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Results for “Complement C3 Convertase, Alternative Pathway”

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At least 19 recordsLinked to original sources

PubMed2007

Decay-accelerating factor must bind both components of the complement alternative pathway C3 convertase to mediate efficient decay.

Decay-accelerating factor (DAF; CD55) inhibits the complement (C) cascade by dissociating the multimolecular C3 convertase enzymes central to amplification. We have previously demonstrated using surface plasmon resonance (Biacore International) that DAF mediates decay of the alternative pathway C3 convertase, C3bBb, but not of the inactive proenzyme, C3bB, and have shown that the major site of interaction is with the larger cleavage subunit factor B (Bb) subunit. In this study, we dissect these interactions and demonstrate that the second short consensus repeat (SCR) domain of DAF (SCR2) interacts only with Bb, whereas SCR4 interacts with C3b. Despite earlier studies that found SCR3 to be critical to DAF activity, we find that SCR3 does not directly interact with either subunit. Furthermore, we demonstrate that properdin, a positive regulator of the alternative pathway, does not directly interact with DAF. Extending from studies of binding to decay-accelerating activity, we show that truncated forms of DAF consisting of SCRs 2 and 3 bind the convertase stably via SCR2-Bb interactions but have little functional activity. In contrast, an SCR34 construct mediates decay acceleration, presumably due to SCR4-C3b interactions demonstrated above, because SCR3 alone has no binding or functional effect. We propose that DAF interacts with C3bBb through major sites in SCR2 and SCR4. Binding to Bb via SCR2 increases avidity of binding, concentrating DAF on the active convertase, whereas more transient interactions through SCR4 with C3b directly mediate decay acceleration. These data provide new insights into the mechanisms involved in C3 convertase decay by DAF.

Biological Assay↗

PubMedDate not supplied

Differences in Cryptococcus neoformans capsular polysaccharide structure influence assembly of alternative complement pathway C3 convertase on fungal surfaces.

Binding of complement component C3 and Factor B to Cryptococcus neoformans serotypes A through D via the alternative complement pathway was measured in a system containing fresh nonimmune human serum. Serotypes B and C (C. neoformans var. gattii) bound approximately half as many molecules of both complement components as serotypes A and D (C. neoformans var. neoformans). In contrast, removal of xylosyl and glucuronyl side chains from the mannan main chain of capsular polysaccharide by the Smith degradation procedure resulted in binding of similar quantities of C3 to each of the four serotypes. We conclude that the relatively high degree of side chain substitution of capsular polysaccharide from C. neoformans variety gattii contributes to inefficient surface assembly of the alternative pathway C3 convertase. Inefficient binding of alternative pathway complement components to serotypes B and C may contribute to the relative difficulty in successfully treating infections caused by these organisms.

Acetylation↗

PubMed1984

Protection of the classical and alternative complement pathway C3 convertases, stabilized by nephritic factors, from decay by the human C3b receptor.

Formation and function of the classical (C4b,2a) and alternative (C3b,Bb) complement pathway C3 convertases are regulated by the intrinsic lability of the enzymes, extrinsic decay by C4bp and H, cleavage of C4b and C3b by I, and by the inhibitory action of the C3b receptor molecule (CR1). Binding of C4 nephritic factor (C4Nef) to C4b and of C3 nephritic factor (C3Nef) to C3b stabilizes the C3 convertases and bypasses inactivation by C4bp, H and/or I. In the present study, binding of C4Nef to the classical C3 convertase was found to prevent decay of C4b,2a by inputs of CR1 that were at least 15 times the amount of CR1 which inactivated 50% unstabilized classical pathway C3 convertase sites in 2.5 min. CR1 could however inhibit lysis of C4b,2a(C4Nef)-bearing cells in a dose-dependent manner. The latter inhibitory effect was directed at the interaction of C5 with the C5 convertase, most likely at C5 binding to cell-bound C3b. In an analogous manner to C4Nef in the classical pathway, stabilization of alternative pathway C3b,Bb convertase sites by C3Nef resulted in a relative protection of C3 convertase sites from decay by CR1. Thus, C4Nef and C3Nef can bypass all mechanisms susceptible to regulate function of the classical and alternative pathway C3 convertases. Because CR1 is essential for degradation of C3b bound to immune complexes in whole blood, stabilization of C4b,2a and C3b,Bb by C4Nef and C3Nef may alter in vivo processing of immune complexes in patients with nephritic factors.

Complement Activating Enzymes↗

PubMed1999

Decay acceleration of the complement alternative pathway C3 convertase.

An ELISA-based method is described for analyzing the mechanism by which the decay of the alternative pathway C3 convertase is accelerated by C3 regulatory proteins. Using this assay, we show that human decay-accelerating factor (DAF) and factor H are active on mature convertase complexes (C3bBb) but not on their nascent precursor (C3bB). This finding has implications on the mechanisms of action of these two regulators. The complement convertases cleave the serum protein C3, and the resulting C3b activation fragments covalently attach to nearby targets where they direct antigen selection, immune clearance, and cell lysis. Several proteins, including the membrane protein DAF, and the serum protein factor H, limit convertase activity by promoting their irreversible dissociation. An understanding of the biochemical mechanisms providing for their activities would be helpful for the therapeutic control of the complement response.

CD55 Antigens↗

PubMed2005

IgG naturally occurring antibodies stabilize and promote the generation of the alternative complement pathway C3 convertase.

Normal human IgG contains naturally occurring anti-C3 antibodies (anti-C3 NAbs) that have been proposed to regulate complement amplification. Here, we report a novel procedure for anti-C3 NAb purification. Pooled human IgG was fractionated on a DEAE column prior to affinity chromatography on IgG and then on C3. Anti-C3 NAbs co-purified with anti-F(ab')2 NAbs. In a refined protocol, IgG fractions were absorbed on Fc, F(ab')2, and C3, which allowed to isolate the directly accessible NAbs and to remove IgG hinge-region-specific NAbs. Since a substantial fraction of total anti-C3 NAbs in whole IgG pre-existed as complexes, IgG that did not bind to the three affinity columns was treated with urea and the affinity chromatography repeated to collect the dissociated NAbs. The urea-accessible anti-F(ab')2 NAbs were rather pure but anti-C3 NAbs yet contained substantial amounts of anti-F(ab')2 NAbs. Anti-C3 NAbs showed up to 400-fold and anti-F(ab')2 NAbs, up to 30-fold enrichment as compared to pooled normal human IgG. Anti-C3 NAb preparations exhibited nephritic factor activity that was up to 60 times stronger than that of total IgG from a patient with membranoproliferative glomerulonephritis type 2. In addition, anti-C3 NAbs promoted C3 convertase generation, when added to the convertase precursor or during convertase assembly, suggesting a non-nephritic-factor mechanism. Factors H and I reduced the overall level of activity but had no influence on the NAb dose-response curve meaning that NAbs did not interfere with factor H binding. Convertase promoting activity during assembly correlated with the content of anti-C3 NAbs in NAb complexes. In conclusion, anti-C3 NAbs associated with framework-specific anti-idiotypic NAbs stabilize C3 convertase and promote its generation but their activity is compensated for in whole IgG.

Antibodies, Anti-Idiotypic↗

PubMed2004

Structural analysis of engineered Bb fragment of complement factor B: insights into the activation mechanism of the alternative pathway C3-convertase.

The C-terminal fragment, Bb, of factor B combines with C3b to form the pivotal C3-convertase, C3bBb, of alternative complement pathway. Bb consists of a von Willebrand factor type A (vWFA) domain that is structurally similar to the I domains of integrins and a serine protease (SP) domain that is in inactive conformation. The structure of the C3bBb complex would be important in deciphering the activation mechanism of the SP domain. However, C3bBb is labile and not amenable to X-ray diffraction studies. We engineered a disulfide bond in the vWFA domain of Bb homologous to that shown to lock I domains in active conformation. The crystal structures of Bb(C428-C435) and its inhibitor complexes reveal that the adoption of the "active" conformation by the vWFA domain is not sufficient to activate the C3-convertase catalytic apparatus and also provide insights into the possible mode of C3-convertase activation.

Animals↗

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

Structural biology of the alternative pathway convertase.

Complement convertases are bimolecular complexes expressing protease activity only against C3 and C5. Their action is necessary for production of the biological activities of the complement system. Formation of these complexes proceeds through sequential protein-protein interactions and proteolytic cleavages of high specificity. Recent structural, mutational and functional data on factors D and B have significantly enhanced our understanding of the assembly, action, and regulation of the alternative pathway convertase. These processes were shown to depend critically on conformational changes, only some of which are reversible. The need for such changes is dictated by the zymogen-like configurations of the active centers of these unique serine proteases. The structural determinants of some of these changes have been defined from structural and mutational analyses of the two enzymes. Transition of factor D from the zymogen-like to the catalytically active conformation is completely reversible, while the active conformation of the catalytic center of the Bb fragment of factor B is irreversibly attenuated to a great extent on dissociation of the convertase complex. Both mechanisms contribute to the regulation of the proteolytic activity of these enzymes. Additional studies are necessary for a complete description of the elegant mechanisms mediating these processes.

Animals↗

PubMed1988

Analysis of the interaction between properdin and factor B, components of the alternative-pathway C3 convertase of complement.

The interactions between Factor B (B), its activation products Ba and Bb, properdin (P) and C3i or C3b, components that together form the alternative-pathway C3 convertase enzyme of human complement, have been analysed. Fluid-phase complexes of the purified components C3i, B and P were probed with the homobifunctional cross-linking reagent disuccinimidyl tartarate, and efficient cross-linking of B to P was observed. The 140 kDa B-P conjugate formed was cleaved by Factor D to yield a single product of 85 kDa. This is consistent with a Ba-P heterodimer, and suggests that the initial interaction of B and P includes an interaction of P with the Ba domain of intact B. (The Ba fragment is not retained in the active P-stabilized complex, C3bBbP). By contrast, no cross-linking of P to the Bb domain of B could be demonstrated. Binding studies on cellular intermediates also provided evidence for a site of interaction between B and P, with high concentrations of B inhibiting P binding to EAC3b (sheep erythrocytes coated with antibody and C3b). Neither isolated Ba nor Bb had any effect on the P-EAC3b interaction. High concentrations of B also accelerated the decay of the functional EAC3bBbP complex. These data indicate that the positive co-operativity of binding to C3i or to C3b between B and P is mediated, at least in part, through a direct interaction between B and P.

Complement Activating Enzymes↗

PubMed1998

Glomerular paramesangial deposits: association with hypocomplementemia in membranoproliferative glomerulonephritis types I and III.

Of 22 subjects previously reported with some form of factor H dysfunction, 12 had a glomerulonephritis that appeared to not be of immune complex origin. Factor H dysfunction results in elevated circulating levels of the C3b-dependent C3 convertase, C3b,Bb. Of the 12 cases with glomerulonephritis, the glomerular deposits in the six whose biopsy specimens were studied were predominately subepithelial on the paramesangial portion of the glomerular basement membrane. In a subsequent study, similar deposits were found in patients with membranoproliferative glomerulonephritis (MPGN) type II, also a nephritis that is probably not of immune complex origin. Paramesangial deposits were found in these patients only in biopsy specimens obtained when the C3 level was low, at which time convertase stabilized by nephritic factor would be present in the circulation. This association of paramesangial deposits with circulating convertase was further tested by correlating these deposits with the level of C3 at the time of biopsy in MPGN types I and III. The results in type III MPGN were similar to those in type II; paramesangial deposits were frequently present when the C3 level was low as a result of circulating nephritic factor of the terminal pathway, NFt, and were usually absent when the C3 level was in the upper two thirds of the normal range. Deposits persisted in those patients with C3 levels that had been low but that had increased during the year before biopsy to within the lower one third of the normal range. The persistence of paramesangial deposits in MPGN type III, as compared with MPGN type II, may be related to the differences in composition and function of the two NF stabilized convertases (C3bn,Bb,P,NFt and C3b,Bb,NFa, respectively) that circulate in these two disorders. In contrast to MPGN type III, the hypocomplementemia in MPGN type I is thought to be, for the most part, the result of classical pathway activation, which is not associated with elevated circulating convertase levels. In agreement with this, paramesangial deposits were found in only two of 34 biopsy specimens. At the time of those two biopsies, both patients had a complement profile indicating that the NFt was circulating, as in MPGN type III. In three other cases with profiles compatible with circulating NFt, paramesangial deposits were not found. In all patients with type I MPGN, electron microscopy and immunofluorescence of the glomeruli gave results typical of an immune complex nephritis. Thus, even though the complement profile in MPGN type I may at times indicate the presence of a nephritic factor, circulating immune complexes appear to be basic to pathogenesis. The observations support the hypothesis that elevated levels of the C3b-dependent convertase, as found in the "experiments of nature" with factor H dysfunction and in MPGN types II and III, are associated with paramesangial deposits. The nature of this association and the role of these deposits in producing the nephritis is not clear.

Biopsy↗

PubMed2005

The role of properdin in the assembly of the alternative pathway C3 convertases of complement.

Complement is a powerful host defense system that contributes to both innate and acquired immunity. There are three pathways of complement activation, the classical pathway, lectin pathway, and alternative pathway. Each generates a C3 convertase, a serine protease that cleaves the central complement protein, C3. Nearly all the biological consequences of complement are dependent on the resulting cleavage products. Properdin is a positive regulator of complement activation that stabilizes the alternative pathway convertases (C3bBb). Properdin is composed of multiple identical protein subunits, with each subunit carrying a separate ligand-binding site. Previous reports suggest that properdin function depends on multiple interactions between its subunits with its ligands. In this study I used surface plasmon resonance assays to examine properdin interactions with C3b and factor B. I demonstrated that properdin promotes the association of C3b with factor B and provides a focal point for the assembly of C3bBb on a surface. I also found that properdin binds to preformed alternative pathway C3 convertases. These findings support a model in which properdin, bound to a target surface via C3b, iC3b, or other ligands, can use its unoccupied C3b-binding sites as receptors for nascent C3b, bystander C3b, or pre-formed C3bB and C3bBb complexes. New C3bP and C3bBP intermediates can lead to in situ assembly of C3bBbP. The full stabilizing effect of properdin on C3bBb would be attained as properdin binds more than one ligand at a time, forming a lattice of properdin: ligand interactions bound to a surface scaffold.

Binding Sites↗

PubMed1987

Alternative complement pathway activation fragment Ba binds to C3b. Evidence that formation of the factor B-C3b complex involves two discrete points of contact.

Alternative complement pathway C3 convertase formation involves the cleavage of C3b-associated factor B into fragments Ba and Bb. Whereas Bb, in complex with C3b, has proteolytic specificity toward native C3, the function of the Ba moiety in the formation and/or decay of alternative complement pathway C3 convertase is uncertain. Therefore, we have examined the effect of purified Ba fragment on both fluid-phase and surface-bound enzymatic activity and showed that whereas Ba could inhibit the rate of C3 convertase formation, the rate of intrinsic decay remained unaffected. A specific, metal ion-independent interaction between Ba and C3b was subsequently demonstrated by use of the cross-linking reagent dithiobis(succinimidyl propionate). When cell-associated 125I-B was activated by D, the dissociation of Bb fragment displayed simple first-order kinetics with a half-time of 2.4 min, this value being in reasonable agreement with the hemolytically determined decay rate of 1.8 min. In contrast, most of the Ba fragment undergoes rapid dissociation, but there is also evidence to suggest the establishment of a new equilibrium due to the ability of Ba to rebind to C3b. Cumulatively, these data are consistent with a model in which the attachment of intact B to C3b is mediated by two points of contact, one being in the Ba domain and the other in the Bb domain. Due to avidity effects, each of these interactions could be of relatively low intrinsic affinity, and the characteristic unidirectionality of alternative complement pathway C3 convertase decay may simply result from the low intrinsic association of "univalent" Bb for the C3b subunit.

Chemical Phenomena↗

PubMed1987

[Formation of classical C3 convertase during the alternative pathway of human complement activation].

The fluid phase C3 convertase of the alternative pathway of human complement activation has been constructed from the isolated C3 component and from purified factors B and D. The enzyme was able to activate the isolated components C4 and C2 in the presence of C4 but had no effect on C2 in the absence of C4. The C4 and C2 activation was monitored by the loss of their hemolytic activity during the incubation with the alternative fluid phase C3 convertase. The activation of C4 and C2 components by the membrane-bound alternative C3 convertase formed on red cells (EC3bBb) was followed by the formation of C3 convertase of the classic pathway--EC4b2a. This resulted in the enhancement of hemolysis.

Animals↗

PubMed1990

Two types of C3 nephritic factor: properdin-dependent C3NeF and properdin-independent C3NeF.

The IgG fraction of serum from patients with membranoproliferative glomerulonephritis (MPGN) types I and II or partial lipodystrophy (PLD) was found to contain C3 nephritic factor (C3NeF) which reacts with the alternative pathway C3 convertase C3bBb and stabilizes it. Two types of C3NeF were detected, of which one was heat sensitive (56 degrees C for 30 min) and properdin dependent (C3NeF:P) but the other was heat stable and properdin independent (C3NeF:nP). C3NeF:P was found in sera from patients with MPGN types I and II and it displayed the properties of properdin and IgG. C3NeF:P was observed in patients with reduced serum concentrations of C3 and terminal complement components (TCC), and the generation of SC5b-9 complex was increased in mixtures with normal human serum. C3NeF:nP was found in sera from patients with MPGN type II and PLD, whose sera revealed a selective decrease in C3 concentrations.

Autoantibodies↗

PubMed1990

Detection of C3bBb-stabilizing activity (C3 nephritic factor) in the serum from patients with membranoproliferative glomerulonephritis.

It is known that membranoproliferative glomerulonephritis (MPGN), hypocomplementaemia and C3 nephritic factor (C3NeF) are closely related to each other, and the presence or absence of C3NeF in the serum is important for evaluating the nature of MPGN. However, some difficulties have been encountered in detecting this factor and therefore a new assay permitting the direct detection of C3NeF without purifying IgG from the patient's serum has been devised. Using this assay method, C3bBb-stabilizing activity was observed even in sera from MPGN patients who were non-hypocomplementaemic. Furthermore, among 98 cases with hypocomplementaemia. C3NeF was found to be absent in 66 cases.

Complement C3 Convertase, Alternative Pathway↗

PubMed2000

Cooperation between decay-accelerating factor and membrane cofactor protein in protecting cells from autologous complement attack.

Decay-accelerating factor (DAF or CD55) and membrane cofactor protein (MCP or CD46) function intrinsically in the membranes of self cells to prevent activation of autologous complement on their surfaces. How these two regulatory proteins cooperate on self-cell surfaces to inhibit autologous complement attack is unknown. In this study, a GPI-anchored form of MCP was generated. The ability of this recombinant protein and that of naturally GPI-anchored DAF to incorporate into cell membranes then was exploited to examine the combined functions of DAF and MCP in regulating complement intermediates assembled from purified alternative pathway components on rabbit erythrocytes. Quantitative studies with complement-coated rabbit erythrocyte intermediates constituted with each protein individually or the two proteins together demonstrated that DAF and MCP synergize the actions of each other in preventing C3b deposition on the cell surface. Further analyses showed that MCP's ability to catalyze the factor I-mediated cleavage of cell-bound C3b is inhibited in the presence of factors B and D and is restored when DAF is incorporated into the cells. Thus, the activities of DAF and MCP, when present together, are greater than the sum of the two proteins individually, and DAF is required for MCP to catalyze the cleavage of cell-bound C3b in the presence of excess factors B and D. These data are relevant to xenotransplantation, pharmacological inhibition of complement in inflammatory diseases, and evasion of tumor cells from humoral immune responses.

Adjuvants, Immunologic↗

PubMed1988

A simple isolation procedure for functionally pure components of the bovine alternative complement pathway (ACP) C3 convertase and bovine conglutinin (K).

A simple multicomponent isolation procedure for bovine C3, factor B, factor D and conglutinin (K) from a single serum sample is described. The components of the alternative pathway C3 convertase were isolated in milligram quantities from 800 ml bovine serum and were found to be functionally pure with respect to each other and to factors H and I.

Animals↗

PubMed1998

Ragweed allergy: correlation between skin reactivity and in vitro complement activation.

Recently we have reported on several observations which indicate that allergen-induced complement activation contributes to the development of the symptoms of ragweed allergy. In the present paper a new finding that supports this assumption is summarized. In 48 ragweed-allergic patients individual skin reactivity to ragweed allergen extract (RWA) was assessed using dilution skin prick testing. Sera of these patients were incubated with 20, 100, and 400 U/ml RWA and generation of two complement activation products, alternative pathway C3-convertase (C3bBbP) and terminal pathway activation complex (C5b-9) was measured by ELISA methods. A strong positive correlation (Spearman correlation coefficient r = 0.495, P = 0.0004, and r = 0.454, P = 0.0012, respectively) was found between individual skin reactivity to RWA and C3bBbP generation induced by 20 and 100 A allergological units/ml (U/ml) RWA. This finding further supports the role of complement activation products in the aggravation of the basic IgE-mediated immunopathology of ragweed allergy.

Complement Activation↗