title: Structure-function analysis of the Abm12 beta mutation using site-directed mutagenesis and DNA-mediated gene transfer.
abstract: The B6.C-H-2bm12 (bm12) mouse possesses a naturally occurring mutation in its class II MHC A beta gene. The three amino acid substitutions at positions 67, 70, and 71 that comprise this mutation lead to changes in both Ia expression and immune recognition of the resultant A beta A alpha molecule. The experiments reported here utilize a combination of oligonucleotide-mediated site-directed mutagenesis and DNA-mediated gene transfer to explore the roles played by each of the three mutant residues in these various phenotypic changes. A beta genes comprising all permutations of the residues distinguishing Ab beta from Abm12 beta were created and were individually co-transfected with Ab beta into mouse L cells. Sublines expressing high levels of membrane Ia were selected by preparative flow cytometry and were studied for reactivity with a panel of monoclonal anti-Ia antibodies, or for their ability to act as antigen-presenting cells (APC) for the stimulation of T cell hybridomas. During the generation of these transfectant lines, it was noted that expression of a high level of Abm12 beta Ab alpha was more difficult to achieve than a similar level of Ab beta Ab alpha. Northern blot analysis of specific A beta and A alpha mRNA levels in these various lines indicated that more class II mRNA, and presumably more A beta and A alpha chains, were required to achieve expression of Abm12 beta Ab alpha equal to that of Ab beta Ab alpha, suggesting that the previously noted reduction of Ia expression on cells from bm12 mice reflects a decreased ability of Abm12 beta Ab alpha chains to pair, or to reach the membrane. Staining of the panel of transfectants with monoclonal antibodies revealed that antibodies which did not distinguish Ab beta Ab alpha from Abm12 beta Ab alpha also reacted equally well with all molecules involving in vitro mutant A beta chains. Monoclonal antibodies reactive with Ab beta Ab alpha but not Abm12 beta Ab alpha were specific for an epitope primarily determined by the presence or absence of Arg 70 in Ab beta. In striking contrast, all three mutant positions were found to play crucial roles in T cell recognition, because all substitutions led to significant or complete loss of antigen-presenting function with all but one of the T hybridomas tested.(ABSTRACT TRUNCATED AT 400 WORDS)
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D000938:Antigen-Presenting Cells / Q000276:immunology; D000945:Antigens, Differentiation, T-Lymphocyte; D000954:Antigens, Surface / Q000502:physiology; D002462:Cell Membrane / Q000502:physiology; D000949:Histocompatibility Antigens Class II / Q000235:genetics* / Q000276:immunology; D008213:Lymphocyte Activation; D051379:Mice; D009154:Mutation; D011971:Receptors, Immunologic / Q000502:physiology; D013329:Structure-Activity Relationship; D013601:T-Lymphocytes / Q000276:immunology*
chemical_list: D000911:Antibodies, Monoclonal; D000945:Antigens, Differentiation, T-Lymphocyte; D000954:Antigens, Surface; D000949:Histocompatibility Antigens Class II; D011971:Receptors, Immunologic