Identification of Residues within the 727–767 Segment of Human Complement Component C3 Important for Its Interaction with Factor H and with Complement Receptor 1 (CR1, CD35)
1999; Elsevier BV; Volume: 274; Issue: 8 Linguagem: Inglês
10.1074/jbc.274.8.5120
ISSN1083-351X
AutoresAlp E. Oran, David E. Isenman,
Tópico(s)Blood Coagulation and Thrombosis Mechanisms
ResumoMapping approaches employing blocking antibodies and synthetic peptides have implicated the 727–767 segment at the NH2 terminus of C3b α′-chain as contributing to the interactions with factor B, factor H, and CR1. Our previous mutagenesis study on the NH2-terminal acidic cluster of this segment identified residues Glu-736 and Glu-737 as contributing to the binding of C3b to factor B and CR1 but not factor H. We have now extended the charged residue mutagenic scan to cover the remainder of the segment (738–767) and have assessed the ability of the C3b-like C3(H2O) form of the mutant molecules to interact with factor H, CR1, and membrane cofactor protein (MCP) using a cofactor-dependent factor I cleavage assay as a surrogate binding assay. We have found that the negatively charged side chains of Glu-744 and Glu-747 are important for the interaction between C3(H2O) and factor H, a result in general agreement with an earlier synthetic peptide study (Fishelson, Z. (1991) Mol. Immunol. 28, 545–552) which implicated residues within the 744–754 segment in H binding. The interactions of the mutants with soluble CR1 (sCR1) revealed two classes of residues. The first are residues required for sCR1 to be an I cofactor for the first two cleavages of α-chain. These are all acidic residues and include the Glu-736/Glu-737 pair, Glu-747, and the Glu-754/Asp-755 pairing. The second class affects only the ability of sCR1 to be a cofactor for the third factor I cleavage and include Glu-744 and the Lys-757/Glu-758 pairing. The dominance of acidic residues in the loss-of-function mutants is striking and suggests that H and CR1 contribute basic residues to the interface. Additionally, although there is partial overlap, the contacts required for CR1 binding appear to extend over a wider portion of the 727–767 segment than is the case for factor H. Finally, none of the mutations had any effect on the interaction between soluble MCP and C3(H2O), indicating that despite its functional homology to H and CR1, MCP differs in its mode of binding to C3b/C3(H2O). Mapping approaches employing blocking antibodies and synthetic peptides have implicated the 727–767 segment at the NH2 terminus of C3b α′-chain as contributing to the interactions with factor B, factor H, and CR1. Our previous mutagenesis study on the NH2-terminal acidic cluster of this segment identified residues Glu-736 and Glu-737 as contributing to the binding of C3b to factor B and CR1 but not factor H. We have now extended the charged residue mutagenic scan to cover the remainder of the segment (738–767) and have assessed the ability of the C3b-like C3(H2O) form of the mutant molecules to interact with factor H, CR1, and membrane cofactor protein (MCP) using a cofactor-dependent factor I cleavage assay as a surrogate binding assay. We have found that the negatively charged side chains of Glu-744 and Glu-747 are important for the interaction between C3(H2O) and factor H, a result in general agreement with an earlier synthetic peptide study (Fishelson, Z. (1991) Mol. Immunol. 28, 545–552) which implicated residues within the 744–754 segment in H binding. The interactions of the mutants with soluble CR1 (sCR1) revealed two classes of residues. The first are residues required for sCR1 to be an I cofactor for the first two cleavages of α-chain. These are all acidic residues and include the Glu-736/Glu-737 pair, Glu-747, and the Glu-754/Asp-755 pairing. The second class affects only the ability of sCR1 to be a cofactor for the third factor I cleavage and include Glu-744 and the Lys-757/Glu-758 pairing. The dominance of acidic residues in the loss-of-function mutants is striking and suggests that H and CR1 contribute basic residues to the interface. Additionally, although there is partial overlap, the contacts required for CR1 binding appear to extend over a wider portion of the 727–767 segment than is the case for factor H. Finally, none of the mutations had any effect on the interaction between soluble MCP and C3(H2O), indicating that despite its functional homology to H and CR1, MCP differs in its mode of binding to C3b/C3(H2O). Stringent regulation of the alternative complement pathway C3 convertase is essential for the prevention of C3 hypercatabolism and for the protection of host tissue from the deleterious effects of alternative pathway propagation. In particular, it is important to regulate alternative pathway propagation that can employ as a nidus C3b molecules that have become adventitiously bound to host cell membrane. In primates, this regulation involves primarily the soluble serum proteins factor H and factor I, as well as the membrane-resident proteins decay-accelerating factor (CD55), membrane cofactor protein (MCP, 1The abbreviations MCPmembrane cofactor proteinCCPcomplement control proteinCRcomplement receptorCVFcobra venom factorDMEMDulbecco's modified Eagle's mediumDMEM/K76DMEM containing K76-COOH-treated fetal calf serumEACsheep erythrocytes coated with antibody and the indicated complement components or fragments thereofELISAenzyme-linked immunosorbent assayFCSfetal calf serumPBSphosphate-buffered salinerC3recombinant C3RCAregulators of complement activationRIAradioimmunoassaySCRshort consensus repeatsCR1soluble CR1sMCPsoluble MCPVBSveronal-buffered salinePAGEpolyacrylamide gel electrophoresisbpbase pairHuhumanTrtroutXeXenopus 1The abbreviations MCPmembrane cofactor proteinCCPcomplement control proteinCRcomplement receptorCVFcobra venom factorDMEMDulbecco's modified Eagle's mediumDMEM/K76DMEM containing K76-COOH-treated fetal calf serumEACsheep erythrocytes coated with antibody and the indicated complement components or fragments thereofELISAenzyme-linked immunosorbent assayFCSfetal calf serumPBSphosphate-buffered salinerC3recombinant C3RCAregulators of complement activationRIAradioimmunoassaySCRshort consensus repeatsCR1soluble CR1sMCPsoluble MCPVBSveronal-buffered salinePAGEpolyacrylamide gel electrophoresisbpbase pairHuhumanTrtroutXeXenopusCD46), and complement receptor 1 (CR1, CD35) (reviewed in Ref. 1Liszewski M.K. Atkinson J.P. Volanakis J.E. Frank M.M. The Human Complement System in Health and Disease. Marcel Dekker, Inc., New York1998: 149-165Crossref Google Scholar). With the exception of the serum protease factor I, the other regulatory molecules are members of a superfamily consisting almost entirely of varying numbers of a sequence motif alternatively referred to as a short consensus repeat (SCR) or complement control protein (CCP) module. Although this motif is also found in many non-complement proteins, the genes encoding the complement regulatory molecules are clustered on the long arm of chromosome 1 at what has been termed the RCA locus, for regulators of complement activation. Structural analyses of single and paired CCP modules have directly demonstrated that they each form independently folded compact globular domains consisting of two interacting layers of anti-parallel β-sheet (2Barlow P.N. Norman D.G. Steinkasserer A. Horne T.J. Pearce J. Driscoll P.C. Sim R.B. Campbell I.D. Biochemistry. 1992; 31: 3626-3634Crossref PubMed Scopus (99) Google Scholar, 3Barlow P.N. Steinkasserer A. Norman D.G. Kieffer B. Wiles A.P. Sim R.B. Campbell I.D. J. Mol. Biol. 1993; 232: 268-284Crossref PubMed Scopus (184) Google Scholar, 4Wiles A.P. Shaw G. Bright J. Perczel A. Campbell I.D. Barlow P.N. J. Mol. Biol. 1997; 272: 253-265Crossref PubMed Scopus (108) Google Scholar). It has been speculated that the protruding loops, some of which are of variable length, are good candidates for mediating interaction with ligand.CCP-containing proteins that bind to C3b exert their regulatory effect via two mechanisms as follows: decay-accelerating activity and factor I-cofactor activity. Specifically, the binding of H, decay-accelerating factor, and CR1 to C3bBb, the alternative pathway C3 convertase, leads to an accelerated rate of unidirectional dissociation of the serine protease Bb from the C3b modulatory subunit of the convertase. I cofactor activity refers to the accessory role of the SCR-containing protein in the I-mediated cleavage of C3b COOH terminus to residues 1281 and 1298 (mature C3 numbering), yielding the major fragmentation product iC3b and a minor fragment termed C3f. Since the iC3b fragment can no longer associate with factor B, this permanently inactivates the C3 molecule with respect to being a nidus for alternative pathway C3 convertase formation. However, this factor I-mediated cleavage can only take place when the C3b is complexed with one of three cofactors, these being the soluble protein factor H and the host membrane-associated proteins CR1 and MCP. Although there is some suggestive evidence in the literature that binding of H to C3b causes a conformational change in the latter (5DiScipio R.G. J. Immunol. 1992; 149: 2592-2599PubMed Google Scholar), thereby increasing the affinity for factor I, there is also recent evidence that factor I can simultaneously bind to both H and C3b, thereby stabilizing the intrinsically weak C3b-I interaction sufficiently to permit cleavage (6Soames C.J. Sim R.B. Biochem. J. 1997; 326: 553-561Crossref PubMed Scopus (69) Google Scholar). Whether factor I similarly binds directly to cofactors CR1 or MCP is as yet unknown. At physiological ionic strength, only CR1 can efficiently act as an I cofactor for the further cleavage of iC3b COOH-terminal to residue 932, yielding fragments C3c and C3dg (7Ross G.D. Lambris J.D. Cain J.A. Newman S.L. J. Immunol. 1982; 129: 2051-2060PubMed Google Scholar).Domain deletion and domain exchange studies have succeeded in identifying CCP/SCR regions within the complement regulatory proteins that are required for their C3b-binding and I cofactor activity (8Kuhn S. Skerka C. Zipfel P.F. J. Immunol. 1995; 155: 5663-5670PubMed Google Scholar, 9Sharma A. Pangburn M.K. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10996-11001Crossref PubMed Scopus (200) Google Scholar, 10Kalli K.R. Hsu P. Bartow T.J. Ahearn J.M. Matsumoto A.K. Klickstein L.B. Fearon D.T. J. Exp. Med. 1991; 174: 1451-1460Crossref PubMed Scopus (74) Google Scholar, 11Krych M. Hauhart R. Atkinson J.P. J. Biol. Chem. 1998; 273: 8623-8629Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 12Adams E.M. Brown M.C. Nunge M. Krych M. Atkinson J.P. J. Immunol. 1991; 147: 3005-3011PubMed Google Scholar, 13Coyne K.E. Hall S.E. Thompson E.S. Arce M.A. Kinoshita T. Fujita T. Anstee D.J. Rosse W. Lublin D.M. J. Immunol. 1992; 149: 2906-2913PubMed Google Scholar). Usually 3–4 SCR domains are required for functional activity. For example, in the case of factor H, SCRs 1–4 are minimally required for the expression of its I cofactor activity (8Kuhn S. Skerka C. Zipfel P.F. J. Immunol. 1995; 155: 5663-5670PubMed Google Scholar, 9Sharma A. Pangburn M.K. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10996-11001Crossref PubMed Scopus (200) Google Scholar). Factor H has also been shown to possess C3b-binding sites involving SCRs 6–10 and 16–20, although these sites do not contribute to the I cofactor activity of the molecule (9Sharma A. Pangburn M.K. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10996-11001Crossref PubMed Scopus (200) Google Scholar). The two C3b-binding sites of human CR1 are contributed by SCRs 8–11 and 15–18, respectively, and these entities are each capable of mediating full I cofactor activity (10Kalli K.R. Hsu P. Bartow T.J. Ahearn J.M. Matsumoto A.K. Klickstein L.B. Fearon D.T. J. Exp. Med. 1991; 174: 1451-1460Crossref PubMed Scopus (74) Google Scholar). A recent study on CR1 has shown that the first three SCR domains in each case account for most of the functional activity (11Krych M. Hauhart R. Atkinson J.P. J. Biol. Chem. 1998; 273: 8623-8629Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar) and critical residues within these functionally important domains have also been identified (11Krych M. Hauhart R. Atkinson J.P. J. Biol. Chem. 1998; 273: 8623-8629Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 14Krych M. Hourcade D. Atkinson J.P. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4353-4357Crossref PubMed Scopus (118) Google Scholar, 15Krych M. Clemenza L. Howdeshell D. Hauhart R. Hourcade D. Atkinson J.P. J. Biol. Chem. 1994; 269: 13273-13278Abstract Full Text PDF PubMed Google Scholar).In contrast to the situation in the complement regulatory molecules where the repeating sequence motifs suggested an obvious experimental approach for functional site-mapping studies, the absence of any identifiable sequence motifs within C3 precludes the domain deletion or domain exchange approach. Nevertheless, several independent groups have used a variety of other approaches to putatively identify segments within C3 that mediate association with its membrane-bound receptors and soluble protein ligands. The approaches have included functional site blocking antibodies against C3, together with the identification of the polypeptide segments to which they bind (16Alsenz J. Becherer J.D. Nilsson B. Lambris J.D. Curr. Top. Microbiol. Immun. 1990; 153: 235-248PubMed Google Scholar, 17Garred P. Mollnes T.E. Kazatchkine M.D. Complement Inflammation. 1989; 6: 205-218Crossref PubMed Scopus (10) Google Scholar) and the combined use of proteolytic fragments and synthetic peptides derived from C3 as functional mimetics of the intact physiologic fragments (18Lambris J.D. Immunol. Today. 1988; 9: 387-393Abstract Full Text PDF PubMed Scopus (167) Google Scholar,19Fishelson Z. Mol. Immunol. 1991; 28: 545-552Crossref PubMed Scopus (46) Google Scholar). Additionally, once one has a candidate site, xenogeneic sequence comparisons, together with knowledge about whether a particular non-human species of C3b can or cannot interact with the human ligands, can provide additional evidence for the involvement of a particular C3 segment in a binding interaction (20Alsenz J. Avila D. Huemer H.P. Esparza I. Becherer J.D. Kinoshita T. Wang Y. Oppermann S. Lambris J.D. Dev. Comp. Immunol. 1992; 16: 63-76Crossref PubMed Scopus (50) Google Scholar). Lambris and co-workers (21Lambris J.D. Lao Z. Oglesby T.J. Atkinson J.P. Hack C.E. Becherer D.J. J. Immunol. 1996; 156: 4821-4832PubMed Google Scholar) have in some cases used this information to construct and assess the ligand binding activities of C3 molecules in which a segment of human C3 has either been deleted or replaced with the homologous segment of the non-human ligand-binding xenogeneic species in order to verify a proposed binding site. Ultimately, one can employ site-directed mutagenesis to test further the validity of proposed binding sites, to identify important residues, and in some cases to establish the chemical nature of the side chains that are required for a particular binding reaction (22Taniguchi-Sidle A. Isenman D.E. J. Immunol. 1994; 153: 5285-5302PubMed Google Scholar, 23Farries T.C. Napper C.M. Harrison R.A. Mol. Immunol. 1996; 33 Suppl. 1 (abstr.): 58Google Scholar).All of the above described approaches have implicated the NH2-terminal segment of C3 α′-chain (residues 727–767) as contributing at least one contact site to the interaction of C3b with factor B, factor H, and CR1. In particular, an anti-peptide antibody directed against this segment recognizes a neoepitope in C3b and can block the interaction with factor B, factor H, and CR1 (24Becherer J.D. Alsenz J. Esparza I. Hack C.E. Lambris J.D. Biochemistry. 1992; 31: 1787-1794Crossref PubMed Scopus (35) Google Scholar). Furthermore, the 727–767 peptide can compete with factor H and CR1 for binding to C3b (24Becherer J.D. Alsenz J. Esparza I. Hack C.E. Lambris J.D. Biochemistry. 1992; 31: 1787-1794Crossref PubMed Scopus (35) Google Scholar, 25Ganu V.S. Müller-Eberhard H.J. Complement. 1985; 2 (abstr.): 27Google Scholar), and in one study (24Becherer J.D. Alsenz J. Esparza I. Hack C.E. Lambris J.D. Biochemistry. 1992; 31: 1787-1794Crossref PubMed Scopus (35) Google Scholar), but not the other (25Ganu V.S. Müller-Eberhard H.J. Complement. 1985; 2 (abstr.): 27Google Scholar), was capable of inhibiting the interaction between C3b and factor B. Finally, a recombinant molecule in which the 727–767 segment was deleted lost the ability to interact with all three proteins (21Lambris J.D. Lao Z. Oglesby T.J. Atkinson J.P. Hack C.E. Becherer D.J. J. Immunol. 1996; 156: 4821-4832PubMed Google Scholar).Fishelson (19Fishelson Z. Mol. Immunol. 1991; 28: 545-552Crossref PubMed Scopus (46) Google Scholar) had analyzed a series of overlapping hexameric and heptameric peptides spanning the 727–767 region for their ability to bind factor B and factor H. The results suggested that the segment730DEDIIAEENI contributed to factor B binding, whereas the segment 744EFPESWLWNVE contributed to the binding of factor H. Site-directed mutagenesis work on intact C3 examined the role of the carboxylate side chains within the 730–739 segment and identified those of glutamic acid residues 736 and 737 as being important not only for the interaction of C3b with factor B but also for the ability of target-bound C3b and iC3b to bind, respectively, to CR1 and CR3 on phagocytes (22Taniguchi-Sidle A. Isenman D.E. J. Immunol. 1994; 153: 5285-5302PubMed Google Scholar). In keeping with the predictions of the Fishelson work, these mutations did not alter the interaction with human factor H. In the present work, we have extended our previous mutagenesis study on human C3 to cover the remainder of the charged residues in the 727–768 segment, initially with the aim of identifying residues crucial for the interaction with factor H. Since like factor H, CR1 and MCP also act as cofactors for the factor I-mediated cleavages of C3, the same series of mutants were also examined for their ability to interact with soluble forms of CR1 (sCR1) and MCP (sMCP). We have identified Glu-744 and Glu-747 within the predicted "Fishelson" segment as residues whose carboxylate side chains contribute to the interaction with factor H. We have also found that the charged residue contacts with CR1, although partially overlapping with factor H, extend over a much larger portion of the segment. In contrast, none of the mutations examined had any effect on the interaction with MCP.DISCUSSIONAs summarized in Table I, the observations in this study have identified critical residues within the hydrophilic 42 amino acid segment at the NH2 terminus of C3b α′-chain that contribute to the binding interaction with factor H and CR1. In contrast, we were unable to show any defect in the association of MCP with any of mutant proteins examined. With respect to a number of criteria including level of expression, biosynthetic processing, reaction with two conformationally sensitive monoclonal antibodies and hemolytic activity, the mutant molecules examined can be considered to be native-like with respect to their conformational state. Although the extent of hemolytic activity, which largely reflected the extent to which the C3 convertase cleavage site was affected, ranged from 73 to 46% of wild-type levels, it is important to note that there was no correlation between the extent of the hemolytic defect and whether or not an I cofactor binding site was affected.Table ISummary of cofactor binding activities of mutant C3 molecules examined in this studyRecombinantFactor HsCR1sMCPWild-type+++aActivity relative to wild-type based on disappearance of α-chain in the cofactor-dependent factor I cleavage assay using the indicated cofactor.++++++R742A+++++++++E744EA+++++bNormal with respect to I cofactor activity for the C3(H2O) to iC3(H2O) conversion but no cleavage by I of iC3(H2O) to C3dg and a C3c-like molecule.+++E747A++++++K761A+++++++++K767A+++++++++E744A/E747A+++++E754A/D755A+++++++K757A/E758A++++++bNormal with respect to I cofactor activity for the C3(H2O) to iC3(H2O) conversion but no cleavage by I of iC3(H2O) to C3dg and a C3c-like molecule.+++E744A/E747A/E754A/D755A+++++E744Q+++++bNormal with respect to I cofactor activity for the C3(H2O) to iC3(H2O) conversion but no cleavage by I of iC3(H2O) to C3dg and a C3c-like molecule.+++E747Q++++++D730N/E731Q+++++++++E736Q/E737Q+++++++D730N/E731Q/E736Q/E737Q++±+++The symbols used are: ±, <25%; +, 25–50%; ++, 50–75%; +++, 75–100%.a Activity relative to wild-type based on disappearance of α-chain in the cofactor-dependent factor I cleavage assay using the indicated cofactor.b Normal with respect to I cofactor activity for the C3(H2O) to iC3(H2O) conversion but no cleavage by I of iC3(H2O) to C3dg and a C3c-like molecule. Open table in a new tab Although the factor H and CR1 sites of interaction within the 727–767 segment partially overlap, the charged residue contacts required by CR1 extend over a wider portion of the segment than is the case for factor H. In particular, whereas the negatively charged side chains of Glu-744 and Glu-747 appear to be most important for the interaction with factor H, for CR1 there were 3 clusters of charged residues, namely Glu-736/Glu-737, Glu-747, and Glu-754/Asp-755 that had approximately equal effects on the CR1-dependent cleavage of C3(H2O) to iC3(H2O) upon replacement of the negative charge by a neutral residue. Additional contacts with Glu-744 and the Lys-757/Glu-758 pair appeared to be required for CR1 to act as a cofactor for the third factor I-mediated cleavage. This additional requirement is reminiscent of previous results using factor H as the I cofactor for the third cleavage. Whereas H is not a cofactor for this cleavage at physiologic ionic strength, it becomes one at low ionic strength (7Ross G.D. Lambris J.D. Cain J.A. Newman S.L. J. Immunol. 1982; 129: 2051-2060PubMed Google Scholar), suggesting a need for additional contacts facilitated by relatively weak ionic bonds in order to alter either the conformation of iC3b/iC3(H2O) or more likely to position the factor I appropriately to enable the third cleavage of C3 α-chain. The recent observation that factors H and I directly interact with one another (6Soames C.J. Sim R.B. Biochem. J. 1997; 326: 553-561Crossref PubMed Scopus (69) Google Scholar) would be consistent with the latter possibility.The observation that residues Glu-744 and Glu-747 are crucial for the binding of factor H to C3(H2O) is fully consistent with the data of the Fishelson (19Fishelson Z. Mol. Immunol. 1991; 28: 545-552Crossref PubMed Scopus (46) Google Scholar) overlapping hexa/heptapeptide study which suggested that C3 residues 744–754 contribute to the binding of factor H. The only other mutant in which the interaction with H appeared to be compromised was the tetra mutant D730N/E731Q/E736Q/E737Q. Since the magnitude of impairment is the same as that seen with either E744Q or E747Q alone, it would suggest that either the contributions of the Asp-730/Glu-731 and Glu-736/Glu-737 charged pairs on their own to the binding of factor H were too small to be detectable in our assay or, more likely, that there is sufficient local conformational distortion caused by the loss of four negative charges over a space of seven amino acids to have had an effect on the nearby segment which now by two independent approaches has been shown to make a measurable contribution to H binding.We believe it noteworthy that all of the mutant proteins for which the I cofactor assay suggested a diminished interaction with either factor H or CR1 had lost at least one negative charge, whereas none of the single positive charge substitutions, representing one-third of the non-overlapping mutations examined, showed any defect. The only ambiguity involves the K757A/E758A mutant which showed resistance to only the third factor I cleavage obtainable with CR1 as the cofactor. It is thus possible that Lys-757 is not involved at all in the interaction with CR1 or that at most it contributes only to the additional cofactor binding site required to mediate the third factor I cleavage. The dominance of negatively charged residues as putative contacts on the C3 side of the interactions with CR1 and factor H, together with the documented ionic strength dependence of these interactions (44DiScipio R.G. Biochem. J. 1981; 199: 485-496Crossref PubMed Scopus (80) Google Scholar, 45Arnaout M.A. Dana M. Melamed J. Medicus R. Colten H.R. Immunology. 1983; 48: 229-237PubMed Google Scholar), strongly suggests that ionic bonds to positively charged residues on the cofactor side of the interaction form an essential component of the binding interface. Indeed, with respect to the extensively studied C3b- and C4b-binding sites within human CR1, Krych et al. (11Krych M. Hauhart R. Atkinson J.P. J. Biol. Chem. 1998; 273: 8623-8629Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar) noted that all loss-of-function mutations either resulted from the loss of a positive charge or the addition of a negative charge. These observations are therefore in keeping with the hypothesis that the ionic interface is formed between positively charged residues of CR1 and negatively charged residues contributed by C3b and C4b. Similar point mutagenesis studies of factor H SCR domains have yet to be done. Based upon other known structures of protein-protein interfaces, it is likely that there is also a hydrophobic component to the interactions of C3b with CR1 and factor H (51Young L. Jernigan R.L. Covell D.G. Protein Sci. 1994; 3: 717-729Crossref PubMed Scopus (343) Google Scholar, 52Jones S. Thornton J.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13-20Crossref PubMed Scopus (2254) Google Scholar, 53Wells J.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1-6Crossref PubMed Scopus (359) Google Scholar). Indeed within the peptide segment suggested by the Fishelson study (19Fishelson Z. Mol. Immunol. 1991; 28: 545-552Crossref PubMed Scopus (46) Google Scholar) to contain a binding site for factor H, there is a fairly hydrophobic stretch of residues, 749WLWNV, some of which may contribute a hydrophobic patch to the binding interfaces for factor H and CR1.Lambris et al. (21Lambris J.D. Lao Z. Oglesby T.J. Atkinson J.P. Hack C.E. Becherer D.J. J. Immunol. 1996; 156: 4821-4832PubMed Google Scholar) have examined the contribution of the NH2-terminal α′-chain segment of human C3 to the interactions with factor H, factor B, sCR1, and sMCP, all of human origin, by either deleting most of the segment or by replacing it with the homologous segments from Xenopus and trout C3 and from the C3-related molecule cobra venom factor (CVF). As summarized in Fig.7, these molecules differ in their abilities to bind to the human ligands (20Alsenz J. Avila D. Huemer H.P. Esparza I. Becherer J.D. Kinoshita T. Wang Y. Oppermann S. Lambris J.D. Dev. Comp. Immunol. 1992; 16: 63-76Crossref PubMed Scopus (50) Google Scholar), although it does not necessarily follow that the NH2-terminal α′-chain segment on its own dictates the inter-species compatibilities. The recombinant molecule in which the 727–767 segment was deleted lost the ability to interact with human H, B, and sCR1 but not sMCP (21Lambris J.D. Lao Z. Oglesby T.J. Atkinson J.P. Hack C.E. Becherer D.J. J. Immunol. 1996; 156: 4821-4832PubMed Google Scholar). Notwithstanding the conformational integrity caveat about the interpretation of loss-of-function data from a molecule containing a 36-residue deletion, there is general agreement between the major conclusions reached from the deletion study and those from our point mutation study. There are, however, some inconsistencies with respect to the results obtained with the homologous replacement mutants as the only deleterious effects seen were on the third factor I-mediated cleavage with sCR1 as the cofactor. In all other respects, the chimeric molecules displayed wild-type behavior. As a point of reference, a minimum gap alignment of the relevant peptide segments from the various species, along with a denotation of point mutants determined in our study to affect the interaction with human H and sCR1, is shown in Fig. 7. One can easily rationalize the lack of effect of the interaction with factor H of the Hu/Xe chimera, since residues Glu-744 and Glu-747 are conserved inXenopus C3. However, in the case of the Hu/Tr and Hu/CVF chimeras, one of the two crucial acidic residues is replaced by either its isosteric amide (Gln-744 in Tr C3) or a positively charged residue (Lys-747 in CVF), and based on our current findings, these changes would have been expected to decrease the extent of cleavage in the H-cofactor assay. One possible technical reason why this was not seen is that the assay performed by Lambris and colleagues (21Lambris J.D. Lao Z. Oglesby T.J. Atkinson J.P. Hack C.E. Becherer D.J. J. Immunol. 1996; 156: 4821-4832PubMed Google Scholar) may not have been performed under limiting conditions of cofactor. We would have also expected compromised CR1-mediated cleavages in the case of the Hu/Tr and Hu/CVF chimeras that were not limited solely to the third factor I cleavage site because of the presence of Lys at residue 747 in the Hu/CVF chimera and the replacement of Glu-754/Asp-755 by a neutral TN pairing in the Hu/Tr chimera. However, in the case of the latter change, the presence of an ED pairing at residues 752 and 753 may compensate for the loss of negative charge at residues 754 and 755. Similarly, it is perhaps not surprising that substitutions of the human Glu-736/Glu-737 pairing in the various chimeric molecules were without effect in the CR1-dependent conversion of C3(H2O) to iC3(H2O) because in each case a triplet composed of two negative side chains and one neutral side chain (EEN in human C3) is replaced by a similarly composed triplet (Xe and CVF have DSD and Tr has SED). At least with respect to factor B binding activity, we have previously shown that the human EEN triplet can be replaced by the CVF-like DSD triplet without effect (22Taniguchi-Sidle A. Isenman D.E. J. Immunol. 1994; 153: 5285-5302P
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