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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1,2-α-Mannosidase catalyzes the specific cleavage of 1,2-α-mannose residues from protein-linked <I>N</I>-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 <I>Aspergillus oryzae</I>. The <I>fmanIB</I> 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 <I>Aspergillus</I> host, though it has rarely been done with other filamentous-fungal mannosidase, was successful with <I>fmanIB</I> 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 Man<SUB>9</SUB>GlcNAc<SUB>2</SUB> as the substrate, Man<SUB>5</SUB>GlcNAc<SUB>2</SUB> 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 <I>Aspergillus</I>, 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 <I>A. oryzae</I> (Yoshida <I>et al.</I>, 2000) support the notion that the filamentous fungus has similar steps of <I>N</I>-linked glycochain trimming to those in mammalian cells.
- Agricultural and Biological Chemistry
Agricultural and Biological Chemistry 70(2), 471-479, 2006-02-23
Japan Society for Bioscience, Biotechnology, and Agrochemistry