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PubMed1982

Characterization of the activation of the human C1r complement molecule.

The proenzyme form of C1r was isolated by sequential chromatography from the euglobulin fraction of human serum on DEAE-Sepharose 6B-CL, CM-Sepharose 6B-CL and Sepharose S-300-CL. This C1r had the tendency to spontaneously activate within 60-90 min of incubation at 37 degrees C in presence of EDTA and more slowly in the presence of Ca2+. The spontaneous activation of C1r was found to be a bimolecular process and could be completely inhibited by DFP in the pH range 6-9 and in the presence of Ca2+ without affecting the hemolytic C1r activity. [14C]DFP bound to trace proteins in the 60-90 kD range, but not to C1r proenzyme. The spontaneous activation of C1r was diminished in the presence of EDTA by DFP, but could not be completely suppressed. EDTA acts by removing Ca2+ from C1r, thereby changing the conformation of the protein and causing an increased digestibility of the C1r H-chain. At temperatures above 0-4 degrees C this influence destroyed the ability of C1r proenzyme and enzyme to form macromolecular C1 and thereby abolished its hemolytic activity. We conclude from these results that the spontaneous C1r activation in the pH range 6-9 and in the presence of Ca2+ is due to contaminant proteases. C1r activated also spontaneously at higher pH values between pH 9 and 13.2, but the spontaneous activation ceased abruptly at pH 13.4. An intramolecular process of activation cannot be excluded at these high pH values. It is, however, not clear, whether this activation is a suitable model for the C1r activation in the C1 molecule, because the hemolytic activity of C1r was substantially diminished under the high pH conditions.

Complement Activating Enzymes↗
PubMed1984

Characterization of the initial C3 convertase of the alternative pathway of human complement.

C3(H2O),Bb, the initial C3 convertase of the alternative complement pathway, was demonstrated to be a metal-containing protein complex by sucrose density gradient ultracentrifugation. Demonstration of this labile enzyme became possible by increasing its half-life with nickel instead of magnesium ions used for enzyme formation. The enzyme was generated from C3, the internal thioester bond of which was hydrolyzed (C3(H2O)), and from 125I-Factor B and Factor D. The sedimentation coefficient of the enzyme complex was 10.7S. By using 63Ni for enzyme formation, the metal ion was detected in the enzyme complex after ultracentrifugation in the presence of 10 mM EDTA. The stoichiometry of the constituents in the C3(H2O),Bb(Ni) complex was 1:1:1. To verify that C3 is incorporated into the enzyme complex in the form of C3(H2O), the enzyme complex was adsorbed to anti-Factor B-Sepharose and subjected to decay-dissociation. Examination of the subsequently eluted protein by SDS gel electrophoresis under reducing conditions demonstrated the presence of an intact C3 alpha-chain. This work provides further evidence that a C3 convertase can be generated from noncleaved C3 that is modified at the thioester site. With the use of a fluorometric assay, the activity (kcat/Km) and the half-life of the initial C3 convertase were determined and compared to those of C3b,Bb.

Chemical Phenomena↗
PubMed2001

Evolution of the initiating enzymes of the complement system.

Analysis of the human MASP-1/3 gene, which encodes two proteases of the lectin-triggered complement cascade, has revealed alternatively used serine-protease-encoding regions for the gene's two protein products. Phylogenetic studies indicate that one arose by retrotransposition early in vertebrate evolution, supporting the idea that the lectin branch of the complement cascade arose earlier than the 'classical' pathway.

Alternative Splicing↗
PubMed1982

Dissociation of C1 and concentration dependence of its activation kinetics.

The activation of the zymogen C1s to the enzyme C1s in the human C1 complex [C1q(c1rC1s)2] was studied as a function of the concentrations of (C1rC1s)2 and C1q which were saturated with oligomers of rabbit IgG. A large concentration dependence of the sigmoidal kinetics was observed in the 2-180 nM concentration range. This was explained by association-dissociation equilibria between the antibody-saturated C1q and various forms of the (C1rC1s)2 complex (unactivated to activated). The establishment of these equilibria (binding constant 2 x 10(7) M-1) was assumed to be fast as compared to the rates of the activation steps (rate constants 10(-3) and 10(-2) sec-1 at 30 degrees C). The fast re-equilibration of the C1 complex explains the finding that small amounts of antibody-saturated C1q catalysed the activation of large amounts of C1s. The interpretation of the kinetic results was supported by a direct demonstration of the dissociation of C1 into C1q and (C1rC1s)2 by analytical and density gradient centrifugation. No difference was found between the rates of activation and the dissociation properties of reconstituted C1 and C1 isolated from serum.

Centrifugation, Density Gradient↗
PubMed1987

Covalent association of C3b with C4b within C5 convertase of the classical complement pathway.

The C5 convertase of the classical complement pathway is a complex enzyme consisting of three complement fragments, C4b, C2a, and C3b. Previous studies have elucidated functional roles of each subunit (4, 6, 7), but little is known about how the subunits associate with each other. In this investigation, we studied the nature of the classical C5 convertase that was assembled on sheep erythrocytes. We found that one of the nascent C3b molecules that had been generated by the C3 convertase directly bound covalently to C4b. C3b bound to the alpha' chain of C4b through an ester bond, which could be cleaved by treatment with hydroxylamine. The ester bond was rather unstable, with a half-life of 7.9 h at pH 7.4 and 37 degrees C. Formation of the C4b-C3b dimer is quite efficient; e.g., 54% of the cell-bound C3b was associated with C4b when 25,000 molecules of C4b and 12,000 molecules of C3b were present per cell. Kinetic analysis also showed the efficient formation of the C4b-C3b dimer; the rate of dimer formation was similar to or even faster than that of cell-bound monomeric C3b molecules. These results indicate that C4b is a highly reactive acceptor molecule for nascent C3b. High-affinity C5-binding sites with an association constant of 2.1 X 10(8) L/M were demonstrated on C4b-C3b dimer-bearing sheep erythrocytes, EAC43 cells. The number of high-affinity C5-binding sites coincided with the number of C4b-C3b dimers, but not with the total number of cell-bound C3b molecules. Anti-C4 antibodies caused 80% inhibition of the binding of C5 to EAC43 cells. These results suggest that only C4b-associated C3b serves as a high-affinity C5 binding site. EAC14 cells had a small amount of high-affinity C5 binding sites with an association constant of 8.1 X 10(7) L/M, 100 molecules of bound C4b being necessary for 1 binding site. In accordance with the hypothesis that C4b-associated C4b might also serve as a high-affinity C5-binding site, a small amount of C4b-C4b dimer was detected on EAC14 cells by SDS-PAGE analysis. Taken together, these observations indicate that the high-affinity binding of C5 is probably divalent, in that C5 recognizes both protomers in the dimers. The high-affinity binding may allow selective binding of C5 to the convertase in spite of surrounding monomeric C3b molecules.

Animals↗
PubMed1982

Circulating immune complexes in patients with progressive systemic sclerosis.

Forty-one patients with progressive systemic sclerosis were studied for the presence of immune complexes by the fluid- and solid-phase C1q binding, C1 activation, and the fluid-phase conglutinin assays. Complement activation and autoantibodies were also studied. Immune complexes were detected in only 6 patients (15%); activation of complement was found in 5 others. The clinical and serologic features of patients with complexes were compared with those in whom complexes were not identified. No significant difference was found with respect to serology. Organ involvement was generally more frequent in the group with immune complexes, but the difference was statistically significant only with respect to lung involvement. The present data suggest that, although complement-fixing immune complexes are infrequently detected in progressive systemic sclerosis, they may play a role in the pathogenesis of lung lesions associated with the disease.

Adult↗
PubMed1984

Lack of activation of C1, despite circulating immune complexes detected by two C1q methods, in patients with rheumatoid arthritis.

The activation of C1 by circulating immune complexes in patients with rheumatoid arthritis was investigated. C1rC1s(C1-In)2 complexes in EDTA-plasma, reflecting C1 activation in vivo, were slightly raised in 35 of 57 patients with rheumatoid arthritis, though most patients had elevated levels of circulating immune complexes as measured with either the 125I-C1q binding test or the C1q solid phase assay. The activation of C1 by circulating immune complexes in vitro was investigated by measuring the generation of C1rC1s(C1-In)2 complexes during 60 minutes at 37 degrees C in diluted recalcified EDTA-plasma. In 16 of the 57 patients, a slightly increased C1 activation in vitro was observed. These patients tended to have high levels of circulating immune complexes. However, the majority of the patients with high levels of circulating immune complexes showed a normal C1 activation in vitro. Therefore, it was concluded that measurement of circulating immune complexes by either of the two C1q methods in patients with rheumatoid arthritis does not imply that these circulating immune complexes are able to activate C1.

Aged↗
PubMed1983

The mechanism of synergistic complement-mediated lysis of rat red cells by monoclonal IgG antibodies.

The mechanism of synergistic complement-mediated lysis of rat red cells was investigated using rat monoclonal antibodies against class I RT1Aa antigens. The increased lytic activity when using two antibodies simultaneously is due to the increase in the number of activated C1 molecules on the cell surface and this results from (a) an increase in the number of binding sites for C1q, (b) an increase in the functional affinity constant for C1q binding and (c) an increase in the rate of activation of C1. Complete lysis of red cells was only achieved if one member of the synergistic pair was of the gamma 2b isotype, and this isotype was the only one to which binding of 125I-labeled C1q could be detected. A partial synergistic effect was seen using an F(ab')2 fragment of antibody. Increased uptake and activation of C1 probably results both from the presence of two antibodies attached to each antigen molecule and from the formation of antigen-antibody catenars.

Animals↗
PubMed1985

Effects of soluble aggregates of IgG on the binding, uptake and degradation of the C1q subcomponent of complement by adherent guinea pig peritoneal macrophages.

Earlier studies have indicated that C1q, the first subcomponent of complement component C1, is bound to lymphocytes via specific C1q receptor sites. We have recently shown that adherent guinea pig peritoneal exudate macrophages express specific receptors for C1q (Veerhuis, R. et al., Immunology 1985. 54: 801). The present studies were performed to determine whether binding of 125I-labeled human C1q (125I-C1qhu) to adherent guinea pig peritoneal exudate macrophages would also result in ingestion and subsequent degradation of 125I-C1qhu. The binding of 125I-C1qhu to adherent peritoneal macrophages at 4 degrees C is inhibited fully not only by C1qhu and guinea pig C1q (C1qgp) but also by pepsin fragments of C1qhu. The amount of trichloroacetic acid nonprecipitable radioactivity that appeared in the supernatant was used as a measure for the degradation of 125I-C1qhu. 125I-C1qhu is degraded initially into fragments of 25 kDa, after which it is degraded further into small molecular weight peptides. Ingestion of 125I-C1q by the macrophages occurs before the 125I-C1q is degraded. In the presence of limited amounts of soluble aggregates of guinea pig IgG2 (AIgG), a known activator of C1, part of the C1q is bound to the AIgG and all of the AIgG in turn is bound to the cellular Fc receptors leading to an enhanced binding of 125I-C1q to the cells, a binding that was maximal at near equimolar concentrations of 125I-C1qhu and 131I-AIgG. In the presence of a 30-fold excess of AIgG, however, only a small percentage of the AIgG binds to cellular Fc receptors and the interaction of C1q with its receptor is decreased due to competitive inhibition. The results presented in this report thus suggest that free C1q may be eliminated by specific interaction with C1q receptors present on circulating and tissue phagocytoses and, in addition, that in the presence of immune complexes modulation of elimination of C1q may be encountered.

Animals↗
PubMed1985

Antibody density on rat red cells determines the rate of activation of the complement component C1.

It is a common observation that there is variability in the rate of activation of C1, the first component of complement, when bound to immune complexes. The cause of this variation has been investigated with experiments designed to assess separately the effect of antibody, antigen and C1 density. Using 125I-labeled C1 and a rat monoclonal antibody specific for the class I antigen, it has been found that the rate of activation is primarily dependent on antibody density on the cell surface and not on antigen or C1 density. This finding supports the suggestion that direct contact between the C1r2C1s2 subcomponent of C1 and antibody may be required for potentiation of C1 activation.

Animals↗
PubMed1986

A model system for the study of the assembly and regulation of human complement C3 convertase (classical pathway).

The formation of classical C3 convertase of complement and its regulation by C4b-binding protein (C4bp) were studied using two different approaches: (a) the analysis was first carried out in fluid phase; a soluble stabilized C3 proconvertase could be assembled from C4b (or C4b-like C4) and iodine-treated C2 in the presence of Ni2+ ions. Upon activation of this complex by C1s, a C3 convertase C4b(C4b-like C4)-C2a was generated which was able to cleave purified C3. C4bp dissociated both C3 proconvertase and C3 convertase, but its effect was more important on C3 convertase. (b) A model system of phospholipid vesicles has been developed to study the assembly of the C3 convertase on a membrane. Among different phospholipid mixtures tested, P-glycerol/P-choline vesicles were found most effective for C4b binding. Optimal conditions were determined for C4b fixation on these vesicles; bound C4b participated in the formation of a functional membrane-associated C3 convertase. C4bp was found to bind to phospholipid vesicles with a higher affinity than C4b; it was able to dissociate the vesicle-associated C3 convertase.

Buffers↗
PubMed1988

Importance of antigen specificity for complement-mediated lysis by monoclonal antibodies.

Lysis of human lymphocytes by autologous complement had been studied using a range of monoclonal antibodies against different antigens. Antigen specificity (and not antibody isotype) was the most important factor which influenced cell lysis and this could not be accounted for merely by differences in surface density between antigens. Three antigens with comparable surface density were studied in detail: CAMPATH-1 (lytic), major histocompatibility complex class I (lytic) and leukocyte common antigen (poorly lytic). C1q binding was roughly proportional to antibody binding and dependent on antibody isotype. However, the lytic antibodies were much better able to bind and activate whole C1 than the poorly lytic ones. This result would not have been predicted from traditional concepts of complement activation but can be interpreted in the light of models for C1 activation which involve Fc-Fc interactions, Fc-C1r2s2 interactions and a critical C1q stem-arm angle for C1 binding and activation.

Antibodies, Monoclonal↗
PubMed1982

Complement components in kidney allograft recipients: relationship to cytomegalovirus infection.

Serum complement profiles of 19 renal transplant recipients were studied serially before and after renal transplantation to determine if there is any relationship between cytomegalovirus (CMV) infection and alteration in the serum complement (C) levels. Most patients had low serum C3 and C4 levels before transplantation, but afterwards these levels increased in some patients. Clq was elevated before and after transplantation. Factor B was low before transplantation, but after surgery, its level approached normal in patients without cytomegalovirus viremia, whereas in those with viremia, Factor B level became even more depressed. Circulating Factor B fragments were found in the sera of five out of seven patients with active cytomegalovirus infection, which suggests activation of the alternative pathway possibly related to active CMV infection.

Adult↗
PubMed1983

The first component of human complement (C1): activation and control.

The first component of human complement (C1) is a 750 000 dalton glycoprotein that requires calcium or other specific metal ions to maintain its native structure and function. Under physiologic conditions, C1 comprises two weakly interacting subunits, C1q and C1r2s2, with C1q containing the binding site(s) for activators and C1r2s2 possessing enzymatic potential. C1 circulates in a precursor state and only after "activation" does it acquire functional activity, manifested as enzymatic activity specific for its natural substrates C2 and C4. C1 activation, which is accompanied by limited proteolysis and conformational changes, can be induced by immune complexes or certain nonimmune substances. With C1 binding to an immune complex, the strength of interaction between C1q and C1r2s2 increases. C1 also spontaneously activates at 37 degrees C by an intramolecular autocatalytic mechanism although at a slower rate than that induced by activators. C1 functions are controlled by the serum glycoprotein C1-inhibitor (C1-In) which blocks the enzymatic activities of activated C1 (C1). Under physiologic conditions, C1 has a half-life of only 13 seconds in the presence of C1-In. C1 is efficiently disassembled by C1-In, thereby releasing two inactive C1rC1s(C1-In)2 complexes per C1 molecule, leaving C1q activator-bound with biologically reactive sites uncovered that are not expressed in macromolecular C1. The most recently recognized function of C1-In is that of controlling the C1 activation process itself. While having only limited effect on immune complex-induced C1 activation, C1-In effectively controls certain nonimmune-induced as well as spontaneous C1 activation. Thus C1-In plays an important role in regulating nonspecific complement activation. The latter observation is relevant for the understanding of the human disease hereditary angioedema. An overabundance of spontaneous C1 autoactivation, due to low C1-In levels, might underlie the abnormal activation of complement via the classical pathway detected in the sera of these patients. Finally, recent studies indicate that C1 may have other important biologic functions in addition to initiating the complement cascade.

Angioedema↗
PubMed1985

Correlation of disease activity with circulating immune complexes (C1qbA) and complement breakdown products (C3D) in patients with systemic lupus erythematosus. A prospective study.

Most biologic effects of immune complexes are mediated through the activation of the complement system. The relationship between lupus disease activity and the presence of C3 breakdown products (C3d) and circulating immune complexes (CIC) as demonstrated with the C1q binding assay (C1qbA), was evaluated. Nearly all 13 systemic lupus erythematosus (SLE) patients had a stable disease course in this prospective study, nevertheless, in each patient the profiles of the serologic parameters were quite different. Despite the small number of investigated patients (13), it is concluded that irrespective of the disease activity, the serologic parameters could be either positive or negative. No relationship could be obtained between disease activity and the presence of C3d and/or CIC. Nor was there any evidence that the presence of CIC would indicate increased levels of C3 breakdown products (C3d). This observation argues against a pathogenetic significance of CIC detected by the C1qbA in SLE. In conclusion, the supposed link between the presence of CIC, consumption and activation of the complement system, and the activity of SLE needs further study.

Adult↗
PubMed1986

Inhibition of C1q binding to antigen-antibody complexes by a factor in rheumatoid arthritis serum.

The sera and synovial fluids of patients with rheumatoid arthritis (RA) contain a factor which decreases the binding of C1q to antigen-antibody complex (IC). Several lines of evidence suggest that this factor is distinct from the documented C1q inhibitor which is a chondroitin sulphate. It binds to IC rather than to C1q. It is resistant to digestion with chondroitinase ABC. The addition of chondroitin sulphate to serum does not inhibit the binding of IC to C1q. The observation that three purified IgM and IgG rheumatoid factors (RF) did not reduce C1q binding to IC indicates that the factor is not RF. The ability of RA sera to reduce IC binding to C1q was inversely correlated with their ability to prevent immune precipitation (PIP), and directly with levels of an inhibitor of PIP. These data suggest that the factor which binds to IC and reduces C1q binding may be responsible for the excessive immune precipitation which occurs in RA sera.

Antigen-Antibody Complex↗