MOLECULAR BIOLOGY OF ASPARAGINE LINKED GLYCOSYLATION

在哺乳动物中，分泌蛋白和细胞表面蛋白被一种或多种碳水化合物修饰，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多种疾病。这些与天冬酰胺相连的碳水化合物会导致多种疾病。天冬酰胺链（N-链）、糖基磷脂酰肌醇（GPI）和c -甘露糖碳水化合物争夺一种常见的猛犸供体甘露糖- p -醇（MPD）。MPD是由dolichol- p和gdp -甘露糖酶法合成的“dolichol途径”的一个组成部分。由于dolichol-P在细胞中是有限的，每一类碳水化合物的生物合成通量是平衡的，以维持细胞的需要。在目前的资助期内，P.I.克隆了一种新的基因，称为Lec35，这对MPD的利用至关重要。Lec35突变体合成MPD，但在合成n -聚糖、GPIs和C-甘露糖方面存在缺陷。因此，Lec35代表了一类具有独特作用机制的新蛋白质。研究人员成功克隆了Lec35的cDNA和基因，并设计了体内和体外的Lec35功能系统。对于下一个资助期，提出了五个目标：1 .确定Lec35蛋白是否参与(a) MPD从内质小叶翻转到管腔小叶，或(b)消除MPD依赖性甘露糖基化的扩散抑制剂。2。确定Lec35蛋白的拓扑结构、必需残基的位置和亚细胞定位，该蛋白被预测为具有两层膜跨度的内质网蛋白。3。确定Lec35蛋白是否在酶促mpd依赖性甘露糖基化之前或期间起作用。IV.确定Lec35蛋白是否与其他蛋白相互作用。V.测量现有的Lec35 cDNA对甘露糖- p -多醇的选择性，而不是葡萄糖- p -多醇（GPD），并分离出一种GPD选择性形式的Lec35 cDNA。

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.