Residue at position 331 in the IgG1 and IgG4 CH2 domains contributes to their differential ability to bind and activate complement.
A conserved proline residue is found at position 331 in the CH2 domains of human IgG subclasses which fix complement. This residue is replaced by a serine in IgG4 which is inactive. To determine the role of residue 331 in the differential ability of human IgGs to activate the complement cascade, a pair of genetically engineered anti-dinitrophenol IgG1 and IgG4 antibodies with reciprocal mutations at position 331 were tested for their hemolytic activity as well as for their ability to bind C1q, activate C1 and cleave C4. The IgG1 Ser331 mutant was virtually unable to mediate the lysis of trinitrobenzene-sulfonic acid-derivatized sheep red blood cells as a result of a marked defect in C1q binding activity. In contrast, the substitution of Pro for Ser331 in IgG4 bestowed partial hemolytic activity (40%) to the IgG4 Pro331 variant. Under low ionic strength conditions, this mutant was found to be approximately 50 and 75% as active as wild-type IgG1 in the C1q binding and C4b deposition assays, respectively. These results indicate that residue Pro331, which folds into close proximity to a previously identified C1q binding motif (Duncan, A. R., and Winter, G. (1988) Nature 332, 738-740), contributes to the architecture of the IgG1 C1q binding site and that its replacement by a serine residue in IgG4 is largely responsible for the functional inactivity of this isotype.
Xu
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Department of Immunology, University of Toronto, Ontario, Canada.
Oomen
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Klein
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D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000378:metabolism; D001483:Base Sequence; D003167:Complement Activation; D015922:Complement C1q / Q000378:metabolism*; D017124:Conserved Sequence; D006461:Hemolysis; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry* / Q000145:classification / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010954:Plasmacytoma; D017354:Point Mutation; D011392:Proline; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015183:Restriction Mapping; D012694:Serine; D014407:Tumor Cells, Cultured
D000906:Antibodies; D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins; D012694:Serine; D015922:Complement C1q; D011392:Proline