Assembly and transfer of N-linked oligosaccharides

描述(由申请人提供)：本提案中描述的研究旨在了解内质网(ER)中天冬酰胺连接的蛋白质通过寡糖转移酶(OST)进行糖基化的机制。将特别强调(I)确定OST的STT3B亚型是否在被OST的STT3A亚型跳过的位点的翻译后糖基化中发挥一般作用，(Ii)阐明这两个OST异构体如何在具有用于N连接糖基化的二元醇连接的寡糖供体的组装缺陷的细胞中促进蛋白质的低糖基化，以及(Iii)在具有翻译核糖体和蛋白质转位通道的复合体中获得OST的冷冻电子显微镜结构。目前的证据表明，OST的STT3A亚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。STT3B亚型可介导未折叠蛋白的翻译后糖基化。通过鉴定其他类别的STT3B底物，将获得对OST的STT3B亚型在体内的作用的新见解。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质将被测试为潜在的STT3B底物。将进行免疫共沉淀实验，以确定低糖基化蛋白是否通过管腔ER伴侣或ER定位的凝集素传递到OST的STT3B亚型。苯二醇寡糖供体(DOL-PP-OS)N-连接糖基化组装途径的遗传缺陷导致先天性糖基化紊乱(CDG-I)。在HeLa细胞中，由于siRNA介导的ALG6葡萄糖基转移酶的缺失，Dol-PP-OS的组装将受到干扰。ALG6和STT3B的同时耗尽将被用来测试在ALG6缺失的细胞中，OST的STT3B亚型是否修改了被OST的STT3A亚型跳过的糖基化位点。最近的报道表明，TUSC3基因突变可导致常染色体隐性遗传性智力低下。SI-RNA介导的TUSC3的缺失将被用来确定这个OST辅助亚基是否是某些蛋白质有效糖基化所必需的。酵母和哺乳动物的OST是大的(240-270kD)的异源低聚膜蛋白，可能直接与内质网中的蛋白质转运通道相互作用。OST、Sec61蛋白转位通道和翻译核糖体的复合体将被制备用于冷冻电子显微镜。对这些复合体的结构分析应该能够深入了解STT3活性部位在OST复合体中的位置，以及新生多肽在蛋白质易位通道的管腔面和OST活性部位之间的路径。

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.