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 digestion of C3b-IgG adducts. In these adducts C3b was radioactively labeled in the free thiol group generated during activation of the internal thioester of C3. Trypsin digestion of ¹⁴C-labeled C3b-IgG adducts degrades C3b to a small antibody-bound ¹⁴C-labeled C3 fragment (¹⁴C-C3frg), whereas the antibody remains unaltered. Papain digestion of trypsin-treated ¹⁴C-C3frg-IgG complexes generated Fc and Fab fragments bearing equivalent amounts of covalently bound ¹⁴C-C3frg (43% and 40%, of the total C3 present in the aggregates, respectively). Hydroxylamine treatment of the ¹⁴C-C3frg-Fab and ¹⁴C-C3frg-Fc complexes released a ¹⁴C-C3frg of similar size (about 3–4 kDa) in which the N-terminal residue was the radiolabeled Cys¹⁰¹⁰. A fragment with the same radioactive N terminus and characteristics was obtained by sequential trypsin and papain digestion of purified C3 labeled with iodo–¹⁴C] acetamide. Affinity-purified ¹⁴C-C3frg-Fc complexes digested with pepsin generated a mixture of radioactive peptides, most probably complexes formed by ¹⁴C-C3frg and Cγ2 or the hinge digestion products, and ¹⁴C-C3frg-pFc' complexes. The latter was also immunoprecipitated with anti-Fc-Sepharose from the pepsin digestion supernatants of ¹⁴C-labeled-C3b-IgG complexes. Taken together these data indicate that, during complement activation through the alternative pathway by IgG immune aggregates, C3 is not bound to a single site on the antibody molecule. Both Fab and Fc regions of IgG are equally efficient targets for C3 anchorage. In addition, the data confirm the pFc' as a region of C3 attachment within the Fc portion, and strongly suggest that C3b is bound either to the Cγ2 domain or the hinge or both.