The binding of human complement proteins C5, factor B, beta 1H and properdin to complement fragment C3b on zymosan.

The covalent binding of complement fragment C3b to zymosan by the action of the alternative-pathway C3 convertase and the reversible binding of several complement proteins (component C5, factor B, beta 1H and properdin) to C3b on zymosan have been investigated. When C3b is deposited on zymosan after activation by a surface-bound C3 convertase, the C3b molecules are deposited in foci around the C3 convertase site, with an average of 30 C3b molecules per site. The association constants of C5, factor B, beta 1H, and properdin for C3b bound to zymosan have been determined. The association constants ranged from 6.5 x 10(-5) M-1 for factor B to 2.9 x 10(7) M-1 for properdin. An approximate stoichiometry of 1 : 1 for C5, factor B, and properdin binding to C3b has been observed. Curvilinear Scatchard plots were observed for beta 1H binding to C3b, with the maximal extrapolated ratio of beta 1H to C3b of 0.32. Physiological amounts of properdin increase by 7-fold the affinity constant for factor B binding to C3b with no alteration in the stoichiometry. Similarly, physiological amounts of factor B increase the affinity constant of properdin to C3b about 4-fold with only a small measured difference in stoichiometry. Competition binding studies and protein modification suggest that C5, factor B, beta 1H, and properdin each bind to a distinct region on C3b.     

Kinetic analysis of the interactions between vaccinia virus complement control protein and human complement proteins C3b and C4b

The vaccinia virus complement control protein (VCP) is an immune evasion protein of vaccinia virus. Previously, VCP has been shown to bind and support inactivation of host complement proteins C3b and C4b and to protect the vaccinia virions from antibody-dependent complement-enhanced neutralization. However, the molecular mechanisms involved in the interaction of VCP with its target proteins C3b and C4b have not yet been elucidated. We have utilized surface plasmon resonance technology to study the interaction of VCP with C3b and C4b. We measured the kinetics of binding of the viral protein to its target proteins and compared it with human complement regulators factor H and sCR1, assessed the influence of immobilization of ligand on the binding kinetics, examined the effect of ionic contacts on these interactions, and sublocalized the binding site on C3b and C4b. Our results indicate that (i) the orientation of the ligand is important for accurate determination of the binding constants, as well as the mechanism of binding; (ii) in contrast to factor H and sCR1, the binding of VCP to C3b and C4b follows a simple 1:1 binding model and does not involve multiple-site interactions as predicted earlier; (iii) VCP has a 4.6-fold higher affinity for C4b than that for C3b, which is also reflected in its factor I cofactor activity; (iv) ionic interactions are important for VCP-C3b and VCP-C4b complex formation; (v) VCP does not bind simultaneously to C3b and C4b; and (vi) the binding site of VCP on C3b and C4b is located in the C3dg and C4c regions, respectively.     

Complement C3b/C3d and cell surface polyanions are recognized by overlapping binding sites on the most carboxyl-terminal domain of complement factor H

Factor H (FH) is a potent suppressor of the alternative pathway of C in plasma and when bound to sialic acid- or glycosaminoglycan-rich surfaces. Of the three interaction sites on FH for C3b, one interacts with the C3d part of C3b. In this study, we generated recombinant constructs of FH and FH-related proteins (FHR) to define the sites required for binding to C3d. In FH, the C3d-binding site was localized by surface plasmon resonance analysis to the most C-terminal short consensus repeat domain (SCR) 20. To identify amino acids of FH involved in binding to C3d and heparin, we compared the sequences of FH and FHRs and constructed a homology-based molecular model of SCR19-20 of FH. Subsequently, we created an SCR15-20 mutant with substitutions in five amino acids that were predicted to be involved in the binding interactions. These mutations reduced binding of the SCR15-20 construct to both C3b/C3d and heparin. Binding of the wild-type SCR15-20, but not the residual binding of the mutated SCR15-20, to C3d was inhibited by heparin. This indicates that the heparin- and C3d-binding sites are overlapping. Our results suggest that a region in the most C-terminal domain of FH is involved in target recognition by binding to C3b and surface polyanions. Mutations in this region, as recently reported in patients with familial hemolytic uremic syndrome, may lead to indiscriminatory C attack against self cells.     

Factor H-related protein 4 activates complement by serving as a platform for the assembly of alternative pathway C3 convertase via its interaction with C3b protein

Human complement factor H-related protein (CFHR) 4 belongs to the factor H family of plasma glycoproteins that are composed of short consensus repeat (SCR) domains. Although factor H is a well known inhibitor of the alternative complement pathway, the functions of the CFHR proteins are poorly understood. CFHR4 lacks SCRs homologous to the complement inhibitory domains of factor H and, accordingly, has no significant complement regulatory activities. We have previously shown that CFHR4 binds C-reactive protein via its most N-terminal SCR, which leads to classical complement pathway activation. CFHR4 binds C3b via its C terminus, but the significance of this interaction is unclear. Therefore, we set out to clarify the functional relevance of C3b binding by CFHR4. Here, we report a novel role for CFHR4 in the complement system. CFHR4 serves as a platform for the assembly of an alternative pathway C3 convertase by binding C3b. This is based on the sustained ability of CFHR4-bound C3b to bind factor B and properdin, leading to an active convertase that generates C3a and C3b from C3. The CFHR4-C3bBb convertase is less sensitive to the factor H-mediated decay compared with the C3bBb convertase. CFHR4 mutants containing exchanges of conserved residues within the C-terminal C3b-binding site showed significantly reduced C3b binding and alternative pathway complement activation. In conclusion, our results suggest that, in contrast to the complement inhibitor factor H, CFHR4 acts as an enhancer of opsonization by promoting complement activation.     

Solution structure of TT30, a novel complement therapeutic agent, provides insight into its joint binding to complement C3b and C3d

A novel therapeutic reagent TT30 was designed to be effective in diseases of the alternative pathway of complement such as paroxysmal nocturnal hemoglobinuria and other diseases. TT30 is constructed from the first four short complement regulator (SCR) domains of complement receptor type 2 (CR2) that bind to complement C3d, followed by the first five SCR domains of complement factor H that bind to complement C3b. In order to assess how TT30 binds to C3d and C3b, we determined the TT30 solution structure by a combination of analytical ultracentrifugation, X-ray scattering and constrained modeling. The sedimentation coefficients and radius of gyration of TT30 were unaffected by citrate or phosphate-buffered saline buffers and indicate an elongated monomeric structure with a sedimentation coefficient of 3.1?S and a radius of gyration R(G) of 6.9?nm. Molecular modeling starting from 3000 randomized TT30 conformations showed that high-quality X-ray curve fits were obtained with extended SCR arrangements, showing that TT30 has a limited degree of inter-SCR flexibility in its solution structure. The best-fit TT30 structural models are readily merged with the crystal structure of C3b to show that the four CR2 domains extend freely into solution when the five complement factor H domains are bound within C3b. We reevaluated the solution structure of the CR2-C3d complex that confirmed its recent crystal structure. This recent CR2-C3d crystal structure showed that TT30 is able to interact readily with C3d ligands in many orientations when TT30 is bound to C3b.     

A complement receptor locus: genes encoding C3b/C4b receptor and C3d/Epstein-Barr virus receptor map to 1q32

The alternative or classical pathways for complement system component C3 may be triggered by microorganisms and antigen-antibody complexes. In particular, an activated fragment of C3, C3b, covalently attaches to microorganisms or antigen-antibody complexes, which in turn bind to the C3b receptor, also known as complement receptor 1. The genes encoding the proteins that constitute the C3-activating enzymes have been cloned and mapped to a "complement activation" locus in the major histocompatibility complex, and we demonstrate in this study such a locus on the long arm of chromosome 1 at band 1q32.     

Regulatory system of guinea-pig complement C3b: two factor I-cofactor proteins on guinea-pig peritoneal granulocytes

Complement factor I is a plasma protease serving for proteolytic inactivation of C3b together with its cofactor. We have identified two factor I-cofactor activities in solubilized extracts of guinea-pig peritoneal granulocytes using guinea-pig factor I (Igp) and fluorescent-labeled methylamine-treated guinea-pig C3 (f-C3(MA)gp). One of these eluted from a chromatofocusing column between pH 7.6-7.1, and the other at about pH 5.7. These two cofactor fractions both interacted with Igp and, to a lesser degree, with human factor I (Ihu) on C3(MA)gp cleaving it into an inactive C3bi analogue, but did not cleave methylamine-treated human C3 (C3(MA)hu) together with Igp or Ihu. These factors are therefore species specific. The neutral and acidic fractions with cofactor activity contained C3(MA)gp-binding proteins with a doublet of 55 kDa and 42 kDa, and a singlet of 160 kDa, respectively, on SDS-PAGE. These proteins may be membrane cofactor protein (MCP) and C3b/C4b receptor (CR1) of guinea-pigs.     

Potential mechanisms for the regulation of growth factor binding by heparin

Heparin and heparan sulfate proteoglycans (HSPG) bind many soluble growth factors and this binding is now recognized as an important mechanism for modulation of cell activity. Fibroblast growth factor-2 (FGF-2) is one of the best characterized of the heparin-binding growth factors and it has been shown experimentally that heparin regulation of FGF-2 activity is dependent on the level of cell HSPG and the concentration of heparin. In this paper, we explore, using mathematical modeling, proposed mechanisms for heparin regulation and determine how they impact FGF receptor binding. We demonstrate that the experimentally observed receptor binding phenomena can be reproduced if cells (1) express heparin-binding cell surface molecules and if either (2) these heparin binding sites are FGFR and bind heparin and FGF-2-heparin complexes or (3) are surface molecules able to bind FGF-2 and couple with FGF-2 receptors to form high-affinity FGF-2-bound surface complexes. The ability of heparin to directly interact with the FGFR and bind FGF-2 in the absence of this coupling function was not sufficient to explain heparin activity. These findings have implications with regard to regulation of heparin-binding growth factors and could help guide the development of highly specific growth regulatory molecules through specific regulation by heparin and HSPG.     

Bivalent and co-operative binding of complement factor H to heparan sulfate and heparin

FH (Factor H) with 20 SCR (short complement regulator) domains is a major serum regulator of complement, and genetic defects in this are associated with inflammatory diseases. Heparan sulfate is a cell-surface glycosaminoglycan composed of sulfated S-domains and unsulfated NA-domains. To elucidate the molecular mechanism of binding of FH to glycosaminoglycans, we performed ultracentrifugation, X-ray scattering and surface plasmon resonance with FH and glycosaminoglycan fragments. Ultracentrifugation showed that FH formed up to 63% of well-defined oligomers with purified heparin fragments (equivalent to S-domains), and indicated a dissociation constant K(d) of approximately 0.5 μM. Unchanged FH structures that are bivalently cross-linked at SCR-7 and SCR-20 with heparin explained the sedimentation coefficients of the FH-heparin oligomers. The X-ray radius of gyration, R(G), of FH in the presence of heparin fragments 18-36 monosaccharide units long increased significantly from 10.4 to 11.7 nm, and the maximum lengths of FH increased from 35 to 40 nm, confirming that large compact oligomers had formed. Surface plasmon resonance of immobilized heparin with full-length FH gave K(d) values of 1-3 μM, and similar but weaker K(d) values of 4-20 μM for the SCR-6/8 and SCR-16/20 fragments, confirming co-operativity between the two binding sites. The use of minimally-sulfated heparan sulfate fragments that correspond largely to NA-domains showed much weaker binding, proving the importance of S-domains for this interaction. This bivalent and co-operative model of FH binding to heparan sulfate provides novel insights on the immune function of FH at host cell surfaces.     

An equilibrium model of the metastable binding sites of alpha 2-macroglobulin and complement proteins C3 and C4

Two cyclic structures, the 15-membered thiolactone A and the 5-membered lactam P, have been proposed for the metastable binding sites of the serum proteins C3, C4, and alpha 2-macroglobulin. Neither structure alone adequately explains two unusual reactions of these sites, namely, covalent attachment to nucleophiles with liberation of a thiol group and spontaneous hydrolysis (autolysis) of an internal peptide bond. The metastable binding sites of these proteins were modeled with the 15-membered thiolactone 1 (Khan, S. A., and Erickson, B. W. (1981) J. Am. Chem. Soc. 103, 7374-7376) and the isomeric 5-membered lactam 2, which contains an internal pyroglutamyl (Glp) residue. Under physiologic conditions (phosphate-buffered saline, pH 7.3, 37 degrees C), thiolactone 1 and lactam 2 exist in dynamic equilibrium. Since the molar ratio of 2/1 is 11:1 at equilibrium, lactam 2 is 15 kcal/mol more stable than thiolactone 1. The activation energy for isomerization of 1 into 2 is 18 kcal/mol, which is about 5 kcal/mol lower than that for hydrolysis of the acyclic thiolester N,S-diacetyl-L-cysteine methylamide. Part of the chemistry of the metastable binding sites can be explained by an analogous equilibrium between protein structures A and B. Lactam B may be a key intermediate in the biosynthesis of thiolactone A. Under denaturating conditions, thiolactone A could either bind covalently to a receptive surface or isomerize into lactam B, which could undergo spontaneous hydrolysis of the Glu-Glp peptide bond.     

Mouse C3b/C4b inactivator: purification and properties

Mouse C3b/C4b inactivator (C3b/C4bINA) was purified approximately 400 times from mouse serum. It is a beta-globulin and consists of 2 disulfide bonded chains of m.w. 60,000 and 35,000. Under nonreducing conditions, its m.w. is 95,000. It cleaves the alpha'-chain of cell-bound C4b into 3 fragments: alpha 2, alpha 3, alpha 4. The alpha 2 fragments remain bound to the cell surface (C4d), and the rest of the molecule (C4c) is released into the fluid phase. In fluid phase, C3b/C4bINA cleaves the alpha'-chain of C4b in a similar manner but only in the presence of mouse or human C4-binding protein (C4-bp). Mouse C4-bp and human C3b/C4bINA do not cleave human C4b, although mouse C4-bp binds to human C4b. This incompatibility suggests that C4-bp and C3b/C4bINA must interact to cleave fluid phase C4b. Mouse C3b/C4bINA also cleaves the alpha'-chain of human C3b in solution into 2 fragments in the presence of human beta 1H. Therefore, it is likely that mouse and human C3b/C4bINA are homologous proteins. A monospecific antiserum to mouse C3b/C4bINA has been prepared in rabbits. By crossed immunoelectrophoresis, this antiserum detects, in addition to the protein described above, a fast beta-globulin with a m.w. of approximately 200,000 and antigenically identical to C3b/C4bINA but enzymatically inactive. This protein could represent a precursor of C3b/C4bINA.     

Tolerance is dependent on complement C3 fragment iC3b binding to antigen-presenting cells

Systemic tolerance can be induced by the introduction of antigen into an immune-privileged site. Here we investigated the role of complement in the induction of tolerance after intraocular injection. We found that the development of antigen-specific tolerance is dependent on a complement activation product. The ligation of the complement C3 activation product iC3b to complement receptor type 3 (the iC3b receptor) on antigen-presenting cells resulted in the sequential production of transforming growth factor-beta2 and interleukin-10, which is essential for the induction of tolerance. These observations may extend to the development of both neonatal tolerance and other forms of acquired tolerance.     

Ligand-loaded but not free complement receptors for C3b/C4b and C3d co-cap with cross-linked B cell surface IgM and IgD

We have performed experiments to investigate possible physical interactions between C receptors (CR) and surface Ig (sIg) on the B cell plasma membrane. These molecules were found to be independent, non-linked, B cell surface structures, because capping CR1, CR2, sIgM, or sIgD with a specific antibody did not affect the distribution of the remainder of these molecules. Both CR1 and CR2, if bound by antibodies that did not independently cap CR, however, became associated with cross-linked sIg because CR that have been bound by intact anti-CR antibodies or their Fab fragments co-capped with sIgM or sIgD that had been bound by divalent anti-IgM or anti-IgD antibody. CR1 that had bound C3b similarly co-capped with sIg when sIg was cross-linked. Ligand-bound or even cross-linked CR did not associate with non-cross-linked sIg because sIgD, bound by a univalent Fab fragment of anti-IgD antibody, did not co-cap with CR that had been cross-linked by a sandwich of mouse anti-CR antibody and goat anti-mouse Ig. Other surface molecules, such as B1 and HLA-DR Ag, when bound by specific antibodies, did not cap with cross-linked sIg, and sIgD, when bound by a univalent Fab fragment of anti-IgD antibody, did not co-cap with cross-linked sIgM. Interactions between CR and sIg were not mediated by an association with IgG FcR because co-capping of CR and sIg was observed when F(ab')2 fragments of both anti-CR and anti-Ig antibodies were used. These results demonstrate that B cell surface CR can become associated with sIg, but only if sIg is cross-linked and CR is bound by anti-CR antibody or has bound its natural ligand.     

The inhibition of covalent binding of the nascent complement component C4b to its target

The inhibition of covalent binding of the nascent C4b fragment of the human complement component to its natural target, immunoglobulin G, was studied. To this end, an immunoenzyme system was developed. In this ELISA method, the complement was activated on the sorbed IgG molecules and the resulting nascent C4b fragment acylated IgG or interacted with a competitive inhibitor added to the system. The inhibition constants for binding of the nascent C4b to its target were determined for immunoglobulins G1, G2, G3, G4, M, and A1, as well as for ferritin, yeast mannan, capsid polysaccharides of theNeisseria meningitidis A, B, and C serotypes, diphtheria anatoxin, epinephrine, and salicylic acid. On the basis of the experimental data, the immunoglobulin role at the activation stage of the complement regulation cascade, the relationship between the antigen immunogenicity and its ability to interact with C4b, and the direct effect of a number of therapeutic agents on the complement system were discussed. Lectins of various specificities were shown to inhibit the enzymic activation of C4 by the first complement component and the subsequent C4b sorption by its target, which allowed us to suggest that some oligosaccharide fragments of the C1s and C4 molecules are spatially close to the C1s active site and to the thioester bond of C4.     

Site-directed mutagenesis of the region around Cys-241 of complement component C2. Evidence for a C4b binding site

We probed the functional significance of the region around Cys-241 in human C2 by testing the hemolytic activity of a series of mutant rC2. Mutant C2 cDNA were constructed by oligonucleotide-directed site-specific mutagenesis and expressed transiently in COS cells. Wild-type rC2 had threefold higher specific hemolytic activity than native serum C2. Substitution of Gly, Ala, or Ser for Cys-241 resulted in a slightly, but significantly, increased activity. In addition, I2 had no effect on the activity of these mutant C2. Substitution of Lys for Gln-243 increased the hemolytic activity by more than two-fold. Increased activity in all cases was due to slower decay rates of the C3 convertase. Finally, substitution of Leu or Ala for Asp-240 or Ser-244, respectively, resulted in more than 100-fold decrease of hemolytic activity. The results suggest that residues 240 to 244 of human C2 represent an important structural determinant of the C4b binding site of C2a. They also confirm that Cys-241 is the residue responsible for the increased activity of C2 reacted with I2.     

Access to the complement factor B scissile bond is facilitated by association of factor B with C3b protein

Factor B is a zymogen that carries the catalytic site of the complement alternative pathway C3 convertase. During convertase assembly, factor B associates with C3b and Mg(2+) forming a pro-convertase C3bB(Mg(2+)) that is cleaved at a single factor B site by factor D. In free factor B, a pair of salt bridges binds the Arg(234) side chain to Glu(446) and to Glu(207), forming a double latch structure that sequesters the scissile bond (between Arg(234) and Lys(235)) and minimizes its unproductive cleavage. It is unknown how the double latch is released in the pro-convertase. Here, we introduce single amino acid substitutions into factor B that preclude one or both of the Arg(234) salt bridges, and we examine their impact on several different pro-convertase complexes. Our results indicate that loss of the Arg(234)-Glu(446) salt bridge partially stabilizes C3bB(Mg(2+)). Loss of the Arg(234)-Glu(207) salt bridge has lesser effects. We propose that when factor B first associates with C3b, it bears two intact Arg(234) salt bridges. The complex rapidly dissociates unless the Arg(234)-Glu(446) salt bridge is released whereupon conformational changes occur that activate the metal ion-dependent adhesion site and partially stabilize the complex. The remaining salt bridge is then released, exposing the scissile bond and permitting factor D cleavage.