Kaliotoxin (KTX) has been originally described as an inhibitor of the intermediate conductance Ca... more Kaliotoxin (KTX) has been originally described as an inhibitor of the intermediate conductance Ca(2+)-activated K+ channel (Crest, M., Jacquet, G., Gola, M., Zerrouk, H., Benslimane, A., Rochat, H., Mansuelle, P., and Martin-Eauclaire, M.-F. (1992) J. Biol. Chem. 267, 1640-1647). However, the radioiodinated 125I-KTX-(1-37) was also able to bind to the dendrotoxin sensitive voltage-dependent K+ channel (Romi, R., Crest, M., Gola, M., Sampieri, F., Jacquet, G., Zerrouk, H., Mansuelle, P., Sorokine, O., Van Dorsselaer, A., Rochat, H., Martin-Eauclaire, M.-F., and Van Rietschoten, J. (1993) J. Biol. Chem. 268, 26302-26309). By following the ability to compete with 125I-KTX-(1-37) for binding to its receptor on rat brain synaptosomes, a new kaliotoxin-like peptide, KTX2, was isolated from Androctonus australis scorpion venom. It is a 37-amino acid residue peptide, and its sequence shares 76% identity with KTX. The differences between the two peptides concern the NH2-terminal region and t...
The resistance of the scorpion Androctonus australis to its own venom, as well as to the venom of... more The resistance of the scorpion Androctonus australis to its own venom, as well as to the venom of other species, was investigated. A comparison of the electrical and pharmacological properties of muscle and nerve fibres from Androctonus australis with those from the crayfish Procambarus clarkii enabled us to understand the lack of effect of scorpion venom (110-180 microg ml-1) and purified toxins, which are active on voltage-gated Na+ and K+ channels, Ca2+-activated K+ channels, on scorpion tissues. Voltage-clamp experiments showed that peptide K+ channel blockers from scorpion and snake have no effect on currents in muscle and nerve fibres from either scorpions or crayfish. The scorpion toxin kaliotoxin (KTX), a specific blocker of Kv1.1 and Kv1.3 K+ channels, had no effect on muscle fibres of A. australis (2 micromol l-1) or P. clarkii (400 nmol l-1). Similarly, charybdotoxin (ChTX) had no effect on the muscle fibres of A. australis (10 micromol l-1) or P. clarkii (200 nmol l-1) a...
The French Ion Channel society has existed since 1989 and its main goal is to annually organize a... more The French Ion Channel society has existed since 1989 and its main goal is to annually organize a scientific meeting. This meeting, which gathers young and senior French scientists, provides a great opportunity for exchange and interaction among the ion channel research community. Additionally, for many years, the French ion channel meeting has attracted a significant number of scientists from different European countries, promoting the discussion of new insights and advances, as well as aiding in the establishment of collaborations. In this report, we summarize the five symposia selected for their novelty and importance in human channelopathies, neuroplasticity, ion channel regulations, intracellular ion channels and plant physiology.
Protein science : a publication of the Protein Society, 2000
HpTX2 is a toxin from the venom of Heteropoda venatoria spider that has been demonstrated to bind... more HpTX2 is a toxin from the venom of Heteropoda venatoria spider that has been demonstrated to bind on Kv4.2 potassium channel. We have determined the solution structure of recombinant HpTX2 by use of conventional two-dimensional NMR techniques followed by distance-geometry and molecular dynamics. The calculated structure belongs to the Inhibitory Cystin Knot structural family that consists in a compact disulfide-bonded core, from which four loops emerge. A poorly defined two-stranded antiparallel beta-sheet (residues 20-23 and 25-28) is detected. Analysis of the electrostatic charge anisotropy allows us to propose a functional map of HpTX2 different from the one described for kappa-conotoxin PVIIA, but strongly related to the one of charybdotoxin. The orientation of the dipole moment of HpTX2 emerges through K27 which could therefore be the critical lysine residue. Close to this lysine are a second basic residue, R23, an aromatic cluster (F7, W25, W30) and an hydrophobic side chain (...
In order to establish a venom fingerprint and a peptide profile of the Lasiodora parahybana taran... more In order to establish a venom fingerprint and a peptide profile of the Lasiodora parahybana tarantula venom gland, we used conventional methods such as reversed phase liquid chromatography coupled to an electrospray-ionisation hybrid quadrupole time of flight mass spectrometer (LC/ESI-QqTOFMS), matrix-assisted laser desorption/ionization time-of-flight-MS (MALDI-TOFMS) and direct study of L. parahybana venom by nanospray-ionization QqTOFMS (nanoESI-QqTOFMS) and a new technology for the direct analysis of fresh tissues using MALDI-TOFMS. The analysis of the crude venom allowed the characterization of specific juvenile and adult biomarkers. In situ MALDI analysis of L. parahybana venom gland sections revealed different peptide expression levels all along the gland and non-processed peptide precursors, demonstrating the power of the method for the dynamic investigation of peptide evolution in the venom gland of spiders.
ABSTRACT The immobilization of biomolecules at interfaces plays a crucial role in the investigati... more ABSTRACT The immobilization of biomolecules at interfaces plays a crucial role in the investigation of structure, dynamics or molecular organization by new techniques like molecular recognition force microscopy (MRFM). The target molecule needs to be presented in a way so that the probe molecule can bind without steric restrictions. The problem is aggravated when a membrane protein is to be studied. We developed a monolayer of phospholipids with disulfide elements at their hydrophobic end to bind covalently to a gold surface and with functional groups like biotin or nitrilotriacetic acid (NTA) at their hydrophilic headgroup [1]. These phospholipids can be spread at the air-water interface, they build a monolayer at the water-surface which can be transferred [2] and covalently bound to plain gold plates. By using a mixture of different phospholipids we are able to adjust the lateral density of surface-bound target molecules. Preliminary studies with atomic force microscopy (AFM, Fig.1) and surface plasmon resonance (SPR, Fig.2) showed the potential of the new concept with biotin- and His6-tagged proteins [3]. As a membrane protein we chose the bacterial KcsA channel with a hexahistidine extension which can be scavenged by a nitrilotriacetic acid functionalized surface. This method of oriented immobilization of proteins shows also potential for the application in SPR biosensors.
We report the use of recombinant scorpion toxins in the form of fusion proteins as antigens for i... more We report the use of recombinant scorpion toxins in the form of fusion proteins as antigens for immunisation in rabbits and mice: the aim was to produce in these animal models protective antisera against the most lethal alpha-type toxins in the venom from the North African scorpion Androctonus australis. The cDNAs encoding AaH I, AaH II and AaH III (the three major alpha-type toxins acting on voltage-sensitive sodium channels) were fused to the sequence encoding the maltose binding protein (MBP). The constructs (MBP-AaH I, MBP-AaH II, MBP-AaH I+II and MBP-AaH III) were expressed in Escherichia coli, and resulting fusion proteins were translocated to the periplasmic space. The recombinant fusion proteins were characterised and used as antigens to generate antibodies in rabbits. These antibodies raised specifically recognised their corresponding radiolabelled-toxin with affinities in the 0.1nM range. In vitro neutralisation assays indicated that 1ml of serum raised against a mixture of fusion proteins was able to neutralise 15 LD(50) of the toxic fraction (AaH-G50) purified from the crude venom by molecular filtration through Sephadex G50. In vivo, the fusion proteins induced a long-term protection in mice against the lethal effects of AaH-G50 or of the native toxins. Ten weeks after the beginning of the immunisation programme, mice were challenged with various toxins or AaH-G50 doses. Mice were fully protected against three LD(50) of AaH-G50. Our work shows that fusion protein constructs can be used as a vaccine providing efficient immune protection against A. australis venom.
In order to find new peptide inhibitors for voltage-dependent potassium (Kv) channels, we examine... more In order to find new peptide inhibitors for voltage-dependent potassium (Kv) channels, we examined the effects of venom from Theraphosa leblondi on Kv channel-mediated currents with the whole-cell patch-clamp technique. Both A-type currents in cultured hippocampal neurons and A-type currents recorded from HEK 293 cells transiently expressing recombinant Kv4.2 channels were selectively inhibited by T. leblondi venom. No venom activity was observed on recombinant Kv1.3, Kv1.4, Kv2.1 or Kv3.4 channels. We purified and sequenced three novel homologous peptides from this venom, which are related to previously identified Kv4 channel-specific peptide inhibitors and were named T. leblondi toxin (TLTx) 1, 2 and 3. The mode of action of TLTx1 on recombinant Kv4.2 channels was studied in more detail. TLTx1 inhibited Kv4.2-mediated currents with an IC50 of approximately 200 nM, and macroscopic current inactivation was slowed in the presence of TLTx1. Notably, TLTx1 also caused a shallower voltage dependence of Kv4.2 peak conductance and a shift of the activation midpoint to more positive potentials (DeltaV1/2 = +35 mV). TLTx1 caused a noticable slowing of Kv4.2 activation kinetics, and Kv4.2 deactivation kinetics were accelerated by TLTx1 as infered from Rb+ tail current measurements. Chimeric Kv2.1(4.2L3-4) channels, in which the linker region between S3 and S4 of the TLTx1-insensitive Kv2.1 channel was replaced by the corresponding Kv4.2 domain, were sensitive to TLTx1. Apparently, TLTx1 can act as a gating modifier of Kv4.2 channels.
The genomic DNA sequence encoding the scorpion toxin Amm VIII was amplified from genomic DNA of t... more The genomic DNA sequence encoding the scorpion toxin Amm VIII was amplified from genomic DNA of the scorpion Androctonus mauretanicus mauretanicus from Morocco, subcloned and sequenced. An intron, with a high A+T content (73.5%), split a Gly codon at the end of the precursor signal peptide and the consensus GT/AG splice junction was identified in the Amm VIII gene. This intron of only 166 bp is the smallest intron described so far for a long-chain scorpion toxin gene. In addition, this study led to the identification of three new toxin-related genes. From the deduced amino acid sequences of the encoded precursor proteins, we found that the mature putative toxins were highly similar to the scorpion toxins Leiurus quinquestriatus quinquestriatus IV and Odonthobuthus doriae 1.
In this study, we have characterized the immunological and pharmacological properties of the thre... more In this study, we have characterized the immunological and pharmacological properties of the three major alpha-type toxins from the scorpion Androctonus amoreuxi, AamH1, AamH2 and AamH3, which were previously described as putative toxins from cDNAs [Chen, T. et al., 2003. Regul. Pept. 115, 115-121]. The immunological tests (ELISA, RIA) have demonstrated that AamH1, AamH2 and AamH3 belong to the immunological groups 3 and 4 of alpha-type toxins. Analysis of the three toxin effects on currents through rat brain (rNav1.2), rat muscle (rNav1.4) and Drosophila (DmNav1) sodium channels expressed in Xenopus oocytes revealed that AamH1 and AamH2, but not AamH3, have anti-insect and anti-mammal activities and can be classified as alpha-like toxins. While AamH1 removes fast inactivation only in neuronal rNav1.2 channel and has no effect on muscular rNav1.4 channel, AamH2 affects both neuronal rNav1.2 and muscular rNav1.4 channels. AamH3 was lethal to mice by intracerebroventricular injection despite its lack of activity on the neuronal rNav1.2 channel. Finally, we have shown that the A. amoreuxi venom was better neutralized by the antiserum raised against the venom of Buthus occitanus tunetanus than by the antisera raised against scorpion venoms from the same genus Androctonus.
The full-length cDNA encoding the scorpion α-toxin Amm V was amplified from a cDNA library produc... more The full-length cDNA encoding the scorpion α-toxin Amm V was amplified from a cDNA library produced from the venom glands of the scorpion Androctonus mauretanicus mauretanicus from Morocco. We deduced the amino acid sequence of the encoded precursor protein and found that the mature toxin was similar to the previously characterised toxin. The genomic DNA sequence encoding the toxin was also amplified, subcloned and sequenced. This also led to the isolation of a new Amm V related-gene. Then, for the first time, we studied changes in the level of toxin mRNA synthesis over time.
Aa1 is a toxin purified from the venom of the North African scorpion Androctonus australis. It bl... more Aa1 is a toxin purified from the venom of the North African scorpion Androctonus australis. It blocks fast K(+) currents in cerebellar granular cells [Biochim. Biophys. Acta 1468 (2000) 203]. Two full-length cDNAs (about 250 bp) encoding the precursors of putative Aa1 isoforms (AaTX1 and AaTX2) were amplified by PCR from a venom gland cDNA library of A. australis. The deduced precursors were composed of 59 amino acid residues including a signal peptide of 22 residues and a mature toxin of 37 residues. The peptides display 94% sequence identity with Aa1. Intron-exon organisation of the gene corresponding to the AaTX1 cDNAs was also depicted.
Mass spectrometric methods, including matrix-assisted laser desorption/ionisation time-of-flight ... more Mass spectrometric methods, including matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS), on-line liquid chromatography/electrospray ionisation mass spectrometry (LC/ESI-MS), and nanospray ionisation/hybrid quadrupole time-of-flight mass spectrometry (nanoESI-QqTOFMS), were applied to characterize by mass fingerprinting the venom of the French Guyanese tarantula Theraphosa leblondi. Of these techniques direct nanoESI-QqTOFMS, which allowed the detection of 65 protonated molecules with high mass accuracy, appeared to give the best results. Three major peptides, TlTx1, TlTx2 and TlTx3, were sequenced using a combination of nanoESI-MS/MS and enzyme digestion/MS and MS/MS experiments. Each sequence was confirmed by automated Edman sequencing. In patch-clamp experiments these peptides were found to have a specific inhibitory effect on the voltage-dependent potassium channel, Kv4.2.
A recombinant peptidic spider toxin, HpTx2, was investigated directly by nanoelectrospray tandem ... more A recombinant peptidic spider toxin, HpTx2, was investigated directly by nanoelectrospray tandem mass spectrometry (MS/MS). This 30-residue toxin possesses a highly knotted structure with cystines arranged in close proximity. The low-energy collision-induced dissociation MS/MS spectrum of the [M+4H](4+) ion permitted characterization of the C-terminal sequence of HpTx2 up to Cys(26) that is involved in a disulfide bridge. Chemical pre-treatment with DTT or TCEP was then investigated, and it was found that an unexpected cleavage reaction of HpTx2 gave two smaller peptides which were completely sequenced by MS/MS experiments using a Qq-TOF mass spectrometer. This unusual hydrolysis reaction facilitated the determination of the complete sequence of the HpTx2 toxin.
The rapid and specific detection of therapeutically important ligands in complex mixtures, that m... more The rapid and specific detection of therapeutically important ligands in complex mixtures, that may bind to membrane proteins, remains challenging for many research laboratories and pharmaceutical industries. Through its use in the development of screening assays, mass spectrometry (MS) is currently experiencing a period of tremendous expansion. In the study presented here, we took advantage of the remarkable stability properties of a bacterial membrane protein, the KcsA K+ channel, produced in E. coli and purified as a tetrameric protein in the presence of a detergent. This membrane protein can subserve as a molecular template to display the pore-forming region of human K+ channels, which are considered as targets in the search for inhibitory ligands. The engineered chimeric proteins were linked to metal-bound magnetic beads, for the screening of complex peptide mixtures, such as that of scorpion venoms. The affinity-captured scorpion toxins were eluted prior to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS), and to nano-electrospray ionization tandem mass QqTOF mass spectrometry (MS/MS) analysis. The de novo sequence of the toxins was deduced by combining the MS/MS fragmentation of the reduced form (up to the 33 first residues) and the trypsin digest peptides of the native toxins. This affinity-capture screening assay led to the isolation and characterization of potent and specific ligands of the human K+ channel, Kv1.3. The affinity-capture procedure is fast and reproducible. When linked to magnetic beads, the chimeric membrane protein can be re-used several times without losing any of its selectivity or specificity. This assay also benefits from the fact that it requires minimal amounts of animal venoms or complex mixtures, which can be expensive or difficult to procure.
Kaliotoxin (KTX) has been originally described as an inhibitor of the intermediate conductance Ca... more Kaliotoxin (KTX) has been originally described as an inhibitor of the intermediate conductance Ca(2+)-activated K+ channel (Crest, M., Jacquet, G., Gola, M., Zerrouk, H., Benslimane, A., Rochat, H., Mansuelle, P., and Martin-Eauclaire, M.-F. (1992) J. Biol. Chem. 267, 1640-1647). However, the radioiodinated 125I-KTX-(1-37) was also able to bind to the dendrotoxin sensitive voltage-dependent K+ channel (Romi, R., Crest, M., Gola, M., Sampieri, F., Jacquet, G., Zerrouk, H., Mansuelle, P., Sorokine, O., Van Dorsselaer, A., Rochat, H., Martin-Eauclaire, M.-F., and Van Rietschoten, J. (1993) J. Biol. Chem. 268, 26302-26309). By following the ability to compete with 125I-KTX-(1-37) for binding to its receptor on rat brain synaptosomes, a new kaliotoxin-like peptide, KTX2, was isolated from Androctonus australis scorpion venom. It is a 37-amino acid residue peptide, and its sequence shares 76% identity with KTX. The differences between the two peptides concern the NH2-terminal region and t...
The resistance of the scorpion Androctonus australis to its own venom, as well as to the venom of... more The resistance of the scorpion Androctonus australis to its own venom, as well as to the venom of other species, was investigated. A comparison of the electrical and pharmacological properties of muscle and nerve fibres from Androctonus australis with those from the crayfish Procambarus clarkii enabled us to understand the lack of effect of scorpion venom (110-180 microg ml-1) and purified toxins, which are active on voltage-gated Na+ and K+ channels, Ca2+-activated K+ channels, on scorpion tissues. Voltage-clamp experiments showed that peptide K+ channel blockers from scorpion and snake have no effect on currents in muscle and nerve fibres from either scorpions or crayfish. The scorpion toxin kaliotoxin (KTX), a specific blocker of Kv1.1 and Kv1.3 K+ channels, had no effect on muscle fibres of A. australis (2 micromol l-1) or P. clarkii (400 nmol l-1). Similarly, charybdotoxin (ChTX) had no effect on the muscle fibres of A. australis (10 micromol l-1) or P. clarkii (200 nmol l-1) a...
The French Ion Channel society has existed since 1989 and its main goal is to annually organize a... more The French Ion Channel society has existed since 1989 and its main goal is to annually organize a scientific meeting. This meeting, which gathers young and senior French scientists, provides a great opportunity for exchange and interaction among the ion channel research community. Additionally, for many years, the French ion channel meeting has attracted a significant number of scientists from different European countries, promoting the discussion of new insights and advances, as well as aiding in the establishment of collaborations. In this report, we summarize the five symposia selected for their novelty and importance in human channelopathies, neuroplasticity, ion channel regulations, intracellular ion channels and plant physiology.
Protein science : a publication of the Protein Society, 2000
HpTX2 is a toxin from the venom of Heteropoda venatoria spider that has been demonstrated to bind... more HpTX2 is a toxin from the venom of Heteropoda venatoria spider that has been demonstrated to bind on Kv4.2 potassium channel. We have determined the solution structure of recombinant HpTX2 by use of conventional two-dimensional NMR techniques followed by distance-geometry and molecular dynamics. The calculated structure belongs to the Inhibitory Cystin Knot structural family that consists in a compact disulfide-bonded core, from which four loops emerge. A poorly defined two-stranded antiparallel beta-sheet (residues 20-23 and 25-28) is detected. Analysis of the electrostatic charge anisotropy allows us to propose a functional map of HpTX2 different from the one described for kappa-conotoxin PVIIA, but strongly related to the one of charybdotoxin. The orientation of the dipole moment of HpTX2 emerges through K27 which could therefore be the critical lysine residue. Close to this lysine are a second basic residue, R23, an aromatic cluster (F7, W25, W30) and an hydrophobic side chain (...
In order to establish a venom fingerprint and a peptide profile of the Lasiodora parahybana taran... more In order to establish a venom fingerprint and a peptide profile of the Lasiodora parahybana tarantula venom gland, we used conventional methods such as reversed phase liquid chromatography coupled to an electrospray-ionisation hybrid quadrupole time of flight mass spectrometer (LC/ESI-QqTOFMS), matrix-assisted laser desorption/ionization time-of-flight-MS (MALDI-TOFMS) and direct study of L. parahybana venom by nanospray-ionization QqTOFMS (nanoESI-QqTOFMS) and a new technology for the direct analysis of fresh tissues using MALDI-TOFMS. The analysis of the crude venom allowed the characterization of specific juvenile and adult biomarkers. In situ MALDI analysis of L. parahybana venom gland sections revealed different peptide expression levels all along the gland and non-processed peptide precursors, demonstrating the power of the method for the dynamic investigation of peptide evolution in the venom gland of spiders.
ABSTRACT The immobilization of biomolecules at interfaces plays a crucial role in the investigati... more ABSTRACT The immobilization of biomolecules at interfaces plays a crucial role in the investigation of structure, dynamics or molecular organization by new techniques like molecular recognition force microscopy (MRFM). The target molecule needs to be presented in a way so that the probe molecule can bind without steric restrictions. The problem is aggravated when a membrane protein is to be studied. We developed a monolayer of phospholipids with disulfide elements at their hydrophobic end to bind covalently to a gold surface and with functional groups like biotin or nitrilotriacetic acid (NTA) at their hydrophilic headgroup [1]. These phospholipids can be spread at the air-water interface, they build a monolayer at the water-surface which can be transferred [2] and covalently bound to plain gold plates. By using a mixture of different phospholipids we are able to adjust the lateral density of surface-bound target molecules. Preliminary studies with atomic force microscopy (AFM, Fig.1) and surface plasmon resonance (SPR, Fig.2) showed the potential of the new concept with biotin- and His6-tagged proteins [3]. As a membrane protein we chose the bacterial KcsA channel with a hexahistidine extension which can be scavenged by a nitrilotriacetic acid functionalized surface. This method of oriented immobilization of proteins shows also potential for the application in SPR biosensors.
We report the use of recombinant scorpion toxins in the form of fusion proteins as antigens for i... more We report the use of recombinant scorpion toxins in the form of fusion proteins as antigens for immunisation in rabbits and mice: the aim was to produce in these animal models protective antisera against the most lethal alpha-type toxins in the venom from the North African scorpion Androctonus australis. The cDNAs encoding AaH I, AaH II and AaH III (the three major alpha-type toxins acting on voltage-sensitive sodium channels) were fused to the sequence encoding the maltose binding protein (MBP). The constructs (MBP-AaH I, MBP-AaH II, MBP-AaH I+II and MBP-AaH III) were expressed in Escherichia coli, and resulting fusion proteins were translocated to the periplasmic space. The recombinant fusion proteins were characterised and used as antigens to generate antibodies in rabbits. These antibodies raised specifically recognised their corresponding radiolabelled-toxin with affinities in the 0.1nM range. In vitro neutralisation assays indicated that 1ml of serum raised against a mixture of fusion proteins was able to neutralise 15 LD(50) of the toxic fraction (AaH-G50) purified from the crude venom by molecular filtration through Sephadex G50. In vivo, the fusion proteins induced a long-term protection in mice against the lethal effects of AaH-G50 or of the native toxins. Ten weeks after the beginning of the immunisation programme, mice were challenged with various toxins or AaH-G50 doses. Mice were fully protected against three LD(50) of AaH-G50. Our work shows that fusion protein constructs can be used as a vaccine providing efficient immune protection against A. australis venom.
In order to find new peptide inhibitors for voltage-dependent potassium (Kv) channels, we examine... more In order to find new peptide inhibitors for voltage-dependent potassium (Kv) channels, we examined the effects of venom from Theraphosa leblondi on Kv channel-mediated currents with the whole-cell patch-clamp technique. Both A-type currents in cultured hippocampal neurons and A-type currents recorded from HEK 293 cells transiently expressing recombinant Kv4.2 channels were selectively inhibited by T. leblondi venom. No venom activity was observed on recombinant Kv1.3, Kv1.4, Kv2.1 or Kv3.4 channels. We purified and sequenced three novel homologous peptides from this venom, which are related to previously identified Kv4 channel-specific peptide inhibitors and were named T. leblondi toxin (TLTx) 1, 2 and 3. The mode of action of TLTx1 on recombinant Kv4.2 channels was studied in more detail. TLTx1 inhibited Kv4.2-mediated currents with an IC50 of approximately 200 nM, and macroscopic current inactivation was slowed in the presence of TLTx1. Notably, TLTx1 also caused a shallower voltage dependence of Kv4.2 peak conductance and a shift of the activation midpoint to more positive potentials (DeltaV1/2 = +35 mV). TLTx1 caused a noticable slowing of Kv4.2 activation kinetics, and Kv4.2 deactivation kinetics were accelerated by TLTx1 as infered from Rb+ tail current measurements. Chimeric Kv2.1(4.2L3-4) channels, in which the linker region between S3 and S4 of the TLTx1-insensitive Kv2.1 channel was replaced by the corresponding Kv4.2 domain, were sensitive to TLTx1. Apparently, TLTx1 can act as a gating modifier of Kv4.2 channels.
The genomic DNA sequence encoding the scorpion toxin Amm VIII was amplified from genomic DNA of t... more The genomic DNA sequence encoding the scorpion toxin Amm VIII was amplified from genomic DNA of the scorpion Androctonus mauretanicus mauretanicus from Morocco, subcloned and sequenced. An intron, with a high A+T content (73.5%), split a Gly codon at the end of the precursor signal peptide and the consensus GT/AG splice junction was identified in the Amm VIII gene. This intron of only 166 bp is the smallest intron described so far for a long-chain scorpion toxin gene. In addition, this study led to the identification of three new toxin-related genes. From the deduced amino acid sequences of the encoded precursor proteins, we found that the mature putative toxins were highly similar to the scorpion toxins Leiurus quinquestriatus quinquestriatus IV and Odonthobuthus doriae 1.
In this study, we have characterized the immunological and pharmacological properties of the thre... more In this study, we have characterized the immunological and pharmacological properties of the three major alpha-type toxins from the scorpion Androctonus amoreuxi, AamH1, AamH2 and AamH3, which were previously described as putative toxins from cDNAs [Chen, T. et al., 2003. Regul. Pept. 115, 115-121]. The immunological tests (ELISA, RIA) have demonstrated that AamH1, AamH2 and AamH3 belong to the immunological groups 3 and 4 of alpha-type toxins. Analysis of the three toxin effects on currents through rat brain (rNav1.2), rat muscle (rNav1.4) and Drosophila (DmNav1) sodium channels expressed in Xenopus oocytes revealed that AamH1 and AamH2, but not AamH3, have anti-insect and anti-mammal activities and can be classified as alpha-like toxins. While AamH1 removes fast inactivation only in neuronal rNav1.2 channel and has no effect on muscular rNav1.4 channel, AamH2 affects both neuronal rNav1.2 and muscular rNav1.4 channels. AamH3 was lethal to mice by intracerebroventricular injection despite its lack of activity on the neuronal rNav1.2 channel. Finally, we have shown that the A. amoreuxi venom was better neutralized by the antiserum raised against the venom of Buthus occitanus tunetanus than by the antisera raised against scorpion venoms from the same genus Androctonus.
The full-length cDNA encoding the scorpion α-toxin Amm V was amplified from a cDNA library produc... more The full-length cDNA encoding the scorpion α-toxin Amm V was amplified from a cDNA library produced from the venom glands of the scorpion Androctonus mauretanicus mauretanicus from Morocco. We deduced the amino acid sequence of the encoded precursor protein and found that the mature toxin was similar to the previously characterised toxin. The genomic DNA sequence encoding the toxin was also amplified, subcloned and sequenced. This also led to the isolation of a new Amm V related-gene. Then, for the first time, we studied changes in the level of toxin mRNA synthesis over time.
Aa1 is a toxin purified from the venom of the North African scorpion Androctonus australis. It bl... more Aa1 is a toxin purified from the venom of the North African scorpion Androctonus australis. It blocks fast K(+) currents in cerebellar granular cells [Biochim. Biophys. Acta 1468 (2000) 203]. Two full-length cDNAs (about 250 bp) encoding the precursors of putative Aa1 isoforms (AaTX1 and AaTX2) were amplified by PCR from a venom gland cDNA library of A. australis. The deduced precursors were composed of 59 amino acid residues including a signal peptide of 22 residues and a mature toxin of 37 residues. The peptides display 94% sequence identity with Aa1. Intron-exon organisation of the gene corresponding to the AaTX1 cDNAs was also depicted.
Mass spectrometric methods, including matrix-assisted laser desorption/ionisation time-of-flight ... more Mass spectrometric methods, including matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS), on-line liquid chromatography/electrospray ionisation mass spectrometry (LC/ESI-MS), and nanospray ionisation/hybrid quadrupole time-of-flight mass spectrometry (nanoESI-QqTOFMS), were applied to characterize by mass fingerprinting the venom of the French Guyanese tarantula Theraphosa leblondi. Of these techniques direct nanoESI-QqTOFMS, which allowed the detection of 65 protonated molecules with high mass accuracy, appeared to give the best results. Three major peptides, TlTx1, TlTx2 and TlTx3, were sequenced using a combination of nanoESI-MS/MS and enzyme digestion/MS and MS/MS experiments. Each sequence was confirmed by automated Edman sequencing. In patch-clamp experiments these peptides were found to have a specific inhibitory effect on the voltage-dependent potassium channel, Kv4.2.
A recombinant peptidic spider toxin, HpTx2, was investigated directly by nanoelectrospray tandem ... more A recombinant peptidic spider toxin, HpTx2, was investigated directly by nanoelectrospray tandem mass spectrometry (MS/MS). This 30-residue toxin possesses a highly knotted structure with cystines arranged in close proximity. The low-energy collision-induced dissociation MS/MS spectrum of the [M+4H](4+) ion permitted characterization of the C-terminal sequence of HpTx2 up to Cys(26) that is involved in a disulfide bridge. Chemical pre-treatment with DTT or TCEP was then investigated, and it was found that an unexpected cleavage reaction of HpTx2 gave two smaller peptides which were completely sequenced by MS/MS experiments using a Qq-TOF mass spectrometer. This unusual hydrolysis reaction facilitated the determination of the complete sequence of the HpTx2 toxin.
The rapid and specific detection of therapeutically important ligands in complex mixtures, that m... more The rapid and specific detection of therapeutically important ligands in complex mixtures, that may bind to membrane proteins, remains challenging for many research laboratories and pharmaceutical industries. Through its use in the development of screening assays, mass spectrometry (MS) is currently experiencing a period of tremendous expansion. In the study presented here, we took advantage of the remarkable stability properties of a bacterial membrane protein, the KcsA K+ channel, produced in E. coli and purified as a tetrameric protein in the presence of a detergent. This membrane protein can subserve as a molecular template to display the pore-forming region of human K+ channels, which are considered as targets in the search for inhibitory ligands. The engineered chimeric proteins were linked to metal-bound magnetic beads, for the screening of complex peptide mixtures, such as that of scorpion venoms. The affinity-captured scorpion toxins were eluted prior to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS), and to nano-electrospray ionization tandem mass QqTOF mass spectrometry (MS/MS) analysis. The de novo sequence of the toxins was deduced by combining the MS/MS fragmentation of the reduced form (up to the 33 first residues) and the trypsin digest peptides of the native toxins. This affinity-capture screening assay led to the isolation and characterization of potent and specific ligands of the human K+ channel, Kv1.3. The affinity-capture procedure is fast and reproducible. When linked to magnetic beads, the chimeric membrane protein can be re-used several times without losing any of its selectivity or specificity. This assay also benefits from the fact that it requires minimal amounts of animal venoms or complex mixtures, which can be expensive or difficult to procure.
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Papers by Christian Legros