Mannosidases in Glycoprotein Biosynthesis & Catabolism

描述（申请人摘要）：本提案的长期目标是 检查一组酶的结构、功能和作用机制 哺乳动物N-连接聚糖的生物合成和催化所需。我们 已经集中于N-聚糖途径中的Cx-甘露聚糖酶，因为它们作为 通过测定复合寡糖合成中的关键步骤， 甘露糖修剪的程度，并且在某些情况下似乎控制了 内质网中未折叠糖蛋白的降解。的 加工和分解代谢α-甘露糖苷酶可被组织成两个多基因 被称为I类和2类甘露聚糖酶的家族，它们在主要功能上不同， 序列和生化特性。该应用程序专注于 专门针对1类甘露聚糖酶。I类的三个亚科 甘露聚糖酶已经从我们的克隆研究中出现。ER甘露糖苷酶I 亚家族从Man 9 GlcNAc 2切割单个残基以产生特异性的 Man 8 GlcNAc 2异构体。高尔基甘露糖苷酶I亚家族切割Man 9 - 8 GlcNAc 2 Man 5GlcNAc 2的结构。HTM亚家族成员，根据其 “与甘露聚糖酶同源”，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蛋白两者都被认为是在细胞凋亡中的关键参与者。 靶向未折叠的糖蛋白以在ER中处置。这 应用程序将检查1类甘露聚糖酶使用的机制， 识别未折叠的糖蛋白进行处理，目的是了解如何 在该过程中的干预可以导致增强的蛋白质稳定性， 遗传性疾病的特点是迅速降解的展开 糖蛋白本建议涉及四个具体目标。第一个目标 将表征水解酶作用的新机制和特异性， 通过诱变、动力学 分析、结合研究和结构分析。第二个目标是评估 HTM蛋白的特异性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ER的新型HTM蛋白的特异性和结合伴侣 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速率的影响 在哺乳动物细胞中的模型未折叠蛋白质。

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.