Identification of the Fc gamma receptor class I binding site in human IgG through the use of recombinant IgG1/IgG2 hybrid and point-mutated antibodies.
To characterize the region on human IgG1 responsible for its high-affinity interaction with the human Fc gamma receptor class I (Fc gamma RI), we have analyzed the binding properties of a series of genetically engineered chimeric antidinitrophenyl antibodies with identical murine antibody combining sites and hybrid IgG1/IgG2 human constant (C) regions. In addition, we have investigated a panel of reciprocally point-mutated IgG1 and IgG2 chimeric antibodies to identify the amino acid residues that confer cytophilic properties to human IgG1. Our data unambiguously indicate that cytophilic activity of IgG1 is an intrinsic property of its heavy-chain C region 2 (CH2) domain. We report that the entire sequence spanning residues 234-237 (LLGG) is required to restore full binding activity to IgG2 and IgG4 and that individual amino acid substitutions failed to render IgG2 active. Nevertheless, the reciprocal single point mutations in IgG1 either significantly lowered its activity or abolished it completely. Finally, we observed that an IgG2 antibody containing the entire ELLGGP sequence (residues 233-238) was more active than wild-type IgG1. This finding suggests that in addition to the primary contact site identified in the N terminus of the gamma 1 CH2 domain, secondary sites involving residues from the C-terminal half of the domain may also contribute to the IgG1-Fc gamma RI interaction.
Chappel
M S
MS
Department of Immunology, University of Toronto, ON, Canada.
Isenman
D E
DE
Everett
M
M
Xu
Y Y
YY
Dorrington
K J
KJ
Klein
M H
MH
D000595:Amino Acid Sequence; D000818:Animals; D000943:Antigens, Differentiation / Q000378:metabolism*; D001666:Binding Sites, Antibody; D001667:Binding, Competitive; D002460:Cell Line; D002678:Chimera; D006801:Humans; D007074:Immunoglobulin G / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010954:Plasmacytoma; D055503:Protein Multimerization; D011961:Receptors, Fc / Q000378:metabolism*; D017452:Receptors, IgG; D011994:Recombinant Proteins / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid
D000943:Antigens, Differentiation; D007074:Immunoglobulin G; D011961:Receptors, Fc; D017452:Receptors, IgG; D011994:Recombinant Proteins