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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↗
PubMed1997

Serum complement activation of SLE patients during plasmapheresis.

The erythrocyte complement receptor 1 (ECR1)-immune complex binding assay is a sensitive method for the determination of complement fragments which can be activated by bovine serum albumin (BSA)-anti-BSA in vitro. When the C3b/C4b containing bovine serum albumin (BSA)-anti-BSA was formed in the presence of the serum of patients with systemic lupus erythematosus (SLE) its binding to ECR1 was found to be lower than that formed in sera of normal volunteers. The plasmapheresis of SLE patients homozygous for the CR1/E high density allele displays a beneficial effect on the formation of C3b/C4b containing BSA-anti-BSA and its binding to ECR1. There was no significant correlation between the serum C3/C4 level and the percentage of C3b/C4b containing BSA-anti-BSA binding to the ECR1 of SLE patients during plasmapheresis. At the same time, there was an inverse correlation between the serum immune complex level and the ECR1 binding, which was significant in 3 of 5 cases. These data suggest that, besides the determination of different components of complement activation, the functional assay of complement activation might be useful in monitoring the effect of plasmapheresis in SLE.

Adult↗
PubMed1989

Complement receptors.

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Antigens, Differentiation, B-Lymphocyte↗
PubMed2004

Measurement of erythrocyte C4d and complement receptor 1 in systemic lupus erythematosus.

OBJECTIVE: C4-derived activation fragments are the only complement ligands present on the surfaces of normal erythrocytes. The significance of this observation is unknown, and the role of erythrocyte-bound C4 (E-C4) in human disease has not been explored. More than any other human disease, the pathogenesis of systemic lupus erythematosus (SLE) has been characterized by defects in clearance of complement-bearing immune complexes via erythrocytes expressing complement receptor 1 (CR1). This study was undertaken to determine whether these functional defects might be reflected by abnormal patterns of E-C4 and E-CR1 expression on erythrocytes of patients with SLE. METHODS: We conducted a cross-sectional study of 100 patients with SLE, 133 patients with other diseases, and 84 healthy controls. Erythrocytes were characterized by indirect immunofluorescence and by flow cytometry for determination of levels of C4d and CR1. RESULTS: Patients with SLE had higher levels of E-C4d and lower levels of E-CR1 than did patients with other diseases (P < or = 0.001) or healthy controls (P < or = 0.001). The test was 81% sensitive and 91% specific for SLE versus healthy controls and 72% sensitive and 79% specific for SLE versus other diseases, and it had an overall negative predictive value of 92%. CONCLUSION: This is the first report of abnormal levels of E-C4d in human disease. We found that abnormally high levels of E-C4d and low levels of E-CR1 are characteristic of SLE, and combined measurement of the 2 molecules has high diagnostic sensitivity and specificity for lupus. Determination of E-C4d/E-CR1 levels may be a useful addition to current tests and criteria for SLE diagnosis.

Adult↗
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↗
PubMed2006

Kaposi's sarcoma-associated herpes virus complement control protein: KCP--complement inhibition and more.

The complement system is an important part of innate immunity providing immediate protection against pathogens without a need for previous exposure, as well as priming the adaptive immune response through opsonisation, leukocyte recruitment and enhancing humoral immune responses. Its importance is not only shown through recurring fulminant infections in individuals with complement component deficiencies, but also through the many complement evasion strategies discovered for a wide range of infectious microbes (including acquisition of endogenous host complement inhibitors and expression of own homologues). Knowledge of these mechanisms at a molecular level may aid development of vaccines and novel therapeutic strategies. Here, we review the structure-function studies of the membrane-bound complement inhibitor KCP that is expressed on the surface of Kaposi's sarcoma-associated herpesvirus (KSHV) virions and infected cells. KCP accelerates the decay of classical C3 convertase and induces the degradation of activated complement factors C4b and C3b by a serine proteinase, factor I. Molecular modeling and site-directed mutagenesis have identified sites on the surface of KCP required for complement inhibition and support the hypothesis that KCP has evolved to mimic the structure and function of endogenous human inhibitors. KCP additionally enhances virion binding to permissive cells through a heparin/heparan sulfate-binding site located at the N-terminus of the protein.

Amino Acids↗
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↗
PubMed2001

Regulation of complement classical pathway by association of C4b-binding protein to the surfaces of SK-OV-3 and Caov-3 ovarian adenocarcinoma cells.

The role of fluid-phase regulators of complement is to inhibit excessive complement activation and maintain homeostasis in blood. By binding to and inactivating complement components on cell surfaces, they can also protect autologous cells from complement-mediated cytotoxicity and phagocytosis. In this study, we wanted to find out whether C4b-binding protein (C4bp), a fluid-phase regulator of the classical complement pathway, could directly bind to cell surfaces in a functionally active form. After screening several malignant cell lines, we observed that the ovarian adenocarcinoma cell lines SK-OV-3, Caov-3, and SW626 were capable of binding C4bp. Binding tests with recombinant deletion mutants suggested that the primary binding site on C4bp is located on the alpha-chain complement control protein 4 domain. Functional tests showed that tumor cell-bound C4bp retained its cofactor activity for factor I-mediated inactivation of C4b, thus increasing the control of classical complement pathway activation on the surfaces of these cells. These results demonstrate a novel mechanism of complement regulation on cell surfaces, particularly on those of malignant ovarian tumor cells.

Adenocarcinoma↗
PubMed1994

Binding of C3b and C4b by the CR1-like site in murine CR1.

We determined whether the six short consensus repeats (SCRs) that are appended to the amino terminus of murine CR2 to form murine CR1 contain a binding site for C4b in addition to that for C3b, and whether these sites overlap or are distinct. Human K562 transfectant cell lines were established that stably expressed constructs encoding variable combinations of these six murine SCRs attached to the amino terminus of a truncated form of human CR2 lacking its iC3b/C3dg binding site. These cell lines, and two others expressing full-length human CR1 and SCRs lacking its iC3b/C3dg binding site. These cell lines, and two others expressing full-length human CR1 and SCRs 8-11 of the C3b binding site of human CR1, respectively, were assessed for their capacity to form rosettes with sheep E bearing rat C4b or guinea pig C3b. K562 cells with full length human CR1 formed rosettes with both EC3b and EC4b, and the cells expressing the construct with human CR1 SCRs 8-11 bound only EC3b. The murine CR1/human CR2 chimera containing murine SCRs 1-6 resembled the full length human CR1 in binding both EC3b and EC4b. Deletion of SCRs 5-6 from the murine CR1/human CR2 chimera diminished in parallel, but did not abolish, binding of EC3b and EC4b. Constructs containing SCRs 2-5, SCRs 3-6, or SCRs 2-6 lacked activity, indicating an absolute requirement for SCR-1 for binding of both C3b and C4b. Therefore, murine CR1 binds both C3b and C4b, and the sites for these ligands have similar, if not identical, amino- and carboxyl-terminal boundaries.

Animals↗
PubMed1988

A physical map of the human regulator of complement activation gene cluster linking the complement genes CR1, CR2, DAF, and C4BP.

We report the organization of the human genes encoding the complement components C4-binding protein (C4BP), C3b/C4b receptor (CR1), decay accelerating factor (DAF), and C3dg receptor (CR2) within the regulator of complement activation (RCA) gene cluster. Using pulsed field gel electrophoresis analysis these genes have been physically linked and aligned as CR1-CR2-DAF-C4BP in an 800-kb DNA segment. The very tight linkage between the CR1 and the C4BP loci, contrasted with the relative long DNA distance between these genes, suggests the existence of mechanisms interfering with recombination within the RCA gene cluster.

CD55 Antigens↗