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Analysis of poly-N-acetyllactosamilie-bearing glycoproteins involved in neural cell differentiation

参与神经细胞分化的聚-N-乙酰乳糖酰胺糖蛋白的分析

基本信息

批准号：
12672133
负责人：
KUROSAKA Akira
金额：
$ 1.34万
依托单位：
Kyoto Sangyou University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

项目摘要

1. Preparation of monoclonal antibodies recognizing glycoproteins containing poly-N-acetyllactosamine carbohydrate chains.(1) There are several glycoproteins containing poly-N-acetyllactosamine carbohydrate chains on the PC12 cell surface. In order to investigate expression of each glycoprotein, preparation of monoclonal antibodies was performed.(2) Each glycoprotein was purified from a large-scale culture of PC12 cells by means of biochemical methods, such as gel filtration.(3) Balb/c mice were immunized by injecting isolated glycoproteins intraperitoneally together with Freund's complete adjuvant.(4) Although the antigenicity of the glycoproteins used for the immunization was low, significant increase in the blood liter was observed after repeated immunization of the mice.(5) Hybridomas were prepared by fusing spleen cells from the immunized mice with myeloma cells. The screening of the hybridomas producing monoclonal antibodies specific for each glycoproteins is under progress.2. Analysis of initial reaction of poly-N-acetyllactosamine biosynthesis.(1) Expression of N-acetylgalactosaminyltransferases in the rat brain was analyzed by in situ hybridization, which catalyze the initial reaction of the biosynthesis of poly-N-acetyllatosamine carbohydrate chains.(2) N-Acetylgalactosaminyltransferases were expressed specifically in the rat brain.(3) They also exhibited characteristic expression in the restricted area of the brain, such as the intermediate layer of cerebral cortex, and hippocampus.(4) With the production of specific monoclonal antibodies, it would be possible to study biosynthetic analysis of each glycoprotein by comparing the expression pattern in the brain between the N-acetygalactosaminyltransferases and the glycoproteins with poly-N-acetyllactosamine chains.3. The results obtained were presented in the International Symposium on Glycoconjugates, and the annual meeting of Japanese Biochemical Society.

1. 识别含有聚n -乙酰乳胺碳水化合物链糖蛋白单克隆抗体的制备。(1) PC12细胞表面存在几种含有聚n -乙酰乳胺碳水化合物链的糖蛋白。为了研究各糖蛋白的表达，制备了单克隆抗体。(2)采用凝胶过滤等生化方法从PC12细胞大规模培养中纯化各糖蛋白。(3)通过腹腔注射分离的糖蛋白和Freund's完全佐剂对Balb/c小鼠进行免疫。(4)虽然用于免疫的糖蛋白抗原性较低，但经小鼠多次免疫后血升明显升高。(5)免疫小鼠脾细胞与骨髓瘤细胞融合制备杂交瘤。对每种糖蛋白产生特异性单克隆抗体的杂交瘤的筛选正在进行中。聚n -乙酰乳胺生物合成初始反应分析。(1)原位杂交分析了n -乙酰半乳糖氨基转移酶在大鼠脑中的表达，该酶催化了生物合成多n -乙酰半乳糖胺碳水化合物链的初始反应。(2) n -乙酰半乳糖氨基转移酶在大鼠脑中特异性表达。(3)在大脑皮层中间层、海马等受限区域也表现出特征性表达。(4)随着特异性单克隆抗体的产生，通过比较n -乙酰半乳糖胺基转移酶与聚n -乙酰乳胺链糖蛋白在脑中的表达模式，可以研究每种糖蛋白的生物合成分析。研究结果发表在国际糖缀合物研讨会和日本生化学会年会上。