Identification and functional characterization of CFD E69K. (A) Sequence alignment of complement factor D (CFD) showing conservation across diverse species of a negatively-charged residue, glutamic acid (E) or aspartic acid (D), at position 69 (arrowhead). Green shading denotes exact sequence conservation, red shading denotes conservation of amino acid charge. (B) Structural modeling of the catalytic triad of CFD WT (left) compared to CFD E69K (right) (Protein Data Bank: 2XWB). (C) Whole cell lysates from HEK-293T cells expressing CFD WT, CFD E69K (E69K), or catalytic-dead CFD (HDS/AAA), in which the catalytic triad residues histidine 66, aspartic acid 114 and serine 208 were replaced with alanines, were subjected to western blot analysis to quantify the total expression of each variant in each cell line. Untransfected (UT) HEK-293T cells were used as a negative control for CFD expression. (D) Red blood cell lysis assay results for human CFD-deficient serum reconstituted with the indicated CFD variants. Data shown are representative results of at least 3 independent experiments.