Pouysségur J, Pastan I
J Biol Chem. 1977 Mar 10;252(5):1639-46.
We hve reported the isolation and preliminary biochemical characterization of two mutants of mouse fibroblasts selected for a decrease incell-to-substrate adhesion (Pouysségur, J., and Pastan, I. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 544-548). We attributed the adhesive defect of these mutants (AD6 and AD8) to the absence of iodinatable cell surface proteins. This study demonstrates that a defect in glycoprotein synthesis is the biochemical basis for the reduction in proteins exposed at the outer surface of the mutant cells. When D-glucosamine or L-fucose was used as radioactive precursor, analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a decrease in the labeling of all the glycoproteins of both clones. This decrease in glycoprotein labeling was not due to a defect in D-glucosamine uptake, since this precursor was taken up at a higher rate in the mutants than in the wild type. In spite of this high uptake, the rate of D-glucosamine incorporation into macromolecules was decreased by 60% and the carbohydrate content of membranes (mannose, galactose, N-acetylglucosamine, N-acetylgalactosamine, and sialic acid) from clone AD6 was diminished by 40 to 60%. When the various cell lines were labeled for 1 to 3 h with glucosamine and the acid-soluble pool analyzed, the wild type cells were found to accumulate UDP-N-acetylhexosamine as the major component. In contrast, clones AD6 and AD8 accumulated glucosamine 6-phosphate as the major component. This last finding suggests that there is a block in the N-acetylation of glucosamine 6-phosphate in both instants. This suggestion is supported by the finding that feeding the mutant cells 10 mM N-acetylglucosamine reverts them to the wild type phenotype (Pouysségur, J., Willingham, M. and Pastan, I. (1977) Proc. Natl. Acad. Sci. U.S.A., in pressy. In wild type cells all of the iodinatable proteins of the cell surface have the same mobility as the glycoproteins when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We conclude that the early block in glycoprotein synthesis accounts for the reduction in iodinatable surface proteins in the mutant cells.
我们已经报道了从小鼠成纤维细胞中分离出的两个突变体，并对其进行了初步的生化特性分析，这些突变体是因细胞与底物的黏附性降低而被筛选出来的（普伊西格尔，J.，和帕斯坦，I.（1976年）《美国国家科学院院刊》73卷，544 - 548页）。我们将这些突变体（AD6和AD8）的黏附缺陷归因于无法碘化的细胞表面蛋白的缺失。本研究表明，糖蛋白合成缺陷是突变细胞外表面暴露蛋白减少的生化基础。当使用D - 葡萄糖胺或L - 岩藻糖作为放射性前体时，通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳分析发现，两个克隆的所有糖蛋白的标记都减少了。糖蛋白标记的这种减少并非由于D - 葡萄糖胺摄取缺陷，因为该前体在突变体中的摄取速率高于野生型。尽管摄取率很高，但D - 葡萄糖胺掺入大分子的速率降低了60%，并且克隆AD6细胞膜的碳水化合物含量（甘露糖、半乳糖、N - 乙酰葡糖胺、N - 乙酰半乳糖胺和唾液酸）减少了40%至60%。当用葡萄糖胺对各种细胞系标记1至3小时并分析酸溶性池时，发现野生型细胞积累UDP - N - 乙酰己糖胺作为主要成分。相比之下，克隆AD6和AD8积累6 - 磷酸葡萄糖胺作为主要成分。这一最终发现表明，在这两种情况下，6 - 磷酸葡萄糖胺的N - 乙酰化均存在阻断。给突变细胞喂食10 mM N - 乙酰葡糖胺可使其恢复到野生型表型这一发现支持了这一推测（普伊西格尔，J.，韦林厄姆，M.和帕斯坦，I.（1977年）《美国国家科学院院刊》，即将发表）。在野生型细胞中，当通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳分析时，细胞表面所有可碘化的蛋白质与糖蛋白具有相同的迁移率。我们得出结论，糖蛋白合成的早期阻断导致了突变细胞中可碘化表面蛋白的减少。
