FORMATION OF COVALENTLY LINKED C3-C3 DIMERS ON IGG IMMUNE AGGREGATES

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 act 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.