Complement deregulation and C3-binding activities of recombinant FHR-1 proteins. (A) Alignment of FH and FHR-1. Numbers in between structures indicate percentages of homology. Proposed FHR-1 dimerization topology is shown. (B) Coomassie-stained sodium dodecyl sulfate–polyacrylamide gel electrophoresis of the recombinant FHR-1 proteins purified from the culture supernatants. (C) FH deregulation assay in sheep erythrocytes (ShEs). Approximately 30% of ShEs are lysed when they are exposed to 20% of a human serum that has been depleted of 75% of the FH. Adding increasing amounts of purified wild-type FHR-1 (FHR-1_WT) or FHR-1_A296V does not significantly increase this percentage of lysis. This is in contrast with the dose-response hemolysis of ShEs that results from the addition of FHR-1_L290S,A296V, FHR-1_L290V, and FHR-1_L290S. Data are means ± standard deviations of triplicates. (D) Binding to immobilized C3b of the different FHR-1 mutants is compared with that of FHR-1_WT using a plate assay. Although FHR-1_L290V and FHR-1_A296V bind similarly to FHR-1_WT (1 ± 0.11 and 0.9 ± 0.4 times, respectively), binding of FHR-1_L290S and FHR-1_L290S,A296V to C3b was estimated in this assay to be 3.65 ± 0.4 and 4.5 ± 1.56 times lower, respectively, than that of FHR-1_WT. (E) FHR-1_WT binds similarly to immobilized native C3 (nC3), C3b, iC3b, and C3dg. The same is also true for mutant FHR-1_L290S,A296V and all other FHR-1 mutants (data not shown). (F) Binding of FHR-1_WT and FH at 300 nM to immobilized C3b and nC3. Only FHR-1 binds to immobilized nC3, indicating that our nC3 preparations were not contaminated with significant amounts of C3(H₂O) forms. OD, optical density.