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Basic research for application of plant-origin N-glycan releasing enzymes to glycotechnology

Research Project

Project/Area Number 07660442
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Applied molecular and cellular biology
Research InstitutionOkayama University

Principal Investigator

KUMURA Yoshinobu  Okayama University, Faculty of Agriculture Associate professor, 農学部, 助教授 (70195387)

Project Period (FY) 1995 – 1996
Project Status Completed (Fiscal Year 1996)
Budget Amount *help
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1996: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1995: ¥1,400,000 (Direct Cost: ¥1,400,000)
Keywordsplant glycoproteins / endo-beta-GlcNAc-ase / peptide : N-glycanase / N-glycan / free N-glycan / MALDI TOF-MS / IS-MS / MS / glycobiology
Research Abstract

Plant-origin endo-beta-N acetylglucosaminidases have been purified or partially purified from pea seeds, soybean seeds, ginkgo seeds, and immature tomato fruit. The molecular weights of endo-GB (Ginkgo biloba), endo-GM (soybean), and endo-LE (tomato) were determined to be about 64 kDa by gel-filtration or SDS-PAGE.On the other hand, the molecular weight of endo-PS (pea) was determined to be 42 kDa by MALDI TOF-MS.All theseendoglycosidases were found to have optimum pH between 6 and 7 and optimum temperature at about 35゚C.And these plant-origin endoglycosidases could hydrolyze the beta-1-4linkage of chitobiose in oligomannose type N-glycans but not the linkage of xylose/fucose containing complex type N-glycans. Using various oligomannose type sugar chains, the detail substrate specificity was further investigated. High mannose type structures bearing alpha1-2 mannosyl residue (s), Man9-6GlcNAc2, could be hydrolyzed at 80-100% relative hydrolysis rate. Man5-3GlcNAc2 stuructures bearing n … More o alpha1-2 mannosyl residue could be also hydrolyzed by these endoglycosidase, however, the relative reaction rates were only 20-60% comparing that for Man6GlcNAc2 structure. These results suggested that (1) plant-origin endoglycosidase has a common subsite for binding or recogmizing alpha1-2 mannosyl residue (s) and the subsite may regulate the hydrolysis rate ; (2) plant-origin endoglycosidase may serve to release high mannose type N-glycans bearing alpha1-2 mannosyl residue (s)
A peptide : N-glycanase (PNGase-GM) has been purified from soybean seed to homogeneity. The molecular weight of PNGase-GM was 90 kDa by gel-filtration and SDS-PAGE.Optimum pH of PNGase-GM was arround 5.0. PNGase-GM could hydrolyze beta-aspartlglycosylamine linkage of glycopeptides having oligomannose type, hybrid type, xylose/fucose containing type, and sialic acid containing complex type sugar chains. However, the reaction rates was depend on N-glycan structures. Assuming that the relative hydrolysis rate for the glycopeptides bearing oligomannose type is 100%, the rate for glycopeptide bearing xylose/fucose containing type was 30%, and that for glycopeptides bearing sialic acid containing complex type were less than 5%.
In epicotyls and hypocotyls of pea seedlings, free N-glycans bearing high mannose type and xylose/fucose containing type structures have been identified by ion-spray tandem mass spectrometry (IS-MS/MS). This result clearly indicates that free N-glycans produced by two kinds of N-glycan releasing enzymes do occur in the developing tissues of plant cells. Less

Report

(3 results)
  • 1996 Annual Research Report   Final Research Report Summary
  • 1995 Annual Research Report
  • Research Products

    (23 results)

All Other

All Publications (23 results)

  • [Publications] Y.Kimura et al.: "Occurrence of free N-glycans in pea (Pisum sativum) seedlings" Biosci.Biotech.Biochem.61(in press). (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Y.Kimura et al.: "Structural elucidation of N-linked sugar chains of storage glycoproteins in mature pea (Pisum sativum) seeds by ion-spray tandem mass spectrometry" Biosci.Biotech Biochem.60. 1841-1850 (1996)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Y.Kimura et al.: "Purification and substrate specificity of an endo-b-N-acetylglucosaminidase from Pea (Pisum sativum) seeds" Biosci.Biotech.Biochem.60. 228-232 (1996)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] K.Hirata et al.: "Purification and characterization of β-xylosidase and α-fucosidase from apple snail (Pomacea canaliculata)" Biosci.Biotech.Biochem.60. 249-254 (1996)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Y.Kimura et al.: "Structural analysis of N-linked oligosaccharide of mitogenic lectin B from the roots of pokeweed (Phytolacca americana)" Biosci.Biotech.Biochem.60. 537-540 (1996)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Y.Kimura et al.: "N-linked sugar chain of 55 kDa royal jelly glycoprotein" Biosci.Biotech.Biochem.60. 2099-2102 (1996)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Y.Kimura et.al.: "Occurrence of free N-glycans in pea (Pisum sativum) seedlings" Biosci.Biotech.Biochem.61 (in press). (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Y.Kimura et.al.: "Structural elucidation of N-linked sugar chains of storage glycoproteins in mature pea (Pisum sativum) seeds by ion-spray tandem mass spectrometry." Biosci.Biotech.Biochem.60 (11). 1841-1850 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Y.Kimura et.al.: "Purification and substrate specificity of an endo-beta-N-acetyl-glucosaminidase from pea (Pisum sativum) seeds" Biosci.Biotech.Biochem.60 (2). 228-232 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Y.Kimura et.al.: "Structural analysis of N-linked oligosaccharide of mitogenic lectin B from the roots of pokeweed (Phytolacca americana)" Biosci.Biotech.Biochem.60 (3). 537-540 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] K.Hirata, Y.Kimura, et.al.: "Purification and characterization of beta-xylosidase and alpha-fucosidase from apple snail (Pomacea canaliculata)" Biosci.Biotech.Biochem.60 (2). 249-254 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Y.Kimura et.al.: "N-linked sugar chain of 55 kDa royal jelly glycoprotein" Biosci.Biotech.Biochem.60 (12). 2099-2102

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Y.Kimura et al.: "Occurrence of free N-glycans in pea (Pisum sativum) seedlings" Biosci.Biotech.Biochem.61(in press). (1997)

    • Related Report
      1996 Annual Research Report
  • [Publications] Y.Kimura et al.: "Strucutral elucidation of N-linked sugar chains of storage glycoproteins in mature pea (Pisum sativum) seeds by ion-spray tandem mass spectrometry" Biosci.Biotech.Biochem.60(11). 1841-1850 (1996)

    • Related Report
      1996 Annual Research Report
  • [Publications] Y.Kimura et al.: "Purification and substrate specificity of an endo-b-N-acetylglucosaminidase from Pea (Pisum sativum) seeds" Biosci.Biotech.Biochem.60(2). 228-232 (1996)

    • Related Report
      1996 Annual Research Report
  • [Publications] K.Hirata et al.: "Purification and characterization of b-xylosidase and a-fucosidase from apple snails (Pomacea canaliculata)" Biosci.Biotech.Biochem.60(2). 249-254 (1996)

    • Related Report
      1996 Annual Research Report
  • [Publications] Y.Kimura et al.: "Structural analysis of N-linked oligosaccharide of mitogenic lectin B from the roots of pokeweed (Phytolacca americana)" Biosci.Biotech.Biochem.60(3). 537-540 (1996)

    • Related Report
      1996 Annual Research Report
  • [Publications] Y.Kimura et al.: "N-linked sugar chain of 55 kDa royal jelly glycoprotein" Biosci.Bitech.biochem.60(12). 2099-2102 (1996)

    • Related Report
      1996 Annual Research Report
  • [Publications] Y.Kimura,K.Iwata,Y.Sumi,S.Takagi.: "Purification and Substrate Specificity ofan Endo-β-N-acetylglucosaminidase from Pea (Pisum sativum) Seeds" Biosci.Biotech.Biochem.60. 228-232 (1996)

    • Related Report
      1995 Annual Research Report
  • [Publications] Y.Kimura,K.Yamaguchi,G.Funatsu: "Structural Analysis of N-linked oligosaccharide of Mitogenic Lectin-B from the Roots of Pokeweed (Phytolacca americana)" Biosci.Biotech.Biochem.60 (in press). (1996)

    • Related Report
      1995 Annual Research Report
  • [Publications] K.Hirata,Y.Nakahara,Y.Kimura,G.Funatsu: "Purification and Characterization of β-Xylosidase anda-Fucosidase from Apple Snails (Pomacea canaliculata)" Biosci.Biotech.Biochem.60 (in press). (1996)

    • Related Report
      1995 Annual Research Report
  • [Publications] Kimura,Y.,Washino,N.Yonekura,M.: "N-linked Sugar Chains of 350 kDa Royal Jelly Glycoprotein" Biosci.Biotech.Biochem.59. 507-509 (1995)

    • Related Report
      1995 Annual Research Report
  • [Publications] Kimura,Y.,and Stadtman,T.C.: "Glycine Reductase Selenoprotein A Is not A Glycoprotein : The Positive Periodic Acid-Schiff Test is the Result of Peptide Bond Cleavage and Garbonyl Group Generation" Proc.Natl.Acad.Sci.USA.92. 2189-2193 (1995)

    • Related Report
      1995 Annual Research Report

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Published: 1995-04-01   Modified: 2016-04-21  

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