Structural Features, Biosynthesis and Processing of Glycolipid-anchored Proteins

糖脂锚定蛋白的结构特征、生物合成和加工

基本信息

批准号：
63570124
负责人：
IKEHARA Yukio
金额：
$ 0.38万
依托单位：
Fukuoka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1988
资助国家：
日本
起止时间：
1988 至 1989
项目状态：
已结题

项目摘要

We have investigated structural-features, biosynthesis, and processing of three membrane- bound proteins including 5'-nucleotidase (5'-NT), Alkaline phosphatase (ALP), and carcinoembryonic antigen (CEA), demonstrating that those proteins are anchored to the plasma membrane via glycosyl- phosphatidylinositol (GPI).1. Common features in primary Structures deduced from their cDNAs we isolated and sequenced cDNAs for 5'-NT and ALP from both the rat liver and human placenta. The two proteins are found to have a common feature in their primary structures deduced from the cDNAs; they contain hydrophobic domains with about 20 amino acids at both the NH2 and COOH termini, which are also observed in the primary structure of CEA.2. Chemical analyses of purified proteins 1) Identification of the NH_2-terminal sequence: 5'-NT and ALP were purified and subjected to determination of their NH_2-terminal sequences. A comparison of the NH_2-terminal sequences between the cDNA-predicted precursors and th … More e purified proteins revealed that the NH_2-terminal hydrophobic domain in each precursor is a signal peptide which is cotranslationally cleaved. 2) Purification and characterization of the COOH-terminal domain: The purified proteins were cleaved into peptide fragments by treatment with protease or BRCN. The hydrophobic COOH- terminal fragment of each protein was purified by sequential chromatographies. Chemical analyses demonstrated that each fragment contains oligopeptide and GPI components such as ethanolamine, mannose, glucosamine, inositol and fatty acids. Each peptide sequence was determined and identified at positions prior to the COOH-terminal hydrophobic domain present in their precursors. The results indicate that the predicted COOH-terminal hydrophobic domain is proteolytically removed and replaced by GPI, which in turn functions as the membrane anchor.3. Labeling experiments in cultured cells The presence of the GPI anchor in these proteins was confirmed by metabolic labeling experiments. Established cell lines of JEG-3 (for ALP) and QGP-1 (CEA) and isolated hepatocytes (5'-NT) were incubated with ^3H-labeled compounds such as ethanolamine, inositol and fatty acids. It was found that all the labeled compounds are incorporated into these proteins. Less

我们已经研究了三种膜结合蛋白的结构功能，生物合成以及包括5'-核苷酸酶（5'-NT），碱性磷酸酶（ALP）和癌细胞蛋白酶抗原（CEA）（CEA）的处理，这些蛋白质通过甘露蛋白固醇固定在氯麦克素（lycyyl-磷酸酯）。从其cDNA推导的一级结构中，我们从大鼠肝脏和人体胎盘中分离出5'-NT和ALP的测序cDNA。发现这两种蛋白质在从cDNA中推导的主要结构中具有共同特征。它们包含在NH2和COOH末端的疏水结构域，在CEA.2的主要结构中也观察到了约20个氨基酸。纯化蛋白质的化学分析1）纯化NH_2-末端序列的鉴定：5'-NT和ALP被纯化并经过确定其NH_2末端序列。比较cDNA预测的前体和Th…更多纯化的蛋白质之间的NH_2末端序列表明，每个前体中的NH_2-末端疏水结构域是一种信号肽，是一种裂解的信号肽。 2）COOH末端结构域的纯化和表征：通过用蛋白酶或BRCN处理将纯化的蛋白质切成肽片段。每种蛋白质的疏水性终末片段通过顺序色谱纯化。化学分析表明，每个片段都包含寡肽和GPI成分，例如乙醇胺，甘露糖，葡萄糖胺，肌醇和脂肪酸。确定每个肽序列并在其前体中存在的COOH末端疏水结构域之前的位置鉴定。结果表明，预测的COOH末端疏水结构域被蛋白水解去除并被GPI取代，而GPI又作为膜锚。3。通过代谢标记实验证实了在培养细胞中的标记实验在这些蛋白质中的存在。将JEG-3（ALP）和QGP-1（CEA）和分离的肝细胞（5'-NT）的细胞系与 ^3H标记的化合物（如乙醇胺，肌醇和脂肪酸）一起孵育。发现所有标记的化合物都掺入了这些蛋白质中。较少的