Efficient in vitro refolding and characterization of a new peptide from the scorpion Buthotus saulcyi venom produced in Escherichia coli

The selective toxicity of depressant scorpion neurotoxins to insects is useful in studying the insect sodium channel gating, as well as being relevant to several other applications. In order to carry out structure/activity studies, the functional expression of such polypeptides is required. In the work reported here, the cDNA of a new peptide from the venom of the scorpion Buthotus saulcyi was cloned and sequenced. It codes for a 64 residues peptide (BsaulI) with 8 highly-conserved cysteines. This peptide shares high sequence similarity with depressant insect toxins of other scorpion species. Large amounts of insoluble BsaulI protein were expressed in Escherichia coli. Purification of this peptide was carried out under denaturing conditions. Renaturation was performed by pulsed dilution of the denatured BsaulI in the refolding buffer. Production of refolded Bsaul1, however, is approximately an order of magnitude higher than that obtained with similar scorpion depressant toxins. Intrinsic fluorescence, far-UV circular dichroism spectra and biological activity assays indicate that the peptide adopts a folded structure.     

Purification, characterization, and primary structure of four depressant insect-selective neurotoxin analogs from scorpion (Buthus sindicus) venom.

Four depressant insect-selective neurotoxin analogs (termed Bs-dprIT1 to 4) from the venom of the scorpion Buthus sindicus were purified to homogeneity in a single step using reverse-phase HPLC. The molecular masses of the purified toxins were 6820.9, 6892.4, 6714.7, and 6657.1 Da, respectively, as determined by mass spectrometry. These long-chain neurotoxins were potent against insects with half lethal dose values of 67, 81, 103, and 78 ng/100 mg larva and 138, 160, 163, and 142 ng/100 mg cockroach, respectively, but were not lethal to mice even at the highest applied dose of 10 microg/20 g mouse. When injected into blowfly larvae (Sarcophaga falculata), Bs-dprIT1 to 4 induced classical manifestations of depressant toxins, i.e., a slow depressant flaccid paralysis. The primary structures of Bs-dprIT 1 to 4 revealed high sequence homology (60-75%) with other depressant insect toxins isolated from scorpion venoms. Despite the high sequence conservation, Bs-dprIT1 to 4 showed some remarkable features such as (i) the presence of methionine (Met(6) in Bs-dprIT1 and Met(24) in Bs-dprIT2 to 4) and histidine (His(53) and His(57) in Bs-dprIT1) residues, i.e., amino acid residues that are uncommon to this type of toxin; (ii) the substitution of two highly conserved tryptophan residues (Trp43 --> Ala and Trp53 --> His) in the sequence of Bs-dprIT1; and (iii) the occurrence of more positively charged amino acid residues at the C-terminal end than in other depressant insect toxins. Multiple sequence alignment, sequence analysis, sequence-based structure prediction, and 3D homology modeling studies revealed a protein fold and secondary structural elements similar to those of other scorpion toxins affecting sodium channel activation. The electrostatic potential calculated on the surface of the predicted 3D model of Bs-dprIT1 revealed a significant positive patch in the region of the toxin that is supposed to bind to the sodium channel.     

Expression of murine poly(A) binding protein II gene in testis

Insect defensin refers to a group of antibacterial peptides derived from a variety of insect species as well as from scorpion and possessing a three-dimensional structure highly similar to that of scorpion toxins. A full-length cDNA encoding an insect defensin-like peptide was isolated from the venom gland cDNA library of the Chinese scorpion Buthus martensii Karsch. The precursor, the overall organization of which is similar to that of insect defensins, consists of 61 amino acid residues with a putative signal peptide of 15 residues, a propeptide of 7 residues, and a mature peptide of 39 residues (named BmTXKS2). The positions of six cysteines and a conserved glycine in mature BmTXKS2 are the same as those in LqDef, the first defensin found in scorpions, which suggests these peptides should present a similar cysteine-stabilized alphabetamotif. Phylogenetic analysis further shows that the structure of BmTXKS2 is closer to that of ancient defensins (e.g., LqDef and AaDef, two insect defensins present in the scorpion hemolymph) than to scorpion toxins.     

BjalphaIT: a novel scorpion alpha-toxin selective for insects--unique pharmacological tool

Long-chain neurotoxins derived from the venom of the Buthidae scorpions, which affect voltage-gated sodium channels (VGSCs) can be subdivided according to their toxicity to insects into insect-selective excitatory and depressant toxins (beta-toxins) and the alpha-like toxins which affect both mammals and insects. In the present study by the aid of reverse-phase HPLC column chromatography, RT-PCR, cloning and various toxicity assays, a new insect selective toxin designated as BjalphaIT was isolated from the venom of the Judean Black Scorpion (Buthotus judaicus), and its full primary sequence was determined: MNYLVVICFALLLMTVVESGRDAYIADNLNCAYTCGSNSYCNTECTKNGAVSGYCQWLGKYGNACWCINLPDKVPIRIPGACR (leader sequence is underlined). Despite its lack of toxicity to mammals and potent toxicity to insects, BjalphaIT reveals an amino acid sequence and an inferred spatial arrangement that is characteristic of the well-known scorpion alpha-toxins highly toxic to mammals. BjalphaITs sharp distinction between insects and mammals was also revealed by its effect on sodium conductance of two cloned neuronal VGSCs heterloguously expressed in Xenopus laevis oocytes and assayed with the two-electrode voltage-clamp technique. BjalphaIT completely inhibits the inactivation process of the insect para/tipE VGSC at a concentration of 100 nM, in contrast to the rat brain Na(v)1.2/beta1 which is resistant to the toxin. The above categorical distinction between mammal and insect VGSCs exhibited by BjalphaIT enables its employment in the clarification of the molecular basis of the animal group specificity of scorpion venom derived neurotoxic polypeptides and voltage-gated sodium channels.     

Molecular characterization of a possible progenitor sodium channel toxin from the Old World scorpion Mesobuthus martensii

Toxins affecting sodium channels widely exist in the venoms of scorpions throughout the world. These molecules comprise an evolutionarily related peptide family with three shared features including conserved three-dimensional structure and gene organization, and similar function. Based on different pharmacological profiles and binding properties, scorpion sodium channel toxins are divided into alpha- and beta-groups. However, their evolutionary relationship is not yet established. Here, we report a gene isolated from the venom gland of scorpion Mesobuthus martensii which encodes a novel sodium channel toxin-like peptide of 64 amino acids, named Mesotoxin. The Mesotoxin gene is organized into three exons and two introns with the second intron location conserved across the family. This peptide is unusual in that it has only three disulfides and a long cysteine-free tail with loop size and structural characteristics close to beta-toxins. Evolutionary analysis favors its basal position in the origin of scorpion sodium channel toxins as a progenitor. The discovery of Mesotoxin will assist investigations into the key issue regarding the origin and evolution of scorpion toxins.     

Purification of two depressant insect neurotoxins and their gene cloning from the scorpion Buthus martensi Karsch.

Insect-specific neurotoxins are important components of scorpion venoms. In this study, two toxins from the scorpion Buthus martensi Karsch (BmK) were purified. They shared high sequence homology with other depressant insect toxins and were designated BmK ITa and BmK ITb, respectively. They were able to suppress the action potential of cockroach isolated axon, which is due to a decrease in the peak sodium current. Furthermore, the effect of BmK ITb was lower than that of BmK ITa, and some of the electrophysiological characteristics of BmK ITb even resemble that of excitatory insect toxins. Their primary structures were determined by N-terminal partial sequence determination and cDNA cloning. The differences in their structures, especially the 31st residues, may result in the unique activity of BmK ITb.     

A scorpion venom neurotoxin paralytic to insects that affects sodium current inactivation: purification, primary structure, and mode of action

A new toxin, Lqh alpha IT, which caused a unique mode of paralysis of blowfly larvae, was purified from the venom of the scorpion Leiurus quinquestriatus hebraeus, and its structural and pharmacological properties were compared to those of three other groups of neurotoxins found in Buthinae scorpion venoms. Like the excitatory and depressant insect-selective neurotoxins, Lqh alpha IT was highly toxic to insects, but it differed from these toxins in two important characteristics: (a) Lqh alpha IT lacked strict selectivity for insects; it was highly toxic to crustaceans and had a measurable but low toxicity to mice. (b) It did not displace an excitatory insect toxin, 125I-AaIT, from its binding sites in the insect neuronal membrane; this indicates that the binding sites for Lqh alpha IT are different from those shared by the excitatory and depressant toxins. However, in its primary structure and its effect on excitable tissues, Lqh alpha IT strongly resembled the well-characterized alpha scorpion toxins, which affect mammals. The amino acid sequence was identical with alpha toxin sequences in 55%-75% of positions. This degree of similarity is comparable to that seen among the alpha toxins themselves. Voltage- and current-clamp studies showed that Lqh alpha IT caused an extreme prolongation of the action potential in both cockroach giant axon and rat skeletal muscle preparations as a result of the slowing and incomplete inactivation of the sodium currents. These observations indicate that Lqh alpha IT is an alpha toxin which acts on insect sodium channels.(ABSTRACT TRUNCATED AT 250 WORDS)     

A depressant insect toxin with a novel analgesic effect from scorpion Buthus martensii Karsch

A new peptide named BmK dITAP3 from scorpion Buthus martensii Karsch (BmK) has been identified to possess a dual bioactivity, a depressant neurotoxicity on insects and an analgesic effect on mice. The bioassays also showed that the peptide was definitely devoid of the neurotoxicity on mammals, which indicated that the analgesic effect of BmK dITAP3 could not be ascribed to the syndromic effects of a mammalian neurotoxicity. BmK dITAP3 exhibited 43.0% inhibition efficiency of the analgesic effect on mice at a dose of 5 mg/kg and the FPU value of 0.5 microg/body (approximately 30 mg) on the fly larvae. The pI value and the molecular mass determined by MALDI-TOF MS for dITAP3 were 6.5 and 6722.7, respectively. Its first 15 N-terminal residues were determined by Edman degradation, based on which the full amino acid sequence was deduced from the cDNA sequence encoding the peptide with 3'-RACE. Circular dichroism and sequence based prediction analyses showed dITAP3 may have a similar molecular scaffold as the most scorpion toxins but with features of the more beta structures and much less of alpha helix. The details of the purification, characterization and sequencing as well as the sequence comparison with other depressant insect toxins and the correlation between the analgesic effect and the insect toxicity will be reported and discussed, respectively.     

Functional duality and structural uniqueness of depressant insect-selective neurotoxins

Depressant insect-selective neurotoxins derived from scorpion venoms (a) induce in blowfly larvae a short, transient phase of contraction similar to that induced by excitatory neurotoxins followed by a prolonged flaccid paralysis and (b) displace excitatory toxins from their binding sites on insect neuronal membranes. The present study was undertaken in order to examine the basis of these similarities by comparing the primary structures and neuromuscular effects of depressant and excitatory toxins. A new depressant toxin (LqhIT2) was purified from the venom of the Israeli yellow scorpion. The effects of this toxin on a prepupal housefly neuromuscular preparation mimic the effects on the intact animal; i.e., a brief period of repetitive bursts of junction potentials is followed by suppression of their amplitude and finally by a block of neuromuscular transmission. Loose patch clamp recordings indicate that the repetitive activity has a presynaptic origin in the motor nerve and closely resembles the effect of the excitatory toxin AaIT. The final synaptic block is attributed to neuronal membrane depolarization, which results in an increase in spontaneous transmitter release; this effect is not induced by excitatory toxin. The amino acid sequences of three depressant toxins were determined by automatic Edman degradation. The depressant toxins comprise a well-defined family of polypeptides with a high degree of sequence conservation. This group differs considerably in primary structure from the excitatory toxin, with which it shares identical or related binding sites, and from the two groups of scorpion toxins that affect sodium conductance in mammals. The two opposing pharmacological effects of depressant toxins are discussed in light of the above data.     

一个新的东亚钳蝎毒素(BmKT_1)全长cDNA的克隆和分析

首先构建了东亚钳蝎毒腺组织 c DNA文库 ;根据已知的东亚钳蝎哺乳动物毒素氨基酸序列保守区设计引物 ,并用 PCR从 c DNA文库中扩增出一个 c DNA片段作为筛选 c DNA文库的探针 ;从 c DNA文库中筛选到二个编码同一个新的蝎毒素多肽的 c DNA,它们除 3′- UTR外 ,其余序列完全一致 .它们均含有 2 55bp长的开放阅读框 ,编码 85肽的前体毒素 ,包括 1 9个氨基酸残基的信号肽 ,66个残基的成熟毒素 (命名为 Bm KT1) ;Bm KT1氨基酸序列与已知的蝎毒素具有较大的同源性 ,与 Bm KM1,Lqq ,Lqhα IT和 Bm K M10 的同源性分别为 77%、67%、67%和 65% .Bm KT1的 C端不存在末端修饰步骤且具有一个与这些毒素不相同的特征结构 ,即在末端延伸了两个氨基酸残基 - P- S,推测 Bm KT1具有新的活性功能特征 .     

Molecular cloning and nucleotide sequence analysis of encoded anti-insect toxin BotIT2 from the scorpion Buthus occitanus tunetanus venom

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.     

An 'Old World' scorpion beta-toxin that recognizes both insect and mammalian sodium channels.

Scorpion toxins that affect sodium channel (NaCh) gating in excitable cells are divided into alpha- and beta-classes. Whereas alpha-toxins have been found in scorpions throughout the world, anti-mammalian beta-toxins have been assigned, thus far, to 'New World' scorpions while anti-insect selective beta-toxins (depressant and excitatory) have been described only in the 'Old World'. This distribution suggested that diversification of beta-toxins into distinct pharmacological groups occurred after the separation of the continents, 150 million years ago. We have characterized a unique toxin, Lqhbeta1, from the 'Old World' scorpion, Leiurus quinquestriatus hebraeus, that resembles in sequence and activity both 'New World'beta-toxins as well as 'Old World' depressant toxins. Lqhbeta1 competes, with apparent high affinity, with anti-insect and anti-mammalian beta-toxins for binding to cockroach and rat brain synaptosomes, respectively. Surprisingly, Lqhbeta1 also competes with an anti-mammalian alpha-toxin on binding to rat brain NaChs. Analysis of Lqhbeta1 effects on rat brain and Drosophila Para NaChs expressed in Xenopus oocytes revealed a shift in the voltage-dependence of activation to more negative membrane potentials and a reduction in sodium peak currents in a manner typifying beta-toxin activity. Moreover, Lqhbeta1 resembles beta-toxins by having a weak effect on cardiac NaChs and a marked effect on rat brain and skeletal muscle NaChs. These multifaceted features suggest that Lqhbeta1 may represent an ancestral beta-toxin group in 'Old World' scorpions that gave rise, after the separation of the continents, to depressant toxins in 'Old World' scorpions and to various beta-toxin subgroups in 'New World' scorpions.     

Structure-activity relationship of an alpha-toxin Bs-Tx28 from scorpion (Buthus sindicus) venom suggests a new alpha-toxin subfamily

Scorpion venoms are among the most widely known source of peptidyl neurotoxins used for callipering different ion channels, e.g., for Na(+), K(+), Ca(+) or Cl(-). An alpha-toxin (Bs-Tx28) has been purified from the venom of scorpion Buthus sindicus, a common yellow scorpion of Sindh, Pakistan. The primary structure of Bs-Tx28 was established using a combination of MALDI-TOF-MS, LC-ESI-MS, and automated Edman degradation analysis. Bs-Tx28 consists of 65 amino acid residues (7274.3+/-2Da), including eight cysteine residues, and shows very high sequence identity (82-94%) with other long-chain alpha-neurotoxins, active against receptor site-3 of mammalian (e.g., Lqq-IV and Lqh-IV from scorpions Leiurus sp.) and insect (e.g., BJalpha-IT and Od-1 from Buthotus judaicus and Odonthobuthus doriae, respectively) voltage-gated Na(+) channels. Multiple sequence alignment and phylogenetic analysis of Bs-Tx28 with other known alpha- and alpha-like toxins suggests the presence of a new and separate subfamily of scorpion alpha-toxins. Bs-Tx28 which is weakly active in both, mammals and insects (LD(50) 0.088 and 14.3microg/g, respectively), shows strong induction of the rat afferent nerve discharge in a dose-dependent fashion (EC(50)=0.01microg/mL) which was completely abolished in the presence of tetrodotoxin suggesting the binding of Bs-Tx28 to the TTX-sensitive Na(+)-channel. Three-dimensional structural features of Bs-Tx28, established by homology modeling, were compared with other known classical alpha-mammal (AaH-II), alpha-insect (Lqh-alphaIT), and alpha-like (BmK-M4) toxins and revealed subtle variations in the Nt-, Core-, and RT-CT-domains (functional domains) which constitute a "necklace-like" structure differing significantly in all alpha-toxin subfamilies. On the other hand, a high level of conservation has been observed in the conserved hydrophobic surface with the only substitution of W43 (Y43/42) and an additional hydrophobic character at position F40 (L40/A/V/G39), as compared to the other mentioned alpha-toxins. Despite major differences within the primary structure and activities of Bs-Tx28, it shares a common structural and functional motif (e.g., transRT-farCT) within the RT-CT domain which is characteristic of scorpion alpha-mammal toxins.     

Neurotoxins active on insects: amino acid sequences, chemical modifications, and secondary structure estimation by circular dichroism of toxins from the scorpion Androctonus australis Hector

Two scorpion neurotoxins active only on insects, the insect toxins AaH IT1 and AaH IT2, were purified from the venom of scorpions Androctonus australis Hector collected in Tozeur (Tunisia) and characterized. AaH IT2 was sequenced and found to differ in four amino acid positions from AaH IT, the single previously sequenced insect toxin [Darbon, H., Zlotkin, E., Kopeyan, C., Van Rietschoten, J., & Rochat, H. (1982) Int. J. Pept. Protein Res. 20, 320-330] which possessed an equal potential for paralyzing fly larvae. The basic amino acid residues of AaH IT1, which differs from AaH IT by one amino acid residue, were selectively chemically modified. Six derivatives were characterized. Their toxicity toward fly larvae and cockroach was determined, and their affinity for the AaH IT1 synaptosomal receptor from cockroach nerve cord was measured. Modification of His-30, Lys-34, and Arg-60 showed no significant effect on biological activity. However, the modification of Lys-28 or Lys-51 demonstrated that these two amino acids are important for toxicity. Furthermore, simultaneous modifications of both Lys-28 and Lys-51 led to a cumulative decrease in biological activity. AaH IT1 and AaH IT2 show similar CD spectra. The secondary structures content of AaH IT2 was estimated from circular dichroism data. Results showed that this class of toxin should possess an additional alpha-helical region and a beta-sheet strand, not found in toxins active on mammals. Attempts to localize these secondary structural features in the amino acid sequence of AaH IT2 indicated that these two regions would be located within the last 20 C-terminal amino acid residues. From these studies on secondary structures, it is possible to consider that toxins active on insects are more structurally constrained than those active on mammals; a decreased molecular flexibility may be, at least partially, responsible for the observed specificity of these toxins for the insect sodium channel. Furthermore, the two alpha-helices found in insect toxins enclosed the two conserved Lys-28 and Lys-51 and might thus be implicated in the toxic site of insect toxins.     

Molecular characterization of an anti-epilepsy peptide from the scorpion Buthus martensi Karsch.

For a long time Asian scorpion Buthus martensi Karsch (BmK) has been used in Chinese traditional medicine to cure many diseases of nervous system. Here we report the purification and characterization of a pharmacologically active neurotoxin from the scorpion BmK. This toxin had little toxicity in mice and insects but was found to have an anti-epilepsy effect in rats, and is thus named as BmK anti-epilepsy peptide (BmK AEP). Its amino-acid sequence was determined by lysylendopeptidase digestion, Edman degradation and mass spectrographic analysis. Based on the determined sequence, the gene coding for this peptide was also cloned and sequenced by the 3' and 5' RACE methods. It encodes a precursor of 85 amino-acid residues including a signal peptide of 21 residues, a mature peptide of 61 residues and three additional residues Gly-Lys-Lys at the C-terminus. The additional Gly sometimes followed by one or two basic residues is prerequisite for the amidation of its C-terminus. C-terminal amidation was also verified by the molecular-mass determination of BmK AEP. This anti-epilepsy peptide toxin shares homology with other depressant insect toxins. The remarkable difference between them was mainly focused at residues 6, 7 and 39; these residues might relate to the unique action of BmK AEP.     

Tityus serrulatus toxin VII bears pharmacological properties of both beta-toxin and insect toxin from scorpion venoms

Some beta-toxins from the South American scorpion Tityus serrulatus (e.g. Ts VII) are highly toxic both for mouse and fly larva. Radioiodinated Ts VII and the insect toxin from the North African scorpion Androctonus australis Hector (AaH IT) bind to the same site on a house fly head synaptosomal fraction. These results reinforce the hypothesis about the existence of a correlated series of scorpion toxins as previously defined by amino acid compositions and sequences, and immunological and circular dichroism studies, in suggesting that Ts VII constitutes a link which may fill the pharmacological gap existing between beta-toxins and insect toxins such as AaH IT.     

Crystal structure of neurotoxin Ts1 from Tityus serrulatus provides insights into the specificity and toxicity of scorpion toxins.

The crystal structure of neurotoxin Ts1, a major component of the venom of the Brazilian scorpion Tityus serrulatus, has been determined at 1.7 A resolution. It is the first X-ray structure of a highly toxic anti-mammalian beta-toxin. The folding of the polypeptide chain of Ts1 is similar to that of other scorpion toxins. A cysteine-stabilised alpha-helix/beta-sheet motif forms the core of the flattened molecule. All residues identified as functionally important by chemical modification and site-directed mutagenesis are located on one side of the molecule, which is therefore considered as the Na+channel recognition site. The distribution of charged and non-polar residues over this surface determines the specificity of the toxin-channel interaction. Comparison to other scorpion toxins shows that positively charged groups at positions 1 and 12 as well as a negative charge at position 2 are likely determinants of the specificity of beta-toxins. In contrast, the contribution of the conserved aromatic cluster to the interaction might be relatively small. Comparison of Ts1 to weak beta-toxins from Centruroides sculpturatus Ewing reveals that a number of basic amino acid residues located on the face of the molecule opposite to the binding surface may account for the high toxicity of Ts1.     

Were arachnids the first to use combinatorial peptide libraries?

Spiders, scorpions, and cone snails are remarkable for the extent and diversity of gene-encoded peptide neurotoxins that are expressed in their venom glands. These toxins are produced in the form of structurally constrained combinatorial peptide libraries in which there is hypermutation of essentially all residues in the mature-toxin sequence with the exception of a handful of strictly conserved cysteines that direct the three-dimensional fold of the toxin. This gene-based combinatorial peptide library strategy appears to have been first implemented by arachnids almost 400 million years ago, long before cone snails evolved a similar mechanism for generating peptide diversity.     

Study of the binding residues between ANEPII and insect sodium channel receptor

The present study aimed at determining the functional characteristics of anti-neuroexcitation peptide II (ANEPII). The depressant insect toxin ANEPII from the Chinese scorpion Buthus martensii Karsch had an effect on insect sodium channels. Previous studies showed that scorpion depressant toxins induce insect flaccid paralysis upon binding to receptor site-4, so we tried to predict the functional residues involved using computational techniques. In this study, three-dimensional structure modeling of ANEPII and site-4 of the insect sodium channel were carried out by homology modeling, and these models were used as the starting point for nanosecond-duration molecular dynamics simulations. Docking studies of ANEPII in the sodium channel homology model were conducted, and likely ANEPII binding loci were investigated. Based on these analyses, the residues Tyr34, Trp36, Gly39, Leu40, Trp53, Asn58, Gly61 and Gly62 were predicted to interact with sodium channel receptor and to act as functional residues.