The C(H)1 domain of IgG is not essential for C3 covalent binding: importance of the other constant domains as targets for C3.

The covalent binding of C3 to antigen-antibody complexes [immune complexes (IC)] plays a pivotal role in the elimination of antigens. C3 prevents the formation of large IC lattices promoting their solubilization. Subsequently, bound C3 fragments determine the efficacy of antigen presentation, and the generation of antibody responses and immunological memory. C3 binding to IgG-IC generates IgG-C3b-C3b complexes which are detected by SDS-PAGE as two major bands: C3alpha65-heavy chain and C3alpha65-C3alpha43 covalent complexes. Using human heat-aggregated IgG1 as a model of IC, a C3b binding site was localized only in the Cgamma1 domain. However, with true IC of ovalbumin and rabbit IgG anti-ovalbumin, C3b binds to both the Fab and Fc regions of IgG. To study the binding of C3b to the different domains of IgG and particularly to evaluate the involvement of the Cgamma1 domain, we have constructed recombinant single-chain antibodies without Cgamma1, which have the structure: V(H)-linker-V(L)-hinge-Cgamma2-Cgamma3 (scAb). The variable domains were from a mouse mAb anti-HSA and the constant region (hinge-C(H)2-C(H)3) from human IgG1 or rabbit IgG. C3 binds very efficiently to IC formed with human (h-scAb) or rabbit (r-scAb) recombinant antibodies (scAb-HSA) and generates also two bands on SDS-PAGE (C3alpha65-scAb and C3alpha65-C3alpha43), which are the counterparts of those of the complete antibody. In addition, IC formed with scAb activate the alternative pathway to a similar extent as IC of the entire IgG. These data indicate that the Cgamma1 domain is a dispensable region for C3b binding and that the remaining constant domains are as efficient as Cgamma1 in C3b binding. Overall these results support the view that C3 does not specifically recognize a unique site in the Cgamma1 domain. Rather it seems to be able to attach along the antibody molecule. Probably this implies an advantage for effective processing of C3b-IC and elimination of antigens in vivo.