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

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。内质网(ER)的质量控制决定了新合成的糖蛋白的命运，要么是正确的折叠，要么是通过ER相关的降解来处置。处置过程的启动包括ERα-甘露糖苷酶I选择性地修剪附着在错误折叠的糖蛋白上的N-糖链，随后被ER定位的凝集素EDEM识别，这两个凝集素都是糖基水解酶家族47的成员。通过对野生型和突变型人ERα-甘露糖苷酶I的动力学和结合分析，以及与未切割的硫代二糖底物类似物的共同络合物的结构分析，研究了该家族成员催化的不寻常的反转水解机制。这些数据揭示了催化酸和碱残基的作用以及结合底物类似物的新的3S1糖构象的鉴定。对这一糖苷酶家族的作用机制进行了讨论。用核磁共振方法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.