SC5b-7, SC5b-8 and SC5b-9 complexes of complement: ultrastructure and localization of the S-protein (vitronectin) within the macromolecules

Abstract

Purified terminal components of the complement system were used together with purified S-protein, the inhibitor of the membrane attack complex, to generate the soluble complexes SC5b-7, SC5b-8 and SC5b-9. These complexes were purified by ultracentrifugation in sucrose density gradients with 50-70% yield, exhibiting sedimentation coefficients of 20 S, 21 S and 23 S, respectively. In Ouchterlony double-diffusion analysis, the purified complexes gave a line of identity against all antisera of the precursor components indicating that complex formation had occurred. The identity of the complexes was also revealed by the appearance of all subunit components after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Since the inhibitor function of S-protein in the terminal complement cascade should also be manifested in the morphology of the macromolecules generated, the ultrastructures of the three complexes were analyzed by electron microscopy. In contrast to aggregated (C5b-7)n and (C5b-8)n, negatively stained SC5b-7 and SC5b-8 imaged mostly as monomeric irregularly shaped cylindrical structures, whereas SC5b-9 less than 27 S) appeared as wedge-shaped structure lacking the tubular polymerized C9. (All three complexes were also generated in the presence of biotinyl-S-protein and labeled with avidin-gold conjugates as electron-dense marker). Analysis of the modified complexes in electron micrographs demonstrated that the complexes were marked exclusively at one site of their ultrastructures, suggesting this region to be the location of S-protein and the critical site for membrane binding of C5b-7 or C5b-8 and for initiation of C9 polymerization. These results support recent findings in which the function of S-protein as complement inhibitor was dependent on conformational changes of the protein molecule with concomitant exposure of the heparin-binding domain.