ALPHA-MANNOSIDASES IN MAMMALIAN GLYCOPROTEIN PROCESSING

这项建议的长期目标是审查以下因素 影响驻留酶的调节、结构和功能 通过检测N-糖链的α-甘露糖苷酶来研究高尔基复合体 加工途径。这些酶决定了 低聚糖对新合成的糖蛋白的影响 加工程度由高甘露糖型结构向复合型结构转变。 人们对这种酶的结构或调节知之甚少。 路径。为了解决这些问题，编码了几个 已分离出加工α-甘露糖苷酶。这些阿尔法- 甘露糖苷酶是第一种哺乳动物加工水解酶 将被克隆，并为研究提供几个独特的实验系统 他们的结构和规则。 这项提案涉及两个具体目标。第一个具体 目的是完成两个亚种的cdna和基因组克隆 加工α-甘露糖苷酶、α-甘露糖苷酶I(Man I)和 α-甘露糖苷酶II(Man II)。这些克隆及其衍生序列 将作为研究的基本信息基础 调节和蛋白质结构。克隆的主要焦点是 对人类II的测序研究将是对 HEMPAS病的分子基础，这是一种人类遗传性疾病 由于MAN II的缺陷。这些研究将利用快速PCR HEMPAS基因cDNAs的扩增和测序 突变和对照Man II结构在COS中的异源表达 细胞来解决每一种突变的功能后果。类似 将进行研究以确定Man I的分子基础 抗蓖麻毒素细胞系克隆6的缺失。 特定目的研究阿尔法的规则和结构- 甘露糖苷酶。组织特异性和发育性表达的研究 酶将在转录本、多肽和酶上进行检测 并将其与其他高尔基人的表达进行比较 糖基转移酶检测酶的协调调节 在这条路上。结构域结构的研究和酶的比较 相关的α-甘露糖苷酶活动也将在 哺乳动物细胞中的过表达结构。 成功完成拟议的研究将为 关于N-糖链的结构、调节和功能的知识 在动物细胞中进行加工。分子基础的测定 人类第二代的遗传缺陷，HEMPAS，也将是巨大的 在确定酶功能所必需的序列和 它们在寡糖结构改变中的后果。 最后，对阿尔法的表达进行详细的检查- 糖基化途径中的甘露糖苷酶 应该会产生一幅协调监管的连贯图景 动物系统中的通路。

The long term goals of this proposal are to examine the factors which influence the regulation, structure, and function of resident enzymes of the Golgi complex by examining the alpha-mannosidases of the N-glycan processing pathway. 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 alpha-mannosidases have been isolated. These alpha- mannosidases represent the first of the mammalian processing hydrolases to be cloned and offer several unique experimental systems for the study of their structure and regulation. Two specific aims are addressed in this proposal. The first specific aim is to complete the isolation of cDNA and genomic clones for two of the processing alpha-mannosidases, alpha-mannosidase I (Man I) and alpha-mannosidase II (Man II). These clones and their derived sequences will act as the fundamental base of information for the studies on regulation and protein structure. The primary focus of the cloning and sequencing studies on Man II will be the characterization of the molecular basis of HEMPAS disease, a human genetic disease characterized by a deficiency in Man II. These studies will utilize the rapid PCR amplification and sequencing of HEMPAS cDNAs in combination with the heterologous expression of mutant and control Man II constructs in COS cells to address the functional consequences of each mutation. Similar studies will be performed to determine the molecular basis of Man I deficiency in the ricin-resistant cell line, clone 6. The second specific aim examines the regulation and structure of the alpha- mannosidases. The tissue-specific and developmental expression of the enzymes will be examined at the transcript, polypeptide, and enzyme level and will be compared with the expression of other Golgi glycosyltransferases to examine the coordinate regulation of the enzymes in the pathway. Domain structure studies and an enzymatic comparison of related alpha-mannosidase activities will also be carried out in overexpression constructs in mammalian cells. Successful completion of the proposed studies will provide fundamental knowledge about the structure, regulation, and function of N-glycan processing in animal cells. The determination of the molecular basis of the human genetic deficiency in Man II, HEMPAS, will also be of great value in determining the sequences essential for enzyme function and their consequences in the alteration of oligosaccharide structures. Finally, a detailed examination of the expression of the alpha- mannosidases in the context of the glycosylation pathway as a whole should yield a cohesive picture of the coordinated regulation of the pathway in animal systems.