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）中低聚糖转移酶（OST）对天冬酰胺连接的蛋白质糖基化的机制。重点将放在(i)确定OST的STT3B异构体是否在OST的STT3A异构体跳过的位点的翻译后糖基化中发挥一般作用，（ii）阐明两个OST异构体如何有助于在具有聚醇连接寡糖供体组装缺陷的细胞中蛋白质的低糖基化，以进行n链糖基化。（iii）获得具有翻译核糖体和蛋白质易位通道的复合物中OST的低温电镜结构。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亚型可以介导未折叠蛋白的翻译后糖基化。通过鉴定STT3B底物的其他类别，将获得对OST STT3B异构体体内作用的新见解。在膜双分子层附近有糖基化位点的完整膜蛋白、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被测试作为潜在的STT3B底物。将进行共免疫沉淀实验，以确定低糖基化蛋白是否通过腔内内质网伴侣或内质网定位凝集素传递到OST的STT3B亚型。先天性糖基化障碍（CDG-I）是由n -链寡糖供体（dolp - os）组装途径的遗传缺陷引起的。在HeLa细胞中，si-RNA介导的ALG6糖基转移酶的缺失会干扰dolp - os的组装。同时去除ALG6和STT3B将用于测试在ALG6缺陷细胞中，OST的STT3B异构体是否修饰了OST的STT3A异构体跳过的糖基化位点。最近的报道表明，TUSC3基因突变可引起常染色体隐性智力低下。Si-RNA介导的TUSC3缺失将用于确定这种OST附属亚基是否需要某些蛋白质的有效糖基化。酵母和哺乳动物的OST是大的（240-270 kD）异聚寡聚膜蛋白，可以直接与内质网中的蛋白质易位通道相互作用。OST复合物，Sec61蛋白易位通道和翻译核糖体将制备用于冷冻电子显微镜。对这些复合物的结构分析将有助于了解STT3活性位点在OST复合物中的位置，以及新生多肽在蛋白质易位通道的管腔面和OST活性位点之间的路径。

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.