MOLECULAR BIOLOGY OF ASPARAGINE LINKED GLYCOSYLATION

天冬酰胺连接糖基化的分子生物学

• 批准号: 6519265
• 负责人: Mark Lehrman
• 金额: $ 31.88万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-07-01 至 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6519265
• 关键词: CHO cells; asparagine; confocal scanning microscopy; diffusion; dolichol; endoplasmic reticulum; expression cloning; fluorescence microscopy; glycosylation; immunoprecipitation; intracellular membranes; laboratory rabbit; mannose; membrane proteins; oligosaccharides; protein localization; protein protein interaction; protein sequence; protein structure function; transfection; western blottings

In mammals, secretory and cell-surface proteins are modified with one or more types of carbohydrates that are important for a spectrum of physiological functions. In humans, defects in these carbohydrates lead to a variety of diseases. Asparagine-linked (N-linked), these carbohydrates lead to a variety of diseases. Asparagine-linked (N- linked), glycosylphosphatidylinositol (GPI), and C-mannose carbohydrates compete for a common mammose donor, mannose-P-dolichol (MPD). MPD is a component of the "dolichol pathway" synthesized enzymatically from dolichol-P and GDP-mannose. Since dolichol-P is limiting in cells, the biosynthetic flux for each class of carbohydrate is balanced to maintain cellular needs. In the current funding period, the P.I. cloned a novel gene, termed Lec35, that is essential for MPD utilization. Lec35 mutants synthesize MPD, but have defects in synthesis of N-glycans, GPIs, and C- mannose. Thus, Lec35 represents a new class of protein with a unique mechanism of action. Both the Lec35 cDNA and gene have been cloned by the P.I., and in vivo and in vitro systems for Lec35 function have been devised. For the next funding period five Aims are proposed: I. Determine whether Lec35 protein is involved in (a) MPD flipping from the cytoplasmic leaflet of the ER to the lumenal leaflet, or (b) elimination of a diffusible inhibitor of MPD-dependent mannosylation. II. Determine the topological arrangement, locations of essential residues, and subcellular localization of Lec35 protein, which is predicted to be a resident ER protein with two membrane spans. III. Determine whether Lec35 protein functions prior to, or during, enzymatic MPD-dependent mannosylation. IV. Determine whether the Lec35 protein interacts with other proteins. V. Measure the selectivity of the existing Lec35 cDNA for mannose-P-dolichol over glucose-P-dolichol (GPD), and isolate a cDNA for a GPD-selective form of Lec35.

在哺乳动物中，分泌蛋白和细胞表面蛋白被一种或多种类型的碳水化合物修饰，这些碳水化合物对一系列生理功能很重要。在人类中，这些碳水化合物的缺陷会导致各种疾病。天冬酰胺连锁（N-连锁），这些碳水化合物导致多种疾病。天冬酰胺连接（N-连接）、糖基磷脂酰肌醇（GPI）和C-甘露糖碳水化合物竞争常见的乳腺糖供体甘露糖-β-多萜醇（MPD）。MPD是由多萜醇-P和GDP-甘露糖酶促合成的“多萜醇途径”的组分。由于长叶醇-P在细胞中是有限的，因此平衡每类碳水化合物的生物合成通量以维持细胞的需要。于本融资期内，P. I.克隆了一个新的基因，命名为Lec 35，这是必需的MPD利用。Lec 35突变体合成MPD，但在N-聚糖、GPI和C-甘露糖的合成中具有缺陷。因此，Lec 35代表了一类具有独特作用机制的新蛋白质。Lec 35 cDNA和基因都已被P.I.克隆，并且已经设计了用于Lec 35功能的体内和体外系统。为下一个供资期提出了五个目标：确定Lec 35蛋白是否参与（a）MPD从ER的胞质小叶翻转到内腔小叶，或（B）MPD依赖性甘露糖基化的扩散性抑制剂的消除。二.确定Lec 35蛋白的拓扑结构、必需残基的位置和亚细胞定位，Lec 35蛋白被预测为具有两个膜跨度的常驻ER蛋白。三.确定Lec 35蛋白是否在酶促MPD依赖性甘露糖基化之前或期间起作用。四.确定Lec 35蛋白是否与其他蛋白相互作用。V.测量现有Lec 35 cDNA对甘露糖-β-多萜醇相对于葡萄糖-β-多萜醇（GPD）的选择性，并分离Lec 35的GPD选择性形式的cDNA。