MECHANISM OF CLASS I ALPHA-MANNOSIDASES INVOLVED IN N-GLYCAN PROCESSING

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。内质网（ER）中的质量控制决定了新合成的糖蛋白的命运，使其正确折叠或通过ER相关降解处理。处置过程的启动涉及通过ER α-甘露糖苷酶I选择性修剪与错误折叠的糖蛋白连接的N-聚糖，并随后通过ER定位的凝集素EDEM（两者都是糖基水解酶家族47的成员）识别。通过结合人ER α-甘露糖苷酶I的野生型和突变形式的动力学和结合分析以及与未裂解的硫代二糖底物类似物的共复合物的结构分析，研究了该家族成员催化的不寻常的转化水解机制。这些数据揭示了催化酸和碱残基的作用，并确定了一种新的3S 1糖构象的结合底物类似物。这个家庭的糖苷酶的作用机制的影响进行了讨论。用核磁共振谱鉴定了硫代二糖的合成中间体，并描述了其溶液构象。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Quality control in the endoplasmic reticulum (ER) determines the fate of newly synthesized glycoproteins toward either correct folding or disposal by ER-associated degradation. Initiation of the disposal process involves selective trimming of N-glycans attached to misfolded glycoproteins by ER alpha-mannosidase I and subsequent recognition by the ER-localized lectin, EDEM, both members of glycosylhydrolase family 47. The unusual inverting hydrolytic mechanism catalyzed by members of this family has been investigated by a combination of kinetic and binding analyses of wild type and mutant forms of human ER alpha-mannosidase I as well as structural analysis of a co-complex with an uncleaved thio-disaccharide substrate analog. The data reveal the roles of catalytic acid and base residues and the identification of a novel 3S1 sugar conformation for the bound substrate analog. Implications for the mechanism of action of this family of glycosidases are discussed. NMR was used in identifying the synthetic intermediates of the thio-disaccharide, as well as describing its solution conformation.