Mutation of residues in the C3dg region of human complement component C3 corresponding to a proposed binding site for complement receptor type 2 (CR2, CD21) does not abolish binding of iC3b or C3dg to CR2.
Most evidence points toward there being a shared binding site in complement receptor type 2 (CR2, CD21) for the complement ligand C3dg and the EBV surface envelope glycoprotein gp350/220. Indeed, synthetic peptide studies have suggested that the CR2-binding sites in human C3dg and EBV gp350/220 share a similar sequence motif. The proposed CR2-binding sequence in C3dg is EDPGKQLYNVEA (residues 1199-1210 of mature C3), whereas that in EBV gp350/220 is EDPGFFNVEI (residues identical to C3dg are underlined). To further examine the role of amino acids 1199-1210 in the binding of the C3 fragments iC3b and C3dg to CR2, the following alanine-substitution variants of human C3 were tested in two independent CR2-binding assays: ED1199,1200AA; KQ1203,1204AA; L1205A; Y1206A; 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 (PVPGGYQLTLEA) from the non-CR2-binding trout C3 molecule was grafted onto a human C3 background. Recombinant C3 proteins were expressed transiently in COS-1 cells, deposited as C3b on C3 convertase-bearing sheep erythrocytes and finally converted to cell-bound iC3b or C3dg using factors H and I. Binding of EAC423bi and EAC423dg to CR2 on Raji cells or EAC423dg to soluble CR2 was assessed. In most cases, the substitutions had little effect on CR2-binding activity and even in the case of the most highly substituted variants, the decrease in CR2-binding activity was less than twofold. Thus, contrary to the results anticipated from synthetic peptide studies, the single and multiple substitutions to the C3 sequence tested failed to corroborate a role for the 1199-1210 sequence in the C3dg-CR2 interaction.
Diefenbach
R J
RJ
Department of Biochemistry, University of Toronto, Ontario, Canada.
Isenman
D E
DE
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D003179:Complement C3b / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010446:Peptide Fragments / Q000235:genetics* / Q000378:metabolism*; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering; D017464:Receptors, Complement 3d / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012397:Rosette Formation
D010446:Peptide Fragments; D010455:Peptides; D017464:Receptors, Complement 3d; D011993:Recombinant Fusion Proteins; C045477:complement C3d,g; D003179:Complement C3b