MANNOSIDASES IN GLYCOPROTEIN BIOSYNTHESIS AND CATABOLISM

糖蛋白生物合成和分解代谢中的甘露糖苷酶

• 批准号: 2910105
• 负责人: KELLEY W. MOREMEN
• 金额: $ 21.8万
• 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 2001-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2910105
• 关键词: biotransformation; complementary DNA; disease /disorder model; enzyme activity; genetically modified animals; glycoprotein biosynthesis; glycoproteins; high performance liquid chromatography; laboratory mouse; laboratory rabbit; mannosidase; mannosidosis; model design /development; oligonucleotides; tissue /cell culture

The long term goals of this proposal are to examine the factors which influence the regulation, structure, and function of enzymes involved in the biosynthesis and catabolism of mammalian N-linked glycans. These enzymes determine the fate of the oligosaccharides on newly synthesized glycoproteins by determining the extent of processing from high mannose- type to complex-type structures. Little is known about the structure or regulation of the enzymes in this pathway. To address these problems the cDNAs encoding several of the processing and catabolic alpha-mannosidases have been isolated and have been organized into two multigene classes. This collection of alpha-mannosidase cDNAs offer several unique experimental systems for the study of the structure, function, and regulation of these enzymes as models for the regulation of glycoprotein biosynthesis and catabolism. Four specific aims are addressed in this proposal. The first specific aim is to complete the isolation of cDNA and genomic clones encoding the mammalian alpha-mannosidases. The second specific aim is to examine the structure, regulation, and function of the mammalian alpha-mannosidase family members. Heterologous expression, purification, determination of substrate specificity, and determination of in vivo tissue and cell- specific expression patterns will be compared among the enzymes in order to define the respective functions of each of members of the various enzyme classes. Enzymes from mammalian and non-mammalian sources will also be isolated for structure, function, and expression pattern studies. An additional focus of the cloning and sequencing studies on the mannosidases will be the characterization of the molecular basis of human genetic diseases characterized by a deficiency in either Golgi Man II or the lysosomal alpha-mannosidase. This is the third specific aim of the grant. The fourth specific aim will be the generation and characterization of murine models for deficiencies in the various processing and catabolic alpha-mannosidases by murine gene ablation techniques. These mouse knockout models will provide a direct demonstration of the in vivo roles of each of the multi-gene family members and generate an animal model for the eventual testing of therapeutic strategies for the lysosomal storage disease, alpha- mannosidosis.

该提案的长期目标是研究影响因素 影响参与酶的调节、结构和功能 哺乳动物 N-连接聚糖的生物合成和分解代谢。这些 酶决定新合成的寡糖的命运 通过确定高甘露糖的加工程度来检测糖蛋白 类型到复杂类型结构。人们对其结构知之甚少 对该途径中酶的调节。为了解决这些问题 编码多种加工和分解代谢 α-甘露糖苷酶的 cDNA 已被分离并被分为两个多基因类。这 α-甘露糖苷酶 cDNA 的收集提供了几种独特的实验 研究这些结构、功能和调节的系统 酶作为糖蛋白生物合成调节的模型 分解代谢。 该提案提出了四个具体目标。第一个具体目标 的目的是完成编码 cDNA 和基因组克隆的分离 哺乳动物α-甘露糖苷酶。第二个具体目标是检查 哺乳动物α-甘露糖苷酶的结构、调节和功能 家庭成员。异源表达、纯化、测定 底物特异性以及体内组织和细胞的测定 将按顺序比较酶之间的特定表达模式 定义各种酶的每个成员各自的功能 类。来自哺乳动物和非哺乳动物来源的酶也将被 分离用于结构、功能和表达模式研究。一个 甘露糖苷酶克隆和测序研究的额外重点 将是人类遗传的分子基础的表征 以高尔基体 II 或高尔基体缺陷为特征的疾病 溶酶体α-甘露糖苷酶。这是该赠款的第三个具体目标。 第四个具体目标是生成和表征 各种加工和分解代谢缺陷的小鼠模型 通过鼠基因消融技术的α-甘露糖苷酶。这些老鼠敲除 模型将直接展示每个的体内作用 多基因家族成员并为最终的动物模型 溶酶体贮积病治疗策略的测试，α- 甘露糖苷沉积症。