In Vivo Expression of UDP-N-Acetylglucosamine : α-3-D-Mannoside β-1,2-N-Acetylglucosaminyltransferase I (Gn T-1) in Aspergillus oryzae and Effects on the Sugar Chain of α-Amylase
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概要
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UDP-N-Acetylglucosamine: α-3-D-mannoside β-1,2-N-acetylglucosaminyltransferase I (GnT-I) is an essential enzyme in the conversion of high mannose type oligosaccharide to the hybrid or complex type. The full length of the rat GnT-I gene was expressed in the filamentous fungus Aspergillus oryzae. A microsomal preparation from a recombinant fungus (strain NG) showed GnT-I activity that transferred N-acetylglucosamine residue to acceptor heptaose, Man5GlcNAc2. The N-linked sugar chain of α-amylase secreted by the strain showed a peak of novel retention on high performance liquid chromatography that was same as a reaction product of in vitro GnT-1 assay. The peak of oligosaccharide disappeared on HPLC after β-N-acetylglucosaminidase treatment. Mass analysis supported the presence of GlcNAcMan5GlcNAc2 as a sugar chain of α-amylase from strain NG. Chimera of GnT-I with green fluorescent protein (GFP) showed a dotted pattern of fluorescence in the mycelia, suggesting localization at Golgi vesicles. We concluded that GnT-1 was functionally expressed in A. oryzae cells and that N-acetylglucosamine residue was transferred to N-glycan of α-amylase in vivo. A. oryzae is expected to be a potential host for the production of glycoprotein with a genetically altered sugar chain.
- 社団法人 日本農芸化学会の論文
- 2006-11-23
著者
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Yoshida Takashi
Laboratory of Bioorganic Chemistry, Tokyo University of Agriculture and Technology
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HIRAI Nobuaki
Laboratory of Infectious Diseases, Department of Disease Control, Graduate School of Veterinary Medi
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Hirai Nobuaki
Laboratory Of Cell Technology Faculty Of Agriculture And Life Science Hirosaki University
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Ohmachi Tetsuo
Faculty Of Agriculture And Life Science Hirosaki University
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YAMAGUCHI Masako
Faculty of Agriculture and Life Science, Hirosaki University
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Yamaguchi Masako
Faculty Of Agriculture And Life Science Hirosaki University
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KASAJIMA Yuya
Laboratory of Cell Technology, Faculty of Agriculture and Life Science, Hirosaki University
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YAMAGUCHI Masako
Laboratory of Cell Technology, Faculty of Agriculture and Life Science, Hirosaki University
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OHMACHI Tetsuo
Laboratory of Cell Technology, Faculty of Agriculture and Life Science, Hirosaki University
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Ohmachi Tetsuo
Dep. Of Biochemistry And Biotechnology Fac. Of Agriculture And Life Sci. Hirosaki Univ.
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Kasajima Yuya
Laboratory Of Cell Technology Faculty Of Agriculture And Life Science Hirosaki University
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Yoshida Takashi
Faculty Of Agriculture And Life Science Hirosaki University
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