Isolation and characterization of two toxins from the Mexican scorpion Centruroides limpidus limpidus Karsch

1. Several toxic polypeptides were found in the venom of the scorpion Centruroides limpidus limpidus. Comparative studies of the potency of the venom in different strains of mice were conducted. 2. A new type of toxin (component II.9), specific for crustaceans (crayfish and isopods), was isolated from this scorpion and was shown to have the following N-terminal amino acid sequence: Lys-Lys-Asp-Gly-Tyr-Leu-Val-Asn-Lys-Tyr-Thr-Gly-Cys-Lys-Val-Asn-Cys- Tyr-Lys-Leu-Gly-Glu-Asn-Lys-Phe-Cys-Asn-Arg-Glu-. 3. A polypeptide toxic to mice (component II.6) from this venom was shown to have the following N-terminal sequence: Lys-Glu-Gly-Tyr-Leu-Val-Asn-His-Ser-Thr-Gly-Cys-Lys-Tyr- Glu-Cys-Tyr-Lys-Leu-Gly-Asp-Asn-Asp-Tyr-Cys-Leu-Arg-Glu-Cys-Lys-. 4. In cultured chick dorsal root ganglion cells, 1 microM of toxin II.6 was shown to reduce the size of sodium currents and to slow-down their activation-inactivation kinetics. The toxin had also a depressive action on the classical Ca2+ current activated at high membrane potentials (greater than 0 mV).     

Isolation of minax toxins from the venom of the scorpion Buthus minax and their metabolic effects

Two neurotoxins, minax toxins 1 and 2, were isolated from venom of the scorpion Buthus minax from the Sudan. Molecular weights of 7000 and 6800 and 66 and 62 amino acids were found for minax toxins 1 and 2, respectively. Both toxins contain four disulfide bonds, 1 mol each of phenylalanine, histidine, and tryptophan, no free sulfhydryl groups, and no methionine. Both minax toxins 1 and 2 are basic polypeptides with isoelectric points of 8.2 and 9.0, respectively. There is a significant increase in the calcium content of rat hearts envenomated with minax toxins 1 and 2 or crude venom. This confirms earlier electron microscopic findings of calcium deposits in the heart following scorpion envenomation. There is a concomitant decrease in the calcium and phosphorus content of rat serum following envenomation. It seems that neither scorpion toxins nor scorpion venoms affect the mineral metabolism of the bone. The present investigation indicates that scorpion toxins have not only a neurotoxic action but also broader biological effects such as mineral metabolism.     

Novel structural class of four disulfide-bridged peptides from Tityus serrulatus venom

A new structural class of short peptides folded by four disulfide-bridges was found in the venom of the Brazilian scorpion Tityus serrulatus. Peptides were put on evidence independently by means of two different approaches of structurally guided prospection. First, a cDNA sequence was obtained using a degenerate primer constructed according to the C-terminal sequence of kaliotoxin (KTx2), from the Androctonus australis venom. Second, MALDI-TOF mass spectrometry analyses of toxic fraction FIII from T. serrulatus venom revealed a family of molecules ranging approximately from 2900 to 3000 Da. Three new peptides were isolated and named TsPep1, TsPep2, and TsPep3. Biochemical characterization showed that they are 29 amino acids long, constrained by a new pattern of four disulfide-bridges. These results enable us to classify these new molecules as part of a novel structural class of short peptides from scorpion venoms.     

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 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.     

SdPI, the first functionally characterized Kunitz-type trypsin inhibitor from scorpion venom

Background

Kunitz-type venom peptides have been isolated from a wide variety of venomous animals. They usually have protease inhibitory activity or potassium channel blocking activity, which by virtue of the effects on predator animals are essential for the survival of venomous animals. However, no Kunitz-type peptides from scorpion venom have been functionally characterized.

Principal Findings

A new Kunitz-type venom peptide gene precursor, SdPI, was cloned and characterized from a venom gland cDNA library of the scorpion Lychas mucronatus. It codes for a signal peptide of 21 residues and a mature peptide of 59 residues. The mature SdPI peptide possesses a unique cysteine framework reticulated by three disulfide bridges, different from all reported Kunitz-type proteins. The recombinant SdPI peptide was functionally expressed. It showed trypsin inhibitory activity with high potency (K_i = 1.6×10⁻⁷ M) and thermostability.

Conclusions

The results illustrated that SdPI is a potent and stable serine protease inhibitor. Further mutagenesis and molecular dynamics simulation revealed that SdPI possesses a serine protease inhibitory active site similar to other Kunitz-type venom peptides. To our knowledge, SdPI is the first functionally characterized Kunitz-type trypsin inhibitor derived from scorpion venom, and it represents a new class of Kunitz-type venom peptides.     

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.     

Radiation protection effectiveness of various zootoxins

A study was made of the effectiveness of a new class of radioprotective agents, polypeptides, obtained from zootoxins of scorpion, tarantula, Lathrodectes tredecimguttatus, and bee under conditions of a short-term and long-term irradiation. The peptide fraction of the scorpion venom, butoxin, was most radioprotective: it provided 65% survival after LD98/30. Butoxin exerted a stimulatory effect on the hypophysis-adrenal system and haemopoiesis of intact and irradiated animals.     

Amino acid sequence of 2 neurotoxins from the scorpion Buthus eupeus venom

Three polypeptides, M10, M14 and M9, toxic to mammals were isolated from the venom of the Central Asian scorpion Buthus eupeus. All the toxins were shown to be homogeneous according to disc-electrophoresis and N-terminal group analyses. The toxin M9 was digested with trypsin, Staphylococcus aureus proteinase and cleaved with BNPS-skatole. The toxin M14 was subjected to tryptic and chymotryptic hydrolyses. The complete amino acid sequences of the toxins M9 and M14 were established and it was shown that each of them consists of 66 amino acid residues with four intramolecular disulfide bonds.     

Precursors of three unique cysteine-rich peptides from the scorpion Buthus martensii Karsch

Scorpion venoms contain a large number of small peptides with diverse primary structures and unique pharmacological functions. From a cDNA library prepared from venom glands of the Chinese scorpion Buthus martensii Karsch, clones encoding precursors of three unique cysteine-rich peptides named BmTXKS3, BmTXLP2 and BmAP1 have been isolated and sequenced. These precursors are composed of 54, 94 and 89 amino acids, respectively, containing a signal peptide in their N-termini. Sequence analysis shows that BmTXKS3 and BmTXLP2 are two novel members of a scorpion toxin family sharing cysteine-stabilized α-helical folds. BmAP1 possesses a distinctive cystine framework, which is similar to some serine protease inhibitors and the segments of several extracellular proteins.     

Tamulustoxin: a novel potassium channel blocker from the venom of the Indian red scorpion Mesobuthus tamulus

We have characterized tamulustoxin, a novel 35-amino-acid peptide found in the venom of the Indian red scorpion (Mesobuthus tamulus). Tamulustoxin was identified through a [125I]toxin I screen, designed to identify toxins that block voltage-activated potassium channels. Tamulustoxin has also been cloned by RT-PCR, using RNA extracted from scorpion venom glands. Tamulustoxin shares no homology with other scorpion venom toxins, although the positions of its six cysteine residues would suggest that it shares the same structural scaffold. Tamulustoxin rapidly inhibited both peak and steady-state currents (18.9 +/- 1.0 and 37 +/- 1.1%, respectively) produced by injecting CHO cells with mRNA encoding the hKv1.6 channel.     

Wide phylogenetic distribution of Scorpine and long-chain beta-KTx-like peptides in scorpion venoms: identification of "orphan" components

Scorpine and toxins specific for potassium channels of the family beta (beta-Ktx) are two types of structurally related scorpion venom components, characterized by an unusually long extended N-terminal segment, followed by a Cys-rich domain with some resemblance to other scorpion toxins. In this communication, we report evidence supporting the ubiquitous presence of Scorpine and beta-KTx-like polypeptides and their precursors in scorpions of the genus Tityus of the family Buthidae, but also included is the first example of such peptides in scorpions from the family Iuridae. Seven new beta-KTxs or Scorpine-like peptides and precursors are reported: five from the genus Tityus (T. costatus, T. discrepans and T. trivittatus) and two from Hadrurus gertschi. The cDNA precursors for all of these peptides were obtained by molecular cloning and their presence in the venoms were confirmed for various peptides. Analysis of the sequences revealed the existence of at least three distinct groups: (1) beta-KTx-like peptides from buthids; (2) Scorpine-like peptides from scorpionid and iurid scorpions; (3) heterogeneous peptides similar to BmTXKbeta of buthids and iurids. The biological function for most of these peptides is not well known; that is why they are here considered "orphan" peptides.     

Solution structure of IsTX. A male scorpion toxin from Opisthacanthus madagascariensis (Ischnuridae).

The novel sex-specific potassium channel inhibitor IsTX, a 41-residue peptide, was isolated from the venom of male Opisthacanthus madagascariensis. Two-dimensional NMR techniques revealed that the structure of IsTX contains a cysteine-stabilized alpha/beta-fold. IsTX is classified, based on its sequential and structural similarity, in the scorpion short toxin family alpha-KTx6. The alpha-KTx6 family contains a single alpha-helix and two beta-strands connected by four disulfide bridges and binds to voltage-gated K(+) channels and apamin-sensitive Ca(2+)-activated K(+) channels. The three-dimensional structure of IsTX is similar to that of Heterometrus spinifer toxin (HsTX1). HsTX1 blocks the Kv1.3 channel at picomolar concentrations, whereas IsTX has much lower affinities (10 000-fold). To investigate the structure-activity relationship, the geometry of sidechains and electrostatic surface potential maps were compared with HsTX1. As a result of the comparison of the primary structures, Lys27 of IsTX was conserved at the same position in HsTX1. The analogous Lys23 of HsTX1, the most critical residue for binding to potassium channels, binds to the channel pore. However, IsTX has fewer basic residues to interact with acidic channel surfaces than HsTX1. MALDI-TOF MS analysis clearly indicated that IsTX was found in male scorpion venom, but not in female. This is the first report that scorpion venom contains sex-specific compounds.     

A mammal toxin derived from the venom of a chactoid scorpion

1. It has been shown that the low toxicity to mammals (LD50 of about 200 mg per kg mice body weight) of the chactoid scorpion venom Scorpio maurus palmatus (Scorpionidae) is due to a single low molecular weight basic protein. 2. This compound was purified by the aid of gel filtration and ion exchange column chromatography, possessed about 80% of the mice lethality of the crude venom with an increase of about 60 fold in its specific toxicity. 3. It is composed of 32 amino acids (mol. wt = 3478) and devoid of isoleucine, leucine, phenylalanine, histidine and tryptophan. 4. The unique amino acid composition of the present toxin is compared to those of the well known buthoid scorpion venom mammal toxins and some other toxins derived from the same venom. 5. It is the first chemically characterized chactoid toxin.     

Purification, cDNA cloning and function assessment of BmK abT, a unique component from the Old World scorpion species

A new neurotoxic component named BmK abT was purified from the venom of Chinese scorpion Buthus martensi Karsch. The molecular weight of BmK abT was determined to be 7212 Da on a mass spectrum. The minimum lethal dose of BmK abT was tested to be about 1.5 microg per mouse by intracerebroventricular injection, and the dose induced significant paralysis effect on cockroach was about 5 microg by i.p. injection. The partial amino acid sequence indicated that it was a distinctive polypeptide in the scorpion neurotoxin family. Thereafter, the whole amino acid sequence of mature BmK abT was deduced from cDNA sequence by 5'- and 3'-rapid amplification of cDNA ends. Finally, it was defined to be composed of 63 residues with amidation at the C-terminal residue. By sequence comparison, BmK abT was found to be most similar to Ts VII, a beta-toxin from the New World scorpion. The patch-clamp recording on DRG neurons, unexpectedly, showed this toxin could prolong the action potential and increase the amplitude of the peak Na+ currents, which are the typical characters of alpha-toxin. These results suggested that BmK abT was a new toxic component found in the Old World scorpion species structurally similar to beta-toxins, but functionally similar to alpha-toxins.     

Antibacterial and antifungal properties of alpha-helical, cationic peptides in the venom of scorpions from southern Africa.

Two novel pore-forming peptides have been isolated from the venom of the South-African scorpion Opistophtalmus carinatus. These peptides, designated opistoporin 1 and 2, differ by only one amino acid and belong to a group of alpha-helical, cationic peptides. For the first time, a comparison of the primary structures of alpha-helical pore-forming peptides from scorpion venom was undertaken. This analysis revealed that peptides in the range of 40-50 amino acids contain a typical scorpion conserved sequence S(x)3KxWxS(x)5L. An extensive study of biological activity of synthesized opistoporin 1 and parabutoporin, a pore-forming peptide previously isolated from the venom of the South-African scorpion Parabuthus schlechteri, was undertaken to investigate an eventual cell-selective effect of the peptides. Opistoporin 1 and parabutoporin were most active in inhibiting growth of Gram-negative bacteria (1.3-25 micro m), while melittin and mastoparan, two well-known cytolytic peptides, were more effective against Gram-positive bacteria in the same concentration range. In addition, the peptides showed synergistic activity with some antibiotics commonly used in therapy. Opistoporin 1 and parabutoporin had hemolytic activity intermediate between the least potent mastoparan and the highly lytic melittin. Furthermore, all peptides inhibited growth of fungi. Experiments with SYTOX green suggested that this effect is related to membrane permeabilization.     

Characterization of two different peptides from the venom of the scorpion Buthus sindicus

Two disulfide-rich, low-molecular mass peptides (approximately 3 kDa and approximately 4 kDa) have been isolated from Buthus sindicus venom using ion-exchange and reverse-phase HPLC. Peptide I has 35 residues with 8 half-cystine residues and is clearly related to four-disulfide core proteins of the neurophysin type and to toxins of other scorpion species (55-63% residue identity). Peptide II, present in low yield, has 28 residues with 6 half-cystine residues and a structure largely dissimilar from that of peptide I and other characterized toxins, although probably still a member of the disulfide core peptide type. Consequently, scorpion venom contains, in addition to toxins characterized before, toxin-like compounds with distant relationships.