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Analvsis of localization of a glycosyltransferase and transfort of glycolipids by gene manipulation

Research Project

Project/Area Number 06670146
Research Category

Grant-in-Aid for General Scientific Research (C)

Allocation TypeSingle-year Grants
Research Field General medical chemistry
Research InstitutionNagasaki University

Principal Investigator

FURUKAWA Keiko  Nagasaki University, School of Medicine, Assistent Professor, 医学部, 助手 (50260732)

Co-Investigator(Kenkyū-buntansha) FURUKAWA Koichi  Nagasaki University, School of Medicine, Assistent Professor, 医学部, 助教授 (80211530)
Project Period (FY) 1994 – 1995
Project Status Completed (Fiscal Year 1995)
Budget Amount *help
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1995: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1994: ¥1,200,000 (Direct Cost: ¥1,200,000)
Keywordsglycosyltransferase / N-glycosylation / localization / gene modification / ganglioside / Golgi局在 / 基質特異性 / Golgi膜
Research Abstract

The amino acid sequence deduced from the cloned human cDNA of beta-1,4-N-acetyl-galactosaminyltransferase (GalNAc-T ; EC2.4.1.92) gene predicted three potential sites for N-linked glycosylation. Although many glycosyltransferases isolated contain from 2 to 6 N-glycosylation sites, their significance has not been adequately demonstrated. To clarify the roles of N-glycosylation in GalNAc-T function, we generated a series of mutant cDNAs, in which some or all of the glycosylation recognition sites were eliminated by polymerase chain reaction( PCR)-mediated site directed mutagenesis. Using transcription/translation in vitro, we confirmed the all potential N-glycosylation sites could be used. Although cell lines transfected with mutant cDNAs showed equivalent levels of GalNAcbeta1*4 (NeuAcalpha2*3) Galbeta1*4GlcCer (G_<M2>) to that of the wild-type, the extracts from mutant cDNA transfectants demonstrated lower enzyme activity than in the wild-type. The decrease in enzyme activity was more evident as the number of deglycosylated sites increased, with about 90% decrease in a totally deglycosylated mutant. The enzyme kinetics analysis revealed no significant change of K_m among wild-type and mutant cDNA products. The intracellular localization of GalNAc-T expressed in transfectants with wild-type or mutant cDNAs also showed a similar perinuclear pattern (Golgi pattern). These results suggest that N-linked carbohydrates on GalNAc-T are required for regulating the stability of the enzyme structure.

Report

(3 results)
  • 1995 Annual Research Report   Final Research Report Summary
  • 1994 Annual Research Report
  • Research Products

    (16 results)

All Other

All Publications (16 results)

  • [Publications] Lutz,M.S.,et al.: "Cloned β1,4 N-acetylgalactosaminyltansferase synthesizes GA2 as well as gangliosides GM2 and GD2.GM3 synthesis haspriority over GA2 synthesis for utilization of lactosylceramide substrate in vivo." The Journal of Biological Chemistry. 269. 29227-29231 (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Yamashiro,S.,et al.: "Substrate specificity of β1,4-N-acetylgactosaminyltransferase in vitro and in cDNA-transfected cells.GM2/DM2 syntase efficiently generates asialo-GM2 in certain cells." J.Biiol.Chem.270. 6149-6155 (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Hamagichi,M.,et al.: "The effects the site-directed removal of N-glycosylation sites from GM2/DM2 synthase on its function." Biochemical J.312. 273-280 (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] RUan,S.,et al.: "Analysis of melanoma cells stably transfected with β1,4GalNAc transferase (GM2/DM2 synthase) cDNA.Relative glycosyltransferase levels play a dominant rolein determining ganglioside expression." Arch.Biochem.Biophys.323. 11-18 (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Lutz, M.S., et al.: "Cloned beta1,4 N-acetylgalactosaminyltransferase synthesizes GA2 as well as gangliosides GM2 and GD2. GM3 synthesis has priority over GA2 synthesis for utilization of lactosylceramide substrate in vivo." The Journal of Biological Chemistry. 269. 29227-29231 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Yamashiro, S., et al.: "Substrate specificity of beta1,4-N-acetylgalactosaminyltransferase in vitro and in cDNA-transfected cells. GM2/GD2 synthase efficiently generates asialo-GM2 in certain cells." The Journal of Biological Chemistry. 270. 6149-6155 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Haraguchi, M., et al.: "The effectrs of the site-directed removal of N-glycosylation sites from GM2/GD2 synthase on its function." Biochemical J.312. 273-280 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] RUan, S., et al.: "Analysis of melanoma cells stably transfected with beta1,4GalNAc transferase (GM2/GD2 synthase) cDNA.Relative glycosyltransferase levels play a dominant role in determining ganglioside expression." Arch.Biochem.Biophys.323. 11-18 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Takamiya,K.: "T cell receptor-mediated stimulation of mouse thymocytes induces up-regulation of the GM2/GD2 synthase gene." FEBS Lett.358. 79-83 (1995)

    • Related Report
      1995 Annual Research Report
  • [Publications] Yamashiro,S.: "Substrate specificity of b 1,4-N-acetyl-galactosaminyltransferase in vitro and in cDNA-transfected cells.GM2/GD2 synthase efficiently generates asialo-GM2 in certain cells." J.Biol.Chem.270. 6149-6155 (1995)

    • Related Report
      1995 Annual Research Report
  • [Publications] Haraguchi,M.: "The effects of the site-directed removal fo N-glycosylation sites from GM2/GD2 synthase on its function." Biochemical J.312. 273-280 (1995)

    • Related Report
      1995 Annual Research Report
  • [Publications] Yamamoto,A.: "Diverse expression of mRNA of b1,4-N-acetylgalactosaminyltransferase gene in the adult mouse brain." J.Neurochem.65. 2417-2424 (1995)

    • Related Report
      1995 Annual Research Report
  • [Publications] Yamashiro,S.: "Expression of a2,8-sialyltransferase (GD3 synthase) gene in human cancer cell lines : High level expression in melanomas and up-regulation in activated T lymphocytes." Glycoconj.J.12. 894-900 (1995)

    • Related Report
      1995 Annual Research Report
  • [Publications] Yamamoto,A.: "Heterogeneity in the expression pattern of two ganglioside synthase genes during mouse brain development." J.Neurochem.66. 26-34 (1996)

    • Related Report
      1995 Annual Research Report
  • [Publications] Kogo Takamiya: "T cell receptor-mediated stimulatoin of mouse thymocytes induces up-regulation of the GM2/GD2 synthase gene" FEBS Letters. 358. 79-83 (1995)

    • Related Report
      1994 Annual Research Report
  • [Publications] Shuji Yamashiro: "Substrate Specificity of B1,4 N-acetylgalactosaminyltransferase in vitro and in cDNA-transfected cells.-GM2/GD2 synthase efficiently generates asialo-GM2 in Cectain cells-." J.Bio.Chem.(1995). (in press.).

    • Related Report
      1994 Annual Research Report

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Published: 1994-04-01   Modified: 2016-04-21  

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