Photolabeling of a hydrophobic domain of the ninth component of human complement.
Recent experiments with membrane-restricted, photoactivatable probes indicated a preferential labeling of C9 within the assembled membrane attack complex (MAC) of complement, suggesting a direct role for C9 in the interaction of the MAC with membrane lipids. To further characterize the lipid-binding sites on C9, we have now used C9 that has been cleaved by alpha-thrombin. This enzyme cleaves C9 at one site but the newly generated peptides, C9a and C9b, respectively, remain noncovalently associated and the cleaved protein suffers no loss in hemolytic activity. When cleaved C9 was incorporated into the MAC during assembly on phospholipid vesicles and photolabeled, subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and fluorography revealed that only the larger fragment C9b, but not the smaller fragment C9a, became labeled. C9 attached alone to vesicles through heat aggregation in the absence of the precursor complex C5b-8 is also accessible to the hydrophobic photolabel. When cleaved C9 is used in the heat-induced assembly on vesicles and the polymerized C9 is photolabeled, the label associates again predominantly with C9b and not C9a. These results not only show that, within C9 polymers or within the assembled MAC, C9 possesses a two-domain structure, but also lend considerable support to the structure proposed for C9 by Biesecker et al. (Biesecker, G., Gerard, C., and Hugli, T. E. (1982) J. Biol. Chem. 257, 2584-2590) who classified C9a as hydrophilic and C9b as hydrophobic.
D001386:Azides; D003186:Complement C9 / Q000378:metabolism*; D015938:Complement Membrane Attack Complex; D003165:Complement System Proteins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D010782:Photolysis; D013917:Thrombin / Q000378:metabolism
D001386:Azides; D003186:Complement C9; D015938:Complement Membrane Attack Complex; C019266:12-(4-azido-2-nitrophenoxy)stearoylglucosamine; D003165:Complement System Proteins; D013917:Thrombin