C4b-binding protein: an update.
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Carrier Proteins↗
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Genetic variability of the MHC class III complement proteins C2, BF, C4A and C4B in southern Brazil.
We describe here the genetic variability of C2, C4 and BF in 225 healthy adult individuals from southern Brazil, including 172 Caucasoids, 47 Mulattos, 3 Blacks and 1 Amerindian. C2 gene frequencies were in accordance with those described for other populations, and two rare variants were observed. The BF allotype frequencies were slightly different between the Caucasoids and Mulattos, the latter having a higher BF*F frequency. A new BF*S variant, identified as S05 was observed in a Caucasoid individual. The frequencies of C4A and C4B in the Caucasoids were similar to other reported Caucasoid populations; a decrease of the silent allele B*Q0, and several rare variants were observed. A higher C4A*3 frequency and a remarkable decrease of C4A*Q0 were observed in the Mulattos. In addition, several C4 heteroduplications and aberrant allotypes were observed. Considering the high genetic variability found in a limited number of individuals, one may conclude that due to genetic admixture much heterogeneity might be expected for the MHC class III region in different Brazilian populations.
Adolescent↗
C4 phenotypic variation suggests an unusual class III gene organization.
The class III gene markers, C4A, C4B, C2 and BF were determined in 26 HLA genotyped families. In 6 of these families the inheritance of the C4A electrophoretic phenotype, C4A 3, A 2 could not be explained assuming the genetic model that the C4A component is controlled by only one locus (C4A) and suggests that both C4A 3 and C4A 2 are encoded by the same chromosome. This apparently duplicated C4A locus haplotype is common in Northern Europeans occurring at a frequency of at least 6%. In 4 families the C4A 3, A 2 'variant' occurred together with HLA-Bw35 and in 2 together with HLA-Bw55. Furthermore, it was striking that in each family the C4A 3, A 2 'variant' was found exclusively with the complotype FC3/20 which encodes no phenotypically expressed C4B locus product.
Alleles↗
DNA polymorphism unique for a complotype with deletion of HLA-linked C4B and 21-hydroxylase B genes causing congenital adrenal hyperplasia.
Defects in the enzyme steroid 21-hydroxylase (21-OH) result in congenital adrenal hyperplasia (CAH), a frequent disorder of steroid biosynthesis. The gene encoding the enzyme, 21-OHB, has been mapped adjacent to the complement component C4B gene in the human HLA gene complex. DNA-level analyses of patients with CAH have shown that the 21-OHB gene has often been deleted, but the detection of 21-OHB deletions in heterozygotes is often problematic because it is based on relative band intensities. We here report a DNA polymorphism in the C4A91 gene unique to one particular type of 21-OHB deletion occurring solely with a complement phenotype BfF C4A91 B null, shown earlier to be frequent in CAH patients. This marker makes direct detection of the 21-OHB deletion in heterozygotes possible.
Adrenal Hyperplasia, Congenital↗
Abnormally high level of C4b binding protein and deficiency of free fraction of protein S in a patient with systemic lupus erythematosus and recurrent thromboses.
A patient with systemic lupus erythematosus and numerous thrombotic accidents was found to have a deficiency of the free fraction of protein S, most likely due to an abnormally high level of C4b binding protein. These abnormalities, unique in comparison to 4 other patients with hypercoagulation states, were partially corrected with cyclophosphamide. There was no evidence of a consumption of complement components. The interactions between the hemostasis system and the complement cascade are discussed.
Adult↗
Protein S and C4b-binding protein levels in patients with stroke: implications for protein S regulation.
Protein S circulates either free or bound to C4b-binding protein (C4b-BP). Only free protein S possesses cofactor activity for protein C, a physiologic anticoagulant. Deficiencies of either protein C or protein S are associated with increased thrombotic risk. Over a 23-month period, 40 patients with low free protein S were identified. Eight of these patients were found to have suffered a stroke. This study examined the relationship between total S, free S, and C4b-BP in 15 healthy adult volunteers, in 20 patients with normal protein S levels, in 40 patients with decreased free protein S levels, and in 8 patients with combined low free S levels and stroke. Total and free protein S and C4b-BP levels were determined using the method of Laurell. In healthy adults, free protein S increased with increasing total protein S (r = 0.60). In patients with normal free S, the total S level increased as C4b-BP increased (r = 0.74), and the free S level remained constant. In patients with low free S, total S did not increase with increasing C4b-BP. In stroke patients, the correlation between free S and total S was actually negative (r = -0.449). Evaluation of dissociation constants for the protein S-C4b-BP complex revealed enhanced binding in patients with low levels of free protein S. A non-C4b-BP protein S binding protein, a previously undescribed regulatory factor which modulates S binding to C4b-BP, or shifts in the amount of non-protein S binding C4b-BP are possible explanations of these results.
Adult↗
Novel androgen-dependent promoters direct expression of the C4b-binding protein alpha-chain gene in epididymis.
C4b-binding protein (C4BP) is a large plasma protein composed of seven alpha-chains and one beta-chain and is involved in the fluid phase regulation of the classical pathway of the complement system. Complement inhibitory activity is located in the alpha-chain, and its mRNA has been detected only in liver to date. Here, we have isolated cDNA clones encoding the alpha-chain of guinea pig C4BP (C4BP alpha) and have demonstrated significant C4BP alpha mRNA expression in epididymis as well as liver. The level of C4BP alpha transcripts increased in the epididymis after birth, while it remained constant in the liver. C4BP alpha mRNA was also detected in the normal murine epididymis at a significant level, but it decreased drastically after castration, suggesting that epididymal expression of the C4BP alpha gene is regulated by androgen. Gene analysis of guinea pig C4BP alpha indicated that liver and epididymis C4BP alpha mRNA share the coding region and 3'-untranslated region, but are transcribed from independent promoters on a single-copy gene. Two novel epididymis-specific promoters were identified in the region corresponding to the first intron of liver transcripts. The binding motif for hepatocyte NF-1 occurs in the promoter used for transcription of liver C4BP alpha, whereas androgen-responsive elements occur in both promoters used in the epididymis. These findings present a novel link between complement regulators and reproduction. Furthermore, variation in the 5'-untranslated regions, arising from alternative splicing of the newly identified exons, is demonstrable in the guinea pig C4BP alpha transcripts.
3' Untranslated Regions↗
Phylogeny of C4b-C3b cleaving activity: similar fragmentation patterns of human C4b and C3b produced by lower animals.
Functional and structural studies of the activated proteins of the complement system C4b and C3b have led to the identification of cleavage products resulting from the effect of the regulatory proteins, factor I, H, and C4b binding protein (bp). In this paper we report the results of studies that investigated the capacity of plasma or serum from a wide range of phylogenetic species to yield similar cleavage products. Sera and plasma from mammals, reptiles, amphibia, and fishes are capable of cleaving fluid phase human C4b and C3b, generating apparently the same fragments as observed using normal human serum: alpha 2, alpha 3, alpha 4 from the alpha' chain of C4b: and alpha-68, alpha-46, alpha-43, and alpha-30 from the alpha' chain of C3b. When C3b bound to a cell membrane is used C3c and C3dg are generated. The generation of these fragments from C3bi is a dose-dependent reaction. There is no correlation between the evolution of the species and the quantitative capability to degrade the substrates. Birds possess only a limited capability to degrade the alpha' chain of C4b and have no cleaving activity for C3b, whereas sera from more primitive vertebrate species (chondrichthyes and agnatha) fail to participate in the reaction. Contrary to other species, the proteins in fish serum or plasma responsible for the degradation of C4b and C3b show a unique requirement for Ca2+ ions. Magnesium and barium are less effective, and in their presence a 65,000 dalton intermediate product is observed. These results demonstrate that protein(s) displaying proteolytic activity for products of complement activation, probably related to I, H, and C4bp, are present in plasma of species whose evolution have preceded humans by 300 million years. Moreover, the recognition of human substrates and the generation of fragments identical to those produced by human serum suggests that human C4b and C3b share structural characteristics with their evolutionary ancestors in the serum or plasma of the species studied.
Animals↗
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.
Antibodies, Monoclonal↗
Flow cytometric assay for phagocytosis of human monocytes mediated via Fc gamma-receptors and complement receptor CR1 (CD35).
We developed a simple flow cytometric assay for phagocytosis by human monocytes that is mediated via Fc gamma receptors and the complement receptor CR1 (CD35), using fluorescent latex beads carrying IgG and complement components C4b and C3b. To prepare fluorescent latex beads carrying IgG(BA), BSA-coated latex beads (B) were incubated with diluted rabbit anti-BSA IgG. To bind complement components, BA-particles were incubated with whole human serum pretreated with K-76 monocarboxylic acid (K-76COOH). K-76COOH inhibits the activities of C5 and factor I (12,13), resulting in the deposition of C1,4b,2a,3b on BA-particles (BAC1,4b,2a,3b). Further incubation of BAC1,4b,2a,3b with EDTA-GVB at 37 degrees C gave particles carrying IgG and C4b,C3b (BAC4b,3b). The C3 fragment, C3b, was confirmed to present on BAC1,4a,2a,3b particles by SDS-PAGE and immunoblot, and these particles were calculated to have approximately 25,000-30,000 C3b molecules per particle. To evaluate the particle attachment, the phagocytic assay was performed with 3 microM cytochalasin D treated cells. The percent cells with ingested particles and the number of ingested particles/100 cells for 60 min were estimated, being 5.1% and 5.4 for B, 12.3% and 26.7 for BA, 42.5% and 108.7 for BAC4b,3b, and 42.6% and 112.5 for BAC1,4b,2a,3b, respectively.
Antigens, Differentiation↗
Immunonephelometric determination of the C4b-binding protein.
A fully mechanised immunonephelometric method for the rapid and specific determination of C4b-binding protein (C4b-BP) in citrated plasma is described. The method utilizes commercially available rabbit antiserum against human C4b-BP and a nephelometer analyser. A single determination can be performed within 6 min, requiring 80 microliters sample volume. The measuring range is about 10 to 200% of normal C4b-BP. Precision is characterized by intraassay coefficients of variation between 1.5% and 2.8%, and interassay coefficients of variation between 4.0% and 4.6% for the same C4b-BP concentrations. The nephelometry of C4b-BP was correlated with electroimmunodiffusion (Laurell technique; r = 0.863, y = 0.909x+7.091, n = 79). C4b-BP concentrations (143%, 96-223%; median and 2.5th-97.5th percentile) from 83 subjects with increased inflammation markers C-reactive protein (> 10 mg/l), and fibrinogen (> 4.5 g/l) showing significantly higher C4b-BP concentrations compared to 151 obviously healthy subjects (97%, 68-141%; p < 0.001). In contrast to 81 patients with therapeutic heparinisation (90%, 60-131%) significant decreased concentrations were found in 90 subjects under oral anticoagulant therapy (OAT) in the stable state (78%, 44-125%; p < 0.001). Depending on different INR levels (< 2.5, n = 40: 71%, 63-85%; > 2.5, n = 50: 81%, 68-92%; median and 25th-75th percentile) no significant differences of C4b-BP concentrations could be measured.
Administration, Oral↗
Hypocomplementaemic urticarial vasculitis, angio-oedema and 'lupus-like' disease: association with C4B null allele.
A patient who presented with urticarial vasculitis, recurrent angio-oedema and gastro-intestinal symptoms in association with a 'lupus-like' disorder is documented. Other 'auto-immune' phenomena such as Sjogren's-like syndrome and polyarthritis were present in association with antibodies to RNP, low C4 and a null allele was demonstrated at the C4B locus. Response to varied therapies was poor and unsustained.
Angioedema↗
Studies of a C2 DNA polymorphism in RA, Felty's and normal subjects.
A restriction fragment length polymorphism at the C2 locus was studied in rheumatoid arthritis (RA), Felty's and control subjects. No association was found between any C2 variant and either RA itself or within the rheumatoid population with Felty's syndrome. The C2 DNA polymorphism can be used to subdivide Bf*S- as well as Bf*F-bearing haplotypes. The 2.65-kb C2 DNA allele showed allelic association with HLA-B44 and C4B*Q0 and may help to further characterize the haplotype B44-Bf*S-C4A*3-C4B*Q0-DR4, which has previously been described in Felty's syndrome.
Alleles↗
Protein S enhances C4b binding protein interaction with neutrophils.
Protein S, an inhibitor of coagulation, interacts reversibly with C4b binding protein (C4bBP). The physiologic role of this complex formation remains unknown. In this study we examined the possibility that protein S would facilitate C4bBP binding to the surface of neutrophils where, in turn, this complex could help protect the cell from complement-mediated damage at the site of inflammation. Neutrophils bind approximately 60,000 protein S molecules per cell (kd = 140 nmol/L) in a Ca(2+)-dependent fashion. C4bBP also binds to neutrophils (23,000 sites per cell at physiologic C4bBP concentration), but this binding is not Ca2+ dependent. Protein S approximately doubled C4bBP binding, but protein S only influenced C4bBP binding in the presence of Ca2+. Protein S binding to neutrophils decreased approximately twofold in the presence of saturating concentrations of C4bBP. Neutrophil activation had only minor effects on the affinity and number of sites for protein S or the protein S-C4bBP complex. These results indicate that the protein S-C4bBP complex binds to the neutrophil surface where it can presumably modulate complement assembly on the cell surface.
Carrier Proteins↗
Some clotting factors in plasma during danazol therapy: free and total protein S, but not C4b-binding protein, are elevated by danazol therapy.
Anabolic steroids are known to increase the plasma concentrations of certain plasma proteins. In four patients given treatment with danazol, an attenuated androgen, the concentrations of heparin cofactor II, Hageman factor (factor XII), protein C, and both free and total protein S increased significantly when tested 39 to 103 days after the start of therapy. The titers of these proteins in samples obtained 21 days to 5 years after therapy was discontinued were similar to those before treatment, except for total protein S, the titer of which remained elevated. No significant changes in the titers of C4b binding protein or plasma plasmin inhibitory activity were found.
Adult↗
Regulation and deregulation of the fluid-phase classical pathway C3 convertase.
Three mechanisms that regulate the formation and function of the fluid-phase classical pathway C3 convertase (C4b2a) have been elucidated: a) a temperature-mediated intrinsic decay of the enzyme; b) an extrinsic accelerated decay mediated by the effect of the serum protein C4b-binding protein (C4-bp); and c) the inactivation of C4b in the C4b-C4b-p complex by the proteolytic action of C3b/C4b inactivator (I), which cleaves the alpha 1-chain of C4b yielding C4d (alpha 2-chain), and C4c (alpha 3-, alpha 4-, beta-, gamma-chains). A fourth mechanism is described based on the observation that the IgG fraction of the serum of certain patients with glomerulonephritis contains a protein that prevents the intrinsic and C4-bp-mediated decay of surface-bound C4b2a. This protein prolongs the half-life of fluid-phase C4b2a from 10 min to more than 5 hr, increasing the utilization of C3. It also inhibits the decay mediated by C4-bp by preventing the dissociation of C2a from the C4b, 2a complex. In addition, I alone or in the presence of C4-bp fails to cleave the alpha 1-chain of C4b in the stabilized C4b, 2a complex. This protective property of the stabilizing factor (NFc) requires the presence of C2a because C4b was not protected unless it was bound to C2a. Therefore, NFc provides a mechanism by which the serum regulatory proteins are bypassed.
Carrier Proteins↗
Human C4 polymorphism: pedigree analysis of qualitative, quantitative, and functional parameters as a basis for phenotype interpretations.
Ten families with 82 members were investigated for C4A- and B polymorphism in a blind trial. Phenotyping was done on neuraminidase treated sera by immunofixation and simultaneously by hemolytic overlay electrophoresis. In addition Rg, Ch, BF, C2, HLA-A, B, C, DR, and GLO were determined. After decoding the samples the reliability of blind typing was found to be 84.4% according to segregation patterns. Inconsistencies occurred mostly when A4, A2, or A92 were present. The detection of silent A*Q0 and B*Q0 alleles was more critical than that of "difficult" allotypes. The quantitation of the C4A/B ratio by densitometry of stained gels or by conventional immunochemical measurements of serum C4 level could not substantially improve the identification of A*Q0 or B*Q0. C4 dependent activity in radial diffusion hemolysis showed satisfactory correspondence with the number of expressed C4B alleles. At least three haplotypes with two C4A genes (duplicated A genes) were observed as ascertained from offspring analysis in accordance with the MHC segregation pattern. Individuals with the duplicated C4A gene (C4A*3, A*2, in the absence of any other expressed A allele or together with C4A*92) showed only partial inhibition of Rodgers antisera. Partial inhibition of Chido antisera was seen in individuals with C4B 2 (in the absence of other B allotypes). The findings support the hypothesis of at least two structural C4 loci. They also demonstrate the inconsistency of quantitative data in the recognition of silent alleles.
Alleles↗
Italian extended HLA haplotypes in congenital adrenal hyperplasia.
In order to complete the data on human 21-Hydroxylase deficiency, we present a study on HLA markers in 35 Italian families (14 from Northern, eight from Central and 13 from Southern Italy) with one affected child. Three children from the issue of first cousin marriages were homozygous for the whole HLA haplotype. Extended haplotypes shared by unrelated patients were not found, and a total absence of the HLA Bw47 allele among the haplotypes carrying the disease as well as normal haplotypes was observed. The absence of A1 Cw7 B8 BfS C4AQ0 C4B1 DR3 extended haplotype was instead confirmed. Allele frequencies in the different clinical forms were analyzed: BfSO7 allele frequency was significantly increased on haplotypes of the salt-wasting form (p less than 0.01). We noticed two duplications (C4B1-2) of C4B genes, on haplotypes involved in the disease. Allele distribution in the regions studied showed that Bw22 (w55), Cw3 and DR2 were characteristic of Northern patients, while B15 was found in patients from Central Italy.
Adrenal Hyperplasia, Congenital↗