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 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.
Antón
L C
LC
Department of Immunology, Fundación Jiménez Diaz, Madrid, Spain.
Ruiz
S
S
Barrio
E
E
Marqués
G
G
Sánchez
A
A
Vivanco
F
F
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
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