Hysteresis of human complement C4A to C4B binding in hemolytic activities and inhibition by immune complex aggregates

Abstract --Human complement C4 is a sophisticated innate immune effector that features two classes of polymorphic proteins with acidic C4A and basic C4B. The thioester carbonyl from Gln-1016 of C4A has high binding affinity towards protein antigens rich in amino groups to form covalent amide bonds; while that of C4B manifests avid preference binding towards carbohydrate antigens rich in hydroxyl groups to form covalent ester bonds. Human subjects with deficiency of C4A are predisposed to increased risk of systemic lupus erythematous (SLE). We performed flow cytometry of activated C4 on red blood cells using monoclonal antibodies against human C4d for both C4A and C4B, and monoclonals specific for C4A. Among human subjects with equal numbers of C4A and C4B genes, the levels of C4d protein from C4B deposited on red cells are consistently 3–10 times higher than that from C4A. We performed in depth kinetic studies of activated C4A and C4B by measuring the time course of Ab-sensitized sheep erythrocyte (EA) binding and cell lysis using plasma C4A or C4B. We tested immune complex (IC) aggregates for inhibition of EA lysis. The results showed both C4A and C4B in plasma can lyse EA but C4A exhibit hysteresis properties by lagging C4B. At dilution to <1% or [C4] < 2 mg/mL, the efficiency of C4B on lysis EA remained significantly higher than that of C4A. Addition of IC aggregates inhibited the course of C4A and C4B on lysis of EA: 1.17 mg/mL IC inhibited 50% EA lysis by 1.20 mg/mL C4A. 5.32 mg/mL IC was required to inhibit 50% EA lysis by 1.01 mg/mL C4B. In conclusion, activated C4B binds quickly to sensitized erythrocytes. Activated complement C4A prefers binding to IC aggregates with hysteresis properties. R21 AR070509, R01 AR073311