The authors updated their report on a randomized trial initiated in 1982 comparing, in early brea... more The authors updated their report on a randomized trial initiated in 1982 comparing, in early breast cancer, high-dose IM Medroxyprogesterone acetate (HD-MPA) adjuvant hormonotherapy during 6 months with no hormonotherapy; node-positive patients also received 6 courses of IV CMF (day 1, day 8; q.4 weeks). 246 node-negative (NN) and 270 node-positive (NP) patients had been followed for a median duration of 13 years. Previous results were confirmed in this analysis on mature data. In NN patients, relapse-free survival (RFS) was improved in the adjuvant hormonotherapy arm, regardless of age while overall survival (OAS) was also increased in younger (less then 50 years) patients. In the whole group of NP patients, no difference was seen regarding RFS or OAS. However, an age-dependant opposite effect was observed: younger patients (< 50) experienced a worse and significant outcome of relapse-free and overall survivals when receiving adjuvant HD-MPA while older patients (> or = 50) e...
A disulfide-rich, low-molecular-mass toxin-like peptide has been isolated from Parabuthus schlech... more A disulfide-rich, low-molecular-mass toxin-like peptide has been isolated from Parabuthus schlechteri venom using gel filtration, ion exchange, and reversed phase chromatography. Partial characterization of this peptide reveals a relationship with four-disulfide bridge proteins belonging to the family of `short' insectotoxins (44% residue identity). In recognition hereof, the peptide was named PBITx1 (sITx10). Our work also reports on the deduced sequences
Peptidyl toxins are used extensively to determine the pharmacology of ion channels. Four families... more Peptidyl toxins are used extensively to determine the pharmacology of ion channels. Four families of peptides have been purified from scorpion venom. In this article, the classification of K + -channel-blocking peptides belonging to family 2 peptides and comprising 30-40 amino acids linked by three or four disulfide bridges, will be discussed. Evidence is provided for the existence of 12 molecular subfamilies, named ␣-KTx1-12, containing 49 different peptides. Because of the pharmacological divergence of these peptides, the principle of classification was based on a primary sequence alignment, combined with maximum parsimony and Neighbour-Joining analysis.
Parabuthus transvaalicus, P. granulatus, and P. villosus are three medically important scorpion s... more Parabuthus transvaalicus, P. granulatus, and P. villosus are three medically important scorpion species occurring in southern Africa which can cause severe envenoming among people. In contrast to many other genera, no data is available on the venom composition of scorpions belonging to the genus Parabuthus. Here we have investigated the components which may contribute to the venomous potential. The constancy of venom composition within each of the three species and between the three species was investigated by means of gel filtration chromatography. The venoms of the three species each were characterized by a constant and typical elution pattern, resulting in a &amp;amp;#39;gel filtration fingerprint&amp;amp;#39; which allows distinction between each species. It appears that certain components in the venoms are common to either all three species, or to two of the three species. This points to a clear interspecies relationship within the genus. We also describe the isolation and characterization of some of the polypeptide toxins present in the venoms of P. villosus, P. transvaalicus and P. granulatus by means of reversed phase chromatography and screening of the toxic components on voltage-activated potassium and sodium channels. Our results confirm that toxins which inhibit potassium channels and alter sodium channel gating are present in the venoms studied.
The first Kv1.3 channel-selective toxin from the venom of the Iranian scorpion Odonthobuthus dori... more The first Kv1.3 channel-selective toxin from the venom of the Iranian scorpion Odonthobuthus doriae (OdK2) was purified, sequenced and characterized physiologically.
The scorpion depressant toxins are a group of evolutionarily conserved polypeptides targeting sod... more The scorpion depressant toxins are a group of evolutionarily conserved polypeptides targeting sodium channels, which show preferential ability to induce flaccid paralysis in insects, making them attractive candidates for the construction of transgenic plants or viral vectors to control pests. In this study, two new depressant toxin-like peptides (BmKITc and BmKITc2) differing only at position 52 (Lys for Thr) were produced in Escherichia coli. Circular dichroism analysis indicated that these two recombinant peptides display a typical structural feature similar to native scorpion toxins. They both cause a maintained current component at the last phase of inactivation of the insect sodium channel DmNav1/tipE expressed in Xenopus oocytes and interestingly, they do not produce a beta effect despite of their primary structure as beta-toxins. Furthermore, an inhibitory effect with BmKITc but not with BmKITc2 was observed on TTX-R sodium currents in rat DRG neurons. We hypothesize that such differential potency highlights a crucial role of lysine 52 in channel selectivity. Our results therefore indicate that, in spite of the general idea, not all scorpion depressant toxins interact with mammalian and/or insect sodium channels in the same manner.
Certain amphibians provide themselves with a chemical defense by accumulating lipophilic alkaloid... more Certain amphibians provide themselves with a chemical defense by accumulating lipophilic alkaloids into skin glands from dietary arthropods. Examples of such alkaloids are pumiliotoxins (PTXs). In general, PTXs are known as positive modulators of voltage-gated sodium channels (VGSCs). Unlike other PTXs, PTX 251D does not share this characteristic. However, mice and insect studies showed that PTX 251D is highly toxic and to date the basis of its toxicity remains unknown.
A novel peptidyl inhibitor of voltage-gated K + channels, named parabutoxin 3 (PBTx3), has been p... more A novel peptidyl inhibitor of voltage-gated K + channels, named parabutoxin 3 (PBTx3), has been purified to homogeneity from the venom of Parabuthus transvaalicus. This scorpion toxin contains 37 residues, has a mass of 4274 Da and displays 41% identity with charybdotoxin (ChTx, also called Ôa-KTx1.1Õ). PBTx3 is the tenth member (called Ôa-KTx1.10Õ) of subfamily 1 of K + channel-blocking peptides known thus far. Electrophysiological experiments using Xenopus laevis oocytes indicate that PBTx3 is an inhibitor of Kv1 channels (Kv1.1, Kv1.2, Kv1.3), but has no detectable effects on Kir-type and ERG-type channels. The dissociation constants (K d ) for Kv1.1, Kv1.2 and Kv1.3 channels are, respectively, 79 lM, 547 nM and 492 nM. A synthetic gene encoding a PBTx3 homologue was designed and expressed as a fusion protein with the maltose-binding protein (MBP) in Escherichia coli. The recombinant protein was purified from the bacterial periplasm compartment using an amylose affinity resin column, followed by a gel filtration purification step and cleavage by factor X a (fX a ) to release the recombinant toxin peptide (rPBTx3). After final purification and refolding, rPBTx3 was shown to be identical to the native PBTx3 with respect to HPLC retention time, mass spectrometric analysis and functional properties. The threedimensional structure of PBTx3 is proposed by homology modelling to contain a double-stranded antiparallel b sheet and a single a-helix, connected by three disulfide bridges. The scaffold of PBTx3 is homologous to most other a-KTx scorpion toxins. Abbreviations: PBTx3, toxin from the venom of the scorpion Parabuthus transvaalicus; AgTx2, toxin from the venom of the scorpion Leiurus quinquestriatus var. Hebraeus; MBP, maltose-binding protein; fXa, factor Xa. Note: a website is available at http://www.toxicology.be (
Two novel pore-forming peptides have been isolated from the venom of the South-African scorpion O... more Two novel pore-forming peptides have been isolated from the venom of the South-African scorpion Opistophtalmus carinatus. These peptides, designated opistoporin 1 and 2, differ by only one amino acid and belong to a group of a-helical, cationic peptides. For the first time, a comparison of the primary structures of a-helical pore-forming peptides from scorpion venom was undertaken. This analysis revealed that peptides in the range of 40-50 amino acids contain a typical scorpion conserved sequence S(x) 3 KxWxS(x) 5 L. An extensive study of biological activity of synthesized opistoporin 1 and parabutoporin, a poreforming peptide previously isolated from the venom of the South-African scorpion Parabuthus schlechteri, was undertaken to investigate an eventual cell-selective effect of the peptides. Opistoporin 1 and parabutoporin were most active in inhibiting growth of Gram-negative bacteria (1.3-25 lM), while melittin and mastoparan, two well-known cytolytic peptides, were more effective against Gram-positive bacteria in the same concentration range. In addition, the peptides showed synergistic activity with some antibiotics commonly used in therapy. Opistoporin 1 and parabutoporin had hemolytic activity intermediate between the least potent mastoparan and the highly lytic melittin. Furthermore, all peptides inhibited growth of fungi. Experiments with SYTOX green suggested that this effect is related to membrane permeabilization.
The inhibitor cystine knot (ICK) fold is an evolutionarily conserved structural motif shared by a... more The inhibitor cystine knot (ICK) fold is an evolutionarily conserved structural motif shared by a large group of polypeptides with diverse sequences and bioactivities. Although found in different phyla (animal, plant, and fungus), ICK peptides appear to be most prominent in venoms of cone snail and spider. Recently, two scorpion toxins activating a calcium release channel have been found to adopt an ICK fold. We have isolated and identified both cDNA and genomic clones for this family of ICK peptides from the scorpion Opistophthalmus carinatus. The gene characterized by three well-delineated exons respectively coding for three structural and functional domains in the toxin precursors illustrates the correlation between exon and module as suggested by the "exon theory of genes." Based on the analysis of precursor organization and gene structure combined with the 3-D fold and functional data, our results highlight a common evolutionary origin for ICK peptides from animals. In contrast, ICK peptides from plant and fungus might be independently evolved from another ancestor.
The aim of the present study was to analyze mass spectra of scorpions belonging to the genus Para... more The aim of the present study was to analyze mass spectra of scorpions belonging to the genus Parabuthus (Pocock 1890) by means of matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOFMS) and to construct a species-specific venom code for species identification. The venom compositions of sixteen Parabuthus species, occurring in southern Africa, were characterized using representative peaks in the molecular mass range of 6400±8400 Da. This mass range is characteristic for the typical long-chain neurotoxins influencing sodium channels. Only a few of these peptides have been sequenced up to now. The impetus for development of these species-specific profiles was the observation of unique, highly reproducible mass spectral peaks within a specific species. An identification label for all the different species could be found using a minimum number of peaks. MALDI-TOFMS is therefore proposed as a complementary method to morphological and behavioural characteristics for species and ultimately subspecies discrimination.
Proteins: Structure, Function, and Bioinformatics, 2004
The gamma-KTx-type scorpion toxins specific for K+ channels were found to interact with ERG chann... more The gamma-KTx-type scorpion toxins specific for K+ channels were found to interact with ERG channels on the turret region, while alpha-KTx3.2 Agitoxin-2 binds to the pore region of the Shaker K+ channel, and alpha-KTx5.3 BmP05 binds to the intermediate region of the small-conductance calcium-activated K-channel (SK(Ca)). In order to explore the critical residues for gamma-KTx binding, we determined the NMR structure of native gamma-KTx1.1 (CnErg1), a 42 amino acid residues scorpion toxin isolated from the venom of the Mexican scorpion Centruroïdes noxius Hoffmann, and we used computational evolutionary trace (ET) analysis to predict possible structural and functional features of interacting surfaces. The 1H-NMR three-dimensional solution structure of native ergtoxin (CnErg1) was solved using a total of 452 distance constraints, 13 3J(NH-Halpha) and 10 hydrogen bonds. The structure is characterized by 2 segments of alpha-helices and a triple-stranded antiparallel beta-sheet stabilized by 4 disulfide bridges. The ET and structural analysis provided indication of the presence of two important amino acid residue clusters, one hydrophobic and the other hydrophilic, that should be involved in the surface contact between the toxin and the channel. Some features of the proposed interacting surface are discussed.
Proteins: Structure, Function, and Bioinformatics, 2003
Scorpion ␣-K ؉ channel toxins are a large family of polypeptides with a similar structure but div... more Scorpion ␣-K ؉ channel toxins are a large family of polypeptides with a similar structure but diverse pharmacological activities. Despite many structural and functional data available at present, little progress has been made in understanding the toxins molecular basis responsible for the functional diversification. In this paper, we report the first complete cDNA sequences of toxins belonging to subfamily 6 and identify five new members, called ␣-KTx 6.6-6.10. By analyzing the rates of mutations that occurred in the corresponding cDNAs, we suggest that accelerated evolution in toxin-coding regions may be associated with the functional diversification of this subfamily. To pinpoint sites probably involved in the functional diversity of ␣-KTx family, we analyzed this family of sequences using the evolutionary trace method. This analysis highlighted one channel-binding surface common for all the members. This surface is composed of one conserved lysine residue at position 29 assisted by other residues at positions 10, 26, 27, 32, 34, and 36. Of them, the positions 29, 32, and 34 have been reported to be the most major determinants of channel specificity. Interestingly, another contrary surface was also observed at a higher evolutionary time cut-off value, which may be involved in the binding of ERG (ether-a-go-go-related gene) channel-specific toxins. The good match between the trace residues and the functional epitopes of the toxins suggested that the evolutionary trace results reported here can be applied to predict channel-binding sites of the toxins. Because, the side-chain variation in the trace positions is strongly linked with the functional alteration and channel-binding surface transfer of ␣-KTx family, we conclude that our findings should also be important for the rational design of new toxins targeting a given potassium channel with high selectivity. Proteins 2004;54:361-370.
In this study, the effect of the scorpion α-like toxin BmK M1 was investigated on isolated DUM ne... more In this study, the effect of the scorpion α-like toxin BmK M1 was investigated on isolated DUM neurons from Locusta migratoria and compared with the effect on para/tipE voltage-gated Na + channels (VGSC), cloned from Drosophila melanogaster. The two insects display different pharmacological properties regarding α-like toxins. Moreover, with the aid of the α-like toxin BmK M1 and 5 of its mutants, the importance of aromatic residues for the interaction of the toxin with the VGSC in L. migratoria and D. melanogaster, is shown.
Neurotoxins have highly specific actions on molecular targets, and thus offer an effective means ... more Neurotoxins have highly specific actions on molecular targets, and thus offer an effective means of characterizing the growing number of identified ion channels and receptors in the nervous system. Separation procedures leading to the identification of neurotoxins almost always include gel filtration chromatography, combined with ion-exchange and/or reversed phase chromatography. We present here an improved fractionation method based on the use of a new Superdex 30 prep grade HiLoad 16/60 FPLC gel filtration column. This single-step gel filtration protocol results in a shortening of the purification process and allows a superior qualitative separation of (neuro-)peptides in crude venoms as compared to any other type of gel filtration column used thus far. Screening of the collected fractions for potential ion channel blocking properties was performed by means of the whole-cell voltage clamp technique. To increase both the amount and speed of expression in Xenopus laevis oocytes of cloned ion channels, we employed a high-expression vector, pGEMHE, wherein the cDNA encoding a neuronal voltage-dependent potassium channel (RCK1) was subcloned. The combination of these techniques represents a fast and efficient identification procedure in the quest for new and selective neurotoxins for cloned channels and receptors.
The subunit stoichiometry of the mammalian K+ channel KVl .l (RCKl) was examined by linking toget... more The subunit stoichiometry of the mammalian K+ channel KVl .l (RCKl) was examined by linking together the coding sequences of 2-5 K+ channel subunits in a single open reading frame and tagging the expression of individual subunits with a mutation (Y379K or Y379R) that altered the sensitivity of the channel to block by external tetraethylammonium ion. Two lines of evidence argue that these constructs lead to K+ channel expression only through the formation of functional tetramers. First, currents expressed by tetrameric constructs containing a single mutant subunit have a sensitivity to tetraethylammonium that is well fitted by a single site binding isotherm. Second, a mutant subunit fY379K) that expresses only as part of a heteromultimer contributes to the expression of functional channels when coexpressed with a trimeric construct but not a tetrameric construct.
The authors updated their report on a randomized trial initiated in 1982 comparing, in early brea... more The authors updated their report on a randomized trial initiated in 1982 comparing, in early breast cancer, high-dose IM Medroxyprogesterone acetate (HD-MPA) adjuvant hormonotherapy during 6 months with no hormonotherapy; node-positive patients also received 6 courses of IV CMF (day 1, day 8; q.4 weeks). 246 node-negative (NN) and 270 node-positive (NP) patients had been followed for a median duration of 13 years. Previous results were confirmed in this analysis on mature data. In NN patients, relapse-free survival (RFS) was improved in the adjuvant hormonotherapy arm, regardless of age while overall survival (OAS) was also increased in younger (less then 50 years) patients. In the whole group of NP patients, no difference was seen regarding RFS or OAS. However, an age-dependant opposite effect was observed: younger patients (< 50) experienced a worse and significant outcome of relapse-free and overall survivals when receiving adjuvant HD-MPA while older patients (> or = 50) e...
A disulfide-rich, low-molecular-mass toxin-like peptide has been isolated from Parabuthus schlech... more A disulfide-rich, low-molecular-mass toxin-like peptide has been isolated from Parabuthus schlechteri venom using gel filtration, ion exchange, and reversed phase chromatography. Partial characterization of this peptide reveals a relationship with four-disulfide bridge proteins belonging to the family of `short' insectotoxins (44% residue identity). In recognition hereof, the peptide was named PBITx1 (sITx10). Our work also reports on the deduced sequences
Peptidyl toxins are used extensively to determine the pharmacology of ion channels. Four families... more Peptidyl toxins are used extensively to determine the pharmacology of ion channels. Four families of peptides have been purified from scorpion venom. In this article, the classification of K + -channel-blocking peptides belonging to family 2 peptides and comprising 30-40 amino acids linked by three or four disulfide bridges, will be discussed. Evidence is provided for the existence of 12 molecular subfamilies, named ␣-KTx1-12, containing 49 different peptides. Because of the pharmacological divergence of these peptides, the principle of classification was based on a primary sequence alignment, combined with maximum parsimony and Neighbour-Joining analysis.
Parabuthus transvaalicus, P. granulatus, and P. villosus are three medically important scorpion s... more Parabuthus transvaalicus, P. granulatus, and P. villosus are three medically important scorpion species occurring in southern Africa which can cause severe envenoming among people. In contrast to many other genera, no data is available on the venom composition of scorpions belonging to the genus Parabuthus. Here we have investigated the components which may contribute to the venomous potential. The constancy of venom composition within each of the three species and between the three species was investigated by means of gel filtration chromatography. The venoms of the three species each were characterized by a constant and typical elution pattern, resulting in a &amp;amp;#39;gel filtration fingerprint&amp;amp;#39; which allows distinction between each species. It appears that certain components in the venoms are common to either all three species, or to two of the three species. This points to a clear interspecies relationship within the genus. We also describe the isolation and characterization of some of the polypeptide toxins present in the venoms of P. villosus, P. transvaalicus and P. granulatus by means of reversed phase chromatography and screening of the toxic components on voltage-activated potassium and sodium channels. Our results confirm that toxins which inhibit potassium channels and alter sodium channel gating are present in the venoms studied.
The first Kv1.3 channel-selective toxin from the venom of the Iranian scorpion Odonthobuthus dori... more The first Kv1.3 channel-selective toxin from the venom of the Iranian scorpion Odonthobuthus doriae (OdK2) was purified, sequenced and characterized physiologically.
The scorpion depressant toxins are a group of evolutionarily conserved polypeptides targeting sod... more The scorpion depressant toxins are a group of evolutionarily conserved polypeptides targeting sodium channels, which show preferential ability to induce flaccid paralysis in insects, making them attractive candidates for the construction of transgenic plants or viral vectors to control pests. In this study, two new depressant toxin-like peptides (BmKITc and BmKITc2) differing only at position 52 (Lys for Thr) were produced in Escherichia coli. Circular dichroism analysis indicated that these two recombinant peptides display a typical structural feature similar to native scorpion toxins. They both cause a maintained current component at the last phase of inactivation of the insect sodium channel DmNav1/tipE expressed in Xenopus oocytes and interestingly, they do not produce a beta effect despite of their primary structure as beta-toxins. Furthermore, an inhibitory effect with BmKITc but not with BmKITc2 was observed on TTX-R sodium currents in rat DRG neurons. We hypothesize that such differential potency highlights a crucial role of lysine 52 in channel selectivity. Our results therefore indicate that, in spite of the general idea, not all scorpion depressant toxins interact with mammalian and/or insect sodium channels in the same manner.
Certain amphibians provide themselves with a chemical defense by accumulating lipophilic alkaloid... more Certain amphibians provide themselves with a chemical defense by accumulating lipophilic alkaloids into skin glands from dietary arthropods. Examples of such alkaloids are pumiliotoxins (PTXs). In general, PTXs are known as positive modulators of voltage-gated sodium channels (VGSCs). Unlike other PTXs, PTX 251D does not share this characteristic. However, mice and insect studies showed that PTX 251D is highly toxic and to date the basis of its toxicity remains unknown.
A novel peptidyl inhibitor of voltage-gated K + channels, named parabutoxin 3 (PBTx3), has been p... more A novel peptidyl inhibitor of voltage-gated K + channels, named parabutoxin 3 (PBTx3), has been purified to homogeneity from the venom of Parabuthus transvaalicus. This scorpion toxin contains 37 residues, has a mass of 4274 Da and displays 41% identity with charybdotoxin (ChTx, also called Ôa-KTx1.1Õ). PBTx3 is the tenth member (called Ôa-KTx1.10Õ) of subfamily 1 of K + channel-blocking peptides known thus far. Electrophysiological experiments using Xenopus laevis oocytes indicate that PBTx3 is an inhibitor of Kv1 channels (Kv1.1, Kv1.2, Kv1.3), but has no detectable effects on Kir-type and ERG-type channels. The dissociation constants (K d ) for Kv1.1, Kv1.2 and Kv1.3 channels are, respectively, 79 lM, 547 nM and 492 nM. A synthetic gene encoding a PBTx3 homologue was designed and expressed as a fusion protein with the maltose-binding protein (MBP) in Escherichia coli. The recombinant protein was purified from the bacterial periplasm compartment using an amylose affinity resin column, followed by a gel filtration purification step and cleavage by factor X a (fX a ) to release the recombinant toxin peptide (rPBTx3). After final purification and refolding, rPBTx3 was shown to be identical to the native PBTx3 with respect to HPLC retention time, mass spectrometric analysis and functional properties. The threedimensional structure of PBTx3 is proposed by homology modelling to contain a double-stranded antiparallel b sheet and a single a-helix, connected by three disulfide bridges. The scaffold of PBTx3 is homologous to most other a-KTx scorpion toxins. Abbreviations: PBTx3, toxin from the venom of the scorpion Parabuthus transvaalicus; AgTx2, toxin from the venom of the scorpion Leiurus quinquestriatus var. Hebraeus; MBP, maltose-binding protein; fXa, factor Xa. Note: a website is available at http://www.toxicology.be (
Two novel pore-forming peptides have been isolated from the venom of the South-African scorpion O... more Two novel pore-forming peptides have been isolated from the venom of the South-African scorpion Opistophtalmus carinatus. These peptides, designated opistoporin 1 and 2, differ by only one amino acid and belong to a group of a-helical, cationic peptides. For the first time, a comparison of the primary structures of a-helical pore-forming peptides from scorpion venom was undertaken. This analysis revealed that peptides in the range of 40-50 amino acids contain a typical scorpion conserved sequence S(x) 3 KxWxS(x) 5 L. An extensive study of biological activity of synthesized opistoporin 1 and parabutoporin, a poreforming peptide previously isolated from the venom of the South-African scorpion Parabuthus schlechteri, was undertaken to investigate an eventual cell-selective effect of the peptides. Opistoporin 1 and parabutoporin were most active in inhibiting growth of Gram-negative bacteria (1.3-25 lM), while melittin and mastoparan, two well-known cytolytic peptides, were more effective against Gram-positive bacteria in the same concentration range. In addition, the peptides showed synergistic activity with some antibiotics commonly used in therapy. Opistoporin 1 and parabutoporin had hemolytic activity intermediate between the least potent mastoparan and the highly lytic melittin. Furthermore, all peptides inhibited growth of fungi. Experiments with SYTOX green suggested that this effect is related to membrane permeabilization.
The inhibitor cystine knot (ICK) fold is an evolutionarily conserved structural motif shared by a... more The inhibitor cystine knot (ICK) fold is an evolutionarily conserved structural motif shared by a large group of polypeptides with diverse sequences and bioactivities. Although found in different phyla (animal, plant, and fungus), ICK peptides appear to be most prominent in venoms of cone snail and spider. Recently, two scorpion toxins activating a calcium release channel have been found to adopt an ICK fold. We have isolated and identified both cDNA and genomic clones for this family of ICK peptides from the scorpion Opistophthalmus carinatus. The gene characterized by three well-delineated exons respectively coding for three structural and functional domains in the toxin precursors illustrates the correlation between exon and module as suggested by the "exon theory of genes." Based on the analysis of precursor organization and gene structure combined with the 3-D fold and functional data, our results highlight a common evolutionary origin for ICK peptides from animals. In contrast, ICK peptides from plant and fungus might be independently evolved from another ancestor.
The aim of the present study was to analyze mass spectra of scorpions belonging to the genus Para... more The aim of the present study was to analyze mass spectra of scorpions belonging to the genus Parabuthus (Pocock 1890) by means of matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOFMS) and to construct a species-specific venom code for species identification. The venom compositions of sixteen Parabuthus species, occurring in southern Africa, were characterized using representative peaks in the molecular mass range of 6400±8400 Da. This mass range is characteristic for the typical long-chain neurotoxins influencing sodium channels. Only a few of these peptides have been sequenced up to now. The impetus for development of these species-specific profiles was the observation of unique, highly reproducible mass spectral peaks within a specific species. An identification label for all the different species could be found using a minimum number of peaks. MALDI-TOFMS is therefore proposed as a complementary method to morphological and behavioural characteristics for species and ultimately subspecies discrimination.
Proteins: Structure, Function, and Bioinformatics, 2004
The gamma-KTx-type scorpion toxins specific for K+ channels were found to interact with ERG chann... more The gamma-KTx-type scorpion toxins specific for K+ channels were found to interact with ERG channels on the turret region, while alpha-KTx3.2 Agitoxin-2 binds to the pore region of the Shaker K+ channel, and alpha-KTx5.3 BmP05 binds to the intermediate region of the small-conductance calcium-activated K-channel (SK(Ca)). In order to explore the critical residues for gamma-KTx binding, we determined the NMR structure of native gamma-KTx1.1 (CnErg1), a 42 amino acid residues scorpion toxin isolated from the venom of the Mexican scorpion Centruroïdes noxius Hoffmann, and we used computational evolutionary trace (ET) analysis to predict possible structural and functional features of interacting surfaces. The 1H-NMR three-dimensional solution structure of native ergtoxin (CnErg1) was solved using a total of 452 distance constraints, 13 3J(NH-Halpha) and 10 hydrogen bonds. The structure is characterized by 2 segments of alpha-helices and a triple-stranded antiparallel beta-sheet stabilized by 4 disulfide bridges. The ET and structural analysis provided indication of the presence of two important amino acid residue clusters, one hydrophobic and the other hydrophilic, that should be involved in the surface contact between the toxin and the channel. Some features of the proposed interacting surface are discussed.
Proteins: Structure, Function, and Bioinformatics, 2003
Scorpion ␣-K ؉ channel toxins are a large family of polypeptides with a similar structure but div... more Scorpion ␣-K ؉ channel toxins are a large family of polypeptides with a similar structure but diverse pharmacological activities. Despite many structural and functional data available at present, little progress has been made in understanding the toxins molecular basis responsible for the functional diversification. In this paper, we report the first complete cDNA sequences of toxins belonging to subfamily 6 and identify five new members, called ␣-KTx 6.6-6.10. By analyzing the rates of mutations that occurred in the corresponding cDNAs, we suggest that accelerated evolution in toxin-coding regions may be associated with the functional diversification of this subfamily. To pinpoint sites probably involved in the functional diversity of ␣-KTx family, we analyzed this family of sequences using the evolutionary trace method. This analysis highlighted one channel-binding surface common for all the members. This surface is composed of one conserved lysine residue at position 29 assisted by other residues at positions 10, 26, 27, 32, 34, and 36. Of them, the positions 29, 32, and 34 have been reported to be the most major determinants of channel specificity. Interestingly, another contrary surface was also observed at a higher evolutionary time cut-off value, which may be involved in the binding of ERG (ether-a-go-go-related gene) channel-specific toxins. The good match between the trace residues and the functional epitopes of the toxins suggested that the evolutionary trace results reported here can be applied to predict channel-binding sites of the toxins. Because, the side-chain variation in the trace positions is strongly linked with the functional alteration and channel-binding surface transfer of ␣-KTx family, we conclude that our findings should also be important for the rational design of new toxins targeting a given potassium channel with high selectivity. Proteins 2004;54:361-370.
In this study, the effect of the scorpion α-like toxin BmK M1 was investigated on isolated DUM ne... more In this study, the effect of the scorpion α-like toxin BmK M1 was investigated on isolated DUM neurons from Locusta migratoria and compared with the effect on para/tipE voltage-gated Na + channels (VGSC), cloned from Drosophila melanogaster. The two insects display different pharmacological properties regarding α-like toxins. Moreover, with the aid of the α-like toxin BmK M1 and 5 of its mutants, the importance of aromatic residues for the interaction of the toxin with the VGSC in L. migratoria and D. melanogaster, is shown.
Neurotoxins have highly specific actions on molecular targets, and thus offer an effective means ... more Neurotoxins have highly specific actions on molecular targets, and thus offer an effective means of characterizing the growing number of identified ion channels and receptors in the nervous system. Separation procedures leading to the identification of neurotoxins almost always include gel filtration chromatography, combined with ion-exchange and/or reversed phase chromatography. We present here an improved fractionation method based on the use of a new Superdex 30 prep grade HiLoad 16/60 FPLC gel filtration column. This single-step gel filtration protocol results in a shortening of the purification process and allows a superior qualitative separation of (neuro-)peptides in crude venoms as compared to any other type of gel filtration column used thus far. Screening of the collected fractions for potential ion channel blocking properties was performed by means of the whole-cell voltage clamp technique. To increase both the amount and speed of expression in Xenopus laevis oocytes of cloned ion channels, we employed a high-expression vector, pGEMHE, wherein the cDNA encoding a neuronal voltage-dependent potassium channel (RCK1) was subcloned. The combination of these techniques represents a fast and efficient identification procedure in the quest for new and selective neurotoxins for cloned channels and receptors.
The subunit stoichiometry of the mammalian K+ channel KVl .l (RCKl) was examined by linking toget... more The subunit stoichiometry of the mammalian K+ channel KVl .l (RCKl) was examined by linking together the coding sequences of 2-5 K+ channel subunits in a single open reading frame and tagging the expression of individual subunits with a mutation (Y379K or Y379R) that altered the sensitivity of the channel to block by external tetraethylammonium ion. Two lines of evidence argue that these constructs lead to K+ channel expression only through the formation of functional tetramers. First, currents expressed by tetrameric constructs containing a single mutant subunit have a sensitivity to tetraethylammonium that is well fitted by a single site binding isotherm. Second, a mutant subunit fY379K) that expresses only as part of a heteromultimer contributes to the expression of functional channels when coexpressed with a trimeric construct but not a tetrameric construct.
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Papers by Jan Tytgat