STRUCTURE, BIOSYNTHESIS & FUNCTION OF GLYCOPROTEINS

描述（由申请人提供）：本研究提案的目的是从分子水平了解磷酸甘露糖基靶向系统，该系统在将新合成的酸性水解酶递送至溶酶体中发挥作用。这种细胞内蛋白质转运途径的缺陷引起严重的溶酶体贮积病。该途径中的关键步骤是通过UDP-GlcNAc：溶酶体酶N-乙酰葡糖胺-1-磷酸转移酶（Ptase）选择性磷酸化酸性水解酶的高甘露糖聚糖上的甘露糖残基。该转移酶是122232六聚体蛋白。具体目标1是针对确定哪些亚基的Ptase介导的识别的共同蛋白质决定簇的酸性水解酶，这是一个过程，这是必不可少的选择性磷酸化这类酶。目的2：鉴定Man-6-P受体3亚基同源结构域（MRH）的功能.我们将测试具有和不具有其3亚基或在MRH结构域中具有突变的重组Ptase在体外测定中磷酸化酸性水解酶和使用表面等离子体共振直接结合到固定化酸性水解酶的能力。这些研究将通过分析表达野生型或β缺陷型Ptase的成纤维细胞磷酸化转染到细胞中的一组酸性水解酶的能力来补充。目的3是确定Ptase是如何定位到高尔基体的顺式亚隔室。目的4是确定GGA（高尔基体定位，3-ear containing，ARF结合）蛋白在包装和运输的Man-6-P受体与酸水解酶在trans-Golgi网络的作用。这些研究将利用编码GGA 1和GGA 3的基因被破坏的小鼠。该目的旨在确定完整动物中的GGA是否多余，以及特定GGA是否存在组织特异性要求。公共卫生相关性这项研究与两种严重的溶酶体贮积病MLII和MLIII的理解直接相关。两者都是由Ptase的1/2和3基因突变引起的。这项工作也与生产用于酶替代疗法的溶酶体酶有关，用于治疗患有溶酶体贮积病（如法布里病和庞贝病）的个体。

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 122232 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 3 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 3 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, 3-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. PUBLIC HEALTH RELEVANCE This research is directly relevant to the understanding of two serious lysosomal storage diseases termed MLII and MLIII. Both are caused by mutations in the 1/2 and 3 genes of Ptase. The work is also relevant to the production of lysosomal enzymes used for enzyme replacement therapy in the treatment of individuals with lysosomal storage diseases such as Fabry and Pompe disease.