Cloning and Expression of 1,2-.ALPHA.-Mannosidase Gene (fmanIB) from Filamentous Fungus Aspergillus oryzae: in Vivo Visualization of the FmanIBp-GFP Fusion Protein

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  • Cloning and Expression of 1,2-α-Mannosidase Gene (fmanIB) from Filamentous Fungus Aspergillus oryzae: in Vivo Visualization of the FmanIBp-GFP Fusion Protein
  • Cloning and Expression of 1 2 アルファ Mannosidase Gene fmanIB from Filamentous Fungus Aspergillus oryzae in Vivo Visualization of the FmanIBp GFP Fusion Protein
  • Cloning and Expression of 1,2-α-Mannosidase Gene (<i>fmanIB</i>) from Filamentous Fungus<i>Aspergillus oryzae</i>:<i>in Vivo</i>Visualization of the FmanIBp-GFP Fusion Protein

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Abstract

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.

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