Mannosidases in Glycoprotein Biosynthesis & Catabolism

糖蛋白生物合成中的甘露糖苷酶

• 批准号: 7126911
• 负责人: KELLEY W. MOREMEN
• 金额: $ 30.9万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-05-01 至 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7126911
• 关键词: endoplasmic reticulum; enzyme activity; enzyme mechanism; enzyme structure; enzyme substrate complex; glycoprotein biosynthesis; hexosan; laboratory rabbit; lectin; mannose; mannosidase; polymerase chain reaction; protein binding; protein engineering; protein folding; protein localization; protein metabolism; recombinant proteins; small interfering RNA; tissue /cell culture

DESCRIPTION (provided by applicant): 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 Class I (family 47) a-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). Three subfamilies of Class I mannosidases have emerged from our prior 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 third subfamily, termed EDEM for ER degradation enhancing a-mannosidase-like protein, 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 EDEM 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 mutant glycoproteins. Four specific aims are addressed in this proposal. The first aim will test hypotheses for the novel hydrolase mechanism and specificity of binding by Class 1 mannosidases by a combination of mutagenesis, kinetic analysis, binding studies, and structural analysis to map the determinants of substrate recognition and catalysis among the hydrolase family members. The second aim will test hypotheses relating to the lectin binding specificity of EDEM subfamily members through in vitro binding studies using wild type and mutant forms of recombinant EDEM or EDEM sub-domains in combination with in vivo functional complementation in model ER-associated degradation (ERAD) systems. The third aim will test hypotheses relating to EDEM function by examining sequences necessary for localization and interactions with components of the ERAD machinery. The fourth aim will test hypotheses for the individual roles and mechanisms of regulation of members of the ERAD targeting machinery by a novel real-time RT-PCR strategy and by siRNA approaches.

描述（由申请方提供）：本提案的长期目标是检查哺乳动物N-连接聚糖生物合成和催化所需的一组酶的结构、功能和作用机制。我们已经集中在N-聚糖途径中的I类（家族47）α-甘露聚糖酶，因为它们通过确定甘露糖修剪的程度而在复杂寡糖的合成中充当关键步骤，并且在某些情况下似乎控制内质网（ER）中未折叠糖蛋白的降解速率。I类甘露聚糖酶的三个亚家族已经从我们先前的克隆研究中出现。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Man 8 GlcNAc 2异构体。高尔基甘露糖苷酶I亚家族将Man 9 - 8 GlcNAc 2结构切割成Man 5GlcNAc 2。第三个亚家族，称为增强α-甘露糖苷酶样蛋白ER降解的EDEM，似乎不具有内在水解酶活性，但可能具有参与糖蛋白降解的凝集素活性。最近，ER Man I和EDEM蛋白都被认为是靶向未折叠糖蛋白以在ER中处置的关键参与者。本申请将研究1类甘露聚糖酶用于识别未折叠糖蛋白以进行处置的机制，目的是了解该过程中的干预如何导致以未折叠突变糖蛋白快速降解为特征的人类遗传疾病中蛋白质稳定性增强。 本建议涉及四个具体目标。第一个目标将测试假设的新型水解酶的机制和特异性结合1类甘露糖苷酶的诱变，动力学分析，结合研究，结构分析相结合，映射底物识别和催化水解酶家族成员之间的决定因素。第二个目的是通过体外结合研究，使用野生型和突变形式的重组EDEM或EDEM亚结构域结合模型ER相关降解（ERAD）系统中的体内功能互补，测试与EDEM亚家族成员的凝集素结合特异性相关的假设。第三个目标将测试有关EDEM功能的假设，通过检查必要的本地化和相互作用的ERAD机制的组成部分的序列。第四个目标是通过一种新的实时RT-PCR策略和siRNA方法来测试ERAD靶向机制成员的个体作用和调节机制的假设。