Identification of an alpha-(1 -> 6)-Mannosyltransferase Contributing To Biosynthesis of the Fungal-Type Galactomannan alpha-Core-Mannan Structure in Aspergillus fumigatus

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. Fungal-type galactomannan, a cell wall component of Aspergillus fumigatus, is composed of alpha-(1 -> 2)-/alpha-(1 -> 6)-linked mannan and beta-(1 -> 5)-/beta-(1 -> 6)-linked galactofuran side chains. Recently, CmsA and CmsB were identified as the alpha-(1 -> 2)-mannosyltransferases involved in the biosynthesis of the alpha-core-mannan. However, the alpha-(1 -> 6)-mannosyltransferase involved in the biosynthesis of the alpha-core-mannan has not been identified yet. In this study, we analyzed 9 putative alpha-(1 -> 6)-mannosyltransferase gene disruption strains of A. fumigatus. The Delta anpA strain resulted in decreased mycelial elongation and reduced conidia formation. Proton nuclear magnetic resonance analysis revealed that the Delta anpA strain failed to produce the alpha-core-mannan of fungal-type galactomannan. We also found that recombinant AnpA exhibited much stronger alpha-(1 -> 6)-mannosyltransferase activity toward alpha-(1 -> 2)-mannobiose than alpha-(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 alpha-(1 -> 6)-mannoside bond formation and that its catalytic activity would be specific for the elongation of the alpha-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 alpha-(1 -> 6)-mannosyltransferase responsible for the biosynthesis of the alpha-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.