General biochemical and immunological characteristics of the venom from Peruvian scorpion Hadruroides lunatus

This communication describes the general biochemical properties and some immunological characteristics of the venom from the Peruvian scorpion Hadruroides lunatus, which is the most medically relevant species in Peru. The soluble venom of this scorpion is toxic to mice, the LD₅₀ determined was 0.1 mg/kg and 21.55 mg/kg when the venom was injected intracranial or intraperitoneally, respectively. The soluble venom displayed proteolytic, hyaluronidasic, phospholipasic and cardiotoxic activities. High performance liquid chromatography of the soluble venom resulted in the separation of 20 fractions. Two peptides with phospholipasic activity were isolated to homogeneity and their molecular masses determined by mass spectrometry (MALDI TOF). Anti-H. lunatus venom sera were produced in rabbits. Western blotting analysis showed that most of the protein content of this venom is immunogenic. H. lunatus anti-venom displayed consistent cross-reactivity with venom antigens from the new World-scorpions Tityus serrulatus and Centruroides sculpturatus venoms; however, a weaker reactivity was observed against the venom antigens from the old World-scorpion Androctonus australis Hector.     

Full Neutralization of Centruroides sculpturatus Scorpion Venom by Combining Two Human Antibody Fragments

A fundamental issue of the characterization of single-chain variable fragments (scFvs), capable of neutralizing scorpion toxins, is their cross-neutralizing ability. This aspect is very important in Mexico because all scorpions dangerous to humans belong to the Centruroides genus, where toxin sequences show high identity. Among toxin-neutralizing antibodies that were generated in a previous study, scFv 10FG2 showed a broad cross-reactivity against several Centruroides toxins, while the one of scFv LR is more limited. Both neutralizing scFvs recognize independent epitopes of the toxins. In the present work, the neutralization capacity of these two scFvs against two medically important toxins of the venom of Centruroides sculpturatus Ewing was evaluated. The results showed that these toxins are recognized by both scFvs with affinities between 1.8 × 10⁻⁹ and 6.1 × 10⁻¹¹ M. For this reason, their ability to neutralize the venom was evaluated in mice, where scFv 10FG2 showed a better protective capacity. A combination of both scFvs at a molar ratio of 1:5:5 (toxins: scFv 10FG2: scFv LR) neutralized the venom without the appearance of any signs of intoxication. These results indicate a complementary activity of these two scFvs during venom neutralization.     

Colombian Scorpion Centruroides margaritatus: Purification and Characterization of a Gamma Potassium Toxin with Full-Block Activity on the hERG1 Channel

The Colombian scorpion Centruroides margaritatus produces a venom considered of low toxicity. Nevertheless, there are known cases of envenomation resulting in cardiovascular disorders, probably due to venom components that target ion channels. Among them, the human ether-à-go-go-Related gene (hERG1) potassium channels are critical for cardiac action potential repolarization and alteration in its functionality are associated with cardiac disorders. This work describes the purification and electrophysiological characterization of a Centruroides margaritatus venom component acting on hERG1 channels, the CmERG1 toxin. This novel peptide is composed of 42 amino acids with a MW of 4792.88 Da, folded by four disulfide bonds and it is classified as member number 10 of the γ-KTx1 toxin family. CmERG1 inhibits hERG1 currents with an IC₅₀ of 3.4 ± 0.2 nM. Despite its 90.5% identity with toxin ɣ-KTx1.1, isolated from Centruroides noxius, CmERG1 completely blocks hERG1 current, suggesting a more stable plug of the hERG channel, compared to that formed by other ɣ-KTx.     

Centruroides sculpturatus venom and platelet reactivity: possible role in scorpion venominduced defibrination syndrome

The effect of Centruroides sculpturatus venom and some purified fractions on the aggregation behavior of dog platelets has been examined. The venom causes a sustained partial aggregation, which proceeds almost instantly to complete aggregation on addition of adenosine diphosphate (ADP): the maximum aggregation to ADP is unaffected. The effects of the venom are not mimicked by any of the currently isolated neurotoxins, but are by a fraction obtained from the primary gradient separation of the venom. The effect of the venom is heat labile and unaffected by prior treatment of the platelets with tetrodotoxin, tetraethylammonium or aspirin. Heat lability rules out effects from most inorganic salts, serotonin or phospholipase A₂, which are heat stable. A defibrination syndrome has been noted following envenomation by a member of the family Buthidae, and possibly occurs for C. sculpturatus venom as well: the effects of the venom on platelets may account in part for the occurrence of this syndrome.     

Characterization of Four Medically Important Toxins from Centruroides huichol Scorpion Venom and Its Neutralization by a Single Recombinant Antibody Fragment

Centruroides huichol scorpion venom is lethal to mammals. Analysis of the venom allowed the characterization of four lethal toxins named Chui2, Chui3, Chui4, and Chui5. scFv 10FG2 recognized well all toxins except Chui5 toxin, therefore a partial neutralization of the venom was observed. Thus, scFv 10FG2 was subjected to three processes of directed evolution and phage display against Chui5 toxin until obtaining scFv HV. Interaction kinetic constants of these scFvs with the toxins were determined by surface plasmon resonance (SPR) as well as thermodynamic parameters of scFv variants bound to Chui5. In silico models allowed to analyze the molecular interactions that favor the increase in affinity. In a rescue trial, scFv HV protected 100% of the mice injected with three lethal doses 50 (LD₅₀) of venom. Moreover, in mix-type neutralization assays, a combination of scFvs HV and 10FG2 protected 100% of mice injected with 5 LD₅₀ of venom with moderate signs of intoxication. The ability of scFv HV to neutralize different toxins is a significant achievement, considering the diversity of the species of Mexican venomous scorpions, so this scFv is a candidate to be part of a recombinant anti-venom against scorpion stings in Mexico.     

Purification and characterization of a mammalian toxin from venom of the Mexican scorpion,Centruroides limpidus tecomanus Hoffmann

The venom from the scorpion Centruroides limpidus tecomanus Hoffmann was obtained by homogenization of entire telsons and electrical stimulation of anaesthetized animals. Both venom preparations contain toxic proteins to mice and were separated in a Sephadex G-50 column followed by carboxymethyl-cellulose chromatography. At least two toxic fractions were resolved from the homogenized telsons and three toxic fractions from the venom obtained by electrical stimulation. Rechromatography of the latter fractions on an ion exchange column (Aminex A-5) gave a homogeneous toxin called II.9.3, which on amino acid analysis was shown to be composed of 65 residues with a calculated mol. wt of 7335. The N-terminal sequence is H-Lys-Glx-Gly-X-Leu-Val-Asx-His-X-Thr-Gly-Cys-…, homologous to other scorpion toxins. The venom obtained by electrical stimulation was further characterized; the fraction I from the Sephadex G-50 chromatography shows hyaluronidase activity, although the fraction III shows at least 15 different components positive to ninhydrin, after separation by peptide mapping techniques.     

A short-chain peptide toxin isolated from Centruroides sculpturatus scorpion venom inhibits ether-à-go-go-related gene K(+) channels.

From the venom of the American scorpion Centruroides sculpturatus Ewing we have isolated a minute peptide fraction (named CsEKerg1) which reversibly inhibits the current through ERG (ether-à-go-go-related gene) K(+) channels. Isolation was done by CM-cellulose column chromatography and reversed phase high-performance liquid chromatography. To test for an effect on ERG channels we used NG108-15 neuroblastomaxglioma hybrid cells voltage-clamped in the whole-cell mode. CsEKerg1 contains 43 amino acids and has a molecular weight of 4833. Its amino acid sequence is similar but not identical to that of ergtoxin, a peptide isolated recently from the venom of the Mexican scorpion Centruroides noxius [FASEB J. 13 (1999) 953]. Half inhibition of ERG current occurs at a peptide concentration of 1.12microg/ml.     

Elevation of blood pressure and plasma renin levels by venom from scorpions, Centruroides sculpturatus and Leiurius quinquestriatus

Rats, anesthetized with pentobarbital, were injected with saline—reconstituted, lyophilized venom from C. sculpturatus (67–134μg/kg) and L. quinquestriatus (35–67 μg/kg). Mean values for systolic, diastolic and mean blood pressure increased by 29, 19 and 22 mm Hg respectively for C. sculpturatus venom and 39, 20 and 26 mm Hg for L. quinquestriatus venom at the maximum dose used. Plasma renin activity increased 2·2 fold for C. sculpturatus venom and 2·8 fold for L. quinquestriatus venom. Blood pressure effects peaked at approximately 5 min and slowly decreased, remaining above control values for up to 60 min. Repeated doses had cumulative effects on blood pressure and plasma renin activity. Phenoxybenzamine (effective blocking dose) blunted but did not completely block the blood pressure response to venom injection whereas plasma renin activity continued to be elevated commensurate with the blood pressure elevation. It is proposed that the renin-angiotensin system participates in the hypertensive response to scorpion venom along with the previously demonstrated elevation in circulating catecholamines.     

The three-dimensional structure of scorpion neurotoxins

The crystal and molecular structure of a toxin from the scorpion Centruroides sculpturatus has been solved by standard x-ray crystallographic methods at 3 Å resolution. Subsequently, the 3 Å model has been refined and the resolution has been extended to 1.8 Å using the gradient-curvature method. The final reliability index is 0.17The structure has two and a half turns of α-helix, a three-strand stretch of antiparallel β-sheet and several β-turns. Three of the four disulfide bridges are found in close interaction with the α-helix and β-sheet structures in what constitutes a very rigid part of the molecule.Examination of available scorpion toxin sequences reveals several sections containing invariant and/or semiinvariant amino acids. Many of these residues are found clustered on a rather large flat surface which is also clearly more hydrophobic than other areas on the molecule. These observations suggest that this surface may play a role in the biological action of scorpion toxins.Secondary structure predictions calculated using the method of Dufton and Hider agree well with the x-ray structure. This is also true for other scorpion toxins and reinforces the idea that scorpion toxins are a family of structurally related proteins.     

Scorpion Species with Smaller Body Sizes and Narrower Chelae Have the Highest Venom Potency

Scorpionism is a global health concern, with an estimation of over one million annual envenomation cases. Despite this, little is known regarding the drivers of scorpion venom potency. One widely held view is that smaller scorpions with less-developed chelae possess the most potent venoms. While this perception is often used as a guide for medical intervention, it has yet to be tested in a formal comparative framework. Here, we use a phylogenetic comparative analysis of 36 scorpion species to test whether scorpion venom potency, as measured using LD₅₀, is related to scorpion body size and morphology. We found a positive relationship between LD₅₀ and scorpion total length, supporting the perception that smaller scorpions possess more potent venoms. We also found that, independent of body size, scorpion species with long narrow chelae have higher venom potencies compared to species with more robust chelae. These results not only support the general perception of scorpion morphology and potency, but also the presence of an ecology trade-off with scorpions either selected for well-developed chelae or more potent venoms. Testing the patterns of venom variations in scorpions aids both our ecological understanding and our ability to address the global health burden of scorpionism.     

Venoms and enzymes: effects of permeability of isolated single electroplax

The isolated single electroplax was mounted between two pools of fluid, one initially non-radioactive, the other containing ¹⁴C choline. Treated cells were exposed for 20 min to 1 mg per ml concentrations of various enzymes and venoms. Cottonmouth moccasin venom (CMV) caused a hundred-fold increase in the control level (～0.01 per cent per 10 min) of penetration. Acid-boiled CMV, phospholipase C and lipase caused only a 2- to 3-fold increase in penetration while the following treatments had no effect on permeability: alkaline-boiled CMV, phospholipase D, hyaluronidase, chymotrypsin, trypsin, venom 1-amino acid oxidase and phosphodiesterase, purified cobra neurotoxin (0.2 mg/ml), cetyltrimethylammonium bromide, Centruroides sculpturatus, Notechis scutatus, Heloderma horridum and Naja naja venoms. The action of acid-boiled CMV is associated with extensive phospholipid splitting, therefore, it does not appear that intact phospholipids are necessary for membrane permeability. The component of unboiled CMV which increases permeability is not known.     

Maurotoxin and the Kv1.1 channel: voltage‐dependent binding upon enantiomerization of the scorpion toxin disulfide bridge Cys31–Cys34

Abstract: Maurotoxin (MTX) is a 34‐amino acid polypeptide cross‐linked by four disulfide bridges that has been isolated from the venom of the scorpion Scorpio maurus palmatus and characterized. Maurotoxin competed with radiolabeled apamin and kaliotoxin for binding to rat brain synaptosomes and blocked K+ currents from Kv1 channel subtypes expressed in Xenopus oocytes. Structural characterization of the synthetic toxin identified half‐cystine pairings at Cys³–Cys²⁴, Cys⁹–Cys²⁹, Cys¹³–Cys¹⁹ and Cys³¹–Cys^34. This disulfide bridge pattern is unique among known scorpion toxins, particularly the existence of a C‐terminal ‘14‐membered disulfide ring’ (i.e. cyclic domain 31–34), We therefore studied structure–activity relationships by investigating the structure and pharmacological properties of synthetic MTX peptides either modified at the C‐terminus {i.e. MTX (1–29), [Abu_31,34]‐MTX and [Cys_31,34, Tyr₃₂]_d ‐MTX} or mimicking the cyclic C–terminal domain [i.e. MTX (31–34)]. Unexpectedly, the absence of a disulfide bridge Cys³¹–Cys³⁴ in [Abu _31,34]‐MTX and MTX (1–29) resulted in MTX‐unrelated half‐cystine pairings of the three remaining disulfide bridges for the two analogs, which is likely to be responsible for their inactivity against Kv1 channel subtypes. Cyclic MTX (31–34) was also biologically inactive. [Cys_31,34, Tyr₃₂]_d ‐MTX, which had a ‘native’, MTX‐related, disulfide bridge organization, but a d ‐residue‐induced reorientation of the C–terminal disulfide bridge, was potent at blocking the Kv1.1 channel. This peptide‐induced Kv1.1 blockage was voltage‐dependent (a property not observed for MTX), maximal in the low depolarization range and associated with on‐rate changes in ligand binding. Thus, the cyclic C–terminal domain of MTX seems to be crucial for recognition of Kv1.3, and to a lesser extent, Kv1.2 channels and it may contribute to the stabilization and strength of the interaction between the toxin and the Kv1.1 channel.     

Binding characteristics of BmK I,an α‐like scorpion neurotoxic polypeptide,on cockroach nerve cord synaptosomes

Abstract: In this study, the binding characteristics of BmK I, an α‐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 (K_d = 16.5 ± 4.4 nm ) and low binding capacity (B_max= 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 α‐like scorpion neurotoxic polypeptides bind to a distinct receptor site on sodium channels, which might be similar to the binding receptor site of α‐type insect toxins, and also related to those of BmK AS type and insect‐selective scorpion toxins on insect sodium channels.     

A selective blocker of Kv1.2 and Kv1.3 potassium channels from the venom of the scorpion Centruroides suffusus suffusus

A novel potassium channel blocker peptide was purified from the venom of the scorpion Centruroides suffusus suffusus by high-performance liquid chromatography and its amino acid sequence was completed by Edman degradation and mass spectrometry analysis. It contains 38 amino acid residues with a molecular weight of 4000.3 Da, tightly folded by three disulfide bridges. This peptide, named Css20, was shown to block preferentially the currents of the voltage-dependent K⁺-channels Kv1.2 and Kv1.3. It did not affect several other ion channels tested at 10 nM concentration. Concentration-response curves of Css20 yielded an IC50 of 1.3 and 7.2 nM for Kv1.2- and Kv1.3-channels, respectively. Interestingly, despite the similar affinities for the two channels the association and dissociation rates of the toxin were much slower for Kv1.2, implying that different interactions may be involved in binding to the two channel types; an implication further supported by in silico docking analyses. Based on the primary structure of Css20, the systematic nomenclature proposed for this toxin is α-KTx 2.13.     

Detoxification with glutaraldehyde of purified scorpion (Centruroides noxius Hoffmann) venom

The venom from the scorpion Centruroides noxius was separated by Sephadex G-50 chromatography. The only toxic fraction (No. II) was detoxified in different conditions with glutaraldehyde and was shown to be immunogenic in rabbits. Purified rabbit antibodies protect mice against the lethal effect of the toxic fraction II. It is also shown that the rabbit serum produces immunoprecipitating bands against the venom from several other Latin American scorpions.     

Effect of maurotoxin,a four disulfide‐bridged toxin from the chactoid scorpion Scorpio maurus,on Shaker K+ channels

Abstract: Maurotoxin is a 34‐residue toxin isolated from the venom of the Tunisian chactoid scorpion Scorpio maurus palmatus and contains four disulfide bridges that are normally found in long‐chain toxins of 60–70 amino acid residues, which affect voltage‐gated sodium channels. However, despite the unconventional disulfide‐bridge pattern of maurotoxin, the conformation of this toxin remains similar to that of other toxins acting on potassium channels. Here, we analyzed the effects of synthetic maurotoxin on voltage‐gated Shaker potassium channels (ShB) expressed in Xenopus oocytes. Maurotoxin produces a strong, but reversible, inhibition of the ShB K⁺ current with an IC₅₀ of 2 nm . Increasing concentrations of the toxin induce a progressively higher block at saturating concentrations. At nonsaturating concentrations of the toxin (5–20 nm ), the channel block appears slightly more pronounced at threshold potentials suggesting that the toxin may have a higher affinity for the closed state of the channel. At the single channel level, the toxin does not modify the unitary current amplitude, but decreases ensemble currents by increasing the number of depolarizing epochs that failed to elicit any opening. A point mutation of Lys²³ to alanine in maurotoxin produces a 1000‐fold reduction in the IC₅₀ of block by the toxin suggesting the importance of this charged residue for the interaction with the channel. Maurotoxin does not affect K⁺ currents carried by Kir2.3 channels in oocytes or Na⁺ currents carried by the α_IIa channel expressed in CHO cells.     

Covalent structure of the insect toxin of the North African scorpion Androctonus australis Hector

The complete covalent structure of the insect toxin purified from the venom of the North-African scorpion Androctonus australis Hector was described. Its amino acid sequence was established by phenylisothiocyanate degradation of several protein derivatives and proteolytic fragments in a liquid protein sequencer using either a “protein” or a “peptide” program. The position of the four disulfide bridges were deduced by analysis of proteolytic peptides before and after performic oxidation, and by partial labeling of the half cystine residues with [¹⁴C]-iodoacetic acid and determining the specific radioactivities of the S-[¹⁴C]-carboxymethylated phenylthiohydantoin cysteines. The sequences of the insect and mammal toxins from scorpions can be aligned with homology with the positions of seven half-cystine residues as registers. The mammal and insect toxins have three disulfide bridges at homologous positions. The fourth bridge is different in that Cys¹² in mammal toxin II is replaced by Cys³⁸ in the insect toxin. It is likely that the position of the disulfide bridges is the same for all scorpion neurotoxins active on mammals. We believe that the shift of one half-cystine residue in the insect toxin may induce a conformational change in the structure of the protein, which, in turn, may partially account for the total specificity of this toxin for insect nervous system.