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PubMed1987

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↗
PubMed1991

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↗
PubMed1991

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↗
PubMed1985

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↗
PubMed1986

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↗
PubMed1987

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↗
PubMed1990

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↗
PubMed1991

Immunoblotting human C4 bound to human erythrocytes in vivo and in vitro.

A study of C4 bound to human erythrocytes in vitro and in vivo has been made by immunoblotting with mouse monoclonal anti-C4c and anti-C4d and human polyclonal anti-C4d (Rodgers and Chido) following SDS-PAGE. Multi-banded patterns differentiated between C4A and C4B isotypes. Treatment of EC4b with trypsin eliminated immunoblotting but not agglutination reactions. Serum inactivation (factor I) of EC4b resulted in banding patterns similar to those obtained from patients' EC4d. Treatment of EC4b membranes with NH2OH affected many of the bands, two were lost, one was markedly reduced and others had altered SDS-PAGE mobility. Interpretation of the bands has been made in terms of C4-acceptor complexes and inactivation fragments of C4. A distinct difference in the banding of C4A and C4B isotypes has been detected.

Ammonium Hydroxide↗
PubMed1990

Homozygous C4A deficiency in systemic lupus erythematosus: analysis of patients from a defined population.

Homozygous C4A deficiency was found at a prevalence of 16% (13/80 patients) in systemic lupus erythematosus (SLE). The patients represented all diagnosed cases retrieved from a defined population in Southern Sweden, which minimizes the influence of patient selection. Photosensitivity was more common among C4A-deficient patients than among other SLE patients (p less than 0.05). Otherwise, clinical features were similar in the two groups. In addition, no differences were found with regard to presence of various autoantibodies (anti-dsDNA, anti-Sm, anti-RNP, anti-SSA, anti-SSB, rheumatoid factors and anti-cardiolipin). In patients expressing both C4A and C4B isotypes, C4B/C4A quotients were fairly stable in plasma irrespective of disease activity. This argues against preferential break-down of either isotype during complement activation in the disease. The increased photosensitivity of C4A-deficient patients partly resembles the findings in patients with complete deficiencies of classical pathway components.

Antibodies, Antinuclear↗
PubMed1984

Biosynthesis of the human C3b/C4b receptor during differentiation of the HL-60 cell line. Identification and characterization of a precursor molecule.

The C3b receptor (C3bR) of the human promyelocytic leukemia cell line (HL-60) was induced by incubating these cells with dimethylsulfoxide (DMSO) or retinoic acid. A majority of differentiated (DMSO- or retinoic acid-treated) but not undifferentiated cells formed rosettes with C3b-coated erythrocytes and were morphologically mature granulocytes. HL-60 cells were surface- or biosynthetically labeled and then solubilized in 1% Nonidet P-40 in the presence of multiple protease inhibitors. The C3bR was isolated either by immunoprecipitation with anti-C3bR antibodies or by affinity chromatography with hemolytically inactive components in which the internal thioester bond within the alpha-chain was cleaved (iC3)- or iC4-Sepharose. Autoradiographs of NaDodSO4-polyacrylamide gels indicated that the surface-labeled C3bR on the differentiated cells had an Mr of 210,000 (nonreduced) or 240,000 (reducing conditions). The bulk (approximately 85%) of the radiolabeled material that was isolated from biosynthetically labeled cells co-migrated with the surface-labeled band. A small fraction (approximately 15%) of the biosynthetically labeled material that was isolated by affinity chromatography or immunoprecipitation had an Mr of 188,000, which did not correspond to any surface-labeled band. This putative precursor molecule was characterized by pulse-chase experiments and by analysis of its carbohydrate. In pulse-chase (15-min pulse) studies of differentiated cells, only the 188,000-mol wt molecule was detected at 0 h. By 2 h, greater than 80% of counts had chased from the 188,000 to the 210,000-mol wt molecule. Treatment of these two molecules with endoglycosidases indicated that the 188,000-mol wt molecule possessed high mannose oligosaccharides, while the mature C3bR had complex oligosaccharides. We conclude from these data that the 188,000-mol wt molecule is a precursor of the C3b receptor of HL-60 cells. Other experiments indicated that the half-maximal time for newly synthesized receptor to attain an Mr of 210,000 was 45 min, and that the t1/2 for the disappearance of the receptor on the surface of differentiated HL-60 cells in tissue culture was approximately 10 h. The ability to observe the induction of the C3b receptor as the HL-60 cell line differentiates is an instructive model system to study the biosynthesis of a human integral membrane receptor glycoprotein.

Cell Differentiation↗
PubMed1989

Complement receptors.

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Antigens, Differentiation, B-Lymphocyte↗
PubMed1990

The ontogenesis of Fc gamma receptors and complement receptors CR1 in human peripheral nerve.

The ontogenesis of Fc gamma receptors (FcR) and C3b/C4b receptors (CR1) was studied in peripheral nerves from ten fetuses aged from 20 to 38 weeks using immunohistochemical and functional assays. Monoclonal antibodies (mAbs) against FcR and CR1 stained nerve fibers at 10 weeks of gestation and the staining intensity increased during nerve maturation. FcR and CR1 are probably expressed on Schwann cells and are early markers during the development of peripheral nerves. Functional FcR activity was detected in nerve sections before initiation of myelination, which occurs at approximately 18-19 weeks, whereas functional CR1 activity was found in the sections after myelination. Functional CR1 activity may, therefore, be related to myelin. The ontogenesis of FcR and CR1 was also studied on Schwann cells in culture from three fetuses aged 14, 16 and 19 weeks, using immunofluorescence technique with mAbs. The FcR and CR1 are lost on cultured Schwann cells. This suggests that the receptors are not intrinsic to the cells or that Schwann cells require axonal contact for the expression of FcR and CR1.

Antibodies, Monoclonal↗
PubMed1990

Differences between C4A and C4B in the handling of immune complexes: the enhancement of CR1 binding is more important than the inhibition of immunoprecipitation.

There are two isotypes of C4--C4A and C4B--, encoded within the major histocompatibility complex with quite different properties. In this study we have compared purified C4A and C4B with regard to their ability to prevent immune complex precipitation and to enhance the binding of both preformed and nascent immune complexes to the receptor CR1 on red cells. C4A was modestly more effective than C4B at inhibiting immunoprecipitation, particularly in antibody excess. In the CR1 binding assay C4A was markedly more effective than C4B in enhancing binding to CR1. This difference was seen with both preformed and nascent immune complexes at equivalence and antibody excess. Thus the major differences between C4A and C4B in regard to immune complex handling is at the level of CR1 binding. Given the strong association of C4A* QO alleles with immune complex-mediated diseases like systemic lupus erythematosus, these findings have important pathogenetic implications.

Antigen-Antibody Complex↗
PubMed1983

The role of the thioester bond in C3 and C4 in the determination of the conformational and functional states of the molecule.

The numerous ligand binding properties expressed by the activated forms of C3 (C3b) and C4 (C4b) are best explained as arising from proteolytic-cleavage-induced conformational changes. The studies described above provide direct physical evidence for such conformational transitions in both complement proteins. Significantly, however, a virtually identical conformational end state was also produced by direct nucleophilic scission of the internal thioester bond in C3 and C4 in the absence of any proteolysis. Clearly, it is the integrity of this thiolactone structure that is the determining factor in maintaining the native conformation in C3 and C4. Recent studies suggest a similar conformational role for the thioester in alpha 2-macroglobulin. Proteolytic activation in all three thioester-containing molecules renders this structure more susceptible to nucleophilic or solvolytic attack. Whether this effect is mediated by a peptide-cleavage-induced increase in the electrophilicity of the reactive carbonyl, or simply by increasing the accessibility of the solvent to this structure, is as yet unknown. In the case of C3 and C4, removal of the activation peptide also has a profound effect on the kinetics of the conformational change initiated by the loss of the thioester. This kinetic constraint has made it possible to correlate the acquisition of ligand binding properties with the spectroscopically detectable conformational changes.

Circular Dichroism↗
PubMedDate not supplied

A monoclonal anti-C4d that demonstrates a specificity related to anti-Ch.

A monoclonal anti-C4d reagent, L003, agglutinated untreated and trypsin-treated C4A Rodgers(Rg)- and C4B Chido(Ch)-coated red cells. Inhibition of the agglutination, regardless of the Rg/Ch phenotype of the C4 coat, showed Ch specificity, more precisely, for the Ch1 determinant. C4A allotypes that express Ch1 and Ch3 rather than Rg1 and Rg2, and C4B allotypes that express Rg1 rather than Ch1, Ch2, or Ch3 confirmed that the inhibition specificity in serologic tests was anti-Ch1 and not anti-C4B.

Animals↗