The effects of mild reduction on the structure and function of C3.

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Abstract
Reduction and alkylation of C3 in nondenaturing buffers by 2 mM DTT (1 hr, 23 degrees C) (C3 R/A) results in a > 99% loss of C3 hemolytic activity. In contrast, sham alkylated protein, or protein allowed to reoxidize from the mildly reduced state, showed full activity. The loss in lytic activity correlated with the alkylation of approximately 6 liberated sulfhydryls, all of which arose from intrachain disulfides in the alpha polypeptide chain of C3. Data from CD experiments indicate that these bonds are essential for maintaining the native conformation of the protein. The loss of hemolytic activity of C3 R/A was neither due to its inability to be cleaved by C3 convertases nor to a dysfunction of the cleavage-induced labile membrane binding site as cells bearing C3 convertases firmly bound C3 R/A to an extent 80% that of native C3. The hemolytic defect in C3 R/A was found to result from its inability, once deposited on C42-bearing cells, to bind C5 and thereby participate in the formation of a C5 convertase. Unlike the effect on hemolytic activity, C3 R/A showed only a partial loss (3-fold) in its ability to bind to C3 receptors on monocytes. Taken together, these studies differentiate in molecular terms several of the functional sites in C3 and stress the significance of intra-alpha-chain disulfides in maintaining the conformation in activated C3 required for binding of C5.