Formation of covalently linked C3-C3 dimers on IgG immune aggregates

Abstract

Upon activation of the complement system by IgG immune aggregates several components become tightly bound to the aggregates. The covalent interaction of C3 with immune complexes is essential for the solubilization and inhibition of immune precipitation of the complexes. It has recently been reported that on erythrocytes that have a fixed complement, activated C3 can become involved in the formation of C3b-C3b covalent dimers, which acts as high-affinity binding sites for C5 (Kinoshita, T., Takata, Y., Kozono, H., Takeda, J., Hong, K. and Inoue, K., J. Immunol. 1988 141: 3895). To characterize the molecular composition of immune aggregates that have fixed complement by the alternative pathway, we have investigated whether such C3b-C3b dimers are formed in IgG immune complexes. For this purpose immune aggregates bearing covalently bound C3 were analyzed by two-dimensional gel electrophoresis and the resolved bands transferred to polyvinylidene difluoride membranes and sequenced. When immune aggregates were incubated with serum for 15 min at 37 degrees C, the major high-molecular mass bands detected by gel electrophoresis corresponded to heavy chain-C3 alpha 65 and C3 alpha 65-C3 alpha 43 (derived from iC3b-iC3b-IgG) covalent complexes. If K76COONa, an inhibitor of factor I, was added to the serum, before incubation with the immune complexes, then the major C3 alpha fragment detected on the complexes corresponded to the C3 alpha' chain (105 kDa) and not C3 alpha 65. Hence C3b-C3b covalent dimers are readily formed on the immune aggregates incubated with normal human serum, and are degraded to iC3b-iC3b by factor I. The second C3b molecule was shown to be bound to the C3 alpha 43 region (C-terminal portion of the C3 alpha' chain) of the first C3b molecule, which was itself covalently bound to the heavy chain of IgG. Covalent complexes of heavy chain-(C3 alpha 65)2 molecular composition were also detected, but their precise bonding pattern has not been established.