C3 BINDS WITH SIMILAR EFFICIENCY TO FAB AND FC REGIONS OF IGG IMMUNE AGGREGATES

The covalent binding reaction of the third component of complement (C3 ) with rabbit IgG immune aggregates has been studied by enzymic digest ion of C3b-IgG adducts. In these adducts C3b was radioactively labeled in the free thiol group generated during activation of the internal t hioester of C3. Trypsin digestion of C-14-labeled C3b-IgG adducts degr ades C3b to a small antibody-bound C-14-labeled C3 fragment (C-14-C3fr g), whereas the antibody remains unaltered. Papain digestion of trypsi n-treated C-14-C3frg-IgG complexes generated Fc and Fab fragments bear ing equivalent amounts of covalently bound C-14-C3frg (43 % and 40 %, of the total C3 present in the aggregates, respectively). Hydroxylamin e treatment of the C-14-C3frg-Fab and C-14-C3frg-Fc complexes released a C-14-C3frg of similar size (about 3-4 kDa) in which the N-terminal residue was the radiolabeled Cys(1010). A fragment with the same radio active N terminus and characteristics was obtained by sequential tryps in and papain digestion of purified C3 labeled with iodo-[C-14] acetam ide. Affinity-purified C-14-C3frg-Fc complexes digested with pepsin ge nerated a mixture of radioactive peptides, most probably complexes for med by C-14-C3frg and C gamma 2 or the hinge digestion products, and C -14-C3frg-pFc' complexes. The latter was also immunoprecipitated with anti-Fc-Sepharose from the pepsin digestion supernatants of C-14-label ed-C3b-IgG complexes. Taken together these data indicate that, during complement activation through the alternative pathway by IgG immune ag gregates, C3 is not bound to a single site on the antibody molecule. B oth Fab and Fc regions of IgG are equally efficient targets for C3 anc horage. In addition, the data confirm the pFc' as a region of C3 attac hment within the Fc portion, and strongly suggest that C3b is bound ei ther to the C gamma 2 domain or the hinge or both.