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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 in Aspergillus oryzae
Aspergillus fungi 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 in Aspergillus fungi. The GPI-anchored α-amylase AmyD in Aspergillus nidulans has been reported to directly suppress...
Autores principales: | , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Frontiers Media S.A.
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10512346/ https://www.ncbi.nlm.nih.gov/pubmed/37746167 http://dx.doi.org/10.3389/ffunb.2022.1061841 |
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author | Koizumi, Ami Miyazawa, Ken Ogata, Makoto Takahashi, Yuzuru Yano, Shigekazu Yoshimi, Akira Sano, Motoaki Hidaka, Masafumi Nihira, Takanori Nakai, Hiroyuki Kimura, Satoshi Iwata, Tadahisa Abe, Keietsu |
author_facet | Koizumi, Ami Miyazawa, Ken Ogata, Makoto Takahashi, Yuzuru Yano, Shigekazu Yoshimi, Akira Sano, Motoaki Hidaka, Masafumi Nihira, Takanori Nakai, Hiroyuki Kimura, Satoshi Iwata, Tadahisa Abe, Keietsu |
author_sort | Koizumi, Ami |
collection | PubMed |
description | Aspergillus fungi 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 in Aspergillus fungi. The GPI-anchored α-amylase AmyD in Aspergillus nidulans has been reported to directly suppress the biosynthesis of cell wall α-1,3-glucan but not to degrade it in 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 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 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 in Pichia pastoris and 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 of agtA in A. nidulans decreased the molecular weight of cell wall α-1,3-glucan. These in vitro and in vivo properties 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 molecular weight of cell wall α-1,3-glucan in vivo. |
format | Online Article Text |
id | pubmed-10512346 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-105123462023-09-22 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 in Aspergillus oryzae Koizumi, Ami Miyazawa, Ken Ogata, Makoto Takahashi, Yuzuru Yano, Shigekazu Yoshimi, Akira Sano, Motoaki Hidaka, Masafumi Nihira, Takanori Nakai, Hiroyuki Kimura, Satoshi Iwata, Tadahisa Abe, Keietsu Front Fungal Biol Fungal Biology Aspergillus fungi 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 in Aspergillus fungi. The GPI-anchored α-amylase AmyD in Aspergillus nidulans has been reported to directly suppress the biosynthesis of cell wall α-1,3-glucan but not to degrade it in 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 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 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 in Pichia pastoris and 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 of agtA in A. nidulans decreased the molecular weight of cell wall α-1,3-glucan. These in vitro and in vivo properties 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 molecular weight of cell wall α-1,3-glucan in vivo. Frontiers Media S.A. 2023-01-10 /pmc/articles/PMC10512346/ /pubmed/37746167 http://dx.doi.org/10.3389/ffunb.2022.1061841 Text en Copyright © 2023 Koizumi, Miyazawa, Ogata, Takahashi, Yano, Yoshimi, Sano, Hidaka, Nihira, Nakai, Kimura, Iwata and Abe https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Fungal Biology Koizumi, Ami Miyazawa, Ken Ogata, Makoto Takahashi, Yuzuru Yano, Shigekazu Yoshimi, Akira Sano, Motoaki Hidaka, Masafumi Nihira, Takanori Nakai, Hiroyuki Kimura, Satoshi Iwata, Tadahisa Abe, Keietsu 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 in Aspergillus oryzae |
title | 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 in Aspergillus oryzae
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title_full | 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 in Aspergillus oryzae
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title_fullStr | 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 in Aspergillus oryzae
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title_full_unstemmed | 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 in Aspergillus oryzae
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title_short | 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 in Aspergillus oryzae
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title_sort | 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 in aspergillus oryzae |
topic | Fungal Biology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10512346/ https://www.ncbi.nlm.nih.gov/pubmed/37746167 http://dx.doi.org/10.3389/ffunb.2022.1061841 |
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