STRUCTURE, BIOSYNTHESIS & FUNCTION OF GLYCOPROTEINS

描述（由申请人提供）：本研究计划的目的是获得磷酰丙氨酸靶向系统的分子理解，该系统在将新合成的酸水解酶递送到溶酶体中起作用。这种细胞内蛋白质运输途径的缺陷导致严重的溶酶体贮积病。该途径的关键步骤是通过溶酶体酶n -乙酰氨基葡萄糖-1-磷酸转移酶（Ptase）对酸性水解酶的高甘露糖聚糖上的甘露糖残基进行选择性磷酸化。这种转移酶是α 2 β 2 γ 2六聚体蛋白。特异性目标1旨在确定哪些Ptase亚基介导酸水解酶的共同蛋白质决定因子的识别，这一过程对于这类酶的选择性磷酸化是必不可少的。目的2旨在鉴定3亚基的Man-6-P受体同源性（MRH）结构域的功能。我们将测试重组Ptase在体外磷酸化酸水解酶的能力，以及使用表面等离子体共振直接与固定化酸水解酶结合的能力。这些研究将通过分析表达野生型或γ缺陷型pase的成纤维细胞磷酸化转染到细胞中的一组酸水解酶的能力来补充。目的3是确定pase如何定位于高尔基体的顺式亚室。目的4是确定GGA（高尔基定位，含γ -耳，arf结合）蛋白在Man-6-P受体的包装和运输中与反式高尔基网络结合的酸水解酶的作用。这些研究将利用编码GGA1和GGA3的基因被破坏的小鼠。该目的旨在确定gga在完整动物中是否多余，以及是否存在对特定gga的组织特异性需求。

DESCRIPTION (provided by applicant): The objective of this research proposal is to obtain a molecular understanding of the phosphomannosyl targeting system which functions in the delivery of newly synthesized acid hydrolases to lysosomes. Defects in this intracellular protein transport pathway give rise to severe lysosomal storage diseases. A key step in this pathway is the selective phosphorylation of mannose residues on the high mannose glycans of the acid hydrolases by UDP-GlcNAc: lysosomal enzyme N-acetylglucosamine-1- phosphotransferase (Ptase). This transferase is an alpha2 beta2 gamma2 hexameric protein. Specific Aim 1 is directed toward identifying which subunits of Ptase mediate the recognition of the common protein determinant of acid hydrolases, a process that is essential for the selective phosphorylation of this class of enzymes. Aim 2 seeks to identify the function of the Man-6-P receptor homology (MRH)-domain of the 3 subunit. We will test the ability of recombinant Ptase with and without its gamma subunit, or with mutations in the MRH domain, to phosphorylate acid hydrolases in in vitro assays and to bind directly to immobilized acid hydrolases using surface plasmon resonance. These studies will be complemented by analyzing the ability of fibroblasts expressing wild type or gamma deficient Ptase to phosphorylate a panel of acid hydrolases transfected into the cells. Aim 3 is to determine how Ptase is localized to the cis-subcompartment of the Golgi. Aim 4 is to define the role of the GGA (for Golgi-localized, gamma-ear containing, ARF-binding) proteins in the packaging and transport of the Man-6-P receptors with bound acid hydrolases at the trans-Golgi network. These studies will utilize mice with disruptions of the genes encoding GGA1 and GGA3. This aim is designed to establish whether the GGAs are redundant in intact animals and whether there are tissue specific requirements for specific GGAs.