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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.
The genes for four components (C) of complement in the human major histocompatibility complex (HLA) have been aligned previously in a series of overlapping cosmid cloned inserts. Those inserts, which contained the two C4 genes C4A and C4B, hybridized with human adrenal mRNA, indicating that they contain a gene expressed in the adrenal. The mRNA fraction of 2.4 kilobases (kb) hybridizes with genomic DNA of 4.5 kb, which is duplicated and lies about 1.5 kb 3' of both the C4A and the C4B complement genes. Sequencing of a 430-base section and comparison with the published cDNA sequence of bovine cytochrome P-450 21-hydroxylase, peptide sequences of porcine 21-hydroxylase, and a cDNA sequence of a rat liver cytochrome P-450 identified the gene as coding for human steroid 21-hydroxylase [steroid,hydrogen-donor:oxygen oxidoreductase (21-hydroxylating), EC 1.14.99.10]. Mapping of the gene was helped by use of a synthetic oligonucleotide based on the bovine cDNA sequence.
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.
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.
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.
Primary Biliary Cirrhosis (PBC) is a female associated disease of unknown aetiology, although there is evidence of immunological abnormalities. There is no known cure; liver damage is progressive and eventually fatal, although transplantation can prevent patient death. Data presented here show, for the first time, a strong association in Caucasoids between PBC and the major histocompatibility complex (MHC). 45% of patients studied, compared with only 17% in a control group, expressed an MHC Class III allotype C4B 2 (pc = 0.014). Polymorphisms of MHC Class I, Class II, and other Class III gene products which flank the C4 genes were not found to be associated with the disease. Although we cannot rule out the involvement of other loci linked to the C4B 2 complement gene, the data provide strong evidence that this genetic area is implicated in the pathogenesis of this disease.
C4A and C4B are closely related homologous complement proteins encoded in the class III region of major histocompatibility complex (MHC). The regulation of their expression is under genetic and hormonal control. In this study we investigated the synovial fluid plasma ratio of C4A and C4B of rheumatoid (RA) and osteoarthritis (OA) patients, and a predominance of the C4B gene expression by the synovial macrophages of RA patients was demonstrated. To clarify the tissue specificity of the expression of C4A and C4B genes, human monocytoid cell line U937 and hepatoma-derived HepG2 cells were studied. The gene expression of C4A and C4B were markedly different in these cells since a relative predominance of C4B mRNA in U937 cells and excess of that of C4A in HepG2 cells were detected. Recombinant interferon-gamma (IFN-gamma) up-regulated the expression of C4A gene in both cells, but had apparently no effect on the C4B gene. Our results demonstrate dissimilar expression patterns for the two human C4 genes, suggesting different tissue specific regulation of human C4A and C4B.
A remarkable difference has been observed between the reactivity of the two forms of human complement component C4. C4B binds twice as effectively as C4A to antibody-coated red cells, but the reverse occurs with protein-antigen complexes. C4B reacts much more effectively with hydroxyl groups than C4A and this is reversed for reaction with amino groups in spite of the very small difference in amino acid sequence between the two forms of C4. No other differences in stability, activation or inactivation were observed. These findings emphasise the biological advantage of the duplication of the C4 gene in its reaction with a wide range of antigenic structures. The correlation of the presence of different forms of C4 with susceptibility to autoimmune diseases may be explicable by these big differences in binding reactivity.
OBJECTIVE: Complement component 4 (C4), encoded by C4A and C4B within the major histocompatibility complex (MHC) on chromosome 6, regulates the immune response and clears immune complexes. The variable copy number (CN) of C4 genes and retroviral human endogenous retrovirus K (HERV-K) element influence its function. Given the relationship of C4 CN with systemic sclerosis (SSc) risk, we assessed associations with SSc clinical and serologic subtypes. METHODS: We compared imputed C4 CNs across SSc subgroups (4,049 anticentromere positive [ACA+]; 2,200 anti-topoisomerase I [ATA+]; 577 anti-RNA polymerase [ARA+]; 1,078 triple-negative [TN] patients; 6,295 limited cutaneous SSc [lcSSc]; and 2,946 diffuse cutaneous SSc [dcSSc]) and 17,991 controls. We evaluated associations with SSc subtypes, identifying C4-independent HLA alleles. RESULTS: Lower C4 CN and higher HERV-K CN were associated with increased risk in all SSc subgroups. ATA+ patients showed the strongest association, particularly with C4A (odds ratio = 1.88), and differences in C4A CN association were more pronounced between autoantibody subgroups (ATA+ vs ACA+, P = 4 × 10-11) than between clinical subgroups (dcSSc vs lcSSc, P = 1 × 10-4). In ACA+ patients, only low C4B CN showed a significant association to SSc risk (P = 1.23 × 10-5). We also observed sex-biased associations: dcSSc, ATA+, and ARA+ male patients showed stronger effects for C4A and ACA+ and lcSSc female patients for C4B. Finally, our results suggest that the HLA alleles associated with SSc subgroups are independent of C4 CN. CONCLUSION: This study highlights distinct genetic contributions of C4A and C4B in SSc subtypes susceptibility. Our findings suggest that lower C4 CNs, particularly C4A, increase the risk of the severe dcSSc subtype, potentially through a mechanism involving immune complex clearance.
The C5 convertase of the classical complement pathway is a complex enzyme consisting of three complement fragments, C4b, C2a, and C3b. Previous studies have elucidated functional roles of each subunit (4, 6, 7), but little is known about how the subunits associate with each other. In this investigation, we studied the nature of the classical C5 convertase that was assembled on sheep erythrocytes. We found that one of the nascent C3b molecules that had been generated by the C3 convertase directly bound covalently to C4b. C3b bound to the alpha' chain of C4b through an ester bond, which could be cleaved by treatment with hydroxylamine. The ester bond was rather unstable, with a half-life of 7.9 h at pH 7.4 and 37 degrees C. Formation of the C4b-C3b dimer is quite efficient; e.g., 54% of the cell-bound C3b was associated with C4b when 25,000 molecules of C4b and 12,000 molecules of C3b were present per cell. Kinetic analysis also showed the efficient formation of the C4b-C3b dimer; the rate of dimer formation was similar to or even faster than that of cell-bound monomeric C3b molecules. These results indicate that C4b is a highly reactive acceptor molecule for nascent C3b. High-affinity C5-binding sites with an association constant of 2.1 X 10(8) L/M were demonstrated on C4b-C3b dimer-bearing sheep erythrocytes, EAC43 cells. The number of high-affinity C5-binding sites coincided with the number of C4b-C3b dimers, but not with the total number of cell-bound C3b molecules. Anti-C4 antibodies caused 80% inhibition of the binding of C5 to EAC43 cells. These results suggest that only C4b-associated C3b serves as a high-affinity C5 binding site. EAC14 cells had a small amount of high-affinity C5 binding sites with an association constant of 8.1 X 10(7) L/M, 100 molecules of bound C4b being necessary for 1 binding site. In accordance with the hypothesis that C4b-associated C4b might also serve as a high-affinity C5-binding site, a small amount of C4b-C4b dimer was detected on EAC14 cells by SDS-PAGE analysis. Taken together, these observations indicate that the high-affinity binding of C5 is probably divalent, in that C5 recognizes both protomers in the dimers. The high-affinity binding may allow selective binding of C5 to the convertase in spite of surrounding monomeric C3b molecules.
HLA-Bw47, a rare human histocompatibility antigen, occurs in strong linkage disequilibrium with HLA-A3,Cw6,DR7 and salt-losing congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency and is associated with a contiguous deletion of the active CA21HB gene and the C4B complement gene. We studied the HLA-A3,Cw6,Bw47,DR7 haplotype in 10 subjects of the Old Order Amish of Lancaster County in Pennsylvania and found that this haplotype, which occurs with a similar frequency in this group as in the general caucasoid population, has C4B and Ca21HB genes. These C4B and CA21HB genes are expressed as assessed by C4 typing and iv ACTH testing, respectively. Serological studies indicate that the HLA-D loci of this Amish haplotype are the same as those in patients with HLA-Bw47 and CAH, but different from HLA-D loci coupled to HLA-B13, which some workers have proposed is the progenitor genotype for HLA-Bw47. Our studies demonstrate that 1) HLA-Bw47 is not an invariant marker for salt-losing CAH due to 21-hydroxylase deficiency, and 2) the HLA-Bw47 phenotype coupled to CAH is not derived from the HLA-B13 genotype by a single mutation.
The complement fragments C3b and C4b are the main ligands for the membrane receptor CR1. We showed elsewhere that CR1 functions as an essential cofactor for the factor I-mediated enzymatic breakdown of membrane-bound C3b (*C3b) into C3c and * C3dg . One of the main findings of the present paper is that CR1 also promotes the degradation of bound C4b (*C4b) into C4c and *C4d. On a weight basis, the cofactor activity of CR1 in the cleavage of *C4b present on the cell intermediate EAC14 is 10(3)-fold greater than that of the serum cofactor C4-binding protein ( C4bp ). An additional finding is that the effect of CR1 on either *C3b or *C4b is modulated by the presence of the other ligand in its vicinity; that is, *C4b degradation by CR1 plus I is enhanced by neighboring *C3b and vice versa. For example, upon uptake of optimal amounts of *C3b onto EAC142 and the assembly of the C3-convertase EAC1423 , the activity of CR1 in generating C4c is enhanced 5-10 times further. Conversely, when the number of *C3b molecules on EAC1423 is relatively small (or when EAC1423 has been converted by I plus H into EAC1423i ), the presence of neighboring *C4b enhances the conversion of *C3b (or *iC3b) into C3c plus * C3dg . The enhancing effect of *C3b on the cleavage of *C4b by I is observed only if the cofactor of this reaction is CR1. Indeed, the activity of I or I plus C4bp on *C4b is significantly inhibited when *C3b is fixed and the main product of the reaction is * iC4b . Taken together, these findings suggest that degradation of *C4b will be more effective when enough C3b molecules are fixed nearby, thus facilitating the interaction of *C4b*3b clusters with CR1-bearing cells, and that under physiological conditions, *C4b activity can be efficiently controlled by CR1.
C4b-binding protein (C4b-BP) is a high molecular weight plasma protein which inhibits the activity of the classical complement pathway C3 convertase. In addition to multiple binding sites for C4b, C4b-BP possesses a single binding site for vitamin K-dependent protein S, an inhibitor of blood coagulation. As protein S bound to C4b-BP has no anticoagulant activity, C4b-BP participates in the regulation of both the complement and the coagulation pathways. We have produced and immunochemically characterized a series of murine monoclonal antibodies to human C4b-BP. A mixture of four monoclonal antibodies precipitating C4b-BP both in agarose gel and in solution was used to develop a highly reproducible radial immunodiffusion method for the measurement of C4b-BP in human serum. C4b-BP levels were measured in sera from 284 patients referred to our central laboratory. Samples from subjects with an increased erythrocyte sedimentation rate (ESR), a1-acid glycoprotein (a1-AGP) or C-reactive protein (CRP) had significantly higher C4b-BP levels (307 mg/l, 292-322 mg/l, geometric mean and 95% confidence limits of the mean) than those from subjects without elevation of the aforementioned established acute phase reactants (231 mg/l, 226-237 mg/l, P less than 0.00001). C4b-BP was significantly (P less than 0.001) correlated with ESR (r = 0.715), a1-AGP (r = 0.692) and CRP (r = 0.567). There was no gender-related difference in C4b-BP levels. In subjects with no increased acute phase reactants there was a significant correlation between C4b-BP levels and age (r = 0.387, P less than 0.001). High C4b-BP might contribute to the increased thrombotic risk associated with inflammation and aging.
A previous study reported an unexpected increased frequency of the uncommon C4B2 allele of complement in a group of patients with senile dementia of the Alzheimer type. We compared the frequency of various C4B types in 25 patients with familial Alzheimer dementia (AD), 22 patients with sporadic AD, and 360 control individuals. Neither Alzheimer group showed any significant increase or decrease in the frequency of C4B alleles compared with controls. We were unable to confirm any association between AD and the C4B2 allele.
The HLA haplotype A3-Cw6-B47-C4A91-BQ0-DR7 is associated with congenital adrenal hyperplasia (CAH), since it only carries the dysfunctional steroid 21-hydroxylase A pseudogene as well as the 5' adjacent complement C4A gene. The recombination site leading to the deletion of the complement C4B and steroid 21-hydroxylase B genes in this haplotype was studied by determining the 21-hydroxylase genomic DNA sequence in comparison to the standard CYP21A- and CYP21B-specific sequences. A 200-bp region between exons 7 and 8 was identified as a possible recombination site. Thus the deleted area comprises the 3' end of the CYP21A pseudogene, the entire C4B gene and the 5' end of the CYP21B gene. The findings were confirmed by PCR amplification of a 1.8-kb fragment of the CYP21 gene. This PCR system is specific for CYP21A/B recombinant genes and may be used for screening among CAH patients carrying this type of deletion.
We determined the 1.8 kb intergenic sequences between the human complement C4B gene and the active steroid 21-hydroxylase gene in two subjects, and between the C4A gene and the steroid 21-hydroxylase pseudogene in one subject. Comparison of these sequences with each other and with published homologues revealed no differences which were unique to either intergenic region. Sequence analysis revealed two copies of an AGGTCA motif in all sequences. This motif is common to steroidogenic enzyme gene promoters and to the response elements for nuclear hormone receptors. Similarities with human enhancers were also found.
Using newly developed techniques, we investigated the complement pathways and the extent of their activation in patients with systemic sclerosis (SSc) of both the diffuse cutaneous and limited cutaneous types. Plasma levels of the fragments C3d, C4d, and Ba were measured in patients with SSc and in matched control subjects. All fragments and ratios were higher in SSc patients than in controls (P less than 0.05), demonstrating that complement activation occurs in SSc. Levels of C3d, C3d:C3, Ba, and Ba:factor B were higher in patients with diffuse cutaneous SSc patients than in controls (P less than 0.01). C3d, C3d:C3, C4d, and C4d:C4 levels were also higher in patients with limited cutaneous SSc than in controls (P less than 0.05). These results show that complement activation occurs in SSc patients and that it reflects clinical severity. Complement activation may therefore have a pathogenetic role in SSc, and its measurement may prove useful in monitoring the disease.