Assembly and transfer of N-linked oligosaccharides

N-连接寡糖的组装和转移

• 批准号: 8186078
• 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/8186078
• 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. PUBLIC HEALTH RELEVANCE: Glycosylation of secretory proteins, lysosomal proteins and integral membrane proteins is of fundamental importance to human health. Hypoglycosylation of glycoproteins in the endoplasmic reticulum is responsible for the family of diseases known as congenital disorders of glycosylation (CDG-I). Mutations in the TUSC3 gene, which encodes a non-catalytic subunit of the oligosaccharyltransferase, can cause autosomal recessive mental retardation. Our studies address the role of oligosaccharyltransferase isoforms in achieving efficient glycosylation of proteins.

描述（由申请人提供）：本提案中描述的研究旨在理解寡糖胆汁含量（OST）内质网（ER）中蛋白质蛋白质蛋白的机理。将特别强调（i）确定OST的STT3b同工型在转移后糖基化中是否扮演一般作用，而OST的STT3A同工型跳过的位点，（ii）阐明两个OST同型如何有助于在蛋白质中溶于降低的蛋白质中的豆类，并在defs液中的豆类溶液组成的细胞中，该细胞中的deftersy deflys defarment in comenty defarment in comenty defarnich Insecy sermin in comention-defarnich Insecy sseption-defarmention defarmention defarmenty defarmention defarmention def的细胞中的demos型，用于N连接的糖基化的寡糖供体，（iii）在具有翻译核糖体和蛋白质易位通道的络合物中获得OST的冷冻电子显微镜结构。当前的证据表明，OST的STT3A同工型主要负责新生多肽通过蛋白质易位通道的新生多肽的共透明N-糖基化。 STT3B同工型可以介导展开的蛋白质的翻译后糖基化。通过识别其他类别的STT3B底物，可以获得OST的STT3B同工型体内角色的新洞察力。膜双层附近的糖基化位点的整体膜蛋白，具有羧基末端糖基化位点的蛋白质或多个紧密间隔的糖基化位点作为潜在的STT3B底物测试。将进行共免疫沉淀实验，以确定降低糖基化蛋白是通过腔内ER伴侣或ER钙化凝集素将降压蛋白传递到OST的OST的。用于N-连接糖基化的Dolichol连接寡糖供体（DOL-PPP-OS）的遗传缺陷导致糖基化的先天性疾病（CDG-I）。 DOL-PP-OS的组装将通过Si-RNA介导的Alg6葡萄糖基转移酶的耗竭在HeLa细胞中受到干扰。 ALG6和STT3B的同时耗竭将用于测试OST的STT3B同工型是否修饰由Alg6缺陷细胞中OST跳过的STT3A同工型的糖基化位点。最近的报道表明，TUSC3基因中的突变会引起常染色体隐性智力障碍。 Si-RNA介导的TUSC3的耗竭将用于确定是否需要有效的某些蛋白质糖基化此OST辅助亚基。酵母和哺乳动物OST是大的（240-270 kD）的杂音膜蛋白，可能与内质网中的蛋白质转运通道直接相互作用。 OST的复合物，SEC61蛋白易位通道和翻译核糖体将准备用于冷冻电子显微镜。这些复合物的结构分析应洞悉OST复合物中STT3活性位点的位置以及在蛋白质易位通道和OST活性位点之间的新生多肽所采取的路径。 公共卫生相关性：分泌蛋白，溶酶体蛋白和整体膜蛋白的糖基化对人类健康至关重要。内质网中糖蛋白的降解性糖基化导致称为糖基化先天性疾病的疾病家族（CDG-I）。 TUSC3基因中的突变编码寡糖胆囊化酶的非催化亚基，可能会引起常染色体隐性智力智力低下。我们的研究探讨了寡糖胆胆胆素促酶同工型在实现蛋白质有效糖基化中的作用。