RESUMO
Numerous toxins from scorpion venoms are much more toxic to insects than to other animal classes, and possess high affinity to Na+ channels. Many of them active on insects were purified from the venom of Buthus occitanus tunetanus. Using amino acid sequences of BotIT2 and RACE-PCR amplification (Rapid amplification of cDNA ends) technique, we isolated, identified and sequenced the nucleotide sequence from the venom glands of the scorpion Buthus occitanus tunetanus. The cDNA encodes a precursor of an insect toxin of 60 amino acid residues. The deduced nucleotide sequence toxin was identical to the determined amino acid sequence of BotIT2. BotIT2 is more similar to the excitatory toxins in its mode of action and to the depressant toxins in its primary structure.
Assuntos
Sequência de Bases/genética , Clonagem Molecular/métodos , Venenos de Escorpião/genética , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Animais , Western Blotting , DNA Complementar/genética , Eletroforese em Gel de Ágar , Eletroforese em Gel de Poliacrilamida , Expressão Gênica/genética , Insetos/efeitos dos fármacos , Dados de Sequência Molecular , Neurotoxinas/efeitos adversos , Neurotoxinas/química , Técnica de Amplificação ao Acaso de DNA Polimórfico , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Venenos de Escorpião/efeitos adversos , Venenos de Escorpião/química , Venenos de Escorpião/classificação , Venenos de Escorpião/isolamento & purificação , Escorpiões , Canais de Sódio/efeitos dos fármacos , Relação Estrutura-Atividade , TunísiaRESUMO
The insect-specific Bothus occitanus tunetanus IT2 toxin is distinguishable from other scorpion toxins by its amino acid sequence and effects on sodium conductance. The present study reveals that Bot IT2 possesses in cockroach neuronal membranes a single class of high affinity (Kd = 0.3 +/- 0.1 nM) and low capacity (Bmax = 2.4 +/- 0.5 pmol/mg) binding sites. Competitive binding experiments with several known sodium channel neurotoxins reveal that the Bot IT2 binding site is in close proximity to the other toxins.
Assuntos
Venenos de Escorpião/metabolismo , Canais de Sódio/metabolismo , Animais , Membrana Celular/metabolismo , Neurônios/metabolismo , Neurotoxinas/metabolismo , Periplaneta , Venenos de Escorpião/química , Venenos de Escorpião/isolamento & purificaçãoRESUMO
One contractive and two depressant toxins active on insect were purified by high-performance liquid chromatography from the venom of Buthus occitanus tunetanus (Bot). The two depressant toxins, BotIT4 and BotIT5, differ only at position 6 (Arg for Lys) and are equally toxic to insects (LD50 to Blatella germanica = 110 ng/100 mg body weight). They show a strong antigenic cross-reaction with a depressive toxin from Leiurus quinquestriatus quinquestriatus (LqqIT2). The two toxins are able to inhibit with high affinity (K0.5 between 2 and 3 nM) the specific binding of the radioiodinated excitatory insect toxin (125I-AaHIT) on its receptor site on Periplaneta americana synaptosomal membranes. These toxins depolarize the cockroach axon, irreversibly block the action potential, and slow down and very progressively block the transmembrane transient Na+ current. The contracturant toxin BotIT1 is highly toxic to B. germanica (LD50 = 60 ng/ 100 mg body weight) and barely toxic to mice (LD50 = 1 microgram/20 g body weight) when injected intracerebroventricularly. It does not compete with 125I-AaHIT for its receptor site on P. americana synaptosomal membranes. On cockroach axon, BotIT1 develops plateau potentials and slows down the inactivation mechanism of the Na+ channels. Thus, BotIT1 belongs to the group of alpha insect-selective toxins and shows a strong sequence identity (> 90%) with Lqh alpha IT and LqqIII, two insect alpha-toxins previously purified from the venom of L. q. hebraeus and L. q. quinquestriatus. respectively.
Assuntos
Neurotoxinas/toxicidade , Venenos de Escorpião/isolamento & purificação , Venenos de Escorpião/toxicidade , Potenciais de Ação/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Cromatografia Líquida de Alta Pressão , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Neurotoxinas/química , Neurotoxinas/isolamento & purificação , Periplaneta/efeitos dos fármacos , Venenos de Escorpião/química , Venenos de Escorpião/genética , Escorpiões , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Neurotoxinas/toxicidade , Venenos de Escorpião/toxicidade , Canais de Sódio/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Antígenos/genética , Antígenos/isolamento & purificação , Axônios/efeitos dos fármacos , Axônios/metabolismo , Eletrofisiologia , Camundongos , Dados de Sequência Molecular , Estrutura Molecular , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurotoxinas/genética , Neurotoxinas/isolamento & purificação , Periplaneta , Venenos de Escorpião/genética , Venenos de Escorpião/isolamento & purificação , Escorpiões/química , Escorpiões/genética , Escorpiões/imunologia , Homologia de Sequência de AminoácidosRESUMO
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.
