Bacterial expression, purification and functional characterization of a recombinant chimeric Fab derived from murine mAb BCF2 that neutralizes the venom of the scorpion Centruroides noxius hoffmann.

The murine monoclonal antibody BCF2 is able to neutralize the venom of the scorpion Centruroides noxius Hoffmann. A chimeric Fab of BCF2 (chFab-BCF2) comprising the variable regions of murine BCF2 and human constant regions was assembled. chFab-BCF2 was expressed as a soluble and functional protein in the periplasmic space of Escherichia coli. An expression yield of 1 mg/l was reached by combination of late-log-phase induction, rich culture medium, low expression temperature and addition of sucrose (0.3 M) to the culture medium. The addition of sucrose induced secretion of 60% of the protein into the medium. After expression for 23 h, a novel process was used to release the remaining periplasmic protein in situ consisting in the addition of lysozyme and sucrose up to 0.6 M (20%) directly to the culture medium. chFab-BCF2 was recovered by ammonium sulfate precipitation and purified in a single step by affinity chromatography using anti-human anti-F(ab')(2) IgG coupled to Sepharose-proteinG. Pure chFab-BCF2 maintained a similar nanomolar affinity as BCF2 to its cognate antigen, the Na(+)-channel-affecting toxin Cn2. Recombinant chFab-BCF2 was able to neutralize Cn2 in vivo even at a molar ratio of 1:1, as well as the whole venom of C. noxius. Thus, it is a promising candidate to be used as a specific and efficient recombinant antidote against scorpion stings.     

Purification and characterization of a novel short-chain insecticidal toxin with two disulfide bridges from the venom of the scorpion Liocheles australasiae.

Scorpion venoms contain a variety of peptides toxic to mammals, insects and crustaceans. Most of the scorpion toxins have been isolated from the venoms of scorpions in the family Buthidae, but little interest has been paid to non-Buthidae scorpions. In this study, we isolated a short-chain insecticidal toxin (LaIT1) from the venom of the scorpion Liocheles australasiae belonging to the Hemiscorpiidae family. This toxin showed insect toxicity against crickets at a dose of 1.0 microg/insect, but no toxicity was observed against mice even after injection of 1.0 microg of LaIT1 via the intracerebroventricular route, suggesting that the effect of the toxin is insect-selective. Edman sequencing and mass spectrometric analysis revealed that the toxin is composed of 36 amino acid residues and cross-linked by only two disulfide bridges. The pattern of the disulfide bridges was assigned by LC/MS analysis after enzymatic digestion. LaIT1 shows no sequence homology to any other known toxins, suggesting that this toxin represents a novel structural motif class.     

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.     

Novel alpha-KTx peptides from the venom of the scorpion Centruroides elegans selectively blockade Kv1.3 over IKCa1 K+ channels of T cells.

From the venom of the Mexican scorpion Centruroides elegans Thorell five peptides were isolated to homogeneity by chromatographic procedures and their full amino acid sequence was determined by automatic Edman degradation. They all belong to the Noxiustoxin subfamily of scorpion toxins and were given the systematic names alpha-KTx 2.8 to 2.12, with trivial names Ce1 to Ce5, respectively. They have 39 amino acid residues, except for Ce3 which has only 38, but all of them have three disulfide bridges, and have molecular weights of 4255, 4267, 4249, 4295 and 4255 atomic mass units, respectively for Ce1 to Ce5. The C-terminal residues of Ce2, Ce4 and Ce5 were found to be amidated. The electrophysiological assay (whole-cell patch-clamp) showed that out of the five peptides, Ce1 (alpha-KTx 2.8), Ce2 (alpha-KTX2.9) and Ce4 (alpha-KTx 2.11) were effective blockers of Kv1.3 channels of human T lymphocytes, whereas these peptides did not inhibit the Ca2+-activated K+ channels (IKCa1) of the same cells. The equilibrium dissociation constants of these peptides for Kv1.3 were 0.70, 0.25 and 0.98nM for Ce1, Ce2 and Ce4, respectively. Furthermore, toxins Ce1, Ce2 and Ce4 practically did not inhibit the related voltage gated Shaker K+ channels, and rKv2.1 channels of the Shab family. The high affinity blockage of Kv1.3 channels by these peptides and their selectivity for Kv1.3 over IKCa1 may have significance in the development of novel tools for suppressing the function of those T cell subsets whose proliferation critically depends on the activity of Kv1.3 channels.     

Pharmacological investigation of the nociceptive response and edema induced by venom of the scorpion Tityus serrulatus.

In this study we characterized the nociceptive response and edema induced by the venom of the scorpion Tityus serrulatus in rats and mice and carried out a preliminary pharmacological investigation of the mechanisms involved in these responses. Intraplantar injection of the venom (1 or 10mug) induced edema and a marked ipsilateral nociceptive response, characterized by thermal and mechanical allodynia and paw licking behaviour. The nociceptive response was inhibited by previous intraperitoneal administration of indomethacin (4mg/kg), dipyrone (200mg/kg), cyproheptadine (10mg/kg) or morphine (5 or 10mg/kg), but not by dexamethasone (1 or 4mg/kg) or promethazine (1 or 5mg/kg). The edema was inhibited by previous treatment with promethazine (5 or 10mg/kg) or cyproheptadine (5 or 10mg/kg), but not by indomethacin (2 or 4mg/kg), dexamethasone (1 or 4mg/kg) or cromolyn (40 or 80mg/kg). Some bioactive amines, including histamine and 5-hydroxytryptamine, were found in the venom in low concentrations. In conclusion, the nociceptive response and edema induced by the venom of T. serrulatus may result from the action of multiple mediators including eicosanoids, histamine and 5-hydroxytryptamine. These results may lead to a better understanding of the host response to potent animal toxins and also give insights into a more rational pharmacological approach to alleviate the intense pain associated with the scorpion envenomation.     

Identification of BmKAPi, a novel type of scorpion venom peptide with peculiar disulfide bridge pattern from Buthus martensii Karsch.

A novel cDNA sequence encoding a new type of scorpion venom peptide (BmKAPi) was first isolated from the venom gland of Buthus martensiiKarsch by cDNA library screening combined with 5′-race. The encoded precursor of BmKAPi consisted of 89 amino acid residues including a signal peptide of 24 residues, a putative mature peptide of 64 residues (BmKAPi) and an extra basic residue at the C-terminus which might be removed in the post-translational processing. BmKAPi is stabilized by five disulfide bridges, whereas all other disulfide-bridged scorpion toxins described are cross-linked by three or four disulfide bridges. It suggested the three-dimensinal scaffold of BmKAPi might be different from other scorpion toxins. The amino acid sequence of BmKAPi showed no homology with other scorpion venom peptides, but shared a little similarity with some anticoagulant peptides and proteinase inhibitors isolated from hookworm, honeybee or European frog, respectively. RT-PCR analysis showed that BmKAPi mRNA could be induced by venom extraction suggesting BmKAPi might be a component of scorpion venom. These results suggest that BmKAPi is a new type of scorpion venom peptide different from other described scorpion toxins in structural and functional aspects.     

HgeTx1, the first K-channel specific toxin characterized from the venom of the scorpion Hadrurus gertschi Soleglad

A novel toxin was identified, purified and characterized from the venom of the Mexican scorpion Hadrurus gertschi (abbreviated HgeTx1). It has a molecular mass of 3950 atomic mass units (a.m.u.) and contains 36 amino acids with four disulfide bridges established between Cys1–Cys5, Cys2–Cys6, Cys3–Cys7 and Cys4–Cys8. It blocks reversibly the Shaker B K⁺-channels with a Kd of 52 nM. HgeTx1 shares 60%, 45% and 40% sequence identity, respectively, with Heterometrus spinnifer toxin1 (HsTX1), Scorpio maurus K⁺-toxin (maurotoxin) and Pandinus imperator toxin1 (Pi1), all four-disulfide bridged toxins. It is 57–58% identical with the other scorpion K⁺-channel toxins that contain only three disulfide bridges. Sequence comparison, chain length and number of disulfide bridges analysis classify HgeTx1 into subfamily 6 of the -KTx scorpion toxins (systematic name: -KTx 6.14).     

Purification and primary structure determination of Tf4, the first bioactive peptide isolated from the venom of the Brazilian scorpion Tityus fasciolatus.

In the present study Tityus fasciolatus crude venom toxicity was evaluated and we also report the purification and characterization of a 6.6 kDa neurotoxin isolated from T. fasciolatus venom. This new toxin, named Tf4, has a molecular mass of 6614Da and its primary structure is homologous to TbIT-I from T. bahiensis and TsTX-VI and TsNTxP from T. serrulatus. Tf4 delays frog sodium channel inactivation reversibly, but it is non-toxic to mammals or crustaceans. An attempt to identify the residues responsible for the partial loss of toxicity in Tf4 was carried out based on homology modeling and sequence comparison.     

Engineering of a recombinant Fab from a neutralizing IgG directed against scorpion neurotoxin AahI, and functional evaluation versus other antibody fragments.

Antibody-based therapy is the only specific treatment for scorpion envenomation. However, there are still major drawbacks associated with its use; mainly because antivenoms are still prepared from immune equine serum raised against crude venoms, whereas only a limited number of neurotoxins are responsible for the lethality of the venom. Using a murine hybridoma that secretes a well-characterized neutralizing IgG directed to neurotoxins AahI and AahIII from the venom of the scorpion Androctonus australis, we constructed a recombinant Fab (rFab) fragment, which was produced and purified from transformed bacteria. It recognized toxin AahI with a high affinity (KD = 8.2 x 10(-11)) equivalent to the homologous pFab prepared by papain digestion of whole IgG. Although the AahI-neutralizing capacity of protein L-purified rFab was low compared to other recombinant antibody formats (scFv and diabody) investigated in parallel, the antibody engineering approach presented here provides an innovative way to synthesize novel toxin-neutralizing molecules. It may serve as a strategy for designing a new generation of antivenoms.     

Immunological characterization of a non-toxic peptide conferring protection against the toxic fraction (AahG50) of the Androctonus australis hector venom.

KAaH1 and KAaH2 are non-toxic peptides, isolated from the venom of the Androctonus australis hector (Aah) scorpion. In a previous study, we showed these peptides to be the most abundant (approximately 10% each) in the toxic fraction (AahG50) of the Aah venom. KAaH1 and KAaH2 showed high sequence identities (approximately 60%) with birtoxin-like peptides, which likewise are the major peptidic components of Parabuthus transvaalicus scorpion venom. Here, we report the immunological characterization of KAaH1 and KAaH2. These peptides were found to be specifically recognized by polyclonal antibodies raised against AahII, the most toxic peptide of Aah venom, and represents the second antigenic group, including toxins from different scorpion species in the world. Moreover, KAaH1 partially inhibits AahII binding to its specific antibody, suggesting some common epitopes between these two peptides. The identification of possible key antigenic residues in KAaH1 was deduced from comparison of its 3-D model with the experimental structure of AahII. Two clusters of putative antigenically important residues were found at the exposed surface; one could be constituted of V3 and D53, the other of D10, T15 and Y16. Polyclonal antibodies raised against KAaH1 in mice were found to cross-react with both AahII and AahG50, and neutralizing 5LD(50)/ml of the toxic fraction. Mice vaccinated with KAaH1 were protected against a challenge of 2LD(50) of AahG50 fraction. All these data suggest that KAaH1 has clear advantages over the use of the whole or part of the venom. KAaH1 is not toxic and could produce sera-neutralizing scorpion toxins, not only from Aah venom, but also toxins of other venoms from Buthus, Leiurus, or Parabuthus scorpion species presenting antigenically related toxins.     

Isolation and primary structure of a potent toxin from the venom of the scorpion Centruroides sculpturatus Ewing

A potent toxin has been purified from the venom of the scorpion Centruroides sculpturatus Ewing using the ion-exchange resin CM-Sepharose CL-6B at basic pH. The toxin, designated CsE M₁, comprised 65 amino acid residues and its primary structure was established as: Lys-Glu-Gly-Tyr-Leu-Val-Asn-Ser-Tyr-Thr¹⁰-Gly-Cys-Lys-Tyr-Glu-Cys-Leu-Lys-Leu-Gly²⁰-Asp-Asn-Asp-Tyr-Cys-Leu-Arg-Glu-Cys-Arg³⁰-Gln-Gln-Tyr-Gly-Lys-Ser-Gly-Gly-Tyr-Cys⁴⁰-Tyr-Ala-Phe-Ala-Cys-Trp-Cys-Thr-His-Leu⁵⁰-Tyr-Glu-Gln-Ala-Val-Val-Trp-Pro-Leu-Pro⁶⁰-Asn-Lys-Thr-Cys-Asn. CsE M₁ is the most lethal protein to be identified in C. sculpturatus venom and the LD₅₀ of the toxin, determined by subcutaneous injection into Swiss mice, is 87 μg/kg. CsE M₁ shows strong structural similarity (92% positional identity) to the most potent β-toxin, Css II, from the Mexican scorpion, Centruroides suffusus suffusus but is quite dissimilar to the previously characterized toxins with low potency isolated from C. sculpturarus Ewing.     

Heterologous expression of a gene that codes for Pg8, a scorpion toxin of Parabuthus granulatus, capable of generating protecting antibodies in mice

A novel peptide named Pg8 was purified from the venom of the South African scorpion Parabuthus granulatus and its primary structure was determined. It contains 63 amino acid residues tightly folded by 4 disulfide bridges. The gene coding for this peptide was cloned from a cDNA library. By recursive PCR strategy a hybrid gene was constructed having a factor X recognition site for proteolysis and a modified sequence for preferential codon usage of E. coli. A pQE30 molecular vector already contained a His-tag was used for expression. This construction was expressed in BL21 and Origami strains. The fusion protein from inclusion bodies was separated by HPLC (yield approximately 5 mg/L) and properly folded in vitro. Lethality tests showed that the recombinant peptide was toxic and was used to immunize mice. A volume of 0.25 ml of the anti-serum produced was capable of protecting up to 3 LD₅₀ doses of pure toxin Pg8 but also, and more importantly, the entire soluble venom.     

A novel short-chain peptide BmKX from the Chinese scorpion Buthus martensi Karsch, sequencing, gene cloning and structure determination.

Scorpion venom is a rich source of bioactive peptides. From the venom of Chinese scorpion Buthus martensi Karsch (BmK), a novel short chain peptide BmKX of 31-amino acid residues was purified, and its amino acid sequence and gene structure were determined. The gene of BmKX was composed of two exons interrupted by an 86-bp intron at the codon-7 upstream of the mature peptide. Although its gene structure is similar to those of other known scorpion toxins, its amino acid sequence, especially the cysteine framework, is different from those of all other known subfamilies of short-chain scorpion toxins. The solution structure of BmKX, determined with two-dimensional NMR spectroscopy, shows that BmKX also forms a typical cysteine-stabilized alpha/beta scaffold adopted by most short-chain scorpion toxins, consisting of a short 3(10)-helix and a two-stranded antiparallel beta-sheet, and the short N-terminal segment forms a pseudo-strand of the beta-sheet. However, the orientation between the helix and the beta-sheet is significantly different from the others, which might be the reason for its unique but still unclear physiological function.     

Identification of BmKAPi, a novel type of scorpion venom peptide with peculiar disulfide bridge pattern from Buthus martensii Karsch

A novel cDNA sequence encoding a new type of scorpion venom peptide (BmKAPi) was first isolated from the venom gland of Buthus martensiiKarsch by cDNA library screening combined with 5′-race. The encoded precursor of BmKAPi consisted of 89 amino acid residues including a signal peptide of 24 residues, a putative mature peptide of 64 residues (BmKAPi) and an extra basic residue at the C-terminus which might be removed in the post-translational processing. BmKAPi is stabilized by five disulfide bridges, whereas all other disulfide-bridged scorpion toxins described are cross-linked by three or four disulfide bridges. It suggested the three-dimensinal scaffold of BmKAPi might be different from other scorpion toxins. The amino acid sequence of BmKAPi showed no homology with other scorpion venom peptides, but shared a little similarity with some anticoagulant peptides and proteinase inhibitors isolated from hookworm, honeybee or European frog, respectively. RT-PCR analysis showed that BmKAPi mRNA could be induced by venom extraction suggesting BmKAPi might be a component of scorpion venom. These results suggest that BmKAPi is a new type of scorpion venom peptide different from other described scorpion toxins in structural and functional aspects.     

Effect of Tityus serrulatus scorpion venom and its major toxin, TsTX-I, on the complement system in vivo.

The effects of Tityus serrulatus venom and TsTX-I (Ts1 or gamma-toxin) on the lytic activity of the complement system (CS) were investigated in vivo. Serum classical pathway (CP) and alternative pathway (AP) activities were determined in sera of rats (200+/-10 g) injected i.p. with soluble venom (150 microg/kg), TsTX-I (150 microg/kg) or saline (control). The animals were sacrificed 0.5, 1, 2, 4, 24 and 48 h after injection. The results showed an increase in serum lytic activity of animals injected with venom, reaching values up to 70% above controls in CP activity and 120% in AP activity. These effects were biphasic with maximum values 1 and 24 h after venom injection. Similar effects were obtained for TsTX-I, but with lower intensity. Hematocrit values of all tested animals were determined to evaluate the effect of hemoconcentration on the lytic activity of the CS. It was observed that the maximum hematocrit value was obtained 1 h after injection and returned to normal values within 24 h. These data indicate that hemoconcentration can play a relevant role in the first peak of complement activity, but we cannot discard a direct action of the venom on the system during this period, since the serum venom concentration is maximal 15-30 min after envenomation. The high lytic activity of the serum observed after 24 h, period in which the hematocrit values are normal and no venom can be detected, may be consequence of the inflammatory process induced by the venom or toxin. The lytic activity of the serum of rats injected with venom, TsTX-I or saline was abolished when the serum was previously adsorbed on zymosan. These data confirm that the increase of the lytic activity of the serum induced by the venom or toxin is dependent on CS.These results show that CS is involved in the inflammatory process induced by the venom or toxin and consequently in the lung edema, hemolysis, leukocytosis, among other clinical manifestations of severe envenomation.     

Characterization of hadrucalcin, a peptide from Hadrurus gertschi scorpion venom with pharmacological activity on ryanodine receptors

Background and purpose:

Members of the calcin family, presently including imperatoxin A, maurocalcin, opicalcins and hemicalcin, are basic, 33-mer peptide activators of ryanodine receptors (RyRs), the calcium channels of the sarcoplasmic reticulum (SR) that provide the majority of calcium for muscle contraction. Here we describe hadrucalcin, a novel member of this family.

Experimental approach:

Hadrucalcin was isolated from the venom of Hadrurus gertschi. Amino acid sequence and mass were determined by Edman degradation and mass spectrometry respectively. A cDNA library was constructed to generate clones for DNA sequence determination. Biological activity of native toxin was confirmed with [³H]ryanodine binding, by using SR vesicles from cardiac and skeletal muscle, and with single skeletal (RyR1) and cardiac (RyR2) channels reconstituted in lipid bilayers. Hadrucalcin was applied to intact ventricular myocytes to investigate effects on calcium transients. The secondary structure of hadrucalcin was computer-modelled by using atomic coordinates from maurocalcin, a structurally similar peptide.

Key results:

Hadrucalcin is distinguished from previously described congeners by two additional amino acids in its primary sequence and the lack of prominent amphipathicity. Hadrucalcin activated RyRs with high affinity (EC₅₀= 37 nmol·L⁻¹), induced a long-lasting subconductance state on RyR1 and RyR2, and rapidly (lag time ∼2 s) penetrated ventricular cardiomyocytes, eliciting discharge of internal calcium stores and spontaneous contractions.

Conclusions and implications:

Hadrucalcin is a cell-permeant, powerful activator of RyRs, which has translational potential for targeted delivery of drugs to RyR as novel therapeutic intervention in arrhythmogenic disease.     

Characterization of venom components from the scorpion Androctonus crassicauda of Turkey: peptides and genes.

The soluble venom from the scorpion Androctonus crassicauda was fractionated by high performance liquid chromatography. At least 44 different sub-fractions were resolved and collected for finger print mass analysis using an electrospray mass spectrometer. This analysis revealed the presence of 80 distinct molecular mass components, from which five were further characterized. A peptide, named Acra1 was fully sequenced. It contains 58 amino acid residues cross-bridged by six cysteines forming three disulfide pairs, with a molecular mass of 6497 Da. A second purified peptide named Acra2 was partially sequenced with a molecular mass of 7849 Da. Acra1 is toxic and Acra2 is lethal to mice, at the dose assayed. Additionally, a cDNA library of the venomous gland of one specimen was prepared and several clones were obtained among which is one that codes for Acra1. Three analog gene sequences were found with point mutations either in the section that corresponds to the mature peptide or to the signal peptide. The signal peptide is 22 amino acid residues long. Several other gene sequences obtained suggest the presence in this venom of three distinct groups of peptides, among which are peptides similar to known Na(+)-channel specific toxins of other scorpions. A new type of peptide was identified with odd number of cysteines (seven), allowing the formation of heterodimers with molecular masses in the range of 16,000 atomic mass units (a.m.u.).     

Purification and pharmacological characterization of BmKK2 (alpha-KTx 14.2), a novel potassium channel-blocking peptide, from the venom of Asian scorpion Buthus martensi Karsch.

BmKK2 (alpha-KTx 14.2) is one of the novel short-chain peptides found in molecular cloning of a venom gland cDNA library from Asian scorpion Buthus martensi Karsch. Based upon its amino acid sequence, the peptide was proposed to adopt a classical alpha/beta-scaffold for alpha-KTxs. In the present study, we purified BmKK2 from the venom of B. martensi Karsch, and investigated its action on voltage-dependent K+ currents in dissociated hippocampal neurons from neonatal rats. BmKK2 (10-100 microM) selectively inhibited the delayed rectifier K+ current, but did not affect the fast transient K+ current. The inhibition of BmKK2 on the delayed rectifier K+ current was reversible and voltage-independent. The peptide did not affect the steady-state activation of the current, but caused a depolarizing shift (about 9 mV) of its steady-state inactivation curve. The results demonstrate that BmKK2 is a novel K+ channel-blocking scorpion peptide.     

The neutralizing effect of a polyclonal antibody raised against the N-terminal eighteen-aminoacid residues of birtoxin towards the whole venom of Parabuthus transvaalicus.

Scorpion venom is composed among other things of a large number of neurotoxic peptides affecting all major types of ion channels. The majority of the toxicity of the venom is attributed to the presence of these peptides. In our previous studies using a combination of HPLC and mass spectrometry, we showed that birtoxin like peptides are the major peptidic components of the venom of Parabuthus transvaalicus. These peptides are quite similar to each other differing by only few amino acid residues. In addition they all share a common N-terminus of eighteen amino acid residues. We hypothesize that neutralization of this domain will decrease the toxicity of the whole venom of P. transvaalicus. Polyclonal antibodies against the common N-terminal region of the peptides are generated. Here we show by bioassays that the polyclonal antibodies neutralize the venom of P. transvaalicus in a dose dependent manner and by mass spectrometry and western blotting that these peptides indeed react with the polyclonal antibodies. Previously antibodies generated against a single major toxic component of venom have proven to be an effective strategy for antivenin production. In the case of P. transvaalicus the generated antibody is against the majority of the peptidic fraction due to the presence of several highly similar and highly toxic components in this venom. We show that using the knowledge obtained through biochemical characterization studies it is possible to design very specific antibodies that will be useful for clinical applications against Parabuthus envenomation.