MUTATION OF RESIDUES IN THE C3DG REGION OF HUMAN-COMPLEMENT COMPONENTC3 CORRESPONDING TO A PROPOSED BINDING-SITE FOR COMPLEMENT RECEPTOR-TYPE-2 (CR-2, CD21) DOES NOT ABOLISH BINDING OF IC3B OR C3DG TO CR-2

Most evidence points toward there being a shared binding site in compl ement receptor type 2 (CR2, CD21) for the complement ligand C3dg and t he EBV surface envelope glycoprotein gp350/220. Indeed, synthetic pept ide studies have suggested that the CR2-binding sites in human C3dg an d EBV gp350/220 share a similar sequence motif. The proposed CR2-bindi ng sequence in C3dg is EDPCKQLYNVEA (residues 1199-1210 of mature C3), whereas that in EBV gp350/220 is EDPGFFNVEI (residues identical to C3 dg are underlined). To further examine the role of amino acids 1199-12 10 in the binding of the C3 fragments iC3b and C3dg to CR2, the follow ing alanine-substitution variants of human C3 were tested in two indep endent CR2-binding assays: ED1199,1200AA; KQ1203,1204AA; L1205A; Y1206 A; NV1207,1208AA; E1209A; and ED-KQ-NV1199,1200-1203,1204-1207,1208AA- AA-AA. Also engineered and tested was a chimeric C3 molecule in which the 1199-1210 sequence (PVPCCYQLTLEA) from the non-CR2-binding trout C 3 molecule was grafted onto a human C3 background. Recombinant C3 prot eins were expressed transiently in COS-1 cells, deposited as C3b on C3 convertase-bearing sheep erythrocytes and finally converted to cell-b ound iC3b or C3dg using factors H and I. Binding of EAC423bi and EAC42 3dg to CR2 on Raji cells or EAC423dg to soluble CR2 was assessed. In m ost cases, the substitutions had little effect on CR2-binding activity and even in the case of the most highly substituted variants, the dec rease in CR2-binding activity was less than twofold. Thus, contrary to the results anticipated from synthetic peptide studies, the single an d multiple substitutions to the C3 sequence tested failed to corrobora te a role for the 1199-1210 sequence in the C3dg-CR2 interaction.