Cargando…
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...
Autores principales: | , , , , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2022
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9024618/ https://www.ncbi.nlm.nih.gov/pubmed/35457172 http://dx.doi.org/10.3390/ijms23084355 |
_version_ | 1784690645109047296 |
---|---|
author | Fabris, Federico Šoštarić, Petra Matak, Ivica Binz, Thomas Toffan, Anna Simonato, Morena Montecucco, Cesare Pirazzini, Marco Rossetto, Ornella |
author_facet | Fabris, Federico Šoštarić, Petra Matak, Ivica Binz, Thomas Toffan, Anna Simonato, Morena Montecucco, Cesare Pirazzini, Marco Rossetto, Ornella |
author_sort | Fabris, Federico |
collection | PubMed |
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. |
format | Online Article Text |
id | pubmed-9024618 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-90246182022-04-23 Detection of VAMP Proteolysis by Tetanus and Botulinum Neurotoxin Type B In Vivo with a Cleavage-Specific Antibody Fabris, Federico Šoštarić, Petra Matak, Ivica Binz, Thomas Toffan, Anna Simonato, Morena Montecucco, Cesare Pirazzini, Marco Rossetto, Ornella Int J Mol Sci Article 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. MDPI 2022-04-14 /pmc/articles/PMC9024618/ /pubmed/35457172 http://dx.doi.org/10.3390/ijms23084355 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Fabris, Federico Šoštarić, Petra Matak, Ivica Binz, Thomas Toffan, Anna Simonato, Morena Montecucco, Cesare Pirazzini, Marco Rossetto, Ornella Detection of VAMP Proteolysis by Tetanus and Botulinum Neurotoxin Type B In Vivo with a Cleavage-Specific Antibody |
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 | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9024618/ https://www.ncbi.nlm.nih.gov/pubmed/35457172 http://dx.doi.org/10.3390/ijms23084355 |
work_keys_str_mv | AT fabrisfederico detectionofvampproteolysisbytetanusandbotulinumneurotoxintypebinvivowithacleavagespecificantibody AT sostaricpetra detectionofvampproteolysisbytetanusandbotulinumneurotoxintypebinvivowithacleavagespecificantibody AT matakivica detectionofvampproteolysisbytetanusandbotulinumneurotoxintypebinvivowithacleavagespecificantibody AT binzthomas detectionofvampproteolysisbytetanusandbotulinumneurotoxintypebinvivowithacleavagespecificantibody AT toffananna detectionofvampproteolysisbytetanusandbotulinumneurotoxintypebinvivowithacleavagespecificantibody AT simonatomorena detectionofvampproteolysisbytetanusandbotulinumneurotoxintypebinvivowithacleavagespecificantibody AT montecuccocesare detectionofvampproteolysisbytetanusandbotulinumneurotoxintypebinvivowithacleavagespecificantibody AT pirazzinimarco detectionofvampproteolysisbytetanusandbotulinumneurotoxintypebinvivowithacleavagespecificantibody AT rossettoornella detectionofvampproteolysisbytetanusandbotulinumneurotoxintypebinvivowithacleavagespecificantibody |