Characterization of an Ig VH idiotope that results in specific homophilic binding and increased avidity for antigen.
mAb against GD3 ganglioside demonstrate homophilic binding in which soluble anti-GD3 mAb bind, through the GD3 binding site, to a VH idiotope (designated IdHOM) on solid phase anti-GD3 mAb. In this way, homophilic binding provides a mechanism for amplifying the binding of mAb to cell surface GD3. We show that serine 52a, within CDR2, is required for IdHOM expression, homophilic binding, and high avidity binding to cell surface GD3. Computer modeling based on the crystal structure of anti-GD3 mAb R24 showed serine 52a situated at the mouth of the GD3 binding pocket, but not directly involved with GD3 binding. Substitutions at position 52a predicted to maintain the GD3 binding pocket (e.g., threonine) resulted in the loss of IdHOM expression and homophilic binding and markedly decreased binding to cell surface GD3, but maintained low avidity GD3 binding as measured by ELISA. All other substitutions at position 52a were predicted to significantly distort the GD3 binding pocket and resulted in the loss of both homophilic binding and any detectable avidity for GD3. We have structurally defined IdHOM and conclude that this idiotope is not required for the GD3 binding pocket, but that the idiotope is necessary for homophilic binding, which is required for high avidity binding to cell surface GD3. We speculate that selection of certain VH genes may result in the expression of idiotopes that allow homophilic binding, and this may represent a general mechanism for increasing the avidity of Abs against T cell-independent Ags.
Yan
X
X
Immunology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA.
Evans
S V
SV
Kaminki
M J
MJ
Gillies
S D
SD
Reisfeld
R A
RA
Houghton
A N
AN
Chapman
P B
PB
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000915:Antibody Affinity; D000937:Antigen-Antibody Reactions; D000941:Antigens / Q000276:immunology* / Q000378:metabolism; D001483:Base Sequence; D001666:Binding Sites, Antibody; D005732:Gangliosides / Q000276:immunology*; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000276:immunology*; D007130:Immunoglobulin Idiotypes / Q000276:immunology*; D007075:Immunoglobulin M / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000276:immunology*; D008546:Melanoma, Experimental / Q000473:pathology; D051379:Mice; D018448:Models, Immunological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000276:immunology / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D000941:Antigens; D005732:Gangliosides; D007143:Immunoglobulin Heavy Chains; D007130:Immunoglobulin Idiotypes; D007075:Immunoglobulin M; D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins; C026226:ganglioside, GD3