Dataset Information

Glucanase-Induced Stipe Wall Extension Shows Distinct Differences from Chitinase-Induced Stipe Wall Extension of Coprinopsis cinerea.

ABSTRACT: This study reports that a high concentration of the endo-?-1,3-glucanase ENG (200??g ml^-1) induced heat-inactivated stipe wall extension of Coprinopsis cinerea, whereas a high concentration of the extracellular ?-glucosidase BGL2 (1,000??g ml^-1) did not; however, in combination, low concentrations of ENG (25??g ml^-1) and BGL2 (260??g ml^-1) induced heat-inactivated stipe cell wall extension. In contrast to the previously reported chitinase-reconstituted stipe wall extension, ?-1,3-glucanase-reconstituted heat-inactivated stipe cell wall extension initially exhibited a fast extension rate that quickly decreased to zero after approximately 60?min; the stipe cell wall extension induced by a high concentration of ?-1,3-glucanase did not result in stipe breakage during measurement, and the inner surfaces of glucanase-reconstituted extended cell walls still remained as amorphous matrices that did not appear to have been damaged. These distinctive features of the ?-1,3-glucanase-reconstituted wall extension may be because chitin chains are cross-linked not only to the nonreducing termini of the side chains and the backbones of ?-1,6 branched ?-1,3-glucans but also to other polysaccharides. Remarkably, a low concentration of either the ?-1,3-glucanase ENG or of chitinase ChiE1 did not induce heat-inactivated stipe wall extension, but a combination of these two enzymes, each at a low concentration, showed stipe cell wall extension activity that exhibited a steady and continuous wall extension profile. Therefore, we concluded that the stipe cell wall extension is the result of the synergistic actions of glucanases and chitinases.IMPORTANCE We previously reported that the chitinase could induce stipe wall extension and was involved in stipe elongation growth of the mushroom Coprinopsis cinerea In this study, we explored that ?-1,3-glucanase also induced stipe cell wall extension. Interestingly, the extension profile and extended ultra-architecture of ?-1,3-glucanase-reconstituted stipe wall were different from those of chitinase-reconstituted stipe wall. However, ?-1,3-glucanase cooperated with chitinase to induce stipe cell wall extension. The significance of this synergy between glucanases and chitinases is that it enables a low concentration of active enzymes to induce wall extension, and the involvement of ?-1,3-glucanases is necessary for the cell wall remodeling and the addition of new ?-glucans during stipe elongation growth.

SUBMITTER: Kang L

PROVIDER: S-EPMC6803297 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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Glucanase-Induced Stipe Wall Extension Shows Distinct Differences from Chitinase-Induced Stipe Wall Extension of Coprinopsis cinerea.

Kang Liqin L Zhou Jiangsheng J Wang Rui R Zhang Xingwei X Liu Cuicui C Liu Zhonghua Z Yuan Sheng S

Applied and environmental microbiology 20191016 21

This study reports that a high concentration of the endo-β-1,3-glucanase ENG (200 μg ml-1) induced heat-inactivated stipe wall extension of Coprinopsis cinerea, whereas a high concentration of the extracellular β-glucosidase BGL2 (1,000 μg ml-1) did not; however, in combination, low concentrations of ENG (25 μg ml-1) and BGL2 (260 μg ml-1) induced heat-inactivated stipe cell wall extension. In contrast to the previously reported chitinase-recons ...[more]

PMID: 31444203

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Project description:The elongation growth of the mushroom stipe is a characteristic but not well-understood morphogenetic event of basidiomycetes. We found that extending native stipe cell walls of Coprinopsis cinerea were associated with the release of N-acetylglucosamine and chitinbiose and with chitinase activity. Two chitinases among all detected chitinases from C. cinerea, ChiE1 and ChiIII, reconstituted heat-inactivated stipe wall extension and released N-acetylglucosamine and chitinbiose. Interestingly, both ChiE1 and ChiIII hydrolyze insoluble crystalline chitin powder, while other C. cinerea chitinases do not, suggesting that crystalline chitin components of the stipe cell wall are the target of action for ChiE1 and ChiIII. ChiE1- or ChiIII-reconstituted heat-inactivated stipe walls showed maximal extension activity at pH 4.5, consistent with the optimal pH for native stipe wall extension in vitro; ChiE1- or ChiIII-reconstituted heat-inactivated stipe wall extension activities were associated with stipe elongation growth regions; and the combination of ChiE1 and ChiIII showed a synergism to reconstitute heat-inactivated stipe wall extension at a low action concentration. Field emission scanning electron microscopy (FESEM) images showed that the inner surface of acid-induced extended native stipe cell walls and ChiE1- or ChiIII-reconstituted extended heat-inactivated stipe cell walls exhibited a partially broken parallel microfibril architecture; however, these broken transversely arranged microfibrils were not observed in the unextended stipe cell walls that were induced by neutral pH buffer or heat inactivation. Double knockdown of ChiE1 and ChiIII resulted in the reduction of stipe elongation, mycelium growth, and heat-sensitive cell wall extension of native stipes. These results indicate a chitinase-hydrolyzing mechanism for stipe cell wall extension.IMPORTANCE A remarkable feature in the development of basidiomycete fruiting bodies is stipe elongation growth that results primarily from manifold cell elongation. Some scientists have suggested that stipe elongation is the result of enzymatic hydrolysis of cell wall polysaccharides, while other scientists have proposed the possibility that stipe elongation results from nonhydrolytic disruption of the hydrogen bonds between cell wall polysaccharides. Here, we show direct evidence for a chitinase-hydrolyzing mechanism of stipe cell wall elongation in the model mushroom Coprinopsis cinerea that is different from the expansin nonhydrolysis mechanism of plant cell wall extension. We presumed that in the growing stipe cell walls, parallel chitin microfibrils are tethered by β-1,6-branched β-1,3-glucans, and that the breaking of the tether by chitinases leads to separation of these microfibrils to increase their spacing for insertion of new synthesized chitin and β-1,3-glucans under turgor pressure in vivo.

Project description:Endo-β-N-acetylglucosaminidase (ENGase), which catalyzes hydrolysis of N-linked oligosaccharides, is a useful tool for analyzing oligosaccharide contents of glycoproteins. However, there are only a few known ENGases that can catalyze the hydrolysis of human complex type oligosaccharides, and although commercially available, they are expensive. Here, we report the cloning of two ENGase encoding cDNAs from the basidiomycete fungus Coprinopsis cinerea, Endo-CC1 and Endo-CC2. We successfully expressed recombinant His6-tagged Endo-CC1 and Endo-CC2 in Escherichia coli and purified them for enzymatic characterization. Both Endo-CC1 and Endo-CC2 showed hydrolytic activity on high-mannose and complex type oligosaccharides. Since Endo-CC1 could be prepared more easily than Endo-CC2 from E. coli cultures, we examined the enzymatic properties of Endo-CC1 in detail. Our results showed that Endo-CC1 acted on both N-linked high-mannose type and sialobiantennary type complex oligosaccharides of glycoproteins RNase B and human transferrin, respectively, but not on the sialotriantennary type complex oligosaccharide of glycoprotein fetuin. Examination of the transglycosylation activity of Endo-CC1 revealed that the wild-type Endo-CC1 could not transfer the sialobiantennary type complex oligosaccharide onto the deglycosylated RNase B. To obtain an Endo-CC1 mutant with desired transglycosylation activity, we performed mutation analysis of the active site residue Asn 180 (N180), known to be important for catalysis, by individually replacing it with the remaining 19 amino acid residues. Transglycosylation analyses of these mutants led us to identify one mutant, namely Endo-CC1N180H, which exhibited the desired transglycosylation activity. Taken together, we suggest that Endo-CC1 would potentially be a valuable tool for analyzing oligosaccharides on glycoproteins, as large quantities of it could be made available more easily and less expensively than the currently used enzyme, Endo-M.

Dataset Information

Glucanase-Induced Stipe Wall Extension Shows Distinct Differences from Chitinase-Induced Stipe Wall Extension of Coprinopsis cinerea.

Publications

Glucanase-Induced Stipe Wall Extension Shows Distinct Differences from Chitinase-Induced Stipe Wall Extension of Coprinopsis cinerea.

Similar Datasets

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