Cleavage of α-1,4-Glycosidic Linkages by the Glycosylphosphatidylinositol-Anchored α-Amylase AgtA Decreases the Molecular Weight of Cell Wall α-1,3-Glucan inAspergillus oryzae

Abstract

AbstractAspergillusfungi contain α-1,3-glucan with a low proportion of α-1,4-glucan as a major cell wall polysaccharide. Glycosylphosphatidylinositol (GPI)-anchored α-amylases are conserved inAspergillusfungi. The GPI-anchored α-amylase AmyD inAspergillus nidulanshas been reported to directly suppress the biosynthesis of cell wall α-1,3-glucan but not to degrade itin vivo. However, the detailed mechanism of cell wall α-1,3-glucan biosynthesis regulation by AmyD remains unclear. Here we focused on AoAgtA, which is encoded by theAspergillus oryzae agtAgene, an ortholog of theA. nidulans amyDgene. Similar to findings inA. nidulans, agtAoverexpression inA. oryzaegrown in submerged culture decreased the amount of cell wall α-1,3-glucan and led to the formation of smaller hyphal pellets in comparison with the wild-type strain. We analyzed the enzymatic properties of recombinant (r)AoAgtA produced inPichia pastorisand found that it degraded soluble starch, but not linear bacterial α-1,3-glucan. Furthermore, rAoAgtA cleaved 3-α-maltotetraosylglucose with a structure similar to the predicted boundary structure between the α-1,3-glucan main chain and a short spacer composed of α-1,4-linked glucose residues in cell wall α-1,3-glucan. Interestingly, rAoAgtA randomly cleaved only the α-1,4-glycosidic bonds of 3-α-maltotetraosylglucose, indicating that AoAgtA may cleave the spacer in cell wall α-1,3-glucan. Consistent with this hypothesis, heterologous overexpression ofagtAinA. nidulansdecreased the molecular weight (MW) of cell wall α-1,3-glucan. Thesein vitroandin vivoproperties of AoAgtA suggest that GPI-anchored α-amylases can degrade the spacer α-1,4-glycosidic linkages in cell wall α-1,3-glucan before its insolubilization, and this spacer cleavage decreases the MW of cell wall α-1,3-glucanin vivo.