A corresponding tyrosine residue in the C2/factor B type A domain is a hot spot in the decay acceleration of the complement C3 convertases

The cleavage of C3 by the C3 convertases (C3bBb and C4b2a) determines whether complement activation proceeds. Dissociation (decay acceleration) of these central enzymes by the regulators decay-accelerating factor (DAF), complement receptor 1 (CR1), factor H, and C4-binding protein (C4BP) controls their function. In a previous investigation, we obtained evidence implicating the alpha4/5 region of the type A domain of Bb (especially Tyr338) in decay acceleration of C3bBb and proposed this site as a potential interaction point with DAF and long homologous repeat A of CR1. Because portions of only two DAF complement control protein domains (CCPs), CCP2 and CCP3, are necessary to mediate its decay of the CP C3 convertase (as opposed to portions of at least three CCPs in all other cases, e.g. CCPs 1-3 of CR1), DAF/C4b2a provides the simplest structural model for this reaction. Therefore, we examined the importance of the C2 alpha4/5 site on decay acceleration of C4b2a. Functional C4b2a complexes made with the C2 Y327A mutant, the C2 homolog to factor B Y338A, were highly resistant to DAF, C4BP, and long homologous repeat A of CR1, whereas C2 substitutions in two nearby residues (N324A and L328A) resulted in partial resistance. Our new findings indicate that the alpha4/5 region of C2a is critical to decay acceleration mediated by DAF, C4BP, and CR1 and suggest that decay acceleration of C4b2a and C3bBb requires interaction of the convertase alpha4/5 region with a CCP2/CCP3 site of DAF or structurally homologous sites of CR1 and C4BP.     

Differences between the binding sites of the complement regulatory proteins DAF, CR1, and factor H on C3 convertases

Binding studies using purified decay-accelerating factor (DAF), CR1, and Factor H indicate that the primary interaction of DAF with C3 convertases is with the Bb or C2a subunits, whereas CR1 and Factor H interact primarily with the C3b or C4b subunits. The ability of soluble DAF, CR1, or Factor H to decay C3b,Bb bound to zymosan was inhibited by various concentrations of fluid-phase competitors (C3b, Bb, C3b,Bb, C3b,B, C4b, or C4b,C2a) in 0.1% NP-40 at 22 degrees C. The apparent association constants (appKa) for DAF were 0.045, 0.067, 0.91, 0.71, 0.00045, and 0.53 microM-1, respectively. The appKa for CR1 were 0.50, 0.0040, 1, 1, 1, and 1.1 microM-1, respectively. The appKa for Factor H were 4.3, 0.0005, 2.9, 6.3, 0.27, and 0.29 microM-1, respectively. Thus, C3b binds to DAF with a 10-fold lower affinity than to CR1 and a 100-fold lower affinity than to Factor H. The appKa of C3b,Bb for the three proteins were more similar: DAF (0.91 microM-1), CR1 (1 microM-1), and Factor H (2.9 microM-1). DAF binds to Bb with a 50% higher affinity than to C3b, and to C4b,C2a with a 1000-fold higher affinity than to C4b alone. In contrast, CR1 and Factor H bind almost equally well to the C3 convertases and to their noncatalytic subunits. The affinity of DAF for CVF,Bb was similar to its affinity for Bb alone, suggesting that DAF does not recognize conformational determinants unique to Bb in C3 convertases.     

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.     

Molecular dissection of interactions between components of the alternative pathway of complement and decay accelerating factor (CD55)

The complement regulatory protein decay accelerating factor (DAF; CD55), inhibits the alternative complement pathway by accelerating decay of the convertase enzymes formed by C3b and factor B. We show, using surface plasmon resonance, that in the absence of Mg(2+), DAF binds C3b, factor B, and the Bb subunit with low affinity (K(D), 14 +/- 0.1, 44 +/- 10, and 20 +/- 7 microm, respectively). In the presence of Mg(2+), DAF bound Bb or the von Willebrand factor type A subunit of Bb with higher affinities (K(D), 1.3 +/- 0.5 and 2.2 +/- 0.1 microm, respectively). Interaction with the proenzyme C3bB was investigated by flowing factor B across a C3b-coated surface in the absence of factor D. The dissociation rate was dependent on the time of incubation, suggesting that a time-dependent conformational transition stabilized the C3b-factor B interaction. Activation by factor D (forming C3bBb) increased the complex half-life; however, the enzyme became susceptible to rapid decay by DAF, unlike the proenzyme, which was unaffected. A convertase assembled with cobra venom factor and Bb was decayed by DAF, albeit far less efficiently than C3bBb. DAF did not bind cobra venom factor, implying that Bb decay is accelerated, at least in part, through DAF binding of this subunit. It is likely that DAF binds the complex with higher affinity/avidity, promoting a conformational change in either or both subunits accelerating decay. Such analysis of component and regulator interactions will inform our understanding of inhibitory mechanisms and the ways in which regulatory proteins cooperate to control the complement cascade.     

Mutations of the type A domain of complement factor B that promote high-affinity C3b-binding

Factor B is a zymogen that carries the catalytic site of the complement alternative pathway convertases. During C3 convertase assembly, factor B associates with C3b and is cleaved at a single site by factor D. The Ba fragment is released, leaving the active complex, C3bBb. During the course of this process, the protease domain becomes activated. The type A domain of factor B, also part of Bb, is similar in structure to the type A domain of the complement receptor and integrin, CR3. Previously, mutations in the factor B type A domain were described that impair C3b-binding. This report describes "gain of function" mutations obtained by substituting factor B type A domain amino acids with homologous ones derived from the type A domain of CR3. Replacement of the betaA-alpha1 Mg2+ binding loop residue D254 with smaller amino acids, especially glycine, increased hemolytic activity and C3bBb stability. The removal of the oligosaccharide at position 260, near the Mg2+ binding cleft, when combined with the D254G substitution, resulted in increased affinity for C3b and iC3b, a C3b derivative. These findings offer strong evidence for the direct involvement of the type A domain in C3b binding, and are suggestive that steric effects of the D254 sidechain and the N260-linked oligosaccharide may contribute to the regulation of ligand binding.     

Segment spanning residues 727-768 of the complement C3 sequence contains a neoantigenic site and accommodates the binding of CR1, factor H, and factor B.

CR1, CR2, DAF, MCP, factor H, C4bp, factor B, and C3 are members of a family of structurally related molecules, the majority of which belong to the complement system. Several of these molecules also share functional features such as cofactor and decay/dissociation activity and compete with one another in binding to C3b. Since factor H appears to bind to multiple sites in C3, we investigated the relationship between the factor H- and CR1-binding sites in C3b. Factor H binding to C3b is inhibited by either the C3c or C3d fragments, and addition of both fragments together augments this inhibition. One monoclonal anti-C3c antibody, anti-C3-9, which recognizes a neoantigenic epitope expressed upon cleavage to C3 to C3b, inhibited both factor H and CR1 binding to EC3b cells. This monoclonal antibody (MoAb) also inhibited factor B binding to EC3b. Two observations further supported our hypothesis that these molecules bind to proximal sites in C3b. First, a synthetic peptide spanning this region of C3b (C3(727-768)) inhibited factor H binding. Second, antibodies raised against this peptide inhibited binding to CR1, factor H, and factor B to C3b. These data show that H binds to at least two sites in C3b: the site in the C3c fragment is within the identified CR1-binding domain while the site in the C3d fragment surrounds the CR2-binding site.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and functional analysis of the polymorphic variants of the C3b/C4b receptor (CR1) and comparison with H, C4b-binding protein (C4bp), and decay accelerating factor (DAF)

Four CR1 variants have been found in the normal population and are designated CR1-A (190,000 daltons), CR1-B (220,000 daltons), CR1-C (160,000 daltons), and CR1-D (250,000 daltons). In the present study, we first developed an improved chromatographic purification scheme for CR1 that does not employ a C3b affinity step. CR1 variants (A, B, and C) were then isolated, and their individual functional activity was assessed. Each possessed similar co-factor activity for I-mediated cleavage of C3(H2O), as well as for the inhibitory activity for fluid phase C3 convertases. These results indicate that, despite relatively large Mr differences, in the purified state these three CR1 variants have similar functional activities. The functional activity of CR1 was also compared with C4bp, H, and decay accelerating factor (DAF) in fluid phase assays designed to assess the inhibition of the C3 convertases and co-factor activity. On a molar basis, CR1 had approximately the same inhibitory activity as C4bp for the classical pathway convertase, and had the same as H for the alternative pathway convertase. These results indicate that CR1 encompasses the functional capabilities of both proteins. They also raise a number of interesting genetic and structural questions in regard to these complement regulatory proteins, because C4bp is thought to have multiple C4b binding domains, whereas H is reported to bind one C3b. DAF was an approximately fourfold better inhibitor of the alternative pathway convertase than CR1 or H, but was a fourfold less efficient inhibitor of the classical pathway convertase than CR1 or C4bp. The effective inhibitory capacity of DAF in these fluid phase assay systems suggests that the DAF substrate specificity is for the convertases. Fluid phase CR1 was twofold less efficient than H in serving as a co-factor for the first cleavage of fluid phase C3b, and hardly mediated the second cleavage. These data are in contrast to the co-factor activity of CR1 on a cell membrane, and provide additional evidence for the local environment being a critical modulator of the function of proteins that regulate the activation of C3.     

Glycoprotein C of herpes simplex virus 1 is an inhibitor of the complement cascade

Mammalian cells in culture express membrane receptors for C3b when infected with HSV-1. C3b binding is mediated by glycoprotein C (gC), a virus-specified membrane glycoprotein. In view of the inhibitory functions of other C3b-binding proteins, we studied the capacity of gC to modulate complement activation. Glycoprotein C was purified from HSV-1-infected cells by immunoaffinity chromatography. Glycoprotein C, but not a control viral glycoprotein, demonstrated dose-dependent acceleration of decay of C3bBb sites. In addition, gC produced a dose-dependent, time-independent depression of the overall hemolytic efficiency of C3bBb sites. Inhibition of C5b6-initiated reactive lysis of cells bearing C3b, but not cells bearing antibody alone, by gC suggests that the second effect represents interference with the C3b-C5/5b interaction. This hypothesis is supported by the failure of gC to inhibit reactive lysis when added after C5b67 insertion into target cells. Glycoprotein C does not accelerate C14b2a decay, nor does it impair classical pathway hemolytic efficiency when excess C5 is present. By limiting available C5/5b, some gC inhibition of C3b-C5/5b interactions can be unmasked in the classical pathway system. Glycoprotein C is devoid of factor I co-factor activity. HSV-1 gC is a modulator of complement activation, especially via the alternative pathway, and may represent a novel viral mechanism for evading host defense processes.     

Activation of the classical pathway of complement by the C3NeF-stabilized cell-bound amplification convertase.

C3 nephritic factor (C3NeF) has been shown to be composed of two heavy and two light chains, like IgG; in addition it shares antigenic determinants with IgG. C3NeF, purified from the sera of eight patients by incorporation of C3NeF into the stabilized fluid phase amplification C3 convertase, C3bBb(C3NeF), followed by its release after decay of convertase function, was investigated for its ability to bind 125I-C1q and to activate 125I-C1. It was found that although fluid phase C3b,Bb(C3NeF) is fully capable of binding 125I-C1q, it is not able to activate 125I-C1 even at concentrations of 1.3 x 10(12) C3bBb(C3NeF) complexs/ml. On the other hand, cell-bound C3bBb(C3NeF) is capable of both binding 125I-C1q and activating 125I-C1. This discrepancy between fluid phase and cell-bound, C3bBb(C3NeF) was found for C3NeF preparations from eight different patients and therefore seems to apply to all C3NeF preparations.     

Evolutionary relationships among proteins encoded by the regulator of complement activation gene cluster

Evolutionary relationships among members of the regulator of complement activation (RCA) gene cluster were analyzed using neighbor-joining and parsimony methods of phylogenetic tree inference. We investigated the structural and functional similarities among short consensus repeats (SCRs) of the following human proteins: the alpha chain of the C4b-binding protein (C4bpalpha), factor H (FH), factor H-related proteins (FHR-1 through FHR-4), complement receptors type 1 (CR1) and type 2 (CR2), the CR1-like protein (CR1L), membrane cofactor protein (MCP), decay accelerating factor (DAF), and the sand bass proteins, the cofactor protein (SBP1) and its homolog, the cofactor-related protein (SBCRP-1). Also included are the beta chain of the human C4b-binding protein (C4bpbeta) and the b subunit of human blood-clotting factor XIII (FXIIIb). Our results indicate that the human plasma complement regulators, FH and C4bpalpha, fall into two distinct groups on the basis of their sequence divergence. Homology among RCA proteins is in agreement with their chromosomal location, with the exception of C4bpbeta. The evolutionary relationships among individual short consensus repeats are confirmed by the exon/intron structure of the RCA members. Structural similarities among repeats of the RCA proteins correlate with their functional activities and demonstrate the importance of the N-terminal SCRs.     

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.     

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.     

Further evidence for the antibody nature of C3 nephritic factor (C3NeF).

C3 nephritic factor (C3NeF), found in the sera of some patients with membranoproliferative glomerulonephritis, has been shown to be composed of two heavy and two light chains, like IgG; in addition it shares antigenic determinants with IgG. Purified C3NeF binds to the amplification convertase of complement, C3b,Bb, and thereby prevents decay of its C3-cleaving potential. The capability of C3NeF to bind to C3b,Bb was used as a means for purifying C3NeF to homogeneity. The investigation described in this report suggests that binding of C3NeF to C3b,Bb occurs via the Fab portion of the molecule. Pepsin treatment of eight C3NeF preparations resulted in an average loss of 76% of C3NeF functional activity. Papain treatment induced a loss of approximately 90%. The decrease in functional activity could be attributed to the accelerated rate of dissociation of 125I-F(ab')2 and 125I-Fab fragments from stabilized cell-bound C3b,Bb. The dissociation rate of 125I-F(ab')2 from C3b,Bb was comparable with the decay of the functional activity of C3b,Bb stabilized by F(ab')2 or Fab fragments of C3NeF. Although these results suggest that the stabilizing activity of C3NeF is mediated by the Fab portion of the molecule, it was found that the Fc portion also contributes to its functional activity.     

Regulation of proteolytic activity of complement factor I by pH: C3b/C4b receptor (CR1) and membrane cofactor protein (MCP) have different pH optima for factor I-mediated cleavage of C3b

C3b/C4b receptor (CR1) and membrane cofactor protein (MCP) are integral membrane glycoproteins with factor I-dependent cofactor activity. They bind to C3b, allowing factor I to cleave C3b at two sites (first and second cleavage), which results in the generation of C3bi, a hemolytically inactive form which is a ligand for complement receptor type three (CR3). C3bi is further degraded by factor I and CR1 (third cleavage) to C3dg (a ligand for complement receptor type two, CR2) and C3c. Using two different substrates, fluid-phase C3b and cell-bound C3b, the cleavage of C3b by MCP and factor I was compared to that by CR1 and factor I under various conditions. The optimal pH for the first and second cleavage of either substrate was 6.0 for MCP and 7.5 for CR1. The third cleavage was mediated only by CR1 and factor I, the optimal pH being 8.0. Low ionic conditions enhanced the C3b binding and cofactor activity of both CR1 and MCP. The efficiency of binding C3b to CR1 or MCP was maximal at pH 6.2. The isoelectric point (pI) of MCP was acidic (approximately 4.0), while that of CR1 was 6.8. Therefore, compared to CR1, MCP possesses distinct functional profiles relative to C3b-binding and factor I-cofactor activity.     

Endogenous association of decay-accelerating factor (DAF) with C4b and C3b on cell membranes

Decay-accelerating factor (DAF) is a membrane glycoprotein found on various cells that are in contact with complement. It inhibits the formation of the C3 convertases of the complement system, both the classic (C4b2a) and alternative (C3bBb) pathways. In this investigation, we used a homobifunctional cross-linking reagent to search for a DAF ligand on the surface of cells subjected to complement attack. We found that DAF forms complexes with C4b and C3b deposited on the same erythrocytes, but not with the physiologic degradation products of these complement fragments, that is, C4d or C3dg. Taken together with prior observations that DAF action is reversible, and DAF does not affect the structure of C4b or C3b, these findings suggest that DAF functions by competitively inhibiting the uptake of C2 or factor B, and preventing the assembly of the C3 convertases.     

Stabilization of the amplification convertase of complement by monoclonal antibodies directed against human factor B

Three IgM mouse monoclonal antibodies (MoAb), 5B5-A, 2D2-B, and 5B12-A, were prepared by fusion of spleen cells from mice immunized with human B with the SP 2/0 myeloma cell line. They were assessed for their effect on cell-bound and fluid-phase amplification convertases of complement (C) with purified proteins in vitro. 5B5-A and 2D2-B were similar in their effects on cell-bound preformed C3bBb in that they bound to cell-bound C3bBb, stabilized the C3bBb convertase, and rendered the C3bBb convertase relatively resistant to the plasma protein H. These two MoAb were also able to enhance C3 consumption in vitro in reaction mixtures containing C3b, C3, B, and D. At the same time, they presumably stabilized the C3 convertase and caused relative sparing of B hemolytic activity in the reaction mixtures. In contrast, 5B12-A, which also bound to Bb and C3bBb, did not induce any stabilization, but rather caused accelerated decay of cell-bound C3bBb. These results indicate that MoAb against B can have C3NeF-like activity. On the other hand, not all MoAb against B have stabilizing activity on the C3bBb convertase.     

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.     

Regulators of complement activity mediate inhibitory mechanisms through a common C3b‐binding mode

Regulators of complement activation (RCA) inhibit complement‐induced immune responses on healthy host tissues. We present crystal structures of human RCA (MCP, DAF, and CR1) and a smallpox virus homolog (SPICE) bound to complement component C3b. Our structural data reveal that up to four consecutive homologous CCP domains (i–iv), responsible for inhibition, bind in the same orientation and extended arrangement at a shared binding platform on C3b. Large sequence variations in CCP domains explain the diverse C3b‐binding patterns, with limited or no contribution of some individual domains, while all regulators show extensive contacts with C3b for the domains at the third site. A variation of ~100° rotation around the longitudinal axis is observed for domains binding at the fourth site on C3b, without affecting the overall binding mode. The data suggest a common evolutionary origin for both inhibitory mechanisms, called decay acceleration and cofactor activity, with variable C3b binding through domains at sites ii, iii, and iv, and provide a framework for understanding RCA disease‐related mutations and immune evasion.     

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.     

Disease-associated N-terminal complement factor H mutations perturb cofactor and decay-accelerating activities

Many mutations associated with atypical hemolytic uremic syndrome (aHUS) lie within complement control protein modules 19-20 at the C terminus of the complement regulator factor H (FH). This region mediates preferential action of FH on self, as opposed to foreign, membranes and surfaces. Hence, speculation on disease mechanisms has focused on deficiencies in regulation of complement activation on glomerular capillary beds. Here, we investigate the consequences of aHUS-linked mutations (R53H and R78G) within the FH N-terminal complement control protein module that also carries the I62V variation linked to dense-deposit disease and age-related macular degeneration. This module contributes to a four-module C3b-binding site (FH1-4) needed for complement regulation and sufficient for fluid-phase regulatory activity. Recombinant FH1-4(V62) and FH1-4(I62) bind immobilized C3b with similar affinities (K(D) = 10-14 μM), whereas FH1-4(I62) is slightly more effective than FH1-4(V62) as cofactor for factor I-mediated cleavage of C3b. The mutant (R53H)FH1-4(V62) binds to C3b with comparable affinity (K(D) ～12 μM) yet has decreased cofactor activities both in fluid phase and on surface-bound C3b, and exhibits only weak decay-accelerating activity for C3 convertase (C3bBb). The other mutant, (R78G)FH1-4(V62), binds poorly to immobilized C3b (K(D) >35 μM) and is severely functionally compromised, having decreased cofactor and decay-accelerating activities. Our data support causal links between these mutations and disease; they demonstrate that mutations affecting the N-terminal activities of FH, not just those in the C terminus, can predispose to aHUS. These observations reinforce the notion that deficiency in any one of several FH functional properties can contribute to the pathogenesis of this disease.