Studies on the glycoproteins expressing neural specific carbohydrate

表达神经特异性碳水化合物的糖蛋白的研究

基本信息

批准号：
03680147
负责人：
KOZUTSUMI Yasunori
金额：
$ 1.09万
依托单位：
KYOTO 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：
1991
资助国家：
日本
起止时间：
1991 至 1992
项目状态：
已结题

项目摘要

With progress in immunological techniques and histochemical methods, neuron-specific carbohydrate moieties have been discovered. These glycoconjugates carrying the neural specific carbohydrates are mainly observed on the cell surface and expressed in region- or stage-specific manner during the development of the nervous system. Therefore, these neuron- specific carbohydrate moieties may play important roles in recognition and/or migration of neural cells. Antiserum raised against horseradish peroxidase(HRP)specifically labels the nervous system of grasshopper and other insects and has been shown to recognize carbohydrate moiety that is expressed on the surface of neurons.In order to determine the structure of the carbohydrate epitope, we isolated the glycoprotein which express the neuron- specific carbohydrate antigen, from grasshopper nervous system using lectin chromatography. The glycoprotein showed apparent molecular weight of 35 kDa on SDS-PAGE under reducing condition. Removal of alpha-linked mannose residues by digestion with alpha-mannosidase completely destroyed the binding activity of the 35 kDa glycoprotein to the anti-HRP antibody. The carbohydrate composition of the 35 kDa glycoprotein was analyzed using a HPLC system with a pulsed amperometry detector. The 35 kDa glycoprotein was composed of Fuc, Man, Gal, Glc, GalNH2, and GlcNH2 with a molecular ratio of 1.0,1.4, 0.4, 1.5, 0.4, and 1.5, respectively.

随着免疫学技术和组织化学方法的进步，已经发现了神经元特异性碳水化合物部分。这些携带神经特异性碳水化合物的糖缀合物主要在细胞表面观察到，并在神经系统发育过程中以区域或阶段特异性方式表达。因此，这些神经元特异性碳水化合物部分可能在神经细胞的识别和/或迁移中发挥重要作用。针对辣根过氧化物酶（HRP）的抗血清特异性标记蚱蜢和其他昆虫的神经系统，并已被证明可以识别神经元表面表达的碳水化合物部分。为了确定碳水化合物表位的结构，我们分离了糖蛋白使用凝集素色谱法表达来自蚱蜢神经系统的神经元特异性碳水化合物抗原。还原条件下的SDS-PAGE显示糖蛋白的表观分子量为35 kDa。通过用 α-甘露糖苷酶消化去除 α-连接的甘露糖残基完全破坏了 35 kDa 糖蛋白与抗 HRP 抗体的结合活性。使用带有脉冲安培检测器的 HPLC 系统分析 35 kDa 糖蛋白的碳水化合物组成。 35 kDa糖蛋白由Fuc、Man、Gal、Glc、GalNH2和GlcNH2组成，分子比分别为1.0、1.4、0.4、1.5、0.4和1.5。