Takuya Onoue1, Yutaka Tanaka2, Daisuke Hagiwara3, Keisuke Ekino1, Akira Watanabe3, Kazuyoshi Ohta1, Katsuhiko Kamei3, Nobuyuki Shibata2, Masatoshi Goto4, Takuji Oka5. 1. Department of Applied Microbial Technology, Faculty of Biotechnology and Life Science, Sojo University, Kumamoto, Japan. 2. Department of Infection and Host Defense, Tohoku Medical and Pharmaceutical University, Sendai, Japan. 3. Medical Mycology Research Center, Chiba University, Chiba, Japan. 4. Department of Applied Biochemistry and Food Science, Saga University, Saga, Japan. 5. Department of Applied Microbial Technology, Faculty of Biotechnology and Life Science, Sojo University, Kumamoto, Japan. oka@bio.sojo-u.ac.jp.
Abstract
Fungal-type galactomannan (FTGM) is a polysaccharide composed of α-(1 → 2)-/α-(1 → 6)-mannosyl and β-(1 → 5)-/β-(1 → 6)-galactofuranosyl residues located at the outer cell wall of the human pathogenic fungus Aspergillus fumigatus. FTGM contains a linear α-mannan structure called core-mannan composed of 9 or 10 α-(1 → 2)-mannotetraose units jointed by α-(1 → 6)-linkages. However, the enzymes involved in core-mannan biosynthesis remain unknown. We speculated that two putative α-1,2-mannosyltransferase genes in A. fumigatus, Afu5g02740/AFUB_051270 (here termed core-mannan synthase A [CmsA]) and Afu5g12160/AFUB_059750 (CmsB) are involved in FTGM core-mannan biosynthesis. We constructed recombinant proteins for CmsA and detected robust mannosyltransferase activity using the chemically synthesized substrate p-nitrophenyl α-D-mannopyranoside as an acceptor. Analyses of CmsA enzymatic product revealed that CmsA possesses the capacity to transfer a mannopyranoside to the C-2 position of α-mannose. CmsA could also transfer a mannose residue to α-(1 → 2)-mannobiose and α-(1 → 6)-mannobiose and showed a 31-fold higher specific activity toward α-(1 → 6)-mannobiose than toward α-(1 → 2)-mannobiose. Proton nuclear magnetic resonance (1H-NMR) spectroscopy and gel filtration chromatography of isolated FTGM revealed that core-mannan structures were drastically altered and shortened in disruptant A. fumigatus strains ∆cmsA, ∆cmsB, and ∆cmsA∆cmsB. Disruption of cmsA or cmsB resulted in severely repressed hyphal extension, abnormal branching hyphae, formation of a balloon structure in hyphae, and decreased conidia formation. The normal wild type core-mannan structure and developmental phenotype were restored by the complementation of cmsA and cmsB in the corresponding disruptant strains. These findings indicate that both CmsA, an α-1,2-mannosyltransferase, and CmsB, a putative mannosyltransferase, are involved in FTGM biosynthesis.
nclass="Disease">Fungal-type galactomannan (class="Chemical">pan class="Chemical">FTGM) is a polysaccharide composed of α-(1 → 2)-/α-(1 → 6)-mannosyl and β-(1 → 5)-/β-(1 → 6)-galactofuranosyl residues located at the outer cell wall of the human pathogenic fungus Aspergillus fumigatus. FTGM contains a linear α-mannan structure called core-mannan composed of 9 or 10 α-(1 → 2)-mannotetraose units jointed by α-(1 → 6)-linkages. However, the enzymes involved in core-mannan biosynthesis remain unknown. We speculated that two putative α-1,2-mannosyltransferase genes in A. fumigatus, Afu5g02740/AFUB_051270 (here termed core-mannan synthase A [CmsA]) and Afu5g12160/AFUB_059750 (CmsB) are involved in FTGM core-mannan biosynthesis. We constructed recombinant proteins for CmsA and detected robust mannosyltransferase activity using the chemically synthesized substrate p-nitrophenyl α-D-mannopyranoside as an acceptor. Analyses of CmsA enzymatic product revealed that CmsA possesses the capacity to transfer a mannopyranoside to the C-2 position of α-mannose. CmsA could also transfer a mannose residue to α-(1 → 2)-mannobiose and α-(1 → 6)-mannobiose and showed a 31-fold higher specific activity toward α-(1 → 6)-mannobiose than toward α-(1 → 2)-mannobiose. Proton nuclear magnetic resonance (1H-NMR) spectroscopy and gel filtration chromatography of isolated FTGM revealed that core-mannan structures were drastically altered and shortened in disruptant A. fumigatus strains ∆cmsA, ∆cmsB, and ∆cmsA∆cmsB. Disruption of cmsA or cmsB resulted in severely repressed hyphal extension, abnormal branching hyphae, formation of a balloon structure in hyphae, and decreased conidia formation. The normal wild type core-mannan structure and developmental phenotype were restored by the complementation of cmsA and cmsB in the corresponding disruptant strains. These findings indicate that both CmsA, an α-1,2-mannosyltransferase, and CmsB, a putative mannosyltransferase, are involved in FTGM biosynthesis.