MUTANT ALTERED IN GLYCOSYLATION OF MEMBRANE PROTEINS

哺乳动物细胞中的一类重要的大分子是 糖蛋白，包括：膜结合酶，如HMG-CoA 还原酶(内质网，ER)、唾液酸基和半乳糖 转移酶(高尔基体)和Na/K-ATPase(质膜)； 生长因子的表面受体(转铁蛋白、胰岛素、EGF、 成纤维细胞生长因子)和低密度脂蛋白；阳离子非依赖性甘露糖 6-磷酸受体参与溶酶体酶的靶向作用 成纤维细胞和可溶性蛋白质，如分泌产物 (激素、纤维连接蛋白、胶原蛋白、层粘连蛋白)和管腔溶酶体 酵素。糖基的作用因蛋白质不同而不同 蛋白质；它对稳定性、溶解性、最终 构象、靶向和/或分子间相互作用。 寡糖通过天冬酰胺或糖蛋白连接到糖蛋白上。 丝氨酸/苏氨酸残基。天冬酰胺的生物合成 低聚糖是由至少50个膜组成的复杂系列- 与四个主要步骤相关的反应。一是综合 聚异戊二烯，磷酸二氢呋喃。第二，一个 低聚糖的合成是通过以下方法合成的 N-乙酰氨基葡萄糖、甘露糖和葡萄糖的逐步添加 从糖核苷酸和单糖化、磷酸化 多里科尔。第三，将低聚糖作为单位转移到 具有ASN-X-Ser(Thr)序列的新生蛋白质。第四, 葡萄糖和一些甘露糖残留物被去除，在某些情况下，N- 乙酰氨基葡萄糖、半乳糖、岩藻糖和唾液酸残基为 添加，给出了成熟时发现的最终多糖结构 糖蛋白。我们正在解剖这种多步骤、多细胞器 利用生化遗传学的生物合成途径。在此期间 项目期内，我们将确定准确的酶缺陷 三个中国仓鼠卵巢细胞突变体。其中一个变种人， F2A8，在二醇的合成过程中有缺陷，并积累 中性聚异戊二烯。我们计划确定这种积累是否， 这导致中性戊烯醇与磷酸化的比率很高 丙烯醇，与细胞内光滑内质网的存在相关。 第二个突变体CHB11-1-3在易位中出现缺陷 从细胞质到细胞质的寡糖-脂中间体 急诊室的腔面。第三个突变体Lec9合成了40倍于 标记较少的(G1c)3(Man)9(G1cNAc)2-P-P-脂 细胞。最后，我们将分离新的糖基化突变体。

An important class of macromolecules in mammalian cells is glycoproteins, including: membrane-bound enzymes such as HMG-CoA reductase (endoplasmic reticulum, ER), sialyl and galactosyl transferases (Golgi), and Na/K ATPase (plasma membrane); cell surface receptors for growth factors (transferrin, insulin, EGF, FGF) and for low density lipoproteins; cation-independent mannose 6-phosphate receptor, involved in targeting of lysosome enzymes in fibroblasts and soluble proteins such as secretory products (hormones, fibronectin, collagen, laminin) and lumenal lysosomal enzymes. The role of the glycose moiety differs from protein to protein; it can be important for stability, solubility, final conformation, targeting, and/or intermolecular interactions. Oligosaccharides are attached to glycoproteins via asparagine or serine/threonine residues. The biosynthesis of asparagine-linked oligosaccharides is a complex series of at least fifty membrane- associated reactions with four major steps. First is the synthesis of the polyisoprene, dolichyl phosphate. Second, an oligosaccharide is synthesized attached to dolichyl phosphate by stepwise additions of N-acetylglucosamine, mannose, and glucose from sugar nucleotides and monoglycosylated, phosphorylated dolichols. Third, the oligosaccharide is transferred as a unit to nascent proteins that have the sequence Asn-X-Ser(Thr). Fourth, glucose and some mannose residues are removed and in some cases N- acetylglucosamine, galactose, fucose, and sialic acid residues are added, giving the final glycan structure found on mature glycoproteins. We are dissecting this multistep, multiorganellar biosynthetic pathway using biochemical genetics. During this project period, we will determine the precise enzymatic defect in three Chinese hamster ovary cell mutants. One of these mutants, F2A8, is defective in the synthesis of dolichol, and accumulates neutral polyisoprenol. We plan to determine if this accumulation, which results in a high ratio of neutral prenols to phosphorylated prenols, correlates with the presence of smooth ER in the cell. A second mutant, CHB11-1-3, appears defective in the translocation of oligosaccharide-lipid intermediates from the cytoplasmic to the lumenal face of the ER. A third mutant Lec9 synthesizes forty-fold less labeled (G1c)3(Man)9(G1cNAc)2-P-P-lipid compared to parental cells. Finally, we will isolate new glycosylation mutants.