The functions of the ninth component of human complement are sustained by disulfide bonds with different susceptibilities to reduction.
Purified C9 with expected hemolytic and polymerizing activities was found to contain approximately 0.2 mol of sulfhydryl groups/mol of C9. By proteolysis of C9 with labeled SH groups, the SH residues on intact C9 were mapped to Cys-359 and Cys-384 which, presumably, form an intra-domain disulfide bond in the intact molecule. The blocking of these sulfhydryl residues by alkylation, however, had minimal influence on the functions of C9. On the other hand, reduction of C9 by 1 mM dithiothreitol (DTT) (6-fold molar excess over Cys residues) followed by alkylation resulted in a complete block of polymerization activity and a 50% loss of C9 hemolytic activity. In contrast, the ability of C9 to bind EAC1-8 remained largely unaffected. The loss of poly-C9 formation activity correlated with the alkylation of approx. 6 liberated sulfhydryl groups. Hemolytic activity was abolished by treatment with > 5 mM DTT which allowed the liberation of approximately 18 sulfhydryl groups. Most of the DTT-susceptible disulfides were within the C9a fragment (an N-terminal peptide derived by thrombin). Thus, three major functions of C9, EAC1-8 binding, polymerization, and hemolytic activity, are sustained by disulfide bond-dependent conformational motifs with different susceptibility to reducing reagents. The maintenance of the N-terminal C9a region is essential for polymerization, but not EAC1-8 binding activity of C9. Taken together, the results of the present study differentiate in molecular terms several of the functional portions of C9, and stress the significance of intra-chain disulfide linkages in maintaining the structural components necessary for the functions of C9.
Hatanaka
M
M
Department of Immunology, Center for Adult Diseases Osaka, Japan.
Seya
T
T
Inai
S
S
Shimizu
A
A
D000818:Animals; D003186:Complement C9 / Q000737:chemistry* / Q000502:physiology*; D004220:Disulfides / Q000737:chemistry*; D004229:Dithiothreitol; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes; D006801:Humans; D066298:In Vitro Techniques; D008301:Maleimides; D051379:Mice; D010084:Oxidation-Reduction; D012756:Sheep; D013329:Structure-Activity Relationship
D003186:Complement C9; D004220:Disulfides; D005456:Fluorescent Dyes; D008301:Maleimides; C019347:N-(7-dimethylamino-4-methylcoumarinyl)maleimide; D004229:Dithiothreitol