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PubMed1993

Associated complement C3b. Towards an understanding of its intracellular modifications.

Covalent Superose microspheres-bound C3b was used as a model system to simplify the analysis of antigen-bound C3b modifications during antigen processing. The model was set up using purified C3 and Superose-bound trypsin. C3b was covalently bound to Superose through an ester link, as indicated by lability to hydroxylamine treatment at alkaline pH. C3b-Superose was incubated with L subcellular fraction, enriched in endosomes/lysosomes, purified from U937 cell line. Two types of limited activities on the C3b-Superose model system were detected: (i) a proteolytic activity cleaving C3b into mainly a C3c-like fragment which was released and a C3d-like fragment of apparent M(r) 32 kDa which remained bound to Superose through the original ester link; (ii) an esterolytic activity cleaving the ester bond and releasing C3b. Inhibition experiments pointed to the involvement of serine, aspartyl and cysteine proteases. Cathepsin B appeared most probably as one of the major proteases of L fraction catalysing the proteolysis of the C3b-bound. Kinetic studies were in favour of a good stability on the ester bond, supporting an effective role of C3b as a chaperone during the extracellular and intracellular travel of C3b-bound antigen.

Cathepsin B↗
PubMed1986

[Immune complexes in allergies: evidence of IgE-containing immune complexes in Churg-Strauss vasculitis].

In five patients with vasculitis, hypereosinophilia and elevated serum-IgE levels, the diagnosis of Churg-Strauss syndrome was established. To identify a possible role of IgE in pathogenic mechanisms leading to vasculitis, a sequential precipitation of patients' sera was performed using various concentrations of polyethylene glycol 6000 (PEG). Using a radioimmunosorbent test, the precipitates obtained were tested for their IgE contents. Considerable amounts of IgE were found in the serum precipitates of all patients. In parallel studies, no IgE-containing precipitates were detected in sera from patients with different allergic diseases and high IgE serum levels. The demonstration of IgE-containing PEG precipitable material in all patients suffering from Churg-Strauss syndrome, together with the finding of an elevated C3d and C4 level and a decreased C3 level in one patient and an increased C3d level in a second patient, suggests that IgE-containing immune complexes play a pathogenic role in the Churg-Strauss syndrome.

Adult↗
PubMed1998

Effect of complement on the rheological properties of the erythrocyte membrane.

The effect of complement (C) on the erythrocyte deformability was investigated. Sheep erythrocytes (E) were sensitized with specific antibodies (A) and treated with various doses of human C. Gravity filtration of unlysed EAC showed a C dose-dependent decrease in whole cell deformability, which was shown to be due to an impairment of the membrane rheological properties. As the fluidity of the lipid bilayer, sensed by a spin label, was not modified, the observed effects should be related to an interaction between C and the cytoskeleton. The influence of C3b and C3d in these processes seems unlikely, as similar hemagglutination titers were found, respectively, for anti-C3c and anti-C3d antisera against the varying EAC. The study of the degree of spontaneous hemolysis of EAC in buffers with solutes of different Stokes radii showed that differing-sized functional C lesions were produced, and that the formation of larger-sized lesions was favored by incubation with higher C doses. These results suggest that the insertion of proteins from the membrane attack complex, C5b-9 (MAC), into the erythrocyte membrane may be responsible for the effect of C on the rheological properties of the erythrocyte membrane.

Animals↗
PubMed1989

The difference between human C3F and C3S results from a single amino acid change from an asparagine to an aspartate residue at position 1216 on the alpha-chain of the complement component, C3.

The third component of the human C system, C3, exhibits two common genetic variants. These variants have been characterized by high voltage agarose electrophoresis and are designated C3 fast (C3F) and C3 slow (C3S). C3F occurs at appreciable frequencies only in Caucasian populations and has been shown to be associated with an increased incidence of certain diseases, such as partial lipodystrophy, IgA nephropathy, and Indian childhood cirrhosis. It has been shown that C3F differs from C3S with regard to isoelectric point as well as its ability to bind macrophages. The availability of a full-length cDNA probe for human C3 has made it possible to study the polymorphism at the genomic level. We have used RNA/RNA hybridization to demonstrate that the difference between C3F and C3S occurs in the C3d region. We subsequently used oligonucleotide-primed DNA amplification to show that C3F arises from a point mutation at codon 1216 converting a deoxyadenosine for a deoxyguanosine. The result of this point mutation at the translational level is the substitution of an asparagine residue in C3S for an aspartic acid residue in C3F. It is known that C3d contains the binding site for CR2 as well as the internal thioester site and multiple protease cleavage sites. The identification of the structural basis of the differences between C3F and C3S will assist our continuing studies of the mechanism of the functional differences between the two alleles and the disease associations of C3F. It also allows us to use DNA based techniques to allotype C3 in subjects with little or no C3 in their serum.

Amino Acid Sequence↗
PubMed2005

Solvent accessibility of native and hydrolyzed human complement protein 3 analyzed by hydrogen/deuterium exchange and mass spectrometry.

Complement protein C3 is a 187-kDa (1641-aa) protein that plays a key role in complement activation and immune responses. Its hydrolyzed form, C3(H2O), is responsible for the initiation of the activation of alternative complement pathway. Previous analyses using mAbs, anilinonaphthalenesulfonate dyes, and functional studies have suggested that C3 is conformationally different from C3(H2O). We have used amide hydrogen/deuterium exchange and MALDI-TOF mass spectrometry to identify and localize structural differences between native C3 and C3(H2O). Both proteins were incubated in D2O for varying amounts of time, digested with pepsin, and then subjected to mass-spectrometric analysis. Of 111 C3 peptides identified in the MALDI-TOF analysis, 31 had well-resolved isotopic mass envelopes in both C3 and C3(H2O) spectra. Following the conversion of native C3 to C3(H2O), 17 of these 31 peptides exhibited a change in deuterium incorporation, suggesting a conformational change in these regions. Among the identified peptides, hydrogen/deuterium exchange data were obtained for peptides 944-967, 1211-1228, 1211-1231, 1259-1270, 1259-1273, 1295-1318, and 1319-1330, which span the factor H binding site on C3d and factor I cleavage sites, and peptides 1034-1048, 1049-1058, 1069-1080, 1130-1143, 1130-1145, 1211-1228, 1211-1231, 1259-1270, and 1259-1273, spanning 30% of the C3d region of C3. Our results suggest that hydrolysis may produce a looser (more open) structure in the C3d region, in which some of the changes affect the conversion of helical segments into coil segments facilitating interactions with factors I and H. This study represents the first detailed study mapping the regions of C3 involved in conformational transition when hydrolyzed to C3(H2O).

Amino Acid Sequence↗
PubMed1990

Molecular basis of hereditary C3 deficiency.

Hereditary deficiency of complement component C3 in a 10-yr-old boy was studied. C3 could not be detected by RIA of serum from the patient. Segregation of C3 S and C3 F allotypes within the family confirmed the presence of a null gene for C3, for which the patient was homozygous. 30 exons have been characterized, spanning the entire beta chain of C3 and the alpha chain as far as the C3d region. Sequence analysis of the exons derived from the C3 null gene showed no abnormalities in the coding sequences. A GT-AT mutation at the 5' donor splice site of the intervening sequence 18 was found in the C3 null gene. Exons 17-21 were amplified by the polymerase chain reaction (PCR) from first-strand cDNA synthesized from mRNA obtained from peripheral blood monocytes stimulated with LPS. This revealed a 61-bp deletion in exon 18, resulting from splicing of a cryptic 5' donor splice site in exon 18 with the normal 3' splice site in exon 19. This deletion leads to a disturbance of the reading frame of the mRNA with a stop codon 17 bp downstream from the abnormal splice in exon 18. His parents had both the normal and abnormal C3 mRNA and were shown to be heterozygous for this mutation by sequence analysis of genomic DNA amplified by PCR. Similar splice mutants have previously been reported in the beta-globin, phenylalanine hydroxylase, and porphobilinogen deaminase genes. This mutation is sufficient to cause the deficiency of C3 in the patient.

Base Sequence↗
PubMed1983

Serum-treated antigen-antibody complexes inhibit the production of C2 and factor B by mononuclear phagocytes.

Antigen-antibody complexes enhanced the synthesis of C2 and factor B by human monocytes and macrophages, and C2 by guinea-pig macrophages. In contrast complexes that had been treated with serum inhibited the production of these components. The inhibitory effect of serum-treated complexes was abrogated by Fab fragments of anti-C3, anti-C3c and anti-C3d. It is therefore probable that inhibition was mediated by a C3 fragment bound to the complex. The enhancing effect of untreated complexes was reversible by serum-treated complexes, and the inhibitory action of serum-treated complexes was counteracted by untreated complexes. Such a system may be important in the regulation of the synthesis of complement components in response to local requirements.

Animals↗
PubMed1987

Quantitation and antigenic characterization of bound C3 of circulating immune complexes in systemic lupus erythematosus, rheumatoid arthritis, and primary biliary cirrhosis.

In recent years defective function of the complement-mediated clearance of immune complexes (IC) has been reported in patients with immune complex disease. The defect has been found at different levels in the clearance system. An important event in this sequential system is the binding of C3-coated particles to C3 receptors on erythrocytes and phagocytes. This study focuses on immunochemical properties of IC-bound C3 that reflect the functional state of the molecule. Sera from patients with primary biliary cirrhosis (PBC), rheumatoid arthritis (RA), and systemic lupus erythematosus (SLE) and from normal subjects were analyzed for their level of C3 precipitable in 2.7% (w/v) polyethylene glycol (PEG). The mean levels for the patient categories were significantly higher than that for the normal subjects. The immunochemical study revealed several differences among the different forms of PEG-precipitable C3. All forms expressed C3(D) antigens which are expressed by immune complex-associated and denatured forms but not by soluble physiological forms of C3. The expression of these antigens was proportionately lower for the complex-associated C3 of PBC compared to that of RA and SLE. Furthermore, employing monoclonal anti-C3(D) antibodies against the C3c and the C3d domain, distinct differences could be detected among all forms of PEG-precipitable C3. Sera from RA and SLE, in particular, contained PEG-precipitable C3 that exhibited distinctive immunochemical features with respect to these epitopes.

Antibodies↗
PubMed1977

[Evaluation of the role of circulating antigen-antibody complexes in the pathogenesis of glomerular nephropathies].

Immunofluorescence studies of the glomeruli in patients suffering from glomerulonephritis (GN) suggest the deposit of immune complexes from the circulating blood. Immune complexes were quantitated using a sensitive radioimmunoassay from sera of 41 patients with GN drawn at the time when renal biopsy was performed. Simultaneously, degradation products of C3 (C3d) were measured. Eleven of 12 patients with GN secondary to a systemic disease (usually LED) presented increased C1q-binding activity of their sera (C1q-BA). C3 was further demonstrated in 9 of these patients, suggesting the presence of circulating immune complexes. By contrast only 2 of 12 patients with idiopathic GN characterized by deposits of complement and IgG in the glomeruli exhibited increased C1q-BA. One of 17 patients with idiopathic GN without deposit of immunoglobulins and C3 in the glomeruli also had increased C1q-BA. The results observed in the group of patients with idiopathic GN with glomerular deposits could be accounted for either by a transitory or minimal presence of immune complexes or by local formation of complexes in the glomerular structures.

Antigen-Antibody Complex↗
PubMed1986

The role of receptors for complement in the induction of polyclonal B-cell proliferation and differentiation.

A panel of monoclonal antibodies and ligands that bind to the CR1 or CR2 complement receptors of B cells has been used to investigate the role of these membrane molecules in regulating B-cell proliferation and differentiation. When CR2 was modulated from the surface of B cells by treatment with the HB-5 antibody and a secondary goat anti-mouse immunoglobulin antibody, Epstein-Barr virus-induced polyclonal B-cell proliferation and immunoglobulin production were inhibited by 83 and 90%, respectively. In contrast, modulation of other cell surface molecules, HB-2, B1, and the C3b receptor (CR1), or pretreatment of B cells with C3d,g (a CR2 ligand) or HB-5 antibody, alone minimally inhibited these responses. Neither the HB-5 antibody C3d,g, nor a monoclonal antibody (YZ-1) reactive with CR1 induced resting B cells to proliferate, nor did they alter anti-mu antibody-induced proliferation. Similarly, treatment with C3d,g or with the HB-5 or YZ-1 antibodies did not induce B cells to secrete immunoglobulin or affect pokeweed mitogen-induced plasma-cell formation. Whereas CR2 appears to be the functionally relevant receptor for Epstein-Barr virus on B cells, the effects of ligand interactions with CR1 and CR2 on normal B-cell proliferation or differentiation remain unidentified.

Adult↗
PubMed1988

CR2 is a complement activator and the covalent binding site for C3 during alternative pathway activation by Raji cells.

Antibody-independent activation of the alternative C pathway by human lymphoblastoid cell lines latently infected with EBV has been recognized for some time, although the mechanisms involved and the specific cell surface molecule(s) recognized by the C system have not been identified. The present studies, carried out with the purified proteins of the alternative pathway have addressed these questions. Activation of the purified proteins of the alternative pathway by Raji lymphoblastoid cells was found to be antibody independent, confirming earlier findings with serum. Surprisingly, activation was highly dependent on properdin. In other models properdin has been found to augment alternative pathway activation and to be required for lysis of virus infected cells. Molecules which activate the alternative pathway provide binding sites on which C3 breakdown by regulatory proteins is impeded; therefore intact C3b accumulates on the activator. Immunoprecipitation studies with either anti-CR2 or anti-C3 have identified CR2, the R for C3d,g and EBV, as a major covalent and noncovalent binding site for C3 deposition on Raji cells during alternative pathway activation. Covalently bound C3b was dissociated from CR2 by hydroxylamine, indicating attachment via an ester bond. C3b binding after activation was not reduced by an anti-CR2 mAb which blocks CR2 R function, indicating that it was probably not mediated by C3d,g R epitopes on CR2. Direct confirmation of the ability of CR2 to trigger the alternative pathway came from studies with purified CR2 which was found to activate the alternative C pathway in serum or in mixtures of the purified proteins of the pathway. This work provides conclusive evidence that CR2 is a C activator and functions in this capacity on Raji cells.

Burkitt Lymphoma↗
PubMed1981

The covalent-binding reaction of complement component C3.

The complement protein C3, when activated by limited proteolysis, forms a short-lived reactive intermediate fragment, 'nascent' C3b, which is known to bind covalently to certain surfaces. The characteristics of the covalent binding reaction have been studied by using Sepharose-trypsin as a combined proteolytic activator and binding surface for C3. Binding of C3 to Sepharose-trypsin is saturable, with a maximum of 25-26 molecules of C3b bound per molecule of trypsin. A minimum life-time of about 60 microseconds for the reactive intermediate has been calculated from binding of C3 at saturation. Initial binding efficiencies of over 30% can be obtained at physiological pH and ionic strength. The efficiency of C3 binding to Sepharose-trypsin decreases as pH increases and also shows a slight decline at high ionic strength. The covalent binding of C3 to Sepharose-trypsin can be inhibited by a range of oxygen and nitrogen nucleophiles. Activation of C3 in the presence of radioactive forms of four such nucleophiles, phenylhydrazine, methylamine, glycerol and glucosamine results in apparent covalent incorporation of the nucleophile into the C3d fragment of C3. The quantity of radioactive nucleophile bound can be predicted from the observed potency of the nucleophile as an inhibitor of the binding of C3 to Sepharose-trypsin. The radioactive nucleophiles may be considered as 'active-site' labels for C3.

Binding Sites↗
PubMed1993

Isolation and biochemical characterization of the iC3b receptor of Candida albicans.

In an effort to identify the protein structure on Candida albicans, pseudohyphal forms which had been shown earlier to bind human iC3b, a protein of about 42 kDa (p42), were obtained from lysates of pseudohyphal forms by absorption with C3(H2O)-Sepharose. An antiserum raised in rabbits against this protein effectively inhibited adherence of sheep erythrocytes carrying iC3b (EAC3bi) to pseudohyphal forms. p42 cross-reacted with OKM-1, a monoclonal antibody directed against the human complement receptor type 3 (CR3, CD11b). This protein, p42, was designated p42-CR3. The antiserum against p42-CR3 was used for further purification of lysates by affinity chromatography. Three proteins of 66, 55, and 42 kDa were isolated. All were recognized by OKM-1 in immunoblots (p66-, p55-, and p42-CR3). The different proteins were separated and treated with neuraminidase and endoglycosidase F. Almost complete deglycosylation of the p66-CR3 protein was obtained after treatment with neuraminidase, indicating a high degree of glycosylation. Neuraminidase also had an effect on p55-CR3, but not on p42-CR3. Endoglycosidase F did not alter any of the three proteins. In ligand blots, p42-CR3 bound C3(H2O), C3b, and iC3b but not C3d; p55-CR3 clearly reacted with C3(H2O) and weakly reacted with C3b and iC3b. p66-CR3 never showed reactivity. It is suggested that p55 and p66 represent glycosylated forms of p42-CR3. Although C. albicans CR3 and human CR3 cross-react and bind identical ligands, the two receptors differ in structure.

Candida albicans↗
PubMed1975

Immunoconglutinins and C3 in human saliva.

High titres of immunoconglutinin activity (antibody to bound complement components) have been found in the parotid, sublingual and submandibular saliva of most healthy subjects. The immunoconglutinin (IK) titre in mixed saliva was substantially lower than in the other samples of saliva. C3 was detectable in only three of 164 samples of parotid, submandibular and sublingual saliva but was present in forty-seven of 117 mixed saliva samples. It is suggested that crevicular fluid is the major source of C3 in mixed saliva. A negative correlation was found in mixed saliva between the C3 concentration and the IK titre, and this suggested that C3 was the inhibitor of IK in mixed saliva. Binding of C3 to IK has been demonstrated in mixed saliva by using highly purified salivary IK and C3. Purified C3, C3i, C3c and C3d inhibited the activity of purified IK. It is suggested that salivary IK represents the secretion of a B-lymphocyte population which has evaded the mechanism responsible for inducing B-cell tolerance to autologous serum proteins. The reason for its persistence in the salivary glands, however, is not known at present.

Adolescent↗
PubMed1986

Inhibition of B cell growth factor (BCGF) by monoclonal antibodies directed against the C3d receptor (CR2).

Normal human B cell proliferation is controlled by various immunoregulatory signals including the T cell-derived lymphokine B cell growth factor (BCGF). Human BCGF provides the final proliferative signal to normal, activated B cells. We herein show that anti-CR2 monoclonal antibodies inhibit human B cell responsiveness to purified BCGF. Addition of anti-CR2 antibody, AB5, was capable of completely inhibiting BCGF-mediated enhancement of either anti-mu or staphylococcal protein A-activated human B cells (191 +/- 21 cpm vs. 3942 +/- 622 cpm, mean +/- SEM). Inhibition of B cell response to BCGF by AB5 occurred in a dose-dependent manner. Monoclonal antibody anti-B2, which recognizes the same 140-kDa glycoprotein as AB5, in comparable concentrations also inhibited B cell responsiveness to BCGF. Monoclonal antibodies of the same subclass (IgG1) showed no inhibitory effect on BCGF enhancement of B cell proliferation. The F(ab')2 fragment of AB5 generated by pepsin digestion was similarly inhibitory as was the intact Ig. AB5-mediated inhibition was independent of the target B cell state of activation. Both resting and activated B cells (anti-mu or staphylococcal protein A activated) incubated with similar concentrations of AB5 were unresponsive to BCGF. The ability of anti-CR2 antibodies to block BCGF-dependent B cell proliferation suggests that occupancy of C3d membrane receptors may result in modulation of B cell proliferation in physiologic or clinical disease states.

Antibodies, Monoclonal↗
PubMed1989

[Clinical significance of the estimations of complement and immune complexes].

Immune complexes (IC) interacting with complement (C) induce tissue injuries and the estimations of their sequential serum levels is clinically significant. In C, among the various parameters, total hemolytic activity (CH 50) is clinically the most useful especially in the diagnosis and treatment of SLE. Elevation of serum CH 50 levels is nonspecifically observed in patients with inflammations and malignancies, although its mechanism remain obscure. Decreased CH 50 levels were observed in various diseases including liver diseases, collagen diseases, kidney diseases, congenital C component deficiencies and so on. Among them, chronic liver disease is the most frequently encountered disease. Liver function should be checked first in cases with low CH 50. In IC, patho-etiological significance has been established and various methods for the estimation have been employed. However, their sensitivities and specificities are various and positivities obtained by these methods vary with the diseases. Clinical significance of IC estimation has been recognized to be limited to a few diseases including SLE, in which some clinical signs parallel the IC level. However, recent developments in the methods employing murine monoclonal antibodies to C1q and C3d and IgG rheumatoid factor are expected to help re-evaluation the significance of serum IC estimation in various diseases. Heat-pretreatment of sera at 56 degrees C or below produced the reactivity of IgG with rheumatoid factors. Therefore, serum samples should be estimated without heat-inactivation.

Antibodies, Monoclonal↗
PubMed1985

Monoclonal antibodies to the 140,000 mol wt glycoprotein of B lymphocyte membranes (CR2 receptor) initiates proliferation of B cells in vitro.

Several mouse monoclonal IgG antibodies (AB1, AB2, AB3, and AB5) were developed that reacted with a 140,000 mol wt glycoprotein on the surface of cultured RAJI B lymphoid cells. The antibodies reacted with purified normal human peripheral blood B cells and CLL Ig+ B cells and showed specific germinal center and mantle zone staining in tissue sections of secondary lymphoid organs. Immunodepletion studies using 125I surface-labeled Raji cell membrane antigens demonstrated that the antigen identified by AB5 is the same 140,000 mol wt glycoprotein detected by anti-B2 that has recently been shown to react with the C3d fragment or CR2 receptor. (Iida et al: J Exp Med 158:1021, 1983). Addition of the AB series and anti-B2 monoclonal antibodies to cultures of purified human peripheral blood B cells resulted in the uptake of 3H-thymidine at two to six times background control levels provided that irradiated autologous T cells were added to the culture. Stimulation was not evoked by other monoclonal antibodies to B cell surface molecules (ie, B1, BA-1, BA-2, and HLA-DR). Pepsin-generated F(ab')2 fragments of anti-CR2 antibodies were essentially as effective as the intact IgG molecule in stimulating B cells. Induction of B cell proliferation by antibody binding to CR2 suggests that the C3d receptor may have an integral role in regulation of humoral immune response.

Animals↗
PubMed1979

Inhibition of lymphocyte blastogenesis by C3c and C3d.

The C3 cleavage products C3c and C3d were tested for their ability to alter the immunoproliferative response of human peripheral mononuclear cells to the antigens SLO and SK-SD, and to the mitogens PHA and PWM. It was found that both C3c (30 to 120 micrograms/ml) and C3d (10 to 40 micrograms/ml) inhibited lymphocyte blastogenesis in the presence on antigens but not mitogens, when cells were cultured in either autologous plasma or FCS. Similarly, the response to antigens of cell populations enriched for T lymphocytes was inhibited, whereas the response to optimal or suboptimal doses of mitogens was unaffected. When nonadherent (NA) cells were reconstituted with increasing numbers of adherent (AD) cells to potentiate the proliferative response of NA cells to the antigen SLO, the addition of either C3c or C3d abolished the potentiation of the response at low levels of reconstitution. However, at given dose of C3c or C3d, addition of excess AD cells could restore the proliferative response. These results suggest that both C3c and C3d can inhibit T cell proliferation in response to antigen and that they may act at the level of the monocyte-T lymphocyte interaction to modulate cellular immune responses.

Antibodies↗