C3b and C4b binding and cofactor activity of K32N compared to wild type MCP.

<p>(A) C4b and C3b binding. CHO cell lysates from transient transfections were incubated with C4b- or C3b-coated wells and binding was assessed using a polyclonal antibody to MCP in an ELISA format. CHO, a mock transfected control. Data represent the mean ± standard deviation at each concentration from three independent experiments. (B) Cofactor activity for CFI-mediated cleavage of C4b and C3b. Cell lysates containing 25 pg of MCP (wild-type or K32N) were incubated with biotinylated C4b or C3b in the presence of purified human CFI in 25 mM NaCl and cleavage was assessed by Western blot. C4b and C3b peptide chains and cleavage fragments are identified. For C4b cofactor activity, C4d fragment generated by cleavage of C4b after 90 min was quantified by densitometric scanning, compared with quantity of β chain, and normalized to wild type. For C3b cofactor activity, α1 fragment generated by cleavage was compared to β chain by densitometry, and normalized to wild type. Data represent the mean ± standard deviation of three independent experiments. (C) Representative Western blots of MCP activity for CFI-mediated cleavage of C4b and C3b. Left panel: MCP K32N has deficient C4b cofactor activity at 20 and 90 min. The C4d fragment generated by cleavage of the α′ chain is not visible at 20 min and is diminished relative to wild type at 90 min. The boxed inserts represent 5-fold longer exposure of the blot. The CHO negative control was a nonadjacent lane (broken line). Right panel: MCP K32N has similar C3b cofactor activity to wild type. Equal amounts of α1 fragments are generated after 20 and 90 min. The CHO negative control was a nonadjacent lane (broken line). In two additional experiments, similar results were obtained in cofactor assays at 150 mM NaCl concentration (unpublished data).