Fungal-type galactomannan, a cell wall component of Aspergillus fumigatus, is composed of α-(1→2)-/α-(1→6)-linked mannan and β-(1→5)-/β-(1→6)-linked galactofuran side chains. Recently, CmsA and CmsB were identified as the α-(1→2)-mannosyltransferases involved in the biosynthesis of the α-core-mannan. However, the α-(1→6)-mannosyltransferase involved in the biosynthesis of the α-core-mannan has not been identified yet. In this study, we analyzed 9 putative α-(1→6)-mannosyltransferase gene disruption strains of A. fumigatus. The ΔanpA strain resulted in decreased mycelial elongation and reduced conidia formation. Proton nuclear magnetic resonance analysis revealed that the ΔanpA strain failed to produce the α-core-mannan of fungal-type galactomannan. We also found that recombinant AnpA exhibited much stronger α-(1→6)-mannosyltransferase activity toward α-(1→2)-mannobiose than α-(1→6)-mannobiose in vitro. Molecular simulations corroborated the fact that AnpA has a structure that can recognize the donor and acceptor substrates suitable for α-(1→6)-mannoside bond formation and that its catalytic activity would be specific for the elongation of the α-core-mannan structure in vivo. The identified AnpA is similar to Anp1p, which is involved in the elongation of the N-glycan outer chain in budding yeast, but the building sugar chain structure is different. The difference was attributed to the difference in substrate recognition of AnpA, which was clarified by simulations based on protein conformation. Thus, even proteins that seem to be functionally identical due to amino acid sequence similarity may be glycosyltransferase enzymes that make different glycans upon detailed analysis. This study describes an example of such a case. IMPORTANCE Fungal-type galactomannan is a polysaccharide incorporated into the cell wall of filamentous fungi belonging to the subphylum Pezizomycotina. Biosynthetic enzymes of fungal-type galactomannan are potential targets for antifungal drugs and agrochemicals. In this study, we identified an α-(1→6)-mannosyltransferase responsible for the biosynthesis of the α-core-mannan of fungal-type galactomannan, which has not been known for a long time. The findings of this study shed light on processes that shape this cellular structure while identifying a key enzyme essential for the biosynthesis of fungal-type galactomannan.
cell wall, galactofuranose, glycosyltransferase, Mannose, Galactomannan, Mannosyltransferase, Aspergillus fumigatus
NCBI PubMed ID: 36445154Publication DOI: 10.1128/msphere.00484-22Journal NLM ID: 101674533Publisher: Washington, DC: American Society for Microbiology
Correspondence: T. Oka
Institutions: Department of Biotechnology and Life Sciences, Sojo Universitygrid.412662.5, Kumamoto, Japan, Division of Infection and Host Defense, Tohoku Medical and Pharmaceutical University, Sendai, Japan, Department of Fungal Infection, National Institute of Infectious Diseasesgrid.410795.e, Tokyo, Japan
Methods: 1H NMR, virulence assays, MD simulations, genetic methods, enzyme assay, phylogenetic analysis, HCl treatment, docking
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