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PubMed1982

A study of a covalent-like interaction between soluble nascent C4b and C4-binding protein.

In the classical pathway of complement, the interaction between C4b and C4bp can be considered as a control of the C3 convertase formation. Purified C4-binding protein (C4bp) interacts with soluble nascent C4b to form covalent-like complexes; the interaction is also possible with nascent C4b-like C4, but not with C4, C4b or C4b-like C4. Formation of the complexes upon incubation of C4bp, C4 and C1s appears to involve a single link between a subunit of C4bp and the alpha' chain of C4b, as observed by SDS-polyacrylamide gel electrophoresis in reducing conditions (160 000 dalton band). In non-reducing conditions, a mixture of C4b-C4bp complexes is observed as a function of the C4b:C4bp molar ratio, with apparent molecular weights differing by a value of 210 000 and reflecting different C4b-C4bp associations. A maximum of five molecules of C4b are bound per molecule of C4bp, which appears to consist of 10 subunits of apparent molecular weight 72 000. The link between C4b and C4bp is partially destroyed by 1 M hydroxylamine at pH 9.0; its formation is strongly inhibited by 3.5 mM hydroxylamine or 60 mM methylamine at pH 9.0. These findings suggest an ester or amide bond between the activated carboxyl group of the thioester bridge in the alpha' or alpha chain of nascent C4b or C4b-like C4 and a hydroxyl or amino group of C4bp. Thus, C4bp might compete with other C4b acceptors such as membranes or IgG.

Carrier Proteins↗
PubMed1983

The reaction of iodine and thiol-blocking reagents with human complement components C2 and factor B. Purification and N-terminal amino acid sequence of a peptide from C2a containing a free thiol group.

Human complement components C2 and Factor B each contain one free thiol group/molecule. Reaction with p-chloromercuribenzoate destroyed the haemolytic activity of C2 but had no effect on Factor B. Reaction of C2 with I2 gave a 16-fold enhancement of its haemolytic activity. The pH optimum for the reaction was 7.0. The I2 reacted at the thiol group in C2 with a stoicheiometry of 1 mol of I2/mol of C2. The product of the reaction was unaffected by millimolar concentrations of dithiothreitol; however, azide and cyanide were inhibitory. Reaction with azide did not result in re-expression of the thiol group. Mild oxidation of the thiol group with m-chloroperbenzoic acid did not enhance the haemolytic activity. The results suggest that reaction with I2 causes intramolecular covalent, but not disulphide, bond formation. I2 reacted with Factor B at the free thiol group without affecting the haemolytic activity. A CNBr-cleavage peptide from C2a (obtained by cleavage of C2 by subcomponent C1s) containing the free thiol group was isolated. Automated Edman degradation of the peptide showed that it was the N-terminal peptide of C2a. The free thiol group was identified at position 18.

Amino Acid Sequence↗
PubMed1980

Analysis of receptor-mediated C1q binding to human peripheral blood mononuclear cells.

The binding of C1q to human peripheral blood leukocytes has been investigated. Studies with fluorescein conjugated F(ab')2 anti-C1q show that few (0 to 4%) normal leukocytes isolated in the presence of EDTA have C1q on their surface. However, approximately 26% of the mononuclear cell population is able to bind added C1q. Quantitative binding studies using 125I-C1q show that the binding to mononuclear cells is specific, saturable, and reversible. Scatchard plot analyses indicate an approximate equilibrium constant of 1.2 times 10(7) M-1. C1q binding appears to be mediated via the collagenous portion of the molecule in that 1) type I collagen inhibits this binding; 2) C1 reconstituted from purified C1q, C1r, and C1s does not bind to mononuclear cells, whereas the same amount of free C1q binds avidly; and 3) C1q enhances the binding of aggregated IgG to mononuclear cells.

Binding Sites↗
PubMed1986

Activation of the first component of human complement, C1, by monoclonal antibodies directed against different domains of subcomponent C1q.

Two monoclonal antibodies directed against C1q, and their (Fab)2 and Fab fragments, were used to study the mechanism of C1 activation. Monoclonal antibody 2A10, an IgG2a, was digested by pepsin to yield fully immunoreactive (Fab')2. Monoclonal antibody 1H11, an IgG1, was digested by papain to yield fully immunoreactive, bivalent (Fab)2. Previously 1H11 had been shown to bind to the C1q "heads," whereas 2A10 bound to stalks. Activation of C1 was followed by the cleavage of 125I-C1s in the presence of C1 inhibitor (C1-Inh) at 37 degrees C. Spontaneous activation was minimal at inhibitor concentrations above 0.4 micron (1.3 X physiologic inhibitor concentration); all results were corrected for the spontaneous activation background. Heat-aggregated IgG activated completely in this system and was taken as 100% activation. Monoclonal antibody 2A10 caused precipitation of C1 and slow activation; neither the (Fab')2 nor the Fab' derived from 2A10-caused activation. Probably, aggregates of intact 2A10 and C1 were serving as immune complexes to activate other molecules of C1. In contrast, both 1H11 and its (Fab)2 activated completely and stoichiometrically; that is, maximal activation was achieved at a ratio of one C1q head to one antibody combining site. The monovalent Fab derived from 1H11 bound well to C1q, but no activation of C1 was observed. Thus, bivalent binding of this head-binding monoclonal is required for C1 activation, but not the presence of the antibody Fc portion. Neither 1H11 nor its (Fab)2 fragments caused C1 precipitation; however, the 1H11 did form complexes composed of two C1q cross-linked by multiple 1H11, which were visualized by electron microscopy. The presence of these dimeric complexes correlated well with activation. A model for C1 activation is proposed in which two C1q subcomponents are held together by multiple (Fab)2 bridging C1q heads. The model is roughly analogous to touching opposing pairs of fingers and thumb tips, the two hands representing the two C1q, forming a cage. C1-Inh, which probably binds to C1r through the open end of the C1 cone, is too long asymmetric to be included within the cage. Thus, according to this model, the dimers of C1 are released from the inhibitory action of C1-Inh, and activation proceeds spontaneously and rapidly at 37 degrees C.

Animals↗
PubMedDate not supplied

Functional effects of domain deletions in a multidomain serine protease, C1r.

The C1r subcomponent of the first component of complement is a complex, multidomain glycoprotein containing five regulatory or binding modules in addition to the serine protease domain. To reveal the functional role of the N-terminal regulatory domains, two deletion mutants of C1r were constructed. One mutant comprises the N-terminal half of domain I joined to the second half of the highly homologous domain III, resulting in one chimeric domain in the N-terminal region, instead of domains I-III. In the second mutant most of the N-terminal portion of domain I was deleted. Both deletion mutants were expressed in the baculovirus-insect cell expression system with yields typical of wild type C1r. Both mutants maintained the ability of the wild type C1r to dimerize. The folding and secretion of the recombinant proteins was not affected by these deletions, and C1-inhibitor binding was not impaired. The stability of the zymogen was significantly decreased however, indicating that the N-terminal region of the C1r molecule contains essential elements involved in the control of activation of the serine protease module. Tetramer formation with C1s in the presence of Ca2+ was abolished by both deletions. We suggest that the first domain of C1r is essential for tetramer formation, since the deletion of domain I from C1r impairs this interaction.

Amino Acid Sequence↗
PubMedDate not supplied

[Role of C1 subcomponents in platelet aggregation induced by aggregated IgG].

Studies have been performed with platelets using C1 haemolytic assays and platelet aggregation induced by anti-C1q, anti-C1s and aggregated IgG in the presence of C1 subcomponents C1q, C1r and C1s. C1q was removed by EDTA or modified by collagenase from human platelets while after the same treatment C1s remained bound to the platelets. EDTA-treated platelets were no longer aggregated by aggregated IgG. The addition of C1q restored the reactivity of the platelets to aggregated IgG while the addition of C1s or C1s was without effect. Furthermore, the addition of C1r or C1s to C1q inhibited the action of C1q in platelet aggregation induced by IgG. The possible association between the different C1 subcomponents and human platelets is discussed.

Complement C1↗
PubMed1993

Recombinant C1 inhibitor P5/P3 variants display resistance to catalytic inactivation by stimulated neutrophils.

Proteolytic inactivation of serine protease inhibitors (serpins) by neutrophil elastase (HNE) is presumed to contribute to the deregulation of plasma cascade systems in septic shock. Here, we report a supplementary approach to construct serpins, in our case C1 inhibitor, that are resistant to catalytic inactivation by HNE. Instead of shifting the specificity of alpha 1-antitrypsin towards the proteases of the contact activation and complement systems, we attempted to obtain a C1 inhibitor species which resists proteolytic inactivation by HNE. 12 recombinant C1 inhibitor variants were produced with mainly conservative substitutions at the cleavage sites for HNE, 440-Ile and/or 442-Val. Three variants significantly resisted proteolytic inactivation, both by purified HNE, as well as by activated neutrophils. The increase in functional half-life in the presence of FMLP-stimulated cells was found to be 18-fold for the 440-Leu/442-Ala variant. Inhibitory function of these variants was relatively unimpaired, as examined by the formation of stable complexes with C1s, beta-Factor XIIa, kallikrein, and plasmin, and as determined by kinetic analysis. The calculated association rate constants (k(on)) were reduced twofold at most for C1s, and appeared unaffected for beta-Factor XIIa. The effect on the k(on) with kallikrein was more pronounced, ranging from a significant ninefold reduction to an unmodified rate. The results show that the reactive centre loop of C1 inhibitor can be modified towards decreased sensitivity for nontarget proteases without loss of specificity for target proteases. We conclude that this approach extends the possibilities of applying recombinant serpin variants for therapeutic use in inflammatory diseases.

Amino Acid Sequence↗
PubMed1976

C1 and human platelets. III. Role of C1 subcomponents in platelet aggregation induced by aggregated IgG.

Studies have been performed with platelets using C1 haemolytic assays and platelet aggregation induced by anti-C1q, anti-C1s and aggregated IgG in the presence of C1 subcomponents C1q, C1r and C1s. C1q was removed by EDTA or modified by collagenase from human platelets while after the same treatment C1s remained bound to the platelets. EDTA treated platelets were no longer aggregated by aggregated IgG. The addition of C1q restored the reactivity of the platelets to aggregated IgG while the addition of C1r or C1s was without effect. Furthermore, the addition of C1r or C1s to C1q inhibited the action of C1q in platelet aggregation induced by IgG.The possible association between the different C1 subcomponents and human platelets is discussed.

Complement C1↗
PubMed1981

Treatment of human complement components C4 and C3 with amines or chaotropic ions. Evidence of a functional and structural change that provides uncleaved C4 and C3 with properties of their soluble activated froms, C4b and C3b.

Treatment of human components C4 and C3 with amines like hydrazine, ammonium hydroxide, and neutral ammonium salts or with chaotropic salts like KSCN and NaBr leads to complete loss of haemolytic activity. The pretreated components are, however, still active in formation of soluble C3 convertases. This activity pattern is reminiscent of the activities of C4 and C3 that have been activated by cleavage in the fluid phase. Indeed, the antigenic properties of pretreated C4 and C3 are similar to soluble C4b and C3b. The polypeptide chain structure of pretreated C4 and C3, is, however, identical to that of the untreated components when investigated by SDS gel electrophoresis. Pretreatment even reduces greatly the susceptibility of C4 to cleavage by C1s and of C3 to cleavage by classical and alternative pathway C3 convertases. Pretreated components have lost the ability to combine with EAC1 and EAC142, respectively; this fact explains their failure to exhibit haemolytic activity. In serum, pretreated C4 and C3 are cleaved in a manner similar to C4b and C3b. Amines and chaotropic ions cause the same functional and structural alterations, which are best explained by assumption of a conformational change. A similar transformation can also occur in C4 and C3 during preparation or storage.

Amines↗
PubMed1991

Mechanism of serpin action: evidence that C1 inhibitor functions as a suicide substrate.

Serpins form a family of structurally related proteins, many of which function in plasma as inhibitors of serine proteases involved in inflammation, blood coagulation, fibrinolysis, and complement activation. To further characterize the mechanism by which serpins inhibit their target enzymes, we have studied the effect of temperature on the reaction of C1 inhibitor and the serine protease plasma kallikrein. At both 38 and 4 degrees C, C1 inhibitor (Mr 105,000) is cleaved by alpha-kallikrein (Mr 85,000 and 88,000) at position P1 (Arg444) of the reactive center, a reaction that leads to the formation of a covalent bimolecular enzyme-serpin complex (Mr 195,000) and cleaved but uncomplexed serpin (Mr 95,000). Between 38 and 4 degrees C, the product distribution is temperature-dependent, with more cleaved C1 inhibitor (Mr 95,000) formed at lower temperatures and correspondingly less Mr 195,000 complex. Studies employing intrinsic tryptophan fluorescence and 1H NMR spectroscopy show that this behavior is not caused by temperature-dependent conformational changes of kallikrein or C1 inhibitor. C1 inhibitor also behaves in this manner with the light chain of kallikrein and, to a lesser extent, with plasmin and C1s. These data are best explained by a branched reaction pathway, identical with the scheme describing the mechanism of action of suicide substrates. This scheme involves the formation of an enzyme-inhibitor intermediate, which can be stabilized into a covalent complex and/or dissociate into free enzyme and cleaved inhibitor, depending on the reaction conditions.

Complement C1 Inactivator Proteins↗
PubMed1978

Biosynthesis of the first component of complement by human and guinea pig peritoneal macrophages: evidence for an independent production of the C1 subunits.

The first component of complement, C1, was produced by human as well as guinea pig macrophages. Supernatants from serum-free cultured macrophage monolayers, tested separately for C1q and C1 activity, showed a ratio of 15:1, respectively, and a different time course of C1q and C1 production, indicating that the subcomponents of C1 are synthesized independently. Heat-inactivated fetal calf or guinea pig serum (2 hr, 56 degrees C), added to the culture medium, were found to be inhibitory for C1 but not for C1q activity, obviously due to still active C1 inhibitor in these sera. De novo synthesis was confirmed by reversible inhibition of C1 and C1q production by cycloheximide (0.5 microgram/ml) and puromycin (1 microgram/ml), by incorporation of radiolabeled amino acids into the C1s subcomponent, and by uptake of incorporated radioactivity by EA, which was sensitive to EDTA. In additional experiments, 2,2'-dipyridyl markedly reduced C1q and also C1 secretion in the supernatants. Inhibition of the prolyl and lysyl hydroxylation and, as a consequence, structural instability of the collagenous region in the C1q molecule, is discussed as the cause of this effect which is analogous to collagen biosynthesis.

2,2'-Dipyridyl↗
PubMed1984

Distinct primary translation products from human liver mRNA give rise to secreted and cell-associated forms of complement protein C2.

The second component of complement (C2), is a class III major histocompatibility complex gene product and a glycoprotein in the classical complement activating system. Synthesis in the human hepatoma-derived cell line HepG2 results in three intracellular forms: an 84-kDa form secreted in 1-2 h; 79-kDa and 70-kDa forms that remain cell-associated for intervals up to 12 h. All three forms are C2 polypeptides as demonstrated by inhibition of immunoprecipitation with unlabeled C2 and the presence of common major peptide fragments following chymotryptic digestion. The cell-associated forms of C2 are not products of proteolysis as demonstrated by experiments with multiple proteinase inhibitors and by observations of the kinetics of synthesis. Inhibition of core glycosylation by tunicamycin and deglycosylation by acid hydrolysis indicate that the three intracellular C2 polypeptides are glycosylated to a similar extent. Although the 84-kDa form of C2 is susceptible to C1s cleavage, the two cell-associated forms are not. Cell-free biosynthesis by mRNA from HepG2 or human liver results in three primary translation products corresponding to the three unglycosylated forms of C2. These results indicate that HepG2 cells synthesize C2 protein in both secreted and cell-associated forms and that each form is derived from a separate primary translation product.

Carcinoma, Hepatocellular↗
PubMed1999

Monomeric complement-activating IgG paraproteins.

Three patients presented a unique syndrome of recurrent panniculitis with an IgGkappa paraprotein and depletion of the early components of the classical pathway of complement. The IgGkappa paraproteins were monomers with a normal structure, and with no evidence for aggregation, as assessed by electron microscopy and ultracentrifugation. Both heavy and light chains were of normal molecular size (SDS-PAGE), and the paraproteins were not heavily glycosylated. However, the paraproteins from all three patients had unusual features that included abnormal behavior on gel filtration chromatography and a heavy chain of high pI. When analyzed by fast protein liquid chromatography (Superdex 200), elution of the paraproteins was retarded, particularly when the ionic strength was increased. This retardation was partially reversed in 20% alcohol, and fully reversed in 6 M guanidine-HCl. Neither anti-C1 inhibitor nor anti-C1q autoantibodies were found in any of the patients' sera. However, the paraproteins bound to the globular heads of C1q at normal ionic strength. They activated C4 in normal human serum, but not in C1q-deficient serum. Activation led to the formation of C1s-C1 inhibitor complexes. Taken together, the data suggest that the unusual paraproteins have the capacity to bind C1q, which then leads to activation of C1. The ability of these paraproteins to activate C1, in spite of their being soluble monomers, is likely to be related to their unique physicochemical features.

Chromatography, Gel↗
PubMed2001

Beta-amyloid fibrils activate the C1 complex of complement under physiological conditions: evidence for a binding site for A beta on the C1q globular regions.

Previous studies based on the use of serum as a source of C have shown that fibrils of beta-amyloid peptides that accumulate in the brain of patients with Alzheimer's disease have the ability to bind C1q and activate the classical C pathway. The objective of the present work was to test the ability of fibrils of peptide Abeta1-42 to trigger direct activation of the C1 complex and to carry out further investigations on the site(s) of C1q involved in the interaction with Abeta1-42. Using C1 reconstituted from purified C1q, C1r, and C1s, it was shown that Abeta1-42 fibrils trigger direct C1 activation both in the absence of C1 inhibitor and at C1 inhibitor:C1 ratios up to 8:0, i.e., under conditions consistent with the physiological context in serum. The truncated peptide Abeta12-42 and the double mutant (D7N, E11Q) of Abeta1-42 did not yield C1 activation, providing further evidence that the C1 binding site of beta-amyloid fibrils is located in the acidic N-terminal 1-11 region of the Abeta1-42 peptide. Binding studies performed using a solid phase assay provided strong evidence that C1q interacts with Abeta1-42 fibrils through its C-terminal globular regions. In contrast to previous studies based on a different experimental design, no significant involvement of the C1q collagen-like domain was detected. These findings were confirmed by additional experiments based on C1 activation and C4 consumption assays. These observations provide direct evidence of the ability of beta-amyloid fibrils to trigger activation of the classical C pathway and further support the hypothesis that C activation may be a component of the pathogenesis of Alzheimer's disease.

Amino Acid Sequence↗
PubMed1987

Kallikrein inhibition and C1-esterase inhibitor levels in patients with the lupus inhibitor.

It has been suggested that kallikrein inhibition may predispose patients with the lupus inhibitor to thrombosis by interfering with the Factor XII-mediated activation of plasminogen. To further investigate this suggestion, the authors measured kallikrein inhibition in 19 patients with the lupus inhibitor. They found that kallikrein inhibition was greater than 100% of that of a normal plasma pool in all patients and greater than 125% in 11 of 19. Kallikrein inhibition was significantly correlated with C1-esterase inhibitor (C1S-INH) concentration, which they measured by rocket immunoelectrophoresis (r = +0.55, P less than 0.05). In three patients the C1S-INH was more than 30% greater than the kallikrein inhibition. Crossed immunoelectrophoresis for C1S-INH in these patients' plasma revealed an electrophoretic mobility identical with that of the normal plasma pool. The authors suggest that C1S-INH-mediated kallikrein inhibition, in conjunction with other coagulation abnormalities, predisposes patients with the lupus inhibitor to thrombosis.

Adolescent↗
PubMed1997

Contribution of the complement control protein modules of C2 in C4b binding assessed by analysis of C2/factor B chimeras.

To identify the complement control protein (CCP) module(s) of C2 that are required for C4b recognition, we constructed a panel of C2/factor B chimeras by substituting intact or partial factor B CCP modules for the corresponding ones of C2. Epitope mapping indicated that the anti-C2b mAb 3A3.3, which inhibits binding of C2 to C4b, reacts with the second CCP of C2 and similarly the anti-Ba mAb HA4-1A, which inhibits binding of factor B to C3b, reacts with the second CCP of factor B. The hemolytic activity of the chimeras CP1, CP2, and CP3a containing CCP1, CCP2, and a fragment of CCP3 of factor B, respectively, was substantially decreased compared with that of wild-type C2. The CP3 and CP1-3 chimeras, in which CCP3 and all three CCP modules of factor B, respectively, were substituted, had no hemolytic activity. Loss of activity could be attributed to the resistance of these two chimeras to C1s cleavage, which was probably due to conformational changes of the cleavage site. The combined results indicate that all three CCP modules of C2 contribute structural elements to the C4b-binding site of C2b. This site has been shown previously to be necessary for the initial binding of C2 to C4b which leads to the formation of the classical pathway C3 convertase.

Antibodies, Monoclonal↗
PubMed1991

Interaction of the envelope glycoprotein of human immunodeficiency virus with C1q and fibronectin under conditions present in human saliva.

Human saliva has been shown to reduce the infectivity of human immunodeficiency virus (HIV) particles in vitro. The factors in human saliva involved in this inhibition of HIV infectivity are unknown, although the salivary sediment of normal individuals has the major HIV neutralizing activity. Interestingly, the first complement component (C1) has been detected on the surface of the salivary sediment in the whole saliva of normal individuals. At the relatively low ionic strength of saliva, we determined that purified human C1q bound with high affinity to the envelope glycoprotein of HIV. Normally, the interaction of the C1q globular heads with immune complexes causes C1 activation. However, direct interactions between C1 and rgp120 (or rgp160) did not lead to C1 fixation, as determined by hemolytic studies with rate-limiting levels of C1, nor did rgp120 cause C1 activation as determined by activated C1s-mediated C4 conversion in normal human serum. Using ELISA, it was observed that intact C1, with the C1r2C1s2 tetramer associated with the collagen-like stem of C1q, did not bind to immobilized rgp120, whereas free C1q did bind. In addition, digestion of the C1q stem portion with collagenase completely eliminated its binding to rgp120. These findings suggest that the collagen-like stem region of C1q, rather than the globular heads, may participate in the binding to the envelope glycoprotein of HIV. Fibronectin, which is present in submandibular saliva, appeared to bind to rgp120 and to enhance the interaction of C1q with rgp120. It is conceivable that C1q and fibronectin, in binding and sequestering HIV particles (i.e. to the salivary sediment), may play an important role in the reduction of HIV transmission via saliva. Further studies will be needed to test the latter speculation.

Blotting, Western↗
PubMed1977

Purification and structural analysis of the fourth component of human complement.

The fourth component of human complement (C4) has been purified in 20% yield from fresh plasma using as starting material the 5-12% poly(ethylene glycol) precipitate which had been depleted of plasminogen by an affinity adsorbent. Sequential ion-exchange chromatography on diethylaminoethylcellulose, QAE-Sephadex, and DEAE-Bio-Gel A resulted in C4 homogeneous by immunological criteria and by polyacrylamide gel electrophoresis, the last chromatographic step achieving separation of native from inactivated C4. Reduction with 20 mM dithiothreitol for 2 h at 37 degrees C in 0.25 M 2-amino-2-hydroxymethyl-1,3-propanediol hydrochloride, pH 8.6, effected cleavage of the interchain disulfide bonds. A three-chain structure for C4 was confirmed, and molecular weight estimates of 93 000 +/- 9300, 75 000 +/- 7500, and 30 000 +/- 3000 determined for the alpha, beta, and gamma chains, respectively. The effects of known inactivators of C4 upon the chains of C4 were investigated, confirming that the inactivations by C1s and trypsin were accompanied by the fragmentation of the alpha chain. Inactivation of C4 by hydrazine, on the other hand, produced no detectable change in chain size. Separation of the chains was accomplished by gel filtration in the presence of 1 M acetic acid. Amino acid compositions of native C4 and the constitutive chains have been performed, and N-terminal sequences of the latter established by automated Edman degradation.

Amino Acid Sequence↗