Abstract
The biosynthesis of the HLA-DR antigens was studied in the B lymphoblastoid cell line BRI 8. Three chains, of molecular weights 33,000 (alpha), 31,000 (p31), and 26,000 (beta) were detected intracellularly in pulse-labeled cells by immunoprecipitation with anti-(HLA-DR) sera. The alpha and beta chains were inserted asymmetrically into the rough endoplasmic reticulum as transmembrane polypeptides with the majority of the polypeptide chains oriented in the lumen. At this stage, both chains carried "high mannose" oligosaccharide units which were processed to the complex form during subsequent intracellular transport to the cell surface. The Mr = 31,000 polypeptide was also glycosylated but was structurally distinct from the alpha chain and was probably oriented differently in the lipid bilayer, with a much greater proportion of its polypeptide chain exposed in the cytoplasm. It ws not, therefore, a precursor of the alpha chain. The mature HLA-DR antigens at the plasma membrane comprised polypeptides of Mr = 34,000 and 28,000. These chains corresponded to the processed alpha and beta chains. Although the Mr = 31,000 component was only detected intracellularly, it was not ruled out that some or all of it may have been processed and exposed on the cell surface with an apparent molecular weight indistinguishable from that of the alpha chain.
Highlights
In the B lymphoblastoid cell line BRI 8
Transmembrane Insertion of HLA-DR Antigens-BRI 8 cells were labeled for 5 min with [:"S]methionine prior to their cells were labeledfor 5 min with100pCi/ml of ["S]methionine before diluting into medium containing an excess of unlabeled methionine
When human B lymphoblastoid cells were labeled biosyntheticallywith ["S]methionine, a t leastthree polypeptide chains were detected, even after early labeling periods b 2 . 5 min), by immunoprecipitation with anti-(HLA-DR) sera and analysis by SDS-polyacrylamide gel electrophoresis. This labeling profde was observed using several B lymphoblastoid cell lines and peripheral blood lymphocytes. It was not an artifactof rabbit antisera since threepolypeptide chains were immunoprecipitated by a variety of monoclonal anti-(HLA-DR) antibodies
Summary
In the B lymphoblastoid cell line BRI 8. In addition to the a and /3 chains, two-dimensional gel electrophoretic analyses of mouse Ia products has demonstrated theexistence of a third chain. This polypeptide chain was fist observed in immunoprecipitatesof mouse I-A and I-. E subregion products as a basic, invariant species of molecular weight about 31,000 (Ii) (Jones et al, 1979).More recently, it proportion of its polypeptide chain exposed in the cy- has been detected in immunoprecipitates of human HLA-. The mature HLA-DR antigens at theplasma Strominger, 1980).In bothmurine and human Iaimmunopremembrane comprised polypeptides ofM, = 34,000 and cipitates, no allelic haplotype differences were detected sug-
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