Effect of glycosylation inhibitors on the structure and function of the murine transferrin receptor.
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 microgram/ml) inhibited mannose incorporation in NS-1 cells by greater than 90%, but also inhibited methionine incorporation by up to 50%. Both swainsonine (1 microgram/ml) and castanospermine (50 micrograms/ml) resulted in mannose incorporation greater than 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-beta-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 microgram/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 micrograms/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 nonreducing conditions showed a single component corresponding to the dimer, indicating that dimerisation of newly synthesised 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.
Ralton
J E
JE
Department of Pathology and Immunology, Monash University Medical School, Melbourne, Australia.
Jackson
H J
HJ
Zanoni
M
M
Gleeson
P A
PA
D000470:Alkaloids / Q000494:pharmacology*; D000818:Animals; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D005643:Fucose / Q000378:metabolism; D006020:Glycopeptides / Q000302:isolation & purification; D006026:Glycoside Hydrolases / Q000037:antagonists & inhibitors*; D006031:Glycosylation; D007212:Indolizines; D008358:Mannose / Q000378:metabolism; D008361:Mannosidases / Q000037:antagonists & inhibitors; D008715:Methionine / Q000378:metabolism; D051379:Mice; D009363:Neoplasm Proteins / Q000096:biosynthesis; D010954:Plasmacytoma; D011865:Radioisotope Dilution Technique; D011990:Receptors, Transferrin / Q000096:biosynthesis / Q000187:drug effects / Q000378:metabolism*; D013462:Sulfur Radioisotopes; D017026:Swainsonine; D014316:Tritium; D014415:Tunicamycin / Q000494:pharmacology*; D054836:Indolizidines
D000470:Alkaloids; D005643:Fucose; D006020:Glycopeptides; D006026:Glycoside Hydrolases; D007212:Indolizines; D008358:Mannose; D008361:Mannosidases; D008715:Methionine; D009363:Neoplasm Proteins; D011990:Receptors, Transferrin; D013462:Sulfur Radioisotopes; D017026:Swainsonine; D014316:Tritium; D014415:Tunicamycin; C037806:castanospermine; D054836:Indolizidines