MUTANT ALTERED IN GLYCOSYLATION OF MEMBRANE PROTEINS

哺乳动物细胞中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），唾液酸和半乳糖 转移酶（高尔基体）和Na/K ATP酶（质膜）;细胞 生长因子（转铁蛋白，胰岛素，EGF， FGF）和低密度脂蛋白;阳离子非依赖性甘露糖 6-磷酸受体，参与靶向溶酶体酶， 成纤维细胞和可溶性蛋白质如分泌产物 （激素、纤连蛋白、胶原蛋白、层粘连蛋白）和管腔溶酶体 内切酶 葡萄糖部分的作用因蛋白质而异， 蛋白质;它可以是重要的稳定性，溶解性，最终 构象、靶向和/或分子间相互作用。 寡聚核苷酸通过天冬酰胺或天冬酰胺与糖蛋白连接。 丝氨酸/苏氨酸残基。 天冬酰胺连接的 寡糖是一个复杂的系列，至少有50个膜- 反应分为四个主要步骤。 首先是综合 聚异戊二烯的一种，长链磷酸酯。 二是 合成的低聚糖附着在磷酸长链酯上， 逐步加入N-乙酰葡糖胺、甘露糖和葡萄糖 从糖核苷酸和单糖基化，磷酸化 长筒大麻酚 第三，将低聚糖作为一个单元转移到 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基被去除，并且在某些情况下N- 乙酰葡糖胺、半乳糖、岩藻糖和唾液酸残基是 添加，得到在成熟的 糖蛋白 我们正在解剖这个多步骤，多细胞器 利用生化遗传学的生物合成途径。 在此 项目期间，我们将确定精确的酶缺陷， 三个中国仓鼠卵巢细胞突变体 其中一个变种人， F2 A8，在合成多萜醇中有缺陷， 中性聚异戊二烯醇 我们计划确定这种积累， 这导致中性异戊烯醇与磷酸化异戊烯醇的高比率， 与细胞中平滑ER的存在相关。 第二个突变体CHB 11 -1-3似乎在易位中有缺陷， 寡糖-脂质中间体从细胞质转移到 急诊室的内腔面。 第三个突变体Lec 9合成了40倍 与亲本相比，标记的（G1 c）3（Man）9（G1 cNAc）2-P-P-脂质较少 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.