Characterization of the Neurospora crassa GH72 family of Laminarin/Lichenin transferases and their roles in cell wall biogenesis
In Neurospora crassa vegetative hyphae, chitin, β-1,3-glucan (laminarin), and a mixed β-1,3−/β-1,4-glucan (lichenin) are the major cell wall polysaccharides. GH72 enzymes have been shown to function as β-1,3-glucanases and glucanosyltransferases and can function in crosslinking β-1,3-glucans togethe...
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Elsevier
2025-06-01
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| Series: | The Cell Surface |
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| author | Apurva Chatrath Pavan Patel Protyusha Dey Stephen J. Free |
| author_facet | Apurva Chatrath Pavan Patel Protyusha Dey Stephen J. Free |
| author_sort | Apurva Chatrath |
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| description | In Neurospora crassa vegetative hyphae, chitin, β-1,3-glucan (laminarin), and a mixed β-1,3−/β-1,4-glucan (lichenin) are the major cell wall polysaccharides. GH72 enzymes have been shown to function as β-1,3-glucanases and glucanosyltransferases and can function in crosslinking β-1,3-glucans together. To characterize the enzymatic activities of the N. crassa enzymes, we expressed GEL-1 with a HIS6 tag in N. crassa. A chimeric maltose binding protein:GEL-2 was produced in E. coli. Purified GEL-1 and GEL-2 were used to characterize their enzymatic activities. We employed thin-layer chromatography (TLC) and polyacrylamide carbohydrate gel electrophoresis (PACE) assays to visualize GEL-1 and GEL-2 hydrolase and transferase activities on lichenin and laminarin substrates. We determined that GEL-1 functions as a laminarinase (β-1,3-glucanase) and as a laminarin transferase. We found that GEL-2 can function as a laminarinase and as a licheninase (β-1,3−/β-1,4-mixed-glucanase) and can crosslink β-1,3-glucans together. We demonstrated that GEL-2 can form enzyme:lichenin intermediates, providing evidence that GEL-2 functions as a lichenin transferase as well as a β-1,3-glucan transferase and crosslinks both types of polysaccharides into the N. crassa cell wall. |
| format | Article |
| id | doaj-art-c534bcbd078049bf818ca5c2b390c97c |
| institution | Kabale University |
| issn | 2468-2330 |
| language | English |
| publishDate | 2025-06-01 |
| publisher | Elsevier |
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| series | The Cell Surface |
| spelling | doaj-art-c534bcbd078049bf818ca5c2b390c97c2025-01-06T04:08:52ZengElsevierThe Cell Surface2468-23302025-06-0113100140Characterization of the Neurospora crassa GH72 family of Laminarin/Lichenin transferases and their roles in cell wall biogenesisApurva Chatrath0Pavan Patel1Protyusha Dey2Stephen J. Free3Department of Biological Sciences, SUNY University at Buffalo, Buffalo, NY 14260, United StatesDepartment of Biological Sciences, SUNY University at Buffalo, Buffalo, NY 14260, United StatesDepartment of Biological Sciences, SUNY University at Buffalo, Buffalo, NY 14260, United StatesCorresponding author at: Department of Biological Sciences, SUNY University at Buffalo, 109 Cooke Hall, Buffalo, NY 14260, United States.; Department of Biological Sciences, SUNY University at Buffalo, Buffalo, NY 14260, United StatesIn Neurospora crassa vegetative hyphae, chitin, β-1,3-glucan (laminarin), and a mixed β-1,3−/β-1,4-glucan (lichenin) are the major cell wall polysaccharides. GH72 enzymes have been shown to function as β-1,3-glucanases and glucanosyltransferases and can function in crosslinking β-1,3-glucans together. To characterize the enzymatic activities of the N. crassa enzymes, we expressed GEL-1 with a HIS6 tag in N. crassa. A chimeric maltose binding protein:GEL-2 was produced in E. coli. Purified GEL-1 and GEL-2 were used to characterize their enzymatic activities. We employed thin-layer chromatography (TLC) and polyacrylamide carbohydrate gel electrophoresis (PACE) assays to visualize GEL-1 and GEL-2 hydrolase and transferase activities on lichenin and laminarin substrates. We determined that GEL-1 functions as a laminarinase (β-1,3-glucanase) and as a laminarin transferase. We found that GEL-2 can function as a laminarinase and as a licheninase (β-1,3−/β-1,4-mixed-glucanase) and can crosslink β-1,3-glucans together. We demonstrated that GEL-2 can form enzyme:lichenin intermediates, providing evidence that GEL-2 functions as a lichenin transferase as well as a β-1,3-glucan transferase and crosslinks both types of polysaccharides into the N. crassa cell wall.http://www.sciencedirect.com/science/article/pii/S2468233024000227GH72 familyLichenin transferaseLaminarin transferaseFungal cell wallNeurospora crassa |
| spellingShingle | Apurva Chatrath Pavan Patel Protyusha Dey Stephen J. Free Characterization of the Neurospora crassa GH72 family of Laminarin/Lichenin transferases and their roles in cell wall biogenesis The Cell Surface GH72 family Lichenin transferase Laminarin transferase Fungal cell wall Neurospora crassa |
| title | Characterization of the Neurospora crassa GH72 family of Laminarin/Lichenin transferases and their roles in cell wall biogenesis |
| title_full | Characterization of the Neurospora crassa GH72 family of Laminarin/Lichenin transferases and their roles in cell wall biogenesis |
| title_fullStr | Characterization of the Neurospora crassa GH72 family of Laminarin/Lichenin transferases and their roles in cell wall biogenesis |
| title_full_unstemmed | Characterization of the Neurospora crassa GH72 family of Laminarin/Lichenin transferases and their roles in cell wall biogenesis |
| title_short | Characterization of the Neurospora crassa GH72 family of Laminarin/Lichenin transferases and their roles in cell wall biogenesis |
| title_sort | characterization of the neurospora crassa gh72 family of laminarin lichenin transferases and their roles in cell wall biogenesis |
| topic | GH72 family Lichenin transferase Laminarin transferase Fungal cell wall Neurospora crassa |
| url | http://www.sciencedirect.com/science/article/pii/S2468233024000227 |
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