Role of the C3b-binding site on C4b-binding protein in regulating classical pathway C5 convertase

A high affinity C5 convertase is generated when a C3 convertase deposits additional Ob molecules on and around itself thereby switching the substrate specificity of C3 convertase from C3 to C5. In the present study the role of the additional Ob molecules in influencing the regulation of classical pathway C5 convertase by CO-binding protein (C4BP) was examined and compared to its precursor, the C3 convertase. Determination Of IC50 for inhibiting formation of the high affinity C5 convertase and for enhancing its decay (72 and 20 nM) were found to be similar to those obtained for the surface-bound C3 convertase (35 and I I nM). No difference was observed in the cofactor activity of C4BP for surface-bound CO alone or when in complex with Ob. Analysis of binding interactions between C4BP and EAC1,C4b cells revealed an average apparent dissociation constant (12 nM) similar to that obtained with EAC1,C4b cells with Ob on them (I I nM). Increasing the C4b or Ob density on the cell surface did not alter the affinity of C4BP. The data suggest that C4BP regulates the C5 convertase by mechanisms similar to those observed for the C3 convertase. Since the IC50 for inhibiting formation of the soluble C3 convertase (5 nM) is 50-80-fold below the normal serum concentration of C4BP (250-400 nM), C4BP in blood effectively prevents formation of classical pathway C3 convertase in the fluid phase. Although deposition of additional Ob molecules is necessary to convert a C3 convertase to a high affinity C5 convertase, the additional Ob molecules play no role in the regulation of C5 convertase by C4BP. (c) 2006 Elsevier Ltd. All rights reserved.