Decay-Accelerating Factor Must Bind Both Components of the Complement Alternative Pathway C3 Convertase to Mediate Efficient Decay

Abstract

Decay-accelerating factor (DAF; CD55) inhibits the complement (C) cascade by dissociating the multimolecular C3 convertase enzymes central to amplification. We have previously demonstrated using surface plasmon resonance (Biacore International) that DAF mediates decay of the alternative pathway C3 convertase, C3bBb, but not of the inactive proenzyme, C3bB, and have shown that the major site of interaction is with the larger cleavage subunit factor B (Bb) subunit. In this study, we dissect these interactions and demonstrate that the second short consensus repeat (SCR) domain of DAF (SCR2) interacts only with Bb, whereas SCR4 interacts with C3b. Despite earlier studies that found SCR3 to be critical to DAF activity, we find that SCR3 does not directly interact with either subunit. Furthermore, we demonstrate that properdin, a positive regulator of the alternative pathway, does not directly interact with DAF. Extending from studies of binding to decay-accelerating activity, we show that truncated forms of DAF consisting of SCRs 2 and 3 bind the convertase stably via SCR2-Bb interactions but have little functional activity. In contrast, an SCR34 construct mediates decay acceleration, presumably due to SCR4-C3b interactions demonstrated above, because SCR3 alone has no binding or functional effect. We propose that DAF interacts with C3bBb through major sites in SCR2 and SCR4. Binding to Bb via SCR2 increases avidity of binding, concentrating DAF on the active convertase, whereas more transient interactions through SCR4 with C3b directly mediate decay acceleration. These data provide new insights into the mechanisms involved in C3 convertase decay by DAF.

Common polymorphisms in C3, factor B, and factor H collaborate to determine systemic complement activity and disease risk

“…To test effects of C3 R102G polymorphism on convertase accelerated decay, different concentrations of sDAF, native fH and rfH1-4 were flowed over C3b 102G Bb or C3b 102R Bb-coated surfaces to catalyze rapid decay of Bb (30,31) (Fig. 3 A-C).…”

“…To explore molecular mechanisms underlying differential hemolytic activity, formation and natural decay of the AP C3 convertase was analyzed using surface plasmon resonance (SPR) (Biacore). AP C3 convertase formed on Biacore chips replicates natural convertase: it is labile, cleaves C3, deposits nascent C3b, and is regulated by decay accelerators (30,31). Either fB alone, or fB and fD together, were flowed over each C3b variant.…”

3D structure of the C3bB complex provides insights into the activation and regulation of the complement alternative pathway convertase

“…These data suggest that DAF interacts with C3bBb through major sites in SCR2 and SCR4. It has been suggested that the high affinity of binding to Bb via SCR2 (compared to little or no binding to fB), concentrates DAF on the active convertase, whereas the weaker interactions through SCR4 with C3b directly mediate decay acceleration (28).…”

Domain Swapping Reveals Complement Control Protein Modules Critical for Imparting Cofactor and Decay-Accelerating Activities in Vaccinia Virus Complement Control Protein

“…5), suggesting a functional role for module 1 in CP DAA. Earlier examination of CP DAA by human complement regulators suggested that dissociation of the protease subunit from the convertase is a result of binding of the regulators to C4b and C2a, followed by a conformational change in the von Willebrand factor type A (vWFA) domain (45,47,63,64). More recently, it has been suggested that dissociation of the convertases could also be a result of displacement of the protease subunit by the regulator owing to a competition posed by the regulator for the protease interaction site on the noncatalytic subunit of the convertase (51).…”