Proteolytic modification of human complement protein C9: loss of poly(C9) and circular lesion formation without impairment of function

Abstract

We have compared the ability of thrombin-cleaved C9 (C9n) with that of native C9 to produce tubular or ring-like poly(C9) and to express the classical complement lesion on target membranes. Three procedures were used to produce poly(C9): (i) limited proteolysis with trypsin, (ii) interaction with small unilamellar lipid vesicles, and (iii) incubation with a 2- to 4-fold molar excess of ZnCl2. In contrast to C9, which could be converted to tubular poly(C9), C9n was converted to smaller peptides by the first procedure and was aggregated into string-like poly(C9) by the other two methods. C9-depleted human serum (R-9 serum) was reconstituted with either C9 or C9n and these sera were then used to lyse sensitized sheep erythrocytes. Numerous classical complement lesions could be detected on ghost membranes obtained from cells lysed by C9-reconstituted R-9 serum but only a few on ghost membranes produced by C9n-reconstituted R-9 serum. C9n was shown to be hemolytically as active as C9 even when tested under "single-hit" conditions and it was about twice as efficient when compared with C9 in releasing sucrose and inulin from resealed ghosts. These results are interpreted to indicate that formation of the classical complement lesion is only incidental to lysis and not an obligatory event and that enlargement of the "functional pore size" of the complement lesion is not linked to formation of a circular membrane attack complex.