Scorpion Buthus Occitanus Tunetanus Research Articles

Kbot55, purified from Buthus occitanus tunetanus venom, represents the first member of a novel α-KTx subfamily

Kbot55 is a 39 amino acid peptide isolated from the venom of the Tunisian scorpion Buthus occitanus tunetanus. This peptide is cross-linked by 3 disulfide bridges and has a molecular mass of 4128.65Da. Kbot55 is very low represented in the venom and thus represents a challenge for biochemical characterization. In this study, Kbot55 has been subjected to a screening on ion channels expressed in Xenopus laevis oocytes. It was found that Kbot55 targets voltage-gated potassium channels with high affinity. Kbot55 shows very low amino acid identity with other scorpion potassium toxins and therefore was considered a bona fide novel type of scorpion toxin. Sequence alignment analysis indicated that Kbot55 is the first representative of the new α-Ktx31 subfamily and therefore was classified as α-Ktx31.1.

Molecular characterization of a new scorpion venom lipolysis activating peptide: Evidence for disulfide bridge-mediated functional switch of peptides

Venoms from scorpions contain extremely rich bioactive peptides that often carry diverse functions and are presumably needed to achieve synergistic effects for rapidly immobilizing prey and defending themselves. BotLVP1 is a unique heterodimer protein recently found in the scorpion Buthus occitanus tunetanus venom that is structurally related to scorpion toxins affecting sodium channels (NaScTxs) but exhibits adipocyte lipolysis activity. We have isolated and identified two cDNA clones encoding subunits α and β of a BotLVP1-like peptide (named BmLVP1) from the Chinese scorpion Buthus martensii venom gland and determined the first complete gene structure of this subfamily. These results highlight a genetic link between these lipolysis activating peptides and NaScTxs. Comparison of cDNA and genomic sequences combined with protein structural and functional analysis provides evidence supporting the existence of RNA editing mechanism in scorpion venom glands, which could mediate functional switch of BmLVP1 gene, from adipocyte lipolysis to neurotoxicity, by altering the wrapper disulfide bridge (WDB) pattern of the peptides.

A novel depolarizing activity of scorpion venom fraction M1 due to activation of skeletal muscle nicotinic receptors

A depolarizing activity following interaction with nicotinic acetylcholine receptors (nAchRs) in skeletal muscle cells, was observed for the first time in the non-toxic venom fraction (M1) of the yellow scorpion Buthus occitanus tunetanus (Bot). The effects of M1 fraction were tested on cultured rat myotubes by recording changes in [Ca 2+]i. When applied, M1 (10 μg/mL) induced a transient increase of [Ca 2+] i which could be blocked by a prior application of α-Bungarotoxin ( α-Bg-Tx).

Insecticidal effects of Buthus occitanus tunetanus BotIT6 toxin expressed in Escherichia coli and baculovirus/insect cells

BotIT6 is a neurotoxin polypeptide derived from the venom of the scorpion Buthus occitanus tunetanus (Bot). Its mature form is composed of 62 amino acids. BotIT6 has been reported to be the most potent toxin from Bot venom that has a strict selectivity for insects. Such toxin may have potential as a potent animal-harmless tool against insects. Using RT-PCR, we isolated and sequenced a cDNA encoding 62 amino acid residues corresponding to the known amino acid sequence of BotIT6. We have expressed a recombinant active form of BotIT6 in significantly high amounts in Escherichia coli. We have also engineered the cDNA into the Autographa californica Nuclear Polyhedrosis Virus (AcMNPV) genome and expressed the protein under control of the polyhedrin promoter. Supernatants of AcIT6-virus infected Sf9 insect cells exhibit a typical intoxication effect when injected to Spodoptera littoralis larvae. Moreover, injection of the recombinant virus showed enhanced insecticidal potency against S. littoralis larvae compared with wild type AcMNPV.

Isolation and molecular characterization of LVP1 lipolysis activating peptide from scorpion Buthus occitanus tunetanus

LVP1, a novel protein inducing lipolytic response in adipose cells, was purified from scorpion Buthus occitanus tunetanus venom. It represented 1% of crude venom proteins, with pHi∼6 and molecular mass of 16 170 Da. In contrast to well-characterized scorpion toxins, reduction and alkylation of LVP1 revealed an heterodimeric structure. Isolated α and β chains of LVP1 have a respective molecular mass of 8877 and 8807 Da as determined by mass spectrometry. The N-terminal and some internal peptide sequences of LVP1α and β were determined by Edman degradation. The full amino acid sequences of both chains were deduced from nucleotide sequences of the corresponding cDNAs prepared based on peptide sequences and the 3′ and 5′ RACE methodologies. LVP1α and β cDNAs encode a signal peptide of 22 residues and a mature peptide of 69 and 73 residues, respectively. Each mature peptide contains seven cysteines, which are compatible with an interchain disulfide bridge. The cDNA deduced protein structures share a high similarity with those of some Na + channel scorpion toxins. LVP1 was not toxic to mice after intracerebro-ventricular injection. LVP1 stimulated lipolysis on freshly dissociated rat adipocytes in a dose-dependent manner with EC50 of ∼1+0.5 μg/ml. LVP1 subunits did not display any lipolytic activity. As previously described for venom, β adrenergic receptor (β AR) antagonists interfere with LVP1 activity. Furthermore, it is shown that LVP1 competes with [ 3H]-CGP 12177 (β 1/β 2 antagonist) for binding to adipocyte plasma membrane with an IC50 of about 10 −7 M. These results demonstrate the existence of a new type of scorpion venom nontoxic peptides that are structurally related to Na + channel toxins but can exert a distinct biological activity on adipocyte lipolysis through a β-type adrenoreceptor pathway.

Interactions of insect sodium channels with Bot IT2, a new toxin extracted from the scorpion Buthus occitanus tunetanus venom

Interactions of insect sodium channels with Bot IT2, a new toxin extracted from the scorpion Buthus occitanus tunetanus venom

A new scorpion venom toxin paralytic to insects that affects Na+ channel activation. Purification, structure, antigenicity and mode of action.

A new toxin, BotIT2, with a unique mode of action on the isolated giant axon of the cockroach Periplaneta americana and DUM (dorsal unpaired median) neurons, has been purified from the venom of the scorpion Buthus occitanus tunetanus. Its structural, antigenic and pharmacological properties are compared to those of three other groups of neurotoxins found in Buthidae scorpion venoms. Like excitatory, depressant and alpha-type insect-selective neurotoxins, BotIT2 is toxic to insects, but shows the following common and distinctive characteristics. (a) As alpha-type toxins, BotIT2 lack strict selectivity to insects; they have measurable but low toxicity to mice. (b) As depressant toxins and unlike alpha-type toxins, BotIT2 is able to displace iodinated AaHIT from its binding sites in insect neuronal membranes. This indicates that the binding site for BotIT2 is identical, contiguous or in allosteric interaction with that of AaHIT and depressant toxins. (c) The BotIT2 amino acid sequence shows strong similarity to depressant toxins. However, unexpectedly, despite this high sequence similarity, BotIT2 shares moderate cross-antigenic reactivity with depressant toxins. (d) Voltage and current-clamp studies show that BotIT2 induces limited depolarization concomitantly with the development of depolarizing after potential, repetitive activity and later plateau potentials terminated by bursts. Under voltage-clamp conditions, BotIT2 specifically acts on Na+ channels by decreasing the peak Na+ current and by simultaneously inducing a new current with very slow activation/deactivation kinetics. The voltage dependence of this slow current is not significantly different from that of the control current. These observations indicate that BotIT2 chiefly modifies the kinetics of axonal and DUM neuronal membrane Na(+)-channel activation.

Lipolytic Action ofButhus occitanus tunetanusVenom: Involvement of the β Adrenergic Pathway

Scorpion venoms contain active neurotoxins known to act selectively at the level of voltage sensitive Na+and K+channels on mammal nervous system. In the present report, we show for the first time that the venom of scorpionButhus occitanus tunetanus(Bot) countains compounds able to activate another cell function in non excitable cells. Addition of this venom to the culture media of 3T3-L1 adipocytes or freshly dissociated rat adipocytes rapidly increases lipolysis as estimated by glycerol release (∼3 to 4 fold over basal values) in a dose-dependent manner (EC50∼12 ± 1.25 μg/ml; n=3). Bot venom effect was lower and not additive to the effect produced by isoproterenol (IPE) (10 μM), a main lipolytic agent, n=3. In Sephadex G-50 size exclusion chromatography, the lipolytic activity was excluded and not associated to the included neurotoxic fraction. Furthermore, no lipolytic effect could be detected in the Na+channel specific toxin II purified fromAndroctonus australis hector(AaHII) or the K+voltage-dependent channel toxin fromAndroctonus mauritanicus mauritanicus(KTx). Propranolol (a non selective β adrenoreceptor (βAR) antagonist), alprenolol and pindolol (selective β1/β2 antagonists) totally inhibited in a dose-dependent manner the lipolytic response to Bot venom (IC50∼1×10−7, 7.5×10−8and 3×10−7M, respectively), suggesting that venom stimulated lipolysis through the β AR pathway. The pharmacological profiles of molecules acting more selectively on β AR subtypes such as CGP 12177 (β1/β2 antagonist with β3 agonist properties), CGP 20712A (β1 antagonist) and ICI 118551 (β2 antagonist) strongly suggest that lipolytic action of venom mainly involves the β2/β1 AR subtypes.

A recombinant insect-specific alpha-toxin of Buthus occitanus tunetanus scorpion confers protection against homologous mammal toxins.

We have constructed a cDNA library from venom glands of the scorpion Buthus occitanus tunetanus and cloned a DNA sequence that encodes an alpha-toxin. This clone was efficiently expressed in Escherichia coli as a fusion protein with two Ig-binding (Z) domains of protein A from Staphylococcus aureus. After CNBr treatment of the fusion protein and HPLC purification, we obtained approximately 1 mg recombinant apha-toxin/l bacterial culture. The toxin, called Bot XIV, displays no toxicity towards mammals but is active towards insects as shown by its paralytic activity against Blatella germanica cockroach and by electrophysiological studies on Periplaneta americana cockroaches. The Bot XIV protein fused to two Z domains is highly immunogenic in mice and induces production of antisera that specifically recognize and neutralize highly toxic components that had been injected into mice. This fusion protein could be very useful for development of potent protective antisera against scorpion venoms.

Bot IT 2, a toxin paralytic to insects from the Buthus occitanus tunetanus venom modifying the activity of insect sodium channels

The effects of insect toxin Bot IT2, purified from the venom of the scorpion Buthus occitanus tunetanus, were investigated on the isolated giant axon and on isolated dorsal unpaired median (DUM) neurone of the cockroach Periplaneta americana under current- and voltage-clamp conditions, using the double-oil-gap technique and the patch-clamp technique, respectively. In both preparations, Bot IT2, induces a limited depolarization together with the development of a repetitive activity in axon and an increase of spontaneous discharge frequency in DUM neurone. After artificial hyperpolarization to the normal resting level, plateau potentials can be evoked in both preparations. Under voltage-clamp conditions, Bot IT2 induces a similar effect in axon and DUM neurone by acting specifically on sodium channels. The peak sodium current is decreased and simultaneously a new current with very slow activation-deactivation kinetics is developed. Voltage dependence of this slow current is not very different from that of the control. The inactivation of the fast component is incomplete because it is masked by the development of the slow component. These results suggest that Bot IT2 modifies the kinetics of insect sodium channel activation, and the transformation of normal fast channels into slow ones is discussed.

Purification and characterization of insect toxins from the venom of the scorpion Buthus occitanus tunetanus

Purification and characterization of insect toxins from the venom of the scorpion Buthus occitanus tunetanus

Evidence that dilevalol induces vasodilation through a ß-2 agonist effect in human coronary artery

Scorpion venoms contain active neurotoxins known to act selectively at the level of voltage sensitive Na+and K+channels on mammal nervous system. In the present report, we show for the first time that the venom of scorpionButhus occitanus tunetanus(Bot) countains compounds able to activate another cell function in non excitable cells. Addition of this venom to the culture media of 3T3-L1 adipocytes or freshly dissociated rat adipocytes rapidly increases lipolysis as estimated by glycerol release (∼3 to 4 fold over basal values) in a dose-dependent manner (EC50∼12 ± 1.25 μg/ml; n=3). Bot venom effect was lower and not additive to the effect produced by isoproterenol (IPE) (10 μM), a main lipolytic agent, n=3. In Sephadex G-50 size exclusion chromatography, the lipolytic activity was excluded and not associated to the included neurotoxic fraction. Furthermore, no lipolytic effect could be detected in the Na+channel specific toxin II purified fromAndroctonus australis hector(AaHII) or the K+voltage-dependent channel toxin fromAndroctonus mauritanicus mauritanicus(KTx). Propranolol (a non selective β adrenoreceptor (βAR) antagonist), alprenolol and pindolol (selective β1/β2 antagonists) totally inhibited in a dose-dependent manner the lipolytic response to Bot venom (IC50∼1×10−7, 7.5×10−8and 3×10−7M, respectively), suggesting that venom stimulated lipolysis through the β AR pathway. The pharmacological profiles of molecules acting more selectively on β AR subtypes such as CGP 12177 (β1/β2 antagonist with β3 agonist properties), CGP 20712A (β1 antagonist) and ICI 118551 (β2 antagonist) strongly suggest that lipolytic action of venom mainly involves the β2/β1 AR subtypes.

Purification of thirteen toxins active on mice from the venom of the North African scorpion Buthus occitanus tunetanus

A combination of several equilibrium chromatographic steps permitted us to purify thirteen proteins from the venom of Buthus occitanus tunetanus using the mouse as the test animal for lethality measurement. These proteins have been characterized by their amino acid composition and, for seven of them, by their N-terminal or complete amino acid sequence. The structural data obtained correlate well with the pharmacological and antigenic properties of the neurotoxins.

Covalent structure of toxins I and II from the scorpion Buthus occitanus tunetanus

The amino acid sequences of neurotoxins I and II, which are active on mammals, purified from the venom of Buthus occitanus tunetanus have been determined using standard methods, including mainly automatic phenylisothiocyanate degradation of S-carboxymethylated derivatives of the two proteins and peptides derived by enzymatic hydrolyses. Both toxins are made of sixty-five amino acid residues cross-linked with four disulfide bridges. For toxin II, the complete covalent structure, including the positions of the four disulfide bridges was determined: the positions are similar to those previously found in toxin II of another scorpion from Africa, Androctonus australis Hector. This finding is in favor of a similar structure for all of the scorpion neurotoxins active on mammals.