Scleroderma: possible significance of silent alleles at the C4B locus.
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Agammaglobulinemia↗
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Results for “Complement C4b”
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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↗
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↗
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↗
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↗
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↗
Site-directed mutagenesis of the region around Cys-241 of complement component C2. Evidence for a C4b binding site.
We probed the functional significance of the region around Cys-241 in human C2 by testing the hemolytic activity of a series of mutant rC2. Mutant C2 cDNA were constructed by oligonucleotide-directed site-specific mutagenesis and expressed transiently in COS cells. Wild-type rC2 had threefold higher specific hemolytic activity than native serum C2. Substitution of Gly, Ala, or Ser for Cys-241 resulted in a slightly, but significantly, increased activity. In addition, I2 had no effect on the activity of these mutant C2. Substitution of Lys for Gln-243 increased the hemolytic activity by more than two-fold. Increased activity in all cases was due to slower decay rates of the C3 convertase. Finally, substitution of Leu or Ala for Asp-240 or Ser-244, respectively, resulted in more than 100-fold decrease of hemolytic activity. The results suggest that residues 240 to 244 of human C2 represent an important structural determinant of the C4b binding site of C2a. They also confirm that Cys-241 is the residue responsible for the increased activity of C2 reacted with I2.
Amino Acid Sequence↗
A flow cytometric assay for measuring complement receptor 1 (CR1) and the complement fragments C3d and C4d on erythrocytes.
A flow cytometric assay (FCA) was developed to measure complement receptor 1 (CR1) and the complement fragments C3d and C4d on erythrocytes. It was possible to measure these parameters accurately with intra- and interassay coefficients of variation of 2.0% and 6.5% respectively. The method was able to discriminate between low and high levels of erythrocyte CR1, C3d and C4d. Comparison with a previously described RIA method gave excellent correlation coefficients with r2 values of 0.94, 0.93 and 0.91 for CR1, C3d and C4d respectively. The flow cytometric assay was used to measure CR1, C3d and C4d on the erythrocytes of 98 healthy individuals and the 95% upper limits for C3d and C4d were established. There was a wide distribution of CR1 levels amongst these individuals but their C3d and C4d levels were low and often not above background. The possible application of this method in clinical medicine is discussed.
Antigens, CD↗
High molecular weight non-immunoglobulin salivary agglutinins (NIA) bind C1Q globular heads and have the potential to activate the first complement component.
Non-Immunoglobulin Salivary Agglutinins (NIA) which directly bind to microbes [including HIV] were studied for their potential to activate the first complement component (C1). It was determined that NIA had the same specific activity as heat aggregated IgG in binding to C1q and in activating C1. In order to determine the region of C1q which bound to NIA, C1q globular heads and C1q stems (collagen-like regions) were prepared and separated via a Western blot procedure. NIA bound principally to the globular heads of C1q and weakly to the collagen-like stem region. NIA were also studied for their potential to activate native C1 in normal human serum. Heat-aggregated IgG and cardiolipin served as positive controls. It was observed that incubation of isolated NIA with fresh normal human serum resulted in the formation of sodium dodecyl sulfate (SDS)-irreversible complexes of activated C1r-C1 inhibitor and activated C1s-C1 inhibitor and in activated C1s mediated C4 conversion. This indicated that isolated NIA had the potential to directly and effectively mediate classical complement pathway activation. Preincubation of NIA with C1q, blocked NIA mediated C1r and C1s activation and C4 conversion. The concn of NIA required to activate C1r and C1s was similar to that of heat-aggregated human IgG. In kinetic ELISA, NIA or aggregated IgG (positive controls) were first immobilized on microtiter plates, blocked with gelatin then incubated with fresh human serum as a source of complement. Depositions of C4b, C3b and iC3b substantiated that the complement system was effectively activated by immobilized NIA. The optimal relative NaCl concn for C4b deposition was 0.11 M. While pre-incubation of NIA with C1q blocked the subsequent C1 fixing potential of NIA, pre-incubation of NIA with rgp160 [HIV-1] or fibronectin did not interfere with the potential of NIA to fix C1.
Agglutinins↗
Effects of LDL-apheresis on serum lipoprotein (a), C4b binding protein, protein C, protein S, and complement components.
The short-term effects of low-density lipoprotein (LDL) apheresis using a dextran sulfate-cellulose (DSC) column equipped with a plasma separator using a polysulfone (PS) membrane filter on the serum total cholesterol, lipoprotein(a) (Lp(a)), C4b binding protein (C4bp), protein C and protein S and complement components levels were examined in a patient with familial hypercholesterolemia (heterozygote, type IIa). PS/DSC-LDL apheresis markedly lowered the total cholesterol by 69.9 +/- 2.9%, serum Lp(a) level by 80.2 +/- 3.4%, and C4bp by 81.1 +/- 2.5% after 8 apheresis sessions. All the above parameters gradually returned to the baseline levels within 10 days. The free protein S level was not significantly changed, while the C4bp/protein S complex level was markedly decreased relative to the decrease in C4bp. The protein C level was moderately reduced by 29%. Almost all serum complements and complement co-factors as well as C4bp were moderately to markedly decreased after LDL apheresis, but returned to the initial levels within a few days. PS/DSC-LDL apheresis effectively eliminated both serum LDL and Lp(a), and did not have an adverse effect on fibrinolysis or complement cascade in the blood.
Adult↗
Binding of C4b-binding protein to porin: a molecular mechanism of serum resistance of Neisseria gonorrhoeae.
We screened 29 strains of Neisseria gonorrhoeae and found 16/21 strains that resisted killing by normal human serum and 0/8 serum sensitive strains that bound the complement regulator, C4b-binding protein (C4bp). Microbial surface-bound C4bp demonstrated cofactor activity. We constructed gonococcal strains with hybrid porin (Por) molecules derived from each of the major serogroups (Por1A and Por1B) of N. gonorrhoeae, and showed that the loop 1 of Por1A is required for C4bp binding. Por1B loops 5 and 7 of serum-resistant gonococci together formed a negatively charged C4bp-binding domain. C4bp-Por1B interactions were ionic in nature (inhibited by high salt or by heparin), whereas the C4bp-Por1A bond was hydrophobic. Only recombinant C4bp mutant molecules containing the NH2-terminal alpha-chain short consensus repeat (SCR1) bound to both Por1A and Por1B gonococci, suggesting that SCR1 contained Por binding sites. C4bp alpha-chain monomers did not bind gonococci, indicating that the polymeric form of C4bp was required for binding. Using fAb fragments against C4bp SCR1, C4bp binding to Por1A and Por1B strains was inhibited in a complement-dependent serum bactericidal assay. This resulted in complete killing of these otherwise fully serum resistant strains in only 10% normal serum, underscoring the importance of C4bp in mediating gonococcal serum resistance.
Amino Acid Sequence↗
Identification of a partial cDNA clone for the human receptor for complement fragments C3b/C4b.
Redundant oligonucleotides were synthesized based on amino acid sequences of tryptic peptides from the purified receptor for human complement fragments C3b/C4b (CR1). These probes were used to screen a size-selected human tonsilar cDNA library. A single positive clone was identified that hybridized to three oligonucleotide probes. The cDNA insert was 1.5 kilobases in length and contained sequences homologous to those of the oligonucleotide probes as well as nucleotide sequences corresponding to another independent CR1 tryptic peptide. Blot-hybridization analysis using fragments of the cDNA insert as probes revealed two distinct species of the CR1 message of 9 and 11 kilobases in human tonsil mRNA. The two EcoRI fragments of the CR1 cDNA insert hybridized to each other, suggesting the presence of homologous sequences. When used as probes in Southern blot analysis of human DNA, each fragment identified similar but not identical patterns of multiple restriction fragments, indicating either a series of homologous domains in a single CR1 gene or the presence of multiple CR1 genes. Furthermore, an additional BamHI fragment was found to segregate with the expression of the S allotype of the CR1 protein in a family. Thus, the molecular weight difference in the polymorphic variants of the CR1 protein is based on differences in nucleotide sequences.
Base Sequence↗
Evaluation of in vivo immune complex formation and complement activation in patients receiving intravenous streptokinase.
The usefulness of several different methods for detecting immune complex formation and complement activation in the circulation were applied to samples from patients receiving intravenous Streptokinase therapy for myocardial infarction. Streptokinase is a foreign antigen and can cause immune reactions. We collected samples from 13 patients, before Streptokinase administration (baseline), at the end of infusion (1 h), 12 h later and on day 7. We measured IgG containing immune complexes (IgG-IC), free C3d and antibodies to Streptokinase by ELISA, and CR1, C3d and C4d on erythrocytes by flow cytometric assay. Antibodies to Streptokinase are common, as all but two of the patients had measurable antibody levels. During Streptokinase treatment there was a drop in antibody levels, most prominent in those patients who had high baseline levels. At the same time increased levels of free C3d and erythrocyte-bound C3d were observed. After 12 h free C3d was usually back to baseline level, but C3d on erythrocytes was still raised. These data indicate the formation of Streptokinase immune complexes in patients with high Streptokinase antibody levels, and show that these complexes are cleared rapidly from the circulation, leaving more persistent signs of complement activation. We conclude that free C3d is a good indicator of ongoing complement activation, whereas C3d on erythrocytes indicates that complement activation has recently taken place.
Antibodies↗
Complement receptors and regulatory proteins: immune adherence revisited and abuse by microorganisms.
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Animals↗
Control of C1 activation by nascent C3b and C4b: a mechanism of feedback inhibition.
We have demonstrated that immune complexes turn over C1, i.e., limiting quantities of immune complexes activate an excess of C1. This was readily apparent in a system of purified C1 and C1-inhibitor (C1-In) but not in normal human serum (NHS). The following results indicate that C3 and C4 are the serum factors responsible for the inhibition of C1 turnover by immune complexes. 1) In a purified protein system composed of C1 and C1-In at pH 7.5, ionic strength 0.14 M, doses of immune complexes that activated all the C1 in 60 min at 37 degrees C yielded no detectable C1 activation when C2, C3, and C4 were also present. All proteins were at their physiologic concentrations. Activation was quantified by SDS-PAGE analysis and hemolytic titration 2) In order to inactivate C3 and C4, NHS was treated with 50 mM methylamine (MeAm) for 15 min at 37 degrees C, after which the MeAm was removed by dialysis. The activities of C1, C2, and C1-In were unaffected by this treatment. Doses of immune complexes that consumed no C1 in NHS, consumed all the C1 in MeAm-treated NHS (MeAm-NHS). 3) Reconstitution of MeAm-NHS with physiologic concentrations of C3 and C4 rendered the serum again resistant to excessive C1 consumption by immune complexes. Immune complexes used in these studies included EA-IgG, EA-IgM, tetanus-human anti-tetanus, and aggregated human IgG. There appeared to be specificity to the inhibition reaction since C4 by itself could inhibit C1 consumption by EA-IgM, whereas the presence of C3 was also required to control EA-IgG. Finally, N-acetyl-L-tyrosine was added to NHS at a final concentration of 30 mM. This nucleophile did not interact with native C3 or C4, nor did it directly activate C1. However, upon the addition of low doses of immune complexes, acetyl tyrosine did yield uncontrolled C1 activation, presumably by binding nascent C3b and C4b and thereby blocking their attachment to the immune complexes. We conclude that in NHS there is a mechanism of feedback inhibition by which nascent C3b and C4b inhibit C1 turnover by immune complexes. This mechanism of control might be physiologically important in that it prevents excessive complement activation by low concentrations of immune complexes.
Antigen-Antibody Complex↗
Heterogeneity of human C4 gene size. A large intron (6.5 kb) is present in all C4A genes and some C4B genes.
In this article we present a study showing that the human C4 genes differ in length because of the presence or absence of a 6.5 kb intron near the 5' end of the gene. DNA from individuals of known HLA, factor B, and C4 haplotypes was analyzed for restriction fragment length polymorphism (RFLP) by Southern blot analysis with C4-specific cDNA probes. The RFLP patterns obtained showed that the C4 genes are either 22.5 kb or 16 kb in length. They are referred to as long and short C4 genes, respectively. A population study was carried out to examine the distribution of the gene size according to C4 allotypes and haplotypes. Long C4 genes included all C4A genes studied and also some C4B allotypes, e.g., B1 on most C4 A3B1 haplotypes. Similarly, C4B null genes were found to be of the long form. Other C4B allotypes tested were found to be coded for by short C4 genes, including B2, B1 in C4 A6B1 and C4 AQOB1 (with a single C4B gene haplotype).
Complement C4↗
Study of quantitative modifications of the plasma protein S system components in non-acute inflammatory syndromes.
Thrombosis frequently accompanies inflammatory disease. There are numerous and frequent modifications of haemostasis parameters during inflammatory disease. Deficiencies in protein S, a protein C cofactor, are predisposing factors for thromboses. In 43 patients with biological inflammatory syndromes (ESR above 80 mm in the first hour found twice in a three-day period and disturbances of other biological inflammatory markers) we studied the variations of the protein S system: total antigenic protein S, free antigenic protein S, C4b BP, all antigenic fractions being assayed by electro-immuno diffusion. The results show an increase in free protein S (mean 114.3%, range 25%-180%, P less than 0.005), the biologically active fraction of protein S. They also evidence the expected increase in C4b BP (mean 188.3%, range 41%-335%, P less than 10(-5]. There is an increase in total protein S Ag (mean 145.3%, range 56%-220%, P less than 10(-5]. The results show a high positive correlation between the increased free protein S Ag and total protein S Ag (r' = 0.78, P less than 0.01), between total protein S and C4b BP (r' = 0.8, P less than 0.01) and between free protein S and C4b BP (r' = 0.73, P less than 0.01). C4b BP concentrations are correlated with haptoglobin (r' = 0.59, P less than 0.01), orosomucoid (r' = 0.53, P less than 0.01), C3 complement component (r' = 0.80, P less than 0.01), C4 (r' = 0.39, P less than 0.02) and platelet count (r' = 0.51, P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)
Adult↗
Complement activation and complement receptors in systemic lupus erythematosus.
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Antigen-Antibody Complex↗