Use of in vitro mutants of H-2 in studying structure-function relationships
Date
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
YU Faculty Profile
Abstract
In these studies, in vitro mutants of the murine Major Histocompatibility Complex (MHC) class I H-2K{dollar}\sp{lcub}\rm b{rcub}{dollar} molecule were used to define some of the structural requirements for normal transport of the molecule to the cell surface, and for recognition by K{dollar}\sp{lcub}\rm b{rcub}{dollar}-specific monoclonal antibodies (mAbs) and cytotoxic T lymphocytes (CTL).;The importance of the disulfide bond in the {dollar}\alpha{dollar}2 domain for transport to the cell surface was demonstrated by construction and analysis of a K{dollar}\sp{lcub}\rm b{rcub}{dollar} mutant gene with nucleotide changes resulting in a Cys 101 to Ser 101 alteration. Indirect immunofluorescence of cells transfected with this construct showed drastically reduced K{dollar}\sp{lcub}\rm b{rcub}{dollar} expression.;Another feature thought to be required for the expression of K{dollar}\sp{lcub}\rm b{rcub}{dollar} is the noncovalent association with {dollar}\beta\sb2{dollar}M. To study what kind of structural alteration in the K{dollar}\sp{lcub}\rm b{rcub}{dollar} molecule might affect {dollar}\beta\sb2{dollar}M binding, putative {dollar}\beta\sb2{dollar}M binding loss mutants were searched by screening a panel of K{dollar}\sp{lcub}\rm b{rcub}{dollar} surface null variant cell lines for the presence of intracellular K{dollar}\sp{lcub}\rm b{rcub}{dollar} heavy chain by immunoprecipitation. A potential {dollar}\beta\sb2{dollar}M binding mutant, MITC 14, was identified and was shown to have Gly 26 to Cys 26 alteration by uncloned cDNA sequencing.;For the purpose of studying the features important for recognition by mAbs and T cells, K{dollar}\sp{lcub}\rm b{rcub}{dollar} structural variants were obtained previously from a chemically mutagenized Abelson virus-induced pre-B cell line, R8. A mutant K{dollar}\sp{lcub}\rm b{rcub}{dollar} gene from one such mutant, R8.313, was cloned and DNA sequence analysis identified a single point mutation resulting in a Leu 82 to Phe 82 substitution. The L cells transfected with the R8.313 K{dollar}\sp{lcub}\rm b{rcub}{dollar} gene were recognized with the same mAb and CTL as the R8.313 cell line, confirming that the altered phenotype of the mutant cell line was due to a point mutation in the K{dollar}\sp{lcub}\rm b{rcub}{dollar} gene.;To further probe structure-function relationships, point mutants were constructed to define the role of each of the three amino acid alterations in an in vivo derived mutant, bm1: K{dollar}\sp{lcub}\rm b152{rcub}{dollar} (Glu 152 to Ala 152), K{dollar}\sp{lcub}\rm b155{rcub}{dollar} (Arg 155 to Tyr 155), and K{dollar}\sp{lcub}\rm b156{rcub}{dollar} (Leu 156 to Tyr 156). Monoclonal antibody mapping of cells transfected with these genes revealed that, of the three changes, only the change at position 155 mimicked the altered mAb binding profile of K{dollar}\sp{lcub}\rm bm1{rcub}{dollar}. Studies done using CTL were particularly interesting as it was found that the K{dollar}\sp{lcub}\rm b{rcub}{dollar}-specific allogeneic CTL recognition is dramatically altered by all the single changes, whereas the Sendai virus-specific K{dollar}\sp{lcub}\rm b{rcub}{dollar}-restricted CTL recognition is not affected.