| Project/Area Number |
11556062
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 展開研究 |
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| Research Field |
生物資源科学
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| Research Institution | OKAYAMA UNIVERSITY |
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Principal Investigator |
KIMUKA Yoshinobu Okayama University, Bioresources Chemistry, Associate Professor, 農学部, 助教授 (70195387)
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| Co-Investigator(Kenkyū-buntansha) |
NAKATA Kengo Kagome Co., LTD., Senior Researcher, 総合研究所, 主任研究員
田中 健吾 カゴメ株式会社, 総合研究所, 研究員
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| Project Period (FY) |
1999 – 2002
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| Project Status |
Completed (Fiscal Year 2002)
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| Budget Amount *help |
¥6,800,000 (Direct Cost: ¥6,800,000)
Fiscal Year 2002: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 2001: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 2000: ¥3,400,000 (Direct Cost: ¥3,400,000)
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| Keywords | plantglycoprotein / free N-glycan / endo-β-N-acetylglucosaminidase / α-mannosidase / peptide : N-glycanase / subcellular distribution / transgenic plant / glyco-signaling molecule / 果実熟成 / 遊離N-グリカン / N-グリカン代謝 / α-マンノシダーゼ / endo-β-N-acetylglucosaminidase / peptide:N-glycanase / free N-glycan / plantglycoproteis / N-glycan structure / N-glycan function |
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| Research Abstract |
In 1999, some endo-β-N-acetylglucosaminidases were purified and characterized from Ginkgo seeds, tomato fruits, pumpkin seedlings, and tobacco cultured cells. The optimum pH of these plant endoglycosidases having the molecular weight of 63 kDa was between 6 to 7 and the enzyme showed strong activity toward the high-mannose type N-glycans bearing the Manα1-2Manα1-3Man unit. This result suggested that the plant endoglycosidase would have a common subsite specific for the structural unit and the resulting free N-glycans might have a critical function for the plant development or differentiation.
In 2000, the subcellular distribution of the plant endoglycosidase was investigated. Subcellular fractionation analysis using ultracentrifugation method showed that the plant endoglycosidase and the high-mannose type free N-glycans are localized in the cytosol but not in the ER or Golgi apparatus.
From 2000 to 2001, we have revealed that at least in every plant material analyzed the high-mannose typ
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e N-glycans with only one GlcNAc residue (Man9〜4GlcNAc1) always existed at the micromolar (μM) concentration. On the other hand, although the plant complex type free glycans with the N-acetylchitobiosyl segment (GlcNAc2〜0Man3Xyl1 Fuc1〜0GlcNAc2) were also detected, the amount of these type structures was about one of the several decade minutes of that of high-mannose type structures. The high-mannose type free N-glycans (Man9〜5GlcNAc1) in plant cells have a common core structure (Manα1-6(Manα1-3)Manα1-6(Manα1-3)Manβ1-4GlcNAc). We also purified and characterized an α-mannosidase from Ginkgo biloba seed. Since this enzyme showed a strong activity against the high-mannos type N-glycan bearing one GlcNAc residue rather than the N-glycan bearing two GlcNAc residues, suggesting this α-mannosidase could be involved in the free N-glycan degradation produced in the cytosol. The molecular cloning of this α-mamosidase and expression of the gene in E. Coli is in progress.
From 2001 to 2002, we have determined the partial amino acid sequences of the endoglycosidase purified from rice cells and succeeded the molecular cloning of the endoglycosidase gene. The DNA sequence determined showed a high homology with a DNA sequence of function-unknown protein in Arabidopsis thaliana, suggesting this Arabidopsis protein may be endo-β-N-acetylglucosaminidase.
Now, we are constructing a transgenic plant (rice plant) in which the endoglycosidase gene is suppressed to confirm the N-glycan function involved in plant growth and differentiation.
Moreover, from 1999 to 2001, using jack bean α-mannosidase as a model glycoprotein, we have revealed that free N-glycans could play a critical role in the formation of quaternary structure of oligomeric proteins. This chaperone-like function of free N-glycans involved in plant cell growth seems to be a new concept on the oligosaccharide's function. Less
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