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At least 19 recordsLinked to original sources
PubMed1990

Independent association of serum amyloid P component, protein S, and complement C4b with complement C4b-binding protein and subsequent association of the complex with membranes.

C4b-binding protein (C4BP) is a large complex assembly of eight subunits that functions as an inhibitor of the complement cascade. A portion of the C4BP in serum exists as a complex with protein S. This study demonstrated that another protein, serum amyloid P component (SAP), also formed a calcium-dependent complex with C4BP. The C4BP.SAP complex was detected by several methods including light scattering intensity, gel filtration, and sucrose density gradient ultracentrifugation. This complex was of high affinity relative to serum levels of these proteins so that no dissociation was detected at 3% of serum protein concentrations. The C4BP.SAP complex was also detected in normal serum and the results suggested that there was virtually no free SAP or uncomplexed C4BP in normal serum. In addition to its complex with C4BP, SAP underwent other calcium-dependent associations such as binding to phospholipid vesicles and self-aggregation. Self-aggregation was highly cooperative with kinetics corresponding to a reaction that was 6th-order with respect to calcium and required about 1.5 mM calcium. In contrast, formation of the SAP.C4BP complex and interaction of SAP with membranes required only about 0.4 and 1.0 mM calcium, respectively. Thus, selection of the correct conditions allowed study of the SAP.C4BP interaction without interference from self-aggregation. All three of these interactions of SAP were mutually exclusive and the SAP. C4BP interaction appeared to be favored over self-aggregation or binding of SAP to phospholipids. It seems likely that the biologically dominant interaction for SAP is with C4BP. The SAP.C4BP complex interacted with protein S and these binding sites appeared to be entirely independent. Furthermore, SAP had little or no effect on the ability of C4BP to bind C4b. Finally, the entire complex of proteins (C4BP, SAP, protein S, and C4b) could associate with membranes in the presence of calcium. Membrane binding occurred through the protein S component. This rather complicated assemblage of proteins probably functions in a regulatory role for the complement cascade or other biological systems. It is possible that elevated levels of SAP or nonequivalent levels of SAP and C4BP could contribute to certain pathological conditions.

Calcium↗
PubMed1991

A covalent dimer of complement C4b serves as a subunit of a novel C5 convertase that involves no C3 derivatives.

A C intermediate, LAC14, was prepared from TNP-aminocaproyl liposomes sensitized with anti-TNP antibody (Ab) and purified human C1 and C4. LAC14, containing radiolabeled C4, was analyzed by SDS-PAGE followed by autoradiography, and yielded a 210-kDa band and a predominant 400-kDa band. The 210-kDa band consisted of monomeric C4b bound to low molecular mass acceptors. The 400-kDa band was comprised of a 200-kDa moiety, as well as beta- and gamma-chains of C4. The 200-kDa moiety contained neither C1 nor sensitizing Ab, but it was largely decreased by treatment with NH2OH to the 90-kDa moiety with the mobility corresponding to the alpha'-chain of C4b. A covalent dimer of C4b, therefore, is the predominant form of C4b deposited on liposomes sensitized with antibody. The C4b-C4b dimer formed rapidly (within 5 min) followed by slow dissociation into monomers. The LAC14 bearing the C4b dimer but not the monomer was lysed, although with relatively low efficiency, by the addition of oxyC2 and EDTA-supplemented C3-deficient serum (C3DS), and, furthermore, LAC142 possessed the ability to convert C5 into C5a and C5b. Moreover, lysis was inhibited not by anti-C3 Ab but by anti-C4 Ab. In other experiments, the dimer served as an element of C3 convertase, as well. These findings imply that the C4b dimer, when complexed with C2, expresses C3/C5 convertase activity without participation of C3, and may provide a molecular mechanism whereby sera from patients with complete C3 deficiency retain the ability to induce C-mediated cytolysis.

Autoradiography↗
PubMed1992

Factor I-dependent inactivation of human complement C4b of the classical pathway by C3b/C4b receptor (CR1, CD35) and membrane cofactor protein (MCP, CD46).

Proteolytic inactivation of C4b is a crucial step for regulation of the classical complement pathway. A plasma protease factor I and membrane cofactors, C3b/C4b receptor (CR1) and membrane cofactor protein (MCP), participate in the regulation of cell-bound C4b although the physiological potency of these cofactors remains unknown. We have examined the optimal conditions of the factor I-mediated C4b regulatory system using purified cofactors. CR1 being a cofactor at a cofactor/C4b ratio less than 0.1 (w/w), fluid phase C4b, and methylamine-treated C4 (C4ma) were degraded by factor I into C4bi: minimal Cd4 was generated in the fluid phase. Liposome-bound C4b (LAC4b), on the other hand, was degraded into C4c and C4d. CR1 showed two optimal pHs (6.0 and 7.5) for fluid phase C4b, but one (6.0) for LAC4b, and in both cases low conductivity conditions enhanced the C4bi generation. CR1 cofactor activity was barely influenced by the NP-40 concentration. On the other hand, MCP degraded C4b and C4ma, as a factor I-cofactor, more efficiently into C4c and C4d. Though MCP cofactor activity, like that of CR1, was enhanced under low conductivity conditions, it has only one optimal pH, 6.0, in both fluid and solid phases. Furthermore, as in the case of C3b cleavage, a sufficient NP-40 concentration to solubilize membrane was needed for MCP to express full cofactor activity for C4b, in contrast to CR1. MCP was less potent for C4b inactivation than for C3b inactivation, while CR1 acted as a slightly more effective cofactor for C4b cleavage than for C3b cleavage.(ABSTRACT TRUNCATED AT 250 WORDS)

Antigens, CD↗
PubMed1990

Substitution of a single amino acid (aspartic acid for histidine) converts the functional activity of human complement C4B to C4A.

The C4B isotype of the fourth component of human complement (C4) displays 3- to 4-fold greater hemolytic activity than does its other isotype C4A. This correlates with differences in their covalent binding efficiencies to erythrocytes coated with antibody and complement C1. C4A binds to a greater extent when C1 is on IgG immune aggregates. The differences in covalent binding properties correlate only with amino acid changes between residues 1101 and 1106 (pro-C4 numbering)--namely, Pro-1101, Cys-1102, Leu-1105, and Asp-1106 in C4A and Leu-1101, Ser-1102, Ile-1105, and His-1106 in C4B, which are located in the C4d region of the alpha chain. To more precisely identify the residues that are important for the functional differences, C4A-C4B hybrid proteins were constructed by using recombinant DNA techniques. Comparison of these by hemolytic assay and binding to IgG aggregates showed that the single substitution of aspartic acid for histidine at position 1106 largely accounted for the change in functional activity and nature of the chemical bond formed (ester vs. amide). Surprisingly, substitution of a neutral residue, alanine, for histidine at position 1106 resulted in an increase in binding to immune aggregates without subsequent reduction in the hemolytic activity. This result strongly suggests that position 1106 is not "catalytic" as previously proposed but interacts sterically/electrostatically with potential acceptor sites and serves to "select" binding sites on potential acceptor molecules.

Animals↗
PubMed1991

Increased frequency of the null allele at the complement C4b locus in autism.

Associations between C4 deficiency and autoimmune disorders have been found over the past several years. Since autism has several autoimmune features, the frequencies of null (no protein produced) alleles at the C4A and C4B loci were studied in 19 subjects with autism and their family members. The autistic subjects and their mothers had significantly increased phenotypic frequencies of the C4B null allele (58% in both the autistic subjects and mothers, compared with 27% in control subjects). The siblings of the autistic subjects also had an increased frequency of the C4B null allele, but this increase was not significant. The fathers had normal frequencies of this null allele. All family members had normal frequencies of the C4A null allele, all normal C4A and C4B alleles and all BF and C2 alleles.

Alleles↗
PubMed1991

A marked drop in the incidence of the null allele of the B gene of the fourth component of complement (C4B*Q0) in elderly subjects: C4B*Q0 as a probable negative selection factor for survival.

The incidence of allotypes of the genes of the fourth component (C4) and factor B of the complement system was compared in 252 persons under 45 years of age ("young" group) with 482 people between 61 and 90 years of age ("old" group). One hundred people older than 90 years of age (nonagenarians) were also investigated. A striking difference was found between the "young" and "old" groups in the incidence (16.1% and 5.4%, respectively) of a silent gene of the C4B allele (C4B*Q0). This difference was even more marked among "young" and "old" men (17.6% vs 3.4%). The incidence of the C4B*Q0 allele in women dropped to the level of the men only in the nonagenarian group. The most probable explanation for this finding is that people carrying the C4B*Q0 allele die from as yet unidentified disease(s) in their middle-age. Therefore, male (and to a lesser extent female) carriers of this allele may have a considerably shorter life expectancy than individuals without a silent gene in the C4B locus.

Adult↗
PubMed1986

Gene conversion in salt-losing congenital adrenal hyperplasia with absent complement C4B protein.

Two of four siblings expressed the salt-losing form of congenital adrenal hyperplasia due to 21-hydroxylase deficiency (CAH) and had identical human lymphocyte antigen (HLA) and complement C4 (fourth component of complement) types (HLA-A3,C4,B35,C4A3,C4BQO,DR1/A2,C-,B18,C4A3, C4BQO,DR6). The father and one unaffected sibling were heterozygous carriers of CAH, as determined by a 30-min iv ACTH stimulation test and HLA typing. In addition, the iv ACTH stimulation test revealed that the mother and the other unaffected sibling also carried an allele for an attenuated form of CAH. Restriction endonuclease digests of genomic DNA obtained from members of this family and from normal unrelated subjects were hybridized with cDNA probes encoding human 21-hydroxylase and C4. With the 21-hydroxylase probe, Southern blots prepared from control DNA samples revealed two major restriction fragments in each of four restriction endonuclease digests; TaqI produced major bands at 3.7 and 3.2 kilobases (kb), KpnI at 4.0 and 2.9 kb, EcoRI at 18 and 13 kb, and BglII at 15 and 12.5 kb. Southern blots prepared from DNA of the two patients lacked the 3.7-kb TaqI and 2.9-kb KpnI fragments, but had increased hybridization intensity (relative to control DNA samples) in the 3.2-kb TaqI and 4.0-kb KpnI fragments. By contrast, blots with EcoRI or BglII had two large hybridization fragments not different from control DNA samples. These data indicate the presence of two different 21-hydroxylase genes. Additional mapping studies revealed that the two genes had the restriction pattern of the inactive 21-hydroxylase gene. When genomic DNA that had been isolated from all members of this family and from normal subjects was hybridized with the human C4 cDNA probe, the restriction fragment hybridization patterns for all four endonuclease digests were similar in the two groups. Hence, our results suggest that the 21-hydroxylase deficiency of our patients is due to conversion of the active 21-hydroxylase gene to the inactive gene. This gene conversion was associated with absence of functional C4B protein, without any detectable alterations in the restriction fragment pattern of the C4 genes.

Adrenal Hyperplasia, Congenital↗
PubMed1988

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.

Binding Sites↗
PubMed1988

Complement C4B-null alleles in Felty's syndrome.

C4A and C4B allotypes were compared in 20 patients with Felty's syndrome (FS), 52 patients with rheumatoid arthritis (RA), and 55 control subjects. Nineteen of the FS patients had HLA-DR4. A C4B-null allele was more frequent in the patients with FS (60%) than in either the RA patients (15%) or the control subjects (26%). Only the differences between patients with FS and those with RA remained statistically significant when DR4 positive subjects were compared. The C4B null allele may identify individuals within the rheumatoid population who are at risk of developing particular systemic complications.

Alleles↗
PubMed1990

Human genes for the alpha and beta chains of complement C4b-binding protein are closely linked in a head-to-tail arrangement.

C4b-binding protein (C4BP) is an important component in the regulation of the complement system and also binds the anticoagulant vitamin K-dependent protein S. These activities are performed by distinct, although structurally related, polypeptides of 70 kDa (alpha chain) and 45 kDa (beta chain), respectively. In this report we have investigated the genetic relationships between these polypeptides. Using pulsed field gel electrophoresis analysis we demonstrate that the genes coding for the alpha (C4BP alpha) and beta (C4BP beta) chains are closely linked within the regulator of complement activation gene cluster. In addition, we have determined that the 3' end of the C4BP beta gene lies 3.5-5 kilobases from the 5' end of the C4BP alpha gene. These findings support the concept that the C4BP alpha and C4BP beta genes are the result of a gene duplication event.

Carrier Proteins↗
PubMed1991

Structure-function studies on human C4b-binding protein using monoclonal antibodies.

Human C4b-binding protein (C4BP) is a multimeric regulatory complement component interacting with vitamin K-dependent protein S and complement C4b. Using hybridoma technology, a panel of monoclonal antibodies (mAb) specific for intact human C4BP and its 160-kDa chymotryptic central core fragment were prepared to study the structure-function relationships of C4BP. By Western blot analysis and competition experiments, four distinct groups of mAb were identified and mapped on the C4BP molecule. By rotary shadowing, spider-like images of C4BP-antibody complexes were obtained and immunoelectron microscopy provided some information on the stoichiometry of the antibody-C4BP interaction. Certain antibodies interacted with C4BP molecules only at a ratio of 1:1. Others formed complexes of two or more antibodies bound to homologous sites on the C4BP molecule. Using an enzyme-linked immunosorbent sandwich assay for the measurement of the complex formation between protein S and C4BP, mAb against the central core and the disulfide-linked beta chain of C4BP were identified that inhibited the binding of protein S to C4BP. In a binding assay using 125I-labeled C4BP and solid-phase C4b, the inhibitory effect of one group of anti-C4BP mAb on the binding of C4BP to C4b was demonstrated.

Antibodies, Monoclonal↗
PubMed1991

Heparin influence on the complex of serum amyloid P component and complement C4b-binding protein.

Serum amyloid P component (SAP) forms a calcium-dependent complex with C4b-binding protein (C4BP) in human serum. this study demonstrated that heparin interacted with SAP in a calcium-dependent manner and prevented formation of the SAP.C4BP complex. Furthermore, the SAP-heparin interaction interfered with SAP binding to membranes. Therefore, all three of these interactions involved similar sites on SAP, or each interaction sterically obstructed the other binding sites. In addition to heparin, SAP bound to heparan sulfate and chondroitin sulfate. In each case, a distinct multimeric species was generated. Gel filtration and sucrose density gradient ultracentrifugation suggested that heparin and heparan sulfate produced a dimer of SAP. The dimer appeared to be the most stable structure since it was not dissociated by excess heparin. While low molecular weight heparin interacted with SAP and inhibited SAP association with membranes, the SAP dimer was not detected in sucrose density gradient ultracentrifugation studies. Polybrene prevented the interaction between SAP and heparin in both a purified system and in human serum that was enriched in SAP and heparin. In contrast, Polybrene did not seem to alter the SAP.C4BP complex. While the function of the SAP.C4BP complex is unknown, it may be important for regulation of complement and/or transport of SAP to sites in the body. Dissociation of the SAP.C4BP complex by sulfated polysaccharides such as heparin may be a physiological response that could be important during tissue damage or complement activation.

Carrier Proteins↗
PubMed1990

Cloning of cDNA coding for the beta chain of human complement component C4b-binding protein: sequence homology with the alpha chain.

The major form of complement component C4b-binding protein, a regulator of the complement system, is composed of seven identical 70-kDa alpha chains, each containing a binding site for the complement protein C4b. We recently showed that C4b-binding protein also contains a unique 45-kDa beta chain. It is disulfide-linked to the central core and contains a binding site for the vitamin K-dependent protein S. We have now isolated and characterized full-length cDNA clones for the beta chain. In addition, 57% of the structure was determined by protein sequencing of tryptic and chymotryptic peptides. Two clones, A8 and C1, isolated from different libraries were sequenced. Except for a deleted triplet encoding Ala-3 in clone A8, the two clones were identical and coded for a leader sequence of 17 amino acids and a mature protein of 235 amino acids (including Ala-3). By N-terminal amino acid sequencing, the Ala-3 heterogeneity was confirmed and a third beta-chain species starting at Glu-4 was identified. The beta chain contains five potential N-linked glycosylation sites, and endoglycosidase digestion suggested that the beta chain contained multiple complex carbohydrate side chains. Northern blot analysis of human liver mRNA, using the beta-chain cDNA as the probe, demonstrated a major mRNA species of approximately 1.0 kilobase. From the N terminus, the beta chain contains three tandem repeat units (60 amino acids long) that are homologous to those present in the alpha chain. The C-terminal region, which was unrelated to the tandem repeats, demonstrated sequence similarity with the corresponding region of the alpha chain. In both alpha and beta chains these regions contain two cysteine residues that probably form the interchain disulfide bridges.

Amino Acid Sequence↗
PubMed1985

Regulatory proteins for the activated third and fourth components of complement (C3b and C4b) in mice. II. Identification and properties of complement receptor type 1 (CR1).

We identified on the membrane of mouse spleen cells a polypeptide of Mr 190,000 (S190), with binding affinity for the mouse third component of the complement system (C3). S190, purified by affinity chromatography on C3-Sepharose, has properties resembling those of the human C3 receptor type 1 (CR1). Thus, S190, like CR1, served as a cofactor for the C3b inactivator (I)-mediated cleavage of fluid-phase C3b into iC3b, and had cofactor activity comparable to that of serum factor H (H). S190 also acted as a cofactor for the cleavages of membrane-bound C3b or membrane-bound iC3b into C3c (Mr 140,000) and C3dg (Mr 40,000) by serum factor I. As is the case with CR1, the specific activity of S190 for the cleavages leading to C3c-C3dg formation was approximately 100-fold greater than that of H. We therefore conclude that S190 and CR1 are analogous proteins.

Animals↗
PubMed1992

Combined total deficiency of C7 and C4B with systemic lupus erythematosus (SLE).

The first inherited combined total deficiency of C7 and C4B complement components associated with SLE is described in a young female. Functional C7 assays showed a homozygous C7 deficiency in the propositus and her sister, and an heterozygous one in their parents. C4 molecular analyses showed that both the propositus and her mother had two HLA haplotypes carrying only C4A-specific DNA sequences and a normal C4 gene number. Thus, only C4A proteins could be expressed, with resultant normal C4 serum levels. The coexistence of a combined complete C7 and C4B deficiency may therefore abrogate essential functions of the complement cascade presumably related to immune complex handling and solubilization despite an excess of circulating C4A. These findings challenge the putative pathophysiological roles of C4A and C4B and stress the need to perform both functional assays and C4 allotyping in patients with autoimmune pathology and low haemolytic activity without low serum levels of a classical pathway complement component.

Adult↗
PubMed1990

Localization of the covalent C3b-binding site on C4b within the complement classical pathway C5 convertase, C4b2a3b.

C5 convertase of the classical complement pathway is a trimolecular protein complex consisting of C4b, C2a, and C3b. In the complex there is an ester bond between C3b and C4b. We analyzed the C5 convertase formed on erythrocytes and localized the covalent binding site of C3b to a small region on C4b. The covalently linked C4b.C3b complex was purified from a detergent extract of the erythrocytes and digested with lysyl endopeptidase. An Mr 17,000 fragment containing the ester linkage between C4b and C3b was purified and its amino-terminal sequence was examined. Two amino acids were obtained at each cycle and identified with those in the sequences of C3 and C4. The sequence derived from C3 corresponded to the thioester region. The sequence derived from C4 started at Ala-1186. Alkali treatment of the fragment yielded an Mr 7,000 peptide derived from C4, which thus appeared to span the region of C4 from Ala-1186 to Lys-1259. Therefore, the covalent C3b-binding site on C4b is located within a 74-residue region of the primary structure. This finding supports the notion that after cleavage of C3 by the C4b2a complex, the covalent binding of metastable C3b to C4b is a specific reaction to form a trimolecular complex with a defined quaternary structure.

Amino Acid Sequence↗