A Glycosylphosphatidylinositol-Anchored α-Amylase Encoded by amyD Contributes to a Decrease in the Molecular Mass of Cell Wall α-1,3-Glucan in Aspergillus nidulans

Abstract

α-1,3-Glucan is one of the main polysaccharides in the cell wall of Aspergillus nidulans. We previously revealed that it plays a role in hyphal aggregation in liquid culture, and that its molecular mass (MM) in an agsA-overexpressing (agsAOE) strain was larger than that in an agsB-overexpressing (agsBOE) strain. The mechanism that regulates its MM is poorly understood. Although the gene amyD, which encodes glycosylphosphatidylinositol (GPI)-anchored α-amylase (AmyD), is involved in the biosynthesis of α-1,3-glucan in A. nidulans, how it regulates this biosynthesis remains unclear. Here we constructed strains with disrupted amyD (ΔamyD) or overexpressed amyD (amyDOE) in the genetic background of the ABPU1 (wild-type), agsAOE, or agsBOE strain, and characterized the chemical structure of α-1,3-glucans in the cell wall of each strain, focusing on their MM. The MM of α-1,3-glucan from the agsBOEamyDOE strain was smaller than that in the parental agsBOE strain. In addition, the MM of α-1,3-glucan from the agsAOE ΔamyD strain was greater than that in the agsAOE strain. These results suggest that AmyD is involved in decreasing the MM of α-1,3-glucan. We also found that the C-terminal GPI-anchoring region is important for these functions.

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

“…In submerged culture, the Δ amyD strain formed tightly aggregated hyphal pellets, but the hyphae of the Δ amyD-agtA OE strain were almost fully dispersed (Figure 4). The latter result is consistent with that for the Δ amyD-amyD OE strain (Figure 4) reported by Miyazawa et al (2022). The flask culture of the agsB OE Δ amyD-agtA OE strain seemed cloudier than those of agsB OE Δ amyD and agsB OE Δ amyD-amyD OE strains, but there was only a slight difference in the hyphal pellets they formed (Figure 4).…”

“…On the other hand, the number-average MW of glucan in the AS2 fraction from agsB OE Δ amyD-agtA OE (95,700 ± 1,300) was significantly lower than that from agsB OE Δ amyD (462,000 ± 38,000; Figure 5B; Table 5, P < 0.01 in Welch’s test). These results indicate that AoAgtA decreases the MW of cell wall α-1,3-glucan, similar to A. nidulans AmyD (Miyazawa et al, 2022).…”

“…Although Agt proteins encoded by the orthologous agtA genes in Aspergillus fungi are thought to be GPI-anchored α-amylases, their function may be related to the biosynthesis of cell wall α-1,3-glucan rather than to starch catabolism (van der Kaaij et al, 2007; He et al, 2014, 2017; Miyazawa et al, 2022). In the present study, similarly to amyD in A. nidulans (He et al, 2014, 2017; Miyazawa et al, 2022), agtA overexpression in A. oryzae decreased the amount of α-1,3-glucan in the cell wall (Figure 2), suggesting that AoAgtA suppresses cell wall α-1,3-glucan biosynthesis in A. oryzae . We also investigated the enzymatic properties of rAoAgtA to further understand its contribution to cell wall α-1,3-glucan biosynthesis.…”

“…rAoAgtA randomly cleaved only the α-1,4-glycosidic bonds of Mal 4 α1,3Glc (Table 3), and the structure of Mal 4 α1,3Glc is similar to the predicted boundary structure between α-1,3-glucan main chain and the spacer in cell wall α-1,3-glucan synthesized by AgsB (Figure 6A). Miyazawa et al (2022) suggested that AmyD of A. nidulans requires a GPI anchor to act on α-1,3-glucan in vivo and that AmyD reacts with cell wall α-1,3-glucan shortly after it is synthesized by α-1,3-glucan synthase on the plasma membrane. Therefore, we speculate that AoAgtA cleaves some of the spacers of cell wall α-1,3-glucan in the process of synthesis in vivo (Figure 6B).…”

Cell Wall Integrity and Its Industrial Applications in Filamentous Fungi

“…These results suggest that AmyD represses α-1,3-glucan biosynthesis. Overexpression of AmyD without the C-terminal GPI-anchor in A. nidulans scarcely affects cell wall α-1,3-glucan, suggesting the importance of the GPI anchor for correct cellular localization and function of AmyD [ 129 ]. In A. fumigatus , treatment with α-1,3-glucanase removes α-1,3-glucan from conidia and leads to their dispersion in medium, indicating the involvement of α-1,3-glucan in conidial aggregation [ 130 ].…”