Sasak V W, Ordovas J M, Elbein A D, Berninger R W
Biochem J. 1985 Dec 15;232(3):759-66. doi: 10.1042/bj2320759.
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.
我们研究了植物生物碱 castanospermine 对人肝癌糖蛋白生物合成和分泌的影响。在有或没有该生物碱的情况下培养 HepG-2 细胞，用 [2-³H]甘露糖进行标记，然后通过链霉蛋白酶消化制备标记的糖肽。在用内切-β-N-乙酰氨基葡糖苷酶 H 处理之前和之后，通过在 Bio-Gel P-4 上进行凝胶过滤对该物质进行分析。Castanospermine 导致高甘露糖寡糖积累，比对照高出 70 - 75%。通过高效液相色谱法鉴定出主要积累产物，该产物也可用 [³H]半乳糖标记，并且仅部分易受α-甘露糖苷酶消化，为 Glc3Man9GlcNAc。因此，该生物碱抑制人肝癌细胞中的葡糖苷酶 I。对细胞分泌到培养基中的总糖蛋白进行分析发现，对照培养物和处理后的培养物中仅存在复杂寡糖，并且处理后的细胞分泌的用放射性甘露糖、半乳糖或 N-乙酰甘露糖胺标记的寡糖量减少了约
