Cloning and Expression of 1,2-α-Mannosidase Gene (fmanIB) from Filamentous Fungus Aspergillus oryzae : in Vivo Visualization of the FmanIBp-GFP Fusion Protein
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概要
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1,2-α-Mannosidase catalyzes the specific cleavage of 1,2-α-mannose residues from protein-linked N-glycan. In this study, a novel DNA sequence homologous to the authentic 1,2-α-mannosidase was cloned from a cDNA library prepared from solid-state cultured Aspergillus oryzae. The fmanIB cDNA consisted of 1530 nucleotides and encoded a protein of 510 amino acids in which all consensus motifs of the class I α-mannosidase were conserved. Expression of the full length of 1,2-α-mannosidase cDNA by the Aspergillus host, though it has rarely been done with other filamentous-fungal mannosidase, was successful with fmanIB and caused an increase in both intracellular and extracellular mannosidase activity. The expressed protein (FmanIBp) specifically hydrolyzed 1,2-α-mannobiose with maximal activity at a pH of 5.5 and a temperature of 45 °C. With Man9GlcNAc2 as the substrate, Man5GlcNAc2 finally accumulated while hydrolysis of the 1,2-α-mannose residue of the middle branch was rate-limiting. To examine the intracellular localization of the enzyme, a chimeric protein of FmanIBp with green fluorescent protein was constructed. It showed a dotted fluorescence pattern in the mycelia of Aspergillus, indicative of the localization in intracellular vesicles. Based on these enzymatic and microscopic results, we estimated that FmanIBp is a fungal substitute for the mammalian Golgi 1,2-α-mannosidase isozyme IB. This and our previous report on the presence of another ER-type mannosidase in A. oryzae (Yoshida et al., 2000) support the notion that the filamentous fungus has similar steps of N-linked glycochain trimming to those in mammalian cells.
- 社団法人 日本農芸化学会の論文
- 2006-02-23
著者
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Akao Takeshi
National Research Institute of Brewing
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YOSHIDA Takashi
Faculty of Pharmaceutical Sciences, Matsuyama University
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Yamada Osamu
National Cardiovascular Center
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AKITA Osamu
National Research Institute of Brewing (NRIB)
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Akita O
Fac. Of Human Life Sciences Dep. Of Food And Health Sciences Jissen Women's Univ.
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Akita Osamu
National Research Institute Of Brewing
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Yamato Osamu
Kagoshima Univ. Kagoshima
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Akao T
National Research Institute Of Brewing
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Ohmachi Tetsuo
Faculty Of Agriculture And Life Science Hirosaki University
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Fujita Hiroya
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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YAHARA Akinori
Hiroshima University, Graduate School of Advanced Sciences of Matter
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YOSHIUCHI Kumi
National Research Institute of Brewing
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ASADA Yoshihiro
Faculty of Agriculture and Life Science, Hirosaki University
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Yahara Akinori
Hiroshima University Graduate School Of Advanced Sciences Of Matter
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Yoshida Takashi
Faculty Of Agriculture Kyusyu University
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Yamaguchi Masako
Faculty Of Agriculture And Life Science Hirosaki University
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Yamada O
Laboratory Of Clinical Pathology Department Of Veterinary Clinical Sciences Faculty Of Agriculture K
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Ohmachi Tetsuo
Dep. Of Biochemistry And Biotechnology Fac. Of Agriculture And Life Sci. Hirosaki Univ.
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Asada Yoshihiro
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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Yahara Akinori
National Research Institute of Brewing
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