Identification of functional regions on the I-Ab molecule by site-directed mutagenesis.
Functional analysis of mutant class II major histocompatibility complex molecules has begun to identify regions important for antibody binding and for T-cell activation. By using in vitro mutagenesis directed at the beta 1 domain of the Ab beta gene we have constructed three structurally distinct mutant Ab beta genes. Each of these genes, as well as the wild-type Ab beta gene, was cotransfected together with the wild-type Ab alpha gene into the Ia-negative B-lymphoma cell line M12.C3. Transfection resulted in the successful synthesis and cell surface expression of three mutant class II antigens that showed serological and functional alterations as compared to the I-Ab antigens from the M12.C3 cell transfected with the wild-type gene. The variable patterns of both I-Ab-specific monoclonal antibody binding and activation of I-Ab-specific T-cell hybridomas show that the mutations result in the loss of structural epitopes required for both monoclonal antibody binding and for T-cell recognition. The data suggest that there are multiple sites on a single Ia molecule that are recognized by T helper cells and also that the tertiary conformation of the Ia molecule can be critical in the formation of such sites.
L E
LE
Cohn
L H
LH
Glimcher
R A
RA
Waldmann
J A
JA
Smith
A
A
Ben-Nun
J G
JG
Seidman
E
E
Choi
D000818:Animals; D001483:Base Sequence; D002469:Cell Separation; D000939:Epitopes; D005802:Genes, MHC Class II; D000949:Histocompatibility Antigens Class II / Q000235:genetics*; D006825:Hybridomas / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D009154:Mutation; D013601:T-Lymphocytes / Q000276:immunology; D006377:T-Lymphocytes, Helper-Inducer / Q000276:immunology
D000939:Epitopes; D000949:Histocompatibility Antigens Class II