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

Research Project

Project/Area Number	12672133
Research Category	Grant-in-Aid for Scientific Research (C)
Allocation Type	Single-year Grants
Section	一般
Research Field	Biological pharmacy
Research Institution	Kyoto Sangyou University
Principal Investigator	KUROSAKA Akira (2001) Kyoto Sangyou University, Faculty of Engineering, Professor, 工学部, 教授 (90186536) 福井成行 (2000) 京都産業大学, 工学部, 助教授 (30218888)
Co-Investigator(Kenkyū-buntansha)	黒坂光京都産業大学, 工学部, 助教授 (90186536)
Project Period (FY)	2000 – 2001
Project Status	Completed (Fiscal Year 2001)
Budget Amount *help	¥2,100,000 (Direct Cost: ¥2,100,000) Fiscal Year 2001: ¥500,000 (Direct Cost: ¥500,000) Fiscal Year 2000: ¥1,600,000 (Direct Cost: ¥1,600,000)
Keywords	poly-N-acetvllactosamine / neurons / monoclonal antibodies / glycosyltransferases / in situ hybridization / ポリラクトサミン / PC12 / PC12D / 糖タンパク質 / 神経突起形成
Research Abstract	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.