Assembly and transfer of N-linked oligosaccharides

N-连接寡糖的组装和转移

• 批准号: 8316232
• 负责人: JAMES REID GILMORE
• 金额: $ 38.99万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-04-01 至 2015-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8316232
• 关键词: Active Sites; Address; Anabolism; Asparagine; Biological Models; C-terminal; Catalytic Domain; Cells; Co-Immunoprecipitations; Collaborations; Complex; Congenital Disorders; Consensus; Cryoelectron Microscopy; Defect; Degradation Pathway; Disease; Dislocations; Dolichol; Endoplasmic Reticulum; Eukaryota; Face; Family; Genes; Glucosidase Inhibitor; Glucosyltransferase; Glycoproteins; Health; Hela Cells; Human; Inherited; Integral Membrane Protein; Label; Lectin; Link; Location; Mammalian Cell; Mediating; Membrane; Membrane Proteins; Mental Retardation; Molecular Chaperones; Monitor; Mutation; N-Glycosylation Site; Oligosaccharides; Pathway interactions; Patients; Physiologic pulse; Property; Protein Binding; Protein Glycosylation; Protein Isoforms; Protein translocation; Proteins; Quality Control; RNA; Reporting; Research; Resolution; Ribosomes; Role; Rough endoplasmic reticulum; Scanning; Site; Small Interfering RNA; Structure; Testing; Translating; Variant; Yeasts; design; dolichyl-diphosphooligosaccharide - protein glycotransferase; glycosylation; glycosyltransferase; in vivo; insight; link protein; lysosomal proteins; novel; polypeptide; protein misfolding; reconstruction; research study; rough endoplasmic reticulum membrane; secretory protein

DESCRIPTION (provided by applicant): The research described in this proposal is directed towards understanding the mechanism of asparagine- linked glycosylation of proteins in the endoplasmic reticulum (ER) by the oligosaccharyltransferase (OST). Particular emphasis will be placed upon (i) determining whether the STT3B isoform of the OST performs a general role in posttranslational glycosylation of sites that are skipped by the STT3A isoform of the OST, (ii) elucidating how the two OST isoforms contribute to hypoglycosylation of proteins in cells with defects in the assembly of the dolichol-linked oligosaccharide donor for N-linked glycosylation, and (iii) obtaining a cryo-electron microscopy structure of the OST in a complex with a translating ribosome and a protein translocation channel. Current evidence indicates that the STT3A isoform of the OST is primarily responsible for cotranslational N-glycosylation of nascent polypeptides as they pass through the protein translocation channel. The STT3B isoform can mediate posttranslational glycosylation of unfolded proteins. Novel insight into the in vivo role of the STT3B isoform of the OST will be obtained by identifying additional classes of STT3B substrates. Integral membrane proteins with glycosylation sites near the membrane bilayer, proteins with carboxyl-terminal glycosylation sites or multiple closely spaced glycosylation sites will be tested as potential STT3B substrates. Co-immunoprecipitation experiments will be performed to determine whether hypoglycosylated proteins are delivered to the STT3B isoform of the OST by lumenal ER chaperones or ER-localized lectins. Inherited defects in the assembly pathway for the dolichol-linked oligosaccharide donor (Dol-PP-OS) for N-linked glycosylation cause congenital disorders of glycosylation (CDG-I). Assembly of Dol-PP-OS will be perturbed in HeLa cells by si-RNA mediated depletion of the ALG6 glucosyltransferase. Simultaneous depletion of ALG6 and STT3B will be used to test whether the STT3B isoform of the OST modifies glycosylation sites that are skipped by the STT3A isoform of the OST in ALG6-deficient cells. Recent reports indicate that mutations in the TUSC3 gene can cause autosomal recessive mental retardation. Si-RNA mediated depletion of TUSC3 will be used to determine whether this OST accessory subunit is required for efficient glycosylation of certain proteins. The yeast and mammalian OST are large (240-270 kD) hetero-oligomeric membrane proteins that may interact directly with the protein translocation channel in the endoplasmic reticulum. Complexes of the OST, the Sec61 protein translocation channel and a translating ribosome will be prepared for cryoelectron microscopy. Structural analysis of these complexes should provide insight into the location of the STT3 active site in the OST complex and the path taken by the nascent polypeptide between the lumenal face of the protein translocation channel and the OST active site.

描述（由申请方提供）：本提案中描述的研究旨在了解寡糖基转移酶（OST）对内质网（ER）中蛋白质进行天冬酰胺连接糖基化的机制。将特别强调（i）确定OST的STT 3B同种型是否在被OST的STT 3A同种型跳过的位点的翻译后糖基化中发挥一般作用，（ii）阐明两种OST同种型如何有助于在具有用于N-连接糖基化的多醇连接寡糖供体的组装缺陷的细胞中蛋白质的低糖基化，和（iii）获得具有翻译核糖体和蛋白质易位通道的复合物中的OST的冷冻电子显微镜结构。目前的证据表明，OST的STT 3A同种型主要负责新生多肽通过蛋白质易位通道时的共翻译N-糖基化。STT 3B同种型可介导未折叠蛋白的翻译后糖基化。新的洞察STT 3B同种型的OST的体内作用将获得通过确定其他类别的STT 3B基板。在膜双层附近具有糖基化位点的完整膜蛋白、具有羧基末端糖基化位点或多个紧密间隔的糖基化位点的蛋白将作为潜在STT 3B底物进行检测。将进行免疫共沉淀实验以确定低糖基化蛋白是否通过内腔ER伴侣或ER定位的凝集素递送至OST的STT 3B同种型。用于N-连接糖基化的多醇连接寡糖供体（Dol-PP-OS）的组装途径中的遗传缺陷引起先天性糖基化病症（CDG-I）。在HeLa细胞中，Dol-PP-OS的组装将通过si-RNA介导的ALG 6葡糖基转移酶的消耗而受到干扰。ALG 6和STT 3B的同时消耗将用于测试OST的STT 3B同种型是否修饰ALG 6缺陷细胞中OST的STT 3A同种型跳过的糖基化位点。最近的报告表明，TUSC 3基因突变可导致常染色体隐性遗传性精神发育迟滞。Si-RNA介导的TUSC 3消耗将用于确定该OST辅助亚基是否是某些蛋白质的有效糖基化所需的。酵母和哺乳动物的OST是大的（240-270 kD）异源寡聚膜蛋白，可以直接与内质网中的蛋白质转运通道相互作用。将制备OST、Sec 61蛋白易位通道和翻译核糖体的复合物用于冷冻电子显微镜检查。这些复合物的结构分析应该提供洞察STT 3活性位点在OST复合物中的位置和蛋白质易位通道的内腔面和OST活性位点之间的新生多肽所采取的路径。