Control of HLA-DR antigen gene expression at the pretranslational level: comparison of an HLA-DR-positive B lymphoblastoid cell line and its HLA-DR-negative variant

Abstract

An HLA-DR-positive human B lymphoblastoid cell line, T5-1, and its HLA-DR negative variant, 6.1.6, were studied to elucidate mechanisms resulting in the nonexpression of HLA-DR genes in 6.1.6. The cell lines were labeled with 35S-methionine in vivo, their proteins immunoprecipitated with a monoclonal HLA-DR-specific antibody, and their two-dimensional gel electrophoresis patterns compared. The T5-1 map showed DR-antigen heavy and light chains, while the 6.1.6 map showed neither chain. When the cells were labeled in the presence of tunicamycin, the two-dimensional map of T5-1 showed nonglycosylated heavy and light chains of DR antigen while that of 6.1.6 did not. RNA was extracted from T5-1 and 6.1.6 cells and translated in rabbit reticulocyte lysates. Two-dimensional gel analysis of the immunoprecipitated proteins from T5-1 revealed spots which were identified as HLA-DR light chain and I invariant on the basis of their precipitation by monoclonal and specific allo- and heteroantibodies, and their molecular weight and pI values. These spots were absent in the 6.1.6 maps, indicating that 6.1.6 has no detectable translatable messenger RNA for HLA-DR light chains. The addition of dog pancreas microsomes to the T5-1 cell-free translation mixture resulted in an increase in the molecular weight of the precursor HLA-DR proteins consistent with glycosylation. Together with earlier cell fusion studies showing that DR structural genes were intact in 6.1.6, these data suggested that the lesion in 6.1.6 is an alteration in a regulatory element required for transcription of DR genes or mRNA processing.