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Sébastien F. Poget - One of the best experts on this subject based on the ideXlab platform.

  • Discovery and characterisation of a novel toxin from Dendroaspis angusticeps, named Tx7335, that activates the potassium channel KcsA
    Scientific Reports, 2016
    Co-Authors: Iván O. Rivera-torres, Martine Cadene, Brian T. Chait, Sébastien F. Poget
    Abstract:

    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.

  • NMR Structural Studies of the Binding of Activating Mamba Toxin Tx7335 on the Potassium Channel KcsA
    Biophysical Journal, 2015
    Co-Authors: Ulfat Shahzad, Sébastien F. Poget
    Abstract:

    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.

  • Discovery and Characterization of a Novel Toxin from Dendroaspis angusticeps, Named TX7335, with an Activating Effect on the Potassium Channel KscA
    Biophysical Journal, 2013
    Co-Authors: Iván O. Rivera-torres, Martine Cadene, Brian T. Chait, Tony B. Jin, Sébastien F. Poget
    Abstract:

    Due to their important role in essential physiological processes such as the propagation of the nerve signal or regulation of the heartbeat, potassium channels are common targets for animal toxins. These toxins provide valuable tools for the study of ion channel function and have potential as lead compounds for drug development. Most toxins affecting potassium channels act as pore blockers, thus inhibiting potassium flow. Using a direct pull-down toxin binding assay with immobilized channel (the bacterial potassium channel KcsA) and crude Dendroaspis angusticeps venom, we identified a novel toxin binder of KcsA, which we called Tx7335.

Alan L. Harvey - One of the best experts on this subject based on the ideXlab platform.

  • Dendrotoxins: structure-activity relationships and effects on potassium ion channels
    Current Medicinal Chemistry, 2004
    Co-Authors: Alan L. Harvey, Boyd Robertson
    Abstract:

    Dendrotoxins are small proteins isolated from mamba (Dendroaspis) snakes. The original dendrotoxin was found in venom of the Eastern green mamba, Dendroaspis angusticeps, and related proteins were subsequently found in other mamba venoms. The dendrotoxins contain 57-60 amino acid residues crosslinked by three disulphide bridges, and they are homologous to Kunitz-type serine protease inhibitors, such as aprotinin (BPTI). The dendrotoxins have little or no anti-protease activity, but they block particular subtypes of voltage-dependent potassium channels of the Kv1 subfamily in neurones. alpha-Dendrotoxin from green mamba Dendroaspis angusticeps and toxin I from the black mamba Dendroaspis polylepis block cloned Kv1.1, Kv1.2 and Kv1.6 channels in the low nanomolar range; toxin K, also from the black mamba Dendroaspis polylepis, preferentially blocks Kv1.1 channels and is active at picomolar concentrations. Structural modifications and mutations to dendrotoxins have helped to define the molecular recognition properties of different types of K+ channels, although more work is needed to characterise the chemical features of the toxins that underlie their selectivity and potency at particular subtypes of channels. Dendrotoxins have been useful markers of subtypes of K+ channels in vivo, and dendrotoxins have become widely used as probes for studying the function of K+ channels in physiology and pathophysiology. With some pathological conditions being associated with voltage-gated K+ channels, analogues of dendrotoxins might have therapeutic potential.

  • effects of muscarinic toxins mt2 and mt7 from green mamba venom on m1 m3 and m5 muscarinic receptors expressed in chinese hamster ovary cells
    Toxicon, 2003
    Co-Authors: Karen N Bradley, E G Rowan, Alan L. Harvey
    Abstract:

    Several small proteins called muscarinic toxins (MTs) have been isolated from venom of green mamba (Dendroaspis angusticeps). They have previously been shown in radioligand binding studies to have high selectivity and affinity for individual muscarinic receptor subtypes, but less is known of their functional effects. This study has examined the actions of two of these MTs, MT2 and MT7, using changes in cytosolic Ca(2+) ([Ca(2+)](i)) measured using the fluorescent indicator fura-2 in Chinese Hamster Ovary (CHO) cells stably transfected with individual muscarinic receptor subtypes, m1, m3 and m5. MT2 activated the m1 receptor: at concentrations above 100 nM it caused significant and concentration-dependent increases in [Ca(2+)](i). From 25 to 800 nM MT2 also produced increases in [Ca(2+)](i) by activating m3 receptors, although these increases in [Ca(2+)](i) were not strictly concentration-dependent with only intermittent responses being recorded (i.e. it was not always possible to obtain a response to the agonist with each application of the compound). MT2 (800-1600 nM) also caused significant increases in [Ca(2+)](i) in CHO cells expressing the m5 muscarinic receptor subtype. MT7 (1 microM) displayed no agonist activity at any of the muscarinic receptors but was a potent non-competitive antagonist (at 20 nM) at the m1 muscarinic receptor subtype. It had no antagonist activity at the m3 or m5 subtypes. These results indicate that MT7 is a highly specific antagonist at the m1 muscarinic receptor subtype as suggested by results from radioligand binding studies. However, MT2 is less selective for the m1 muscarinic receptor than previously described as it also exhibits agonist activity at the m3 and m5 muscarinic receptors, which was not detected in radioligand binding studies.

  • Twenty years of dendrotoxins.
    Toxicon : official journal of the International Society on Toxinology, 2001
    Co-Authors: Alan L. Harvey
    Abstract:

    Dendrotoxins are small proteins that were isolated 20 years ago from mamba (Dendroaspis) snake venoms (Harvey, A.L., Karlsson, E., 1980. Dendrotoxin from the venom of the green mamba, Dendroaspis angusticeps: a neurotoxin that enhances acetylcholine release at neuromuscular junctions. Naunyn-Schmiedebergs Arch. Pharmacol. 312, 1-6.). Subsequently, a family of related proteins was found in mamba venoms and shown to be homologous to Kunitz-type serine protease inhibitors, such as aprotinin. The dendrotoxins contain 57-60 amino acid residues cross-linked by three disulphide bridges. The dendrotoxins have little or no anti-protease activity, but they were demonstrated to block particular subtypes of voltage-dependent potassium channels in neurons. Studies with cloned K(+) channels indicate that alpha-dendrotoxin from green mamba Dendroaspis angusticeps blocks Kv1.1, Kv1.2 and Kv1.6 channels in the nanomolar range, whereas toxin K from the black mamba Dendroaspis polylepis preferentially blocks Kv1.1 channels. Structural analogues of dendrotoxins have helped to define the molecular recognition properties of different types of K(+) channels, and radiolabelled dendrotoxins have also been useful in helping to discover toxins from other sources that bind to K(+) channels. Because dendrotoxins are useful markers of subtypes of K(+) channels in vivo, dendrotoxins have become widely used as probes for studying the function of K(+) channels in physiology and pathophysiology.

  • Isolation of a postsynaptic blocker from the venom of the green mamba, Dendroaspis angusticeps
    Naunyn-schmiedebergs Archives of Pharmacology, 1998
    Co-Authors: G. Poorheidari, Edward G. Rowan, Alan L. Harvey
    Abstract:

    A blocker of postsynaptic acetylcholine receptors was isolated from venom of the Eastern green mam ba Dendroaspis angusticeps, by gel filtration, ion-exchange and reverse phase high performance liquid chromatography. The isolated component blocks neuromuscular transmission and responses to exoge nously applied acetylcholine in isolated chick biventer cer vices preparations. These results suggest that postsynaptic blockers exist in venoms of all members of the Dendroaspis genus. Iran. Biomed. J. 3 (1 & 2): 53-57, 1999

  • binding of muscarinic toxins mtx1 and mtx2 from the venom of the green mamba Dendroaspis angusticeps to cloned human muscarinic cholinoceptors
    Toxicon, 1995
    Co-Authors: Edgar Kornisiuk, Carlos Cervenansky, Diana Jerusalinsky, Alan L. Harvey
    Abstract:

    Muscarinic toxins MTx1 and MTx2 are 7500 mol. wt polypeptides isolated from the venom of the green mamba snake Dendroaspis angusticeps. Previous competition binding studies indicate that the MTxs may be selective for the M1 subtype of muscarinic acetylcholine receptors. The present work was undertaken in order to clarify the muscarinic subtype specificity and functional effects of MTx1 and MTx2. Binding interactions were determined using 3H-N-methyl scopolamine (NMS) and cloned human muscarinic receptor subtypes m1, m2, m3 and m4. Some preliminary functional studies were performed on rabbit vas deferens preparations, which contain M1 cholinoceptors. MTx1 and MTx2 inhibited 3H-NMS binding to m1 and m3 receptors, with little effect on binding to m2 and m4 receptors. Affinity was higher for m1 receptors: Ki for MTx1 were 48 nM at m1 receptors and 72 nM at m3 receptors, and Ki for MTx2 were 364 nM at m1 and 1.2 microM at m3 receptors. At m1 receptors, about 90% of the binding of MTx1 and MTx2 appears to be irreversible. On rabbit vas deferens preparations, MTx1 and MTx2 at concentrations above 50 nM behaved in a similar way to the relatively selective M1-agonists McN-A-343 and CPCP (4-[N-(chlorophenyl)carbamoyloxy]-4-20-ynyl-trimethylammoniu m iodide) by reducing responses to nerve stimulation. The results confirm that MTx1 and MTx2 bind to m1 receptors rather than to m2 or m4 receptors, but they also reveal a slightly weaker effect at m3 receptors. The interaction at m1 receptors appears to be essentially irreversible, implying that the toxins could be useful tools in studies of the functional role of m1 muscarinic receptors.

Evert Karlsson - One of the best experts on this subject based on the ideXlab platform.

  • Weak toxin WTX from Naja kaouthia cobra venom interacts with both nicotinic and muscarinic acetylcholine receptors
    FEBS Journal, 2009
    Co-Authors: Dmitry Y. Mordvintsev, Victor I. Tsetlin, Dmitry I. Rodionov, Yakov L. Polyak, Jan Jakubík, Vladimir Dolezal, Evert Karlsson, Yuri N. Utkin
    Abstract:

    Iodinated [125I] weak toxin from Naja kaouthia (WTX) cobra venom was injected into mice, and organ-specific binding was monitored. Relatively high levels of [125I]WTX were detected in the adrenal glands. Rat adrenal membranes were therefore used for analysis of [125I]WTX-binding sites. Specific [125I]WTX binding was partially inhibited by both alpha-cobratoxin, a blocker of the alpha7 and muscle-type nicotinic acetylcholine receptors (nAChRs), and by atropine, an antagonist of the muscarinic acetylcholine receptor (mAChR). Binding to rat adrenal nAChR had a Kd of 2.0+/-0.8 microM and was inhibited by alpha-cobratoxin but not by a short-chain alpha-neurotoxin antagonist of the muscle-type nAChR, suggesting a specific interaction with the alpha7-type nAChR. WTX binding was reduced not only by atropine but also by other muscarinic agents (oxotremorine and muscarinic toxins from Dendroaspis angusticeps), indicating an interaction with mAChR. This interaction was further characterized using individual subtypes of human mAChRs expressed in Chinese hamster ovary cells. WTX concentrations up to 30 microM did not inhibit binding of [3H]acetylcholine to any subtype of mAChR by more than 50%. Depending on receptor subtype, WTX either increased or had no effect on the binding of the muscarinic antagonist [3H]N-methylscopolamine, which binds to the orthosteric site, a finding indicative of an allosteric interaction. Furthermore, WTX alone activated G-protein coupling with all mAChR subtypes and reduced the efficacy of acetylcholine in activating G-proteins with the M1, M4, and M5 subtypes. Our data demonstrate an orthosteric WTX interaction with nAChR and an allosteric interaction with mAChRs.

  • Muscarinic Toxins from the Black Mamba Dendroaspis polylepis
    European journal of biochemistry, 1995
    Co-Authors: Mikael Jolkkonen, Abdu Adem, Paul L.m. Van Giersbergen, Ulf Hellman, Christer Wernstedt, Aldo Oras, Nisamanee Satyapan, Evert Karlsson
    Abstract:

    Three new toxins acting on muscarinic receptors were isolated from the venom of the black mamba Dendroaspis polylepis. They were called muscarinic toxins alpha, beta, and gamma (MT alpha, MT beta, and MT gamma). All of the toxins have four disulphide bonds and 65 or 66 amino acids. The sequences of MT alpha and MT beta were determined. The muscarinic toxins, of which about 12 have been isolated from venoms of green and black mambas, have 60-98% sequence identity with each other, and are similar to many (about 180) other snake venom components, such as alpha-neurotoxins, cardiotoxins, and fasciculins. In contrast to the alpha-neurotoxins, muscarinic toxins do not bind to nicotinic acetylcholine receptors. The binding constants of MT alpha and MT beta were determined for human muscarinic receptors of subtypes m1-m5 stably expressed in Chinese hamster ovary cells. The toxins are less selective than the earlier discovered muscarinic toxins from the green mamba Dendroaspis angusticeps. MT alpha and the muscarinic toxin MT4 from D. angusticeps differ only in a region of three amino acids (residues 31-33), which are Leu-Asn-His in MT alpha and Ile-Val-Pro in MT4. This difference causes a pronounced shift in subtype selectivity. MT alpha has high affinity to all subtypes, with Ki (inhibition constant) values of 23 nM (m1; pKi = 7.64 +/- 0.10), 44 nM (m2; pKi = 7.36 +/- 0.06), 3 nM (m3; pKi = 8.46 +/- 0.14), 5 nM (m4; pKi = 8.32 +/- 0.07), and 8 nM (m5; pKi = 8.09 +/- 0.07). MT4 has high affinity only to m1 (Ki = 62 nM) and m4 (87 nM) receptors, and low (Ki > 1 microM) affinity to m2, m3, and m5. The region at positions 31-33 evidently plays an important role in the toxin-receptor interaction. MT beta has low affinity for m1 and m2 receptors (Ki > 1 microM) and intermediate affinity for m3 (140 nM; pKi = 6.85 +/- 0.03), m4 (120 nM; pKi = 6.90 +/- 0.06), and m5 (350 nM; pKi = 6.46 +/- 0.01). The low affinity of MT beta may reflect a tendency for spontaneous inactivation.

  • Purification and sequence determination of a new muscarinic toxin (MT4) from the venom of the green mamba (Dendroaspis angusticeps)
    Toxicon : official journal of the International Society on Toxinology, 1995
    Co-Authors: André Vandermeers, Mikael Jolkkonen, Aldo Oras, Marie-claire Vandermeers-piret, Jean Rathe, Magali Waelbroeck, Evert Karlsson
    Abstract:

    A toxin which partially inhibited [3H]N-methylscopolamine binding to rat brain muscarinic receptors was purified from the venom of green mamba, Dendroaspis angusticeps. The N-terminal sequence (up to 45 amino acids) was determined by automated Edman degradation of the whole molecule. The complete sequence was elucidated after enzymatic cleavage with endoproteinase Arg-C or endoproteinase Lys-C and peptide fragments purification. The identity of the C-terminal amino acid was confirmed by hydrazinolysis. The new toxin (MT4) had eight half-cystines and 66 amino acids. It differed from muscarinic toxin MT1 by a single substitution in position 57 (arginine in MT1, histidine in MT4), proximal to the sixth half-cystine.

  • A toxin from the green mamba Dendroaspis angusticeps: amino acid sequence and selectivity for muscarinic m4 receptors.
    FEBS letters, 1994
    Co-Authors: Mikael Jolkkonen, Paul L.m. Van Giersbergen, Ulf Hellman, Christer Wernstedt, Evert Karlsson
    Abstract:

    Abstract Muscarinic toxin 3 (MT3) (65 amino acids, four disulphides, Mr 7379) was isolated from the venom of the African snake Dendroaspis angusticeps (green mamba) and its amino acid sequence determined. Its ability to inhibit the binding of [3H]N-methylscopolamine ([3H]NMS) to Chinese hamster ovary cells stably expressing subtypes of muscarinic receptors was studied. MT3 displayed high affinity for the m4 receptor (pKi = 8.7 ± 0.06), 40-fold lower affinity at m1 receptors (pKi = 7.11 ± 0.17) whereas no inhibition of [3H]NMS binding to m2, m3 and m5 receptors was observed at concentrations up to 1 μM. This makes MT3 the most selective m4 receptor ligand known to date.

Juan J. Calvete - One of the best experts on this subject based on the ideXlab platform.

  • 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, 2016
    Co-Authors: Daniel Petras, Paul Heiss, Roderich D. Süssmuth, Robert A Harrison, Juan J. Calvete
    Abstract:

    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.

  • Peptides with in vitro anti-tumor activity from the venom of the Eastern green mamba, Dendroaspis angusticeps (Elapidae)
    2015
    Co-Authors: Michael J Conlonα, Manju Prajeepα, Milena Mechkarskaα, Kholoud Arafatβ, Samir Attoubβ, Davinia Pla, Juan J. Calvete
    Abstract:

    by-nc/3.0). This license permits non-commercial use, distribution and reproduction of the article, provided the original work is appropriately acknowledged with correct citation details. 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 three-finger 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 cyto-toxic against breast adenocarcinoma MDA-MB-231 cells (LC 50 = 62±2µg/ml) and colorectal adenocarcinom

  • peptides with in vitro anti tumor activity from the venom of the eastern green mamba Dendroaspis angusticeps elapidae
    Journal of venom research, 2014
    Co-Authors: J. M. Conlon, Kholoud Arafat, Manju Prajeep, Milena Mechkarska, Abdu Adem, Samir Attoub, Juan J. Calvete
    Abstract:

    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).

Loic Quinton - One of the best experts on this subject based on the ideXlab platform.

  • In-Depth Glyco-Peptidomics Approach Reveals Unexpected Diversity of Glycosylated Peptides and Atypical Post-Translational Modifications in Dendroaspis angusticeps Snake Venom.
    International Journal of Molecular Sciences, 2017
    Co-Authors: Michel Degueldre, Julien Echterbille, Christian Damblon, Charlotte Gouin, Nicolas Gilles, Nicolas Smargiasso, Gilles Mourier, Edwin De Pauw, Loic Quinton
    Abstract:

    Animal venoms represent a valuable source of bioactive peptides that can be derived into useful pharmacological tools, or even innovative drugs. In this way, the venom of Dendroaspis angusticeps (DA), the Eastern Green Mamba, has been intensively studied during recent years. It mainly contains hundreds of large toxins from 6 to 9 kDa, each displaying several disulfide bridges. These toxins are the main target of venom-based studies due to their valuable activities obtained by selectively targeting membrane receptors, such as ion channels or G-protein coupled receptors. This study aims to demonstrate that the knowledge of venom composition is still limited and that animal venoms contain unexpected diversity and surprises. A previous study has shown that Dendroaspis angusticeps venom contains not only a cocktail of classical toxins, but also small glycosylated peptides. Following this work, a deep exploration of DA glycopeptidome by a dual nano liquid chromatography coupled to electrospray ionization mass spectrometry (nanoLC-ESI-MS) and Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) analyses was initiated. This study reveals unsuspected structural diversity of compounds such as 221 glycopeptides, displaying different glycan structures. Sequence alignments underline structural similarities with natriuretic peptides already characterized in Elapidae venoms. Finally, the presence of an S-cysteinylation and hydroxylation of proline on four glycopeptides, never described to date in snake venoms, is also revealed by proteomics and affined by nuclear magnetic resonance (NMR) experiments.

  • An Unusual Family of Glycosylated Peptides Isolated from Dendroaspis angusticeps Venom and Characterized by Combination of Collision Induced and Electron Transfer Dissociation
    Journal of The American Society for Mass Spectrometry, 2011
    Co-Authors: Loic Quinton, Nicolas Gilles, Nicolas Smargiasso, Andrea Kiehne, Edwin Pauw
    Abstract:

    This study describes the structural characterization of a totally new family of peptides from the venom of the snake green mamba ( Dendroaspis angusticeps) . Interestingly, these peptides differ in several points from other already known mamba toxins. First of all, they exhibit very small molecular masses, ranging from 1.3 to 2.4 kDa. The molecular mass of classical mamba toxins is in the range of 7 to 25 kDa. Second, the new peptides do not contain disulfide bonds, a post-translational modification commonly encountered in animal toxins. The third difference is the very high proportion of proline residues in the sequence accounting for about one-third of the sequence. Finally, these new peptides reveal a carbohydrate moiety, indicating a glycosylation in the sequence. The last two features have made the structural characterization of the new peptides by mass spectrometry a real analytical challenge. Peptides were characterized by a combined use of MALDI- TOF/TOF and nanoESI-IT-ETD experiments to determine not only the peptide sequence but also the composition and the position of the carbohydrate moiety. Anyway, such small glycosylated and proline-rich toxins are totally different from any other known snake peptide and form, as a consequence, a new family of peptides.

  • G protein-coupled receptors, an unexploited animal toxin targets: Exploration of green mamba venom for novel drug candidates active against adrenoceptors.
    Toxicon, 2011
    Co-Authors: Arhamatoulaye Maïga, Loic Quinton, Gilles Mourier, Céline Rouget, Céline Gales, Colette Denis, Philippe Lluel, Jean-michel Sénard, Stefano Palea, Denis Servent
    Abstract:

    At a time when pharmaceutical companies are having trouble finding new low MW drugs and when biologics are becoming more common, animal venoms could constitute an underexploited source of novel drug candidates. We looked for identifying novel animal toxins active against G protein-coupled receptors (GPCR), the most frequently exploited class of treatment targets, with the aim to develop novel research tools and drug candidates. Screening of green mamba (Dendroaspis angusticeps) venom against adrenoceptors identified two novel venom peptides. ρ-Da1a shown an affinity of 0.35 nM for the α1a-AR while ρ-Da1b displayed affinities between 14 and 73 nM for the three α2-ARs. These two venom peptides have sequences similar to those of muscarinic toxins and belong to the three-finger-fold protein family. α1a-AR is the primary target for the treatment of prostate hypertrophy. In vitro and in vivo tests demonstrated that ρ-Da1a reduced prostatic muscle tone as efficiently as tamsulosin (an antagonist presently used), but with fewer cardiovascular side effects. α2-ARs are the prototype of GPCRs not currently used as treatment targets due to a lack of specific ligands. Blockage of these receptors increases intestinal motility, which may be compromised by abdominal surgery and reduces orthosteric hypotension. In vitro and in vivo tests demonstrated that ρ-Da1b antagonizes α2-ARs in smooth muscles and increased heart rate and blood catecholamine concentrations. These results highlight possible exploitation of ρ-Da1a and ρ-Da1b in important pathologies.