STRUCTURE, BIOSYNTHESIS & FUNCTION OF GLYCOPROTEINS

描述(申请人提供)：这项研究计划的目的是从分子上了解在将新合成的酸性水解酶输送到溶酶体中起作用的磷酸芒果糖基靶向系统。这种细胞内蛋白运输途径的缺陷会导致严重的溶酶体储存性疾病。该途径的一个关键步骤是通过UDP-GlcNAc：溶酶体酶N-乙酰氨基葡萄糖-1-磷酸转移酶(Ptase)选择性地将甘露糖残基磷酸化到酸性水解酶的高甘露聚糖上。这种转移酶是一种122232的六聚体蛋白。具体目标1旨在确定Ptase的哪些亚基介导对酸性水解酶的共同蛋白质决定因素的识别，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鉴定3亚基的Man-6-P受体同源(MRH)结构域的功能。我们将在体外实验中测试带有和不带有其3个亚基的重组Ptase，或具有MRH结构域突变的重组Ptase对酸性水解酶的磷酸化能力，并利用表面等离子共振技术直接与固定化酸性水解酶结合。这些研究将通过分析表达野生型或3缺失Ptase的成纤维细胞将一组导入细胞的酸性水解酶磷酸化的能力来补充。目的3是确定Ptase如何定位于高尔基体的顺式小室。目的4是确定GGA(用于高尔基体定位的，含3-EAR的ARF结合)蛋白在跨高尔基体网络结合酸性水解酶的Man-6-P受体的包装和运输中的作用。这些研究将利用编码GGA1和GGA3基因中断的小鼠。这一目的是为了确定GGA在完整动物中是否多余，以及是否对特定GGA有组织特异性要求。公共卫生相关性这项研究直接关系到对两种严重的溶酶体储存疾病MLII和MLIII的理解。两者都是由Ptase的1/2和3基因突变引起的。这项工作还与溶酶体酶的生产有关，用于酶替代疗法治疗患有溶酶体储存疾病的个人，如Fabry和Pompe病。

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.