The Experts below are selected from a list of 69 Experts worldwide ranked by ideXlab platform
Sébastien F. Poget - One of the best experts on this subject based on the ideXlab platform.
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Discovery and characterisation of a novel toxin from Dendroaspis angusticeps, named Tx7335, that activates the potassium channel KcsA
Scientific Reports, 2016Co-Authors: Iván O. Rivera-torres, Martine Cadene, Brian T. Chait, Sébastien F. PogetAbstract:Due to their central role in essential physiological processes, potassium channels are common targets for animal toxins. These toxins in turn are of great value as tools for studying channel function and as lead compounds for drug development. Here, we used a direct toxin pull-down assay with immobilised KcsA potassium channel to isolate a novel KcsA-binding toxin (called Tx7335) from Eastern Green Mamba snake ( Dendroaspis angusticeps ) venom. Sequencing of the toxin by Edman degradation and mass spectrometry revealed a 63 amino acid residue peptide with 4 disulphide bonds that belongs to the three-finger toxin family, but with a unique modification of its disulphide-bridge scaffold. The toxin induces a dose-dependent increase in both open probabilities and mean open times on KcsA in artificial bilayers. Thus, it unexpectedly behaves as a channel activator rather than an inhibitor. A charybdotoxin-sensitive mutant of KcsA exhibits similar susceptibility to Tx7335 as wild-type, indicating that the binding site for Tx7335 is distinct from that of canonical pore-blocker toxins. Based on the extracellular location of the toxin binding site (far away from the intracellular pH gate), we propose that Tx7335 increases potassium flow through KcsA by allosterically reducing inactivation of the channel.
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NMR Structural Studies of the Binding of Activating Mamba Toxin Tx7335 on the Potassium Channel KcsA
Biophysical Journal, 2015Co-Authors: Ulfat Shahzad, Sébastien F. PogetAbstract:We have recently identified a novel 63 amino acid residue three-finger toxin (called Tx7335) from Eastern Green Mamba snake (Dendroaspis angusticeps) which interacts with KcsA and induces an increase in frequency and duration of individual channel openings when added to the outside of the channel. The toxin exerts this activating effect both on wild-type KcsA as well as on an agitoxin2-sensitive mutant form of the channel, indicating a mode of action and binding site that are different from the classic pore-blocker toxins. We are currently using NMR spectroscopy to unravel the structural underpinnings of this mechanism of action. High yield of purified 15N labeled KcsA and excellent NMR spectral quality have been achieved. Currently the characterization of toxin binding using 1H15N correlation spectra of 15N labeled KcsA in the absence and presence of toxin is ongoing. Experiments are conducted in different membrane mimetics including DMPC/DHPC bicelles and DPC or DM micelles at different pH, temperature and salt concentration. Some chemical shifts and peak intensity changes upon toxin addition have been observed. Continuing NMR structural studies will further elucidate the mechanism of how Tx7335 interacts with KcsA and shed light on the conformational and dynamic changes of C-type inactivation in KcsA and on a novel mechanism of ion channel regulation.
Jose Maria Gutierrez - One of the best experts on this subject based on the ideXlab platform.
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toxicovenomics and antivenom profiling of the Eastern Green Mamba snake dendroaspis angusticeps
Journal of Proteomics, 2016Co-Authors: Line Praest Lauridsen, Andreas Hougaard Laustsen, Bruno Lomonte, Jose Maria GutierrezAbstract:Abstract A toxicovenomic study was performed on the venom of the Green Mamba, Dendroaspis angusticeps. Forty-two different proteins were identified in the venom of D. angusticeps , in addition to the nucleoside adenosine. The most abundant proteins belong to the three-finger toxin (3FTx) (69.2%) and the Kunitz-type proteinase inhibitor (16.3%) families. Several sub-subfamilies of the 3FTxs were identified, such as Orphan Group XI (Toxin F-VIII), acetylcholinesterase inhibitors (fasciculins), and aminergic toxins (muscarinic toxins, synergistic-like toxins, and adrenergic toxins). Remarkably, no α-neurotoxins were identified. Proteins of the Kunitz-type proteinase inhibitor family include dendrotoxins. Toxicological screening revealed a lack of lethal activity in all RP-HPLC fractions, except one, at the doses tested. Thus, the overall toxicity depends on the synergistic action of various types of proteins, such as dendrotoxins, fasciculins, and probably other synergistically-acting toxins. Polyspecific antivenoms manufactured in South Africa and India were effective in the neutralization of venom-induced lethality. These antivenoms also showed a pattern of broad immunorecognition of the different HPLC fractions by ELISA and immunoprecipitated the crude venom by gel immunodiffusion. The synergistic mechanism of toxicity constitutes a challenge for the development of effective recombinant antibodies, as it requires the identification of the most relevant synergistic toxins. Biological significance Envenomings by elapid snakes of the genus Dendroaspis , collectively known as Mambas, represent a serious medical problem in sub-Saharan Africa. The development of novel antivenoms and of recombinant neutralizing antibodies demands the identification of the most relevant toxins in these venoms. In this study, a bottom-up approach was followed for the study of the proteome of the venom of the Eastern Green Mamba, D. angusticeps . Forty-two different proteins were identified, among which the three-finger toxin (3FTx) family, characteristic of elapid venoms, was the most abundant, followed by the Kunitz-type proteinase inhibitor family. In addition, several other protein families were present in the venom, together with the nucleoside adenosine. No α-neurotoxins were identified within the family of 3FTxs in the venom of D. angusticeps , in contrast to the venom of Dendroaspis polylepis , in which α-neurotoxins are largely responsible for the toxicity. With one exception, HPLC fractions from D. angusticeps venom did not kill mice at the doses tested. This underscores that the toxicity of the whole venom is due to the synergistic action of various components, such as fasciculins and dendrotoxins, and probably other synergistically-acting toxins. Thus, the venoms of these closely related species ( D. angusticeps and D. polylepis ) seem to have different mechanisms to subdue their prey, which may be related to different prey preferences, as D. angusticeps is predominantly arboreal, whereas D. polylepis lives mostly in open bush country and feeds mainly on mammals. It is therefore likely that the predominant clinical manifestations of human envenomings by these species also differ, although in both cases neurotoxic manifestations predominate. Polyspecific antivenoms manufactured in South Africa and India were effective in the neutralization of venom-induced lethality in mice and showed a pattern of broad immunorecognition of the various venom fractions. It is necessary to identify the toxins responsible for the synergistic mode of toxicity in this venom, since they are the targets for the development of recombinant antibodies for the treatment of envenomings.
Juan J Calvete - One of the best experts on this subject based on the ideXlab platform.
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Top-down venomics of the East African Green Mamba, Dendroaspis angusticeps, and the black Mamba, Dendroaspis polylepis, highlight the complexity of their toxin arsenals
Journal of Proteomics, 2016Co-Authors: Daniel Petras, Paul Heiss, Robert A. Harrison, Roderich D. Süssmuth, Juan J CalveteAbstract:Abstract We report the characterization, by combination of high-resolution on-line molecular mass and disulfide bond profiling and top-down MS/MS analysis, of the venom proteomes of two congeneric African snake species of medical importance, Dendroaspis angusticeps (Green Mamba) and D. polylepis (black Mamba). Each of these Mamba venoms comprised more than two-hundred polypeptides belonging to just a few toxin families. Both venom proteomes are overwhelmingly composed of post-synaptically-acting short- and long-chain neurotoxins that potently inhibit muscle- and neuronal-type nicotinic acetylcholine receptors; muscarinic cardiotoxins; and dendrotoxins, that block some of the Kv1, n-class of K+ channels. However, the identity of the major proteins and their relative abundances exhibit marked interspecific variation. In addition, the greater resolution of the top-down venomic analytical approach revealed previously undetected protein species, isoforms and proteoforms, including the identification and precise location of modified lysine residues in a number of proteins in both venoms, but particularly in Green Mamba toxins. This comparative top-down venomic analysis unveiled the untapped complexity of Dendroaspis venoms and lays the foundations for rationalizing the notably different potency of Green and black Mamba lethal arsenals at locus resolution. Significance paragraph Dendroaspis angusticeps (Eastern Green Mamba) and D. polylepis (black Mamba) are African snake species of medical concern. Their venoms comprise a high diversity of pharmacologically active peptides, including extremely rapid-acting neurotoxins. Studies on the venoms of D. polylepis and D. angusticeps have focused on the biochemical and pharmacological characterization of their most relevant toxins to rationalize the common neurological and neuromuscular symptoms of envenomings caused by these species. Only very recently an overview of the composition of the venom of a Dendroaspis species, D. polylepis, has been reported through a bottom-up venomics strategy. Peptide-centric approaches provide incomplete sequence coverage, and in general do not allow to distinguish between different proteoforms or closely related toxin isoforms. To overcome this shortcoming we have now applied top-down venomics to unveil the complexity of the toxin arsenals of the black Mamba and the Eastern Green Mamba at locus resolution. Our data show that the Green and the black Mamba venom contain, respectively, ≥ 232 and ≥ 268 protein species, highlighting that D. angusticeps and D. polylepis venom comprise a much higher complexity than the 20 and the 27 toxin sequences available, respectively for these snake species, in the non-redundant NCBI database. On the other hand, 36 (D. angusticeps) and 3 (D. polylepis) minor venom proteins showed mass shifts of + 42 Da modifications, suggesting the presence of monoacetyl lysine residues. Noteworthy, although both venoms have highly similar global molecular compositions, the identity of the major proteins and their relative abundances vary between D. angusticeps and D. polylepis. Our data lay the foundation for rationalizing the notably different venom toxicity profiles of the Green and the black Mamba.
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peptides with in vitro anti tumor activity from the venom of the Eastern Green Mamba dendroaspis angusticeps elapidae
Journal of venom research, 2014Co-Authors: J M Conlon, Manju Prajeep, Milena Mechkarska, Kholoud Arafat, Samir Attoub, Abdu Adem, Juan J CalveteAbstract:Two structurally related (48.6% amino acid sequence identity) peptides with cytotoxic activity against human non-small cell lung adenocarcinoma A549 cells were purified from the venom of the Eastern Green Mamba Dendroaspis angusticeps using reversed phase HPLC. The peptides were identified as members of the threefinger superfamily of snake toxins by mass fingerprinting of tryptic digests. The more potent peptide (LC 50 against A549 cells = 56±4µg/ml) was identical to the previously described toxin C13S1C1 and the less active peptide (LC 50 against A549 cells = 106±5µg/ml) was identical to toxin F-VIII. Toxin C13S1C1 was also cytotoxic against breast adenocarcinoma MDA-MB-231 cells (LC 50 = 62±2µg/ml) and colorectal adenocarcinoma HT-29 cells (LC 50 = 110±4µg/ml). Although the peptide was appreciably less hemolytic activity against human erythrocytes (LC 50 >600µg/ml), it was cytotoxic to human umbilical vein endothelial HUVEC cells (57±3µg/ ml) indicating no differential activity against cell lines derived from neoplastic tissues. Toxin F-VIII was not cytotoxic to MDA-MB-231, HT-29 cells, and HUVEC cells at concentrations up to 300µg/ml and was not hemolytic at concentrations up to 1mg/ml. Neither peptide inhibited growth of reference strains of Escherichia coli or Staphylococcus aureus (MIC values >200µg/ml).
Iván O. Rivera-torres - One of the best experts on this subject based on the ideXlab platform.
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Discovery and characterisation of a novel toxin from Dendroaspis angusticeps, named Tx7335, that activates the potassium channel KcsA
Scientific Reports, 2016Co-Authors: Iván O. Rivera-torres, Martine Cadene, Brian T. Chait, Sébastien F. PogetAbstract:Due to their central role in essential physiological processes, potassium channels are common targets for animal toxins. These toxins in turn are of great value as tools for studying channel function and as lead compounds for drug development. Here, we used a direct toxin pull-down assay with immobilised KcsA potassium channel to isolate a novel KcsA-binding toxin (called Tx7335) from Eastern Green Mamba snake ( Dendroaspis angusticeps ) venom. Sequencing of the toxin by Edman degradation and mass spectrometry revealed a 63 amino acid residue peptide with 4 disulphide bonds that belongs to the three-finger toxin family, but with a unique modification of its disulphide-bridge scaffold. The toxin induces a dose-dependent increase in both open probabilities and mean open times on KcsA in artificial bilayers. Thus, it unexpectedly behaves as a channel activator rather than an inhibitor. A charybdotoxin-sensitive mutant of KcsA exhibits similar susceptibility to Tx7335 as wild-type, indicating that the binding site for Tx7335 is distinct from that of canonical pore-blocker toxins. Based on the extracellular location of the toxin binding site (far away from the intracellular pH gate), we propose that Tx7335 increases potassium flow through KcsA by allosterically reducing inactivation of the channel.
Line Praest Lauridsen - One of the best experts on this subject based on the ideXlab platform.
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toxicovenomics and antivenom profiling of the Eastern Green Mamba snake dendroaspis angusticeps
Journal of Proteomics, 2016Co-Authors: Line Praest Lauridsen, Andreas Hougaard Laustsen, Bruno Lomonte, Jose Maria GutierrezAbstract:Abstract A toxicovenomic study was performed on the venom of the Green Mamba, Dendroaspis angusticeps. Forty-two different proteins were identified in the venom of D. angusticeps , in addition to the nucleoside adenosine. The most abundant proteins belong to the three-finger toxin (3FTx) (69.2%) and the Kunitz-type proteinase inhibitor (16.3%) families. Several sub-subfamilies of the 3FTxs were identified, such as Orphan Group XI (Toxin F-VIII), acetylcholinesterase inhibitors (fasciculins), and aminergic toxins (muscarinic toxins, synergistic-like toxins, and adrenergic toxins). Remarkably, no α-neurotoxins were identified. Proteins of the Kunitz-type proteinase inhibitor family include dendrotoxins. Toxicological screening revealed a lack of lethal activity in all RP-HPLC fractions, except one, at the doses tested. Thus, the overall toxicity depends on the synergistic action of various types of proteins, such as dendrotoxins, fasciculins, and probably other synergistically-acting toxins. Polyspecific antivenoms manufactured in South Africa and India were effective in the neutralization of venom-induced lethality. These antivenoms also showed a pattern of broad immunorecognition of the different HPLC fractions by ELISA and immunoprecipitated the crude venom by gel immunodiffusion. The synergistic mechanism of toxicity constitutes a challenge for the development of effective recombinant antibodies, as it requires the identification of the most relevant synergistic toxins. Biological significance Envenomings by elapid snakes of the genus Dendroaspis , collectively known as Mambas, represent a serious medical problem in sub-Saharan Africa. The development of novel antivenoms and of recombinant neutralizing antibodies demands the identification of the most relevant toxins in these venoms. In this study, a bottom-up approach was followed for the study of the proteome of the venom of the Eastern Green Mamba, D. angusticeps . Forty-two different proteins were identified, among which the three-finger toxin (3FTx) family, characteristic of elapid venoms, was the most abundant, followed by the Kunitz-type proteinase inhibitor family. In addition, several other protein families were present in the venom, together with the nucleoside adenosine. No α-neurotoxins were identified within the family of 3FTxs in the venom of D. angusticeps , in contrast to the venom of Dendroaspis polylepis , in which α-neurotoxins are largely responsible for the toxicity. With one exception, HPLC fractions from D. angusticeps venom did not kill mice at the doses tested. This underscores that the toxicity of the whole venom is due to the synergistic action of various components, such as fasciculins and dendrotoxins, and probably other synergistically-acting toxins. Thus, the venoms of these closely related species ( D. angusticeps and D. polylepis ) seem to have different mechanisms to subdue their prey, which may be related to different prey preferences, as D. angusticeps is predominantly arboreal, whereas D. polylepis lives mostly in open bush country and feeds mainly on mammals. It is therefore likely that the predominant clinical manifestations of human envenomings by these species also differ, although in both cases neurotoxic manifestations predominate. Polyspecific antivenoms manufactured in South Africa and India were effective in the neutralization of venom-induced lethality in mice and showed a pattern of broad immunorecognition of the various venom fractions. It is necessary to identify the toxins responsible for the synergistic mode of toxicity in this venom, since they are the targets for the development of recombinant antibodies for the treatment of envenomings.