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P E Bougis

Publications and source records attributed to P E Bougis.

At least 19 recordsLinked to original sources
PubMed2005

A natural anatoxin, Amm VIII, induces neutralizing antibodies against the potent scorpion alpha-toxins.

In this study, we have used Amm VIII, a natural anatoxin from the scorpion Androctonus mauretanicus mauretanicus, to elicit specific polyclonal antibodies in rabbit. Using liquid-phase radioimmunoassay, we have studied its selectivity and its neutralizing activity both in vitro and in vivo for the most lethal scorpion alpha-toxins described, in particular the alpha-toxin of reference AaH II. We have shown that the anti-Amm VIII serum prevents the association of 125I-AaH II with its receptor and is able to remove 125I-AaH II already bound to its site (the half-life of the complex 125I-AaH II-receptor site was 12 min in the absence of anti-Amm VIII serum but decreased to only 2 min in the presence of anti-Amm VIII serum). In vivo, the serum also has a protective effect in mice: 42 LD50 of AaH II by millilitre are neutralized, measured by subcutaneous injection.

Animals↗
PubMed2001

Use of fusion protein constructs to generate potent immunotherapy and protection against scorpion toxins.

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.

Animals↗
PubMed2001

A new class of scorpion toxin binding sites related to an A-type K+ channel: pharmacological characterization and localization in rat brain.

A new scorpion toxin (3751.8 Da) was isolated from the Buthus martensi venom, sequenced and chemically synthesized (sBmTX3). The A-type current of striatum neurons in culture completely disappeared when 1 microM sBmTX3 was applied (Kd=54 nM), whereas the sustained K+ current was unaffected. 125I-sBmTX3 specifically bound to rat brain synaptosomes (maximum binding=14 fmol x mg(-1) of protein, Kd=0.21 nM). A panel of toxins yet described as specific ligands for K+ channels were unable to compete with 125I-sBmTX3. A high density of 125I-sBmTX3 binding sites was found in the striatum, hippocampus, superior colliculus, and cerebellum in the adult rat brain.

Amino Acid Sequence↗
PubMed2001

Moving pieces in a proteomic puzzle: mass fingerprinting of toxic fractions from the venom of Tityus serrulatus (Scorpiones, Buthidae).

Scorpion venoms are very complex mixtures of molecules, most of which are peptides that display different kinds of biological activity. These venoms have been studied in the light of their pharmacological targets and their constituents are able to bind specifically to a variety of ionic channels located in prey tissues, resulting in neurotoxic effects. Toxins that modulate Na(+), K(+), Ca(++) and Cl(-) currents have been described in scorpion venoms. Mass spectrometry was employed to analyze toxic fractions from the venom of the Brazilian scorpion Tityus serrulatus in order to shed light on the molecular composition of this venom and to facilitate the search for novel pharmacologically active compounds. T. serrulatus venom was first subjected to gel filtration to separate its constituents according to their molecular size. The resultant fractions II and III, which account for 90 and 10% respectively of the whole venom toxic effect, were further analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS), on-line liquid chromatography/electrospray mass spectrometry (LC/ESMS) and off-line LC/MALDI-TOFMS in order to establish their mass fingerprints. The molecular masses in fraction II were predominantly between 6500 and 7500 Da. This corresponds to long-chain toxins that mainly act on voltage-gated Na(+) channels. Fraction III is more complex and predominantly contained molecules with masses between 2500 and 5000 Da. This corresponds to the short-chain toxin family, most of which act on K(+) channels, and other unknown peptides. Finally, we were able to measure the molecular masses of 380 different compounds present in the two fractions investigated. To our knowledge, this is the largest number of components ever detected in the venom of a single animal species. Some of the toxins described previously from T. serrulatus venom could be detected by virtue of their molecular masses. The interpretation of this large set of data has provided us with useful proteomic information on the venom, and the implications of these findings are discussed.

Amino Acid Sequence↗
PubMed2001

Characterisation of the gene encoding the alpha-toxin Amm V from the scorpion Androctonus mauretanicus mauretanicus.

The full-length cDNA encoding the scorpion alpha-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.

Amino Acid Sequence↗
PubMed2000

Generating a high affinity scorpion toxin receptor in KcsA-Kv1.3 chimeric potassium channels.

The crystal structure of the bacterial K(+) channel, KcsA (Doyle, D. A., Morais, C. J., Pfuetzner, R. A., Kuo, A., Gulbis, J. M., Cohen, S. L., Chait, B. T., and MacKinnon, R. (1998) Science 280, 69-77), and subsequent mutagenesis have revealed a high structural conservation from bacteria to human (MacKinnon, R., Cohen, S. L., Kuo, A., Lee, A., and Chait, B. T. (1998) Science 280, 106-109). We have explored this conservation by swapping subregions of the M1-M2 linker of KcsA with those of the S5-S6 linker of the human Kv-channel Kv1.3. The chimeric K(+) channel constructs were expressed in Escherichia coli, and their multimeric state was analyzed after purification. We used two scorpion toxins, kaliotoxin and hongotoxin 1, which bind specifically to Kv1.3, to analyze the pharmacological properties of the KcsA-Kv1.3 chimeras. The results demonstrate that the high affinity scorpion toxin receptor of Kv1.3 could be transferred to KcsA. Our biochemical studies with purified KcsA-Kv1.3 chimeras provide direct chemical evidence that a tetrameric channel structure is necessary for forming a functional scorpion toxin receptor. We have obtained KcsA-Kv1.3 chimeras with kaliotoxin affinities (IC(50) values of approximately 4 pm) like native Kv1.3 channels. Furthermore, we show that a subregion of the S5-S6 linker may be an important determinant of the pharmacological profile of K(+) channels. Using available structural information on KcsA and kaliotoxin, we have developed a structural model for the complex between KcsA-Kv1.3 chimeras and kaliotoxin to aid future pharmacological studies of K(+) channels.

Amino Acid Sequence↗
PubMed1999

Conformational flexibility of the acetylcholinesterase tetramer suggested by x-ray crystallography.

Acetylcholinesterase, a polymorphic enzyme, appears to form amphiphilic and nonamphiphilic tetramers from a single splice variant; this suggests discrete tetrameric arrangements where the amphipathic carboxyl-terminal sequences can be either buried or exposed. Two distinct, but related crystal structures of the soluble, trypsin-released tetramer of acetylcholinesterase from Electrophorus electricus were solved at 4.5 and 4.2 A resolution by molecular replacement. Resolution at these levels is sufficient to provide substantial information on the relative orientations of the subunits within the tetramer. The two structures, which show canonical homodimers of subunits assembled through four-helix bundles, reveal discrete geometries in the assembly of the dimers to form: (a) a loose, pseudo-square planar tetramer with antiparallel alignment of the two four-helix bundles and a large space in the center where the carboxyl-terminal sequences may be buried or (b) a compact, square nonplanar tetramer that may expose all four sequences on a single side. Comparison of these two structures points to significant conformational flexibility of the tetramer about the four-helix bundle axis and along the dimer-dimer interface. Hence, in solution, several conformational states of a flexible tetrameric arrangement of acetylcholinesterase catalytic subunits may exist to accommodate discrete carboxyl-terminal sequences of variable dimensions and amphipathicity.

Acetylcholinesterase↗
PubMed1999

Crystal structure of mouse acetylcholinesterase. A peripheral site-occluding loop in a tetrameric assembly.

The crystal structure of mouse acetylcholinesterase at 2.9-A resolution reveals a tetrameric assembly of subunits with an antiparallel alignment of two canonical homodimers assembled through four-helix bundles. In the tetramer, a short Omega loop, composed of a cluster of hydrophobic residues conserved in mammalian acetylcholinesterases along with flanking alpha-helices, associates with the peripheral anionic site of the facing subunit and sterically occludes the entrance of the gorge leading to the active center. The inverse loop-peripheral site interaction occurs within the second pair of subunits, but the peripheral sites on the two loop-donor subunits remain freely accessible to the solvent. The position and complementarity of the peripheral site-occluding loop mimic the characteristics of the central loop of the peptidic inhibitor fasciculin bound to mouse acetylcholinesterase. Tetrameric forms of cholinesterases are widely distributed in nature and predominate in mammalian brain. This structure reveals a likely mode of subunit arrangement and suggests that the peripheral site, located near the rim of the gorge, is a site for association of neighboring subunits or heterologous proteins with interactive surface loops.

Acetylcholinesterase↗
PubMed1998

Evidence for a new class of scorpion toxins active against K+ channels.

cDNAs encoding novel long-chain scorpion toxins (64 amino acid residues, including only six cysteines) were isolated from cDNA libraries produced from the venom glands of the scorpions Androctonus australis from Old World and Tityus serrulatus from New World. The encoded peptides were very similar to a recently identified toxin from T. serrulatus, which is active against the voltage-sensitive 'delayed-rectifier' potassium channel, but they were completely different from the long-chain and short-chain scorpion toxins already characterised. However, there was some sequence similarity (42%) between these new toxins, Aa TX Kbeta and Ts TX Kbeta, and scorpion defensins purified from the hemolymph of Buthidae scorpions Leiurus quinquestriatus and A. australis. Thus, according to a multiple sequence alignment using CLUSTAL, these new toxins seem to be related to the scorpion defensins.

Amino Acid Sequence↗
PubMed1998

Inhibition of mouse acetylcholinesterase by fasciculin: crystal structure of the complex and mutagenesis of fasciculin.

Fasciculins are members of the superfamily of three-fingered peptidic toxins from Elapidae venoms. They selectively inhibit mammalian and electric fish acetylcholinesterases (AChE) with Ki values in the pico- to nanomolar range. Kinetic studies performed in solution indicate that fasciculin does not totally occlude ligand access to the active site of AChE, but rather binds to a peripheral site of the enzyme to inhibit catalysis, perhaps allosterically. The crystal structure of the Fas2-mouse AChE complex delineated a large contact area consistent with the low dissociation constant of the complex; the Fas2 and AChE residues participating in the binding interface were unambiguously established, and major hydrophobic interactions were identified. The structure however suggests that fasciculin totally occludes substrate entry into the catalytic site of AChE, and does not reveal to what extent each contact between Fas2 and AChE contributes to the overall binding energy. New probes, designed to delineate the individual contributions of the fasciculin residues to the complex formation and conformation, were generated by site-directed mutagenesis of a synthetic Fas2 gene. A fully processed recombinant fasciculin, rFas2, that is undistinguishable from the natural, venom-derived Fas2, was expressed in a mammalian system; fourteen mutants, encompassing 16 amino acid residues distributed among the three loops (fingers) of Fas2, were developed from both the kinetic and structural data and analyzed for inhibition of mouse AChE. The determinants identified by the structural and the functional approaches do coincide. However, only a few of the many residues which make up the overall interactive site of the Fas2 molecule provide the strong interactions required for high affinity binding and enzyme inhibition. Potential drug design from the fasciculin molecule is discussed.

Acetylcholine↗
PubMed1997

Influence of a NH2-terminal extension on the activity of KTX2, a K+ channel blocker purified from Androctonus australis scorpion venom.

A cDNA encoding a short polypeptide blocker of K+ channels, kaliotoxin 2 (KTX2), from the venom of the North African scorpion Androctonus australis was expressed in the periplasmic space of Escherichia coli. KTX2 was produced as a fusion protein with the maltose binding protein followed by the recognition site for factor Xa or enterokinase preceding the first amino acid residue of the toxin. The fully refolded recombinant KTX2 (rKTX2) was obtained (0.15-0.30 mg/l of culture) and was indistinguishable from the native toxin according to chemical and biological criteria. An N-extended analogue of KTX2 exhibiting three additional residues was also expressed. This analogue had 1000-fold less affinity for the 125I-kaliotoxin binding site on rat brain synaptosomes than KTX2. Conformational models of KTX2 and its mutant were designed by amino acid replacement using the structure of agitoxin 2 from Leiurus quinquestriatus as template, to try to understand the decrease in affinity for the receptor.

ATP-Binding Cassette Transporters↗
PubMed1997

Expression and activity of mutants of fasciculin, a peptidic acetylcholinesterase inhibitor from mamba venom.

Fasciculin, a selective peptidic inhibitor of acetylcholinesterase, is a member of the three-fingered peptide toxin superfamily isolated from snake venoms. The availability of a crystal structure of a fasciculin 2 (Fas2)-acetylcholinesterase complex affords an opportunity to examine in detail the interaction of this toxin with its target site. To this end, we constructed a synthetic fasciculin gene with an appropriate leader peptide for expression and secretion from mammalian cells. Recombinant wild-type Fas2, expressed and amplified in Chinese hamster ovary cells, was purified to homogeneity and found to be identical in composition and biological activities to the venom-derived toxin. Sixteen mutations at positions where the crystal structure of the complex indicates a significant interfacial contact point or determinant of conformation were generated. Two mutants of loop I, T8A/T9A and R11Q, ten mutants of the longest loop II, R24T, K25L, R27W, R28D, H29D, DeltaPro30, P31R, K32G, M33A, and V34A/L35A, and two mutants of loop III, D45K and K51S, were expressed transiently in human embryonic kidney cells. Inhibitory potencies of the Fas2 mutants toward mouse AChE were established, based on titration of the mutants with a polyclonal anti-Fas2 serum. The Arg27, Pro30, and Pro31 mutants each lost two or more orders of magnitude in Fas2 activity, suggesting that this subset of three residues, at the tip of loop II, dominates the loop conformation and interaction of Fas2 with the enzyme. The Arg24, Lys32, and Met33 mutants lost about one order of magnitude, suggesting that these residues make moderate contributions to the strength of the complex, whereas the Lys25, Arg28, Val34-Leu35, Asp45, and Lys51 mutants appeared as active as Fas2. The Thr8-Thr9, Arg11, and His29 mutants showed greater ratios of inhibitory activity to immunochemical titer than Fas2. This may reflect immunodominant determinants in these regions or intramolecular rearrangements in conformation that enhance the interaction. Of the many Fas2 residues that lie at the interface with acetylcholinesterase, only a few appear to provide substantial energetic contributions to the high affinity of the complex.

Amino Acid Sequence↗
PubMed1997

Genomic organization of the KTX2 gene, encoding a 'short' scorpion toxin active on K+ channels.

A single intron of 87 bp, close to the region encoding the C-terminal part of the signal peptide, was found in the gene of the 'short' scorpion toxin kaliotoxin 2 of Androctonus australis acting on various types of K+ channels. Its A+T content was particularly high (up to 86%). By walking and ligation-mediated PCR, the promoter sequences of the kaliotoxin 2 gene of Androctonus australis were studied. The transcription unit of the gene is 390 bp long. Consensus sequences were identified. The genes of 'short' scorpion toxins active on K+ channels are organized similarly to those of the 'long' scorpion toxins active on Na+ channels and not like those of structurally related insect defensins, which are intronless.

Amino Acid Sequence↗
PubMed1996

Characterization of a new peptide from Tityus serrulatus scorpion venom which is a ligand of the apamin-binding site.

A new ligand (Ts kappa) of the apamin binding site on rat brain synaptosomes (K0.5 = 300 pM) was purified and characterized from the venom of Tityus serrulatus. It is a polypeptide toxin of 35 amino acid residues, with three disulfide bridges. Its cDNA was amplified from a venom gland cDNA library and the nucleotide sequence determined. A model of Ts kappa was constructed by amino acid replacement using charybdotoxin structure as determined by 1H nuclear magnetic resonance as starting model.

Amino Acid Sequence↗
PubMed1996

Characterization of alpha-neurotoxin and phospholipase A2 activities from Micrurus venoms. Determination of the amino acid sequence and receptor-binding ability of the major alpha-neurotoxin from Micrurus nigrocinctus nigrocinctus.

New World elapids are coral snakes that belong to the genus Micrurus, and for which the venom biochemistry is mostly unknown. Analysis has been difficult because the coral snakes produce small quantities of venom. Clinical observations following bites show mainly neurotoxic effects. Experimentally, cardiotoxic, haemolytic and myotoxic activities are also reported. An experimental approach, using reverse-phase high-performance liquid chromatography and specific assays for alpha-neurotoxin and phospholipase A2 activities, was conducted on milligram quantities of venoms from three Micrurus species from Costa Rica; M. nigrocinctus nigrocinctus, M. alleni yatesi and M. multifasciatus. Neurotoxicity was determined by competition binding experiments with the Torpedo marmorata acetylcholine receptor. Phospholipase A2 activity was measured by fluorimetry using a pyrene lipid substrate. In this way, we purified and characterized seven alpha-neurotoxins, five phospholipases A2 and four toxin homologs. The amino acid sequence of the major alpha-neurotoxin from M. nigrocinctus nigrocinctus venom was fully determined and compared to Old Word representatives. Distance matrix data were generated to set up phylogeny relationships among elapid short-chain alpha-neurotoxins, which proved to be in accordance with the taxonomic classification and geographical distribution of snake species.

Americas↗
PubMed1996

Structure of fasciculin 2 from green mamba snake venom: evidence for unusual loop flexibility.

The crystal structure of the snake toxin fasciculin 2, a potent acetylcholinesterase inhibitor from the venom of the green mamba (Dendroaspis angusticeps), has been determined by the molecular-replacement method, using the fasciculin 1 model and refined to 2.0 A resolution. The introduction of an overall anisotropic temperature factor improved significantly the quality of the electron-density map. It suggests, as it was also indicated by the packing, that the thermal motion along the unique axis direction is less pronounced than on the (ab) plane. The final crystallographic R factor is 0.188 for a model having r.m.s. deviations from ideality of 0.016 A for bond lengths and 2.01 degrees for bond angles. As fasciculin 1, fasciculin 2 belongs to the three-finger class of Elapidae toxins, a structural group that also contains the alpha-neurotoxins and the cardiotoxins. Although the two fasciculins have, overall, closely related structures, the conformation of loop I differs appreciably in the two molecules. The presence of detergent in crystallization medium in the case of fasciculin 2 appears to be responsible for the displacement of the loop containing Thr9. This conformational change also results in the formation of a crystallographic dimer that displays extensive intermolecular interactions.

Journal Article↗
PubMed1996

Model of interaction between a cardiotoxin and dimyristoylphosphatidic acid bilayers determined by solid-state 31P NMR spectroscopy.

The interaction of cardiotoxin IIa, a small basic protein extracted from Naja mossambica mossambica venom, with dimyristoylphosphatidic acid (DMPA) membranes has been investigated by solid-state 31P nuclear magnetic resonance spectroscopy. Both the spectral lineshapes and transverse relaxation time values have been measured as a function of temperature for different lipid-to-protein molar ratios. The results indicate that the interaction of cardiotoxin with DMPA gives rise to the complete disappearance of the bilayer structure at a lipid-to-protein molar ratio of 5:1. However, a coexistence of the lamellar and isotropic phases is observed at higher lipid contents. In addition, the number of phospholipids interacting with cardiotoxin increases from about 5 at room temperature to approximately 15 at temperatures above the phase transition of the pure lipid. The isotropic structure appears to be a hydrophobic complex similar to an inverted micellar phase that can be extracted by a hydrophobic solvent. At a lipid-to-protein molar ratio of 40:1, the isotropic structure disappears at high temperature to give rise to a second anisotropic phase, which is most likely associated with the incorporation of the hydrophobic complex inside the bilayer.

Aniline Compounds↗