Nucleophilic modification of human complement protein C3: correlation of conformational changes with acquisition of C3b-like functional properties

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Abstract
Inactivation of [complement] C3 by enzymatic cleavage, nucleophilic addition or slow freezing and thawing resulted in the acquisition of similar end-state conformations as judged by near-UV circular dichroism. Although inactivation by the 2 nonenzymatic processes involves no peptide bond scission, the inactivated C3 resembled C3b; it possessed a free SH group not present in the native protein and an increased surface hydrophobicity as evidenced by enhanced binding of the fluorophore 8-anilino-1-naphthalenesulfonate (ANS). The C3b-like functional properties of modified C3 [Pangburn, M.K., and Mueller-Eberhard, H.J.] may thus be understood in terms of the similarity of its conformation to that of C3b. The rate of the conformational change following proteolytic cleavage was fast and appeared to be limited by the rate of the enzymatic reaction. The rate of conformational change following addition of methylamine was slow and ratelimited by the conformational rearrangement itself, not by the chemical modification. A kinetic analysis of the changes in circular dichroism and ANS fluorescence enhancement suggested that the nucleophilic addition was spectroscopically undetectable and was followed by a minimally biphasic, spectroscopically demonstrable conformational rearrangment. The appearance of C3b-like functional activity in nucleophile-modified C3 largely parallels the time course of the spectroscopically detectable conformational change, but it is distinctly slower than the rate at which hemolytic activity is lost. While fully transconformed methylamine-inactivated C3 can bind factor B and is susceptible to cleavage by C3b inactivator and its cofactor .beta.1H, this cleavage occurs at a substantially slower rate than the equivalent process in C3b. The implications of these findings in terms of the mechanism through which the alternative pathway of C is initiated are discussed.