Assembly and transfer of N-linked oligosaccharides

 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）不表达STT 3A或STT 3B复合物的特定亚基的HEK 293衍生细胞系的产生和分析，（ii）STT 3A复合物在富含半胱氨酸的糖蛋白的共翻译糖基化中的作用的分析，（iii）阐明负责STT 3A复合物邻近蛋白质易位通道定位的分子机制，和（iv）STT 3B复合物识别蛋白质中非糖基化受体位点的机制分析。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同种型可以介导在新生糖蛋白折叠之前已经被STT 3A复合物跳过的受体位点的易位后糖基化。CRISPR-Cas9基因编辑系统将用于产生缺乏STT 3A、STT 3B或STT 3A或STT 3B复合物的辅助亚基的HEK 293衍生的细胞系。将使用质谱法对HEK 293糖蛋白组进行分类，并鉴定当细胞缺乏功能性STT 3A或STT 3B复合物时低糖基化的糖基化受体位点。糖肽序列分析应确定促进STT 3A复合物跳跃的糖基化位点中的基序，并指出每个OST同种型修饰的总受体位点的比例。当STT 3A复合物被siRNA处理耗尽时，富含半胱氨酸的糖蛋白如鞘脂激活蛋白原和颗粒蛋白原被低糖基化。生物信息学分析将用于鉴定模式生物中富含半胱氨酸的糖蛋白，并确定糖基化受体位点和半胱氨酸残基之间的间距。将使用野生型和突变体细胞中的体内脉冲标记来确定STT 3A复合物是否负责由多个富含半胱氨酸的重复结构域组成的蛋白质的糖基化。已通过天然凝胶电泳检测到由STT 3A复合物、蛋白质易位通道（Sec 61复合物）和易位子相关蛋白（TRAP）复合物组成的大复合物。将探讨TRAP复合物的作用和STT 3A特异性辅助亚基在这些大复合物的组装和稳定性中的作用，以确定STT 3A复合物如何定位在蛋白质易位通道附近。将进行免疫共沉淀实验，以确定低糖基化蛋白与OST的STT 3B亚型的结合是否由MagT 1或TUSC 3氧化还原酶介导。总之，这项研究将有助于揭示STT 3A和STT 3B复合物之间的合作如何增强人类细胞中蛋白质的糖基化效率。

 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) the generation and analysis of HEK293 derived cell lines that do not express specific subunits of the STT3A or STT3B complexes, (ii) the analysis of the role of the STT3A complex in cotranslational glycosylation of cysteine-rich glycoproteins, (iii) elucidation of the molecular mechanism responsible for localization of the STT3A complex adjacent to the protein translocation channel and (iv) analysis of the mechanism of recognition of nonglycosylated acceptor sites in proteins by the STT3B complex. 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 posttranslocational glycosylation of acceptor sites that have been skipped by the STT3A complex prior to folding of the nascent glycoprotein. The CRISPR-Cas9 gene- editing system will be used to generate HEK293 derived cell lines that lack STT3A, STT3B or accessory subunits of the STT3A or STT3B complexes. Mass spectrometry will be used to catalogue the HEK293 glycoproteome and to identify glycosylation acceptor sites that are hypoglycosylated when cells lack a functional STT3A or STT3B complex. Glycopeptide sequence analysis should identify motifs in glycosylation sites that promote skipping by the STT3A complex, and indicate what fraction of the total acceptor sites are modified by each OST isoform. Cysteine rich glycoproteins like prosaposin and progranulin are hypoglycosylated when the STT3A complex is depleted by siRNA treatment. Bioinformatics analysis will be used to identify cysteine-rich glycoproteins in model organisms and to determine the spacing between glycosylation acceptor sites and cysteine residues. In vivo pulse labeling in wild type and mutant cells will be used to determine whether the STT3A complex is responsible for glycosylation of proteins that are comprised of multiple cysteine-rich repeat domains. Large complexes consisting of the STT3A complex, the protein translocation channel (Sec61 complex) and the translocon- associated protein (TRAP) complex have been detected by native gel electrophoresis. The role of the TRAP complex and role of STT3A-specific accessory subunits in the assembly and stability of these large complexes will be explored to determine how the STT3A complex is positioned adjacent to the protein translocation channel. Co-immunoprecipitation experiments will be performed to determine whether binding of hypoglycosylated proteins to the STT3B isoform of the OST is mediated by the MagT1 or TUSC3 oxidoreductases. Together, this research will help reveal how cooperation between the STT3A and STT3B complexes enhances the glycosylation efficiency of proteins in human cells.