Modulation of the formation of the human C3 amplification convertase of complement by polyelectrolytes

Heparin in the fluid phase inhibits generation of the C3 amplification convertase of complement C3b,Bb. The anticomplementary activity requires the presence of O-sulfate groups on the molecule and is suppressed when the negative charges of heparin are neutralized with positive charges on polycations. In the absence of heparin, polycations inhibit generation of the cell-bound or fluid phase amplification convertase at final concentrations of 1 to 2×10^–8 M for poly-l-lysine 50,000 (PLL). PLL is more active on the D-dependent convertase C3b,Bb than in preventing generation of C3b,B; it does not alter the stabilizing effect of properdin. As for heparin, the major site of the inhibitory action of polycations is on the binding capacity of C3b for B. The low affinity interaction of C3b and B is a privileged site for potential pharmacologic modulation of the amplification convertase of complement by polyelectrolytes.     

Modulation of the formation of the human amplification C3 convertase of complement by polycations

Neutralization of the negative charges of heparin by polycations in the fluid phase suppressed the inhibitory effect of heparin on the generation of the C3b-dependent amplification convertase of complement C3b,Bb. Polymeric polycations alone, whether natural or synthetic, prevented formation of the cell-bound amplification convertase and of the fluid-phase interaction of C3b, B and D, in a dose-related fashion in the concentration range of 1 to 2 X 10(-8)M for poly-L-lysine (PLL) 50,000. The inhibitory effect of PLL on formation of the cell-bound convertase was independent of the presence of P. Percent inhibition of C3b,Bb,P and C3b,B,P formation was constant when the convertases were formed with a fixed concentration of PLL and increasing amounts of B; PLL was more effective in preventing convertase formation on cells bearing low numbers of C3b and developed with high doses of B. The decay of the preformed P stabilized convertase was not altered by PLL whether in the presence or absence of H. Thus, polycations in the fluid phase specifically inhibit formation of the amplification C3 convertase by preventing the association between C3b and B, most likely by acting on C3b. The low-affinity interaction between C3b and B is a privileged site for natural or pharmacological modulation of complement by polyelectrolytes.     

Regulation of the C3 nephritic factor stabilized C3/C5 convertase of complement by purified human erythrocyte C3b receptor

Activation of complement may result in the generation of the amplification convertase C3bBb. This convertase can be stabilized by properdin (P) or C3NeF. C3bBbP is susceptible to inactivation by beta 1H, while C3bBbNeF is relatively resistant. Since it has been shown that the human erythrocyte C3b receptor (CR1) is able to inactivate C3bBbP, the inactivating action of CR1 on C3bBbNeF was investigated CR1 is at least five times more efficient than beta 1H in inactivating C3bBbNeF. Kinetic studies revealed that CR1 induces an enhanced biphasic kinetics of decay of C3bBbNeF; further purification of this C3NeF preparation by cation exchange chromatography showed that this phenomenon is dependent on the population of C3NeF. Finally CR1 is also able to inactivate fluid phase C3bBbNeF.     

Modulation of classical C3 convertase of complement by tear lactoferrin.

Lactoferrin isolated fom normal human tears was shown to inhibit the complement-mediated lysis of antibody-coated red cells. The anti-complementary effect of lactoferrin on serum complement could be reversed by adding Fe3+ but not by Mg++ or Ca++. Lactoferrin did not inhibit the formation of EAC14 but markedly blocked the assembly of the EAC142 enzyme. Once the C3 convertase was formed lactoferrin did not affect the function of the enzyme and only had a minor effect on the intrinsic decay of C2 from the convertase. Inhibition of the C3 convertase formation was not seen by preincubating EAC14 intermediates with lactoferrin, but only occurred when lactoferrin and C2 were incubated together with EAC14 cells. Our findings suggest that lactoferrin may play an anti-inflammatory role by modulating activation of the complement system.     

Inhibition of the alternative C3 convertase and classical C5 convertase of complement by group A streptococcal M protein

When Streptococcus pyogenes group A type 3 strain C203 (M+) and its M-protein-lacking derivative, strain C203S (M-), were treated with normal human serum in the presence of magnesium-EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid], virulent M+ bacteria bound only 10 to 30% as much C3 and factors B and P as did avirulent M- bacteria. After treatment of M+ bacteria with trypsin, which inactivates M protein, their binding of these substances was similar to that of M- bacteria. Pretreatment of M+ bacteria with the Fab fragment of rabbit immunoglobulin G anti-M antibody also increased their binding of C3 in the absence of Ca2+. Therefore, M protein inhibits the alternative C3 convertase. In contrast, in the presence of Ca2+ and Mg2+, M+ bacteria bound 75% as much C3 as M- bacteria. This binding was mostly mediated by classical pathway activation, because M+ bacteria bound much smaller amounts of factors B and P than did M- bacteria but consumed amounts of C4 and C2 comparable to those consumed by M- bacteria. On the other hand, the amount of C5 bound to M+ bacteria was much less than that bound to M- bacteria, and the consumption of C5 and C8 by M+ bacteria was also much less than that by M- bacteria. Therefore, M protein does not inhibit the classical C3 convertase but does inhibit the classical C5 convertase. When M+ and M- streptococci were incubated with normal human serum containing radiolabeled C3 in the presence of Ca2+ and Mg2+, more than 85% of the C3 bound to either type of streptococcus was extractable by sodium dodecyl sulfate and alkali treatment. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the C3 extracted from both strains showed that it was mostly C3b and iC3b. The proportions of C3b and iC3b, respectively, were 7.5 and 71.9% on M+ bacteria and 18.9 and 58.4% on M- bacteria. These results support and extend previous findings that the antiphagocytic activity of streptococcal M protein may be due to complement inhibition mediated by the binding of factor H.     

补体经典激活途径C3转化酶的体外组装及活性观察

目的：体外组装包含人C4分子的补体经典激活途径C3转化酶，并对其转化酶活性及衰变特性进行观察。方法：利用豚鼠血清功能纯C1、C2及溶血中间体EAC4^hu体外组装经典途径C3转化酶，观察不同C1、C2用量及孵育温度对C3转化酶形成和自发性衰变的影响，以及人红细胞膜抽提蛋白对C3转化酶衰变化的影响。结果：高剂量和低剂量的C1均会影响C3转化酶的形成，增加C2用量可增加C3转化酶的形成数量，C3转化酶的自发性衰变随孵育温度的升高而加速，人红细胞膜抽提蛋白可抑制C3转化酶的自发性衰变过程，结论：C1、C2用量及孵育温度是影响C3转化酶形成和自发性衰变的主要因素，体外组装的补体经典激活途径C3转化酶可应用于相关补体调控蛋白的活性检测。     

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.     

Stringent regulation of complement lectin pathway C3/C5 convertase by C4b-binding protein (C4BP)

The complement lectin pathway, an essential component of the innate immune system, is geared for rapid recognition of infections as each C4b deposited via this pathway is capable of forming a C3/C5 convertase. In the present study, role of C4b-binding protein (C4BP) in regulating the lectin pathway C3/C5 convertase assembled on zymosan and sheep erythrocytes coated with mannan (E_Man) was examined. While the C4BP concentration for inhibiting 50% (IC₅₀) formation of surface-bound C3 convertase on the two surfaces was similar to that obtained for the soluble C3 convertase (1.05 nM), 3- and 41-fold more was required to inhibit assembly of the C5 convertase on zymosan (2.81 nM) and E_Man (42.66 nM). No difference in binding interactions between C4BP and surface-bound C4b alone or in complex with C3b was observed. Increasing the C4b density on zymosan (14,000–431,000 C4b/Zym) increased the number of C4b bound per C4BP from 2.87 to 8.23 indicating that at high C4b density all seven α-chains of C4BP are engaged in C4b-binding. In contrast, the number of C4b bound per C4BP remained constant (3.79 ± 0.60) when the C4b density on E_Man was increased. The data also show that C4BP regulates assembly and decay of the lectin pathway C3/C5 convertase more stringently than the classical pathway C3/C5 convertase because of a 7- to 13-fold greater affinity for C4b deposited via the lectin pathway than the classical pathway. C4BP thus regulates efficiently the four times greater potential of the lectin pathway than the classical pathway in generating the C3/C5 convertase and hence production of pro-inflammatory products, which are required to fight infections but occasionally cause pathological inflammatory reactions.     

Engineering of human complement component C3 for catalytic inhibition of complement

As a novel therapeutic approach in complement-mediated pathologies, we recently developed a human C3 derivative capable of obliterating functional complement by a catalytic, non-inhibitory mechanism. In this derivative, the C-terminal region of hC3 was substituted by a 275 amino acid sequence derived from the corresponding sequence of cobra venom factor (CVF), a complement-activating C3b homologue from snake venom. In this study, we replaced shorter C-terminal sequences of hC3 by corresponding CVF sequences to further reduce potential immunogenicity and to identify domains essential for the formation of functionally stable C3 convertases. In one of these derivatives that is still capable of obliterating functional complement in vitro, the non-human portion could be reduced to a small domain located in the C-terminus of different complement proteins. This conserved NTR/C345C motif is known to be involved in assembly of different convertases of the complement system. These results suggest a major role of the C345C domain in the regulation of the half-life of the C3 convertase. Moreover, its overall identity of 96% to human C3 renders this derivative a promising candidate for therapeutic intervention in complement-mediated pathologies.     

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.     

Aurin tricarboxylic acid self-protects by inhibiting aberrant complement activation at the C3 convertase and C9 binding stages

Aberrant complement activation is known to exacerbate the pathology in a spectrum of degenerative diseases of aging. We previously reported that aurin tricarboxylic acid (ATA) is an orally effective agent which prevents formation of the membrane attack complex of complement. It inhibits C9 attachment to tissue bound C5b678 and thus prevents bystander lysis of host cells. In this study, we investigated the effects of ATA on the alternative complement pathway. We found that ATA prevented cleavage of the tissue bound properdin-C3b-Factor B complex into the active C3 convertase enzyme properdin-C3b-Factor Bb. This inhibition was reversed by adding Factor D to the serum. Using enzyme-linked immunosorbent type assays, we established that ATA binds directly to Factor D and C9 but not to properdin or other complement proteins. We conclude that ATA, by inhibiting at two stages of the alternative pathway, might be a particularly effective therapeutic agent in conditions such as macular degeneration, paroxysmal nocturnal hemoglobinemia, and rheumatoid arthritis, in which activation of the alternative complement pathway initiates self damage.     

FUT-175 as a potent inhibitor of C5/C3 convertase activity for production of C5a and C3a

We examined the inhibitory effect of FUT-175 on the C3/C5 convertase activity of the cobra venom factor-derived enzyme CVF,Bb by measuring C5b6-mediated reactive lysis of unsensitized guinea pig erythrocytes and by measuring directly the released fragments C3-des-Arg and C5a-des-Arg. In this study, we showed that the concentration of 4.5 X 10(-6) M of FUT-175 caused 50% inhibition of C5 convertase activity of CVF,Bb in reactive hemolysis assays, and that 4.0 X 10(-6) M FUT-175 caused 50% inhibition of the production of C3a and C5a generated by the C3/C5 convertase activity of CVF,Bb.     

C3 convertase of the human alternative pathway of complement: modulation of enzyme activity and stability by mouse monoclonal antibodies to Bb

To further characterize functional sites on C3b,Bb, and C3 convertase of the alternative pathway of complement, we examined the effect of four monoclonal antibodies on its activity and stability. These antibodies recognized antigenic epitopes on human Bb which were not fully exposed in intact factor B. Three of the monoclonal antibodies inhibited lysis of rabbit erythrocytes by normal human serum in the presence of Mg2+ and EGTA. Two of these antibodies markedly inhibited the activity of purified C3b,Bb deposited on rabbit erythrocytes. However, all four monoclonal antibodies increased the half-life of the C3 convertase. Thus, these results demonstrate that binding of antibodies to Bb may concomitantly stabilize C3b,Bb and abate its activity. It is likely that such antibodies induce in Bb conformational changes which increase the C3b,Bb complex stability but may also hinder its catalytic site.     

Compstatin,a peptide inhibitor of complement,exhibits species-specific binding to complement component C3

Although activation of complement protein C3 is essential for the generation of normal inflammatory responses against pathogens, its unregulated activation during various pathological conditions leads to host cell damage. Previously we have identified a 13-residue cyclic peptide, Compstatin, that inhibits C3 activation. In this study, we have examined the species-specificity of Compstatin. Bimolecular interaction analysis using a real-time surface plasmon resonance-based assay showed that Compstatin exhibits exclusive specificity for primate C3s and does not bind either to C3s from lower mammalian species or to two structural homologs of C3, human C4 and C5. Furthermore, it showed that although the kinetics of binding of Compstatin to non-human primate C3s were distinctly different from those to human C3, like human C3 its mechanism of binding to non-human primate C3 was biphasic and did not follow a simple 1:1 interaction, suggesting that this binding mechanism could be important for its inhibitory activity. Analysis of Ala substitution analogs of Compstatin for their inhibitory activities against mouse and rat complement suggested that the lack of binding of Compstatin to mouse and rat C3s was not a result of sterically hindered access to the binding pocket due to individual bulky side chains or the presence of charge on the Compstatin molecule. These results suggest that Compstatin's exclusive specificity for primate C3s could be exploited for the development of species-specific complement inhibitors.     

Inhibition of classical C5 convertase in the complement system by factor H

This paper described the influence of factor H on the haemolytic activity of the classical C5 convertase. Factor H showed little effect on the interaction of C5 with EAC1,4b,2a,3b cells bearing low numbers of C3b sites, but displayed the inhibitory effect on the interaction of C5 with the intermediate cells bearing high numbers of C3b sites. The higher the number of C3b sites on the cells, the greater the degree of the inhibition by factor H. The inhibition by factor H was accompanied by the inhibition of consumption of C5 from the fluid phase, indicating that factor H inhibits the activity of C5 convertase, not the binding of activated C5 to the cells.     

Isolation of human complement factors C3, C5 and H

An improved method for simultaneous purification of complement factors C3, C5 and H from human plasma has been developed. Using an initial batch separation technique with QAE-Sephadex, followed by chromatography on SP-Sephadex and gel filtration in Sephadex G-200, 600 mg of highly pure C3 can be prepared from 1600 ml of plasma. Simultaneously about 70 mg of highly pure factor H and 30 mg of C5 are obtained by chromatography of post SP-Sephadex material on DEAE-Sephacel. A small amount of C3 in the C5 pool is removed by anti-C3-Sepharose. By maleylation or citraconylation of reduced and alkylated C3, the constitutive polypeptide chains are modified in a way that made them separable by ion exchange chromatography.     

The C3/C5 convertase of the alternative pathway of complement: stabilization and restriction of control by lanthanide ions

The alternative pathway C3 convertase (C3b,Bb) is a Mg-dependent, labile enzyme with a t/2 of 3 min at 37 degrees C and of 14 min at 24 degrees C (at half physiological ionic strength). To stabilize the enzyme, metal ions of the lanthanide series were tested. Formation and decay of the enzyme as well as binding of radiolabeled Factor B, Factor H or properdin to C3b were measured using C3b-bearing sheep erythrocytes (EC3b). Binding of Factor B to EC3b in presence of 40 microM gadolinium (Gd) was two to three times greater than in presence of 1 mM Mg. Binding of Factor H and of properdin to EC3b was partially inhibited by Gd. Although it enhanced Factor B uptake by EC3b, Gd was unable to substitute for Mg in enzyme formation by Factor D and completely inhibited (at 10 microM) Mg-dependent enzyme activation. However, the preformed enzyme was not inhibited by Gd. Instead, exposure of EC3b,Bb to 40-100 microM Gd increased the t/2 at 37 degrees C from 3 min to 12-28 min, and at 24 degrees C from 14 min to 32 min. The slow decay of the enzyme correlated with slow release of Bb. Similar enzyme stabilization was observed using terbium, ytterbium, dysprosium and lanthanum. The Gd stabilized enzyme was also less susceptible to control by Factor H and properdin than the unstabilized enzyme. Furthermore, Gd protected surface bound-C3b from being cleaved by Factor I. The Gd effects were instantaneously reversed upon addition of 10 mM EDTA. Thus, Gd is able to stabilize preformed C3b,Bb and to render the enzyme refractory to control by Factors H and I.     

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.     

Characterization of tryptic fragments of human complement factor C3

C3c and C3d fragments were prepared in pure form from trypsin-digested human C3, and the individual chains of tryptic C3c were isolated by gel filtration on Sepharose 4B in 6M guanidinium hydrochloride. No low mol. wt (Mr) fragments were identified. The polypeptide chains were characterized with regard to Mr, amino acid composition and N-terminal amino acid sequence. Tryptic C3c consisted of one fragment from the beta-chain (Mr 64,000) and two from the alpha'-chain (Mr 40,000 and 23,000). The beta-chain fragment was derived from the C-terminal part of the chain, and the 23,000-Mr component constituted the amino terminal end of the alpha-chain. The 40,000-Mr fragment emanated from the C-terminal end of the alpha-chain. Tryptic C3d displayed microheterogeneity on polyacrylamide gel electrophoresis in sodium dodecyl sulfate, but possessed a homogeneous N-terminal, identical to that described by Tack et al. (1980) (Proc. natn. Acad. Sci. U.S.A. 77, 5764-5768). By utilization of antisera against subunits of C3 and C3c in immunoblotting a degradation scheme for C3 by trypsin was proposed and the positions of the fragments in the intact molecule indicated.