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.

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钙网蛋白，crt），特别识别单糖基化糖蛋白。该机制可防止未正确折叠糖蛋白到高尔基体并提高糖蛋白折叠效率，并与所谓的“构型疾病”直接相关。为了继续我们表征确定GT参与QC机制的结构特征，我们现在建议研究：（i）微妙的构象差异，包括最终导致淀粉样蛋白纤维形成的细微构象差异，这些差异确定糖蛋白是否在gt中识别出糖蛋白是在Vivo中识别的； （ii）N-聚糖与结构变形之间的最大距离，该糖基化葡萄糖素化以及（iii）糖蛋白结构稳定性是否决定了其在ER中的命运，即成功地通过QC或转移到ER相关的降解（ERAD）。为此，我们将表达几种N-糖基化和稳定性溶菌酶突变体（包括一些导致淀粉样链蛋白酶的产生），并遵循它们是否被GT识别，以及它们是否成功折叠还是否则，它们是否被衍生为Erad。这些数据与溶菌酶突变体的结构表征结合在一起，将提供有关在某些情况下细胞在某些情况下失败的信息，从而导致危险的，易受聚集的糖蛋白降解以及糖蛋白的结构稳定性是否决定其在ER中的命运。为了继续表征N-聚糖在ERAD上的作用，我们将进一步研究ER甘露糖苷酶，推定的凝集素HTM1P/MNL1P/EDEM和YOS9P的机制，以及构型传感器GT参与推动不可误误折叠的糖蛋白以降低化力的方法。作为模型系统，我们选择了S. pombe，这是一种酵母，与酿酒酵母中发生的情况相反，糖蛋白折叠的QC与哺乳动物细胞中发生的糖蛋白折叠相似。 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-糖蛋白相互作用。