Ray M K, Yang J, Sundaram S, Stanley P
Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York 10461.
J Biol Chem. 1991 Dec 5;266(34):22818-25.
Lec23 Chinese hamster ovary (CHO) cells have been shown to possess a unique lectin resistance phenotype and genotype compared with previously isolated CHO glycosylation mutants (Stanley, P., Sallustio, S., Krag, S. S., and Dunn, B. (1990) Somatic Cell Mol. Genet. 16, 211-223). In this paper, a biochemical basis for the lec23 mutation is identified. The carbohydrates associated with the G glycoprotein of vesicular stomatitis virus (VSV) grown in Lec23 cells (Lec23/VSV) were found to possess predominantly oligomannosyl carbohydrates that bound strongly to concanavalin A-Sepharose, eluted 3 sugar eq beyond a Man9GlcNAc marker oligosaccharide on ion suppression high pressure liquid chromatography, and were susceptible to digestion with jack bean alpha-mannosidase. Monosaccharide analyses revealed that the oligomannosyl carbohydrates contained glucose, indicating a defect in alpha-glucosidase activity. This was confirmed by further structural characterization of the Lec23/VSV oligomannosyl carbohydrates using purified rat mammary gland alpha-glucosidase I, jack bean alpha-mannosidase, and 1H NMR spectroscopy at 500 MHz. [3H]Glucose-labeled Glc3Man9GlcNAc was prepared from CHO/VSV labeled with [3H]galactose in the presence of the processing inhibitors castanospermine and deoxymannojirimycin. Subsequently, [3H]Glc2Man9GlcNAc was prepared by purified alpha-glucosidase I digestion of [3H]Glc3Man9GlcNAc. When these oligosaccharides were used as alpha-glucosidase substrates it was revealed that Lec23 cells are specifically defective in alpha-glucosidase I, a deficiency not previously identified among mammalian cell glycosylation mutants.
已证明Lec23中国仓鼠卵巢（CHO）细胞与先前分离的CHO糖基化突变体相比，具有独特的凝集素抗性表型和基因型（斯坦利，P.，萨卢斯蒂奥，S.，克拉格，S.S.，和邓恩，B.（1990年）《体细胞与分子遗传学》16，211 - 223）。在本文中，确定了lec23突变的生化基础。发现在Lec23细胞（Lec23/VSV）中生长的水泡性口炎病毒（VSV）的G糖蛋白相关碳水化合物主要具有寡甘露糖基碳水化合物，它们与伴刀豆球蛋白A - 琼脂糖强烈结合，在离子抑制高压液相色谱上洗脱至超出Man9GlcNAc标记寡糖3个糖当量，并且易被刀豆α - 甘露糖苷酶消化。单糖分析表明寡甘露糖基碳水化合物含有葡萄糖，表明α - 葡萄糖苷酶活性存在缺陷。使用纯化的大鼠乳腺α - 葡萄糖苷酶I、刀豆α - 甘露糖苷酶和500 MHz的1H NMR光谱对Lec23/VSV寡甘露糖基碳水化合物进行进一步结构表征，证实了这一点。[3H]葡萄糖标记的Glc3Man9GlcNAc是在加工抑制剂栗精胺和脱氧甘露基野茉莉霉素存在下，由用[3H]半乳糖标记的CHO/VSV制备的。随后，通过用纯化的α - 葡萄糖苷酶I消化[3H]Glc3Man9GlcNAc制备[3H]Glc2Man9GlcNAc。当这些寡糖用作α - 葡萄糖苷酶底物时，发现Lec23细胞在α - 葡萄糖苷酶I方面存在特异性缺陷，这是哺乳动物细胞糖基化突变体中先前未发现的缺陷。
