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PubMedDate not supplied

Complement polymorphism in Greece.

The polymorphisms of the complement components C2, C3, C4 and BF have been studied in a sample of 166 unrelated individuals from Northern Greece. The C3*F and BF*F allele frequencies of Greeks are within the range of frequencies reported from Europe. A single individual with a rare heterozygote variant C2C/C2A was found in Greeks. This C2*A allele was found for the first time in European Caucasoids. For the C4 system six different alleles were found at both C4A and C4B loci. There were a low frequency of the null alleles at the C4A locus and a relatively high incidence of gene duplications in this system.

Complement C2↗
PubMed2001

Improved method for the allelic definition of C4A and C4B polymorphism (HLA class III).

The polymorphism of the fourth component of human serum complement (C4) is well established at the proteinic level; at the DNA level in the analysis of C4A and C4B gene polymorphism, the PCR technique is not widely and routinely used because it is time consuming and still presents reproducibility problems. This is a serious problem because only PCR genotyping allows the establishment of Rodgers-Chido reverse antigenicity without the need for classical family segregation studies, whose samples are not always easy to obtain. The most commonly used protocol requires an initial PCR followed by nested amplification of all the products supposed positive or negative. The two reactions are set up using differing cycling conditions, primers, and magnesium chloride concentrations. We developed a simplified procedure to easily obtain reproducible results and used a single protocol for all reactions. Nested PCR is made using only the positive samples, so we decrease the number of samples to handle, the time spent for the work, and the reagents used for the reactions. Moreover, we increased the reproducibility of the experiments.

Alleles↗
PubMed1987

Determination of C4b.C4-bp complex formed by the activation of classical complement pathway using an enzyme-linked immunosorbent assay.

We developed a quantitative enzyme-linked immunosorbent assay (ELISA) for the detection of C4b.C4-bp complex by incubating the sample on anti-C4-bp-coated plate and then developing with HRP-labeled anti-C4. The amount of C4b.C4-bp complex, generated in vivo by the interaction of purified C4b with C4-bp or normal human serum with aggregated human IgG, was measured by the ELISA. The complex, however, rapidly decreased in serum by the action of factor I. Six out of the 100 plasma samples from patients with various diseases were found positive in the ELISA. One plasma sample from a patient with SLE showed high level of C4b.C4-bp complex with decreased levels of factor I, C4, C4-bp and CH50. These results suggest that the detection of C4b.C4-bp complex is useful for monitoring the diseases in which the classical pathway activation is expected.

Arthritis, Rheumatoid↗
PubMed1989

The low C5 convertase activity of the C4A6 allotype of human complement component C4.

We have compared the C5-convertase-forming ability of different C4 allotypes, including the C4A6 allotype, which has low haemolytic activity and which has previously been shown to be defective in C5-convertase formation. Recent studies suggest that C4 plays two roles in the formation of the C5 convertase from the C3 convertase. Firstly, C4b acts as the binding site for C3 which, upon cleavage by C2, forms a covalent linkage with the C4b. Secondly, C4b with covalently attached C3b serves to form a high-affinity binding site for C5. Purified allotypes C4A3, C4B1 and C4A6 were used to compare these two activities of C4. Covalently linked C4b-C3b complexes were formed on sheep erythrocytes with similar efficiency by using C4A3 and C4B1, indicating that the two isotypes behave similarly as acceptors for covalent attachment of C3b. C4A6 showed normal efficiency in this function. However, cells bearing C4b-C3b complexes made from C4A6 contained only a small number of high-affinity binding sites for C5. Therefore a lack of binding of C5 to the C4b C3b complexes is the reason for the inefficient formation of C5 convertase by C4A6. The small number of high-affinity binding sites created, when C4A6 was used, were tested for inhibition by anti-C3 and anti-C4. Anti-C4 did not inhibit C5 binding, whereas anti-C3 did. This suggests that the sites created when C4A6 is used to make C3 convertase may be C3b-C3b dimers, and hence the low haemolytic activity of C4A6 results from the creation of low numbers of alternative-pathway C5-convertase sites.

Alleles↗
PubMed2001

Genetics of human complement component C4 and evolution the central MHC.

The two classes of human complement component C4 proteins C4A and C4B manifest differential chemical reactivities and binding affinities towards target surfaces and complement receptor CR1. There are multiple, polymorphic allotypes of C4A and C4B proteins. A complex multiplication pattern of C4A and C4B genes with variations in gene size, gene dosage and flanking genes exists in the population. This is probably driven by the selection pressure to respond to a great variety of parasites efficiently and effectively, which the bony fish achieved through the multiplication and diversification of the related complement C3 proteins. Complement C4, C3 and C5 belong to the alpha2 macroglobulin protein family but acquired specific features that include an anaphylatoxin domain, a netrin (NTR) domain, and stretches of basic residues for proteolytic processings to form multiple chain structures. Complement C3 and C4 are important in the innate immune response as they opsonize parasites for phagocytosis. The emergence of complement C3 predates proteins involved in the adaptive immune response as C3 is present in deuterostome invertebrates such as echinoderms. The human C4 genes are located in the central MHC at chromosome 6p21.3. C3 and C5 are located at chromosome 19 and 9, respectively, with representatives of the other groups of genes paralogous to the MHC at 19p13.1-p13.3, 1q21-25, and 9q33-34. The central MHC also contains genes for complement components C2 and Bf. These genes appear to have similar evolutionary histories to C3/C4/C5 and are used here to illustrate stepwise processes resulting in co-location of diverse domains, chromosomal duplication, local segmental duplication and divergence of sequence and function. This model of evolution is useful in the investigation of innate and acquired immunity and in seeking explanations for diseases associated with MHC ancestral haplotypes.

Amino Acid Sequence↗
PubMed1992

The relative roles of C4A and C4B in prevention of immune precipitation, solubilisation and immune adherence.

C4A and C4B levels were measured in serum from 246 normal individuals. Complement-mediated solubilisation, assayed using alkaline phosphatase anti-alkaline phosphatase immune complexes (IC), correlated with both C4A and C4B levels. However, C4A and C4B levels showed no correlation with solubilisation of bovine serum albumin (BSA) ICs, or with the prevention of immune precipitation of BSA or alkaline phosphatase ICs, nor with immune adherence assayed using thyroglobulin and BSA ICs.

Antigen-Antibody Complex↗
PubMed2006

The Kaposi's sarcoma-associated herpesvirus complement control protein (KCP) binds to heparin and cell surfaces via positively charged amino acids in CCP1-2.

The Kaposi's sarcoma-associated herpesvirus (KSHV) complement control protein (KCP) inhibits the human complement system, and is similar in structure and function to endogenous complement inhibitors. Other inhibitors such as C4b-binding protein and factor H, as well as the viral homologue vaccinia virus complement control protein are known to bind heparin and, for the two latter, also to glycosaminoglycans at the surface of cells. We report here that KCP also binds to heparin at physiological ionic strength. With help of site directed mutagenesis, positively charged amino acids in the two N-terminal complement control protein (CCP) domains 1-2 were found to be necessary for heparin binding. In silico molecular docking of heparin to KCP confirmed the experimental data, and further explored the heparin binding site, enabling us to present a model of the KCP-heparin interaction. Furthermore, the docking analysis also yielded insights of the KCP structure, by indicating that the angle between CCP domains 1-2 during the initial binding of heparin is more extended than in the model we have previously presented. We also found that KCP binds to heparan sulfate and weakly to glycosaminoglycans at the surface of cells. This might indicate that KCP at the surface of viral particles aids in the primary attachment to the target cells, which is known to involve binding to heparan sulfate. Therefore, the present study contributes to the knowledge of heparin-protein interactions in general as well as to the understanding of the biology of KSHV.

Amino Acids, Basic↗
PubMed1989

Molecular genetics of C4B deficiency in IgA nephropathy.

The fourth component of complement (C4) occurs in two functionally distinct isotypes, C4A and C4B. The two closely linked genes are located on chromosome 6p, between HLA-B and -DR. Several reports have established complete C4B deficiency as the major genetic risk factor for IgA nephropathy (RR = 6.5; p = 0.0004). It is not clear whether this association derives from immune dysfunction related to the absent isotype or from another disease susceptibility gene closely linked to C4B. To help distinguish between these mechanisms, we examined the molecular basis of complete C4B deficiency in five patients with IgA nephropathy and eight healthy individuals. C4 and Bf protein typing were performed by immunofixation electrophoresis of plasma. Genomic DNA was digested with several restriction enzymes, chosen to produce informative restriction fragment length polymorphisms (RFLPs). After electrophoresis and Southern blotting, digests were hybridized to a series of cDNA probes specific to the 5' and 3' ends of the C4 genes, the C4d region, and the adjacent 21-hydroxylase genes. Availability of DNA from family members allowed assignment of RFLPs to specific haplotypes. The 10 C4B-deficient IgA nephropathy-associated haplotypes displayed seven different protein phenotype/RFLP patterns. Three haplotypes consisted of the common C4B/21-hydroxylase deletion on the Bf*S, C4A*3, C4B*Q0 complotype. Two haplotypes were characterized by the C4A*3,2 duplication, with two C4 genes present but a C4A protein being produced by the gene at the usual C4B locus. All of the remaining haplotypes had unique Bf, C4, and 21-hydroxylase patterns. C4B-deficient IgA nephropathy patients display a variety of molecular genetic bases for their protein deficiency. This observation speaks against linkage of C4B deficiency with a locus encoding disease susceptibility and supports a primary role for the complement abnormality in this disease.

Child↗
PubMed1991

Decreased inhibition of immune precipitation by sera with the C2 B allotype.

Complement-mediated precipitation inhibiting (CMPI) activity of sera of 5 individuals homozygous for C2 B was compared to that of sera of 20 individuals carrying the common C2 C allotype. Sera with the rare C2 B allotype had a depressed CMPI capacity in both the early (5 min) and the late (60 min) stages of the reaction. We have also compared the CMPI activity of seven homozygous C4A deficient (C4A*Q0) and eight C4B deficient (C4B*Q0) serum samples and did not find significant differences from the controls (no C4 null alleles) in any stage of the reaction. These results indicate that C2 is the critical component in the CMPI reaction of the two constituents of the classical pathway C3 convertase and that C2 B is less active than C2 C.

Antigen-Antibody Reactions↗
PubMed1994

Inhibition of complement activation by natural sulfated polysaccharides (fucans) from brown seaweed.

In the present study, we demonstrate that natural sulfated polysaccharides (fucans) isolated from brown seaweed are potent inhibitors of human complement activation. A fucan fraction of chromatographic molecular weight 22,600, termed BS8, was found to inhibit classical and alternative pathway activation in whole serum in a dose-dependent fashion. Fucan BS8 inhibited formation of the classical pathway C3 convertase by interfering with C1 activation or by inhibiting C4 cleavage and the interaction between C4b and C2. The fucan also inhibited formation/function of the alternative pathway C3 convertase by suppressing the binding of B to C3b and by interfering with the stabilizing function of Properdin. The inhibitory effect of fucans on formation of the C3 convertases was dependent on the molecular weight of the polysaccharide for compounds of chromatographic molecular weight below 16,600. Fucan had no effect on the function of the terminal complex. Since fucans were more efficient than heparin in inhibiting activation of the classical pathway in whole serum and exhibited a lesser specific anticoagulant activity on a molar basis, our results suggest that these natural sulfated polysaccharides have a potential for use as anti-complementary and anti-inflammatory agents.

Complement Activation↗
PubMed1984

The structural basis of the multiple forms of human complement component C4.

cDNA clones of human complement components C4A and C4B alleles were prepared from mRNA obtained from the liver of a donor heterozygous at both loci. cDNA from one C4A allele was sequenced to give the derived complete amino acid sequence of 1722 amino acid residues of the C4 single chain precursor molecule and the estimated sequences of the three peptide chains of secreted C4. Comparison with partial sequences of a second C4A allele and a C4B allele has led to the tentative identification of some class differences in nucleotide sequences between C4A and C4B and of allelic differences between C4A alleles in this highly polymorphic system.

Amino Acid Sequence↗
PubMed1988

Epstein-Barr virus regulates activation and processing of the third component of complement.

Serum incubated with purified EBV was found to contain C3 cleavage fragments characteristic of C3c. Since the cofactors necessary for such cleavage of C3b by factor I are not normally present in serum, EBV was tested for factor I cofactor activity. Purified EBV from both human and marmoset EBV-producing cell lines was found to act as a cofactor for the factor I-mediated breakdown C3b to iC3b and iC3b to C3c and C3dg. EBV also acted as a cofactor for the factor I-mediated cleavage of C4b to iC4b and iC4b to C4c and C4d. EBV from both the human and marmoset cell lines accelerated the decay of the alternative pathway C3 convertase. The classical pathway C3 convertase was unaffected. Multiple lines of evidence eliminated the possibility that marmoset or human CR1 was responsible for the functional activities of EBV preparations. The spectrum of activities was different from CR1 in that EBV and EBV-expressing cell lines failed to rosette with C3b or particles bearing C3b, the primary functional assay for CR1, and EBV did not accelerate classical pathway C3 convertase decay, another property of CR1. In addition, CR1 could not be detected immunologically on marmoset or human EBV-expressing cells and mAbs to CR1 failed to alter EBV-produced decay acceleration and factor I cofactor activities, although the antibodies blocked the same CR1-dependent functional activities. The multiple complement regulatory activities exhibited by purified EBV derived from human and marmoset cells differ from those of any of the known C3 or C4 regulatory proteins. These various activities would be anticipated to provide survival value for the virus by subverting complement- and cell-dependent host defense mechanisms.

Animals↗
PubMedDate not supplied

Cryopreservation of complement-coated erythrocytes.

Cells coated with complement components (C3b-C4b and C4b cells) were prepared by various methods and stored in liquid nitrogen using a low-glycerol, rapid freeze technique. Freshly coated cells, and cells coated and then frozen were tested versus various antiglobulin and anticomplement reagents to evaluate the reactivity of such frozen, stored, complement-coated human red blood cells. Liquid nitrogen preservation of such coated cells proved to be feasible when such cells were compared with freshly coated control cells.

Animals↗
PubMed1999

Complement and Reactants of Acute Phase of Inflammation in the Processes of Functional Activity of Non-Specific Resistance and Immunoregulation.

Complement system is a family comprising of 20 plasma and membrane proteins, acting in concord by cascade principle. The investigators display great interest to C4b-binding protein (C4bp) which is the main regulatory protein of complement system, regulating of C3 convertase activity in classical way of complement activation. The major regulatory function of C4bp is related to its interaction with activated form of the forth complement component, C4b. C4bp may also interact with one of pentraxins, serum amyloid P component (SAP) that inhibits complement-regulatory functions of C4bp. C4bp forms tight complexes with protein S and SAP. Pentraxin family (proteins with five-ray symmetry, such as C-reactive protein and SAP) became more numerous. Protein PTX3, synthesized by endothelial cells, macrophages and leukocytes, and considered to be local regulator of reaction of non-specific resistance in tissues; serum acute phase protein TSG-14; neuronal pentraxin - protein of cerebellum, hippocampus and brain cortex neurons, binding typoxin of snake venom; Narp (neuronal activity-regulated pentraxin) protein; apexin, acrosomal protein of spermatozoa; female hamster protein; pentraxin XL-PXN1 from Xenopus laevis - are all examples of new pentraxins. Pro-inflammatory cytokines, IL-1 and IL-6, may induce expression of pentraxin genes. Pentraxins are closely integrated not only to non-specific resistance, but to the system of immunoregulation and take part in its key events.

Journal Article↗
PubMed1984

The effects of iodine and thiol-blocking reagents on complement component C2 and on the assembly of the classical-pathway C3 convertase.

I2 can react with complement component C2 in a two-stage process. In the first stage, a form of C2 with enhanced haemolytic activity is produced. This form of C2 is cleaved to C2a and C2b by C1s at the same rate as native C2. The enhanced C2 haemolytic activity correlates with the ability to form a stable fluid-phase C3 convertase on addition of the C2 to C4b and C1s. It reflects an increased affinity for C4b of C2a formed from I2-treated C2, although the affinity for C4b of I2-treated C2 itself is not markedly increased. The specific activity of C3 convertase formed from I2-treated C2 is the same as that formed from native C2. The second stage of the reaction with I2, which is favoured at high pH or in the presence of excess I2, inactivates C2 on production of a species that cannot be cleaved by C1s. The presence of a single free thiol group in C2, which is the site of modification by I2, was confirmed by titration with p-chloromercuribenzoate, iodoacetamide and 5,5'-dithiobis-(2-nitrobenzoic acid). A single thiol group is also present in Factor B, and the cysteine residue, like that in C2, requires denaturation of the protein before reaction with iodoacetamide and 5,5'-dithiobis-(2-nitrobenzoic acid) but not p-chloro- mercuribenzoate .

Chloromercuribenzoates↗
PubMed1986

Evidence that a nicked C4b, C4b', is a functionally active C4b derivative.

Factor I-catalyzed C4b cleavage is a regulatory reaction for the classical pathway of the complement system. Although the reaction was shown to be a two-step reaction, production of a nicked form of C4b, C4b', as an intermediate cleavage product and subsequent splitting of C4b' into C4c and C4d, it is not known which of the two steps represents the inactivation of the C4b function in the assembly of C3 convertase, C4b,2a. We have purified C4b' and assessed the ability of C4b' to assemble C3 convertase with C2 by utilizing size exclusion high performance liquid chromatography. Evidence was obtained demonstrating that C4b' still retains the function of C4b to assemble C3 convertase. Thus, the substantial step for the inactivation of the C4b function appears to be the second cleavage reaction, that is, the cleavage of C4b' into C4c and C4d.

Catalysis↗
PubMed1996

Mouse complement component C4 is devoid of classical pathway C5 convertase subunit activity.

It has long been known that mouse C4 has unusually low hemolytic activity relative to the C4 of other mammalian species (e.g. human and guinea pig), the measurements being done in most cases using a C4-deficient guinea pig serum reagent in a one-step assay with EA. This low activity for mouse C4 previously had been attributed to "technical" difficulties such as lability of the protein during blood collection and partial species incompatibilities with guinea pig components. Recently, we presented evidence for the involvement of human C4 beta-chain residues 455-469, a putatively exposed hydrophilic segment, in contributing to a C5 binding site in the C4b subunit of the classical pathway C5 convertase, C4b3b2a. Given that there were five sequence differences between the human and mouse protein within this segment, we hypothesized that these substitutions may have compromised the C5 convertase subunit activity of mouse C4, thereby resulting in its low hemolytic activity. Using a multi-step hemolytic assay which was totally dependent upon C5 cleavage by the classical pathway, we found that mouse C4 was completely devoid of classical pathway C5 convertase subunit activity. We have been able to rule out the most obvious potential species incompatibilities (e.g. between C4mo and C5gp) as being responsible for this lack of activity. Moreover, we found that the low level of hemolytic activity of mouse C4 measured in the one-step assay can be ascribed totally to C5 cleavage, and subsequent terminal component assembly, by the alternative pathway C5 convertase, (C3b)2Bb. However, the assembly of the latter enzyme complex is dependent upon the presence of C3b molecules deposited initially via the classical pathway C3 convertase in which mouse C4b is a subunit. Finally, whereas conversion of human residues 458RP to the mouse-like sequence PL was sufficient to abrogate classical pathway C5 convertase subunit activity in human C4, the five substitutions which "humanized" the 452-466 segment of mouse C4 (corresponding to human residues 455-469) were on their own insufficient to impart this activity to mouse C4. This implies that, in addition to the 455-469 beta-chain segment of human C4, there are other regions of the molecule contributing to C5 binding which are also non-conserved between human and mouse C4.

Amino Acid Sequence↗
PubMed1996

Major histocompatibility complex class II and complement polymorphisms in postpartum thyroiditis.

The objective was to re-evaluate the association between class II HLA-DR and DQ MIIC antigens and postpartum thyroiditis (PPT) and to determine the prevalence of the class III complement allotypes of Properdin factor B (Bf), C4A and C4B in this condition. Two hundred and sixty-five (of 2897) pregnant women screened positive for thyroid autoantibody activity took part. Further blood samples were obtained for HLA class II (185) and complement (193) typing. The severity of the ensuing PPT was assessed by measuring thyroid function during the postpartum year. The HLA-DR and DQ phenotypes were assigned from restriction fragment length polymorphism analysis, and Bf, C4A and C4B allotypes were determined by immunofixation with anti-Bf or anti-C4 antibodies after electrophoresis. A weak association between the HLA class II antigens and PPT, as indicated by a reduced frequency of DR15 and DQ6 together with an increased frequency of-DR5 and DQ7, was confirmed. However, only the change in DR5 frequency remained significant after correction (corrected p < 0.05). Postpartum thyroiditis was also associated with frequency disturbances in BI and C4A allotypes but not C4B allotypes. Whilst this study has not provided evidence of a strong marker gene for PPT, it does not preclude the involvement of the MIIC in this condition. These data show disturbances in complement allotype frequencies, suggesting that the class III region may provide a useful focus for further study of this pathology.

Complement C4a↗