Enhancement of antibodies to the human immunodeficiency virus type 1 envelope by using the molecular adjuvant C3d

DNA vaccines expressing the envelope (Env) protein of the human immunodeficiency virus have been relatively ineffective at generating high-titer, long-lasting, neutralizing antibodies in a variety of animal models. In this study, the murine and human homologues of the complement component, C3d, were used in a DNA vaccine to enhance the titers of antibody to Env. Initially, plasmids expressing a secreted form of Env (sgp120) fused to one, two, or three copies of the murine homologue of C3d (mC3d) were constructed. Mice were inoculated with four vaccinations of DNA or two DNA vaccinations, followed by two boosts of affinity-purified gp120 protein. Analyses of titers demonstrated that multiple copies of mC3d coupled to sgp120 induced long-lasting, high-titer anti-Env antibody. Priming mice with sgp120-mC3d-DNA, followed by inoculation of purified gp120 protein, elicited the strongest antibody titers; however, the avidity maturation of the antibody was accelerated in the mice inoculated with sgp120-mC3d(3)-DNA. In addition, DNAs expressing sgp120 fused to three copies of the human homologue of C3d (hC3d(3)) efficiently enhanced the anti-Env antibody in rabbits. Lastly, antisera from both mice and rabbits vaccinated with DNA expressing sgp120-C3d(3) elicited higher titers of neutralizing antibody than did nonfused forms of Env. These results indicate that C3d, conjugated to sgp120, enhances the antibody responses to Env compared to non-C3d fused forms of Env, and this approach may be one way to overcome the poor ability of DNA vaccines to generate antibodies to Env.     

Evidence that C4b-binding protein is an acute phase protein

C4b-binding protein is a regulatory factor for both complement and coagulation systems. We found that a human hepatoma cell line, Hep G2, was capable of synthesizing C4b-binding protein and that the secretion of C4b-binding protein was enhanced by interleukin-6 and tumor necrosis factor, which are known to be modulators of acute phase proteins. In addition, the plasma content of C4b-binding protein was found to increase in patients of acute pneumonia. These results suggest that C4b-binding protein is an acute phase protein.     

Expression and immunogenicity of the extracellular domain of the human immunodeficiency virus type 1 envelope glycoprotein, gp160.

The envelope glycoprotein of human immunodeficiency virus type 1 is synthesized as a precursor, gp160, that subsequently is cleaved to yield mature gp120 and gp41. In these studies, the gene encoding gp160 was mutagenized so as direct the synthesis of a truncated protein consisting of the extracellular domains of both gp120 and gp41. The variant protein, termed sgp160, consisted of 458 amino acids of gp120 and 172 amino acids of gp41. To facilitate protein purification, the normal polyglycoprotein processing site between gp120 and gp41 was deleted through the use of site-directed mutagenesis. This allowed for the synthesis of a molecule that could be purified by affinity chromatography, using acid elution, without dissociation of the gp120 polypeptide from the gp41 polypeptide. The conformation of the sgp160 variant appeared to be functionally relevant, as reflected by its ability to bind to CD4 with an affinity comparable to that of the variant rgp120. The structure of the sgp160-containing polypeptide differed from that of rgp120 in that it tended to form high-molecular-weight aggregates that could be dissociated to monomers and dimers in the presence of reducing agents. Antibodies against the sgp160 protein reacted with authentic virus-derived gp160, gp120, and gp41; neutralized viral infectivity; and inhibited the binding of rgp120 to CD4. Rabbit antibodies to the sgp160 protein differed from those raised against rgp120 in that they were enriched for populations that blocked CD4 binding but did not prevent human immunodeficiency virus type 1-induced syncytium formation.     

Inhibition of cofactor activity of protein S by a complex of protein S and C4b-binding protein. Evidence for inactive ternary complex formation between protein S, C4b-binding protein, and activated protein C

To elucidate the mechanism by which C4b-binding protein inhibits the cofactor activity of protein S for anticoagulant-activated protein C, the interactions between protein S, activated protein C, and C4b-binding protein were studied using solid-phase enzyme immunoassays. Both activated protein C and C4b-binding protein bound to protein S fixed to microplate wells. C4b-binding protein did not inhibit the binding of activated protein C to protein S, nor did activated protein C inhibit the binding of C4b-binding protein to protein S. Activated protein C bound to a protein S-C4b-binding protein complex which was cross-linked with a chemical reagent as well as it bound to free protein S. Protein S-C4b-binding protein complex competitively inhibited activated protein C-binding to free protein S and also the cofactor activity of free protein S. Immunoblotting analysis showed ternary complex formation with protein S, C4b-binding protein, and activated protein C in the liquid phase by treatment with the cross-linking reagent. These findings suggest that the protein S-C4b-binding protein complex inhibits the cofactor activity of free protein S probably by inhibition of functionally active protein S-activated protein C complex formation by the apparent competitive formation of an inactive ternary complex with protein S, C4b-binding protein, and activated protein C.     

Characterization of a synthetic peptide that inhibits the interaction between protein S and C4b-binding protein

Protein S is unique among the vitamin K-dependent proteins found in blood plasma because it is a cofactor rather than a zymogen of a serine protease. Instead of a trypsin-like domain, protein S contains a domain that has sequence homology with steroid binding proteins. In order to understand the function of this structural domain, peptides have been synthesized with amino acid sequences that are homologous between human protein S and rat androgen binding protein. Two peptides, corresponding to amino acids 400-407 (PINPRLDG) and 605-614 (GVQLDLDEAI) of the protein S sequence have been tested for their effects on protein S function. Neither peptide altered the clotting of bovine or human plasma. The peptide GVQLDLDEAI enhanced the anticoagulant activity of human-activated protein C in human plasma while the peptide PINPRLDG had no effect. The peptide GVQLDLDEAI was observed to inhibit the binding of protein S to C4b-binding protein in plasma, resulting in increased concentrations of free protein S. GVQLDLDEAI was also observed to enhance the disassociation of the protein S.C4b-binding protein complex when purified complex was used. Finally, C4b-binding protein was observed to bind to GVQLDLDEAI. These results suggest that the carboxyl-terminal region of protein S, which contains the sequence GVQLDLDEAI, is involved in the interaction between protein S and C4b-binding protein.     

Molecular cloning and characterization of the cDNA coding for C4b-binding protein, a regulatory protein of the classical pathway of the human complement system.

By using synthetic oligonucleotides as probes, plasmid clones containing portions of cDNA coding for human C4b-binding protein were isolated from a liver cDNA library. The entire amino acid sequence of the C4b-binding protein can be predicted from this study of the cloned cDNA when allied to a previous sequence study at the protein level [Chung, Gagnon & Reid (1985) Mol. Immunol. 22, 427-435], in which over 55% of the amino acid sequence, including the N-terminal 62 residues, was obtained. The plasmid clones isolated allowed the unambiguous determination of 1717 nucleotides of cDNA sequence between the codon for the 32nd amino acid in the sequence of C4b-binding protein and the 164th nucleotide in the 3' non-translated region. The sequence studies show that the secreted form of C4b-binding protein, found in plasma, is composed of chains of apparent Mr 70 000 that contains 549 amino acid residues. Examination of the protein and cDNA sequence results show that there are at least two polymorphic sites in the molecule. One is at position 44, which can be glutamine or threonine, and the other is at position 309, which can be tyrosine or histidine. Northern-blot analysis indicated that the mRNA for C4b-binding protein is approx. 2.5 kilobases long. The N-terminal 491 amino acids of C4b-binding protein can be divided into eight internal homologous regions, each approx. 60 amino acids long, which can be aligned by the presence in each region of four half-cystine, one tryptophan and several other conserved residues. These regions in C4b-binding protein are homologous with the three internal-homology regions that have been reported to be present within the Ba region of the complement enzyme factor B and also to the internal-homology regions found in the non-complement beta 2-glycoprotein I.     

Purification of human C4b-binding protein and formation of its complex with vitamin K-dependent protein S.

C4b-binding protein was purified from human plasma in high yield by a simple procedure involving barium citrate adsorption and two subsequent chromatographic steps. Approx. 80% of plasma C4b-binding protein was adsorbed on the barium citrate, presumably because of its complex-formation with vitamin K-dependent protein S. The purified C4b-binding protein had a molecular weight of 570 000, as determined by ultracentrifugation, and was composed of about eight subunits (Mr approx. 70 000). Uncomplexed plasma C4b-binding protein was purified from the supernatant after barium citrate adsorption. On sodium dodecyl sulphate/polyacrylamide-gel electrophoresis in non-reducing conditions and on agarose-gel electrophoresis it appeared as a doublet, indicating two forms differing slightly from each other in molecular weight and net charge. The protein band with the higher molecular weight in the doublet corresponded to the C4b-binding protein purified from the barium citrate eluate. Complex-formation between protein S and C4b-binding protein was studied in plasma, and in a system with purified components, by an agarose-gel electrophoresis technique. Protein S was found to form a 1:1 complex with the higher-molecular-weight form of C4b-binding protein, whereas the lower-molecular-weight form of C4b-binding protein did not bind protein S. The KD for the C4b-binding protein-protein S interaction in a system with purified components was approx. 0.9 X 10(-7) M. Rates of association and dissociation at 37 degrees C were low, namely about 1 X 10(3) M-1 . S-1 and 1.8 X 10(-4)-4.5 X 10(-4) S-1 respectively. In human plasma free protein S and free higher-molecular-weight C4b-binding protein were in equilibrium with the C4b-binding protein-protein S complex. Approx. 40% of both proteins existed as free proteins. From equilibrium data in plasma a KD of about 0.7 X 10(-7) M was calculated for the C4b-binding protein-protein S interaction.     

Analysis of the interaction of the human immunodeficiency virus type 1 gp120 envelope glycoprotein with the gp41 transmembrane glycoprotein.

The human immunodeficiency virus type 1 (HIV-1) gp120 exterior envelope glycoprotein interacts with the viral receptor (CD4) and with the gp41 transmembrane envelope glycoprotein. To study the interaction of the gp120 and gp41 envelope glycoproteins, we compared the abilities of anti-gp120 monoclonal antibodies to bind soluble gp120 and a soluble glycoprotein, sgp140, that contains gp120 and gp41 exterior domains. The occlusion or alteration of a subset of gp120 epitopes on the latter molecule allowed the definition of a gp41 "footprint" on the gp120 antibody competition map. The occlusion of these epitopes on the sgp140 glycoprotein was decreased by the binding of soluble CD4. The gp120 epitopes implicated in the interaction with the gp41 ectodomain were disrupted by deletions of the first (C1) and fifth (C5) conserved gp120 regions. These deletions did not affect the integrity of the discontinuous binding sites for CD4 and neutralizing monoclonal antibodies. Thus, the gp41 interface on the HIV-1 gp120 glycoprotein, which elicits nonneutralizing antibodies, can be removed while retaining immunologically desirable gp120 structures.     

Unusual ultrastructure of complement-component-C4b-binding protein of human complement by synchrotron X-ray scattering and hydrodynamic analysis.

Solution X-ray-scattering experiments with the use of synchrotron radiation on the human complement-component-C4b-binding protein showed that its RG is 13 nm and that its Mr is 550,000. From the known primary amino acid sequence and estimated carbohydrate content, C4b-binding protein is inferred to have a total of 7.4 +/- 1 subunits. Heptameric computer models for C4b-binding protein were based on the X-ray-scattering curve to a resolution of 6.4 nm, and literature values for sedimentation coefficients and electron-microscopy images. The macromolecule was represented by a bundle of seven arms held together at the C-terminal end and spaced out by a base containing 23% of C4b-binding protein by volume. If the overall length of each arm is assumed to be 33 nm as seen in electron microscopy, the solution data indicate an average arm-axis angle of 5-10 degrees. The seven arms of C4b-binding protein are found to be close together, in distinction to the splayed-out images seen in electron micrographs.     

Hemopexin joins transferrin as representative members of a distinct class of receptor-mediated endocytic transport systems

Receptor-mediated transport of heme by hemopexin in vivo and in vitro results in catabolism of heme but not the protein, suggesting that intact apohemopexin recycles from cells. However, until now, the intracellular transport of hemopexin by receptor-mediated endocytosis remained to be established. Biochemical studies on cultured human HepG2 and mouse Hepa hepatoma cells demonstrate that hemopexin is transported to an intracellular location and, after endocytosis, is subsequently returned intact to the medium. During incubation at 37 degrees C, hemopexin accumulated intracellularly for ca. 15 min before reaching a plateau while surface binding was saturated by 5 min. No internalization of ligand took place during incubation at 4 degrees C. These and other data suggest that hemopexin receptors recycle, and furthermore, incubation with monensin significantly inhibits the amount of cell associated of heme-[125I]hemopexin during short-term incubation at 37 degrees C, consistent with a block in receptor recycling. Ammonium chloride and methylamine were less inhibitory. Electron microscopic autoradiography of heme-[125I]hemopexin showed the presence of hemopexin in vesicles of the classical pathway of endocytosis in human HepG2 hepatoma cells, confirming the internalization of hemopexin. Colloidal gold-conjugated hemopexin and electron microscopy showed that hemopexin bound to receptors at 4 degrees C is distributed initially over the entire cell surface, including microvilli and coated pits. After incubation at 37 degrees C, hemopexin-gold is located intracellularly in coated vesicles and then in small endosomes and multivesicular bodies. Colocalization of hemopexin and transferrin intracellularly was shown in two ways. Radioiodinated hemopexin was observed in the same subcellular compartment as horseradish peroxidase conjugates of transferrin using the diaminobenzidine-induced density shift assay. In addition, colloidal gold derivatives of heme-hemopexin and diferric transferrin were found together in coated pits, coated vesicles, endosomes and multivesicular bodies. Therefore, hemopexin and transferrin act by a similar receptor-mediated mechanism in which the transport protein recycles after endocytosis from the cell to undergo further rounds of intracellular transport.     

Ultrastructure of C4b-binding protein fragments formed by limited proteolysis using chymotrypsin

C4b-binding protein is a regulator of the classical pathway of the complement system, acting as a cofactor to the serine protease factor I in the degradation of C4b. Its molecular weight is approximately 570,000 and it is composed of multiple, disulfide-linked 70-kDa subunits. Visualized by electron microscopy (Dahlb?ck, B., Smith, C. A., and Muller-Eberhard, H. J. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 3641-3645), it has an unusual spider-like structure with multiple thin (30 A), elongated (330 A) tentacles. The number of tentacles was estimated to be seven. Limited proteolysis by chymotrypsin produces fragments of approximately 50- and 160-kDa, the latter composed of multiple, disulfide-linked, 25-kDa polypeptides. We now have isolated the undenatured C4b-binding protein fragments formed by treatment of the protein with chymotrypsin and have visualized them by electron microscopy. The 160-kDa fragment comprises the central portion of the C4b-binding protein, which appears as a ringlike structure with an inner diameter of 13 A and an outer diameter of 60 A and having attached an approximately 40-A long piece of each tentacle. The liberated 50-kDa fragment constitutes the major part (290-A long) of the tentacles. Chymotrypsin digestion of C4b-binding protein was also monitored as a function of time by polyacrylamide gel electrophoresis and the number of subunits cleaved was found to be seven, supporting our previous ultrastructural data which suggested that C4b-binding protein contains seven identical tentacle-like subunits.     

The localization of heparin-binding fragments on human C4b-binding protein

C4b-binding protein (C4BP) is a multimeric plasma protein, which regulates the classical pathway of the C system. C4BP interacts with C C4b on a domain located in a 48-kDa chymotryptic fragment. We now demonstrate that C4BP contains heparin-binding fragments, which are located within the C4b binding domain. We have used an assay using heparin coupled to Sepharose CL-6B to show that 125I-C4BP binds to heparin in a time-dependent, saturable, and reversible manner. Binding could be inhibited by purified 48-kDa fragments and direct binding on the 48-kDa fragments to heparin-Sepharose was demonstrated by SDS-PAGE. mAb against native C4BP and the isolated 160-kDa central core fragment were evaluated for their ability to block the binding of 125I-C4BP to heparin and C4b. The relative efficacy of mAb against intact C4BP in blocking C4BP binding to heparin-Sepharose was similar to that for blocking 125I-C4BP binding to C4b. In addition, heparin blocked the binding of 125I-C4BP to C4b and vice versa. It is therefore likely that the heparin-binding fragments are localized on or close to the C4b-binding site of C4BP.     

A conserved hairpin structure in Alfamovirus and Bromovirus subgenomic promoters is required for efficient RNA synthesis in vitro

The coat protein gene in RNA 3 of alfalfa mosaic virus (AMV; genus Alfamovirus, family Bromoviridae) is translated from the subgenomic RNA 4. Analysis of the subgenomic promoter (sgp) in minus-strand RNA 3 showed that a sequence of 37 nt upstream of the RNA 4 start site (nt +1) was sufficient for full sgp activity in an in vitro assay with the purified viral RNA-dependent RNA-polymerase (RdRp). The sequence of nt -6 to -29 could be folded into a potential hairpin structure with a loop represented by nt -16, -17, and -18, and a bulge involving nt -23. By introducing mutations that disrupted base pairing and compensatory mutations that restored base pairing, it was shown that base pairing in the top half of the putative stem (between the loop and bulge) was essential for sgp activity, whereas base pairing in the bottom half of the stem was less stringently required. Deletion of the bulged residue A-23 or mutation of this residue into a C strongly reduced sgp activity, but mutation of A-23 into U or G had little effect on sgp activity. Mutation of loop residues A-16 and A-17 affected sgp activity, whereas mutation of U-18 did not. Using RNA templates corresponding to the sgp of brome mosaic virus (BMV; genus Bromovirus, family Bromoviridae) and purified BMV RdRp, evidence was obtained indicating that also in BMV RNA a triloop hairpin structure is required for sgp activity.     

Characterization of the interaction between human protein S and C4b-binding protein (C4bp)

C4b-binding protein, C4bp, is a regulatory factor of the complement system and is also known to be a binding protein of vitamin K-dependent coagulation factor, protein S. Whereas the C4b-binding site is known to be located in the middle part of the subunit chains of C4bp, the location and properties of protein S-binding site are uncertain. Therefore, we have examined the characteristics of the interaction between human protein S and C4bp. Proteolysis of C4bp-protein S complex with chymotrypsin yielded N-terminal-derived 48-kDa fragments of C4bp subunit chains and a C-terminal-derived 160-kDa core fragment of C4bp, to which protein S was still bound. This result suggested that the protein S-binding site is located in the core domain of C4bp. Gel filtration of guanidine-treated C4bp-protein S complex in the absence of guanidine resulted in the separation of C4bp and protein S. Binding assay with 125I-labeled protein S showed that the guanidine-treated C4bp lacked the protein S-binding activity. This result suggests that the protein S-binding site in C4bp is denatured irreversibly by guanidine treatment and therefore seems to be dependent on a specific conformation of C4bp. The C4bp-binding site of protein S was lost upon thrombin treatment, suggesting that the N-terminal thrombin-sensitive region of protein S may be related to the C4bp-binding site. Although free protein S was susceptible to chymotrypsin, leukocyte elastase, and cathepsin G, C4bp-bound protein S was found to be resistant to these proteases.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the baboon erythrocyte C3b-binding protein

E from primates demonstrate type 1 CR (CR1) with binding specificities for C3b and C4b. In the present study we characterized the E C3b-binding protein of baboons. We showed that three out of four mouse mAb and one polyclonal antiserum, raised against human E CR1, cross-reacted with baboon E. In addition, one anti-human CR1 mAb (1B4) and a polyclonal anti-human CR1 inhibited the binding of C3b opsonized immune complexes to baboon E. Finally, a mAb to human CR1 (E11) recognized epitopes on E of a variety of nonhuman primates, including baboons. SDS-PAGE analysis of biochemically purified baboon E membrane fractions reactive with E11 demonstrated a 65-kDa protein as a major component. Affinity absorption and elution experiments verified this protein to be E11 reactive as well as a C3b binding protein. E surface radiolabeling, followed by C3i affinity purification, confirmed that this 65-kDa protein is the only C3b-binding protein present on the baboon E membrane. We postulate that the baboon E 65-kDa protein is the equivalent of the human E CR1. In addition, there appear to be antigenic similarities between the baboon E 65-kDa protein and the human E CR1.     

Covalent binding of C3b to C4b within the classical complement pathway C5 convertase. Determination of amino acid residues involved in ester linkage formation.

C5 convertase of the classical complement pathway is a protein complex consisting of C4b, C2a, and C3b. Within this complex C3b binds to C4b via an ester linkage. We now present evidence that the covalent C3b-binding site on human C4b is Ser at position 1217 of C4. We also show that formation of the covalently linked C4b.C3b complex occurs in the mouse complement system and that the C3b-binding site on mouse C4b is Ser at position 1213 which is homologous to Ser-1217 of human C4. Therefore, covalent binding of C3b to a single specific site on C4b within the classical pathway C5 convertase is likely a common phenomenon in the mammalian complement system. Specific noncovalent association of metastable C3b with C4b would occur first, leading to reaction of the thioester with a specific hydroxy group. This is supported by two lines of experimental evidence, one which shows that a mutant C4 that does not make a covalent linkage with C3b is still capable of forming C5 convertase and a second in which the C4b.C3b complex has been demonstrated by cross-linking erythrocytes bearing this C5 convertase.     

Identification of the streptococcal M protein binding site on membrane cofactor protein (CD46)

Adherence of group A streptococcus (GAS) to keratinocytes is mediated by an interaction between human CD46 (membrane cofactor protein) with streptococcal cell surface M protein. CD46 belongs to a family of proteins that contain structurally related short consensus repeat (SCR) domains and regulate the activation of the complement components C3b and/or C4b. CD46 possesses four SCR domains and the aim of this study was to characterize their interaction with M protein. Following confirmation of the M6 protein-dependent interaction between GAS and human keratinocytes, we demonstrated that M6 protein binds soluble recombinant CD46 protein and to a CD46 construct containing only SCRs 3 and 4. M6 protein did not bind to soluble recombinant CD46 chimeric proteins that had the third and/or fourth SCR domains replaced with the corresponding domains from another complement regulator, CD55 (decay-accelerating factor). Homology-based molecular modeling of CD46 SCRs 3 and 4 revealed a cluster of positively charged residues between the interface of these SCR domains similar to the verified M protein binding sites on the plasma complement regulators factor H and C4b-binding protein. The presence of excess M6 protein did not inhibit the cofactor activity of CD46 and the presence of excess C3b did not inhibit the ability of CD46 to bind M6 protein by ELISA. In conclusion, 1) adherence of M6 GAS to keratinocytes is M protein dependent and 2) a major M protein binding site is located within SCRs 3 and 4, probably at the interface of these two domains, at a site distinct from the C3b-binding and cofactor site of CD46.     

Binding site for vitamin K-dependent protein S on complement C4b-binding protein

Half of the protein S in plasma is present as a complex with a C4b-binding protein (C4bp), a complement component (Mr 570,000). In this study, the protein S-binding site on C4bp was examined by using monoclonal anti-C4bp-IgGs. C4bp was cleaved by chymotryptic digestion into seven NH2-terminal arm fragments (Mr 48,000) and a COOH-terminal core fragment (Mr 160,000). The COOH-terminal fragment inhibited the cofactor activity of protein S and its binding to C4bp in a dose-dependent manner. A monoclonal anti-C4bp-IgG (MFbp16), which binds to the COOH-terminal fragment, inhibited the binding of protein S to C4bp. The chymotryptic digest of the reduced and carboxymethylated COOH-terminal fragment was subjected to MFbp16-Sepharose 4B column affinity chromatography, and a peptide of Mr 2,500 was obtained. Protein S bound to the Mr 2,500 peptide, and this binding was inhibited by C4bp in a dose-dependent manner. The sequence of this peptide corresponded to Ser447-Tyr467 near the COOH terminus of the C4bp subunit. MFbp16, which bound to Mr 570,000 C4bp (C4bp-high), did not bind to Mr 510,000 C4bp (C4bp-low) in human plasma that does not form a complex with protein S. This suggests that C4bp-low lacks the protein S-binding site present in the COOH-terminal region of C4bp-high. Since C4bp-low also dissociates into identical subunits when reduced, the interchain disulfide bond region that links the seven subunits of C4bp appears to be closer to the NH2-terminal end than the protein S-binding site.     

Mapping of the sites responsible for factor I-cofactor activity for cleavage of C3b and C4b on human C4b-binding protein (C4bp) by deletion mutagenesis

Human C4b-binding protein (C4bp) facilitates the factor I-mediated proteolytic cleavage of the active forms of complement effectors C3b and C4b into their inactive forms. C4bp comprises a disulfide-linked heptamer of alpha-chains with complement (C) regulatory activity and a beta-chain. Each alpha-chain contains 8 short consensus repeat (SCR) domains. Using SCR-deletion mutants of recombinant multimeric C4bp, we identified the domains responsible for the C3b/C4b-binding and C3b/C4b-inactivating cofactor activity. The C4bp mutant with deletion of SCR2 lost the C4b-binding ability, as judged on C3b/C4b-Sepharose binding assaying and ELISA. In contrast, the essential domains for C3b-binding extended more to the C-terminus, exceeding SCR4. Using fluid phase cofactor assaying and deletion mutants of C4bp, SCR2 and 3 were found to be indispensable for C4b cleavage by factor I, and SCR1 contributed to full expression of the factor I-mediated C4b cleaving activity. On the other hand, SCR1, 2, 3, 4, and 5 participated in the factor I-cofactor activity for C3b cleavage, and SCR2, 3, and 4 were absolutely required for C3b inactivation. Thus, different sets of SCRs participate in C3b and C4b inactivation, and the domain repertoire supporting C3b cofactor activity is broader than that supporting C4b inactivation by C4bp and factor I. Furthermore, the domains participating in C3b/C4b binding are not always identical to those responsible for cofactor activity. The necessity of the wide range of SCRs in C3b inactivation compared to C4b inactivation by C4bp and factor I may reflect the physiological properties of C4bp, which is mainly directed to C4b rather than C3b.