Delta-short consensus repeat 4-decay accelerating factor (DAF: CD55) inhibits complement-mediated cytolysis but not NK cell-mediated cytolysis

NK cells play a critical role in the rejection of xenografts. In this study, we report on an investigation of the effect of complement regulatory protein, a decay accelerating factor (DAF: CD55), in particular, on NK cell-mediated cytolysis. Amelioration of human NK cell-mediated pig endothelial cell (PEC) and pig fibroblast cell lyses by various deletion mutants and point substitutions of DAF was tested, and compared with their complement regulatory function. Although wild-type DAF and the delta-short consensus repeat (SCR) 1-DAF showed clear inhibition of both complement-mediated and NK-mediated PEC lyses, delta-SCR2-DAF and delta-SCR3-DAF failed to suppress either process. However, delta-SCR4-DAF showed a clear complement regulatory effect, but had no effect on NK cells. Conversely, the point substitution of DAF (L147 x F148 to SS and KKK(125-127) to TTT) was half down-regulated in complement inhibitory function, but the inhibition of NK-mediated PEC lysis remained unchanged. Other complement regulatory proteins, such as the cell membrane-bound form factor H, fH-PI, and C1-inactivator, C1-INH-PI, and CD59 were also assessed, but no suppressive effect on NK cell-mediated PEC lysis was found. These data suggest, for DAF to function on NK cells, SCR2-4 is required but no relation to its complement regulatory function exists.     

Cell surface expression of the C3b/C4b receptor (CR1) protects Chinese hamster ovary cells from lysis by human complement.

The C3b/C4b receptor, also known as complement receptor type 1 (CR1, CD35), is a single chain glycoprotein consisting of 30 repeating homologous protein domains known as short consensus repeats (SCR) followed by transmembrane and cytoplasmic domains. A series of recombinant proteins derived from CR1 has been prepared and assessed for the capacity to inhibit complement lysis of the host Chinese hamster ovary (CHO) cells. The full-length recombinant CR1 inhibited human complement-mediated CHO cell lysis, and the efficiency of inhibition was directly proportional to the number of receptors/cell. The SCR 15-18 of CR1, but not SCR 15-16, inhibited complement lysis of the host CHO cell, bound monomeric C3b (Kd,app = 6.5 x 10(-7) M), and dimeric C3b (Kd = 1.8 x 10(-8) M), and served as a cofactor in the proteolysis of C3b by factor I, confirming and extending the observations of Fearon and colleagues (Kalli, K. R., Hsu, P., Bartow, T. J., Ahearn, J. M., Matsumoto, A. K., Klickstein, L. B., and Fearon, D. T. (1991) J. Exp. Med. 174, 1451-1460). The SCR 1-4 of CR1, but not SCR 1-2, also inhibited complement lysis of the host CHO cell, indicating that more than two SCR are necessary and that four SCR are sufficient for optimal C4b binding to CR1. Thus, the structural requirements for C4b binding are analogous to those for C3b binding, namely, four SCR of CR1 form the binding sites for each of these proteins. CR1 has long been recognized to regulate extrinsic complement activation, that is, to bind to and promote the degradation of fluid phase C3b and of C3b attached to immune complex. These results demonstrate that CR1 is also an intrinsic regulator of complement activation in that, under appropriate conditions, CR1 inhibits complement-mediated lysis of the cell on which it is expressed.     

Isotyping of component C4 of human complement using differences in the functional activity of isotypes C4A and C4B

The difference in the functional activity of the isotypes A and B of component C4 of human complement was used to determine their ratio and to detect the inherited deficiency of the isotypes. ELISA methods were developed for the quantitative assay of component C4 (conventional sandwich method) and its functional activity. When determining the functional activity, the classic pathway of the complement and therefore of component C4 was activated on activators sorbed on ELISA microplates: immunoglobulin IgG3 or liposaccharide of theShigella sonnei cell walls, which activates the complement by binding component C1. The nascent fragment C4b is covalently bound to the target activator; C4Ab binds better to the target protein (immunoglobulin), and C4Bb to the target carbohydrate (liposaccharide). Therefore, when immunoglobulin is a target activator, isotype C4A is bound and determined; and when the complement is activated with liposaccharide, isotype C4B is determined. The radio of the activities determined by the two methods indicates the deficiency in the individual isotypes of component C4 or its absence. The rabbit polyclonal monospecific antibodies against the human component C4 and the conjugates of these antibodies with horseradish peroxidase were used in the methods described.     

Recombinant generation of two fragments of the rat complement inhibitory factor H [FH(SCR1-7) and FH(SCR1-4)] and their structural and functional characterization in comparison to FH isolated from rat serum

Factor H (FH) is the predominant soluble inhibitor of the complement system. With a concentration of 200-800 microg/ml in human and rat plasma it acts as a cofactor for the soluble factor I (FI)-mediated cleavage of the component C3b to iC3b. Furthermore it competes with factor B for binding to C3b and C3(H2O) and promotes the dissociation of the C3bBb complex. FH is a monomer of about 155 kDa which comprises 20 short consensus repeats (SCR), each of which is composed of approximately 60 amino acid (aa) residues. Two functional fragments of FH comprising the SCR1-4 or SCR1-7 were generated using either the Baculovirus system or stably transfected human embryonal kidney cells, respectively. These fragments, as well as FH purified from rat serum, were first analyzed for their relative molecular weights (Mr) using non-reducing or reducing SDS-PAGE. The Mr of the FH variants differed by about 20% depending on the experimental conditions employed. Only the Mr of proteins separated under reducing conditions were in accordance with the MW calculated from the aa sequence. Analyses of the glycosylation patterns using PAS-staining showed a lack of staining of the recombinant variants (SCR1-4 and SCR1-7) in contrast to FH(SCR1-20) from serum. Using a complement hemolysis assay (CH50-assay) all three variants exhibited a molar complement inhibitory activity of FH(1-20)/FH(1-7)/FH(1-4) of about 3/1/1. These data support the postulated model of FH bearing three binding sites for its ligand C3b, from which one is located in the SCR1-4, whereas the other two are located in the SCR8-20.     

The pH-regulated antigen 1 of Candida albicans binds the human complement inhibitor C4b-binding protein and mediates fungal complement evasion

Candida albicans binds and utilizes human complement inhibitors, such as C4b-binding protein (C4BP), Factor H, and FHL-1 for immune evasion. Here, we identify Candida pH-regulated antigen 1 (Pra1) as the first fungal C4BP-binding protein. Recombinant Pra1 binds C4BP, as shown by ELISA and isothermal titration calorimetry, and the Pra1-C4BP interaction is ionic in nature. The Pra1 binding domains within C4BP were localized to the complement control protein domain 4 (CCP4), CCP7, and CCP8. C4BP bound to Pra1 maintains complement-inhibitory activity. C4BP and Factor H bind simultaneously to Candida Pra1 and do not compete for binding at physiological levels. A Pra1-overexpressing C. albicans strain, which had about 2-fold Pra1 levels at the surface acquired also about 2-fold C4BP to the surface, compared with the wild type strain CAI4. A Pra1 knock-out strain showed ～22% reduced C4BP binding. C4BP captured by C. albicans from human serum inhibits C4b and C3b surface deposition and also maintains cofactor activity. In summary, Candida Pra1 represents the first fungal C4BP-binding surface protein. Pra1, via binding to C4BP, mediates human complement control, thereby favoring the immune and complement evasion of C. albicans.     

Pig complement regulator factor H: molecular cloning and functional characterization

Complement is an efficient defense mechanism of innate immunity. Factor H is the central complement regulator of the alternative pathway, acting in the fluid-phase and on self surfaces. Pigs are considered a suitable source for xenotransplantation and thus several membrane-bound pig complement regulators with importance for the acute rejection phase have been investigated. However, pig fluid-phase regulators have not been described so far. We report the cloning, expression and functional characterization of pig factor H. After constructing a pig liver cDNA library, a full-length factor H cDNA was isolated and sequenced. The predicted protein is organized in 20 short consensus repeat (SCR) domains and has an overall identity of 62% to the human protein. For functional characterization, three deletion constructs of pig factor H were expressed in insect cells. Pig factor H construct SCR 1–4 has cofactor activity for factor I-mediated cleavage of human C3b, which is similar to the human regulator. In addition, this N-terminal construct binds to human C3b, while a construct consisting of SCR 15–20 showed a weaker binding to human C3b/C3d. Pig factor H has two major binding sites for heparin, as the two constructs representing SCR 1–7 and SCR 15–20 proteins, but not the SCR 1–4 protein, bind heparin. The C-terminal construct is able to bind to human endothelial cells, as assayed by FACS. We show that pig and human factor H share functional characteristics in complement regulation and cell surface binding. Possible consequences of using pig livers for xenotransplantation are discussed.The nucleotide sequence data reported are available in the EMBL database (accession number AJ278470)     

Complete sequencing and expression of three complement components,C1r,C4 and C1 inhibitor,of the classical activation pathway of the complement system in rainbow trout<Emphasis Type="Italic"> Oncorhynchus mykiss</Emphasis>

Three complement components, C1r, C4 and C1 inhibitor, of the classical activation pathway have been fully sequenced and their expression investigated in rainbow trout (Oncorhynchus mykiss). Trout C1r cDNA encodes a 707-amino-acid (aa) protein with a theoretical M _r of 77,200. The trout translation shows highest homology with carp C1r/s, and lower, equal homologies to mammalian C1r and C1s, and MASPs from other vertebrate species. However, phylogenetic analysis and structural features suggest that the trout sequence, together with the two carp sequences, are the orthologues of mammalian C1r. The trout C4 cDNA encodes a 1,724-aa protein with a theoretical M _r of 192,600. The trout translation shows higher homologies to the carp C4B and medaka C4, but lower homologies to C4 from other species and the carp C4A. It has a predicted signal peptide of 22 aa, a -chain of 773 aa, a -chain of 635 aa and a -chain of 288 aa. Trout C1 inhibitor cDNA encodes a 611-aa protein with a theoretical M _r of 68,700. The trout translation has a C-terminal serpin domain with high homologies with mammalian counterparts (~37% identities), and a longer N-terminus, with no significant homology to other serpins, which contains two Ig-like domains. A molecule containing two Ig-like domains followed by a serpin domain, has also been found in an EST clone from another bony fish, the Japanese flounder. This suggests a unique structural feature of C1 inhibitor in fish. The functional significance of the Ig domains is discussed. The liver is the major site of expression of the three trout complement components, C1r, C4 and C1 inhibitor, although their expression is also detectable in other tissues. The extra-hepatic expression of complement genes may be important for local protection and inflammatory responses. Low-level constitutive expression of the three components was also detectable in a trout monocyte/macrophage cell line RTS-11, but only the expression of C4 could be upregulated by LPS.The nucleotide sequence data will appear in the EMBL/DDBJ/GenBank nucleotide sequence database under the following accession numbers: AJ519929 (trout C1r), AJ519930 (trout C1 inhibitor), AJ544262 (trout C4) and BN000290 (flounder C1 inhibitor)     

Adiponectin binds C1q and activates the classical pathway of complement

The adipose-specific protein adiponectin binds to a number of target molecules, including damaged endothelium and the surface of apoptotic cells. However, the significance of this binding remains unclear. This study demonstrates the binding of purified C1q to recombinant adiponectin under physiological conditions, and the dependence of this upon Ca⁺⁺ and Mg⁺⁺. Binding was enhanced by metaperiodate-mediated destruction of glucosylgalactosyl sugars on adiponectin. Adiponectin was bound by the globular domain of the A chain of collagenase-digested C1q, and C1q binding induced deposition of C4 and C3 through activation of the classical complement pathway. After Western blotting, affinity-purified adiponectin from human serum bound C1q, whereas adiponectin in whole serum did not, unless pre-treated with metaperiodate. These results suggest adiponectin is member of the pattern-recognition family of defence collagens, able to bind target molecules and activate complement. It may therefore play an important role in innate immunity and autoimmune phenomena.     

Complement C3 and C4 expression in C1q sufficient and deficient mouse models of Alzheimer's disease

Alzheimer's disease (AD) is a neurodegenerative disease resulting in progressive cognitive decline. Amyloid plaque deposits consisting specifically of β-amyloid peptides that have formed fibrils displaying β-pleated sheet conformation are associated with activated microglia and astrocytes, are colocalized with C1q and other complement activation products, and appear at the time of cognitive decline in AD. Amyloid precursor protein (APP) transgenic mouse models of AD that lack the ability to activate the classical complement pathway display less neuropathology than do the APPQ+/+ mice, consistent with the hypothesis that complement activation and the resultant inflammation may play a role in the pathogenesis of AD. Further investigation of the presence of complement proteins C3 and C4 in the brain of these mice demonstrate that both C3 and C4 deposition increase with age in APPQ+/+ transgenic mice, as expected with the age-dependent increase in fibrillar β-amyloid deposition. In addition, while C4 is predominantly localized on the plaques and/or associated with oligodendrocytes in APPQ+/+ mice, little C4 is detected in APPQ−/− brains consistent with a lack of classical complement pathway activation because of the absence of C1q in these mice. In contrast, plaque and cell associated C3 immunoreactivity is seen in both animal models and, surprisingly, is higher in APPQ−/− than in APPQ+/+ mice, providing evidence for alternative pathway activation. The unexpected increase in C3 levels in the APPQ−/− mice coincident with decreased neuropathology provides support for the hypothesis that complement can mediate protective events as well as detrimental events in this disease. Finally, induced expression of C3 in a subset of astrocytes suggests the existence of differential activation states of these cells.     

Evasion of complement-mediated lysis and complement C3 deposition are regulated by Francisella tularensis lipopolysaccharide O antigen

The bacterium Francisella tularensis (Ft) is a potential weapon of bioterrorism when aerosolized. Macrophage infection is necessary for disease progression and efficient phagocytosis by human macrophages requires serum opsonization by complement. Microbial complement activation leads to surface deposition of a highly regulated protein complex resulting in opsonization or membrane lysis. The nature of complement component C3 deposition, i.e., C3b (opsonization and lysis) or C3bi (opsonization only) fragment deposition, is central to the outcome of activation. In this study, we examine the mechanisms of Ft resistance to complement-mediated lysis, C3 component deposition on the Ft surface, and complement activation. Upon incubation in fresh nonimmune human serum, Schu S4 (Ft subsp. tularensis), Fn (Ft subsp. novicida), and LVS (Ft subsp. holarctica live vaccine strain) were resistant to complement-mediated lysis, but LVSG and LVSR (LVS strains altered in surface carbohydrate structures) were susceptible. C3 deposition, however, occurred on all strains. Complement-susceptible strains had markedly increased C3 fragment deposition, including the persistent presence of C3b compared with C3bi, which indicates that C3b inactivation results in survival of complement-resistant strains. C1q, an essential component of the classical activation pathway, was necessary for lysis of complement-susceptible strains and optimal C3 deposition on all strains. Finally, use of Francisella LPS mutants confirmed O Ag as a major regulator of complement resistance. These data provide evidence that pathogenic Francisella activate complement, but are resistant to complement-mediated lysis in part due to limited C3 deposition, rapid conversion of surface-bound C3b to C3bi, and the presence of LPS O Ag.     

Heterocomplexes of mannose-binding lectin and the pentraxins PTX3 or serum amyloid P component trigger cross-activation of the complement system

The long pentraxin 3 (PTX3), serum amyloid P component (SAP), and C-reactive protein belong to the pentraxin family of pattern recognition molecules involved in tissue homeostasis and innate immunity. They interact with C1q from the classical complement pathway. Whether this also occurs via the analogous mannose-binding lectin (MBL) from the lectin complement pathway is unknown. Thus, we investigated the possible interaction between MBL and the pentraxins. We report that MBL bound PTX3 and SAP partly via its collagen-like domain but not C-reactive protein. MBL-PTX3 complex formation resulted in recruitment of C1q, but this was not seen for the MBL-SAP complex. However, both MBL-PTX3 and MBL-SAP complexes enhanced C4 and C3 deposition and opsonophagocytosis of Candida albicans by polymorphonuclear leukocytes. Interaction between MBL and PTX3 led to communication between the lectin and classical complement pathways via recruitment of C1q, whereas SAP-enhanced complement activation occurs via a hitherto unknown mechanism. Taken together, MBL-pentraxin heterocomplexes trigger cross-activation of the complement system.     

Identification of functional domains in kaposica, the complement control protein homolog of Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8)

Recently it has been shown that kaposica, an immune evasion protein of Kaposi's sarcoma-associated herpesvirus, inactivates complement by acting on C3-convertases by accelerating their decay as well as by acting as a cofactor in factor I-mediated inactivation of their subunits C3b and C4b. Here, we have mapped the functional domains of kaposica. We show that SCRs 1 and 2 (SCRs 1-2) and 1-4 are essential for the classical and alternative pathway C3-convertase decay-accelerating activity (DAA), respectively, while the SCRs 2-3 are required for factor I cofactor activity (CFA) for C3b and C4b. SCR 3 and SCRs 1 and 4, however, contribute to optimal classical pathway DAA and C3b CFA, respectively. Binding data show that SCRs 1-4 and SCRs 1-2 are the smallest structural units required for measuring detectable binding to C3b and C4b, respectively. The heparin-binding site maps to SCR 1.     

The extended multidomain solution structures of the complement protein Crry and its chimeric conjugate Crry-Ig by scattering,analytical ultracentrifugation and constrained modelling: implications for function and therapy

Complement receptor-related gene/protein y (Crry) is a cell membrane-bound regulator of complement activation found in mouse and rat. Crry contains only short complement/consensus repeat (SCR) domains. X-ray and neutron scattering was performed on recombinant rat Crry containing the first five SCR domains (rCrry) and mouse Crry with five SCR domains conjugated to the Fc fragment of mouse IgG1 (mCrry-Ig) in order to determine their solution structures at medium resolution. The radius of gyration R(G) of rCrry was determined to be 4.9-5.0 nm, and the R(G) of the cross-section was 1.2-1.5 nm as determined by X-ray and neutron scattering. The R(G) of mCrry-Ig was 6.6-6.7 nm, and the R(G) of the cross-section were 2.3-2.4 nm and 1.3 nm. The maximum dimension of rCrry was 18 nm and that for mCrry-Ig was 26 nm. The neutron data indicated that rCrry and mCrry-Ig have molecular mass values of 45,000 Da and 140,000 Da, respectively, in agreement with their sequences, and sedimentation equilibrium data supported these determinations. Time-derivative velocity experiments gave sedimentation coefficients of 2.4S for rCrry and 5.4S for mCrry-Ig. A medium-resolution model of rCrry was determined using homology models that were constructed for the first five SCR domains of Crry from known crystal and NMR structures, and linked by randomly generated linker peptide conformations. These trial-and-error calculations revealed a small family of extended rCrry structures that best accounted for the scattering and ultracentrifugation data. These were shorter than the most extended rCrry models as the result of minor bends in the inter-SCR orientations. The mCrry-Ig solution data were modelled starting from a fixed structure for rCrry and the crystal structure of mouse IgG1, and was based on conformational searches of the hinge peptide joining the mCrry and Fc fragments. The best-fit models showed that the two mCrry antennae in mCrry-Ig were extended from the Fc fragment. No preferred orientation of the antennae was identified, and this indicated that the accessibility of the antennae for the molecular targets C4b and C3b was not affected by the covalent link to Fc. A structural comparison between Crry and complement receptor type 1 indicated that the domain arrangement of Crry SCR 1-3 is as extended as that of the CR1 SCR 15-17 NMR structure.     

Solution structure of the complex between CR2 SCR 1-2 and C3d of human complement: an X-ray scattering and sedimentation modelling study

Complement receptor type 2 (CR2, CD21) forms a tight complex with C3d, a fragment of C3, the major complement component. Previous crystal structures of the C3d-CR2 SCR 1-2 complex and free CR2 SCR 1-2 showed that the two SCR domains of CR2 form contact with each other in a closed V-shaped structure. SCR 1 and SCR 2 are connected by an unusually long eight-residue linker peptide. Medium-resolution solution structures for CR2 SCR 1-2, C3d, and their complex were determined by X-ray scattering and analytical ultracentrifugation. CR2 SCR 1-2 is monomeric. For CR2 SCR 1-2, its radius of gyration R(G) of 2.12(+/-0.05) nm, its maximum length of 10nm and its sedimentation coefficient s20,w(o) of 1.40(+/-0.03) S do not agree with those calculated from the crystal structures, and instead suggest an open structure. Computer modelling of the CR2 SCR1-2 solution structure was based on the structural randomisation of the eight-residue linker peptide joining SCR 1 and SCR 2 to give 9950 trial models. Comparisons with the X-ray scattering curve indicated that the most favoured arrangements for the two SCR domains corresponded to an open V-shaped structure with no contacts between the SCR domains. For C3d, X-ray scattering and sedimentation velocity experiments showed that it exists as a monomer-dimer equilibrium with a dissociation constant of 40 microM. The X-ray scattering curve for monomeric C3d gave an R(G) value of 1.95 nm, and this together with its s20,w(o) value of 3.17 S gave good agreement with the monomeric C3d crystal structure. Modelling of the C3d dimer gave good agreements with its scattering and ultracentrifugation parameters. For the complex, scattering and ultracentrifugation experiments showed that there was no dimerisation, indicating that the C3d dimerisation site was located close to the CR2 SCR 1-2 binding site. The R(G) value of 2.44(+/-0.1) nm, its length of 9 nm and its s20,w(o) value of 3.45(+/-0.01) S showed that its structure was not much more elongated than that of C3d. Calculations with 9950 models of CR2 SCR 1-2 bound to C3d through SCR 2 showed that SCR 1 formed an open V-shaped structure with SCR 2 and was capable of interacting with the surface of C3d. We conclude that the open V-shaped structures formed by CR2 SCR 1-2, both when free and when bound to C3d, are optimal for the formation of a tight two-domain interaction with its ligand C3d.     

Restoration of complement-enhanced neutralization of vaccinia virus virions by novel monoclonal antibodies raised against the vaccinia virus complement control protein

The vaccinia virus complement control protein (VCP) is secreted by infected cells and has been shown to inhibit complement activation through interactions with C3b/C4b. It contains four short consensus repeat (SCR) domains. It has been suggested that all four SCRs are required for VCP's activity. To elucidate which SCR domains are involved in abolishing complement-enhanced neutralization of vaccinia virus virions, we generated and characterized a panel of mouse monoclonal antibodies (MAbs) raised against VCP. Ten MAbs were isolated and all recognized VCP on Western blots under reducing conditions as well as native-bound VCP in a sandwich enzyme-linked immunosorbent assay. Three of the 10 MAbs (2E5, 3D1, and 3F11) inhibited VCP's abolition of complement-enhanced neutralization of vaccinia virus virions. These MAbs blocked the interaction of VCP with C3b/C4b. The seven remaining MAbs did not alter VCP function in the complement neutralization assay and recognized VCP bound to C3b/C4b. To understand MAb specificity and mode of interaction with VCP, we mapped the MAb binding regions on VCP. The seven nonblocking MAbs all bound to the first SCR of VCP. One of the blocking MAbs recognized SCR 2 while the other two recognized either SCR 4 or the junction between SCRs 3 and 4, indicating that structural elements involved in the interaction of VCP with C3b/C4b are located within SCR domains 2 and 3 and 4. These anti-VCP MAbs may have clinical significance as therapeutic inhibitors of VCP's complement control activity and may also offer a novel approach to managing vaccinia virus vaccine complications that occur from smallpox vaccination.     

Identification of the streptococcal M protein binding site on membrane cofactor protein (CD46)

Adherence of group A streptococcus (GAS) to keratinocytes is mediated by an interaction between human CD46 (membrane cofactor protein) with streptococcal cell surface M protein. CD46 belongs to a family of proteins that contain structurally related short consensus repeat (SCR) domains and regulate the activation of the complement components C3b and/or C4b. CD46 possesses four SCR domains and the aim of this study was to characterize their interaction with M protein. Following confirmation of the M6 protein-dependent interaction between GAS and human keratinocytes, we demonstrated that M6 protein binds soluble recombinant CD46 protein and to a CD46 construct containing only SCRs 3 and 4. M6 protein did not bind to soluble recombinant CD46 chimeric proteins that had the third and/or fourth SCR domains replaced with the corresponding domains from another complement regulator, CD55 (decay-accelerating factor). Homology-based molecular modeling of CD46 SCRs 3 and 4 revealed a cluster of positively charged residues between the interface of these SCR domains similar to the verified M protein binding sites on the plasma complement regulators factor H and C4b-binding protein. The presence of excess M6 protein did not inhibit the cofactor activity of CD46 and the presence of excess C3b did not inhibit the ability of CD46 to bind M6 protein by ELISA. In conclusion, 1) adherence of M6 GAS to keratinocytes is M protein dependent and 2) a major M protein binding site is located within SCRs 3 and 4, probably at the interface of these two domains, at a site distinct from the C3b-binding and cofactor site of CD46.     

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.     

Structure of the extracellular portion of CD46 provides insights into its interactions with complement proteins and pathogens

The human membrane cofactor protein (MCP, CD46) is a central component of the innate immune system. CD46 protects autologous cells from complement attack by binding to complement proteins C3b and C4b and serving as a cofactor for their cleavage. Recent data show that CD46 also plays a role in mediating acquired immune responses, and in triggering autophagy. In addition to these physiologic functions, a significant number of pathogens, including select adenoviruses, measles virus, human herpes virus 6 (HHV-6), Streptococci, and Neisseria, use CD46 as a cell attachment receptor. We have determined the crystal structure of the extracellular region of CD46 in complex with the human adenovirus type 11 fiber knob. Extracellular CD46 comprises four short consensus repeats (SCR1-SCR4) that form an elongated structure resembling a hockey stick, with a long shaft and a short blade. Domains SCR1, SCR2 and SCR3 are arranged in a nearly linear fashion. Unexpectedly, however, the structure reveals a profound bend between domains SCR3 and SCR4, which has implications for the interactions with ligands as well as the orientation of the protein at the cell surface. This bend can be attributed to an insertion of five hydrophobic residues in a SCR3 surface loop. Residues in this loop have been implicated in interactions with complement, indicating that the bend participates in binding to C3b and C4b. The structure provides an accurate framework for mapping all known ligand binding sites onto the surface of CD46, thereby advancing an understanding of how CD46 acts as a receptor for pathogens and physiologic ligands of the immune system.     

Mapping of the sites responsible for factor I-cofactor activity for cleavage of C3b and C4b on human C4b-binding protein (C4bp) by deletion mutagenesis

Human C4b-binding protein (C4bp) facilitates the factor I-mediated proteolytic cleavage of the active forms of complement effectors C3b and C4b into their inactive forms. C4bp comprises a disulfide-linked heptamer of alpha-chains with complement (C) regulatory activity and a beta-chain. Each alpha-chain contains 8 short consensus repeat (SCR) domains. Using SCR-deletion mutants of recombinant multimeric C4bp, we identified the domains responsible for the C3b/C4b-binding and C3b/C4b-inactivating cofactor activity. The C4bp mutant with deletion of SCR2 lost the C4b-binding ability, as judged on C3b/C4b-Sepharose binding assaying and ELISA. In contrast, the essential domains for C3b-binding extended more to the C-terminus, exceeding SCR4. Using fluid phase cofactor assaying and deletion mutants of C4bp, SCR2 and 3 were found to be indispensable for C4b cleavage by factor I, and SCR1 contributed to full expression of the factor I-mediated C4b cleaving activity. On the other hand, SCR1, 2, 3, 4, and 5 participated in the factor I-cofactor activity for C3b cleavage, and SCR2, 3, and 4 were absolutely required for C3b inactivation. Thus, different sets of SCRs participate in C3b and C4b inactivation, and the domain repertoire supporting C3b cofactor activity is broader than that supporting C4b inactivation by C4bp and factor I. Furthermore, the domains participating in C3b/C4b binding are not always identical to those responsible for cofactor activity. The necessity of the wide range of SCRs in C3b inactivation compared to C4b inactivation by C4bp and factor I may reflect the physiological properties of C4bp, which is mainly directed to C4b rather than C3b.