Effect of glycosylation inhibitors on the structure and function of the murine transferrin receptor

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Abstract
The murine transferrin receptor is a disulphide-linked dimer with three N-glycosylation sites. We have investigated the structural and functional properties of the transferrin receptor from murine plasmacytoma cells (NS-1 cells) treated with the glycosylation inhibitor, tunicamycin and the glycosylation-processing inhibitors, swainsonine and castanospermine. 1 Tunicamycin (1 μg/ml) inhibited mannose incorporation in NS-1 cells by > 90%, but also inhibited me-thionine incorporation by up to 50%. Both swainsonine (1 μg/ml) and castanospermine (50 μg/ml) resulted in mannose incorporation > 100% of untreated cells and neither drug affected methionine incorporation. 2 Incubation of NS-1 cells with tunicamycin resulted in a shift in the apparent molecular mass of the transferrin receptor from 96 kDa and 94 kDa to approximately 82 kDa. 3 Peptide N-glycosidase F digestion of the receptor from untreated cells resulted in the fully deglycosylated 82 kDa component as well as an 87 kDa component which represents partially deglycosylated receptor resistant to peptide N-glycosidase F digestion. 4 The receptor from swainsonine-treated cells was equally sensitive to peptide N-glycosidase F and endo-β-N-acetylglucosaminidase H (endo H; resulting in both 87-kDa and 82-kDa components), whereas the receptor from castanospermine-treated cells was only partially sensitive to endo H. 5 Analysis of mannose- and fucose-labelled cellular glycopeptides by concanavalin-A - Sepharose chromatography showed that swainsonine (1 μg/ml) treatment resulted in approximately 90% inhibition of the synthesis of complex N-glycans and an accumulation of fucosylated hybrid structures. In contrast, castanospermine (100 μg/ml) treatment resulted in only partial inhibition (60%) of the synthesis of complex N-glycans. 6 Analysis of the receptor from tunicamycin, swainsonine and castanospermine treated cells under non-reducing conditions showed a single component corresponding to the dimer, indicating that dimerisation of newly synthesisted murine receptor is independent of carbohydrate. 7 The non-glycosylated receptor from tunicamycin-treated cells appears to bind transferrin as demonstrated by interaction with transferrin-Sepharose. 8 Surface expression of the receptor was not significantly altered in the presence of either swainsonine or castanospermine as judged by flow cytometry.