Tetanus toxin fragments and Bcl-2 fusion proteins: cytoprotection and retrograde axonal migration
Abstract Background Tetanus neurotoxin (TeNT) is taken up at nerve terminals and undergoes retrograde migration. The toxic properties of TeNT reside in the toxin light chain (L), but like complete TeNT, the TeNT heavy chain (TTH) and the C-terminal domain (TTC) alone can bind and enter into neurons....
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2018-06-01
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| Series: | BMC Biotechnology |
| Subjects: | |
| Online Access: | http://link.springer.com/article/10.1186/s12896-018-0452-z |
| _version_ | 1818061405677420544 |
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| author | Yasuhiro Watanabe Takashi Matsuba Mami Nakanishi Mio Une Ritsuko Hanajima Kenji Nakashima |
| author_facet | Yasuhiro Watanabe Takashi Matsuba Mami Nakanishi Mio Une Ritsuko Hanajima Kenji Nakashima |
| author_sort | Yasuhiro Watanabe |
| collection | DOAJ |
| description | Abstract Background Tetanus neurotoxin (TeNT) is taken up at nerve terminals and undergoes retrograde migration. The toxic properties of TeNT reside in the toxin light chain (L), but like complete TeNT, the TeNT heavy chain (TTH) and the C-terminal domain (TTC) alone can bind and enter into neurons. Here, we explored whether atoxic fragments of TeNT could act as drug delivery vehicles in neurons. In this study, we used Bcl-2, a protein known to have anti-apoptotic properties in vivo and in vitro, as a parcel to couple to TeNT fragments. Results We expressed Bcl-2 and the TTC fragments alone, and also attempted to express fusion proteins with the Bcl-2 coupled at the N-terminus of TTH (Bcl2-TTH) and the N- and C-terminus of TTC (TTC-Bcl2 and Bcl2-TTC) in mammalian (Cos7 cells) and Escherichia coli systems. TTC and Bcl-2 were efficiently expressed in E. coli and Cos7 cells, respectively, but Bcl-2 and the fusion proteins did not express well in E. coli. The fusion proteins were also not expressed in Cos7 cells. To improve the yield and purity of the fusion protein, we genetically deleted the N-terminal half of TTC from the Bcl2-TTC fusion to yield Bcl2-hTTC. Purified Bcl2-hTTC exhibited neuronal binding and prevented cell death of neuronal PC12 cells induced by serum and NGF deprivation, as evidenced by the inhibition of cytochrome C release from the mitochondria. For in vivo assays, Bcl2-hTTC was injected into the tongues of mice and was seen to selectively migrate to hypoglossal nuclei mouse brain stems via retrograde axonal transport. Conclusions These results indicate that Bcl2-hTTC retains both Bcl-2 and TTC functions and therefore could be a potent therapeutic agent for various neurological conditions. |
| first_indexed | 2024-12-10T13:47:48Z |
| format | Article |
| id | doaj.art-18fafcfe3f68453f8c85f7a5355d4c13 |
| institution | Directory Open Access Journal |
| issn | 1472-6750 |
| language | English |
| last_indexed | 2024-12-10T13:47:48Z |
| publishDate | 2018-06-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Biotechnology |
| spelling | doaj.art-18fafcfe3f68453f8c85f7a5355d4c132022-12-22T01:46:20ZengBMCBMC Biotechnology1472-67502018-06-0118111010.1186/s12896-018-0452-zTetanus toxin fragments and Bcl-2 fusion proteins: cytoprotection and retrograde axonal migrationYasuhiro Watanabe0Takashi Matsuba1Mami Nakanishi2Mio Une3Ritsuko Hanajima4Kenji Nakashima5Division of Neurology, Department of Brain and Neurosciences, Faculty of Medicine, Tottori UniversityDivision of Bacteriology, Department of Microbiology and immunology, Faculty of Medicine, Tottori UniversityDivision of Neurology, Department of Brain and Neurosciences, Faculty of Medicine, Tottori UniversityDivision of Neurology, Department of Brain and Neurosciences, Faculty of Medicine, Tottori UniversityDivision of Neurology, Department of Brain and Neurosciences, Faculty of Medicine, Tottori UniversityDivision of Neurology, Department of Brain and Neurosciences, Faculty of Medicine, Tottori UniversityAbstract Background Tetanus neurotoxin (TeNT) is taken up at nerve terminals and undergoes retrograde migration. The toxic properties of TeNT reside in the toxin light chain (L), but like complete TeNT, the TeNT heavy chain (TTH) and the C-terminal domain (TTC) alone can bind and enter into neurons. Here, we explored whether atoxic fragments of TeNT could act as drug delivery vehicles in neurons. In this study, we used Bcl-2, a protein known to have anti-apoptotic properties in vivo and in vitro, as a parcel to couple to TeNT fragments. Results We expressed Bcl-2 and the TTC fragments alone, and also attempted to express fusion proteins with the Bcl-2 coupled at the N-terminus of TTH (Bcl2-TTH) and the N- and C-terminus of TTC (TTC-Bcl2 and Bcl2-TTC) in mammalian (Cos7 cells) and Escherichia coli systems. TTC and Bcl-2 were efficiently expressed in E. coli and Cos7 cells, respectively, but Bcl-2 and the fusion proteins did not express well in E. coli. The fusion proteins were also not expressed in Cos7 cells. To improve the yield and purity of the fusion protein, we genetically deleted the N-terminal half of TTC from the Bcl2-TTC fusion to yield Bcl2-hTTC. Purified Bcl2-hTTC exhibited neuronal binding and prevented cell death of neuronal PC12 cells induced by serum and NGF deprivation, as evidenced by the inhibition of cytochrome C release from the mitochondria. For in vivo assays, Bcl2-hTTC was injected into the tongues of mice and was seen to selectively migrate to hypoglossal nuclei mouse brain stems via retrograde axonal transport. Conclusions These results indicate that Bcl2-hTTC retains both Bcl-2 and TTC functions and therefore could be a potent therapeutic agent for various neurological conditions.http://link.springer.com/article/10.1186/s12896-018-0452-zApoptosisBcl-2TeNTTTCFusion proteinNeuron |
| spellingShingle | Yasuhiro Watanabe Takashi Matsuba Mami Nakanishi Mio Une Ritsuko Hanajima Kenji Nakashima Tetanus toxin fragments and Bcl-2 fusion proteins: cytoprotection and retrograde axonal migration BMC Biotechnology Apoptosis Bcl-2 TeNT TTC Fusion protein Neuron |
| title | Tetanus toxin fragments and Bcl-2 fusion proteins: cytoprotection and retrograde axonal migration |
| title_full | Tetanus toxin fragments and Bcl-2 fusion proteins: cytoprotection and retrograde axonal migration |
| title_fullStr | Tetanus toxin fragments and Bcl-2 fusion proteins: cytoprotection and retrograde axonal migration |
| title_full_unstemmed | Tetanus toxin fragments and Bcl-2 fusion proteins: cytoprotection and retrograde axonal migration |
| title_short | Tetanus toxin fragments and Bcl-2 fusion proteins: cytoprotection and retrograde axonal migration |
| title_sort | tetanus toxin fragments and bcl 2 fusion proteins cytoprotection and retrograde axonal migration |
| topic | Apoptosis Bcl-2 TeNT TTC Fusion protein Neuron |
| url | http://link.springer.com/article/10.1186/s12896-018-0452-z |
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