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PubMed1991

Amino acid residues 1101-1105 of the isotypic region of human C4B is important to the covalent binding activity of complement component C4.

The C4A and C4B isotypes of human C4 show certain functional differences that stem from their relative preference for transacylation to amino (-NH2) vs hydroxyl (-OH) nucleophiles, respectively, on complement-activating surfaces. Comparison of amino acid sequences of the alpha-chain fragment of C4, C4d, has shown C4A- and C4B-specific sequences at residues 1101-1106 are the only consistent structural difference between isotype, i.e., Pro, Cys, Pro, Val, Leu, Asp in C4A and Leu, Ser, Pro, Val Ile, His in C4B. These residues may be responsible either in part or entirely for properties associated with isotype. To examine the functional role of residues 1101-1106 in C4B-mediated hemolysis, whole serum or immunopurified human C4 with allotypes, A3B1, A3, B2B1, or B1 were preincubated in the presence or absence of an antipeptide mAb (BII-1) specific for amino acid residues 1101-1105 of C4B. Sensitized sheep E and C4-deficient guinea pig serum was then added and lysis measured by absorbance at 415 nm. Our results show lysis of antibody-sensitized sheep E is inhibited by antibody and C4B2B1, C4B1, or C4A3B1 but not antibody and C4A3. The interference of hemolysis by BII-1 could not be explained by inhibition of activation of C4B or inhibition of C3 or C5 convertase activity. Furthermore, results from uptake experiments show that BII-1 interferes with the covalent binding activity of C4B, indicating residues 1101-1105 play a role in the covalent binding reaction of C4B to the target E-antibody complex.

Amino Acid Sequence↗
PubMed1999

Both G-type domains of protein S are required for the high-affinity interaction with C4b-binding protein.

Anticoagulant protein S interacts with the complement regulatory protein C4b-binding protein (C4BP) via its sex-hormone-binding globulin (SHB6)-like region, which contains two globular (G) domains. Similar G domains are found in Gas6, a protein homologous to protein S, which is not known to bind C4BP or to have any anticoagulant activity. To determine the relative importance of the two G domains in protein S for C4BP protein binding, three recombinant protein S chimeras were produced having either of the two globular domains, or the whole SHB6-like globulin region, replaced by corresponding parts from Gas6. The chimeras were tested for binding to immobilized C4BP using surface-plasmon-resonance technology and microtiter plate-based assays. In both systems, chimeras containing either only globular domains G1 or G2 from protein S were found to bind C4BP. Binding was stimulated by Ca2+ in a manner similar to that found for wild-type protein S. The affinities for C4BP of both chimeras containing individual G domains from protein S, were lower than that of wild-type protein S. Chimera II, containing the G1 domain from protein S, consistently bound C4BP more efficiently than chimera I, which had the protein S-derived G2 domain. The chimera containing the whole SHB6-like globulin region from Gas6 interacted considerably more weakly with C4BP. Our results demonstrate that both G domains of protein S are involved in the interaction between protein S and C4BP and that full affinity binding is dependent on contributions from both domains.

Anticoagulants↗
PubMed2003

The binding of protein S and the protein S-C4BP complex to neutrophils is apoptosis dependent.

Vitamin K-dependent protein S and complement regulator C4b-binding protein (C4BP) form a high-affinity complex in plasma. We have previously shown that both free protein S and the C4BP-protein S complex can bind to apoptotic Jurkat cells. It has been demonstrated in the past that protein S and C4BP can bind to neutrophils. We now show that it is only the apoptotic neutrophil population that binds these proteins. In addition, we also show that binding is mediated through the Gla domain on protein S, which binds negatively charged phospholipids, since a monoclonal antibody directed against this domain blocks the binding. Thus, we conclude that binding of protein S and the C4BP-protein S complex to neutrophils is not cell specific, but rather apoptosis dependent.

Antibodies, Monoclonal↗
PubMed2003

Vaccinia virus complement control protein increases early bacterial clearance during experimental peritonitis.

BACKGROUND: Complement is one of the first immunological pathways activated in peritonitis. It functions to initiate and augment the innate immune response. Complement activation has also been shown to contribute to multiple organ failure after sepsis. Vaccinia virus complement control protein (VCP) is an immunomodulatory protein encoded by vaccinia virus and binds complement components C3b and C4b of the complement cascade to inhibit both the classical and alternative pathways of complement activation. This study investigates the effect of complement inhibition by recombinant (r) VCP on bacterial clearance after cecal ligation and puncture (CLP). METHODS: Swiss Webster mice were intravenously given either 20 mg/kg rVCP in 0.2 mL of normal saline, or 0.2 mL of normal saline alone, at the time of CLP. After 4 and 18 h, samples of peritoneal washout, blood, liver, and lung were collected for bacteriology, myeloperoxidase (MPO) assay for neutrophil accumulation, differential cell counts, and interleukin (IL)12 ELISA. Statistical analysis was by Mann-Whitney U test for bacteriology, and analysis of variance (ANOVA) for MPO and IL-12 concentrations. RESULTS: Aerobic and anaerobic bacterial levels were significantly lower at 4 h after treatment with rVCP (p < 0.05) in peritoneal lavage, blood, and liver compared with controls. There were no differences in bacterial levels at 18 h. There were no differences in myeloperoxidase concentrations or in the differential cell counts between the groups at either 4 or 18 h after CLP. IL-12 concentrations in serum or peritoneal washout were also not different. CONCLUSIONS: rVCP enhances early bacterial clearance in mice after CLP, although not through neutrophil recruitment, as MPO concentrations and cell counts were not different. rVCP may, however, increase neutrophil function potentially by prevention of accumulation of complement factors that inhibit leukocytes. Further studies will be needed to elucidate this pathway.

Animals↗
PubMed1995

Interaction of complement and specific antibodies with the external glycoprotein 120 of HIV-1.

Previously we have investigated the interaction of human complement as well as one polyclonal and three human monoclonal antibody preparations with the human immunodeficiency virus type-1 (HIV-1) transmembrane recombinant glycoprotein (rgp41). A strong competition was found between the antibodies and deposited complement proteins for the same binding sites located within the immunodominant region of rgp41. The aim of the present experiments was to see if the same type of antibody-complement-HIV-1 interactions could be observed with the outer envelope glycoprotein (rgp120) of HIV-1. Three different glycosylated rgp120 preparations, as well as a synthetic peptide corresponding to the V3 loop of the MN strain, were adsorbed to enzyme-linked immunosorbent assay (ELISA) plates and incubated with mixtures of anti-rgp120 antibodies and normal human serum (NHS) as a complement source. Fixed complement proteins and antibodies were detected with specific, peroxidase-labelled antibodies against different complement proteins (C1q, C4b, C3b) and the gamma-chain of antibodies. In the absence of anti-rgp120, high amounts of C3 were deposited to each rgp120 preparation tested (including the V3 peptide) but significant differences in the amounts of bound C1q and C4b were observed. Using sera deficient in different complement proteins, we found that both the classical and the alternative pathways contributed to the C3 binding to rgp120. Addition of specific antibodies did not increase complement activation by rgp120 and only in the case of a monoclonal antibody to the V3-loop could we see complement-dependent inhibition of antibody binding.

Antibodies, Monoclonal↗
PubMed1998

Rapid activation of the complement system by cuprophane depends on complement component C4.

Hemodialysis with cuprophane dialyzer membranes promotes rapid activation of the complement system, which is thought to be mediated by the alternative pathway. Complete hereditary deficiency of complement C4, a classical pathway component, in two hemodialysis patients provided the opportunity to investigate a possible role of the classical pathway. In two hemodialysis patients with both C4 isotypes, C4A and C4B, and in one patient with C4B deficiency complement activation occurred immediately after the onset of hemodialysis, with peak levels of C3a and terminal complement complex (TCC) after ten to fifteen minutes. In patients with complete C4 deficiency, C3a and TCC remained unchanged for fifteen minutes and increased thereafter, reaching the highest level after thirty minutes. The leukocyte nadir was also delayed from fifteen to thirty minutes. In vitro incubation of normal, C4A- or C4B-deficient serum with cuprophane caused complement activation after fifteen minutes. In contrast, no activation was observed in sera of four C4-deficient patients. The addition of normal serum or purified human C4 restored the capacity for rapid complement activation. In one patient with severe immunoglobulin deficiency, C3a and TCC levels increased only moderately after 25 minutes of cuprophane dialysis. This patient's serum also exhibited delayed complement activation in vitro, which was normalized after pretreatment of cuprophane with immunoglobulins. Preincubation of normal serum with MgEGTA, a blocker of the classical pathway, inhibited rapid complement activation through cuprophane. As basal levels of C4a are markedly increased in hemodialysis patients (3450 +/- 850 ng/ml) compared to healthy controls (224 +/- 81 ng/ml), no further elevation of C4a was detectable during cuprophane hemodialysis. Incubation of normal serum with cuprophane, however, caused a slight increase in C4a after five minutes. These results indicate that the initial deposition of complement C3b on the cuprophane membrane, necessary for activation of the amplification loop of the alternative pathway, is mediated by the classical pathway C3-convertase C4b2a. We propose an extended concept of complement activation through cuprophane, which is based on four steps: (a) binding of anti-polysaccharide antibodies, (b) classical pathway activation, (c) alternative pathway activation and (d) terminal pathway activation.

Adolescent↗
PubMed1975

Isolation and analysis of the mechanism of action of an inactivator of C4b in normal human serum.

A complement regulatory principle, C4b inactivator, was isolated in a partially purified form from normal human serum. The C4b inactivator, a beta1-globulin with an approximate mol wt of 88,000 daltons, and which may be identical to C3b inactivator, cleaved C4b in free solution or on the surface of cells and rendered it unable to participate in hemolytic reactions or to interact with cells, having receptors for C4b. C/b inactivator functioned by cleaving the alpha-polypeptide chain of C4b at a single site which was sufficient to dissociate the molecule into two fragments, C4c and C4d, and to inactivate it biological function. Certain structural correlates of C4 functions deriving from these studies are discussed and a model for C4 structure based on these findings is presented.

Animals↗
PubMed1989

Protein S binding in relation to the subunit composition of human C4b-binding protein.

The human regulatory complement component C4b-binding protein (C4BP) circulates in plasma either as a free protein or in a bimolecular complex with the vitamin K-dependent protein S. The major form of C4BP is composed of 7 identical, disulfide-linked 70 kDa subunits (alpha-chains), the arrangement of which gives the C4BP molecule a spider-like appearance. Recently, we identified a unique 45 kDa subunit (beta-chain) in C4BP. We have now isolated a subpopulation of C4BP, which does not bind protein S. This C4BP species, which had a molecular weight slightly lower than that of the predominant form, was found to lack the beta-chain. Another lower molecular weight form of C4BP was also purified. It contained the beta-chain and was efficient in binding protein S. Its subunit composition was judged to comprise six alpha-chains and one beta-chain. These results indicate C4BP in plasma to be heterogeneous at a molecular level vis-a-vis subunit composition and/or protein S binding ability and provide support for the concept that the beta-chain of C4BP contains the single protein S binding site.

Blotting, Western↗
PubMed1986

Structural and functional studies on the human C3b/C4b receptor (CR1) purified by affinity chromatography using a monoclonal antibody.

A procedure was devised that has several advantages over previously described methods to purify CR1 from both erythrocytes (E) and the HL-60 promyelocytic cell line. Using a monoclonal antibody immunoaffinity column, CR1 was purified to homogeneity as assessed by silver staining and 2-D gel analysis. Protein purified by this method comigrates on SDS-PAGE with 125I surface-labeled CR1 isolated by immunoprecipitation or iC3-Sepharose affinity chromatograhy and can be specifically immunoblotted with a second monoclonal anti-CR1 antibody. Employing this method, CR1 can be purified to homogeneity in amounts adequate for both functional studies and biochemical microanalysis. Purified E CR1 is functionally active as assessed by its ability to specifically rebind to an iC3-Sepharose affinity column, act as a cofactor for I-mediated cleavage of C3b to C3c and C3d, g and to accelerate decay of both the classical and alternative pathway C3 convertases. Its specific activity is similar to that of CR1 purified by a method not employing the potentially denaturing washing and eluting conditions of immunoaffinity chromatography. The pIs of the two major E CR1 allotypes are both approximately 7.1. Using pooled human E CR1, an amino acid composition was derived which revealed a relatively high proline content. This has also been found in two functionally related and genetically linked complement-regulatory proteins, H and C4-binding protein, NH2-terminal sequencing of E CR1 and HL-60 CR1 was unsuccessful indicating that the NH2-terminus is blocked.

Amino Acid Sequence↗
PubMed1986

Structural analysis of human complement protein H: homology with C4b binding protein, beta 2-glycoprotein I, and the Ba fragment of B2.

We report here a partial primary structure for human complement protein H. Tryptic peptides comprising 27% of the H molecule were isolated by conventional techniques and were sequenced (333 amino acid residues). Several mixed-sequence oligonucleotide probes were constructed, based on the peptide sequence data, and were used to screen a human liver cDNA library. The largest recombinant plasmid (pH1050), which hybridized with two probes, was further characterized. The cDNA insert of this plasmid contained coding sequence (672 bp) for 224 amino acids of H. The 3' end of this clone had a polyadenylated tail preceded by a polyadenylation recognition site (ATTAAA) and a 3'-untranslated region (229 bp). Four regions of internal homology, each about 60 amino acids in length, were observed in the derived protein sequence from this cDNA clone, and a further seven from the tryptic peptide sequences. The consensus sequence for each of the repetitive units of H was four cysteines, two prolines, three glycines, one tryptophan, and two tyrosines/phenylalanines. Based on the mole percent values for each of these amino acids, it is likely that H is composed of about 20 repetitive units of this nature. Furthermore, the repetitive unit of H shows pronounced homology with the Ba fragment of B, the C4b binding protein, and beta 2-glycoprotein I. Therefore, it seems that at least portions of these proteins have evolved from a common ancestral DNA element.

Amino Acid Sequence↗
PubMed1995

Expression and characterization of a recombinant C4b-binding protein lacking the beta-chain.

C4b-binding protein (C4BP) is a high-molecular-mass glycoprotein which contains binding sites for complement component C4b, anti-coagulant vitamin K-dependent protein S and serum amyloid P component (SAP). The major form of C4BP in plasma is composed of seven identical alpha-chains and a single beta-chain. We have expressed full-length cDNA for the alpha-chain in a eukaryotic expression system and characterized functional properties of non-beta-chain-containing C4BP. During synthesis, recombinant alpha-chains polymerized into two different high-molecular-mass C4BP forms which were composed of seven or eight alpha-chains. Recombinant C4BP bound C4(H2O) (used instead of C4b) equally as well as native C4BP, functioned equally as well as factor I cofactor in the degradation of C4(H2O) and bound to SAP. In contrast, the recombinant C4BP did not bind protein S and therefore did not inhibit the ability of protein S to function as a cofactor to activated protein C. Tunicamycin treatment of the transfected cells prevented N-linked glycosylation, but did not affect polymerization of the alpha-chains into a high-molecular-mass C4BP. The non-glycosylated C4BP had comparable properties to glycosylated C4BP in several functional assays. These results demonstrate polymerization of C4BP alpha-chains to be independent both of the beta-chain and of the N-linked carbohydrates. Moreover, N-linked carbohydrates and the beta-chain were neither required for the ability of C4BP to bind C4b and to function as factor I cofactor nor for the interaction with SAP.

Binding, Competitive↗
PubMed1983

Interaction of C4-binding protein with cell-bound C4b. A quantitative analysis of binding and the role of C4-binding protein in proteolysis of cell-bound C4b.

Purified C4-binding protein (C4-bp) was shown to bind to cell-bound C4b by radioactive tracer techniques. With EAC4 bearing greater than 3,000 C4b-molecules/cell, the number of C4-bp molecules bound was directly proportional to the number of C4b molecule on the cell surface; EAC4 bearing less than 3,000 C4b-molecules/cell bound a very small amount of C4-bp. Scatchard analysis of binding of C4-bp indicated an equilibrium constant of 4.6 X 10(8) L/M and a maximum of 0.43 C4-bp molecules bound per C4b molecule, equivalent to an average of one molecule of C4-bp per two or three molecules of C4b. Fluid-phase C4b inhibited the binding of C4-bp to cell-bound C4b in a dose-dependent manner, whereas native C4 had little effect. C2 inhibited this binding and also released C4-bp from EAC4,C4-bp. However, C2 was 27 times less effective than unlabeled C4-bp on a molar basis and a considerable amount of C4-bp remained bound to C4b on the cell surface even in the presence of a large excess of C2. We also examined the cofactor activity of C4-bp in the cleavage of cell-bound C4b by C3b/C4b inactivator (I). Cleavage of the alpha' chain of C4b on the cell surface by I alone was incomplete and an intermediate cleavage product, alpha-75, was observed. When C4-bp bound to C4b on the cell surface, the alpha' chain of the C4b cleaved into three fragments, alpha 2, alpha 3, and alpha 4. The alpha 3, alpha 4, beta, and gamma peptides (C4c) were released into the fluid phase, and the alpha 2 fragment (C4d) remained linked covalently to the cell membrane via an ester bond. In some situations, therefore, C4-bp enhances the proteolytic activity of I on cell-bound C4b.

Animals↗
PubMed1993

Clonal variations in complement activation and deposition of C3b and C4b on model immune complexes.

This study examined the relationship between complement activation and the deposition of C3b and C4b on a panel of model immune complexes (IC). IC were constructed by combining murine monoclonal IgM, IgA, IgG1, IgG2a or IgG3 anti-dinitrophenyl (DNP) antibodies with DNP-bovine serum albumin (DNP-BSA). The IC were incubated with human plasma as a complement source and the formation of C4a and C3a, as well as the deposition of C4b and C3b on the IC, measured by radioimmunoassay. The results indicate that there were isotype-independent variations in the capacity of different types of IC to activate the classical pathway, especially for isotype-matched pairs of IC containing IgG1, IgG2a and IgG3 antibodies. In most cases, there was a direct relationship between classical pathway activation and the cleavage of C3. There was, for most of the IC, a direct correlation between cleavage of C4 and C3 and the subsequent deposition of C4b and C3b on the IC. However, a pair of IC constructed with independently derived IgG1 antibodies was virtually identical with respect to C3 cleavage and yet differed in the number of C3b molecules deposited on the IC. Collectively, these data suggest that the immunoglobulin variable region can play a significant role in both complement activation and the deposition of C3b and C4b on IC.

Antigen-Antibody Complex↗
PubMed2003

CCP1-4 of the C4b-binding protein alpha-chain are required for factor I mediated cleavage of complement factor C3b.

C4b-binding protein (C4BP) is a potent regulator of the complement system because it strongly inhibits the classical pathway of complement. Furthermore, C4BP serves as a cofactor to factor I (FI) in the cleavage of fluid phase C3b and can, therefore, influence the alternative pathway of complement. The major form of C4BP in plasma consists of seven identical alpha-chains and one beta-chain. Both types of subunits are composed of complement control protein (CCP) domains, eight such domains make up one alpha-chain. To elucidate the structural requirements for the interaction between C3b and the alpha-chain, nineteen recombinant C4BP variants were used: six truncated monomeric variants, nine polymeric variants in which individual CCPs were deleted, and finally four variants in which double alanine residues were introduced between CCPs. We found that C4BP requires all four N-terminal CCPs of the alpha-chain, with CCP2 and 3 being the most important, to act as a cofactor in the cleavage of C3b. Also, a cluster of positively charged amino acids on the interface between CCP1 and 2 is involved in the binding. Compared to the interaction with C4b, we conclude that binding of C3b to C4BP requires larger molecular surface on C4BP. We found that C4BP was able to act as cofactor in degradation of surface bound C3b and to accelerate decay of alternative C3-convertase. However, in both cases 1,000-fold molar excess of C4BP over factor H (FH), well known inhibitor of the alternative pathway, was required to obtain the same effect.

Amino Acid Substitution↗
PubMed1987

Complement-mediated adherence of immune complexes to human erythrocytes. Difference in the requirements for C4A and C4B.

The classical pathway of complement is required for the adherence of soluble tetanus toxoid (TT)-human anti-TT complexes to erythrocytes. Using human C4-deficient serum we compared the capacity of the two forms of human C4 (C4A and C4B) to mediate this function: C4A was shown to be 1.5-fold more efficient than C4B. In contrast, haemolysis by C4B was 3.7-fold more efficient than by C4A. Such large differences suggest that both forms are complementary, and that C4A is preferentially involved in the processing of immune complexes in humans.

Antibodies↗