C3 binds covalently to the Cγ3 domain of IgG immune aggregates during complement activation by the alternative pathway

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Abstract
Ovalbumin-antiovalbumin IgG immune aggregates were incubated with normal human serum in the presence of iodo[1-14C]acetamide, in conditions in which only the alternative pathway of complement was activated. The [14C]C3b-IgG covalent complexes formed were digested with pepsin, and analysed by SDS/polyacrylamide-gel electrophoresis and fluorography. Covalent complexes of [14C]C3-Fd and [14C]C3-pFc'' were visualized, demonstrating that, during complement activation by the alternative pathway, C3 is covalently incorporated into the C.gamma.3 domain of IgG, as well as into the Fd region. The C.gamma.2 domain becomes protected from pepsin action by the bound C3b. All the covalent linkages between C3 and the IgG were sensitive to hydroxylamine. When [14C]C3-pFc'' covalent complexes were treated with 1 M-NH2OH and loaded onto a Bio-Gel P-4 column, a radioactive peak of 3 kDa was obtained. The material released from [14C]C3-pFc'' and [14C]C3-F(ab'')2 complexes after treatment with 1 M-NH2OH was mixed and analysed in the Bio-Gel P-4 column. A similar radioactive peak of 3 kDa was obtained. When this peak, either from [14C]C3-pFc'' alone or from the mixture of [14C]C3-F(ab'')2 and [14C]C3-pFc'', was fractionated by h.p.l.c., virtually the same radioactive peptide profile was obtained, indicating that very similar C3 peptides remained covalently bound to both regions (Fab and C.gamma.3) of the antibody molecule. It is suggested that C3 bound to the C.gamma.3 domain of IgG interfere with the Fc-Fc interactions of immune aggregates and thus may be involved in several biological properties displayed by these complement-activating aggregates.

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