MANNOSIDASES IN GLYCOPROTEIN BIOSYNTHESIS AND CATABOLISM

这项建议的长期目标是审查以下因素 影响参与的酶的调节、结构和功能 哺乳动物N-连接多聚糖的生物合成和分解代谢。这些 酶决定了新合成的寡糖的命运 通过测定高甘露糖的加工程度来确定糖蛋白- 从类型到复杂类型的结构。人们对这个结构知之甚少 调节这一途径中的酶。为了解决这些问题， 编码几种加工和分解的α-甘露糖苷酶的cDNA 已经被分离出来，并被组织成两个多基因类。这 收集的α-甘露糖苷酶cDNA提供了几个独特的实验 研究这些物质的结构、功能和调节的系统 酶作为糖蛋白生物合成和调控的模型 分解代谢。 这项提案涉及四个具体目标。第一个具体目标 完成编码该基因的cDNAs和基因组克隆的分离 哺乳动物的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、检测 底物专一性和体内组织和细胞的测定- 特定的表达模式将在酶之间按顺序进行比较 以确定各种酶的每个成员的各自功能 上课。来自哺乳动物和非哺乳动物来源的酶也将 分离出来用于结构、功能和表达模式的研究。一个 甘露糖苷酶克隆和测序研究的新热点 将是人类基因的分子基础的表征 一种疾病，其特征是高尔基人II或 溶酶体α-甘露糖苷酶。这是这笔赠款的第三个具体目标。 第四个具体目标将是产生和表征 小鼠在各种加工和分解代谢方面的缺陷模型 用小鼠基因消融技术研究α-甘露糖苷酶。这些鼠标敲除 模型将提供每一个在体内的角色的直接演示 多基因家族成员并为最终的 溶酶体储存性疾病治疗策略的测试 甘露糖沉着症。

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.