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 compl exes (IC)] plays a pivotal role in the elimination of antigens. C3 pre vents the formation of large IC lattices promoting their solubilizatio n. Subsequently, bound C3 fragments determine the efficacy of antigen presentation, and the generation of antibody responses and immunologic al memory. C3 binding to IgG-IC generates IgG-C3b-C3b complexes which are detected by SDS-PAGE as two major bands: C3 alpha 65-heavy chain a nd C3 alpha 65-C3 alpha 43 covalent complexes. Using human heat-aggreg ated IgG1 as a model of IC, a C3b binding site was localized only in t he C(gamma)1 domain. However, with true IC of ovalbumin and rabbit IgG anti-ovalbumin, C3b binds to both the Fab and Fe regions of IgG. To s tudy the binding of C3b to the different domains of IgG and particular ly to evaluate the involvement of the C(gamma)1 domain, we have constr ucted recombinant single-chain antibodies without C(gamma)1, which hav e the structure: V-H-linker-V-L-hinge-C(gamma)2-C(gamma)3 (scAb). The variable domains were from a mouse mAb anti-HSA and the constant regio n (hinge-C(H)2-C(H)3) from human IgG1 or rabbit IgG. C3 binds very eff iciently to IC formed with human (h-scAb) or rabbit (r-scAb) recombina nt antibodies (scAb-HSA) and generates also two bands on SDS-PAGE (C3 alpha 65-scAb and C3 alpha 65-C3 alpha 43), which are the counterparts of those of the complete antibody. In addition, IC formed with scAb a ctivate the alternative pathway to a similar extent as IC of the entir e IgG. These data indicate that the C(gamma)1 domain is a dispensable region for C3b binding and that the remaining constant domains are as efficient as C(gamma)1 in C3b binding. Overall these results support t he view that C3 does not specifically recognize a unique site in the C (gamma)1 domain. Rather it seems to be able to attach along the antibo dy molecule. Probably this implies an advantage for effective processi ng of C3b-IC and elimination of antigens in vivo.