The Experts below are selected from a list of 67938 Experts worldwide ranked by ideXlab platform
Giampietro Schiavo - One of the best experts on this subject based on the ideXlab platform.
-
Uptake and transport of clostridial Neurotoxins
The Comprehensive Sourcebook of Bacterial Protein Toxins, 2020Co-Authors: Nathalie Schmieg, Kinga Bercsenyi, Giampietro SchiavoAbstract:Clostridium Neurotoxins (CNT) are the most toxic substances known. Different animals show a great range of sensitivity to Clostridium tetani, named Tetanus Neurotoxin (TeNT) and Clostridium botulinum and its Neurotoxins (Botulinum Neurotoxins, BoNTs). After entering the general circulation, CNT bind to the presynaptic membrane of the neuromuscular junction (NMJ). Following binding, CNT are internalized into the neuron. BoNTs remain at the NMJ and block the release of the excitatory neurotransmitter acetylcholine (ACh), causing a flaccid paralysis. TeNT can bind sensory as well as adrenergic neurons, which exhibit a similar retrograde uptake. Although excitatory synapses appear not to be compromised in the early stages of the disease, they may be inhibited at later periods of TeNT intoxication. CNT binding has been examined using morphological and biochemical techniques in different systems like primary neuronal cultures, cell lines, and various membrane preparations. CNT bind to the presynaptic membrane of cholinergic nerve terminals. TeNT may also bind to sympathetic and adrenergic nerve fibres. The complexity of the CNT receptors, which are composed of multiple lipid and protein components, together with the high affinity of CNT for the neuronal membrane has led to the recent proposal that arrays of presynaptic receptors (APRs) are involved in CNT binding. © 2006 Elsevier Inc. All rights reserved.
-
botulinum Neurotoxins mechanism of action
Toxicon, 2013Co-Authors: Ann P Tighe, Giampietro SchiavoAbstract:Botulinum Neurotoxins are used clinically for conditions characterized by hyperexcitability of peripheral nerve terminals and hypersecretory syndromes. These Neurotoxins are synthesized as precursor proteins with low activity, but their effects are mediated by the active form of the neurotoxin through a multistep mechanism. Following a high-affinity interaction with a protein receptor and polysialogangliosides on the synaptic membrane, botulinum Neurotoxins enter the neuron and causes a sustained inhibition of synaptic transmission. The active neurotoxin is part of a high-molecular-weight complex that protects the neurotoxin from proteolytic degradation. Although complexing proteins do not affect diffusion of therapeutic neurotoxin, they may lead to the development of neutralizing antibodies that block responsiveness to it. Nerve terminal intoxication is reversible and its duration varies for different BoNT serotypes. Although it was previously assumed that botulinum Neurotoxins exert effects only on the peripheral synapses, such as the neuromuscular junction, there is now substantial evidence that these Neurotoxins affect neurotransmission at distal central nervous system sites as well.
-
presynaptic receptor arrays for clostridial Neurotoxins
Trends in Microbiology, 2004Co-Authors: Cesare Montecucco, Ornella Rossetto, Giampietro SchiavoAbstract:Tetanus and botulinum Neurotoxins act at femtomolar concentrations and are specific for the presynaptic membrane of neurons. Their mode of binding is still poorly defined. The exceptional potency and specificity of these Neurotoxins raise unprecedented questions about the nature of their receptor(s) and the mode of their membrane binding. We propose a presynaptic binding process for tetanus and botulinum Neurotoxins based on a capture step performed by an antenna, consisting of a lipid- or a protein-linked oligosaccharide, which brings about a very large membrane concentration effect; this is followed by additional interactions with arrays of receptor molecules, arranged in membrane microdomains, which render the neurotoxin binding practically irreversible and triggers endocytosis.
-
tetanus and botulinum Neurotoxins turning bad guys into good by research
Toxicon, 2001Co-Authors: Ornella Rossetto, Giampietro Schiavo, M Seveso, Paola Caccin, Cesare MontecuccoAbstract:Abstract The neuroparalytic syndromes of tetanus and botulism are caused by Neurotoxins produced by bacteria of the genus Clostridium . They are 150 kDa proteins consisting of three-domains, endowed with different functions: neurospecific binding, membrane translocation and specific proteolysis of three key components of the neuroexocytosis apparatus. After binding to the presynaptic membrane of motoneurons, tetanus neurotoxin (TeNT) is internalized and transported retroaxonally to the spinal cord, where it blocks neurotransmitter release from spinal inhibitory interneurons. In contrast, the seven botulinum Neurotoxins (BoNT) act at the periphery and inhibit acetylcholine release from peripheral cholinergic nerve terminals. TeNT and BoNT-B, -D, -F and -G cleave specifically at single but different peptide bonds, VAMP/synaptobrevin, a membrane protein of small synaptic vesicles. BoNT types -A, -C and -E cleave SNAP-25 at different sites within the COOH-terminus, whereas BoNT-C also cleaves syntaxin. BoNTs are increasingly used in medicine for the treatment of human diseases characterized by hyperfunction of cholinergic terminals.
-
functional characterisation of tetanus and botulinum Neurotoxins binding domains
Journal of Cell Science, 1999Co-Authors: Giovanna Lalli, Cesare Montecucco, Judit Herreros, Shona L Osborne, Ornella Rossetto, Giampietro SchiavoAbstract:Tetanus and botulinum Neurotoxins constitute a family of bacterial protein toxins responsible for two deadly syndromes in humans (tetanus and botulism, respectively). They bind with high affinity to neurons wherein they cause a complete inhibition of evoked neurotransmitter release. Here we report on the cloning, expression and use of the recombinant fragments of the heavy chains of tetanus neurotoxin and botulinum neurotoxin serotypes A, B and E as tools to study the neurospecific binding of the holotoxins. We found that the recombinant 50 kDa carboxy-terminal domains of tetanus and botulinum Neurotoxins alone are responsible for the specific binding and internalisation into spinal cord cells in culture. Moreover, we provide evidence that the recombinant fragments block the internalization of the parental holotoxins in a dose-dependent manner, as determined by following the neurotoxin-dependent cleavage of their targets VAMP/synaptobrevin and SNAP-25. In addition, the recombinant binding fragments cause a significant delay in the paralysis induced by the corresponding holotoxin on the mouse phrenic nerve-hemidiaphragm preparation. Taken together, these results show that the carboxy-terminal domain of tetanus and botulinum Neurotoxins is necessary and sufficient for the binding and internalisation of these proteins in neurons and open the possibility to use them as tools for the functional characterisation of the intracellular transport of clostridial Neurotoxins.
Cesare Montecucco - One of the best experts on this subject based on the ideXlab platform.
-
Botulinum neurotoxin A1 likes it double sweet
Nature Structural & Molecular Biology, 2016Co-Authors: Cesare Montecucco, Giuseppe ZanottiAbstract:The pathogenesis of the nerve paralysis induced by botulinum Neurotoxins begins with their specific and high-affinity binding to peripheral nerve terminals. The new crystal structure of the toxin bound to its glycosylated receptor, presented in this issue, represents a major step forward in the understanding of how botulinum neurotoxin type A1, the toxin used in human therapy and cosmetics, binds its protein receptor.
-
On Botulinum Neurotoxin Variability
Mbio, 2015Co-Authors: Cesare Montecucco, Maria B. RasottoAbstract:ABSTRACT The rapidly growing number of botulinum neurotoxin sequences poses the problem of the possible evolutionary significance of the variability of these superpotent Neurotoxins for toxin-producing Clostridium species. To progress in the understanding of this remarkable phenomenon, we suggest that researchers should (i) abandon an anthropocentric view of these Neurotoxins as human botulism-causing agents or as human therapeutics, (ii) begin to investigate in depth the role of botulinum Neurotoxins in animal botulism in the wilderness, and (iii) devote large efforts to next-generation sequencing of soil samples to identify novel botulinum Neurotoxins. In order to compare the fitness of the different toxins, we suggest that assays of all the steps from toxin production to animal death should be performed.
-
presynaptic receptor arrays for clostridial Neurotoxins
Trends in Microbiology, 2004Co-Authors: Cesare Montecucco, Ornella Rossetto, Giampietro SchiavoAbstract:Tetanus and botulinum Neurotoxins act at femtomolar concentrations and are specific for the presynaptic membrane of neurons. Their mode of binding is still poorly defined. The exceptional potency and specificity of these Neurotoxins raise unprecedented questions about the nature of their receptor(s) and the mode of their membrane binding. We propose a presynaptic binding process for tetanus and botulinum Neurotoxins based on a capture step performed by an antenna, consisting of a lipid- or a protein-linked oligosaccharide, which brings about a very large membrane concentration effect; this is followed by additional interactions with arrays of receptor molecules, arranged in membrane microdomains, which render the neurotoxin binding practically irreversible and triggers endocytosis.
-
tetanus and botulinum Neurotoxins turning bad guys into good by research
Toxicon, 2001Co-Authors: Ornella Rossetto, Giampietro Schiavo, M Seveso, Paola Caccin, Cesare MontecuccoAbstract:Abstract The neuroparalytic syndromes of tetanus and botulism are caused by Neurotoxins produced by bacteria of the genus Clostridium . They are 150 kDa proteins consisting of three-domains, endowed with different functions: neurospecific binding, membrane translocation and specific proteolysis of three key components of the neuroexocytosis apparatus. After binding to the presynaptic membrane of motoneurons, tetanus neurotoxin (TeNT) is internalized and transported retroaxonally to the spinal cord, where it blocks neurotransmitter release from spinal inhibitory interneurons. In contrast, the seven botulinum Neurotoxins (BoNT) act at the periphery and inhibit acetylcholine release from peripheral cholinergic nerve terminals. TeNT and BoNT-B, -D, -F and -G cleave specifically at single but different peptide bonds, VAMP/synaptobrevin, a membrane protein of small synaptic vesicles. BoNT types -A, -C and -E cleave SNAP-25 at different sites within the COOH-terminus, whereas BoNT-C also cleaves syntaxin. BoNTs are increasingly used in medicine for the treatment of human diseases characterized by hyperfunction of cholinergic terminals.
-
functional characterisation of tetanus and botulinum Neurotoxins binding domains
Journal of Cell Science, 1999Co-Authors: Giovanna Lalli, Cesare Montecucco, Judit Herreros, Shona L Osborne, Ornella Rossetto, Giampietro SchiavoAbstract:Tetanus and botulinum Neurotoxins constitute a family of bacterial protein toxins responsible for two deadly syndromes in humans (tetanus and botulism, respectively). They bind with high affinity to neurons wherein they cause a complete inhibition of evoked neurotransmitter release. Here we report on the cloning, expression and use of the recombinant fragments of the heavy chains of tetanus neurotoxin and botulinum neurotoxin serotypes A, B and E as tools to study the neurospecific binding of the holotoxins. We found that the recombinant 50 kDa carboxy-terminal domains of tetanus and botulinum Neurotoxins alone are responsible for the specific binding and internalisation into spinal cord cells in culture. Moreover, we provide evidence that the recombinant fragments block the internalization of the parental holotoxins in a dose-dependent manner, as determined by following the neurotoxin-dependent cleavage of their targets VAMP/synaptobrevin and SNAP-25. In addition, the recombinant binding fragments cause a significant delay in the paralysis induced by the corresponding holotoxin on the mouse phrenic nerve-hemidiaphragm preparation. Taken together, these results show that the carboxy-terminal domain of tetanus and botulinum Neurotoxins is necessary and sufficient for the binding and internalisation of these proteins in neurons and open the possibility to use them as tools for the functional characterisation of the intracellular transport of clostridial Neurotoxins.
Thomas Binz - One of the best experts on this subject based on the ideXlab platform.
-
thioredoxin and its reductase are present on synaptic vesicles and their inhibition prevents the paralysis induced by botulinum Neurotoxins
Cell Reports, 2014Co-Authors: Marco Pirazzini, Thomas Binz, Clifford C. Shone, Domenico Azarnia Tehran, Giulia Zanetti, Aram Megighian, Michele Scorzeto, Silvia Fillo, O Rossetto, Florigio ListaAbstract:Summary Botulinum Neurotoxins consist of a metalloprotease linked via a conserved interchain disulfide bond to a heavy chain responsible for neurospecific binding and translocation of the enzymatic domain in the nerve terminal cytosol. The metalloprotease activity is enabled upon disulfide reduction and causes neuroparalysis by cleaving the SNARE proteins. Here, we show that the thioredoxin reductase-thioredoxin protein disulfide-reducing system is present on synaptic vesicles and that it is functional and responsible for the reduction of the interchain disulfide of botulinum neurotoxin serotypes A, C, and E. Specific inhibitors of thioredoxin reductase or thioredoxin prevent intoxication of cultured neurons in a dose-dependent manner and are also very effective inhibitors of the paralysis of the neuromuscular junction. We found that this group of inhibitors of botulinum Neurotoxins is very effective in vivo. Most of them are nontoxic and are good candidates as preventive and therapeutic drugs for human botulism.
-
Exchanging the minimal cell binding fragments of tetanus neurotoxin in botulinum neurotoxin A and B impacts their toxicity at the neuromuscular junction and central neurons.
Toxicon, 2013Co-Authors: Markus Höltje, Thomas Binz, Sebastian Schulze, J. Strotmeier, Stefan Mahrhold, Karin Richter, Hans Bigalke, Gudrun Ahnert-hilger, Andreas RummelAbstract:Abstract The modular four domain structure of clostridial Neurotoxins supports the idea to reassemble individual domains from tetanus and botulinum Neurotoxins to generate novel molecules with altered pharmacological properties. To treat disorders of the central nervous system drug transporter molecules based on catalytically inactive clostridial Neurotoxins circumventing the passage of the blood–brain-barrier are desired. Such molecules can be produced based on the highly effective botulinum neurotoxin serotype A incorporating the retrograde axonal sorting property of tetanus neurotoxin which is supposed to be encoded within its C-terminal cell binding domain H C . The corresponding exchange of the tetanus neurotoxin H C -fragment in botulinum neurotoxin A yielded the novel hybrid molecule AATT which displayed decreased potency at the neuromuscular junction like tetanus neurotoxin but exerted equal activity in cortical neurons compared to botulinum neurotoxin A wild-type. Minimizing the tetanus neurotoxin cell binding domain to its N- or C-terminal half drastically reduced the potencies of AATA and AAAT in cortical neurons indicating that the structural motif mediating sorting of tetanus neurotoxin is predominantly encoded within the entire H C -fragment. However, the reciprocal exchange resulted in TTAA which showed a similar potency as tetanus neurotoxin at the neuromuscular junction indicating that the tetanus neurotoxin portion prevents a high potency as observed for botulinum Neurotoxins. In conclusion, clostridial neurotoxin based inactivated drug transporter for targeting central neurons should contain the cell binding domain of tetanus neurotoxin to exert its tropism for the central nervous system.
-
Exocytosis at the hair cell ribbon synapse apparently operates without neuronal SNARE proteins
Nature Neuroscience, 2011Co-Authors: Régis Nouvian, Thomas Binz, Jakob Neef, Anna V. Bulankina, Ellen Reisinger, Tina Pangršič, Stefan Sikorra, Nils Brose, Thomas Frank, Tobias MoserAbstract:SNARE proteins mediate membrane fusion. Neurosecretion depends on neuronal soluble NSF attachment protein receptors (SNAREs; SNAP-25, syntaxin-1, and synaptobrevin-1 or synaptobrevin-2) and is blocked by neurotoxin-mediated cleavage or genetic ablation. We found that exocytosis in mouse inner hair cells (IHCs) was insensitive to Neurotoxins and genetic ablation of neuronal SNAREs. mRNA, but no synaptically localized protein, of neuronal SNAREs was present in IHCs. Thus, IHC exocytosis is unconventional and may operate independently of neuronal SNAREs.
-
botulinum Neurotoxins c e and f bind gangliosides via a conserved binding site prior to stimulation dependent uptake with botulinum neurotoxin f utilising the three isoforms of sv2 as second receptor
Journal of Neurochemistry, 2009Co-Authors: Andreas Rummel, Stefan Mahrhold, Hans Bigalke, Tino Karnath, Kirstin Hafner, Natallia Darashchonak, Matthew Holt, Reinhard Jahn, Silke Beermann, Thomas BinzAbstract:The high toxicity of clostridial Neurotoxins primarily results from their specific binding and uptake into neurons. At motor neurons, the seven botulinum neurotoxin serotypes A–G (BoNT/A–G) inhibit acetylcholine release, leading to flaccid paralysis, while tetanus neurotoxin blocks neurotransmitter release in inhibitory neurons, resulting in spastic paralysis. Uptake of BoNT/A, B, E and G requires a dual interaction with gangliosides and the synaptic vesicle (SV) proteins synaptotagmin or SV2, whereas little is known about the entry mechanisms of the remaining serotypes. Here, we demonstrate that BoNT/F as wells depends on the presence of gangliosides, by employing phrenic nerve hemidiaphragm preparations derived from mice expressing GM3, GM2, GM1 and GD1a or only GM3. Subsequent site-directed mutagenesis based on homology models identified the ganglioside binding site at a conserved location in BoNT/E and F. Using the mice phrenic nerve hemidiaphragm assay as a physiological model system, cross-competition of full-length neurotoxin binding by recombinant binding fragments, plus accelerated neurotoxin uptake upon increased electrical stimulation, indicate that BoNT/F employs SV2 as protein receptor, whereas BoNT/C and D utilise different SV receptor structures. The co-precipitation of SV2A, B and C from Triton-solubilised SVs by BoNT/F underlines this conclusion.
-
cell entry strategy of clostridial Neurotoxins
Journal of Neurochemistry, 2009Co-Authors: Thomas Binz, Andreas RummelAbstract:Tetanus neurotoxin and botulinum Neurotoxins are the causative agents of tetanus and botulism. They block the release of neurotransmitters from synaptic vesicles in susceptible animals and man and act in nanogram quantities because of their ability to specifically attack motoneurons. They developed an ingenious strategy to enter neurons. This involves a concentration step via complex polysialo gangliosides at the plasma membrane and the uptake and ride in recycling synaptic vesicles initiated by binding to a specific protein receptor. Finally, the Neurotoxins shut down the synaptic vesicle cycle, which they had misused before to enter their target cells, via specific cleavage of protein core components of the cellular membrane fusion machinery. The uptake of four out of seven known botulinum Neurotoxins into synaptic vesicles has been demonstrated to rely on binding to intravesicular segments of the synaptic vesicle proteins synaptotagmin or synaptic vesicle protein 2. This review summarizes the present knowledge about the cell receptor molecules and the mode of toxin-receptor interaction that enables the toxins’ sophisticated access to their site of action.
Andreas Rummel - One of the best experts on this subject based on the ideXlab platform.
-
Exchanging the minimal cell binding fragments of tetanus neurotoxin in botulinum neurotoxin A and B impacts their toxicity at the neuromuscular junction and central neurons.
Toxicon, 2013Co-Authors: Markus Höltje, Thomas Binz, Sebastian Schulze, J. Strotmeier, Stefan Mahrhold, Karin Richter, Hans Bigalke, Gudrun Ahnert-hilger, Andreas RummelAbstract:Abstract The modular four domain structure of clostridial Neurotoxins supports the idea to reassemble individual domains from tetanus and botulinum Neurotoxins to generate novel molecules with altered pharmacological properties. To treat disorders of the central nervous system drug transporter molecules based on catalytically inactive clostridial Neurotoxins circumventing the passage of the blood–brain-barrier are desired. Such molecules can be produced based on the highly effective botulinum neurotoxin serotype A incorporating the retrograde axonal sorting property of tetanus neurotoxin which is supposed to be encoded within its C-terminal cell binding domain H C . The corresponding exchange of the tetanus neurotoxin H C -fragment in botulinum neurotoxin A yielded the novel hybrid molecule AATT which displayed decreased potency at the neuromuscular junction like tetanus neurotoxin but exerted equal activity in cortical neurons compared to botulinum neurotoxin A wild-type. Minimizing the tetanus neurotoxin cell binding domain to its N- or C-terminal half drastically reduced the potencies of AATA and AAAT in cortical neurons indicating that the structural motif mediating sorting of tetanus neurotoxin is predominantly encoded within the entire H C -fragment. However, the reciprocal exchange resulted in TTAA which showed a similar potency as tetanus neurotoxin at the neuromuscular junction indicating that the tetanus neurotoxin portion prevents a high potency as observed for botulinum Neurotoxins. In conclusion, clostridial neurotoxin based inactivated drug transporter for targeting central neurons should contain the cell binding domain of tetanus neurotoxin to exert its tropism for the central nervous system.
-
double receptor anchorage of botulinum Neurotoxins accounts for their exquisite neurospecificity
Current Topics in Microbiology and Immunology, 2012Co-Authors: Andreas RummelAbstract:The high potency of the botulinum Neurotoxins (BoNT) and tetanus neurotoxin (TeNT) is mainly due to their neurospecific binding which is mediated by the interaction with two receptor components. TeNT and all BoNT bind first to complex polysialo-gangliosides abundantly present on the outer leaflet of neuronal membranes. The ganglioside binding occurs in BoNT/A, B, E, F and G via a conserved ganglioside binding pocket within the most carboxyl-terminal 25 kDa domain HCC whereas TeNT, BoNT/C and D display two different ganglioside binding sites within their HCC-domain. Subsequently, upon exocytosis the intraluminal domains of synaptic vesicle proteins are exposed and can be accessed by the surface accumulated Neurotoxins. BoNT/B and G bind with their HCC-domain to a 20-mer membrane juxtaposed segment of the intraluminal domain of synaptotagmin-I and -II, respectively. BoNT/A and E employ the intraluminal domain 4 of the synaptic vesicle glycoprotein 2 (SV2) as protein receptor. Whereas the 50 kDa cell binding domain HC of BoNT/A interacts with all three SV2 isoforms, BoNT/E HC only binds SV2A and SV2B. Also, BoNT/D, F, and TeNT employ SV2 for binding and uptake. Thereafter, the synaptic vesicle is recycled and the anchored neurotoxin is endocytosed. Acidification of the vesicle lumen triggers membrane insertion of the translocation domain followed by pore formation and finally translocation of the enzymatically active light chain to its site of action leading to block of neurotransmitter release.
-
botulinum Neurotoxins c e and f bind gangliosides via a conserved binding site prior to stimulation dependent uptake with botulinum neurotoxin f utilising the three isoforms of sv2 as second receptor
Journal of Neurochemistry, 2009Co-Authors: Andreas Rummel, Stefan Mahrhold, Hans Bigalke, Tino Karnath, Kirstin Hafner, Natallia Darashchonak, Matthew Holt, Reinhard Jahn, Silke Beermann, Thomas BinzAbstract:The high toxicity of clostridial Neurotoxins primarily results from their specific binding and uptake into neurons. At motor neurons, the seven botulinum neurotoxin serotypes A–G (BoNT/A–G) inhibit acetylcholine release, leading to flaccid paralysis, while tetanus neurotoxin blocks neurotransmitter release in inhibitory neurons, resulting in spastic paralysis. Uptake of BoNT/A, B, E and G requires a dual interaction with gangliosides and the synaptic vesicle (SV) proteins synaptotagmin or SV2, whereas little is known about the entry mechanisms of the remaining serotypes. Here, we demonstrate that BoNT/F as wells depends on the presence of gangliosides, by employing phrenic nerve hemidiaphragm preparations derived from mice expressing GM3, GM2, GM1 and GD1a or only GM3. Subsequent site-directed mutagenesis based on homology models identified the ganglioside binding site at a conserved location in BoNT/E and F. Using the mice phrenic nerve hemidiaphragm assay as a physiological model system, cross-competition of full-length neurotoxin binding by recombinant binding fragments, plus accelerated neurotoxin uptake upon increased electrical stimulation, indicate that BoNT/F employs SV2 as protein receptor, whereas BoNT/C and D utilise different SV receptor structures. The co-precipitation of SV2A, B and C from Triton-solubilised SVs by BoNT/F underlines this conclusion.
-
cell entry strategy of clostridial Neurotoxins
Journal of Neurochemistry, 2009Co-Authors: Thomas Binz, Andreas RummelAbstract:Tetanus neurotoxin and botulinum Neurotoxins are the causative agents of tetanus and botulism. They block the release of neurotransmitters from synaptic vesicles in susceptible animals and man and act in nanogram quantities because of their ability to specifically attack motoneurons. They developed an ingenious strategy to enter neurons. This involves a concentration step via complex polysialo gangliosides at the plasma membrane and the uptake and ride in recycling synaptic vesicles initiated by binding to a specific protein receptor. Finally, the Neurotoxins shut down the synaptic vesicle cycle, which they had misused before to enter their target cells, via specific cleavage of protein core components of the cellular membrane fusion machinery. The uptake of four out of seven known botulinum Neurotoxins into synaptic vesicles has been demonstrated to rely on binding to intravesicular segments of the synaptic vesicle proteins synaptotagmin or synaptic vesicle protein 2. This review summarizes the present knowledge about the cell receptor molecules and the mode of toxin-receptor interaction that enables the toxins’ sophisticated access to their site of action.
-
identification of the protein receptor binding site of botulinum Neurotoxins b and g proves the double receptor concept
Proceedings of the National Academy of Sciences of the United States of America, 2007Co-Authors: Andreas Rummel, Stefan Mahrhold, Timo Eichner, Tanja Weil, Tino Karnath, Aleksandrs Gutcaits, Konrad Sandhoff, Richard L Proia, Ravi K Acharya, Hans BigalkeAbstract:Botulinum Neurotoxins (BoNTs) cause muscle paralysis by selectively cleaving core components of the vesicular fusion machinery within motoneurons. Complex gangliosides initially bind into a pocket that is conserved among the seven BoNTs and tetanus neurotoxin. Productive neurotoxin uptake also requires protein receptors. The interaction site of the protein receptor within the neurotoxin is currently unknown. We report the identification and characterization of the protein receptor binding site of BoNT/B and BoNT/G. Their protein receptors, synaptotagmins I and II, bind to a pocket at the tip of their HCC (C-terminal domain of the C-terminal fragment of the heavy chain) that corresponds to the unique second carbohydrate binding site of tetanus neurotoxin, the sialic acid binding site. Substitution of amino acids in this region impaired binding to synaptotagmins and drastically decreased toxicity at mouse phrenic nerve preparations; CD-spectroscopic analyses evidenced that the secondary structure of the mutated Neurotoxins was unaltered. Deactivation of the synaptotagmin binding site by single mutations led to virtually inactive BoNT/B and BoNT/G when assayed at phrenic nerve preparations of complex-ganglioside-deficient mice. Analogously, a BoNT B mutant with deactivated ganglioside and synaptotagmin binding sites lacked appreciable activity at wild-type mouse phrenic nerve preparations. Thus, these data exclude relevant contributions of any cell surface molecule other than one ganglioside and one protein receptor to the entry process of BoNTs, which substantiates the double-receptor concept. The molecular characterization of the synaptotagmin binding site provides the basis for designing a novel class of potent binding inhibitors.
Ornella Rossetto - One of the best experts on this subject based on the ideXlab platform.
-
presynaptic receptor arrays for clostridial Neurotoxins
Trends in Microbiology, 2004Co-Authors: Cesare Montecucco, Ornella Rossetto, Giampietro SchiavoAbstract:Tetanus and botulinum Neurotoxins act at femtomolar concentrations and are specific for the presynaptic membrane of neurons. Their mode of binding is still poorly defined. The exceptional potency and specificity of these Neurotoxins raise unprecedented questions about the nature of their receptor(s) and the mode of their membrane binding. We propose a presynaptic binding process for tetanus and botulinum Neurotoxins based on a capture step performed by an antenna, consisting of a lipid- or a protein-linked oligosaccharide, which brings about a very large membrane concentration effect; this is followed by additional interactions with arrays of receptor molecules, arranged in membrane microdomains, which render the neurotoxin binding practically irreversible and triggers endocytosis.
-
tetanus and botulinum Neurotoxins turning bad guys into good by research
Toxicon, 2001Co-Authors: Ornella Rossetto, Giampietro Schiavo, M Seveso, Paola Caccin, Cesare MontecuccoAbstract:Abstract The neuroparalytic syndromes of tetanus and botulism are caused by Neurotoxins produced by bacteria of the genus Clostridium . They are 150 kDa proteins consisting of three-domains, endowed with different functions: neurospecific binding, membrane translocation and specific proteolysis of three key components of the neuroexocytosis apparatus. After binding to the presynaptic membrane of motoneurons, tetanus neurotoxin (TeNT) is internalized and transported retroaxonally to the spinal cord, where it blocks neurotransmitter release from spinal inhibitory interneurons. In contrast, the seven botulinum Neurotoxins (BoNT) act at the periphery and inhibit acetylcholine release from peripheral cholinergic nerve terminals. TeNT and BoNT-B, -D, -F and -G cleave specifically at single but different peptide bonds, VAMP/synaptobrevin, a membrane protein of small synaptic vesicles. BoNT types -A, -C and -E cleave SNAP-25 at different sites within the COOH-terminus, whereas BoNT-C also cleaves syntaxin. BoNTs are increasingly used in medicine for the treatment of human diseases characterized by hyperfunction of cholinergic terminals.
-
functional characterisation of tetanus and botulinum Neurotoxins binding domains
Journal of Cell Science, 1999Co-Authors: Giovanna Lalli, Cesare Montecucco, Judit Herreros, Shona L Osborne, Ornella Rossetto, Giampietro SchiavoAbstract:Tetanus and botulinum Neurotoxins constitute a family of bacterial protein toxins responsible for two deadly syndromes in humans (tetanus and botulism, respectively). They bind with high affinity to neurons wherein they cause a complete inhibition of evoked neurotransmitter release. Here we report on the cloning, expression and use of the recombinant fragments of the heavy chains of tetanus neurotoxin and botulinum neurotoxin serotypes A, B and E as tools to study the neurospecific binding of the holotoxins. We found that the recombinant 50 kDa carboxy-terminal domains of tetanus and botulinum Neurotoxins alone are responsible for the specific binding and internalisation into spinal cord cells in culture. Moreover, we provide evidence that the recombinant fragments block the internalization of the parental holotoxins in a dose-dependent manner, as determined by following the neurotoxin-dependent cleavage of their targets VAMP/synaptobrevin and SNAP-25. In addition, the recombinant binding fragments cause a significant delay in the paralysis induced by the corresponding holotoxin on the mouse phrenic nerve-hemidiaphragm preparation. Taken together, these results show that the carboxy-terminal domain of tetanus and botulinum Neurotoxins is necessary and sufficient for the binding and internalisation of these proteins in neurons and open the possibility to use them as tools for the functional characterisation of the intracellular transport of clostridial Neurotoxins.
-
tetanus and botulinum Neurotoxins mechanism of action and therapeutic uses
Philosophical Transactions of the Royal Society B, 1999Co-Authors: Rossella Pellizzari, Giampietro Schiavo, Ornella Rossetto, Cesare MontecuccoAbstract:The clostridial Neurotoxins responsible for tetanus and botulism are proteins consisting of three domains endowed with different functions: neurospecific binding, membrane translocation and proteolysis for specific components of the neuroexocytosis apparatus. Tetanus neurotoxin (TeNT) binds to the presynaptic membrane of the neuromuscular junction, is internalized and transported retroaxonally to the spinal cord. The spastic paralysis induced by the toxin is due to the blockade of neurotransmitter release from spinal inhibitory interneurons. In contrast, the seven serotypes of botulinum Neurotoxins (BoNTs) act at the periphery by inducing a flaccid paralysis due to the inhibition of acetylcholine release at the neuromuscular junction. TeNT and BoNT serotypes B, D, F and G cleave specifically at single but different peptide bonds, of the vesicle associated membrane protein (VAMP) synaptobrevin, a membrane protein of small synaptic vesicles (SSVs). BoNT types A, C and E cleave SNAP-25 at different sites located within the carboxyl-terminus, while BoNT type C additionally cleaves syntaxin. The remarkable specificity of BoNTs is exploited in the treatment of human diseases characterized by a hyperfunction of cholinergic terminals.
-
Tetanus and Botulism Neurotoxins
Advances in Experimental Medicine and Biology, 1996Co-Authors: Fiorella Tonello, Giampietro Schiavo, Ornella Rossetto, Silvia Morante, Cesare MontecuccoAbstract:Tetanus and botulinum Neurotoxins are produced by bacteria of the genus Clostridium and cause the paralytic syndromes of tetanus and botulism with a persistent inhibition of neurotransmitter release at central and peripheral synapses, respectively. These Neurotoxins consist of two disulfide-linked polypeptides: H (100 kDa) is responsible for neurospecific binding and cell penetration of L (50 kDa), a zinc-endopeptidase specific for three protein subunits of the neuroexocytosis apparatus. Tetanus neurotoxin and botulinum Neurotoxins serotypes B, D, F and G cleave at single sites, which differ for each neurotoxin, VAMP/synaptobrevin, a membrane protein of the synaptic vesicles. Botulinum A and E Neurotoxins cleave SNAP-25, a protein of the presynaptic membrane, at two different carboxyl-terminal peptide bonds. Serotype C cleaves specifically syntaxin, another protein of the nerve plasmalemma. The target specificity of these metallo-proteinases relies on a double recognition of their substrates based on interactions with the cleavage site and with a non contiguous segment that contains a structural motif common to VAMP, SNAP-25 and syntaxin.
