Cloning and Expression of 1,2-α-Mannosidase Gene (fmanIB) from Filamentous FungusAspergillus oryzae:in VivoVisualization of the FmanIBp-GFP Fusion Protein

Abstract

1,2-alpha-Mannosidase catalyzes the specific cleavage of 1,2-alpha-mannose residues from protein-linked N-glycan. In this study, a novel DNA sequence homologous to the authentic 1,2-alpha-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 alpha-mannosidase were conserved. Expression of the full length of 1,2-alpha-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-alpha-mannobiose with maximal activity at a pH of 5.5 and a temperature of 45 degrees C. With Man(9)GlcNAc(2) as the substrate, Man(5)GlcNAc(2) finally accumulated while hydrolysis of the 1,2-alpha-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-alpha-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.

The Golgi apparatus: insights from filamentous fungi

“…In one of the early studies with endogenous proteins, CopA, the A. nidulans alpha subunit of the heptameric CopI coat mediating intra-Golgi and Golgi-to-ER retrograde transport (Popoff et al 2011), was used as a reporter fused to GFP. CopA::GFP was seen to decorate hollow "doughnut-like" structures scattered in the cytoplasm (Breakspear et al 2007), consistent with cisternae morphology in electron micrographs and resembling the 1,2-alpha-mannosidase-labeled structures that had been observed in Aspergillus oryzae (Akao et al 2006). CopA::GFP was shown to colocalize with the red fluorescent protein (RFP)-tagged transmembrane domain of alpha-2, 6-sialyltransferase (Hubbard and Kaminskyj 2008), a Golgi enzyme.…”

Rapid tip-directed movement of Golgi equivalents in growing Aspergillus nidulans hyphae suggests a mechanism for delivery of growth-related materials

“…Evidence from A. nidulans (Breakspear et al, 2007), Aspergillus oryzae (Akao et al, 2006), Candida albicans (Rida et al, 2006) and Saccharomyces cerevisiae (Matsuura-Tokita et al, 2006) suggests that GE distribution in fungi is related to tip growth.…”

Purification and biochemica characterisation of endoplasmic reticulum α 1,2-mannosidase from Sporothrix schenckiil

“…Enzymes from family 47 are α1,2-mannosidases that participate in the trimming of N-glycans and in ER-associated degradation (Herscovics 1999a, b, 2001, Helenius & Aebi 2004. Two subgroups of enzymes comprise family 47: the ER α1,2-mannosidases that trim only one mannose residue from Man 9 GlcNAc 2 (M 9 ) generating Man 8 GlcNAc isomer B (M 8 B) (Ziegler & Trimble 1991, Tremblay & Herscovics 1999, Mora-Montes et al 2004, Movsichoff et al 2005; and Golgi α1,2-mannosidases from mammalian cells and filamentous fungi, which act after the ER enzyme to remove three α1,2-linked mannose units, generating Man 5 GlcNAc 2 , an intermediate required for the biosynthesis of complex and hybrid N-glycans (Yoshida et al 1993, Lal et al 1998, Ichishima et al 1999, Eades & Hintz 2000, Tremblay & Herscovics 2000, Lobsanov et al 2002, Akao et al 2006. Lower eukaryotes, such as Saccharomyces cerevisiae, lack Golgi α1,2-mannosidases and synthesise only high-mannose N-glycans (Herscovics 1999b).…”