc atc aga ttt aga tgt cag ccc ggg ttt gtc atg 4752Ser Leu Thr Glu Ile Ile Arg Phe Arg Cys Gln Pro Gly Phe Val Met1570 1575 1580gta ggg tcc cac act gtg cag tgc cag acc aat ggc aga tgg ggg ccc 4800Val Gly Ser His Thr Val Gln Cys Gln Thr Asn Gly Arg Trp Gly Pro1585 1590 1595 1600aag ctg cca cac tgc tcc agg gtg tgt cag ccg cct cca gaa atc ctg 4848Lys Leu Pro His Cys Ser Arg Val Cys Gln Pro Pro Pro Glu Ile Leu1605 1610 1615cat ggt gag cat acc cta agc cat cag gac aac ttt tca cct ggg cag 4896His Gly Glu His Thr Leu Ser His Gln Asp Asn Phe Ser Pro Gly Gln1620 1625 1630gaa gtg ttc tac agc tgt gag ccc agc tat gac ctc aga ggg gct gcg 4944Glu Val Phe Tyr Ser Cys Glu Pro Ser Tyr Asp Leu Arg Gly Ala Ala1635 1640 1645tct ctg cac tgc acg ccc cag gga gac tgg agc cct gaa gcc cct aga 4992Ser Leu His Cys Thr Pro Gln Gly Asp Trp Ser Pro Glu Ala Pro Arg1650 1655 1660tgt aca gtg aaa tcc tgt gat gac ttc ctg ggc caa ctc cct cat ggc 5040Cys Thr Val Lys Ser Cys Asp Asp Phe Leu Gly Gln Leu Pro His Gly1665 1670 1675 1680cgt gtg cta ctt cca ctt aat ctc cag ctt ggg gca aag gtg tcc ttt 5088Arg Val Leu Leu Pro Leu Asn Leu Gln Leu Gly Ala Lys Val Ser Phe1685 1690 1695gtt tgc gat gaa ggg ttc cga tta aaa ggc agg tct gct agt cat tgt 5136Val Cys Asp Glu Gly Phe Arg Leu Lys Gly Arg Ser Ala Ser His Cys1700 1705 1710gtc ttg gct gga atg aaa gcc ctt tgg aat agc agt gtt cca gtg tgt 5184Val Leu Ala Gly Met Lys Ala Leu Trp Asn Ser Ser Val Pro Val Cys1715 1720 1725gaa caa atc ttt tgt cca aat cct cca gct atc ctt aat ggg aga cac 5232Glu Gln Ile Phe Cys Pro Asn Pro Pro Ala Ile Leu Asn Gly Arg His1730 1735 1740aca gga act ccc ttt gga gat att ccc tat gga aaa gaa ata tct tac 5280Thr Gly Thr Pro Phe Gly Asp Ile Pro Tyr Gly Lys Glu Ile Ser Tyr1745 1750 1755 1760gca tgc gac acc cac cca gac aga ggg atg acc ttc aac ctc att ggg 5328Ala Cys Asp Thr His Pro Asp Arg Gly Met Thr Phe Asn Leu Ile Gly1765 1770 1775gag agc tcc atc cgc tgc aca agt gac cct caa ggg aat ggg gtt tgg 5376Glu Ser Ser Ile Arg Cys Thr Ser Asp Pro Gln Gly Asn Gly Val Trp1780 1785 1790agc agc cct gcc cct cgc tgt gaa ctt tct gtt cct gct gcc tgc cca 5424Ser Ser Pro Ala Pro Arg Cys Glu Leu Ser Val Pro Ala Ala Cys Pro1795 1800 1805cat cca ccc aag atc caa aac ggg cat tac att gga gga cac gta tct 5472His Pro Pro Lys Ile Gln Asn Gly His Tyr Ile Gly Gly His Val Ser1810 1815 1820cta tat ctt cct ggg atg aca atc agc tac act tgt gac ccc ggc tac 5520Leu Tyr Leu Pro Gly Met Thr Ile Ser Tyr Thr Cys Asp Pro Gly Tyr1825 1830 1835 1840ctg tta gtg gga aag ggc ttc att ttc tgt aca gac cag gga atc tgg 5568Leu Leu Val Gly Lys Gly Phe Ile Phe Cys Thr Asp Gln Gly Ile Trp1845 1850 1855agc caa ttg gat cat tat tgc aaa gaa gta aat tgt agc ttc cca ctg 5616Ser Gln Leu Asp His Tyr Cys Lys Glu Val Asn Cys Ser Phe Pro Leu1860 1865 1870ttt atg aat gga atc tcg aag gag tta gaa atg aaa aaa gta tat cac 5664Phe Met Asn Gly Ile Ser Lys Glu Leu Glu Met Lys Lys Val Tyr His1875 1880 1885tat gga gat tat gtg act ttg aag tgt gaa gat ggg tat act ctg gaa 5712Tyr Gly Asp Tyr Val Thr Leu Lys Cys Glu Asp Gly Tyr Thr Leu Glu1890 1895 1900ggc agt ccc tgg agc cag tgc cag gcg gat gac aga tgg gac cct cct 5760Gly Ser Pro Trp Ser Gln Cys Gln Ala Asp Asp Arg Trp Asp Pro Pro1905 1910 1915 1920ctg gcc aaa tgt acc tct cgt gca cat gat gct ctc ata gtt ggc act 5808Leu Ala Lys Cys Thr Ser Arg Ala His Asp Ala Leu Ile Val Gly Thr1925 1930 1935tta tct ggt acg atc ttc ttt att tta ctc atc att ttc ctc tct tgg 5856Leu Ser Gly Thr Ile Phe Phe Ile Leu Leu Ile Ile Phe Leu Ser Trp1940 1945 1950ata att cta aag cac aga aaa ggc aat aat gca cat gaa aac cct aaa 5904Ile Ile Leu Lys His Arg Lys Gly Asn Asn Ala His Glu Asn Pro Lys1955 1960 1965gaa gtg gct atc cat tta cat tct caa gga ggc agc agc gtt cat ccc 5952Glu Val Ala Ile His Leu His Ser Gln Gly Gly Ser Ser Val His Pro1970 1975 1980cga act ctg caa aca aat gaa gaa aat agc agg gtc ctt cct 5994Arg Thr Leu Gln Thr Asn Glu Glu Asn Ser Arg Val Leu Pro1985 1990 1995tgacaaagta ctatacagct gaagaacatc tcgaatacaa ttttggtggg aaaggagcca 6054attgatttca acagaatcag atctgagctt cataaagtct ttgaagtgac ttcacagaga 6114cgcagacatg tgcacttgaa gatgctgccc cttccctggt acctagcaaa gctcctgcct 6174ctttgtgtgc gtcactgtga aacccccacc cttctgcctc gtgctaaacg cacacagtat 6234ctagtcaggg gaaaagactg catttaggag atagaaaata gtttggatta cttaaaggaa 6294taaggtgttg cctggaattt ctggtttgta aggtggtcac tgttcttttt taaaatattt 6354gtaatatgga atgggctcag taagaagagc ttggaaaatg cagaaagtta tgaaaaataa 6414gtcacttata attatgctac ctactgataa ccactcctaa tattttgatt cattttctgc 6474ctatcttctt tcacatatgt gtttttttac atacgtactt ttcccccctt agtttgtttc 6534cttttatttt atagagcaga accctagtct tttaaacagt ttagagtgaa atatatgcta 6594tatcagtttt tactttctct agggagaaaa attaatttac tagaaaggca tgaaatgatc 6654atgggaagag tggttaagac tactgaagag aaatatttgg aaaataagat ttcgatatct 6714tctttttttt tgagatggag tctggctctg tctcccaggc tggagtgcag tggcgtaatc 6774tcggctcact gcaacgtccg cctcccg 6801131998PRTHomo sapiensCR-1 13Gln Cys Asn Ala Pro Glu Trp Leu Pro Phe Ala Arg Pro Thr Asn Leu 1 5 10 15Thr Asp Glu Phe Glu Phe Pro Ile Gly Thr Tyr Leu Asn Tyr Glu Cys 20 25 30Arg Pro Gly Tyr Ser Gly Arg Pro Phe Ser Ile Ile Cys Leu Lys Asn 35 40 45Ser Val Trp Thr Gly Ala Lys Asp Arg Cys Arg Arg Lys Ser Cys Arg 50 55 60Asn Pro Pro Asp Pro Val Asn Gly Met Val His Val Ile Lys Gly Ile65 70 75 80Gln Phe Gly Ser Gln Ile Lys Tyr Ser Cys Thr Lys Gly Tyr Arg Leu 85 90 95Ile Gly Ser Ser Ser Ala Thr Cys Ile Ile Ser Gly Asp Thr Val Ile 100 105 110Trp Asp Asn Glu Thr Pro Ile Cys Asp Arg Ile Pro Cys Gly Leu Pro 115 120 125Pro Thr Ile Thr Asn Gly Asp Phe Ile Ser Thr Asn Arg Glu Asn Phe 130 135 140His Tyr Gly Ser Val Val Thr Tyr Arg Cys Asn Pro Gly Ser Gly Gly145 150 155 160Arg Lys Val Phe Glu Leu Val Gly Glu Pro Ser Ile Tyr Cys Thr Ser 165 170 175Asn Asp Asp Gln Val Gly Ile Trp Ser Gly Pro Ala Pro Gln Cys Ile 180 185 190Ile Pro Asn Lys Cys Thr Pro Pro Asn Val Glu Asn Gly Ile Leu Val 195 200 205Ser Asp Asn Arg Ser Leu Phe Ser Leu Asn Glu Val Val Glu Phe Arg 210 215 220Cys Gln Pro Gly Phe Val Met Lys Gly Pro Arg Arg Val Lys Cys Gln225 230 235 240Ala Leu Asn Lys Trp Glu Pro Glu Leu Pro Ser Cys Ser Arg Val Cys 245 250 255Gln Pro Pro Pro Asp Val Leu His Ala Glu Arg Thr Gln Arg Asp Lys 260 265 270Asp Asn Phe Ser Pro Gly Gln Glu Val Phe Tyr Ser Cys Glu Pro Gly 275 280 285Tyr Asp Leu Arg Gly Ala Ala Ser Met Arg Cys Thr Pro Gln Gly Asp 290 295 300Trp Ser Pro Ala Ala Pro Thr Cys Glu Val Lys Ser Cys Asp Asp Phe305 310 315 320Met Gly Gln Leu Leu Asn Gly Arg Val Leu Phe Pro Val Asn Leu Gln 325 330 335Leu Gly Ala Lys Val Asp Phe Val Cys Asp Glu Gly Phe Gln Leu Lys 340 345 350Gly Ser Ser Ala Ser Tyr Cys Val Leu Ala Gly Met Glu Ser Leu Trp 355 360 365Asn Ser Ser Val Pro Val Cys Glu Gln Ile Phe Cys Pro Ser Pro Pro 370 375 380Val Ile Pro Asn Gly Arg His Thr Gly Lys Pro Leu Glu Val Phe Pro385 390 395 400Phe Gly Lys Ala Val Asn Tyr Thr Cys Asp Pro His Pro Asp Arg Gly 405 410 415Thr Ser Phe Asp Leu Ile Gly Glu Ser Thr Ile Arg Cys Thr Ser Asp 420 425 430Pro Gln Gly Asn Gly Val Trp Ser Ser Pro Ala Pro Arg Cys Gly Ile 435 440 445Leu Gly His Cys Gln Ala Pro Asp His Phe Leu Phe Ala Lys Leu Lys 450 455 460Thr Gln Thr Asn Ala Ser Asp Phe Pro Ile Gly Thr Ser Leu Lys Tyr465 470 475 480Glu Cys Arg Pro Glu Tyr Tyr Gly Arg Pro Phe Ser Ile Thr Cys Leu 485 490 495Asp Asn Leu Val Trp Ser Ser Pro Lys Asp Val Cys Lys Arg Lys Ser 500 505 510Cys Lys Thr Pro Pro Asp Pro Val Asn Gly Met Val His Val Ile Thr 515 520 525Asp Ile Gln Val Gly Ser Arg Ile Asn Tyr Ser Cys Thr Thr Gly His 530 535 540Arg Leu Ile Gly His Ser Ser Ala Glu Cys Ile Leu Ser Gly Asn Ala545 550 555 560Ala His Trp Ser Thr Lys Pro Pro Ile Cys Gln Arg Ile Pro Cys Gly 565 570 575Leu Pro Pro Thr Ile Ala Asn Gly Asp Phe Ile Ser Thr Asn Arg Glu 580 585 590Asn Phe His Tyr Gly Ser Val Val Thr Tyr Arg Cys Asn Pro Gly Ser 595 600 605Gly Gly Arg Lys Val Phe Glu Leu Val Gly Glu Pro Ser Ile Tyr Cys 610 615 620Thr Ser Asn Asp Asp Gln Val Gly Ile Trp Ser Gly Pro Ala Pro Gln625 630 635 640Cys Ile Ile Pro Asn Lys Cys Thr Pro Pro Asn Val Glu Asn Gly Ile 645 650 655Leu Val Ser Asp Asn Arg Ser Leu Phe Ser Leu Asn Glu Val Val Glu 660 665 670Phe Arg Cys Gln Pro Gly Phe Val Met Lys Gly Pro Arg Arg Val Lys 675 680 685Cys Gln Ala Leu Asn Lys Trp Glu Pro Glu Leu Pro Ser Cys Ser Arg 690 695 700Val Cys Gln Pro Pro Pro Asp Val Leu His Ala Glu Arg Thr Gln Arg705 710 715 720Asp Lys Asp Asn Phe Ser Pro Gly Gln Glu Val Phe Tyr Ser Cys Glu 725 730 735Pro Gly Tyr Asp Leu Arg Gly Ala Ala Ser Met Arg Cys Thr Pro Gln 740 745 750Gly Asp Trp Ser Pro Ala Ala Pro Thr Cys Glu Val Lys Ser Cys Asp 755 760 765Asp Phe Met Gly Gln Leu Leu Asn Gly Arg Val Leu Phe Pro Val Asn 770 775 780Leu Gln Leu Gly Ala Lys Val Asp Phe Val Cys Asp Glu Gly Phe Gln785 790 795 800Leu Lys Gly Ser Ser Ala Ser Tyr Cys Val Leu Ala Gly Met Glu Ser 805 810 815Leu Trp Asn Ser Ser Val Pro Val Cys Glu Gln Ile Phe Cys Pro Ser 820 825 830Pro Pro Val Ile Pro Asn Gly Arg His Thr Gly Lys Pro Leu Glu Val 835 840 845Phe Pro Phe Gly Lys Ala Val Asn Tyr Thr Cys Asp Pro His Pro Asp 850 855 860Arg Gly Thr Ser Phe Asp Leu Ile Gly Glu Ser Thr Ile Arg Cys Thr865 870 875 880Ser Asp Pro Gln Gly Asn Gly Val Trp Ser Ser Pro Ala Pro Arg Cys 885 890 895Gly Ile Leu Gly His Cys Gln Ala Pro Asp His Phe Leu Phe Ala Lys 900 905 910Leu Lys Thr Gln Thr Asn Ala Ser Asp Phe Pro Ile Gly Thr Ser Leu 915 920 925Lys Tyr Glu Cys Arg Pro Glu Tyr Tyr Gly Arg Pro Phe Ser Ile Thr 930 935 940Cys Leu Asp Asn Leu Val Trp Ser Ser Pro Lys Asp Val Cys Lys Arg945 950 955 960Lys Ser Cys Lys Thr Pro Pro Asp Pro Val Asn Gly Met Val His Val 965 970 975Ile Thr Asp Ile Gln Val Gly Ser Arg Ile Asn Tyr Ser Cys Thr Thr 980 985 990Gly His Arg Leu Ile Gly His Ser Ser Ala Glu Cys Ile Leu Ser Gly 995 1000 1005Asn Thr Ala His Trp Ser Thr Lys Pro Pro Ile Cys Gln Arg Ile Pro 1010 1015 1020Cys Gly Leu Pro Pro Thr Ile Ala Asn Gly Asp Phe Ile Ser Thr Asn1025 1030 1035 1040Arg Glu Asn Phe His Tyr Gly Ser Val Val Thr Tyr Arg Cys Asn Leu 1045 1050 1055Gly Ser Arg Gly Arg Lys Val Phe Glu Leu Val Gly Glu Pro Ser Ile 1060 1065 1070Tyr Cys Thr Ser Asn Asp Asp Gln Val Gly Ile Trp Ser Gly Pro Ala 1075 1080 1085Pro Gln Cys Ile Ile Pro Asn Lys Cys Thr Pro Pro Asn Val Glu Asn 1090 1095 1100Gly Ile Leu Val Ser Asp Asn Arg Ser Leu Phe Ser Leu Asn Glu Val1105 1110 1115 1120Val Glu Phe Arg Cys Gln Pro Gly Phe Val Met Lys Gly Pro Arg Arg 1125 1130 1135Val Lys Cys Gln Ala Leu Asn Lys Trp Glu Pro Glu Leu Pro Ser Cys 1140 1145 1150Ser Arg Val Cys Gln Pro Pro Pro Glu Ile Leu His Gly Glu His Thr 1155 1160 1165Pro Ser His Gln Asp Asn Phe Ser Pro Gly Gln Glu Val Phe Tyr Ser 1170 1175 1180Cys Glu Pro Gly Tyr Asp Leu Arg Gly Ala Ala Ser Leu His Cys Thr1185 1190 1195 1200Pro Gln Gly Asp Trp Ser Pro Glu Ala Pro Arg Cys Ala Val Lys Ser 1205 1210 1215Cys Asp Asp Phe Leu Gly Gln Leu Pro His Gly Arg Val Leu Phe Pro 1220 1225 1230Leu Asn Leu Gln Leu Gly Ala Lys Val Ser Phe Val Cys Asp Glu Gly 1235 1240 1245Phe Arg Leu Lys Gly Ser Ser Val Ser His Cys Val Leu Val Gly Met 1250 1255 1260Arg Ser Leu Trp Asn Asn Ser Val Pro Val Cys Glu His Ile Phe Cys1265 1270 1275 1280Pro Asn Pro Pro Ala Ile Leu Asn Gly Arg His Thr Gly Thr Pro Ser 1285 1290 1295Gly Asp Ile Pro Tyr Gly Lys Glu Ile Ser Tyr Thr

Cys Asp Pro His 1300 1305 1310Pro Asp Arg Gly Met Thr Phe Asn Leu Ile Gly Glu Ser Thr Ile Arg 1315 1320 1325Cys Thr Ser Asp Pro His Gly Asn Gly Val Trp Ser Ser Pro Ala Pro 1330 1335 1340Arg Cys Glu Leu Ser Val Arg Ala Gly His Cys Lys Thr Pro Glu Gln1345 1350 1355 1360Phe Pro Phe Ala Ser Pro Thr Ile Pro Ile Asn Asp Phe Glu Phe Pro 1365 1370 1375Val Gly Thr Ser Leu Asn Tyr Glu Cys Arg Pro Gly Tyr Phe Gly Lys 1380 1385 1390Met Phe Ser Ile Ser Cys Leu Glu Asn Leu Val Trp Ser Ser Val Glu 1395 1400 1405Asp Asn Cys Arg Arg Lys Ser Cys Gly Pro Pro Pro Glu Pro Phe Asn 1410 1415 1420Gly Met Val His Ile Asn Thr Asp Thr Gln Phe Gly Ser Thr Val Asn1425 1430 1435 1440Tyr Ser Cys Asn Glu Gly Phe Arg Leu Ile Gly Ser Pro Ser Thr Thr 1445 1450 1455Cys Leu Val Ser Gly Asn Asn Val Thr Trp Asp Lys Lys Ala Pro Ile 1460 1465 1470Cys Glu Ile Ile Ser Cys Glu Pro Pro Pro Thr Ile Ser Asn Gly Asp 1475 1480 1485Phe Tyr Ser Asn Asn Arg Thr Ser Phe His Asn Gly Thr Val Val Thr 1490 1495 1500Tyr Gln Cys His Thr Gly Pro Asp Gly Glu Gln Leu Phe Glu Leu Val1505 1510 1515 1520Gly Glu Arg Ser Ile Tyr Cys Thr Ser Lys Asp Asp Gln Val Gly Val 1525 1530 1535Trp Ser Ser Pro Pro Pro Arg Cys Ile Ser Thr Asn Lys Cys Thr Ala 1540 1545 1550Pro Glu Val Glu Asn Ala Ile Arg Val Pro Gly Asn Arg Ser Phe Phe 1555 1560 1565Ser Leu Thr Glu Ile Ile Arg Phe Arg Cys Gln Pro Gly Phe Val Met 1570 1575 1580Val Gly Ser His Thr Val Gln Cys Gln Thr Asn Gly Arg Trp Gly Pro1585 1590 1595 1600Lys Leu Pro His Cys Ser Arg Val Cys Gln Pro Pro Pro Glu Ile Leu 1605 1610 1615His Gly Glu His Thr Leu Ser His Gln Asp Asn Phe Ser Pro Gly Gln 1620 1625 1630Glu Val Phe Tyr Ser Cys Glu Pro Ser Tyr Asp Leu Arg Gly Ala Ala 1635 1640 1645Ser Leu His Cys Thr Pro Gln Gly Asp Trp Ser Pro Glu Ala Pro Arg 1650 1655 1660Cys Thr Val Lys Ser Cys Asp Asp Phe Leu Gly Gln Leu Pro His Gly1665 1670 1675 1680Arg Val Leu Leu Pro Leu Asn Leu Gln Leu Gly Ala Lys Val Ser Phe 1685 1690 1695Val Cys Asp Glu Gly Phe Arg Leu Lys Gly Arg Ser Ala Ser His Cys 1700 1705 1710Val Leu Ala Gly Met Lys Ala Leu Trp Asn Ser Ser Val Pro Val Cys 1715 1720 1725Glu Gln Ile Phe Cys Pro Asn Pro Pro Ala Ile Leu Asn Gly Arg His 1730 1735 1740Thr Gly Thr Pro Phe Gly Asp Ile Pro Tyr Gly Lys Glu Ile Ser Tyr1745 1750 1755 1760Ala Cys Asp Thr His Pro Asp Arg Gly Met Thr Phe Asn Leu Ile Gly 1765 1770 1775Glu Ser Ser Ile Arg Cys Thr Ser Asp Pro Gln Gly Asn Gly Val Trp 1780 1785 1790Ser Ser Pro Ala Pro Arg Cys Glu Leu Ser Val Pro Ala Ala Cys Pro 1795 1800 1805His Pro Pro Lys Ile Gln Asn Gly His Tyr Ile Gly Gly His Val Ser 1810 1815 1820Leu Tyr Leu Pro Gly Met Thr Ile Ser Tyr Thr Cys Asp Pro Gly Tyr1825 1830 1835 1840Leu Leu Val Gly Lys Gly Phe Ile Phe Cys Thr Asp Gln Gly Ile Trp 1845 1850 1855Ser Gln Leu Asp His Tyr Cys Lys Glu Val Asn Cys Ser Phe Pro Leu 1860 1865 1870Phe Met Asn Gly Ile Ser Lys Glu Leu Glu Met Lys Lys Val Tyr His 1875 1880 1885Tyr Gly Asp Tyr Val Thr Leu Lys Cys Glu Asp Gly Tyr Thr Leu Glu 1890 1895 1900Gly Ser Pro Trp Ser Gln Cys Gln Ala Asp Asp Arg Trp Asp Pro Pro1905 1910 1915 1920Leu Ala Lys Cys Thr Ser Arg Ala His Asp Ala Leu Ile Val Gly Thr 1925 1930 1935Leu Ser Gly Thr Ile Phe Phe Ile Leu Leu Ile Ile Phe Leu Ser Trp 1940 1945 1950Ile Ile Leu Lys His Arg Lys Gly Asn Asn Ala His Glu Asn Pro Lys 1955 1960 1965Glu Val Ala Ile His Leu His Ser Gln Gly Gly Ser Ser Val His Pro 1970 1975 1980Arg Thr Leu Gln Thr Asn Glu Glu Asn Ser Arg Val Leu Pro1985 1990 1995143036DNAHomo sapiensCR2 14att tct tgt ggc tct cct ccg cct atc cta aat ggc cgg att agt tat 48Ile Ser Cys Gly Ser Pro Pro Pro Ile Leu Asn Gly Arg Ile Ser Tyr1 5 10 15tat tct acc ccc att gct gtt ggt acc gtg ata agg tac agt tgt tca 96Tyr Ser Thr Pro Ile Ala Val Gly Thr Val Ile Arg Tyr Ser Cys Ser20 25 30ggt acc ttc cgc ctc att gga gaa aaa agt cta tta tgc ata act aaa 144Gly Thr Phe Arg Leu Ile Gly Glu Lys Ser Leu Leu Cys Ile Thr Lys35 40 45gac aaa gtg gat gga acc tgg gat aaa cct gct cct aaa tgt gaa tat 192Asp Lys Val Asp Gly Thr Trp Asp Lys Pro Ala Pro Lys Cys Glu Tyr50 55 60ttc aat aaa tat tct tct tgc cct gag ccc ata gta cca gga gga tac 240Phe Asn Lys Tyr Ser Ser Cys Pro Glu Pro Ile Val Pro Gly Gly Tyr65 70 75 80aaa att aga ggc tct aca ccc tac aga cat ggt gat tct gtg aca ttt 288Lys Ile Arg Gly Ser Thr Pro Tyr Arg His Gly Asp Ser Val Thr Phe85 90 95gcc tgt aaa acc aac ttc tcc atg aac gga aac aag tct gtt tgg tgt 336Ala Cys Lys Thr Asn Phe Ser Met Asn Gly Asn Lys Ser Val Trp Cys100 105 110caa gca aat aat atg tgg ggg ccg aca cga cta cca acc tgt gta agt 384Gln Ala Asn Asn Met Trp Gly Pro Thr Arg Leu Pro Thr Cys Val Ser115 120 125gtt ttc cct ctc gag tgt cca gca ctt cct atg atc cac aat gga cat 432Val Phe Pro Leu Glu Cys Pro Ala Leu Pro Met Ile His Asn Gly His130 135 140cac aca agt gag aat gtt ggc tcc att gct cca gga ttg tct gtg act 480His Thr Ser Glu Asn Val Gly Ser Ile Ala Pro Gly Leu Ser Val Thr145 150 155 160tac agc tgt gaa tct ggt tac ttg ctt gtt gga gaa aag atc att aac 528Tyr Ser Cys Glu Ser Gly Tyr Leu Leu Val Gly Glu Lys Ile Ile Asn165 170 175tgt ttg tct tcg gga aaa tgg agt gct gtc ccc ccc aca tgt gaa gag 576Cys Leu Ser Ser Gly Lys Trp Ser Ala Val Pro Pro Thr Cys Glu Glu180 185 190gca cgc tgt aaa tct cta gga cga ttt ccc aat ggg aag gta aag gag 624Ala Arg Cys Lys Ser Leu Gly Arg Phe Pro Asn Gly Lys Val Lys Glu195 200 205cct cca att ctc cgg gtt ggt gta act gca aac ttt ttc tgt gat gaa 672Pro Pro Ile Leu Arg Val Gly Val Thr Ala Asn Phe Phe Cys Asp Glu210 215 220ggg tat cga ctg caa ggc cca cct tct agt cgg tgt gta att gct gga 720Gly Tyr Arg Leu Gln Gly Pro Pro Ser Ser Arg Cys Val Ile Ala Gly225 230 235 240cag gga gtt gct tgg acc aaa atg cca gta tgt gaa gaa att ttt tgc 768Gln Gly Val Ala Trp Thr Lys Met Pro Val Cys Glu Glu Ile Phe Cys245 250 255cca tca cct ccc cct att ctc aat gga aga cat ata ggc aac tca cta 816Pro Ser Pro Pro Pro Ile Leu Asn Gly Arg His Ile Gly Asn Ser Leu260 265 270gca aat gtc tca tat gga agc ata gtc act tac act tgt gac ccg gac 864Ala Asn Val Ser Tyr Gly Ser Ile Val Thr Tyr Thr Cys Asp Pro Asp275 280 285cca gag gaa gga gtg aac ttc atc ctt att gga gag agc act ctc cgt 912Pro Glu Glu Gly Val Asn Phe Ile Leu Ile Gly Glu Ser Thr Leu Arg290 295 300tgt aca gtt gat agt cag aag act ggg acc tgg agt ggc cct gcc cca 960Cys Thr Val Asp Ser Gln Lys Thr Gly Thr Trp Ser Gly Pro Ala Pro305 310 315 320cgc tgt gaa ctt tct act tct gcg gtt cag tgt cca cat ccc cag atc 1008Arg Cys Glu Leu Ser Thr Ser Ala Val Gln Cys Pro His Pro Gln Ile325 330 335cta aga ggc cga atg gta tct ggg cag aaa gat cga tat acc tat aac 1056Leu Arg Gly Arg Met Val Ser Gly Gln Lys Asp Arg Tyr Thr Tyr Asn340 345 350gac act gtg ata ttt gct tgc atg ttt ggc ttc acc ttg aag ggc agc 1104Asp Thr Val Ile Phe Ala Cys Met Phe Gly Phe Thr Leu Lys Gly Ser355 360 365aag caa atc cga tgc aat gcc caa ggc aca tgg gag cca tct gca cca 1152Lys Gln Ile Arg Cys Asn Ala Gln Gly Thr Trp Glu Pro Ser Ala Pro370 375 380gtc tgt gaa aag gaa tgc cag gcc cct cct aac atc ctc aat ggg caa 1200Val Cys Glu Lys Glu Cys Gln Ala Pro Pro Asn Ile Leu Asn Gly Gln385 390 395 400aag gaa gat aga cac atg gtc cgc ttt gac cct gga aca tct ata aaa 1248Lys Glu Asp Arg His Met Val Arg Phe Asp Pro Gly Thr Ser Ile Lys405 410 415tat agc tgt aac cct ggc tat gtg ctg gtg gga gaa gaa tcc ata cag 1296Tyr Ser Cys Asn Pro Gly Tyr Val Leu Val Gly Glu Glu Ser Ile Gln420 425 430tgt acc tct gag gtg tgg aca ccc cct gta ccc caa tgc aaa gtg gca 1344Cys Thr Ser Glu Val Trp Thr Pro Pro Val Pro Gln Cys Lys Val Ala435 440 445gcg tgt gaa gct aca gga agg caa ctc ttg aca aaa ccc cag cac caa 1392Ala Cys Glu Ala Thr Gly Arg Gln Leu Leu Thr Lys Pro Gln His Gln450 455 460ttt gtt aga cca gat gtc aac tct tct tgt ggt gaa ggg tac aag tta 1440Phe Val Arg Pro Asp Val Asn Ser Ser Cys Gly Glu Gly Tyr Lys Leu465 470 475 480agt ggg agt gtt tat cag gag tgt caa ggc aca att cct tgg ttt atg 1488Ser Gly Ser Val Tyr Gln Glu Cys Gln Gly Thr Ile Pro Trp Phe Met485 490 495gag att cgt ctt tgt aaa gaa atc acc tgc cca cca ccc cct gtt atc 1536Glu Ile Arg Leu Cys Lys Glu Ile Thr Cys Pro Pro Pro Pro Val Ile500 505 510tac aat ggg gca cac acc ggg agt tcc tta gaa gat ttt cca tat gga 1584Tyr Asn Gly Ala His Thr Gly Ser Ser Leu Glu Asp Phe Pro Tyr Gly515 520 525acc acg gtc act tac aca tgt aac cct ggg cca gaa aga gga gtg gaa 1632Thr Thr Val Thr Tyr Thr Cys Asn Pro Gly Pro Glu Arg Gly Val Glu530 535 540ttc agc ctc att gga gag agc acc atc cgt tgt aca agc aat gat caa 1680Phe Ser Leu Ile Gly Glu Ser Thr Ile Arg Cys Thr Ser Asn Asp Gln545 550 555 560gaa aga ggc acc tgg agt ggc cct gct ccc ctg tgt aaa ctt tcc ctc 1728Glu Arg Gly Thr Trp Ser Gly Pro Ala Pro Leu Cys Lys Leu Ser Leu565 570 575ctt gct gtc cag tgc tca cat gtc cat att gca aat gga tac aag ata 1776Leu Ala Val Gln Cys Ser His Val His Ile Ala Asn Gly Tyr Lys Ile580 585 590tct ggc aag gaa gcc cca tat ttc tac aat gac act gtg aca ttc aag 1824Ser Gly Lys Glu Ala Pro Tyr Phe Tyr Asn Asp Thr Val Thr Phe Lys595 600 605tgt tat agt gga ttt act ttg aag ggc agt agt cag att cgt tgc aaa 1872Cys Tyr Ser Gly Phe Thr Leu Lys Gly Ser Ser Gln Ile Arg Cys Lys610 615 620gct gat aac acc tgg gat cct gaa ata cca gtt tgt gaa aaa gaa aca 1920Ala Asp Asn Thr Trp Asp Pro Glu Ile Pro Val Cys Glu Lys Glu Thr625 630 635 640tgc cag cat gtg aga cag agt ctt caa gaa ctt cca gct ggt tca cgt 1968Cys Gln His Val Arg Gln Ser Leu Gln Glu Leu Pro Ala Gly Ser Arg645 650 655gtg gag cta gtt aat acg tcc tgc caa gat ggg tac cag ttg act gga 2016Val Glu Leu Val Asn Thr Ser Cys Gln Asp Gly Tyr Gln Leu Thr Gly660 665 670cat gct tat cag atg tgt caa gat gct gaa aat gga att tgg ttc aaa 2064His Ala Tyr Gln Met Cys Gln Asp Ala Glu Asn Gly Ile Trp Phe Lys675 680 685aag att cca ctt tgt aaa gtt att cac tgt cac cct cca cca gtg att 2112Lys Ile Pro Leu Cys Lys Val Ile His Cys His Pro Pro Pro Val Ile690 695 700gtc aat ggg aag cac aca ggc atg atg gca gaa aac ttt cta tat gga 2160Val Asn Gly Lys His Thr Gly Met Met Ala Glu Asn Phe Leu Tyr Gly705 710 715 720aat gaa gtc tct tat gaa tgt gac caa gga ttc tat ctc ctg gga gag 2208Asn Glu Val Ser Tyr Glu Cys Asp Gln Gly Phe Tyr Leu Leu Gly Glu725 730 735aaa aaa ttg cag tgc aga agt gat tct aaa gga cat gga tct tgg agc 2256Lys Lys Leu Gln Cys Arg Ser Asp Ser Lys Gly His Gly Ser Trp Ser740 745 750ggg cct tcc cca cag tgc tta cga tct cct cct gtg act cgc tgc cct 2304Gly Pro Ser Pro Gln Cys Leu Arg Ser Pro Pro Val Thr Arg Cys Pro755 760 765aat cca gaa gtc aaa cat ggg tac aag ctc aat aaa aca cat tct gca 2352Asn Pro Glu Val Lys His Gly Tyr Lys Leu Asn Lys Thr His Ser Ala770 775 780tat tcc cac aat gac ata gtg tat gtt gac tgc aat cct ggc ttc atc 2400Tyr Ser His Asn Asp Ile Val Tyr Val Asp Cys Asn Pro Gly Phe Ile785 790 795 800atg aat ggt agt cgc gtg att agg tgt cat act gat aac aca tgg gtg 2448Met Asn Gly Ser Arg Val Ile Arg Cys His Thr Asp Asn Thr Trp Val805 810 815cca ggt gtg cca act tgt atc aaa aaa gcc ttc ata ggg tgt cca cct 2496Pro Gly Val Pro Thr Cys Ile Lys Lys Ala Phe Ile Gly Cys Pro Pro820 825 830ccg cct aag acc cct aac ggg aac cat act ggt gga aac ata gct cga 2544Pro Pro Lys Thr Pro Asn Gly Asn His Thr Gly Gly Asn Ile Ala Arg835 840 845ttt tct cct gga atg tca atc ctg tac agc tgt gac caa ggc tac ctg 2592Phe Ser Pro Gly Met Ser Ile Leu Tyr Ser Cys Asp Gln Gly Tyr Leu850 855 860ctg gtg gga gag gca ctc ctt ctt tgc aca cat gag gga acc tgg agc 2640Leu Val Gly Glu Ala Leu Leu Leu Cys Thr His Glu Gly Thr Trp Ser865 870 875 880caa cct gcc cct cat tgt aaa gag gta aac tgt agc tca cca gca gat 2688Gln Pro Ala Pro His Cys Lys Glu Val Asn Cys Ser Ser Pro Ala Asp885 890 895atg gat gga atc cag aaa ggg ctg gaa cca agg aaa atg tat cag tat 2736Met Asp Gly Ile Gln Lys Gly Leu Glu Pro Arg Lys Met Tyr Gln Tyr900 905 910gga gct gtt gta act ctg gag tgt gaa gat ggg tat atg ctg gaa ggc 2784Gly Ala Val Val Thr Leu Glu Cys Glu Asp Gly Tyr Met Leu Glu Gly915 920 925agt ccc cag agc cag tgc caa tcg gat cac caa tgg aac cct ccc ctg 2832Ser Pro Gln Ser Gln Cys Gln Ser Asp His Gln Trp Asn Pro Pro Leu930 935 940gcg gtt tgc aga tcc cgt tca ctt gct cct gtc ctt tgt ggt att gct 2880Ala Val Cys Arg Ser Arg Ser Leu Ala Pro Val Leu Cys Gly Ile Ala945 950 955 960gca ggt ttg ata ctt ctt acc ttc ttg att gtc gtt acc tta tac gtg 2928Ala Gly Leu Ile Leu Leu Thr Phe Leu Ile Val Val Thr Leu Tyr Val965 970 975ata tca aaa cac aga gca cgc aat tat tat aca gat aca agc cag aaa 2976Ile Ser Lys His Arg Ala Arg Asn Tyr Tyr Thr Asp Thr Ser Gln Lys980 985 990gaa gct ttt cat tta gaa gca cga gaa gta tat tct gtt gat cca tac 3024Glu Ala Phe His Leu Glu Ala Arg Glu Val Tyr Ser Val Asp Pro Tyr995 1000 1005aac cca gcc agc 3036Asn Pro Ala Ser1010151012PRTHomo sapiensCR2 15Ile Ser Cys Gly Ser Pro Pro Pro Ile Leu Asn Gly Arg Ile Ser Tyr1 5 10 15Tyr Ser Thr Pro Ile Ala Val Gly Thr Val Ile Arg Tyr Ser Cys Ser 20 25 30Gly Thr Phe Arg Leu Ile Gly Glu Lys Ser Leu Leu Cys Ile Thr Lys 35 40 45Asp Lys Val Asp Gly Thr Trp Asp Lys Pro Ala Pro Lys Cys Glu Tyr 50 55 60Phe Asn Lys Tyr Ser Ser Cys Pro Glu Pro Ile Val Pro Gly Gly Tyr65 70 75 80Lys Ile Arg Gly Ser Thr Pro Tyr Arg His Gly Asp Ser Val Thr Phe 85 90 95Ala Cys Lys Thr Asn Phe Ser Met Asn Gly Asn Lys Ser Val Trp Cys 100 105 110Gln Ala Asn Asn Met Trp Gly Pro Thr Arg Leu Pro Thr Cys Val Ser 115 120 125Val Phe Pro Leu Glu Cys Pro Ala Leu Pro Met Ile His Asn Gly His 130 135 140His Thr Ser Glu Asn Val Gly Ser Ile Ala Pro Gly Leu Ser Val Thr145 150 155 160Tyr Ser Cys Glu Ser Gly Tyr Leu Leu Val Gly Glu Lys Ile Ile Asn 165 170 175Cys Leu Ser Ser Gly Lys Trp Ser Ala Val Pro Pro Thr Cys Glu Glu 180 185 190Ala Arg Cys Lys Ser Leu Gly Arg Phe Pro Asn Gly Lys Val Lys Glu 195

200 205Pro Pro Ile Leu Arg Val Gly Val Thr Ala Asn Phe Phe Cys Asp Glu 210 215 220Gly Tyr Arg Leu Gln Gly Pro Pro Ser Ser Arg Cys Val Ile Ala Gly225 230 235 240Gln Gly Val Ala Trp Thr Lys Met Pro Val Cys Glu Glu Ile Phe Cys 245 250 255Pro Ser Pro Pro Pro Ile Leu Asn Gly Arg His Ile Gly Asn Ser Leu 260 265 270Ala Asn Val Ser Tyr Gly Ser Ile Val Thr Tyr Thr Cys Asp Pro Asp 275 280 285Pro Glu Glu Gly Val Asn Phe Ile Leu Ile Gly Glu Ser Thr Leu Arg 290 295 300Cys Thr Val Asp Ser Gln Lys Thr Gly Thr Trp Ser Gly Pro Ala Pro305 310 315 320Arg Cys Glu Leu Ser Thr Ser Ala Val Gln Cys Pro His Pro Gln Ile 325 330 335Leu Arg Gly Arg Met Val Ser Gly Gln Lys Asp Arg Tyr Thr Tyr Asn 340 345 350Asp Thr Val Ile Phe Ala Cys Met Phe Gly Phe Thr Leu Lys Gly Ser 355 360 365Lys Gln Ile Arg Cys Asn Ala Gln Gly Thr Trp Glu Pro Ser Ala Pro 370 375 380Val Cys Glu Lys Glu Cys Gln Ala Pro Pro Asn Ile Leu Asn Gly Gln385 390 395 400Lys Glu Asp Arg His Met Val Arg Phe Asp Pro Gly Thr Ser Ile Lys 405 410 415Tyr Ser Cys Asn Pro Gly Tyr Val Leu Val Gly Glu Glu Ser Ile Gln 420 425 430Cys Thr Ser Glu Val Trp Thr Pro Pro Val Pro Gln Cys Lys Val Ala 435 440 445Ala Cys Glu Ala Thr Gly Arg Gln Leu Leu Thr Lys Pro Gln His Gln 450 455 460Phe Val Arg Pro Asp Val Asn Ser Ser Cys Gly Glu Gly Tyr Lys Leu465 470 475 480Ser Gly Ser Val Tyr Gln Glu Cys Gln Gly Thr Ile Pro Trp Phe Met 485 490 495Glu Ile Arg Leu Cys Lys Glu Ile Thr Cys Pro Pro Pro Pro Val Ile 500 505 510Tyr Asn Gly Ala His Thr Gly Ser Ser Leu Glu Asp Phe Pro Tyr Gly 515 520 525Thr Thr Val Thr Tyr Thr Cys Asn Pro Gly Pro Glu Arg Gly Val Glu 530 535 540Phe Ser Leu Ile Gly Glu Ser Thr Ile Arg Cys Thr Ser Asn Asp Gln545 550 555 560Glu Arg Gly Thr Trp Ser Gly Pro Ala Pro Leu Cys Lys Leu Ser Leu 565 570 575Leu Ala Val Gln Cys Ser His Val His Ile Ala Asn Gly Tyr Lys Ile 580 585 590Ser Gly Lys Glu Ala Pro Tyr Phe Tyr Asn Asp Thr Val Thr Phe Lys 595 600 605Cys Tyr Ser Gly Phe Thr Leu Lys Gly Ser Ser Gln Ile Arg Cys Lys 610 615 620Ala Asp Asn Thr Trp Asp Pro Glu Ile Pro Val Cys Glu Lys Glu Thr625 630 635 640Cys Gln His Val Arg Gln Ser Leu Gln Glu Leu Pro Ala Gly Ser Arg 645 650 655Val Glu Leu Val Asn Thr Ser Cys Gln Asp Gly Tyr Gln Leu Thr Gly 660 665 670His Ala Tyr Gln Met Cys Gln Asp Ala Glu Asn Gly Ile Trp Phe Lys 675 680 685Lys Ile Pro Leu Cys Lys Val Ile His Cys His Pro Pro Pro Val Ile 690 695 700Val Asn Gly Lys His Thr Gly Met Met Ala Glu Asn Phe Leu Tyr Gly705 710 715 720Asn Glu Val Ser Tyr Glu Cys Asp Gln Gly Phe Tyr Leu Leu Gly Glu 725 730 735Lys Lys Leu Gln Cys Arg Ser Asp Ser Lys Gly His Gly Ser Trp Ser 740 745 750Gly Pro Ser Pro Gln Cys Leu Arg Ser Pro Pro Val Thr Arg Cys Pro 755 760 765Asn Pro Glu Val Lys His Gly Tyr Lys Leu Asn Lys Thr His Ser Ala 770 775 780Tyr Ser His Asn Asp Ile Val Tyr Val Asp Cys Asn Pro Gly Phe Ile785 790 795 800Met Asn Gly Ser Arg Val Ile Arg Cys His Thr Asp Asn Thr Trp Val 805 810 815Pro Gly Val Pro Thr Cys Ile Lys Lys Ala Phe Ile Gly Cys Pro Pro 820 825 830Pro Pro Lys Thr Pro Asn Gly Asn His Thr Gly Gly Asn Ile Ala Arg 835 840 845Phe Ser Pro Gly Met Ser Ile Leu Tyr Ser Cys Asp Gln Gly Tyr Leu 850 855 860Leu Val Gly Glu Ala Leu Leu Leu Cys Thr His Glu Gly Thr Trp Ser865 870 875 880Gln Pro Ala Pro His Cys Lys Glu Val Asn Cys Ser Ser Pro Ala Asp 885 890 895Met Asp Gly Ile Gln Lys Gly Leu Glu Pro Arg Lys Met Tyr Gln Tyr 900 905 910Gly Ala Val Val Thr Leu Glu Cys Glu Asp Gly Tyr Met Leu Glu Gly 915 920 925Ser Pro Gln Ser Gln Cys Gln Ser Asp His Gln Trp Asn Pro Pro Leu 930 935 940Ala Val Cys Arg Ser Arg Ser Leu Ala Pro Val Leu Cys Gly Ile Ala945 950 955 960Ala Gly Leu Ile Leu Leu Thr Phe Leu Ile Val Val Thr Leu Tyr Val 965 970 975Ile Ser Lys His Arg Ala Arg Asn Tyr Tyr Thr Asp Thr Ser Gln Lys 980 985 990Glu Ala Phe His Leu Glu Ala Arg Glu Val Tyr Ser Val Asp Pro Tyr 995 1000 1005Asn Pro Ala Ser 1010161930DNAHomo sapiensDAF 16gac tgt ggc ctt ccc cca gat gta cct aat gcc cag cca gct ttg gaa 48Asp Cys Gly Leu Pro Pro Asp Val Pro Asn Ala Gln Pro Ala Leu Glu1 5 10 15ggc cgt aca agt ttt ccc gag gat act gta ata acg tac aaa tgt gaa 96Gly Arg Thr Ser Phe Pro Glu Asp Thr Val Ile Thr Tyr Lys Cys Glu20 25 30gaa agc ttt gtg aaa att cct ggc gag aag gac tca gtg acc tgc ctt 144Glu Ser Phe Val Lys Ile Pro Gly Glu Lys Asp Ser Val Thr Cys Leu35 40 45aag ggc atg caa tgg tca gat att gaa gag ttc tgc aat cgt agc tgc 192Lys Gly Met Gln Trp Ser Asp Ile Glu Glu Phe Cys Asn Arg Ser Cys50 55 60gag gtg cca aca agg cta aat tct gca tcc ctc aaa cag cct tat atc 240Glu Val Pro Thr Arg Leu Asn Ser Ala Ser Leu Lys Gln Pro Tyr Ile65 70 75 80act cag aat tat ttt cca gtc ggt act gtt gtg gaa tat gag tgc cgt 288Thr Gln Asn Tyr Phe Pro Val Gly Thr Val Val Glu Tyr Glu Cys Arg85 90 95cca ggt tac aga aga gaa cct tct cta tca cca aaa cta act tgc ctt 336Pro Gly Tyr Arg Arg Glu Pro Ser Leu Ser Pro Lys Leu Thr Cys Leu100 105 110cag aat tta aaa tgg tcc aca gca gtc gaa ttt tgt aaa aag aaa tca 384Gln Asn Leu Lys Trp Ser Thr Ala Val Glu Phe Cys Lys Lys Lys Ser115 120 125tgc cct aat ccg gga gaa ata cga aat ggt cag att gat gta cca ggt 432Cys Pro Asn Pro Gly Glu Ile Arg Asn Gly Gln Ile Asp Val Pro Gly130 135 140ggc ata tta ttt ggt gca acc atc tcc ttc tca tgt aac aca ggg tac 480Gly Ile Leu Phe Gly Ala Thr Ile Ser Phe Ser Cys Asn Thr Gly Tyr145 150 155 160aaa tta ttt ggc tcg act tct agt ttt tgt ctt att tca ggc agc tct 528Lys Leu Phe Gly Ser Thr Ser Ser Phe Cys Leu Ile Ser Gly Ser Ser165 170 175gtc cag tgg agt gac ccg ttg cca gag tgc aga gaa att tat tgt cca 576Val Gln Trp Ser Asp Pro Leu Pro Glu Cys Arg Glu Ile Tyr Cys Pro180 185 190gca cca cca caa att gac aat gga ata att caa ggg gaa cgt gac cat 624Ala Pro Pro Gln Ile Asp Asn Gly Ile Ile Gln Gly Glu Arg Asp His195 200 205tat gga tat aga cag tct gta acg tat gca tgt aat aaa gga ttc acc 672Tyr Gly Tyr Arg Gln Ser Val Thr Tyr Ala Cys Asn Lys Gly Phe Thr210 215 220atg att gga gag cac tct att tat tgt act gtg aat aat gat gaa gga 720Met Ile Gly Glu His Ser Ile Tyr Cys Thr Val Asn Asn Asp Glu Gly225 230 235 240gag tgg agt ggc cca cca cct gaa tgc aga gga aaa tct cta act tcc 768Glu Trp Ser Gly Pro Pro Pro Glu Cys Arg Gly Lys Ser Leu Thr Ser245 250 255aag gtc cca cca aca gtt cag aaa cct acc aca gta aat gtt cca act 816Lys Val Pro Pro Thr Val Gln Lys Pro Thr Thr Val Asn Val Pro Thr260 265 270aca gaa gtc tca cca act tct cag aaa acc acc aca aaa acc acc aca 864Thr Glu Val Ser Pro Thr Ser Gln Lys Thr Thr Thr Lys Thr Thr Thr275 280 285cca aat gct caa gca aca cgg agt aca cct gtt tcc agg aca acc aag 912Pro Asn Ala Gln Ala Thr Arg Ser Thr Pro Val Ser Arg Thr Thr Lys290 295 300cat ttt cat gaa aca acc cca aat aaa gga agt gga acc act tca ggt 960His Phe His Glu Thr Thr Pro Asn Lys Gly Ser Gly Thr Thr Ser Gly305 310 315 320act acc cgt ctt cta tct ggg cac acg tgt ttc acg ttg aca ggt ttg 1008Thr Thr Arg Leu Leu Ser Gly His Thr Cys Phe Thr Leu Thr Gly Leu325 330 335ctt ggg acg cta gta acc atg ggc ttg ctg act tagccaaaga agagttaaga 1061Leu Gly Thr Leu Val Thr Met Gly Leu Leu Thr340 345agaaaataca cacaagtata cagactgttc ctagtttctt agacttatct gcatattgga 1121taaaataaat gcaattgtgc tcttcattta ggatgctttc attgtcttta agatgtgtta 1181ggaatgtcaa cagagcaagg agaaaaaagg cagtcctgga atcacattct tagcacacct 1241gcgcctcttg aaaatagaac aacttgcaga attgagagtg attcctttcc taaaagtgta 1301agaaagcata gagatttgtt cgtattaaga atgggatcac gaggaaaaga gaaggaaagt 1361gatttttttc cacaagatct gaaatgatat ttccacttat aaaggaaata aaaaatgaaa 1421aacattattt ggatatcaaa agcaaataaa aacccaattc agtctcttct aagcaaaatt 1481gctaaagaga gatgaccaca ttataaagta atctttggct aaggcatttt catctttcct 1541tcggttggca aaatatttta aaggtaaaac atgctggtga accagggtgt tgatggtgat 1601aagggaggaa tatagaatga aagactgaat cttcctttgt tgcacaaata gagtttggaa 1661aaagcctgtg aaaggtgtct tctttgactt aatgtcttta aaagtatcca gagatactac 1721aatattaaca taagaaaaga ttatatatta tttctgaatc gagatgtcca tagtcaaatt 1781tgtaaatctt attcttttgt aatatttatt tatatttatt tatgacagtg aacattctga 1841ttttacatgt aaaacaagaa aagttgaaga agatatgtga agaaaaatgt atttttccta 1901aatagaaata aatgatccca ttttttggt 193017347PRTHomo sapiensDAF 17Asp Cys Gly Leu Pro Pro Asp Val Pro Asn Ala Gln Pro Ala Leu Glu 1 5 10 15Gly Arg Thr Ser Phe Pro Glu Asp Thr Val Ile Thr Tyr Lys Cys Glu 20 25 30Glu Ser Phe Val Lys Ile Pro Gly Glu Lys Asp Ser Val Thr Cys Leu 35 40 45Lys Gly Met Gln Trp Ser Asp Ile Glu Glu Phe Cys Asn Arg Ser Cys 50 55 60Glu Val Pro Thr Arg Leu Asn Ser Ala Ser Leu Lys Gln Pro Tyr Ile65 70 75 80Thr Gln Asn Tyr Phe Pro Val Gly Thr Val Val Glu Tyr Glu Cys Arg 85 90 95Pro Gly Tyr Arg Arg Glu Pro Ser Leu Ser Pro Lys Leu Thr Cys Leu 100 105 110Gln Asn Leu Lys Trp Ser Thr Ala Val Glu Phe Cys Lys Lys Lys Ser 115 120 125Cys Pro Asn Pro Gly Glu Ile Arg Asn Gly Gln Ile Asp Val Pro Gly 130 135 140Gly Ile Leu Phe Gly Ala Thr Ile Ser Phe Ser Cys Asn Thr Gly Tyr145 150 155 160Lys Leu Phe Gly Ser Thr Ser Ser Phe Cys Leu Ile Ser Gly Ser Ser 165 170 175Val Gln Trp Ser Asp Pro Leu Pro Glu Cys Arg Glu Ile Tyr Cys Pro 180 185 190Ala Pro Pro Gln Ile Asp Asn Gly Ile Ile Gln Gly Glu Arg Asp His 195 200 205Tyr Gly Tyr Arg Gln Ser Val Thr Tyr Ala Cys Asn Lys Gly Phe Thr 210 215 220Met Ile Gly Glu His Ser Ile Tyr Cys Thr Val Asn Asn Asp Glu Gly225 230 235 240Glu Trp Ser Gly Pro Pro Pro Glu Cys Arg Gly Lys Ser Leu Thr Ser 245 250 255Lys Val Pro Pro Thr Val Gln Lys Pro Thr Thr Val Asn Val Pro Thr 260 265 270Thr Glu Val Ser Pro Thr Ser Gln Lys Thr Thr Thr Lys Thr Thr Thr 275 280 285Pro Asn Ala Gln Ala Thr Arg Ser Thr Pro Val Ser Arg Thr Thr Lys 290 295 300His Phe His Glu Thr Thr Pro Asn Lys Gly Ser Gly Thr Thr Ser Gly305 310 315 320Thr Thr Arg Leu Leu Ser Gly His Thr Cys Phe Thr Leu Thr Gly Leu 325 330 335Leu Gly Thr Leu Val Thr Met Gly Leu Leu Thr 340 345181050DNAHomo sapiensMCP 18tgt gag gag cca cca aca ttt gaa gct atg gag ctc att ggt aaa cca 48Cys Glu Glu Pro Pro Thr Phe Glu Ala Met Glu Leu Ile Gly Lys Pro1 5 10 15aaa ccc tac tat gag att ggt gaa cga gta gat tat aag tgt aaa aaa 96Lys Pro Tyr Tyr Glu Ile Gly Glu Arg Val Asp Tyr Lys Cys Lys Lys20 25 30gga tac ttc tat ata cct cct ctt gcc acc cat act att tgt gat cgg 144Gly Tyr Phe Tyr Ile Pro Pro Leu Ala Thr His Thr Ile Cys Asp Arg35 40 45aat cat aca tgg cta cct gtc tca gat gac gcc tgt tat aga gaa aca 192Asn His Thr Trp Leu Pro Val Ser Asp Asp Ala Cys Tyr Arg Glu Thr50 55 60tgt cca tat ata cgg gat cct tta aat ggc caa gca gtc cct gca aat 240Cys Pro Tyr Ile Arg Asp Pro Leu Asn Gly Gln Ala Val Pro Ala Asn65 70 75 80ggg act tac gag ttt ggt tat cag atg cac ttt att tgt aat gag ggt 288Gly Thr Tyr Glu Phe Gly Tyr Gln Met His Phe Ile Cys Asn Glu Gly85 90 95tat tac tta att ggt gaa gaa att cta tat tgt gaa ctt aaa gga tca 336Tyr Tyr Leu Ile Gly Glu Glu Ile Leu Tyr Cys Glu Leu Lys Gly Ser100 105 110gta gca att tgg agc ggt aag ccc cca ata tgt gaa aag gtt ttg tgt 384Val Ala Ile Trp Ser Gly Lys Pro Pro Ile Cys Glu Lys Val Leu Cys115 120 125aca cca cct cca aaa ata aaa aat gga aaa cac acc ttt agt gaa gta 432Thr Pro Pro Pro Lys Ile Lys Asn Gly Lys His Thr Phe Ser Glu Val130 135 140gaa gta ttt gag tat ctt gat gca gta act tat agt tgt gat cct gca 480Glu Val Phe Glu Tyr Leu Asp Ala Val Thr Tyr Ser Cys Asp Pro Ala145 150 155 160cct gga cca gat cca ttt tca ctt att gga gag agc acg att tat tgt 528Pro Gly Pro Asp Pro Phe Ser Leu Ile Gly Glu Ser Thr Ile Tyr Cys165 170 175ggt gac aat tca gtg tgg agt cgt gct gct cca gag tgt aaa gtg gtc 576Gly Asp Asn Ser Val Trp Ser Arg Ala Ala Pro Glu Cys Lys Val Val180 185 190aaa tgt cga ttt cca gta gtc gaa aat gga aaa cag ata tca gga ttt 624Lys Cys Arg Phe Pro Val Val Glu Asn Gly Lys Gln Ile Ser Gly Phe195 200 205gga aaa aaa ttt tac tac aaa gca aca gtt atg ttt gaa tgc gat aag 672Gly Lys Lys Phe Tyr Tyr Lys Ala Thr Val Met Phe Glu Cys Asp Lys210 215 220ggt ttt tac ctc gat ggc agc gac aca att gtc tgt gac agt aac agt 720Gly Phe Tyr Leu Asp Gly Ser Asp Thr Ile Val Cys Asp Ser Asn Ser225 230 235 240act tgg gat ccc cca gtt cca aag tgt ctt aaa gtg tcg act tct tcc 768Thr Trp Asp Pro Pro Val Pro Lys Cys Leu Lys Val Ser Thr Ser Ser245 250 255act aca aaa tct cca gcg tcc agt gcc tca ggt cct agg cct act tac 816Thr Thr Lys Ser Pro Ala Ser Ser Ala Ser Gly Pro Arg Pro Thr Tyr260 265 270aag cct cca gtc tca aat tat cca gga tat cct aaa cct gag gaa gga 864Lys Pro Pro Val Ser Asn Tyr Pro Gly Tyr Pro Lys Pro Glu Glu Gly275 280 285ata ctt gac agt ttg gat gtt tgg gtc att gct gtg att gtt att gcc 912Ile Leu Asp Ser Leu Asp Val Trp Val Ile Ala Val Ile Val Ile Ala290 295 300ata gtt gtt gga gtt gca gta att tgt gtt gtc ccg tac aga tat ctt 960Ile Val Val Gly Val Ala Val Ile Cys Val Val Pro Tyr Arg Tyr Leu305 310 315 320caa agg agg aag aag aaa ggg aaa gca gat ggt gga gct gaa tat gcc 1008Gln Arg Arg Lys Lys Lys Gly Lys Ala Asp Gly Gly Ala Glu Tyr Ala325 330 335act tac cag act aaa tca acc act cca gca gag cag aga ggc 1050Thr Tyr Gln Thr Lys Ser Thr Thr Pro Ala Glu Gln Arg Gly340 345 35019350PRTHomo sapiensMCP 19Cys Glu Glu Pro Pro Thr Phe Glu Ala Met Glu Leu Ile Gly Lys Pro1 5 10 15Lys Pro Tyr Tyr Glu Ile Gly Glu Arg Val Asp Tyr Lys Cys Lys Lys 20 25 30Gly Tyr Phe Tyr Ile Pro Pro Leu Ala Thr His Thr Ile Cys Asp Arg 35 40 45Asn His Thr Trp Leu Pro Val Ser Asp Asp Ala Cys Tyr Arg Glu Thr 50 55 60Cys Pro Tyr Ile Arg Asp Pro Leu Asn Gly Gln Ala Val Pro Ala Asn65 70

75 80Gly Thr Tyr Glu Phe Gly Tyr Gln Met His Phe Ile Cys Asn Glu Gly 85 90 95Tyr Tyr Leu Ile Gly Glu Glu Ile Leu Tyr Cys Glu Leu Lys Gly Ser 100 105 110Val Ala Ile Trp Ser Gly Lys Pro Pro Ile Cys Glu Lys Val Leu Cys 115 120 125Thr Pro Pro Pro Lys Ile Lys Asn Gly Lys His Thr Phe Ser Glu Val 130 135 140Glu Val Phe Glu Tyr Leu Asp Ala Val Thr Tyr Ser Cys Asp Pro Ala145 150 155 160Pro Gly Pro Asp Pro Phe Ser Leu Ile Gly Glu Ser Thr Ile Tyr Cys 165 170 175Gly Asp Asn Ser Val Trp Ser Arg Ala Ala Pro Glu Cys Lys Val Val 180 185 190Lys Cys Arg Phe Pro Val Val Glu Asn Gly Lys Gln Ile Ser Gly Phe 195 200 205Gly Lys Lys Phe Tyr Tyr Lys Ala Thr Val Met Phe Glu Cys Asp Lys 210 215 220Gly Phe Tyr Leu Asp Gly Ser Asp Thr Ile Val Cys Asp Ser Asn Ser225 230 235 240Thr Trp Asp Pro Pro Val Pro Lys Cys Leu Lys Val Ser Thr Ser Ser 245 250 255Thr Thr Lys Ser Pro Ala Ser Ser Ala Ser Gly Pro Arg Pro Thr Tyr 260 265 270Lys Pro Pro Val Ser Asn Tyr Pro Gly Tyr Pro Lys Pro Glu Glu Gly 275 280 285Ile Leu Asp Ser Leu Asp Val Trp Val Ile Ala Val Ile Val Ile Ala 290 295 300Ile Val Val Gly Val Ala Val Ile Cys Val Val Pro Tyr Arg Tyr Leu305 310 315 320Gln Arg Arg Lys Lys Lys Gly Lys Ala Asp Gly Gly Ala Glu Tyr Ala 325 330 335Thr Tyr Gln Thr Lys Ser Thr Thr Pro Ala Glu Gln Arg Gly 340 345 350

Claims

1-34. (canceled)

35. A modified form of a regulator of complement activation protein (RCA protein), wherein said RCA protein is selected from the group consisting of complement receptor 1, decay accelerating factor, membrane cofactor protein, C4 binding protein, factor H and any one of the foregoing RCA proteins wherein the carboxy terminus is removed to allow the RCA protein to be secreted; wherein said modified form is selected from the group consisting of: a) a hybrid RCA protein comprising short consensus repeats (SCRs) of said RCA protein and SCRs from a different RCA protein selected from the group consisting of complement receptor 1, decay accelerating factor, membrane cofactor protein, C4 binding protein and factor H; b) a recombined RCA protein in which the SCRs of said RCA protein are rearranged; c) a truncated RCA protein consisting of three SCRs of said RCA protein; and d) a modified RCA protein comprising any combination of a), b) and c), wherein the modified form has complement regulatory activity.

36. The modified form of a RCA protein of claim 35, wherein the complement regulatory activity is one or more activities selected from the group consisting of C3b binding activity, C4b binding activity, C3b and C4b binding activity, C3b cofactor activity, C4b cofactor activity, and decay accelerating activity.

37. The modified form of a RCA protein of claim 35, wherein said modified form is a recombined RCA protein in which the SCRs of said RCA protein are rearranged.

38. The modified form of a RCA protein of claim 35, wherein said modified form is a truncated RCA protein consisting of three SCRs of said RCA protein.

39. A hybrid regulator of complement activation protein (RCA protein), wherein said hybrid RCA protein comprises: a) short consensus repeats (SCRS) of a first RCA protein selected from the group consisting of complement receptor 1, decay accelerating factor, membrane cofactor protein, C4 binding protein and factor H; and b) SCRs from a different RCA protein selected from the group consisting of complement receptor 1, decay accelerating factor, membrane cofactor protein, C4 binding protein and factor H, wherein said hybrid RCA protein has complement regulatory activity.

40. The hybrid RCA protein of claim 39, wherein said first RCA protein is decay accelerating factor, and said different RCA protein is selected from the group consisting of complement receptor 1, membrane cofactor protein, C4 binding protein and factor H.

41. The hybrid RCA protein of claim 40, wherein said different RCA protein is membrane cofactor protein.

42. The hybrid RCA protein of claim 41, wherein said hybrid RCA protein comprises SCRs 2-3 of decay accelerating factor, and SCRs 1, 2, 3 and 4 of membrane cofactor protein.

43. The hybrid RCA protein of claim 42, wherein said hybrid RCA protein has decay accelerating activity, and one or more additional complement regulatory activities selected from the group consisting of C3b binding activity, C4b binding activity, C3b and C4b binding activity, C3b cofactor activity, and C4b cofactor activity.

44. The hybrid RCA protein of claim 39, wherein said hybrid RCA protein is soluble.

45. An isolated DNA molecule that encodes the modified form of a RCA protein of claim 35.

46. An expression vector comprising a DNA molecule that encodes the modified form of a RCA protein of claim 35 operably linked to a control sequence compatible with a host cell.

47. A recombinant host cell comprising the expression vector of claim 46.

48. A nonhuman transgenic animal having a DNA molecule that encodes the modified form of a RCA protein of claim 35 stably integrated into its genome.

49. A method for making the modified form of a RCA protein any one of claims 35, 39 and 40, comprising expressing a DNA encoding said modified form of a RCA protein in a host cell.

50. A pharmaceutical composition comprising the modified form of an RCA protein of claim 35 and a pharmaceutically acceptable carrier.

51. An isolated DNA molecule that encodes the hybrid RCA protein of claim 39.

52. An expression vector comprising a DNA molecule that encodes the hybrid RCA protein of claim 39 operably linked to a control sequence compatible with a host cell.

53. A recombinant host cell comprising the expression vector of claim 52.

54. A nonhuman transgenic animal having a DNA molecule that encodes the hybrid RCA protein of claim 39 stably integrated into its genome.

55. A method for making the hybrid RCA protein of claim 39, comprising expressing a DNA encoding said hybrid RCA protein in a host cell.

56. A pharmaceutical composition comprising the hybrid RCA protein of claim 39 and a pharmaceutically acceptable carrier.

57. A hybrid regulator of complement activation protein (RCA protein), wherein said hybrid RCA protein comprises: a) short consensus repeats (SCRs) 2, 3 and 4 of decay accelerating factor; and b) SCRs of a RCA protein selected from the group consisting of complement receptor 1, complement receptor 2, membrane cofactor protein, C4 binding protein and factor H, wherein said hybrid RCA protein has decay accelerating activity and one or more activities selected from the group consisting of C3b binding activity, C4b binding activity, C3b and C4b binding activity, C3b cofactor activity, and C4b cofactor activity.

58. The hybrid RCA protein of claim 57, wherein said hybrid RCA protein comprises SCRs of membrane cofactor protein.

59. The hybrid RCA protein of claim 58, wherein said hybrid RCA protein comprises SCRs 3 and 4 of membrane cofactor protein.

60. The hybrid RCA protein of claim 59, wherein said hybrid RCA protein comprises SCRs 2, 3 and 4 of membrane cofactor protein.

61. The hybrid RCA protein of claim 60, wherein said hybrid RCA protein comprises SCRs 1, 2, 3 and 4 of membrane cofactor protein.

62. The hybrid RCA protein of claim 57, wherein the hybrid RCA protein binds C3b and C4b and has C3b cofactor activity, C4b cofactor activity, and decay accelerating activity.

63. The hybrid RCA protein of claim 57, wherein said hybrid RCA protein is soluble.

64. An isolated DNA molecule that encodes the hybrid RCA protein of claim 57.

65. An expression vector comprising a DNA molecule that encodes the hybrid RCA protein of claim 57 operably linked to a control sequence compatible with a host cell.

66. A recombinant host cell comprising the expression vector of claim 65.

67. A method for making the hybrid RCA protein of claim 57, comprising expressing a DNA encoding said hybrid RCA protein in a host cell.

68. A pharmaceutical composition comprising the hybrid RCA protein of claim 57 and a pharmaceutically acceptable carrier.