ALPHA-MANNOSIDASES IN GLYCOPROTEIN PROCESSING

糖蛋白加工中的α-甘露糖苷酶

• 批准号: 2185001
• 负责人: KELLEY W. MOREMEN
• 金额: $ 19.33万
• 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 1997-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2185001
• 关键词: Golgi apparatus; carbohydrate metabolism; enzyme activity; enzyme mechanism; enzyme structure; gene expression; genetic disorder; glycoproteins; glycosyltransferase; inborn carbohydrate metabolism disorder; laboratory rabbit; laboratory rat; mannosidase; molecular cloning; molecular pathology; nucleic acid sequence; oligosaccharides; polymerase chain reaction; protein metabolism

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.

本提案的长期目标是审查 影响的调节，结构和功能的常驻酶 高尔基复合体通过检查N-聚糖的α-甘露聚糖酶 加工路径 这些酶决定了 新合成的糖蛋白上的寡糖， 从高甘露糖型到复合型结构的加工程度。 关于这一过程中酶的结构或调节知之甚少。 通路 为了解决这些问题，编码几种 已经分离出加工α-甘露糖苷酶。 这些阿尔法- 甘露聚糖酶代表哺乳动物加工水解酶中的第一种 并为这项研究提供了几个独特的实验系统 他们的结构和规则。 本建议涉及两个具体目标。 第一特定 目的是完成两个的cDNA和基因组克隆的分离， 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 将作为以下研究的基本信息基础： 调节和蛋白质结构。 克隆的主要焦点和 对人II的测序研究将是人类的特征， HEMPAS病的分子基础，HEMPAS病是一种人类遗传疾病， 人类二号的缺陷 这些研究将利用快速PCR HEMPAS cDNA的扩增和测序， 突变型和对照Man II构建体在COS中异源表达 细胞来解决每个突变的功能后果。 类似 将进行研究以确定Man I的分子基础 蓖麻毒素抗性细胞系克隆6中的缺陷。 第二 具体目的是检查阿尔法的调节和结构， 甘露聚糖酶。 组织特异性和发育性表达， 将在转录物、多肽和酶处检查酶。 水平，并将与其他高尔基体的表达进行比较 糖基转移酶，以检查酶的协调调节 在通道中。 结构域研究和酶促比较 相关的α-甘露糖苷酶活动也将在 在哺乳动物细胞中的过表达构建体。 成功完成拟议的研究将提供基本的 了解N-聚糖的结构、调节和功能 在动物细胞中加工。 分子基础的确定 人类II的人类遗传缺陷，HEMPAS，也将是巨大的， 在确定酶功能所必需的序列方面的价值， 它们在寡糖结构改变中的后果。 最后，详细研究了阿尔法的表达， 在整个糖基化途径的背景下， 应该产生一个协调监管的连贯画面， 动物系统中的路径。