Construction of a Yeast Cell-Based Assay System to Analyze SNAP25-Targeting Botulinum Neurotoxins
Herein, we describe a yeast cell-based assay system to analyze SNAP25-targeting botulinum neurotoxins (BoNTs). BoNTs are protein toxins, and, upon incorporation into neuronal cells, their light chains (BoNT-LCs) target specific synaptosomal N-ethylmaleimide-sensitive attachment protein receptor (SNA...
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MDPI AG
2023-04-01
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| Series: | Microorganisms |
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| Online Access: | https://www.mdpi.com/2076-2607/11/5/1125 |
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| author | Shilin Chen Feng Li Guoyu Liu Yuqing Li Zijie Li Yishi Liu Hideki Nakanishi |
| author_facet | Shilin Chen Feng Li Guoyu Liu Yuqing Li Zijie Li Yishi Liu Hideki Nakanishi |
| author_sort | Shilin Chen |
| collection | DOAJ |
| description | Herein, we describe a yeast cell-based assay system to analyze SNAP25-targeting botulinum neurotoxins (BoNTs). BoNTs are protein toxins, and, upon incorporation into neuronal cells, their light chains (BoNT-LCs) target specific synaptosomal N-ethylmaleimide-sensitive attachment protein receptor (SNARE) proteins, including synaptosomal-associated protein 25 (SNAP25). BoNT-LCs are metalloproteases, and each BoNT-LC recognizes and cleaves conserved domains in SNAREs termed the SNARE domain. In the budding yeast <i>Saccharomyces cerevisiae</i>, the SNAP25 ortholog Spo20 is required for production of the spore plasma membrane; thus, defects in Spo20 cause sporulation deficiencies. We found that chimeric SNAREs in which SNARE domains in Spo20 are replaced with those of SNAP25 are functional in yeast cells. The Spo20/SNAP25 chimeras, but not Spo20, are sensitive to digestion by BoNT-LCs. We demonstrate that <i>spo20</i>∆ yeasts harboring the chimeras exhibit sporulation defects when various SNAP25-targeting BoNT-LCs are expressed. Thus, the activities of BoNT-LCs can be assessed by colorimetric measurement of sporulation efficiencies. Although BoNTs are notorious toxins, they are also used as therapeutic and cosmetic agents. Our assay system will be useful for analyzing novel BoNTs and BoNT-like genes, as well as their manipulation. |
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| institution | Directory Open Access Journal |
| issn | 2076-2607 |
| language | English |
| last_indexed | 2024-03-11T03:28:35Z |
| publishDate | 2023-04-01 |
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| record_format | Article |
| series | Microorganisms |
| spelling | doaj.art-95a8eb020d9e4b1f8436dcfe20e9b5fe2023-11-18T02:32:11ZengMDPI AGMicroorganisms2076-26072023-04-01115112510.3390/microorganisms11051125Construction of a Yeast Cell-Based Assay System to Analyze SNAP25-Targeting Botulinum NeurotoxinsShilin Chen0Feng Li1Guoyu Liu2Yuqing Li3Zijie Li4Yishi Liu5Hideki Nakanishi6The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214126, ChinaThe Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214126, ChinaThe Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214126, ChinaThe Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214126, ChinaThe Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214126, ChinaThe Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214126, ChinaThe Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214126, ChinaHerein, we describe a yeast cell-based assay system to analyze SNAP25-targeting botulinum neurotoxins (BoNTs). BoNTs are protein toxins, and, upon incorporation into neuronal cells, their light chains (BoNT-LCs) target specific synaptosomal N-ethylmaleimide-sensitive attachment protein receptor (SNARE) proteins, including synaptosomal-associated protein 25 (SNAP25). BoNT-LCs are metalloproteases, and each BoNT-LC recognizes and cleaves conserved domains in SNAREs termed the SNARE domain. In the budding yeast <i>Saccharomyces cerevisiae</i>, the SNAP25 ortholog Spo20 is required for production of the spore plasma membrane; thus, defects in Spo20 cause sporulation deficiencies. We found that chimeric SNAREs in which SNARE domains in Spo20 are replaced with those of SNAP25 are functional in yeast cells. The Spo20/SNAP25 chimeras, but not Spo20, are sensitive to digestion by BoNT-LCs. We demonstrate that <i>spo20</i>∆ yeasts harboring the chimeras exhibit sporulation defects when various SNAP25-targeting BoNT-LCs are expressed. Thus, the activities of BoNT-LCs can be assessed by colorimetric measurement of sporulation efficiencies. Although BoNTs are notorious toxins, they are also used as therapeutic and cosmetic agents. Our assay system will be useful for analyzing novel BoNTs and BoNT-like genes, as well as their manipulation.https://www.mdpi.com/2076-2607/11/5/1125botulinum neurotoxinsyeast cell-based assaySNARESNAP25sporulation |
| spellingShingle | Shilin Chen Feng Li Guoyu Liu Yuqing Li Zijie Li Yishi Liu Hideki Nakanishi Construction of a Yeast Cell-Based Assay System to Analyze SNAP25-Targeting Botulinum Neurotoxins Microorganisms botulinum neurotoxins yeast cell-based assay SNARE SNAP25 sporulation |
| title | Construction of a Yeast Cell-Based Assay System to Analyze SNAP25-Targeting Botulinum Neurotoxins |
| title_full | Construction of a Yeast Cell-Based Assay System to Analyze SNAP25-Targeting Botulinum Neurotoxins |
| title_fullStr | Construction of a Yeast Cell-Based Assay System to Analyze SNAP25-Targeting Botulinum Neurotoxins |
| title_full_unstemmed | Construction of a Yeast Cell-Based Assay System to Analyze SNAP25-Targeting Botulinum Neurotoxins |
| title_short | Construction of a Yeast Cell-Based Assay System to Analyze SNAP25-Targeting Botulinum Neurotoxins |
| title_sort | construction of a yeast cell based assay system to analyze snap25 targeting botulinum neurotoxins |
| topic | botulinum neurotoxins yeast cell-based assay SNARE SNAP25 sporulation |
| url | https://www.mdpi.com/2076-2607/11/5/1125 |
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