Cleavage of -1,4-glycosidic linkages by the glycosylphosphatidylinositol-anchored -amylase AgtA decreases the molecular weight of cell wall -1,3-glucan in Aspergillus oryzae

Aspergillus fungi contain alpha-1,3-glucan with a low proportion of alpha-1,4-glucan as a major cell wall polysaccharide. Glycosylphosphatidylinositol (GPI)-anchored a-amylases are conserved in Aspergillus fungi. The GPI-anchored a-amylase AmyD in Aspergillus nidulans has been reported to directly suppress the biosynthesis of cell wall Aspergillus fungi contain alpha-1,3-glucan with a low proportion of alpha 1,4-glucan as a major cell wall polysaccharide. alpha-1,3-glucan but not to degrade it in vivo. However, the detailed mechanism of cell wall alpha-1,3-glucan biosynthesis regulation by AmyD remains unclear. Here we focused on AoAgtA, which is encoded by the Aspergillus oryzae agtA gene, an ortholog of the A. nidulans amyD gene. Similar to findings in A. nidulans, agtA overexpression in A. oryzae grown in submerged culture decreased the amount of cell wall alpha-1,3-glucan and led to the formation of smaller hyphal pellets in comparison with the wildtype strain. We analyzed the enzymatic properties of recombinant (r)AoAgtA produced in Pichia pastoris and found that it degraded soluble starch, but not linear bacterial alpha-1,3-glucan. Furthermore, rAoAgtA cleaved 3-amaltotetraosylglucose with a structure similar to the predicted boundary structure between the alpha-1,3-glucan main chain and a short spacer composed of alpha-1,4- linked glucose residues in cell wall alpha-1,3-glucan. Interestingly, rAoAgtA randomly cleaved only the alpha-1,4- glycosidic bonds of 3-amaltotetraosylglucose, indicating that AoAgtA may cleave the spacer in cell wall alpha- 1,3- glucan. Consistent with this hypothesis, heterologous overexpression of agtA in A. nidulans decreased the molecular weight of cell wall alpha-1,3-glucan. These in vitro and in vivo properties of AoAgtA suggest that GPI-anchored a- amylases can degrade the spacer alpha- 1,4-glycosidic linkages in cell wall alpha-1,3-glucan before its insolubilization, and this spacer cleavage decreases the molecular weight of cell wall alpha-1,3-glucan in vivo.