Roberts W L, Santikarn S, Reinhold V N, Rosenberry T L
Department of Pharmacology, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106.
J Biol Chem. 1988 Dec 15;263(35):18776-84.
The glycoinositol phospholipid membrane anchor of human erythrocyte acetylcholinesterase (EC 3.1.1.7) is composed of a glycan linked through a glucosamine residue to an inositol phospholipid that is resistant to the action of phosphatidylinositol-specific phospholipase C. Deamination cleavage of the glucosamine with nitrous acid released the inositol phospholipid which was purified by high performance liquid chromatography. Analysis by fast atom bombardment mass spectrometry with negative ion monitoring and by the complementary technique of collision-induced dissociation revealed molecular and daughter ions that indicated a plasmanylinositol with a palmitoyl group on an inositol hydroxyl. The intact membrane anchor was released from reductively methylated human erythrocyte acetylcholinesterase by proteolysis with papain or Pronase, deacylated by base hydrolysis, and purified by high performance liquid chromatography. Positive and negative ion fast atom bombardment mass spectrometry of the major products isolated by high performance liquid chromatography indicated the following structure for the complete glycoinositol phospholipid anchor. (formula; see text) Methylation of free amino groups by reduction with deuterium instead of hydrogen permitted determination of the number of free amino groups in individual fragment ions as further confirmation of structural assignments. The structure of the glycan portion of the human erythrocyte acetylcholinesterase membrane anchor appears to be similar to that described for Trypanosome brucei variant surface glycoprotein MITat 1.4 (variant 117) (Ferguson, M.A.J., Homans, S.W., Dwek, R.A., and Rademacher, T.W. (1988) Science 239, 753-759) except for the absence of a galactose antenna and the presence of a phosphorylethanolamine on the hexose adjacent to glucosamine.
人红细胞乙酰胆碱酯酶（EC 3.1.1.7）的糖基肌醇磷脂膜锚由一个通过葡糖胺残基连接到肌醇磷脂上的聚糖组成，该肌醇磷脂对磷脂酰肌醇特异性磷脂酶C的作用具有抗性。用亚硝酸对葡糖胺进行脱氨裂解，释放出肌醇磷脂，通过高效液相色谱法对其进行纯化。采用负离子监测的快原子轰击质谱分析以及碰撞诱导解离的互补技术分析表明，分子离子和子离子表明存在一种在肌醇羟基上带有棕榈酰基的质膜肌醇。完整的膜锚可通过木瓜蛋白酶或链霉蛋白酶进行蛋白水解从还原甲基化的人红细胞乙酰胆碱酯酶中释放出来，通过碱水解进行脱酰基处理，并通过高效液相色谱法进行纯化。通过高效液相色谱法分离得到的主要产物的正离子和负离子快原子轰击质谱表明了完整糖基肌醇磷脂锚的以下结构。（分子式；见正文）用氘而非氢进行还原对游离氨基进行甲基化，从而能够确定各个碎片离子中的游离氨基数量，进一步证实结构归属。人红细胞乙酰胆碱酯酶膜锚聚糖部分的结构似乎与布氏锥虫变异表面糖蛋白MITat 1.4（变异体117）所描述的结构相似（弗格森，M.A.J.，霍曼斯，S.W.，德韦克，R.A.，和拉德马赫，T.W.（1988年）《科学》239，753 - 759），只是缺少一个半乳糖天线，并且在与葡糖胺相邻的己糖上存在磷酸乙醇胺。
