STRUCTURE, BIOSYNTHESIS & FUNCTION OF GLYCOPROTEINS

结构、生物合成

基本信息

批准号：
8444665
负责人：
STUART A KORNFELD
金额：
$ 70.26万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1979
资助国家：
美国
起止时间：
1979-09-01 至 2014-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8444665
关键词：
Acetylglucosamine Acids Affinity Alpha-mannosidase Anabolism Animals Binding Binding Proteins Capsid Proteins Cell Separation Cells Clathrin Complement Defect Ear Endosomes Enzymes Eukaryotic Cell Fabry Disease Family Fibroblasts Genes Glycogen storage disease type II Glycoproteins Golgi Apparatus Grant Hydrolase Individual Lysosomal Storage Diseases Lysosomes Mannose Mediating Missense Mutation Modeling Molecular Molecular Conformation Mus Mutation Nature Oligosaccharides Pathway interactions Patients Phosphorylation Phosphotransferase Gene Phosphotransferases Polysaccharides Process Production Proteins Recombinants Relative (related person)Research Research Proposals Role Structure Surface Plasmon Resonance System Testing Time Tissues Transferase Transport Vesicles Work design enzyme replacement therapy enzyme substrate gene cloning golgi associated, gamma adaptin homologous, ADP-ribosylation factor interacting protein in vitro Assay intracellular protein transport man receptor tissue/cell culture trans-Golgi Network

项目摘要

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.

描述（由申请人提供）：该研究建议的目的是获得对磷洋糖基靶向系统的分子理解，该系统在将新合成的酸水解酶传递到溶酶体中起作用。这种细胞内蛋白转运途径的缺陷导致严重的溶酶体储存疾病。该途径的一个关键步骤是UDP-GLCNAC的高甘露糖甘露酶在酸水解酶高甘露糖中的选择性磷酸化：溶酶体酶N-乙酰葡萄糖胺-1-磷酸转移酶（PTase）。该转移酶是Alpha2 beta2 gamma2六聚体蛋白。具体目标1用于鉴定PTase的哪些亚基介导了对酸水解酶的常见蛋白决定因素的识别，该过程对于此类酶的选择性磷酸化至关重要。 AIM 2试图识别3个亚基的MAN-6-P受体同源性（MRH）的功能。我们将测试有或没有其伽马亚基的重组PTase，或在MRH结构域中突变的重组PTase在体外测定中磷酸化酸水解酶的磷酸化，并使用表面等离子体共振直接与固定的酸水解酶结合。这些研究将通过分析表达野生型或γ不足PTase磷酸化的成纤维细胞的能力来补充，从而磷酸化了转染到细胞中的一组酸水解酶。 AIM 3是确定PTase如何定位于高尔基体的顺式贝克派对。 AIM 4是定义GGA（对于GOLGI定位的，含有ARF的γ-结合）蛋白在MAN-6-P受体的包装和运输中，在反式高尔基网络上使用结合的酸水解酶的包装和运输。这些研究将利用与编码GGA1和GGA3的基因中断的小鼠。该目标旨在确定完整动物中的GGA是否是多余的，以及是否对特定的GGA有特定的要求。