title: C3 binds with similar efficiency to Fab and Fc regions of IgG immune aggregates.
abstract: 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 C gamma 2 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 C gamma 2 domain or the hinge or both.
authors.affiliation: Department of Immunology, Fundación Jiménez Diaz, Madrid, Spain.
authors.forename: L C
authors.initials: LC
authors.lastname: Antón
authors.forename: S
authors.initials: S
authors.lastname: Ruiz
authors.forename: E
authors.initials: E
authors.lastname: Barrio
authors.forename: G
authors.initials: G
authors.lastname: Marqués
authors.forename: A
authors.initials: A
authors.lastname: Sánchez
authors.forename: F
authors.initials: F
authors.lastname: Vivanco
mesh_terms: D000818:Animals; D000936:Antigen-Antibody Complex / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D003176:Complement C3 / Q000378:metabolism*; D003179:Complement C3b / Q000378:metabolism; D003170:Complement Pathway, Alternative; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000737:chemistry; D007140:Immunoglobulin Fab Fragments / Q000378:metabolism*; D007141:Immunoglobulin Fc Fragments / Q000378:metabolism*; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D010449:Peptide Mapping; D011485:Protein Binding; D011817:Rabbits
chemical_list: D000936:Antigen-Antibody Complex; D003176:Complement C3; D006898:Hydroxylamines; D007140:Immunoglobulin Fab Fragments; D007141:Immunoglobulin Fc Fragments; D046911:Macromolecular Substances; D019811:Hydroxylamine; D003179:Complement C3b