Mannosidases in Glycoprotein Biosynthesis & Catabolism

描述(申请人摘要)：这项建议的长期目标是 研究一组酶的结构、功能和作用机制 哺乳动物N-连接的糖链的生物合成和分解代谢所必需的。我们 一直专注于N-糖链途径中的CX-甘露糖苷酶，因为它们起着 在合成复杂低聚糖中的承诺步骤通过测定 甘露糖修剪的程度，在某些情况下似乎控制了 内质网(ER)中未折叠糖蛋白的降解。这个 加工和分解α-甘露糖苷酶可以组织成两个多基因 原生性不同的家族，称为I类和2类甘露糖苷酶 序列和生化特征。此应用程序的重点是 独家报道1类甘露糖苷酶。第I类的三个亚族 从我们的克隆研究中已经出现了甘露糖苷酶。内质网甘露糖苷酶I 亚家族切割Man9GlcNAc2中的单个残基以产生特定的 Man8GlcNAc2异构体。高尔基甘露糖苷酶I亚家族切割Man9-8GlcNAc2 结构设置为Man5GlcNAc2。HTM子家族成员，根据其 “甘露糖苷酶的同源物”似乎没有内在的水解酶。 活性，但可能具有参与糖蛋白降解的凝集素活性。 最近，ER Man I和HTM蛋白都被认为是 靶向未折叠的糖蛋白以在内质网中处置。这 应用程序将检查1类甘露糖苷酶用于 识别待处理的未折叠糖蛋白，目的是了解如何 对这一过程的干预可以增强人类蛋白质的稳定性 遗传性疾病的特征是未折叠的细胞迅速退化 糖蛋白。这项提案涉及四个具体目标。第一个目标 将表征水解酶作用的新机制和特异性 内质网和高尔基体甘露糖苷酶联合诱变，动力学 分析、约束研究和结构分析。第二个目标将评估 HtM蛋白的特定结构域和必需残基的作用 酵母模型系统。第三个目标将确定底物/配体 参与内质网的新型HTM蛋白的特异性和结合伙伴 糖蛋白降解。第四个目标将决定改变的效果。 ER Man I和HTM蛋白的表达水平对降解率的影响 哺乳动物细胞中未折叠蛋白质的模型。

DESCRIPTION (Applicant's abstract): The long-term goals of this proposal are to examine the structure, function, and mechanism of action for a set of enzymes required for the biosynthesis and catabolism of mammalian N-linked glycans. We have focused on the cx-mannosidases in the N-glycan pathway since they act as committed steps in the synthesis of complex oligosaccharides by determining the extent of mannose trimming, and in some cases appear to control the rate of degradation of unfolded glycoproteins in the endoplasmic reticUlum (ER). The processing and catabolic a-mannosidases can be organized into two multigene families, termed Class I and Class 2 mannosidases, that differ in primary sequence and biochemical characteristics. This application is focused exclusively on the Class 1 mannosidases. Three subfamilies of Class I mannosidases have emerged from our cloning studies. The ER mannosidase I subfamily cleaves a single residue from Man9GlcNAc2 to generate a specific Man8GlcNAc2 isomer. The Golgi mannosidase I subfamily cleaves Man9-8GlcNAc2 structures to Man5GlcNAc2. The HTM subfamily members, designated based on their "homology to mannosidases," do not appear to have an intrinsic hydrolase activity, but may have a lectin activity involved in glycoprotein degradation. Recently, both ER Man I and HTM proteins have been implicated as key players in the targeting of unfolded glycoproteins for disposal in the ER. This application will examine the mechanism used by the Class 1 mannosidases to recognize unfolded glycoproteins for disposal with a goal of understanding how intervention in the process can lead to enhanced protein stability in human genetic diseases characterized by the rapid degradation of unfolded glycoproteins. Four specific aims are addressed in this proposal. The first aim will characterize the novel mechanism of hydrolase action and specificity of the ER and Golgi mannosidases by a combination of mutagenesis, kinetic analysis, binding studies, and structural analysis. The second aim will assess the roles of specific domains and essential residues of HTM proteins using a yeast model system. The third aim will determine the substrate/ligand specificity and binding partners of the novel HTM proteins involved in ER glycoprotein degradation. The fourth aim will determine the effects of altering the expression levels of ER Man I and HTM proteins on the rates of degradation of model unfolded proteins in mammalian cells.