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The studies of glycotechnology on the restriction of carbohydrate chain bearing physiological functions of proteoglycans

Research Project

Project/Area Number 04454153
Research Category

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

Allocation TypeSingle-year Grants
Research Field General medical chemistry
Research InstitutionHIROSAKI UNIVERSITY

Principal Investigator

ENDO Masahiko  Hirosaki University School of Medicine, Department of Biochemistry. Professor, 医学部, 教授 (20004616)

Co-Investigator(Kenkyū-buntansha) IZUMI Jun  Hirosaki University School of Medicine, Department of Biochemistry. Research ass, 医学部, 助手 (20241458)
TAKAGI Keiichi  Hirosaki University School of Medicine, Department of Biochemistry. Assistant Pr, 医学部, 助教授 (70163160)
Project Period (FY) 1992 – 1994
Project Status Completed (Fiscal Year 1994)
Budget Amount *help
¥7,300,000 (Direct Cost: ¥7,300,000)
Fiscal Year 1994: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1993: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 1992: ¥4,900,000 (Direct Cost: ¥4,900,000)
KeywordsProteoglycan / glycotechnology / carbohydrate chain reconstruction / glycosaminoglycan / physiological domain / extracellular matrix / endo-type glycosidase / 糖鎖の切り出し / 生理活性ドメイン糖鎖 / 糖転移反応 / 糖鎖の再構築
Research Abstract

Purpose : As recombinant proteins produced by the techniques of genetic engineering often have incomplete carbohydrate chains, there is a need to control artificially the structure of the carbohydrate chains as well as the process of genetic engineering. Therefore, using proteoglycan of extracellular matrix as a model, we have planned a series of studies aimed at the establishment of glycotechnology techniques of manipulate the glycosaminoglycans bearing physiological domain structures.
Experimental Methods and Results :
Enzymes that act on glycosaminoglycan chains, equivalent to the restriction enzymes and ligases used in genetic engineering, have not been available untill now. Therefore, we have focused on the transglycosylation reaction as a reverse reaction of the hydrolysis that is catalyzed by all glycosidases. Using chondroitin sulfate and hyaluronic acid as glycosaminoglycan chains, the optimum conditions for the transglycosylation reaction of testicular hyaluronidase as a repres … More entative endo-type glycosidase were investigated, and some conditions which efficiently accelerated the reaction were founded.
Subsequently, it was found that some oligosaccharides longer than octasaccharide labeled with a fluorescent compound (2-aminopyridine) at the reducing terminals were available as donors, whereas certain oligosaccharides longer than hexasaccharide, which were produced from 4-methylumbelliferyl-beta-D xyloside as a primer in the incubation medium of cultured human skin fibroblasts, were available as for acceptors.
After incubation of these donors and acceptors with hyaluronidase under the optimum conditions, it was observed that the chain length of glycosaminoglycans increased until at least docosaccharide, and that unnatural glycosaminoglycan chains, previously unknown, were synthesized as planned.
Discussion : The success of this random reconstruction method for glycosaminoglycan chains is considered to be a step foward the establishment of a protocol for glycotechnological procedures. Less

Report

(4 results)
  • 1994 Annual Research Report   Final Research Report Summary
  • 1993 Annual Research Report
  • 1992 Annual Research Report
  • Research Products

    (35 results)

All Other

All Publications (35 results)

  • [Publications] Takagaki,K.: "Characterization of hydrolysis and transglycosylation by testicular hyaluronidase using ion-spray mass spectrometry." Biochemistry. 33. 6503-6507 (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Takagaki,K.: "Analysis of glycosaminoglycans by high-performance liquid chromatography." J.Biochem.Biophys.Methods. 28. 313-320 (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Tanaka,K.: "Hyaluronate depolymerization activity induced by progesterone in cultured fibroblasts derived from human uterine cervix." FEBS Lett.347. 95-98 (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Izumi,J.: "A novel oligosaccharide,xylosylβ1-4xylosy1β1-(4-methyl-umbelliferone),synthesized by cultured human skin fibroblasts." J.Biochem.116. 524-529 (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Higuchi,T.: "A method for determination of galactosyltransferase I activity synthesizing the proteoglycan linkage region." J.Biochem.Biophys.Methods. 29. 135-142 (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Nakamura,T.: "A novel oligosaccharide,GlcA β1-4Xyl β1-(4-methylumbelliferone),synthesized by human cultured skin fibroblasts." Biochem.J.304. 731-736 (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Saitoh,H.: "Enzymic reconstruction of glycosaminoglycan oligosaccharide chaine using the transglycosylation reaction of bovine testicular hyaluronidase." J.Biol.Chem.270. (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Shibata,S.: "HNK-1-reactive novel oligosaccharide,sulfate-0-3GalcA β-1-4Xy β1-(4-methylumbelliferone),synthesized by cultured human skin fibroblasts." J.Biol.Chem.(in press).

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Takagaki, K., Nakamura, T., Izumi, J., Saitoh, H., and Endo, M.: "Characterization of hydrolysis and transglycosylation by testicular hyaluronidase using ion-spray mass spectrometry." Biochemistry. 33. 6503-6507 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Takagaki, K., Takeda, Y., Nakamura, T., Daidouji, K., Narita, H., and Endo, M.: "Analysis of glycosaminoglycans by high-performance liquid chromatography." J.Biochem.Biophys.Methods. 28. 313-320 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Tanaka, K., Nakamura, T., Ikeya, H., Higuchi, T., Tanaka, A., Morikawa, A., Saito, Y., Takagaki, K., and Endo, M.: "Hyaluronate depolymerization activity induced by progesterone in cultured fibroblasts derived from human uterine cervix." FEBS Lett.347. 95-98 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Izumi, J., Takagaki, K., Nakamura, T., Shibata, S., Kojima, K., Kato, I., and Endo, M: "A novel oligosaccharide, xylosyl beta 1-4xylosyl beta 1- (4-methylumbelliferone), synthesized by cultured human skin fibroblasts." J.Biochem.116. 524-529 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Higuchi, T., Tamura, S., Takagaki, K., Nakamura, T., Morikawa, A., Tanaka, K., Tanaka, A., Saito, Y., and Endo, M.: "A method for determination of galactosyltransferase I activity synthesizing the proteoglycan linkage region." J.Biochem.Biophys.Methods. 29. 135-142 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Nakamura, T., Izumi, J., Takagaki, K., Shibata, S., Kojima, K., kato, I., and Endo, M.: "A novel oligosaccharide, GlcA beta 1-4Xyl beta 1- (4-methyl-umbelliferone), synthesized by human cultured skin fibroblasts." Biochem.J.304. 731-736 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Saitoh, H., Takagaki, K., Majima, M., Nakamura, T., Matsuki, A., Kasai, M., Narita, H., and Endo, M.: "Enzymic reconstruction of glycosaminoglycan oligo-saccharide chain usingthe trasnglycosylation reactoin of bovine testicular hyaluronidase." J.Biol.Chem.270. 3741-3747 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Shibata, S., Takagaki, K., Nakamura, T., Izumi, J., Kojima K., Kato, I.and Endo, M.: "HNK-1-reactive novel oligosaccharide, sulfate-0-3GalcA beta-1-4Xyl beta 1- (4-methylumbelliferone), synthesized by cultured human skin fibroblasts." J.Biol.Chem.(in press).

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Takagaki,K.: "Characterization of hydrolysis and transglycosylation by testicular hyaluronidase using ion-spray mass spectrometry." Biochemistry. 33. 6503-6507 (1994)

    • Related Report
      1994 Annual Research Report
  • [Publications] Takagaki,K.: "Analysis og glycosaminoglycans by high-performance liquid chromatography." J.Biochem.Biophys.Methods. 28. 313-320 (1994)

    • Related Report
      1994 Annual Research Report
  • [Publications] Tanaka,K.: "Hyaluronate depolymerization activity induced by progesterone in cultured fibrolasts derived from human uterine cervix." FEBS Lett.347. 95-98 (1994)

    • Related Report
      1994 Annual Research Report
  • [Publications] Izumi,J.: "A novel oligosaccharide,xylosl β1-4xylosy1β1-(4-methyl-umbelliferone),synthesized by cultured human skin fibroblasts." J.Biochem.116. 524-529 (1994)

    • Related Report
      1994 Annual Research Report
  • [Publications] Higuchi,T.: "A method for determination of galactosyltransferase I activity synthesizing the proteoglycan linkage region." J.Biochem.Biophys.Methods. 29. 135-142 (1994)

    • Related Report
      1994 Annual Research Report
  • [Publications] Nakamura,T.: "A novel oligosaccharide,GlcAβ1-4Xylβ1-(4-methylumbelliferone),synthesized by human cultured skin fibroblasts." Biochem.J.304. 731-736 (1994)

    • Related Report
      1994 Annual Research Report
  • [Publications] Saitoh,H.: "Enzymic reconstruction of glycosaminoglycan oligosaccharide chain using the transglycosylation reaction of bovine testicular hyaluronidase." J.Biol.Chem.,. (in press).

    • Related Report
      1994 Annual Research Report
  • [Publications] Shibata,S.: "HNK-1-reactive novel oligosaccharide,sulfate-0-3GalcAβ-1-4Xylβ1-(4-methylumbelliferone),synthesized by cultured human skin fibroblasts." J.Biol.Chem.,. (in press).

    • Related Report
      1994 Annual Research Report
  • [Publications] Tanaka,A.: "Simple measurement of glycosaminoglycan produced by cultured fibloblasts using 4-methyllumbelliferyl β-D-xyloside." J.Biochem.Biophys.Methods. 26. 309-316 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] Kubo,K.: "Depolymerization of hyaluronan by sonication." Glycoconjugate J.10. 435-439 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] Nakamura,T.: "Serum hyaluronidase assay using fluorogenic hyaluronate as a substrate." Connect.Tissue(受理番号; 25-E0-3). (in press).

    • Related Report
      1993 Annual Research Report
  • [Publications] Takagaki,K.: "Characterization of hydrolysis and transglylation by testicular hyaluronidase using ion-spray mass spectrometry." Biochemistry (受理番号; B1932904L). (in press).

    • Related Report
      1993 Annual Research Report
  • [Publications] 高垣 啓一,中村 敏也,武田 裕介,大道寺 浩一,遠藤 正彦: "A new endo-β-galactosidase acting on the Galβ1→3Gal linkage of proteoglycan linkage region (プロテオグリカンの結合部Gal→Galに作用するエンド-β-ガラクトシダーゼ)" Journal of Biological Chemistry. 267. 18558-18563 (1992)

    • Related Report
      1992 Annual Research Report
  • [Publications] 高垣 啓一,児島 薫,真嶋 光雄,加藤 郁之進,遠藤 正彦: "Ion-spray mass spectrometric analysis of glycosaminoglycan oligosaccharide (グリコサミノグリカン・オリゴ糖のイオンスプレー型質量分析計による分析)" Glycoconnjugate Journal. 9. 174-179 (1992)

    • Related Report
      1992 Annual Research Report
  • [Publications] 武田 裕介,原田 征行,高垣 啓一,成田 穂積,遠藤 正彦: "Characterization of glycosaminoglycsns in ossified human yellow ligament (ヒト骨化黄色靭帯のグリコサミノグリカンの性状)" Connective Tissue. 24. 109-114 (1992)

    • Related Report
      1992 Annual Research Report
  • [Publications] 中村 敏也,高垣 啓一,久保 恒明,斉藤 太郎,遠藤 正彦: "Hyaluronic acid synthesized by cultured skin fibroblasts derived from patients with Werner's syndrome (ウエルナー症候群患者皮膚線維芽細胞により産生されるヒアルロン酸)" Biochimia et Biophysica Acta. 1139. 84-90 (1992)

    • Related Report
      1992 Annual Research Report
  • [Publications] 田中 昭宏,森川 晶子,斉藤 良治,田村 真理,中村 敏也,高垣 啓一,遠藤 正彦: "Simple measurement of glycosaminoglycan produced by cultured fibroblasts using 4-methylumbelliferyl β-D-xyloside (4-メチルウンベルフェリルキシロシドを用いた培養線維芽細胞の産生するグリコサミノグリカンの定量法)" Journal of Biochemical and Biophysical Methods. (1993)

    • Related Report
      1992 Annual Research Report
  • [Publications] 吉原 秀一,佐々木 睦男,横山 慎,遠藤 正彦,今 充: "Enzymic determination of glycoconjugates in human pancreatic juice (ヒト膵液中の複合糖質の酵素的定量)" International Journal of Pancreatology. (1993)

    • Related Report
      1992 Annual Research Report
  • [Publications] 遠藤 正彦,高垣 啓一,中村 敏也: "Endoglycosidases acting on the linkage region between the core protein and glycosaminoglycan chains of proteoglycans" CRC Press, 26 (1992)

    • Related Report
      1992 Annual Research Report

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Published: 1992-04-01   Modified: 2016-04-21  

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