Mutant HLA-A201 heavy chains with lowered affinity for beta 2m are transported after growth at reduced temperatures.
Forty-five site-directed mutants bearing single amino acid substitutions in the alpha 3 domain of the class I molecule HLA-A201 were previously transfected into CIR cells and screened for surface expression by antibody binding. Eight mutants are expressed at significantly reduced levels relative to HLA-A201, including two with substitutions at residues contacting beta 2m. One of the latter mutations, position 242 gln > lys (242K), is now shown to block assembly with beta 2m and prevent intracellular transport at 37 degrees C. At temperatures ranging from 21 degrees C to 30 degrees C, 242K heavy chains and beta 2m form dimers that are exported to the cell surface. Surface expression at 26 degrees C is not blocked by cycloheximide pretreatment, but is completely inhibited by Brefeldin A, suggesting that at 37 degrees C preformed heavy chains accumulate in the ER. Glycans on the retained heavy chains are sensitive to digestion by Endo H, but become Endo H resistant after cells are grown at 26 degrees C. Preincubation of 242K cells with synthetic peptides shown previously to bind HLA-A201 does not increase reactivity with anti-HLA-A2 antibodies, suggesting that the defective phenotype is not due to instability of cell surface mutant class I dimers, but derives instead from impaired assembly of 242K heavy chains with beta 2m inside the cell. This contrasts with mutant cells such as .174, T2 and RMA-S, which exhibit defects in internal peptide transporters, but assemble and export "empty" dimers to their surfaces that can be stabilized subsequently by exogenous peptides. 242K mutants may therefore be suited uniquely for studying assembly and peptide binding to class I molecules in the ER.
Salter
R D
RD
Department of Pathology, University of Pittsburgh School of Medicine, Pennsylvania 15261.
D000596:Amino Acids / Q000235:genetics; D000911:Antibodies, Monoclonal; D000954:Antigens, Surface / Q000378:metabolism; D001692:Biological Transport; D002460:Cell Line; D002478:Cells, Cultured; D004721:Endoplasmic Reticulum / Q000378:metabolism; D015234:HLA-A Antigens / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D016297:Mutagenesis, Site-Directed; D011134:Polysaccharides / Q000378:metabolism; D014162:Transfection; D001613:beta 2-Microglobulin / Q000378:metabolism*
D000596:Amino Acids; D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D015234:HLA-A Antigens; D011134:Polysaccharides; D001613:beta 2-Microglobulin