STRUCTURE, BIOSYNTHESIS AND FUNCTION OF GLYCOPROTEINS

该研究计划的目的是获得一种分子 了解磷酸甘露糖基靶向系统的功能， 将新合成的酸性水解酶递送到溶酶体。网站的缺陷 细胞内蛋白质转运途径引起严重溶酶体 贮藏病害具体目标包括：（1） DNA酶I上的蛋白质识别结构域， 与磷酸转移酶相互作用，磷酸转移酶催化第一步， Man-6-P识别标记的产生。我们将使用网站- 定向诱变以鉴定DNA酶I表面上 确定与磷酸转移酶的结合，并分析这些残基 在选定的糖基化位点直接磷酸化。(2)完成 磷酸二酯α-GlcNAc酶的克隆及蛋白质的鉴定 决定其高尔基体定位的结构域。这种酶可以去除 从酸性水解酶寡糖中覆盖GlcNAc以暴露Man-6-P 它们与Man-6-p受体结合所必需的残基。cDNA克隆 将编码野生型和突变体酶的基因转染到COS细胞中 并利用脉冲追踪技术分析了高尔基体的定位和运输 实验和E/M免疫金定位。(3)探讨 酸性水解酶（而不是IGF-II）与胰岛素受体结合的机制 Man-6-P/IGF-II受体通过以下途径加速内化速率： 网格蛋白包被的凹坑(4)为了识别高尔基体膜对接 与异源三聚体AP-1衔接子复合物相互作用的蛋白质 并用作随后组装的成核位点， 网格蛋白包被的囊泡在高尔基体网络上。这些囊泡 在其第一条腿上携带Man-6-P受体配体复合物， 胞内行程我们已经证明了coat中的初始事件 组装涉及ADP-核糖基化因子介导的 细胞质中的AP-1。在高尔基体膜上组装的复合体 用温和的洗涤剂溶解并通过凝胶过滤纯化， 亲和层析一旦对接蛋白被识别， 并克隆，我们将使用分子技术来定义结构， 决定其细胞内定位和解剖的特异性 蛋白质之间的相互作用是AP-1募集的基础。这 研究旨在详细了解其性质， 负责网格蛋白包被囊泡的蛋白质的功能 形成于高尔基体膜上。

The objective of the research proposal is to obtain a molecular understanding of the phosphomannosyl targeting system which functions to deliver newly synthesized acid hydrolases to lysosomes. Defects in this intracellular protein transport pathway give rise to severe lysosomal storage diseases. The specific aims include: (1) The characterization of the protein recognition domain on DNase I that is necessary for interaction with phosphotransferase which catalyzes the first step in the generation of the Man-6-P recognition marker. We will use site- directed mutagenesis to identify residues on the surface of DNase I that determine binding to phosphotransferase and analyze how these residues direct phosphorylation at selected glycosylation sites. (2) To complete the cloning of phosphodiester alpha-GlcNAcase and define the protein domains that determine its Golgi localization. This enzymes removes the covering GlcNAc from acid hydrolase oligosaccharides to expose Man-6-P residues essential for their binding to Man-6-p receptors. cDNA clones encoding wild-type and mutant enzyme will be transfected into COS cells and analyzed for Golgi localization and trafficking using pulse-chase experiments and E/M immunogold localization. (3) To investigate the mechanism whereby binding of acid hydrolases (but not IGF-II) to the Man-6-P/IGF-II receptor accelerates the rate of internalization via clathrin-coated pits. (4) To identify the Golgi membrane docking protein(s) that interacts with the heterotrimeric AP-1 adaptor complex and serves as the nucleation site for the subsequent assembly of Clathrin-coated vesicles on the trans-Golgi network. These vesicles carry the Man-6-P receptor.ligand complex on the first leg of its intracellular itinerary. We have shown that the initial event in coat assembly involves the ADP-ribosylation factor mediated recruitment of AP-1 from the cytosol. The assembled complex on the Golgi membrane will be solubilized with mild detergent and purified by gel filtration and affinity chromatography. Once the docking protein(s) has been identified and cloned, we will use molecular techniques to define the structural determinants that specificity its intracellular localization and dissect the protein-protein interactions that underlie AP-1 recruitment. This study is designed to provide a detailed understanding of the nature and function of the proteins responsible for clathrin-coated vesicle formation on Golgi membranes.