STRUCTURE, BIOSYNTHESIS AND FUNCTION OF GLYCOPROTEINS

研究提案的目标是获得一种分子 了解具有以下功能的磷甘露醇靶向系统 将新合成的酸性水解酶输送到溶酶体。这里面的瑕疵 细胞内蛋白转运途径导致严重溶酶体 储藏疾病。具体目标包括：(1)刻画 DNase I上的蛋白质识别结构域，它是 与磷酸转移酶的相互作用，它催化了 Man-6-P识别标记的产生。我们将使用Site- 定向突变鉴定DNA酶I表面残基 确定与磷酸转移酶的结合并分析这些残基是如何 在选定的糖基化位点直接磷酸化。(2)完成 磷酸二酯α-GlcNAcase的克隆及蛋白鉴定 决定其高尔基本地化的区域。这种酶去除了 酸性水解酶寡糖覆盖GlcNAc暴露Man-6-P 它们与Man-6-p受体结合所必需的残基。CDNA克隆 编码野生型和突变型酶将导入COS细胞 利用脉冲追踪法对高尔基体定位和贩运进行了分析 实验和E/M免疫金定位。(3)调查 酸性水解酶(但不是IGF-II)与 MAN-6-P/IGF-II受体通过 笼罩着网状蛋白的坑。(4)确定高尔基体膜对接 与异三聚体AP-1接头复合体相互作用的蛋白质(S) 并作为后续组装的成核位置 跨高尔基体网上包裹着网状蛋白的小泡。这些小泡 在其第一条腿上携带Man-6-P受体.配体复合体 胞内行程。我们已经证明了Coat中的初始事件 组装涉及ADP-核糖化因子介导的募集 AP-1从胞浆中分离出来。高尔基体膜上组装的复合体将 用温和的洗涤剂溶解，并通过凝胶过滤和纯化 亲和层析。一旦确定了对接蛋白(S) 和克隆，我们将使用分子技术来定义结构 特异性其细胞内定位和解剖的决定因素 AP-1招募背后的蛋白质-蛋白质相互作用。这 研究的目的是提供对自然界和 负责包膜囊泡的蛋白质的功能 在高尔基体膜上形成。

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.