Molecular organization of C9 within the membrane attack complex of complement. Induction of circular C9 polymerization by the C5b-8 assembly.

Evidence has been presented suggesting that during assembly of the membrane attack complex (MAC) of complement, the C5b-8 complex induces polymerization of C9. The C9 polymer was detected by sodium dodecyl sulfate (SDS) gel electrophoresis of MAC isolated from complement-lysed erythrocytes. It resembled the previously described polymerized C9 (poly C9) produced from isolated monomeric C9 by prolonged incubation at 37 degrees C in that it was resistant to dissociation by SDS and reducing agents and had an apparent molecular weight of approximately 1.1 million. The presence of poly C9 in the MAC was further supported by the expression of identical neoantigens by the MAC and poly C9 and by the high C9 content of the MAC relative to its other constituents. Isolated C8 in solution was found to have a single C9-binding site. In mixture, the two proteins formed a reversible equimolar complex that had a sedimentation coefficient of 10.5S. In contrast, a single, cell-bound C5b-8 complex was found to bind up to 12-15 C9 molecules and clusters of C5b- 8 bound 6-8 C9 molecules per C8 molecule. In either case, typical ultrastructural membrane lesions were observed, suggesting that the membrane lesion is identical with the tubular poly C9 consisting of 12-16 C9 molecules, and that the MAC can have either the composition (C5b-8)polyC9 or (CSb-8)(2)polyC9. When C9 input was restricted so that the molar C9/C8 ratio was less than or equal to 3, C9-induced aggregates of C5b-8 were observed but virtually no circular membrane lesions were found. We suggest, therefore, that C9, at low dosage, causes cross-linking of multiple C5b-8 complexes within the target membrane and that, at high dosage, C9 is polymerized by C5b-8 to form a transmembrane channel within the MAC assembly. It is primarily the C9 polymer that evokes the ultrastructural image of the MAC or of membrane lesions caused by complement.