STRUCTURE AND FUNCTION OF GLYCOCYL PHOSPHATIDYLINOSITOL ANCHORED PROTEIN

糖基磷脂酰肌醇锚定蛋白的结构和功能

基本信息

批准号：
07672372
负责人：
TAGUCHI Ryo
金额：
$ 1.41万
依托单位：
NAGOYA CITY UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

项目摘要

Screening of new GPI-anchored proteins were carried out in mouse B cell myelome, NS-1 and bovine erythrocytes. At least eight GPI-anchored proteins (170/150kD,96kD,80/76kD,66kD,63kD,59kD,44kD and 27kD) were detected on the outer surface of plasma memberane of mouse myeloma NS-1 cells. From bovine erythrocyte menbrane, we also detected several GPI-anchored proteins, 55kD,42kD,40kD and 30kD,other than acetylcholinesterase. These preteins were confirmrd by detecting myo-inositol moeity in their structures by gas chromatography (GC) -mass spectrometry.We applied the improved sensitivity and soft Ionization characteristic of electrospray ionization mass/mass spectrometry (ESI-MS/MS) and matrix-assisted laser dissorption/ionization (MALDI) -time of fright (TOF) mass spectrometry (MS)to the analysis of GPI-anchored structure. Heterogeneous peaks of GPI-anchored peptides and their caracteristic fragment ions were detected in positive mode analysis. ESI-MS/MS and MALDI-TOF-MS is very effective and sensitive methods in analyzing small amount of GPI-anchored structure less than 10pmol.chiro-Inositol, an inositol isomer other than myo-inositol was identified in hydrolytic products from several GPI-anchored proteins. The chiro-inositol contents in several different GPI-anchored proteins varied with hydrolytic conditions of these GPI anchor. Isomerization of 20-60% of myo-inositol occurred on the hydrolysis in 6N HCl solution. In the hydrolytic condition under HCl gas stream in place of solution, however, isomerization was very low (less than 0.1%) . In the hydrolysis of phosphatidylinositol or myo-inositol-1-phosphate, however, significant amount of chiro-inositol was not detected in 6N HCl solution. These facts indicated that isomerization occurred during the hydrolysis of GPI anchor, where myo-inositol is substituted by glucosamine at 6-OH and is substituted by phosphate at 1-OH.

在小鼠B细胞骨髓，NS-1和牛红细胞中筛选新的GPI锚定蛋白。在小鼠NS-1细胞的血浆表面上检测到至少八种GPI锚定蛋白（170/150KD，96KD，80/76KD，66KD，63KD，59KD，44KD和27KD）。从牛红细胞造物构成牛，我们还检测到了几种GPI锚定的蛋白质，55KD，42KD，40KD和30KD，除了乙酰胆碱酯酶以外。通过气相色谱法（GC） - 质量光谱法检测其结构中的肌醇素的经历，以确认这些假蛋白。我们应用了电弹性电离质量/质量光谱法（ESI-MS/MS）的敏感性和软电离特征的提高敏感性和柔和电离特征，以及迫使激光（MALD）群（MALDIINERITITIEN-MALDIINITIES-MALDI-IINTIRE） - 范围（T）（MS）分析GPI锚定结构。在阳性模式分析中检测到GPI锚定肽的异质峰及其特性片段离子。 ESI-MS/MS和MALDI-TOF-MS在分析小于10 pmol的GPI锚定结构的少量GPI-Chiro-Inositol（Chiro-Inositol）中是非常有效且灵敏的方法，这是一种肌醇异构醇，除了从几种GPI锚蛋白的水解产物中鉴定出肌醇以外的肌醇异构醇。几种不同GPI锚定蛋白中的Chiro肌醇含量随这些GPI锚的水解条件而变化。在6N HCl溶液中的水解上发生了20-60％的肌醇的异构化。但是，在HCl气流下的水解条件下，代替溶液，异构化非常低（小于0.1％）。然而，在6N HCl溶液中未检测到磷脂酰肌醇或肌醇1-磷酸磷脂酰肌醇1-磷酸的水解。这些事实表明，异构化发生在GPI锚的水解过程中，在6-OH处将肌氨醇用葡萄糖胺取代，并在1-OH处被磷酸盐取代。