Assembly and transfer of N-linked oligosaccharides

N-连接寡糖的组装和转移

基本信息

批准号：
7389488
负责人：
JAMES REID GILMORE
金额：
$ 36.08万
依托单位：
UNIV OF MASSACHUSETTS MED SCH WORCESTER
依托单位国家：
美国
项目类别：
财政年份：
1990
资助国家：
美国
起止时间：
1990-04-01 至 2011-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7389488
关键词：
Active Sites Address Affinity Amino Acid Sequence Databases Anabolism Asparagine Binding Binding Sites Biochemical Biochemical Reaction Biological Assay Biological Models Biological Process Biosensor Canis familiaris Catalytic Domain Cell Line Cell model Cells Complex Condition Congenital Disorders Cryptococcus neoformans Databases Defect Detergents Disease Dissociation Dolichol Donor Selection Electrons Entamoeba histolytica Eukaryotic Cell Face Family Funding Glucosamine Glucosyltransferase Glucosyltransferases Glycoconjugates Glycoproteins Goals Hela Cells Human Immunoblotting In Vitro Inherited Insecta Label Ligands Link Mammalian Cell Mannose Mediating Membrane Nature Numbers Oligosaccharides Organism Pathway interactions Patients Pattern Peptides Phenotype Physiologic pulse Plant Genome Property Protein Analysis Protein Binding Protein Glycosylation Protein Isoforms Proteins Pulse taking Quality Control Reporter Reporting Research Resolution Role Rough endoplasmic reticulum Small Interfering RNA Solutions Structure System Testing Transfection Trichomonas vaginalis Trypanosoma Trypanosoma cruzi Variant Yeasts base dimer dolichyl-diphosphooligosaccharide - protein glycotransferase electron crystallography fungus genome sequencing glycosylation glycosyltransferase human tissue in vivo inhibitor/antagonist insight lipooligosaccharide novel polypeptide response ribophorin I rough endoplasmic reticulum membrane two-dimensional

项目摘要

DESCRIPTION (provided by applicant): The long-term objective of this of this project is to understand the biological function, mechanism and structure of the eukaryotic oligosaccharyltransferase (OST). The OST transfers a preassembled high mannose oligosaccharide onto asparagine residues of nascent proteins in the lumen of the rough endoplasmic reticulum. During the proposed funding period particular emphasis will be placed on elucidating the in vivo roles of two isoforms of the human oligosaccharyltransferase that are composed of an active site subunit (STT3A or STT3B) and a shared set of non-catalytic subunits. The two OST isoforms differ greatly both with respect to specific activity and stringency of dolichol-linked oligosaccharide donor selection. STT3A and STT3B are widely expressed in human tissues, and are coexpressed in all cell lines analyzed to date. We will test the hypothesis that mammalian OST isoforms have overlapping but non-identical roles in N-linked glycosylation of proteins. Hypoglycosylation of glycoprotein reporters will be analyzed in cells that have been manipulated to express a single OST isoform. Simultaneous siRNA mediated knockdowns of an OST active site subunit (STT3A or STT3B) and the ALG6 glucosyltransferase will provide a model system to analyze the influence of OST isoform expression on protein hypoglycosylation in cells that are impaired in dolichol-linked oligosaccharide assembly. Protein sequence database searches predict that most protist organisms assemble monomeric or hetero-tetrameric OST complexes instead of the heptameric or octameric OST complexes assembled by fungi and metazoan organisms. Novel assays and biochemical probes will be used to analyze the binding of dolichol-linked oligosaccharide donor and peptide acceptors to the OST from diverse eukaryotic organisms including the pathogenic protists Trypanosoma cruzi, Entamoeba histolytica and Trichomonas vaginalis and the pathogenic yeast Cryptococcus neoformans. A final goal of this project is to obtain a mid to high-resolution structure of the mammalian OST by electron crystallography. The research proposed here will provide insight into the family of inherited human autosomal diseases known as congenital disorders of glycosylation (CDG-I). Analysis of protein N-glycosylation in pathogenic protists may reveal differences in enzymatic reaction mechanism that could potentially be exploited to produce useful pharmacological agents.

描述（由申请人提供）：该项目的长期目标是了解真核寡素胆汁含量的生物学功能，机制和结构（OST）。 OST将预组装的高甘露糖寡糖转移到粗糙内质网的腔内中新生蛋白的天冬酰胺残基上。在拟议的资金期间，将特别强调阐明由活性位点亚基（STT3A或STT3B）和一组非催化亚基的人类寡素胆囊核层转移酶的体内作用。在特异性活性和多甲基连接的寡糖供体选择方面，两个OST同工型都大不相同。 stt3a和stt3b在人体组织中广泛表达，并在迄今为止分析的所有细胞系中共表达。我们将检验以下假设：哺乳动物OST同工型在蛋白质的N连接糖基化中具有重叠但非相同的作用。将在被操纵以表达单个OST同工型的细胞中分析糖蛋白报告蛋白的降节化。 OST活性位点亚基（STT3A或STT3B）的同时siRNA介导的敲低和ALG6葡萄糖基转移酶将提供一个模型系统，以分析OST同工型表达对在多乙醇连接的寡糖糖基团体中受损的细胞中OST同工型的影响。蛋白质序列数据库搜索预测，大多数原生物有机体组装单体或异光聚体OST复合物，而不是由真菌和后生动物组装的七聚体或八聚体OST复合物。 Novel assays and biochemical probes will be used to analyze the binding of dolichol-linked oligosaccharide donor and peptide acceptors to the OST from diverse eukaryotic organisms including the pathogenic protists Trypanosoma cruzi, Entamoeba histolytica and Trichomonas vaginalis and the pathogenic yeast Cryptococcus neoformans.该项目的最终目标是通过电子晶体学获得哺乳动物OST的高分辨率结构。这里提出的研究将洞悉被称为糖基化先天性疾病的遗传性人常染色体疾病（CDG-I）。蛋白质N-糖基化在致病性生物中的分析可能会揭示酶促反应机制的差异，该机制可能被利用以产生有用的药理学剂。