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 细胞骨髓组、NS-1 和牛红细胞中筛选新的 GPI 锚定蛋白。在小鼠骨髓瘤NS-1细胞的血浆膜外表面检测到至少8种GPI锚定蛋白（170/150kD、96kD、80/76kD、66kD、63kD、59kD、44kD和27kD）。从牛红细胞膜中，除乙酰胆碱酯酶外，我们还检测到了几种GPI锚定蛋白，55kD、42kD、40kD和30kD。这些蛋白质通过气相色谱（GC）-质谱检测其结构中的肌醇部分来证实。我们应用电喷雾电离质谱/质谱（ESI-MS/MS）和基质辅助的改进的灵敏度和软电离特性激光解吸/电离 (MALDI) - 惊吓时间 (TOF) 质谱 (MS) 来分析 GPI 锚定结构。在正模式分析中检测到 GPI 锚定肽及其特征碎片离子的异质峰。 ESI-MS/MS 和 MALDI-TOF-MS 是分析小于 10pmol 的少量 GPI 锚定结构的非常有效和灵敏的方法。手性肌醇是一种除肌醇以外的肌醇异构体，在多种 GPI 的水解产物中被鉴定出-锚定蛋白。几种不同的 GPI 锚定蛋白中的手性肌醇含量随这些 GPI 锚定蛋白的水解条件而变化。在6N HCl溶液中水解时，发生20-60%肌醇的异构化。然而，在HCl气流代替溶液的水解条件下，异构化率非常低(小于0.1％)。然而，在磷脂酰肌醇或肌醇-1-磷酸的水解中，在6N HCl溶液中没有检测到显着量的手性肌醇。这些事实表明，在 GPI 锚的水解过程中发生了异构化，其中肌醇在 6-OH 处被葡萄糖胺取代，在 1-OH 处被磷酸盐取代。