Mapping of the C3b-binding site of CR1 and construction of a (CR1)2-F(ab')2 chimeric complement inhibitor.

Abstract

SummaryCR1/CR2 chimeric receptors in which various short consensus repeats (SCRs) of CR1 were attached to CR2 were transiently expressed on COS cells, and assessed for the binding ofpolymerized C3b (pC3b) and anti-CR2 by immunofluorescence. Of COS cells expressing chimeras containing SCR 1-4, 1-3, 2-4, 1-2, and 2-3 of the long homologous repeats (LHRs) -B or -C, 96%, 66%, 23%, 0%, and 0%, respectively, bound pC3b. K562 cells were stably transfected with wild-type CR1, deletion mutants of CR1, and the CR1/CR2 chimeras, respectively, and assayed for binding of 12SI-pC3b. The dissociation constants (Kd) for pC3b of wild-type CR1 and the LHR-BD and -CD constructs were in the range of 1.0-2.7 nM, and of the CR1/CR2 chimeras containing SCRs 1-4, 1-3, and 2-4 of LHR-B or -C were 1.8-2.4, 6-9, and 22-36 nM, respectively. The factor I-cofactor function of the CR1/CR2 chimeras paralleled the C3b-binding function of the constructs. A CR1/immunoglobulin (Ig) chimeric protein was prepared by fusing SCRs 1-4 of LHR-B to the heavy chains of a murine F(ab')2 anti-nitrophenacetyl (NP) monoclonal antibody. The (CR1)2-F(ab')a chimera, which retained its specificity for NP, was as effective as soluble, full-length CR1 in binding pC3b, serving as a cofactor for factor I-mediated cleavage of C3b, and inhibiting activation of the alternative pathway, indicating that the bivalent expression of these SCRs reconstitutes the alternative pathway inhibitory function of CR1. The feasibility of creating CR1/Ig chimeras makes possible a new strategy of targeting complement inhibition by the use of Ig fusion partners having particular antigenic specificities.

Soluble complement receptor type 1 (sCR1) in chronic liver diseases: serum levels at different stages of liver diseases

“…CR1 acts as a cofactor of I in C3b degradation, and this makes it a potent inhibitor of complement activation. A recombinant soluble form of CR1 (rCR1) was shown to be 100-fold more efficient as a regulator of complement activation than factor H, the physiological cofactor for the inactivation of C3b in plasma, and its possible therapeutic use as an antiinflammatory agent is at present being investigated [7,8].…”

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

“…A similar mechanism could also be operative for the interaction of VCP with C4b and factor I. Because domain requirements for ligand binding and CFAs in viral (34,48,49,54) and human complement regulators (55)(56)(57)(58)(59)(60)(61)(62) are conserved, it could be inferred that the recognition sites for C3b/C4b and factor I are spatially conserved in both human and viral complement regulators, and that they employ a common mechanism to inactivate C3b and C4b.…”

Identification of Complement Regulatory Domains in Vaccinia Virus Complement Control Protein

“…FUNCTIONAL DOMAINS OF VCP 12389 14,16,19,20,44,52). There is now a consensus that factor I-mediated inactivation of C3b and C4b involves two steps: (i) recognition of C3b or C4b by the cofactor and (ii) interaction of factor I with C3b or C4b and the cofactor (48,54).…”