Transient Glucosylation of Glycoproteins

糖蛋白的瞬时糖基化

• 批准号: 7348825
• 负责人: ARMANDO JOSE PARODI
• 金额: $ 7.78万
• 依托单位: FUNDACION INSTITUTO LELOIR
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-08-15 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7348825
• 关键词: Amyloid; Amyloid fibers; Automobile Driving; Binding; Binding Proteins; Biological Models; Bos taurus structural-GP protein; Calnexin; Cell physiology; Cells; Characteristics; Complex; Coupled; Data; Disease; Endoplasmic Reticulum; Ensure; Fission Yeast; Glucosidase II; Glucosyltransferase; Glucosyltransferases; Glycoproteins; Golgi Apparatus; Laboratories; Lead; Lectin; Life; Link; Mammalian Cell; Mannosidase; Mediating; Membrane; Molecular Chaperones; Muramidase; Polysaccharides; Process; Production; Proteins; Purpose; Quality Control; Relative (related person); Role; Saccharomyces cerevisiae; Yeasts; amyloid formation; calreticulin; cell type; conformer; glycosylation; in vivo; insight; interest; microorganism; mutant; novel; polypeptide; prevent; protein folding; sensor; tool; yeast genetics

DESCRIPTION (provided by applicant): The quality control (QC) of glycoprotein folding in the endoplasmic reticulum (ER) involves the interplay of a glucosyltransferase (GT) that only glycosylates not properly folded glycoprotein conformers, glucosidase II that removes residues added by GT and two ER resident lectins (calnexin, CNX and calreticulin, CRT) that specifically recognize monoglucosylated glycoproteins. This mechanism prevents exit of not properly folded glycoproteins to the Golgi and enhances glycoprotein folding efficiency and is directly related to the so-called "conformational diseases". To continue our characterization of the structural features that determine the participation of GT in the QC mechanism we now propose to study: (i) the subtle conformational differences, including those that may eventually result in the formation of amyloid fibers, that determine whether glycoproteins are recognized or not in vivo by GT; (ii) the maximum distance between the N-glycan and the structural distortion that allows GT-mediated glucosylation and (iii) whether glycoprotein structural stability determines its fate in the ER, i.e. successfully passing through the QC or being diverted to ER associated degradation (ERAD). For this purpose, we will express several N-glycosylation and stability lysozyme mutants (including some ones leading to the production of amyloids) in S. pombe cells and to follow whether they are recognized by GT, and whether they successfully fold or if, alternatively, they are derived to ERAD. These data, coupled to a structural characterization of the lysozyme mutants will provide information on why cells fail, in certain instances, to derive dangerous, aggregation-prone glycoproteins to degradation and on whether the structural stability of glycoproteins determines their fate in the ER. To continue our characterization of the role of N-glycans on ERAD we will further study the mechanism by which the ER mannosidase, the putative lectins Htm1p/Mnl1p/EDEM and Yos9p and the conformational sensor GT participate in driving irreparably misfolded glycoproteins to degradation. As a model system we chose S. pombe, a yeast that, contrary to what happens in S. cerevisiae, has a QC of glycoprotein folding similar to that occurring in mammalian cells. We plan to further study the proposal, derived from our own results, that the role of ER 1-mannosidase in ERAD is related to a putative lectin capacity of the protein and not to its enzymatic activity and to evaluate the relative importance in both productive glycoprotein folding and in diversion to ERAD of the first, partial glycan deglucosylation-dependent and of the following, reglucosylation, GT-dependent, CNX-glycoprotein interactions.

描述（由申请人提供）：内质网（ER）中糖蛋白折叠的质量控制（QC）涉及葡萄糖基转移酶（GT）的相互作用，该酶仅糖基化未正确折叠的糖蛋白构象异构体，葡萄糖苷酶 II 去除 GT 添加的残基，以及两种内质网驻留凝集素（钙联蛋白，CNX 和钙网蛋白，CRT）专门识别单葡萄糖基化糖蛋白。这种机制可以防止未正确折叠的糖蛋白退出高尔基体并提高糖蛋白折叠效率，并且与所谓的“构象疾病”直接相关。为了继续表征决定 GT 参与 QC 机制的结构特征，我们现在建议研究：（i）细微的构象差异，包括最终可能导致淀粉样纤维形成的差异，这些差异决定了糖蛋白是否是GT 在体内识别或不识别； (ii) N-聚糖和允许 GT 介导的糖基化的结构变形之间的最大距离，以及 (iii) 糖蛋白结构稳定性是否决定其在 ER 中的命运，即成功通过 QC 或转向 ER 相关的降解（ ERAD）。为此，我们将在粟酒裂殖酵母细胞中表达几种 N-糖基化和稳定性溶菌酶突变体（包括一些导致淀粉样蛋白产生的突变体），并追踪它们是否被 GT 识别，以及它们是否成功折叠或是否折叠。 ，它们派生至 ERAD。这些数据与溶菌酶突变体的结构特征相结合，将提供关于为什么细胞在某些情况下无法产生危险的、易于聚集的糖蛋白以进行降解的信息，以及糖蛋白的结构稳定性是否决定它们在内质网中的命运。为了继续表征 N-聚糖对 ERAD 的作用，我们将进一步研究 ER 甘露糖苷酶、假定的凝集素 Htm1p/Mnl1p/EDEM 和 Yos9p 以及构象传感器 GT 参与驱动不可挽回的错误折叠糖蛋白降解的机制。作为模型系统，我们选择了粟酒裂殖酵母，这种酵母与酿酒酵母相反，其糖蛋白折叠的质量控制类似于哺乳动物细胞中发生的情况。我们计划进一步研究根据我们自己的结果得出的建议，即 ER1-甘露糖苷酶在 ERAD 中的作用与蛋白质的假定凝集素能力有关，而不是与其酶活性有关，并评估在生产性糖蛋白中的相对重要性第一个、部分聚糖脱糖基依赖性的折叠和转向 ERAD 以及随后的、重新糖基化、GT 依赖性、CNX-糖蛋白相互作用。