Cloning and expression of 1,2-alpha-mannosidase gene (fmanIB) from filamentous fungus Aspergillus oryzae: in vivo visualization of the FmanIBp-GFP fusion protein.

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.

Thiamine-regulated gene expression of Aspergillus oryzae thiA requires splicing of the intron containing a riboswitch-like domain in the 5'-UTR.

Exogenous thiamine regulates Aspergillus oryzae thiA, which is involved in thiamine synthesis. One of the two introns in its 5'-untranslated region (5'-UTR) contains motifs (regions A and B) highly conserved among fungal thiamine biosynthesis genes. Deletion of either region relieved the repression by thiamine and thiamine inhibited intron splicing, suggesting that regions A and B are required for efficient splicing. Furthermore, transcript splicing was essential for thiA gene expression. These observations suggest a novel gene expression regulatory mechanism in filamentous fungi, in which exogenous thiamine controls intron splicing to regulate gene expression. Interestingly, regions A and B constitute a part of a thiamine pyrophosphate-binding riboswitch-like domain that has been quite recently found in the 5'-UTR of thiA.