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

1.制备识别含有聚-N-乙酰乳糖胺糖链的糖蛋白的单克隆抗体。(1)在PC 12细胞表面存在几种含有聚N-乙酰乳糖胺糖链的糖蛋白。为了研究每种糖蛋白的表达，进行单克隆抗体的制备。(2)每种糖蛋白都是通过生物化学方法（例如凝胶过滤）从PC 12细胞的大规模培养物中纯化的。(3)通过将分离的糖蛋白与弗氏完全佐剂一起腹腔内注射免疫Balb/c小鼠。(4)尽管用于免疫的糖蛋白的抗原性低，但在小鼠重复免疫后观察到血升的显著增加。(5)将免疫小鼠的脾细胞与骨髓瘤细胞融合制备杂交瘤。产生针对每种糖蛋白的特异性单克隆抗体的杂交瘤的筛选正在进行中。聚N-乙酰乳糖胺生物合成起始反应的分析。(1)通过原位杂交分析了N-乙酰氨基半乳糖转移酶在大鼠脑中的表达，其催化聚-N-乙酰氨基乳糖糖链生物合成的初始反应。(2)N-乙酰氨基半乳糖转移酶在大鼠脑内特异性表达。(3)它们在大脑的限制区域，如大脑皮层的中间层和海马中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-乙酰氨基半乳糖转移酶和具有聚-N-乙酰氨基乳糖链的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.所获得的结果在糖缀合物国际研讨会和日本生物化学学会年会上发表。

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.