Castanospermine inhibits glucosidase I and glycoprotein secretion in human hepatoma cells.
We studied the effect of the plant alkaloid castanospermine on the biosynthesis and secretion of human hepatoma glycoproteins. The HepG-2 cells, grown in the presence or absence of the alkaloid, were labelled with [2-3H]mannose and then the labelled glycopeptides were prepared by Pronase digestion. This material was analysed by gel filtration on Bio-Gel P-4 before and after treatment with endo-beta-N-acetylglucosaminidase H. Castanospermine caused an accumulation of high-mannose oligosaccharides, by 70-75% over control. The major accumulated product, which could also be labelled with [3H]galactose and was only partially susceptible to alpha-mannosidase digestion, was identified by h.p.l.c. as a Glc3Man9GlcNAc. Thus the alkaloid inhibits glucosidase I in the human hepatoma cells. Analysis of total glycoproteins secreted by the cells into the medium revealed the presence of only complex oligosaccharides in both control and treated cultures, and the amount of the oligosaccharides labelled with radioactive mannose, galactose or N-acetylmannosamine, secreted by treated cells, was decreased by about 60%. The rate of secretion of total protein labelled with [35S]methionine and precipitated from the medium with trichloroacetic acid was inhibited by up to 40% in the presence of castanospermine. Pulse-chase studies utilizing [35S]methionine labelling were performed to study the effect of the alkaloid on secretion of individual plasma proteins. Immunoprecipitation at different chase times with monospecific antisera showed that castanospermine markedly decreased the secretion rates of alpha 1-antitrypsin, caeruloplasmin and, to a lesser extent, that of antithrombin-III. Secretions of apolipoprotein E, a glycoprotein containing only O-linked oligosaccharide(s), and albumin, a non-glycosylated protein, were not affected by the drug. It is suggested that castanospermine inhibits secretion of at least some glycoproteins containing N-linked oligosaccharides, owing to the inhibition of oligosaccharide processing.
D000470:Alkaloids / Q000494:pharmacology*; D006528:Carcinoma, Hepatocellular / Q000378:metabolism*; D002478:Cells, Cultured; D011232:Chemical Precipitation; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D005959:Glucosidases / Q000037:antagonists & inhibitors*; D006023:Glycoproteins / Q000276:immunology / Q000378:metabolism*; D065089:Glycoside Hydrolase Inhibitors; D006801:Humans; D007212:Indolizines; D008113:Liver Neoplasms; D009844:Oligosaccharides / Q000096:biosynthesis; D011506:Proteins / Q000378:metabolism; D000520:alpha-Glucosidases; D054836:Indolizidines
D000470:Alkaloids; D005959:Glucosidases; D006023:Glycoproteins; D065089:Glycoside Hydrolase Inhibitors; D007212:Indolizines; D009844:Oligosaccharides; D011506:Proteins; D000520:alpha-Glucosidases; C042059:glucosidase I; C037806:castanospermine; D054836:Indolizidines