Classical Pathway C3 Research Articles

P053 The intestinal complement system and its role in dysregulation of the intestinal B-cell compartment in Crohn’s Disease patients

Abstract Background For several decades, there is growing evidence that a hyperactive B-cell compartment may be involved in the disturbed mucosal immune response in Crohn’s disease (CD) patients. This is reflected by approximately one third of all CD patients displaying an excessive production of autoantibodies against the intestinal M-cell expressed FimH receptor glycoprotein 2 (GP2) which inhibits induction of adaptive immunity against adherent-invasive flagellated bacteria. However, underlying mechanisms of an imbalanced B-cell compartment in CD patients still remain elusive. Given that the complement system plays a critical role in the detection and clearance of bacteria and promotes B-cell immunity, we asked if the complement system contributes to a dysregulated B-cell response in CD. Methods Systematical expression analyses of the intestinal complement components were performed in IBD patients, including CD and ulcerative colitis (UC) patients, versus non-IBD controls. Additionally, activity of the three activation pathways of complement system was studied in serum samples of CD patients and controls. Further, immunoglobulin levels in intestinal biopsies, sera and fecal samples were analyzed. Results CD patients, but no UC patients, showed increased expression of the early classical complement pathway components C1QA, C1QB, C1S, C2, C3, C4A, CR1 and CR2 in sigmoidal biopsy samples that does not differ significantly between remission and active disease state. This could be verified by immunohistochemistry, displaying an over-representation of mucosal C1q, C3 and B-cell expressed CR2 (also CD21) in colon biopsies of CD patients in remission. In line with this, CD patients in remission present with enhanced activity of the classical as well as alternative pathway of complement activation in serum compared to non-IBD controls. Accordingly, high protein levels of C3 cleavage fragments were detected in fecal samples from CD patients in contrast to non-IBD controls. While there were no differences in systemic immunoglobulin levels in serum, intestinal IgM was increased in CD patients in remission. Notably, fecal IgA levels were strongly diminished in these patients, while expression of the polymeric immunoglobulin receptor was not affected. Conclusion In summary, our findings demonstrate an augmented activation of the intestinal complement system that may potentially be involved in the etiology of CD. Expansion of mucosal IgM+/CR2+ B cells together with a reduced fecal IgA level, points to a locally disturbed induction of intestinal IgA secreting plasma cells in CD patients.

The human complement receptor type 2 (CR2)/CR1 fusion protein TT32, a novel targeted inhibitor of the classical and alternative pathway C3 convertases, prevents arthritis in active immunization and passive transfer mouse models

Complement activation in human diseases is characterized by the local covalent deposition of the long-lived C3 fragments iC3b/C3dg/C3d. Previously, TT30, a complement alternative pathway (AP)-selective inhibitor, was designed as a fusion protein linking the first four short consensus repeats (SCRs) of human complement receptor type 2 (CR2) with the first five SCRs of human factor H (fH). TT30 acts by utilizing CR2 SCR1–4 to bind the initially formed iC3b/C3dg/C3d fragments and delivering surface-targeted inhibition of AP C3 and C5 convertases through fH SCR 1-5. In order to combine classical (CP) and lectin (LP) pathway inhibitory abilities employing CR2-mediated targeting, TT32 was developed. TT32 is a CR2-CR1 fusion protein using the first ten SCRs of CR1, chosen because they contain both C3 and C5 convertase inhibitory activity through utilization of decay-acceleration and cofactor activity for both AP and CP. In Wieslab assays, TT32 showed potent inhibition of the CP and AP with IC50 of 11 and 46 nM, respectively. The TT32 inhibitory activity is partially blocked with a molar excess of a competing anti-CR2 mAb, thus demonstrating the importance of the CR2 targeting. TT32 was studied in the type II (CII) collagen-induced arthritis (CIA), an active immunization model, and the CII antibody-induced arthritis (CAIA) passive transfer model. In CIA, injection of 2.0 mg TT32 at day 21 and 28 post disease induction, but not untargeted CR1 alone, resulted in a 51.5% decrease in clinical disease activity (CDA). In CAIA, treatment with TT32 resulted in a 47.4% decrease in CDA. Therefore, a complement inhibitor that targets both the AP and CP/LP C3/C5 convertases was shown to limit complement-mediated tissue damage and inflammation in disease models in which all three complement activation pathways are implicated.

Genome-wide expression differences in anti-Vegf and dexamethasone treatment of inflammatory angiogenesis in the rat cornea

Angiogenesis as a pathological process in the eye can lead to blindness. In the cornea, suppression of angiogenesis by anti-VEGF treatment is only partially effective while steroids, although effective in treating inflammation and angiogenesis, have broad activity leading to undesirable side effects. In this study, genome-wide expression was investigated in a suture-induced corneal neovascularization model in rats, to investigate factors differentially targeted by dexamethasone and anti-Vegf. Topical treatment with either rat-specific anti-Vegf, dexamethasone, or normal goat IgG (sham) was given to sutured corneas for 48 hours, after which in vivo imaging, tissue processing for RNA microarray, and immunofluorescence were performed. Dexamethasone suppressed limbal vasodilation (P < 0.01) and genes in PI3K-Akt, focal adhesion, and chemokine signaling pathways more effectively than anti-Vegf. The most differentially expressed genes were confirmed by immunofluorescence, qRTPCR and Western blot. Strong suppression of Reg3g and the inflammatory chemokines Ccl2 and Cxcl5 and activation of classical complement pathway factors C1r, C1s, C2, and C3 occurred with dexamethasone treatment, effects absent with anti-Vegf treatment. The genome-wide results obtained in this study provide numerous potential targets for specific blockade of inflammation and angiogenesis in the cornea not addressed by anti-Vegf treatment, as possible alternatives to broad-acting immunosuppressive therapy.

The structural basis for inhibition of the classical and lectin complement pathways by S. aureus extracellular adherence protein.

The extracellular adherence protein (Eap) plays a crucial role in pathogenesis and survival of Staphylococcus aureus by inhibiting the classical and lectin pathways of complement. We have previously shown that Eap binds with nanomolar affinity to complement C4b and disrupts the initial interaction between C4b and C2, thereby inhibiting formation of the classical and lectin pathway C3 pro-convertase. Although an underlying mechanism has been identified, the structural basis for Eap binding to C4b is poorly understood. Here, we show that Eap domains 3 and 4 each contain a low-affinity, but saturable binding site for C4b. Taking advantage of the high lysine content of Eap, we used a zero-length crosslinking approach to map the Eap binding site to both the α'- and γ-chains of C4b. We also probed the C4b/Eap interface through a chemical footprinting approach involving lysine modification, proteolytic digestion, and mass spectrometry. This identified seven lysines in Eap that undergo changes in solvent exposure upon C4b binding. We found that simultaneous mutation of these lysines to either alanine or glutamate diminished C4b binding and complement inhibition by Eap. Together, our results provide insight into Eap recognition of C4b, and suggest that the repeating domains that comprise Eap are capable of multiple ligand-binding modes.

Regulation of the alternative pathway of complement modulates injury and immunity in a chronic model of dextran sulphate sodium-induced colitis.

The role of complement in inflammatory bowel disease (IBD) has been studied primarily using acute models, and it is unclear how complement affects processes in more relevant chronic models of IBD in which modulation of adaptive immunity and development of fibrosis have pathogenic roles. Using mice deficient in C1q/mannose-binding lectin (MBL) or C3, we demonstrated an important role for these opsonins and/or the classical pathway C3 convertase in providing protection against mucosal injury and infection in a model of chronic dextran sulphate sodium (DSS)-induced colitis. In contrast, deficiency of the alternative pathway (fB(-/-) mice) had significantly less impact on injury profiles. Consequently, the effect of a targeted inhibitor of the alternative pathway was investigated in a therapeutic protocol. Following the establishment of colitis, mice were treated with CR2-fH during subsequent periods of DSS treatment and acute injury (modelling relapse). CR2-fH significantly reduced complement activation, inflammation and injury in the colon, and additionally reduced fibrosis. Alternative pathway inhibition also altered the immune response in the chronic state in terms of reducing numbers of B cells, macrophages and mature dendritic cells in the lamina propria. This study indicates an important role for the alternative pathway of complement in the pathogenesis and the shaping of an immune response in chronic DSS-induced colitis, and supports further investigation into the use of targeted alternative pathway inhibition for the treatment of IBD.

The role of the lys628 (192) residue of the complement protease, c1s, in interacting with Peptide and protein substrates.

The C1s protease of the classical complement pathway propagates the initial activation of this pathway of the system by cleaving and thereby activating the C4 and C2 complement components. This facilitates the formation of the classical pathway C3 convertase (C4bC2a). C1s has a Lys residue located at position 628 (192 in chymotrypsin numbering) of the SP domain that has the potential to partially occlude the S2–S2′ positions of the active site. The 192 residue of serine proteases generally plays an important role in interactions with substrates. We therefore investigated the role of Lys628 (192) in interactions with C4 by altering the Lys residue to either a Gln (found in many other serine proteases) or an Ala residue. The mutant enzymes had altered specificity profiles for a combinatorial peptide substrate library, suggesting that this residue does influence the active site specificity of the protease. Generally, the K628Q mutant had greater activity than wild type enzyme against peptide substrates, while the K628A residue had lowered activity, although this was not always the case. Against peptide substrates containing physiological substrate sequences, the K628Q mutant once again had generally higher activity, but the activity of the wild type and mutant enzymes against a C4 P4–P4′ substrate were similar. Interestingly, alteration of the K628 residue in C1s had a marked effect on the cleavage of C4, reducing cleavage efficiency for both mutants about fivefold. This indicates that this residue plays a different role in cleaving protein versus peptide substrates and that the Lys residue found in the wild type enzyme plays an important role in interacting with the C4 substrate. Understanding the basis of the interaction between C1s and its physiological substrates is likely to lead to insights that can be used to design efficient inhibitors of the enzyme for use in treating diseases caused by inflammation as result of over-activity of the classical complement pathway.

Autoantibody stabilization of the classical pathway C3 convertase leading to C3 deficiency and Neisserial sepsis: C4 nephritic factor revisited

C3 deficiency is a rare disorder that leads to recurrent pyogenic infections. Here we describe a previously healthy 18 y/o Caucasian male with severe meningococcal disease. Total hemolytic activity was zero secondary to an undetectable C3. The C3 gene was normal by sequencing. Mixing the patient's serum with normal human serum led to C3 consumption. An IgG autoantibody in the patient's serum was identified that stabilized the classical pathway C3 and C5 convertases, thus preventing decay of these enzyme complexes. This autoantibody is an example of a C4 nephritic factor, with an additional feature of stabilizing the C5 convertase. Previous patients with C4 nephritic factor had membranoproliferative glomerulonephritis. Two years after presentation, this patient's C3 remains undetectable with no evidence of renal disease. We revisit the role of autoantibodies to classical pathway convertases in disease, review the literature on C4-NeF and comment on its detection in the clinical laboratory.

The human complement receptor type 2 (CR2)/CR1 fusion protein TT32, a targeted inhibitor of the classical and alternative pathway C3 convertases, prevents arthritis in active immunization and passive transfer models and acts by CR2-dependent targeting of CR1 regulatory activity

The human complement receptor type 2 (CR2)/CR1 fusion protein TT32, a targeted inhibitor of the classical and alternative pathway C3 convertases, prevents arthritis in active immunization and passive transfer models and acts by CR2-dependent targeting of CR1 regulatory activity

Domain Swapping Reveals Complement Control Protein Modules Critical for Imparting Cofactor and Decay-Accelerating Activities in Vaccinia Virus Complement Control Protein

Vaccinia virus encodes a structural and functional homolog of human complement regulators named vaccinia virus complement control protein (VCP). This four-complement control protein domain containing secretory protein is known to inhibit complement activation by supporting the factor I-mediated inactivation of complement proteins, proteolytically cleaved form of C3 (C3b) and proteolytically cleaved form of C4 (C4b) (termed cofactor activity), and by accelerating the irreversible decay of the classical and to a limited extent of the alternative pathway C3 convertases (termed decay-accelerating activity [DAA]). In this study, we have mapped the VCP domains important for its cofactor activity and DAA by swapping its individual domains with those of human decay-accelerating factor (CD55) and membrane cofactor protein (MCP; CD46). Our data indicate the following: 1) swapping of VCP domain 2 or 3, but not 1, with homologous domains of decay-accelerating factor results in loss in its C3b and C4b cofactor activities; 2) swapping of VCP domain 1, but not 2, 3, or 4 with corresponding domains of MCP results in abrogation in its classical pathway DAA; and 3) swapping of VCP domain 1, 2, or 3, but not 4, with homologous MCP domains have marked effect on its alternative pathway DAA. These functional data together with binding studies with C3b and C4b suggest that in VCP, domains 2 and 3 provide binding surface for factor I interaction, whereas domain 1 mediates dissociation of C2a and Bb from the classical and alternative pathway C3 convertases, respectively.

Ectromelia virus inhibitor of complement enzymes protects intracellular mature virus and infected cells from mouse complement.

Poxviruses produce complement regulatory proteins to subvert the host's immune response. Similar to the human pathogen variola virus, ectromelia virus has a limited host range and provides a mouse model where the virus and the host's immune response have coevolved. We previously demonstrated that multiple components (C3, C4, and factor B) of the classical and alternative pathways are required to survive ectromelia virus infection. Complement's role in the innate and adaptive immune responses likely drove the evolution of a virus-encoded virulence factor that regulates complement activation. In this study, we characterized the ectromelia virus inhibitor of complement enzymes (EMICE). Recombinant EMICE regulated complement activation on the surface of CHO cells, and it protected complement-sensitive intracellular mature virions (IMV) from neutralization in vitro. It accomplished this by serving as a cofactor for the inactivation of C3b and C4b and by dissociating the catalytic domain of the classical pathway C3 convertase. Infected murine cells initiated synthesis of EMICE within 4 to 6 h postinoculation. The levels were sufficient in the supernatant to protect the IMV, upon release, from complement-mediated neutralization. EMICE on the surface of infected murine cells also reduced complement activation by the alternative pathway. In contrast, classical pathway activation by high-titer antibody overwhelmed EMICE's regulatory capacity. These results suggest that EMICE's role is early during infection when it counteracts the innate immune response. In summary, ectromelia virus produced EMICE within a few hours of an infection, and EMICE in turn decreased complement activation on IMV and infected cells.

Complement Component C2, Inhibiting a Latent Serine Protease in the Classical Pathway of Complement Activation

The innate immune response to infection or injury involves an antigen-antibody triggered classical pathway (CP) of complement activation, in which soluble and cell surface plasma proteins cooperatively effect elimination of foreign organisms and damaged host cells. However, protracted or dysfunctional complement activation can lead to inflammatory diseases. Complement component 2 bound to C4b is cleaved by classical (C1s) or lectin (MASP2) proteases to produce C4bC2a, a very short-lived C3 convertase (t(1/2) 2 min) that in turn cleaves C3 to C3a and C3b, leading ultimately to formation of Membrane Attack Complex (MAC) and lysis of bacteria and damaged cells. C2 has the same serine protease domain as C4bC2a but in an inactive zymogen-like conformation, requiring cofactor-induced conformational change for activity. Here, we show that C2 has catalytic protease activity in its own right above pH 7, in the absence of cofactor, processing C3 and C3-derived chromogenic peptide fragments. In contrast to the instability of C3 convertase (t(1/2) 2 min, pH 7), the C2 enzyme is indefinitely stable under alkaline conditions, facilitating studies of its catalytic properties and development of small molecule inhibitors. We characterize the catalytic activity of C2 against C3 and short paranitroanilide peptide substrates, and identify potent small molecule inhibitors of C2 that also inhibit classical pathway C3 convertase, MAC formation, and hemolysis of sensitized sheep erythrocytes. These results provide a new avenue and valuable new insights to inhibiting CP complement activation relevant to inflammatory diseases.

An engineered complement receptor 1 composed of two functional domains can protect against immune-mediated hemolysis

Complement receptor type 1 (CR1) is a versatile inhibitor of both classical and alternative pathway C3 and C5 convertases with an ability to accelerate decay activity and act as a co-factor in C3b/C4b cleavage. In order to develop a short form of CR1 with similar biological activities to the full-length human CR1, we combined functional domain 1, located in the long homologous repeat (LHR) A, with functional domain 2, located in LHR C. We expressed the two-domain, two-function protein with an enterokinase site at the N-terminus and a termination codon at the C-terminus in Escherichia coli. The fusion protein was purified on a Ni–NTA–agarose column. After subsequent refolding, the recombinant CR1-derivative protein was obtained by enterokinase cleavage and subsequent purification. In vitro, the recombinant CR1-derivative reduced hemolysis, C5a release and surface C3 deposition. It was also effective in prolonging survival of transfused incompatible red blood cells in vivo. Our results indicate that the CR1-based protein may be a model for developing smaller and more potent complement inhibitors for future therapeutics.

Regulated complement deposition on the surface of human endothelial cells: Effect of tobacco smoke and shear stress

Cigarette smoke and hemodynamic stress both contribute to vascular inflammation and associated atherosclerosis. We recently demonstrated direct activation of complement components C4 and C3 on human endothelial cells (EC). The present study was designed to explore complement activation on bone marrow microvascular endothelial cells (BMEC) and human umbilical vein endothelial cells (HUVEC) in response to endothelial cell injury by tobacco smoke extract, shear stress, or other known inflammatory and atherogenic mediators, lipopolysaccharide (LPS) and INF-γ. Following treatment, confluent EC monolayers were exposed to plasma (60 min, 37 °C), and cell surface deposition of stable complement derivatives C4d, iC3b and SC5b-9 was measured in situ using an ELISA approach. Consistent with previous results, moderate levels of C4d, iC3b and SC5b-9 deposition were observed on native EC monolayers exposed to human plasma. Tobacco smoke and shear stress enhanced EC C4d deposition. In contrast, LPS and INF-γ failed to affect EC mediated complement activation, despite evidence of EC activation illustrated by ICAM-1 expression. The combination of tobacco smoke and shear stress nearly doubled EC C4d expression. No increases in iC3b or SC5b-9 were noted, suggesting inhibition of classical and alternative pathway C3 convertase assembly or activity. Indeed, concomitantly increased surface expression of complement regulatory proteins CD35 (CR1) and CD55 was observed following EC exposure to tobacco smoke and shear stress. These results suggest that a balance between complement activation and regulation exists at the EC surface, and may impact vascular injury leading to thrombosis, arteriosclerosis, and atherogenesis.

Complement Protein C1q Inhibits Antibody-Dependent Enhancement of Flavivirus Infection in an IgG Subclass-Specific Manner

Severe dengue virus infection can occur in humans with pre-existing antibodies against the virus. This observation led to the hypothesis that a subneutralizing antibody level in vivo can increase viral burden and cause more severe disease. Indeed, antibody-dependent enhancement of infection (ADE) in vitro has been described for multiple viruses, including the flaviviruses dengue virus and West Nile virus. Here, we demonstrate that the complement component C1q restricts ADE by anti-flavivirus IgG antibodies in an IgG subclass-specific manner in cell culture and in mice. IgG subclasses that avidly bind C1q induced minimal ADE in the presence of C1q. These findings add a layer of complexity for the analysis of humoral immunity and flavivirus infection.

The Crystal Structure of C2a, the Catalytic Fragment of Classical Pathway C3 and C5 Convertase of Human Complement

The multi-domain serine protease C2 provides the catalytic activity for the C3 and C5- convertases of the classical and lectin pathways of complement activation. Formation of these convertases requires the Mg 2+-dependent binding of C2 to C4b, and the subsequent cleavage of C2 by C1s or MASP2, respectively. The C-terminal fragment C2a consisting of a serine protease (SP) and a von Willebrand factor type A (vWFA) domain, remains attached to C4b, forming the C3 convertase, C4b2a. Here, we present the crystal structure of Mg 2+-bound C2a to 1.9 Å resolution in comparison to its homolog Bb, the catalytic subunit of the alternative pathway C3 convertase, C3bBb. Although the overall domain arrangement of C2a is similar to Bb, there are certain structural differences. Unexpectedly, the conformation of the metal ion-dependent adhesion site and the position of the α7 helix of the vWFA domain indicate a co-factor-bound or open conformation. The active site of the SP domain is in a zymogen-like inactive conformation. On the basis of these structural features, we suggest a model for the initial steps of C3 convertase assembly.

Role of the C3b-binding site on C4b-binding protein in regulating classical pathway C5 convertase

A high affinity C5 convertase is generated when a C3 convertase deposits additional C3b molecules on and around itself thereby switching the substrate specificity of C3 convertase from C3 to C5. In the present study the role of the additional C3b molecules in influencing the regulation of classical pathway C5 convertase by C4b-binding protein (C4BP) was examined and compared to its precursor, the C3 convertase. Determination of IC 50 for inhibiting formation of the high affinity C5 convertase and for enhancing its decay (72 and 20 nM) were found to be similar to those obtained for the surface-bound C3 convertase (35 and 11 nM). No difference was observed in the cofactor activity of C4BP for surface-bound C4b alone or when in complex with C3b. Analysis of binding interactions between C4BP and EAC1,C4b cells revealed an average apparent dissociation constant (12 nM) similar to that obtained with EAC1,C4b cells with C3b on them (11 nM). Increasing the C4b or C3b density on the cell surface did not alter the affinity of C4BP. The data suggest that C4BP regulates the C5 convertase by mechanisms similar to those observed for the C3 convertase. Since the IC 50 for inhibiting formation of the soluble C3 convertase (5 nM) is 50–80-fold below the normal serum concentration of C4BP (250–400 nM), C4BP in blood effectively prevents formation of classical pathway C3 convertase in the fluid phase. Although deposition of additional C3b molecules is necessary to convert a C3 convertase to a high affinity C5 convertase, the additional C3b molecules play no role in the regulation of C5 convertase by C4BP.

Mannan-binding lectin activates C3 and the alternative complement pathway without involvement of C2

Lectin pathway activation of C3 is known to involve target recognition by mannan-binding lectin (MBL) or ficolins and generation of classical pathway C3 convertase via cleavage of C4 and C2 by MBL-associated serine protease 2 (MASP-2). We investigated C3 activation in C2-deficient human sera and in sera with other defined defects of complement to assess other mechanisms through which MBL might recruit complement. The capacity of serum to support C3 deposition was examined by ELISA using microtiter plates coated with O antigen-specific oligosaccharides derived from Salmonella typhimurium, S. thompson, and S. enteritidis corresponding to serogroups B, C, and D (BO, CO, and DO). MBL bound to CO, but not to BO and DO, and efficiently supported C3 deposition in the absence of C2, C4, or MASP-2. The existence of an MBL-dependent C2 bypass mechanism for alternative pathway-mediated C3 activation was clearly demonstrated using CO, solid-phase mannan, and E. coli LPS. MASP-1 might contribute, but was not required for C3 deposition in the model used. Independent of MBL, specific antibodies to CO supported C3 deposition through classical and alternative pathways. MBL-dependent C2 bypass activation could be particularly important in various inherited and acquired complement deficiency states.

Mannan-binding lectin activates C3 and the alternative complement pathway without involvement of C2

Lectin pathway activation of C3 is known to involve target recognition by mannan-binding lectin (MBL) or ficolins and generation of classical pathway C3 convertase via cleavage of C4 and C2 by MBL-associated serine protease 2 (MASP-2). We investigated C3 activation in C2-deficient human sera and in sera with other defined defects of complement to assess other mechanisms through which MBL might recruit complement. The capacity of serum to support C3 deposition was examined by ELISA using microtiter plates coated with O antigen-specific oligosaccharides derived from Salmonella typhimurium, S. thompson, and S. enteritidis corresponding to serogroups B, C, and D (BO, CO, and DO). MBL bound to CO, but not to BO and DO, and efficiently supported C3 deposition in the absence of C2, C4, or MASP-2. The existence of an MBL-dependent C2 bypass mechanism for alternative pathway-mediated C3 activation was clearly demonstrated using CO, solid-phase mannan, and E. coli LPS. MASP-1 might contribute, but was not required for C3 deposition in the model used. Independent of MBL, specific antibodies to CO supported C3 deposition through classical and alternative pathways. MBL-dependent C2 bypass activation could be particularly important in various inherited and acquired complement deficiency states.

CRIT peptide interacts with factor B and interferes with alternative pathway activation

Complement C2 receptor inhibitor trispanning (CRIT) inhibits the classical pathway (CP) C3 convertase formation by competing with C4b for the binding of C2. The C-terminal 11-amino-acid of the first CRIT-extracellular domain (CRIT-H17) has a strong homology with a sequence in the C4β chain, which is responsible for the binding of C2. Since the CP and alternative pathway (AP) C3 convertases have many functional and structural similarities, we further investigated the effects of CRIT-H17 on the AP. The factor D-mediated cleavage of factor B (FB) was blocked by CRIT-H17. By ELISA and immunoblot, CRIT-H17 was shown to bind FB. CRIT-H17 had no decay activity on the C3bBb complex as compared to decay-accelerating factor. Binding of CRIT-H17 to FB did not interfere with the assembly of C3bB complex. In a haemolytic assay using C2-deficient serum, CRIT-H17 interfered with AP complement activation.

Synergy between Two Active Sites of Human Complement Receptor Type 1 (CD35) in Complement Regulation: Implications for the Structure of the Classical Pathway C3 Convertase and Generation of More Potent Inhibitors

The extracellular domain of the complement receptor type 1 (CR1; CD35) consists entirely of 30 complement control protein repeats (CCPs). CR1 has two distinct functional sites, site 1 (CCPs 1-3) and two copies of site 2 (CCPs 8-10 and CCPs 15-17). In this report we further define the structural requirements for decay-accelerating activity (DAA) for the classical pathway (CP) C3 and C5 convertases and, using these results, generate more potent decay accelerators. Previously, we demonstrated that both sites 1 and 2, tandemly arranged, are required for efficient DAA for C5 convertases. We show that site 1 dissociates the CP C5 convertase, whereas the role of site 2 is to bind the C3b subunit. The intervening CCPs between two functional sites are required for optimal DAA, suggesting that a spatial orientation of the two sites is important. DAA for the CP C3 convertase is increased synergistically if two copies of site 1, particularly those carrying DAA-increasing mutations, are contained within one protein. DAA in such constructs may exceed that of long homologous repeat A (CCPs 1-7) by up to 58-fold. To explain this synergy, we propose a dimeric structure for the CP C3 convertase on cell surfaces. We also extended our previous studies of the amino acid requirements for DAA of site 1 and found that the CCP 1/CCP 2 junction is critical and that Phe82 may contact the C3 convertases. These observations increase our understanding of the mechanism of DAA. In addition, a more potent decay-accelerating form of CR1 was generated.