Amino acid sequence of neurotoxin I from Centruroides sculpturatus Ewing

The further characterization of toxin I from venom of the scorpion Centruroides sculpturatus Ewing (region, Southwestern United States) is reported. Toxin I is a single polypeptide chain of 64 amino acid residues crosslinked by four disulfide bridges. The complete amino acid sequence of toxin I was deduced from the sequence of its tryptic peptides and overlaps provided by its chymotryptic peptides. Toxin I has an amino terminal lysyl residue and a carboxyl terminal threonyl residue.The amino acid sequences of toxin I and neurotoxic variants 1, 2, and 3, likewise isolated from C. sculpturatus venom, differ at 26 positions.The sequences of toxin I from C. sculpturatus and toxins I and II from the North African scorpion, Androctonus australis Hector, are also compared.     

Action of different toxins from the scorpion Androctonus australis on a locust nerve-muscle preparation

The neuromuscular effects of four purified toxins and crude venom from the scorpion Androctonus australis were investigated in the extensor tibiae nerve-muscle preparation of the locust Locusta migratoria. Insect and crustacean toxin and the mammal toxins I and II which have previously been shown to act on fly larvae, isopods, and mice all paralyse locust larvae. The paralytic potencies decrease in the following order: insect toxin → mammal toxin I → crustacean toxin → mammal toxin II.The toxins and crude venom cause repetitive activity of the motor axons. This leads to long spontaneous trains of junction potentials in the case of crude venom and insect toxin. The other toxins chiefly cause short bursts of action and junction potentials following single stimuli.The ‘slow’ excitatory motor axon invariably is affected sooner than the inhibitory or the ‘fast’ excitatory one. The minimal doses of toxins required to affect the ‘slow’ motor axon decrease in an order somewhat different from that established for their paralytic potencies: insect toxin → crustacean toxin → mammal toxin I → mammal toxin II.Crude venom depolarises and destabilises the muscle membrane potential at low doses. At high doses it decreases the membrane resistance, whereas insect toxin leads to an increase.Crude venom and insect toxin enhance the frequency of mejps, whereas mammal toxin I leads to the occurrence of ‘giant’ mejps.The pattern of axonal activities indicates that the various peripheral branches of the motor nerve are the primary target of the toxins.The time course of nerve action potentials is affected by mammal toxin I and crustacean toxin which cause anomalous shapes and prolongations not caused by insect toxin.The results with other animals suggest that only the insect toxin is selective in its activity. The way it affects the axon might be quite different from that previously reported for scorpion venoms or toxins.     

The complete amino acid sequence of toxin TsTX-VI isolated from the venom of the scorpionTityus serrulatus

The complete sequence of the toxin TsTX-VI from the venom of the scorpionTityus serrulatus Lutz and Mello is presented. The sequence has been determined by automated Edman analysis of the reduced and carboxymethylated protein as well as of the resulting peptides, obtained fromS. aureus protease and tryptic digestions. TsTX-VI is composed of 62 residues and has a calculated molecular weight of 6717. Homology studies with other scorpion toxins show that TsTX-VI is more similar to the Old World than to the North American scorpion toxins. The hydropathic index indicates that TsTX-VI is more hydrophobic than Ts-γ. Toxicity studies carried out in mice demonstrate that i.v. injection of TsTX-VI is unable to evoke the usual symptoms induced by the typical neurotoxins of this venom, but only a generalized allergic reaction. These properties are important in clarifying the relationship between primary structure and biological function of scorpion toxins.     

Rapid purification and molecular modeling of AaIT peptides from venom of Androctonus australis

As recombinant viruses expressing scorpion toxins are moving closer toward the market, it is important to obtain large amounts of pure toxin for biochemical characterization and the evaluation of biological activity in nontarget organisms. In the past, we purified a large amount of Androctonus australis anti-insect toxin (AaIT) present in the venom of A. australis with an analytical reversed-phase column by repeated runs of crude sample. We now report 20 times improved efficiency and speed of the purification by employing a preparative reversed-phase column. In just two consecutive HPLC steps, almost 1 mg of AaIT was obtained from 70 mg crude venom. Furthermore, additional AaIT was obtained from side fractions in a second HPLC run. Recently discovered insect selective toxin, AaIT5, was isolated simultaneously from the same venom batch. It shows different biological toxicity symptoms than the known excitatory and depressant insect toxins. AaIT5 gave 100% mortality with a dose of less than 1.3 μg against fourth-instar tobacco budworms Heliothis virescens 24 h after injection. During the purification process, we implemented mass spectrometry in addition to bioassays to monitor the presence of AaIT and AaIT5 in the HPLC fractions. Mass spectrometric screening can unambiguously follow the purification process and can greatly facilitate and expedite the downstream purification of AaIT and AaIT5 eliminating the number of bioassays required. Further, electrospray ionization was compared with matrix-assisted desorption/ionization and evaluated as a method of choice for mass spectrometric characterization of fractions from the venom purification for it provided higher mass accuracy and relative quantitation capability. Molecular models were built for AaIT5, excitatory toxin AaIT4, and depressant toxin LqhIT2. Three-dimensional structure of AaIT5 was compared with structures of the other two toxins, suggesting that AaIT5 is similar to depressant toxins. Arch. Insect Biochem. Physiol. 38:53–65, 1998. © 1998 Wiley-Liss, Inc.     

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.     

Functional and immuno-reactive characterization of a previously undescribed peptide from the venom of the scorpion Centruroides limpidus

A previously undescribed toxic peptide named Cl13 was purified from the venom of the Mexican scorpion Centruroides limpidus. It contains 66 amino acid residues, including four disulfide bonds. The physiological effects assayed in 7 different subtypes of voltage gated Na⁺-channels, showed that it belongs to the β-scorpion toxin type. The most notorious effects were observed in subtypes Nav1.4, Nav1.5 and Nav1.6. Although having important sequence similarities with two other lethal toxins from this scorpion species (Cll1m and Cll2), the recently developed single chain antibody fragments (scFv) of human origin were not capable of protecting against Cl13. At the amino acid sequence level, in 3 stretches of peptide Cl13 (positions 7–9, 30–38 and 62–66) some differences with respect to other similar toxins are observed. Some of these differences coincide with contact points with the human antibody fragments.     

In vivo and in vitro protection against lethal activity of Buthus occitanus tunetanus venom with a recombinant protein

We report the use of recombinant scorpion toxin in the form of fusion protein as antigen for mice immunisation. The aim is to produce protective antisera against lethal activity of the venom from Tunisian scorpion Buthus occitanus tunetanus, responsible for several annually reported human cases of scorpion stings. The gene encoding Bot III (the most toxic alpha toxin of Buthus occitanus tunetanus) was fused to the sequence encoding synthetic ZZ domains of staphylococcal protein A. The construct ZZ-Bot III was expressed in the periplasm of E. coli as a fusion protein and purified by affinity chromatography. The recombinant fusion protein was characterized and used as antigen to generate antibodies in mice. The antibodies against the recombinant protein neutralize the toxic venom (10 LD50/ml) and also confer protection for immunized mice against antigenically related mammal toxins.     

Amino acid sequence of toxin VII,A β-toxin from the venom of the scorpton Tityus serrulatus

The sequence of the 61 amino acids of toxin VII, a β-toxin from the venom of the South American scorpion Tityus serrulatus, has been determined by automatic sequencing of the reduced and S-[¹⁴C] car?ymethylated protein and of tryptic peptides obtained before or after citraconylation of this protein. This toxin, the most active β-toxin from this venom, is the first Tityus toxin to be fully sequenced. The results clearly show that toxin VII belongs to the structural group of scorpion toxins originating from Central and North America.     

Primary Structure of a Short Toxin from the Venom of a Vietnamese Scorpion (Buthus sp.)

The complete amino acid sequence of an important toxin (toxin 14) from the venom of a Vietnamese scorpion (Buthus occitanus sp.) has been determined, which includes 35 amino acid residues and three disulfide bridges (molecular weight, 3843 Da). The comparison of the sequence with known sequences of short scorpion toxins led to the conclusion that toxin 14 belongs to a novel group of toxins affecting the excitability of myelinated nerves.     

A positive charge at the N-terminal segment of Discrepin increases the blocking effect of K channels responsible for the IA currents in cerebellum granular cells

Discrepin is a scorpion peptide that blocks preferentially the I_A currents of the voltage-dependent K⁺ channel of rat cerebellum granular cells. It was isolated from the venom of the buthid scorpion Tityus discrepans and contains 38 amino acid residues with a pyroglutamic acid at the N-terminal site. Discrepin has the lowest sequence identity (approx. 50%) among the six members of the α-KTx15 sub-family of scorpion toxins. In order to find out which residues are important for the blocking effects of Discrepin, six mutants were chemically synthesized (V6K, I19R, D20K, T35V, I19R-D20K, I19R-D20K-R21V), correctly folded and their physiological properties were examined. Substitution of residues V6 and D20 for basically charged amino acids increases the blocking activity of Discrepin, specially the mutation V6K at the N-terminal segment of the toxin. Analysis of 3D-structure models of the mutants V6K and D20K supports the idea that basic residues improve their blocking activities, similarly to what happens with BmTx3, a toxic peptide obtained from Buthus martensi scorpion, which has the highest known blocking effects of I_A currents in K⁺ channels of rat cerebellum granular cells.     

The binding of the insect selective neurotoxin (AaIT) from scorpion venom to locust synaptosomal membranes

The binding of the radioiodinated insect selective neurotoxin from the venom of the scorpion Androctonus australis (AaIT), to synaptic plasma membrane vesicles derived from osmotically shocked insect synaptosomes was studied under kinetic and equilibrium conditions. The integrity of these vesicles and the existence of membrane potential and its modifiability were demonstrated by assays of the uptake of the lipophilic cation tetraphenylphosphonium. It has been shown that ¹²⁵I-labeled AaIT binds specifically and reversibly to a single class of noninteracting binding sites of high affinity ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}{}^1\text{= 1.2–3}\text{nM}$) and low capacity (1.2–2.0 pmol/mg protein). The values of the rate association and dissociation constants k₁ and k_?1 are, respectively, 1.36 · 10⁶ M^?1 · s^?1 and 1.9 · 10^?3 s^?1, and are in a good accordance with the equilibrium constant. The use of various ionophores and changes in external potassium concentration shown to modify the membrane potential of the present neuronal preparation, did not affect the binding of ¹²⁵I-AaIT, thus indicating its voltage-independence. Veratridine, tetrodotoxin, sea anemone toxin and the α and β scorpion toxins specific for vertebrates did not affect the binding of ¹²⁵I-AaIT. Furthermore, the above scorpion toxins were devoid of specific binding to the present insect neuronal preparation. Two additional insect toxins derived from the venom of the scorpion Buthotus judaicus, BjIT₁ (spastic-excitatory toxin, homologus to the AaIT) and BjIT₂ (flaccidity inducing-depressory toxin), were both shown to displace the ¹²⁵I-AaIT with a high affinity (K_d = 2.2 and 1.3 nM, respectively). These data are compared and discussed in light of the information concerning the interaction of scorpion venom toxins affecting vertebrates with mammalian neuronal tissues.     

东亚钳蝎蝎毒素BmKBT基因组序列的克隆及其分析

东亚钳蝎 (ButhusmartensiiKarsch ,BmK)蝎毒素BmKBT(又名BmKabT)是一个在初级结构上相似于β类哺乳动物毒素和功能接近于α类哺乳动物毒素的Na+ 通道毒素 .基于从毒腺cDNA文库中筛选得到的全长BmKBT前体核苷酸序列设计引物 ,以蝎基因组总DNA为模板进行聚合酶链式反应 (PCR) ,将PCR产物克隆至T载体、测序 .序列分析表明 :在BmKBT信号肽编码区的 3′端的- 4位Gly密码子的第 1位与第 2位碱基中有 1个长 2 2 5nt的内含子 ,插入位点距离该基因的起始密码子 4 6nt ,AT含量为 78 7% ,其内含子可能的剪接分枝位点距离 3′剪接受体位点 4 7nt.内含子的大小及其基因组织结构分析表明 :BmKBT具有与α类哺乳动物毒素类似的基因组织结构 ,进一步说明BmKBT是一个介于α类和β类Na+ 通道毒素之间的中间型蝎毒素 ,可以作为研究蝎毒素分子进化的合适材料     

Binding characteristics of BmK I, an alpha-like scorpion neurotoxic polypeptide, on cockroach nerve cord synaptosomes.

In this study, the binding characteristics of BmK I, an alpha-like neurotoxic polypeptide purified from the venom of the Chinese scorpion Buthus martensi Karsch, were investigated on rat brain and cockroach nerve cord synaptosomes. The results showed that BmK I can bind to a single class of noninteracting binding sites on cockroach nerve cord synaptosomes with medium affinity (Kd = 16.5 +/ - 4.4 nM) and low binding capacity (Bmax = 1.05 +/- 0.23 pmol/mg protein), but lacks specific binding on rat brain synaptosomes. BmK AS, BmK AS-1 (two novel sodium channel-blocking ligands), BmK IT (an excitatory insect-selective toxin) and BmK IT2 (a depressant insect-selective toxin) from the same venom were found to be capable of depressing BmK I binding in cockroach nerve cord synaptosomes, which might be attributed to either allosteric modulation of voltage-gated Na+ channels by these toxic polypeptides or partial overlapping between the receptor binding sites of BmK I and these toxins. This thus supported the notion that alpha-like scorpion neurotoxic polypeptides bind to a distinct receptor site on sodium channels, which might be similar to the binding receptor site of alpha-type insect toxins, and also related to those of BmK AS type and insect-selective scorpion toxins on insect sodium channels.     

An anti-insect toxin purified from the scorpion Androctonus australis Hector also acts on the alpha- and beta-sites of the mammalian sodium channel: sequence and circular dichroism study

A new anti-insect neurotoxin, AaH IT4, has been isolated from the venom of the North African scorpion Androctonus australis Hector. This polypeptide has a toxic effect on insects and mammals and is capable of competing with anti-insect scorpion toxins for binding to the sodium channel of insects; it also modulates the binding of alpha-type and beta-type anti-mammal scorpion toxins to the mammal sodium channel. This is the first report of a scorpion toxin able to exhibit these three kinds of activity. The molecule is composed of 65 amino acid residues and lacks methionine and, more unexpectedly, proline, which until now has been considered to play a role in the folded structure of all scorpion neurotoxins. The primary structure showed a poor homology with the sequences of other scorpion toxins; however, it had features in common with beta-type toxins. In fact, radioimmunoassays using antibodies directed to scorpion toxins representative of the main structural groups showed that there is a recognition of AaH IT4 via anti-beta-type toxin antibodies only. A circular dichroism study revealed a low content of regular secondary structures, particularly in beta-sheet structures, when compared to other scorpion toxins. This protein might be the first member of a new class of toxins to have ancestral structural features and a wide toxic range.     

Purification and chemical and biological characterizations of seven toxins from the Mexican scorpion, Centruroides suffusus suffusus

Seven polypeptides highly toxic to mice were isolated from the venom of the scorpion, Centruroides suffusus suffusus (Css), and their chemical and toxic properties were characterized. It was shown that the most active toxins by intracerebroventricular injection are less active when injected subcutaneously. The complete amino acid sequence (66 residues) of toxin II (Css II) has been determined. The C-terminal end is amidated as found for most other scorpion toxins. Css II is a beta-type toxin, previously used to define the binding site for activation of the sodium channel. Using rat brain synaptosomes, we demonstrated that all Css toxins compete with 125I-Css II to bind to site 4 and should be considered as beta-scorpion toxins. Specific binding parameters for Css VI, one of the most active toxins, were determined: KD = 100 pM; capacity in binding sites, 2.2 pmol of toxin/mg of synaptosomal protein. Css VI was shown to inhibit gamma-aminobutyric acid uptake by synaptosomes: K 0.5 = 100 pM, which agrees with its KD. Competition experiments between the seven Css toxins and 125I-Css II for antiserum raised against Css II demonstrated that all these toxins have common antigenic properties.     

The complete amino acid sequence of toxin TsTX-VI isolated from the venom of the scorpion Tityus serrulatus

The complete sequence of the toxin TsTX-VI from the venom of the scorpionTityus serrulatus Lutz and Mello is presented. The sequence has been determined by automated Edman analysis of the reduced and carboxymethylated protein as well as of the resulting peptides, obtained fromS. aureus protease and tryptic digestions. TsTX-VI is composed of 62 residues and has a calculated molecular weight of 6717. Homology studies with other scorpion toxins show that TsTX-VI is more similar to the Old World than to the North American scorpion toxins. The hydropathic index indicates that TsTX-VI is more hydrophobic than Ts-. Toxicity studies carried out in mice demonstrate that i.v. injection of TsTX-VI is unable to evoke the usual symptoms induced by the typical neurotoxins of this venom, but only a generalized allergic reaction. These properties are important in clarifying the relationship between primary structure and biological function of scorpion toxins.     

马氏蝎毒毒素的分离纯化及其对红细胞膜作用的初步研究

本文用山东产马氏蝎(Buthus martensii kashi)粗毒为材料,经SephadexG-50和Sp-Sephadex C-25二次柱层析,分离纯化获得三个毒峰部分,毒性比粗毒分别提高40—100倍。 纯度鉴定表明三个毒峰的聚丙烯酰胺凝胶电泳和等电聚焦电泳均为一条带,等电点分别为8.7,9.1,9.1,分子量用SDS-不连续聚丙烯酰胺凝胶电泳测定分别为6,600,5,000和8,500。对纯化蝎毒毒素的氨基酸组分也作了分析。 蝎毒毒素对人红细胞膜作用的初步探索结果表明:它使人红细胞膜的Na.K-ATP酶活性和膜脂流动性有所降低。     

Proteins toxic to arthropods in the venom of elapid snakes.

It has been found that the lethal action of elapid snake venoms to arthropods (fly larvae and isopods) is due to proteic factors differing from the toxins which are strongly and specifically active on mammals.This conclusion was based on the following: (1) Lack of any correlation between the toxic activity on larvae, isopods, and mice of ten elapid snake venoms. (2) Absence of any toxicity to arthropods in pure toxins isolated and purified from several elapid snake venoms according to their lethality. (3) Electrophoretical separation of the venom of the snake Naja mossambica mossambica (= N. nigricollis mossambica) resulted in fractions active either to arthropods and/or to mice. (4) Separation of the above venom by gel filtration on Sephadex G-50 enabled the isolation of fractions highly toxic to arthropods. (5) The above fractions demonstrated a high phospholipase activity corresponding to about 80 per cent of the total activity of the whole venom. The link between phospholipase and toxicity to arthropods will serve as a target for further investigation.It appears that the phenomenon of diversity in toxic activities of different proteins to different groups of organism, as previously demonstrated in scorpion venoms, is equally shared by elapid snake venoms.     

New weak toxins from the cobra venom

A protein with M 7485 Da containing five disulfide bonds was isolated from the venom of cobra Naja oxiana using various types of liquid chromatography. The complete amino acid sequence of the protein was determined by protein chemistry methods, which permitted us to assign it to the group of weak toxins. This is the first weak toxin isolated from the venom of N. oxiana. In a similar way, two new toxins with M 7628 and 7559 Da, which fall into the range of weak toxin masses, were isolated from the venom of the cobra N. kaouthia. The characterization of these proteins using Edman degradation and MALDI mass spectrometry has shown that one of these proteins is a novel weak toxin, and the other is the known weak toxin WTX with an oxidized methionine residue in position 9. Such a modification was detected in weak toxins for the first time. A study of the biological activity of the toxin from N. oxiana showed that, like other weak toxins, it can be bound by α7 and muscle-type nicotinic acetylcholine receptors.