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 C3b 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 C3b molecules in influencing the regulation of classical pathway C5 convertase by C4b-binding protein (C4BP) was examined and compared to its precursor, the C3 convertase. Determination of IC(50) 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 11 nM). No difference was observed in the cofactor activity of C4BP for surface-bound C4b alone or when in complex with C3b. 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 C3b on them (11 nM). Increasing the C4b or C3b 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 IC(50) 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 C3b molecules is necessary to convert a C3 convertase to a high affinity C5 convertase, the additional C3b molecules play no role in the regulation of C5 convertase by C4BP.
Rawal
Nenoo
N
Department of Biochemistry, University of Texas Health Science Center, 11937 US Highway 271, Tyler, TX 75708-3154, USA. nenoo.rawal@uthct.edu
Pangburn
Michael K
MK
D000818:Animals; D001665:Binding Sites / Q000276:immunology; D002645:Chickens; D003179:Complement C3b / Q000378:metabolism* / Q000502:physiology; D050716:Complement C4b-Binding Protein / Q000378:metabolism* / Q000502:physiology; D051577:Complement C5 Convertase, Classical Pathway / Q000378:metabolism*; D006801:Humans; D011485:Protein Binding / Q000276:immunology
D050716:Complement C4b-Binding Protein; D003179:Complement C3b; D051577:Complement C5 Convertase, Classical Pathway