W1206R mutation alters cell surface binding, but not cofactor activity, of mouse FH. (A) Alignment of amino acid sequences showing W1206 of mouse FH is equivalent to W1183 in human FH. (B) Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of purified recombinant mouse FH SCR 19-20 and its W1206R variant (Coomassie blue staining). (C) ELISA plate-based heparin and (D) C3b-binding assay of WT and mutant mouse FH SCR 19-20. Heparin and C3b were coated onto plates at 0.1 to 10 μg per well, and recombinant mouse FH SCR 19-20 was added at 0.5 μg per well. (E) When added to 50% mouse serum, WT but not the W1206R mutant form of mouse FH SCR 19-20 caused lysis of complement-susceptible RBCs of DAF^−/−Crry^−/−C3^−/− mice. Hypotonic lysis in pure water was used as reference control (100% lysis). (F) Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of purified recombinant full-length mouse FH and its W1206R mutant variant (Coomassie blue). (G) Fast protein liquid chromatography analysis showing that the W1206R mutant FH had strong longer retention on a heparin column than WT mouse FH. Recombinant mouse FH proteins (100 μg) were loaded onto the heparin column in PBS and eluted off with 0.5 M NaCl in PBS. (H) Western blot analysis of products from a cofactor activity assay. The W1206R mutant did not affect the cofactor activity of mouse FH in factor I–mediated cleavage of C3b. Data shown in panels C-E represent mean ± standard deviation (SD) of the results. *P < .05; **P < .01; NS, not significant (Student t test).