Quantitative analysis of C4Ab and C4Bb binding to the C3b/C4b receptor (CR1, CD35)

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.

Section: Vcp Domains Critical For Factor I Cfasmentioning

confidence: 99%

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

Ahmad

Raut

Pyaram

et al. 2010

“…A C4b dimer was also a predominant C4b form during the CP C3 convertase assembly on the sensitized liposome surface (38). Formation of C4b dimers could also explain why site 2, which by itself has low DAA, becomes much more efficient if combined with site 1 in the protein LHR A/LHR C. Higher avidity of CR1 to C3b or C4b dimers compared with monomers is well documented (39)(40)(41)(42). Moreover, on E, CR1's cofactor activity for C4b is higher if it forms C4b-C3b dimers (43).…”

Section: C3 Convertasementioning

confidence: 99%

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

Krych-Goldberg

Hauhart

Porzukowiak

et al. 2005

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.

“…The C4b binding site in SCR domains 1-3 became known as functional site 1, and the C3b binding sites in the nearly identical three domain combinations of SCRs 8 -10 and SCRs 15-17 of LHR-B and LHR-C, respectively, each became known as functional site 2. It later became clear that site 2 was also capable of binding C4b (9,10), although there is dispute about whether the site 2 interaction with C4b is weaker (9) or stronger (10) than that of C4b with site 1.…”

mentioning

confidence: 99%

“…Other functional properties, including cleavage rate by C1 s, classical pathway C3 convertase subunit activity, regulation by complement factor I (fI) in the presence of C4b-binding protein (C4BP), and ability to act as a transacylation acceptor for C3b in the formation of the classical pathway C5 convertase, were found not to differ substantially between C4A and C4B (13,14,22). However, there have been three reports concluding that C4Ab (or, in one case, ammonia-treated C4A, a thioester-cleaved C4b-like species) displayed higher binding to CR1 than did C4Bb, or ammonia-treated C4B (23)(24)(25). The study by Reilly and Mold (25) used isotypic forms of covalent dimers of human C4b fragment, in which the cross-link was via the liberated thioester cysteine, to actually measure equilibrium-binding constants.…”

mentioning

confidence: 99%

“…At present, nothing is known about the relative transacylation efficiency of C4Ab vs C4Bb to apoptotic bodies. However, both the greater ability of C4Ab relative to C4Bb to bind covalently to IgG, and thereby inhibit large and insoluble immune complexes from forming (20,21), as well as the reported 3-to 4-fold higher affinity of C4Ab for CR1 (25) can be envisaged as preferentially facilitating the clearance of C4Ab-opsonized autoantigens. This, in turn, has been invoked as an explanation for the SLE association with C4A, but not C4B deficiency states.…”

mentioning

confidence: 99%

See 1 more Smart Citation

The C4A and C4B Isotypic Forms of Human Complement Fragment C4b Have the Same Intrinsic Affinity for Complement Receptor 1 (CR1/CD35)

Clemenza

Isenman

2004

Several previous reports concluded that the C4b fragment of human C4A (C4Ab) binds with higher affinity to CR1 than does C4Bb. Because the isotypic residues, 1101PCPVLD and 1101LSPVIH in C4A and C4B, respectively, are located within the C4d region, one may have expected a direct binding contribution of C4d to the interaction with CR1. However, using surface plasmon resonance as our analytical tool, with soluble rCR1 immobilized on the biosensor chip, we failed to detect significant binding of C4d of either isotype. By contrast, binding of C4c was readily detectable. C4A and C4B, purified from plasma lacking one of the isotypes, were C1̄s converted to C4Ab and C4Bb. Spontaneously formed disulfide-linked dimers were separated from monomers and higher oligomers by sequential chromatographic steps. The binding sensorgrams of C4Ab and C4Bb monomers as analytes reached steady state plateaus, and these equilibrium data yielded essentially superimposable saturation curves that were well fit by a one-site binding model. Although a two-site model was required to fit the equilibrium-binding data for the dimeric forms of C4b, once again there was little difference in the KD values obtained for each isotype. Independent verification of our surface plasmon resonance studies came from ELISA-based inhibition experiments in which monomers of C4Ab and C4Bb were equipotent in inhibiting the binding of soluble CR1 to plate-bound C4b. Although divergent from previous reports, our results are consistent with recent C4Ad structural data that raised serious doubts about there being a conformational basis for the previously reported isotypic differences in the C4b-CR1 interaction.