Detection of VAMP Proteolysis by Tetanus and Botulinum Neurotoxin Type B In Vivo with a Cleavage-Specific Antibody
Tetanus and Botulinum type B neurotoxins are bacterial metalloproteases that specifically cleave the vesicle-associated membrane protein VAMP at an identical peptide bond, resulting in inhibition of neuroexocytosis. The minute amounts of these neurotoxins commonly used in experimental animals are no...
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MDPI AG
2022-04-01
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| Series: | International Journal of Molecular Sciences |
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| author | Federico Fabris Petra Šoštarić Ivica Matak Thomas Binz Anna Toffan Morena Simonato Cesare Montecucco Marco Pirazzini Ornella Rossetto |
| author_facet | Federico Fabris Petra Šoštarić Ivica Matak Thomas Binz Anna Toffan Morena Simonato Cesare Montecucco Marco Pirazzini Ornella Rossetto |
| author_sort | Federico Fabris |
| collection | DOAJ |
| description | Tetanus and Botulinum type B neurotoxins are bacterial metalloproteases that specifically cleave the vesicle-associated membrane protein VAMP at an identical peptide bond, resulting in inhibition of neuroexocytosis. The minute amounts of these neurotoxins commonly used in experimental animals are not detectable, nor is detection of their VAMP substrate sensitive enough. The immune detection of the cleaved substrate is much more sensitive, as we have previously shown for botulinum neurotoxin type A. Here, we describe the production in rabbit of a polyclonal antibody raised versus a peptide encompassing the 13 residues C-terminal with respect to the neurotoxin cleavage site. The antibody was affinity purified and found to recognize, with high specificity and selectivity, the novel N-terminus of VAMP that becomes exposed after cleavage by tetanus toxin and botulinum toxin type B. This antibody recognizes the neoepitope not only in native and denatured VAMP but also in cultured neurons and in neurons in vivo in neurotoxin-treated mice or rats, suggesting the great potential of this novel tool to elucidate tetanus and botulinum B toxin activity in vivo. |
| first_indexed | 2024-03-09T13:32:00Z |
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| id | doaj.art-470fb1f84e3d4b83bf2a47cb73d2522d |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-09T13:32:00Z |
| publishDate | 2022-04-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-470fb1f84e3d4b83bf2a47cb73d2522d2023-11-30T21:16:07ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672022-04-01238435510.3390/ijms23084355Detection of VAMP Proteolysis by Tetanus and Botulinum Neurotoxin Type B In Vivo with a Cleavage-Specific AntibodyFederico Fabris0Petra Šoštarić1Ivica Matak2Thomas Binz3Anna Toffan4Morena Simonato5Cesare Montecucco6Marco Pirazzini7Ornella Rossetto8Department of Biomedical Sciences, University of Padova, Via Ugo Bassi 58/B, 35131 Padova, ItalyDepartment of Pharmacology, School of Medicine, University of Zagreb, Šalata 11, 10000 Zagreb, CroatiaDepartment of Pharmacology, School of Medicine, University of Zagreb, Šalata 11, 10000 Zagreb, CroatiaInstitute of Cellular Biochemistry, Hannover Medical School, Carl-Neuberg-Straße 1, 30625 Hannover, GermanyIstituto Zooprofilattico Sperimentale delle Venezie, Viale dell’Università 10, 35020 Legnaro, ItalyInstitute of Neuroscience, Italian Research Council, University of Padova, Via Ugo Bassi 58/B, 35131 Padova, ItalyDepartment of Biomedical Sciences, University of Padova, Via Ugo Bassi 58/B, 35131 Padova, ItalyDepartment of Biomedical Sciences, University of Padova, Via Ugo Bassi 58/B, 35131 Padova, ItalyDepartment of Biomedical Sciences, University of Padova, Via Ugo Bassi 58/B, 35131 Padova, ItalyTetanus and Botulinum type B neurotoxins are bacterial metalloproteases that specifically cleave the vesicle-associated membrane protein VAMP at an identical peptide bond, resulting in inhibition of neuroexocytosis. The minute amounts of these neurotoxins commonly used in experimental animals are not detectable, nor is detection of their VAMP substrate sensitive enough. The immune detection of the cleaved substrate is much more sensitive, as we have previously shown for botulinum neurotoxin type A. Here, we describe the production in rabbit of a polyclonal antibody raised versus a peptide encompassing the 13 residues C-terminal with respect to the neurotoxin cleavage site. The antibody was affinity purified and found to recognize, with high specificity and selectivity, the novel N-terminus of VAMP that becomes exposed after cleavage by tetanus toxin and botulinum toxin type B. This antibody recognizes the neoepitope not only in native and denatured VAMP but also in cultured neurons and in neurons in vivo in neurotoxin-treated mice or rats, suggesting the great potential of this novel tool to elucidate tetanus and botulinum B toxin activity in vivo.https://www.mdpi.com/1422-0067/23/8/4355botulinum neurotoxinstetanus neurotoxinsvesicle-associated membrane protein VAMPSNARE proteinspolyclonal antibodies |
| spellingShingle | Federico Fabris Petra Šoštarić Ivica Matak Thomas Binz Anna Toffan Morena Simonato Cesare Montecucco Marco Pirazzini Ornella Rossetto Detection of VAMP Proteolysis by Tetanus and Botulinum Neurotoxin Type B In Vivo with a Cleavage-Specific Antibody International Journal of Molecular Sciences botulinum neurotoxins tetanus neurotoxins vesicle-associated membrane protein VAMP SNARE proteins polyclonal antibodies |
| title | Detection of VAMP Proteolysis by Tetanus and Botulinum Neurotoxin Type B In Vivo with a Cleavage-Specific Antibody |
| title_full | Detection of VAMP Proteolysis by Tetanus and Botulinum Neurotoxin Type B In Vivo with a Cleavage-Specific Antibody |
| title_fullStr | Detection of VAMP Proteolysis by Tetanus and Botulinum Neurotoxin Type B In Vivo with a Cleavage-Specific Antibody |
| title_full_unstemmed | Detection of VAMP Proteolysis by Tetanus and Botulinum Neurotoxin Type B In Vivo with a Cleavage-Specific Antibody |
| title_short | Detection of VAMP Proteolysis by Tetanus and Botulinum Neurotoxin Type B In Vivo with a Cleavage-Specific Antibody |
| title_sort | detection of vamp proteolysis by tetanus and botulinum neurotoxin type b in vivo with a cleavage specific antibody |
| topic | botulinum neurotoxins tetanus neurotoxins vesicle-associated membrane protein VAMP SNARE proteins polyclonal antibodies |
| url | https://www.mdpi.com/1422-0067/23/8/4355 |
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