Proteomic analysis of Tityus discrepans scorpion venom and amino acid sequence of novel toxins

The Venezuelan scorpion Tityus discrepans is known to cause human fatalities. We describe the first complete proteomic analysis of its venom. By HPLC 58 different fractions were obtained and 205 different components were identified by MS analysis. Components having molecular masses from 272 to 57 908 amu were found. Forty homogeneous components had their N-terminal amino acid sequence determined by Edman degradation, from which two new peptides named TdK2 and TdK3 (meaning T. discrepans (Td) K(+) channel toxins 2 and 3) were fully characterized. The first contains 34 amino acid residues with a molecular mass of 3451 amu, and the second has 36 amino acids with 3832 amu. Both peptides are tightly bound by three disulfide bridges. TdK2 was shown to block reversibly the Shaker B K(+)-channel expressed heterologously in Sf9 cells. The systematic number assigned to TdK2 is alpha-KTx-18.2 and that of TdK3 is alpha-KTx-18.3. Comparative analysis of the amino acid sequences found suggests that this venom contains peptides highly similar to those that block K(+) channels, as well as those that modify the gating mechanisms of Na(+) channels, found in other scorpions. Additionally, peptides similar to defensins were also identified.     

Discrepin, a new peptide of the sub-family alpha-ktx15, isolated from the scorpion Tityus discrepans irreversibly blocks K+ -channels (IA currents) of cerebellum granular cells

A new peptide was purified from the venom of the Venezuelan scorpion Tityus discrepans, 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 4177.7 atomic mass units, tightly folded by three disulfide bridges, and has a pyroglutamic acid at the N-terminal region. This peptide, named Discrepin, was shown to block preferentially the IA currents of the voltage-dependent K+ -channel of rat cerebellum granular cells in culture. The K+ -currents are inhibited in an apparently irreversible manner, whose 50% inhibitory effect is reached with a 190 nM toxin concentration. The systematic nomenclature proposed for this toxin is alpha-KTx15.6.     

Molecular dissection of venom from Chinese scorpion Mesobuthus martensii: identification and characterization of four novel disulfide-bridged venom peptides

Scorpion venom is composed of a large repertoire of biologically active polypeptides. However, most of these peptides remain to be identified and characterized. In this paper, we report the identification and characterization of four novel disulfide-bridged venom peptides (named BmKBTx, BmKITx, BmKKx1 and BmKKx2, respectively) from the Chinese scorpion, Mesobuthus martensii (also named Buthus martensii Karsch). BmKBTx is composed of 58 amino acid residues and cross-linked by three disulfide bridges. The sequence of BmKBTx shows some similarities to that of the toxin, birtoxin, and its analogs. It is likely that BmKBTx is a beta-toxin active on Na+ channels, which is toxic to either insects or mammals. BmKITx is composed of 71 amino acid residues with four disulfide bridges. It is the longest venom peptide identified from M. martensii so far. BmKITx shows little sequence identity with scorpion alpha-toxins toxic to insects. It is likely that BmKITx is a new type of Na+ -channel specific toxin active on both insects and mammals. BmKKx1 contains 38 amino acid residues cross-linked by three disulfide bridges and shows 84% sequence identity with BmTx3, an inhibitor of A-type K+ channel and HERG currents. BmKKx1 has been classified as alpha-KTx-15.8. BmKKx2 is composed of 36 residues and stabilized by three disulfide bridges. BmKKx2 is a new member of the gamma-K+ -channel toxin subfamily (classified as gamma-KTx 2.2). The venoms of scorpions thus continue to provide novel toxins with potential novel actions on targets.     

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 sequences of short scorpion toxins led us to conclude that toxin 14 belongs to a novel group of toxins affecting the excitability of myelinated nerves.     

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.     

Molecular cloning and characterization of four scorpion K(+)-toxin-like peptides: a new subfamily of venom peptides (alpha-KTx14) and genomic analysis of a member.

Four full-length cDNAs encoding the precursors of four K(+)-toxin-like peptides (named BmKK(1), BmKK(2), BmKK(3) and BmmKK(4), respectively) were first isolated from a venom gland cDNA library of the Chinese scorpion Buthus martensii Karsch. The deduced precursors of BmKK(1), BmKK(2) and BmKK(3) are all made of 54 amino acid residues including a signal peptide of 23 residues, and a mature toxin of 31 residues with three disulfide bridges. The precursor of BmKK(4) is composed of 55 amino acid residues including a signal peptide of 23 residues, a mature toxin of 30 residues cross-linked by three disulfide bridges, and an extra Gly-Lys tail which should be removed in the processing step. The four peptides displayed 24-97% sequence identity with each other, and less than 27% homology with any other scorpion toxins described. However, they shared a common disulfide bridge pattern, which was consistent with that of most short-chain K(+)-toxins, suggesting they represent a new class of scorpion toxins and their target receptors may be a subfamily of K(+) channels. We classified the BmKK toxin subfamily as alpha-KTx14 according to the classification rules. The genomic sequence of BmKK(2) was also cloned and sequenced. It consisted of two exons, disrupted by an intron of 79 bp inserted in the region encoding the C-terminal part of the signal peptide. This structure was very similar to that of other K(+)-toxins described previously.     

Solution structure of a K+-channel blocker from the scorpion Tityus cambridgei

A new K(+)-channel blocking peptide identified from the scorpion venom of Tityus cambridgei (Tc1) is composed of 23 amino acid residues linked with three disulfide bridges. Tc1 is the shortest known toxin from scorpion venom that recognizes the Shaker B K(+) channels and the voltage-dependent K(+) channels in the brain. Synthetic Tc1 was produced using solid-phase synthesis, and its activity was found to be the same as that of native Tc1. The pairings of three disulfide bridges in the synthetic Tc1 were identified by NMR experiments. The NMR solution structures of Tc1 were determined by simulated annealing and energy-minimization calculations using the X-PLOR program. The results showed that Tc1 contains an alpha-helix and a 3(10)-helix at N-terminal Gly(4)-Lys(10) and a double-stranded beta-sheet at Gly(13)-Ile(16) and Arg(19)-Tyr(23), with a type I' beta-turn at Asn(17)-Gly(18). Superposition of each structure with the best structure yielded an average root mean square deviation of 0.26 +/- 0.05 A for the backbone atoms and of 1.40 +/- 0.23 A for heavy atoms in residues 2 to 23. The three-dimensional structure of Tc1 was compared with two structurally and functionally related scorpion toxins, charybdotoxin (ChTx) and noxiustoxin (NTx). We concluded that the C-terminal structure is the most important region for the blocking activity of voltage-gated (Kv-type) channels for scorpion K(+)-channel blockers. We also found that some of the residues in the larger scorpion K(+)-channel blockers (31 to 40 amino acids) are not involved in K(+)-channel blocking activity.     

Proteomics of the venom from the Amazonian scorpion Tityus cambridgei and the role of prolines on mass spectrometry analysis of toxins

Scorpion venom are complex mixtures of peptides, known to cause impairment of ion-channel function in biological membranes. This report describes the separation of approximately 60 different components by high performance liquid chromatography and the characterization by Edman degradation and mass spectrometry of 26 peptides from the soluble venom of the Amazonian scorpion Tityus cambridgei. One of these peptides, named Tc48a, was fully characterized. It contains 65 amino acid residues, the C-terminal residue is amidated and it affects Na(+)-channels with a K(d) of about 82 nM. Furthermore, this report shows the thermo-instability of scorpion toxins subjected to electron spray ionization-mass spectrometry (ESI-MS). When a proline residue is located near the N-terminal region of the toxin, not stabilized by disulfide bridges, artificial components are generated by the mass spectrometer conditions, due to the cleavage of the peptide bond at the proline positions. This phenomenon was confirmed by using four model proteins (variable regions of immunoglobulins) studied by ESI-MS and matrix assisted laser desorption ionization-time of flight (MALDI-TOF)/MS.     

Phaiodactylipin, a glycosylated heterodimeric phospholipase A from the venom of the scorpion Anuroctonus phaiodactylus.

Phaiodactylipin was purified from the venom of the scorpion Anuroctonus phaiodactylus. It is the first protein to be purified from a scorpion of the family Iuridae and has a molecular mass of 19 172 atomic mass units. The mature protein is composed of two subunits, the large one consisting of 108 amino acid residues, whereas the small subunit has only 18 residues, and the structure is stabilized by five disulfide bridges. The heterodimer is expressed from a single message containing 769 base pairs and a signal peptide with 16 and/or 25 amino acid residues. During maturation an internal hexapeptide is excised. There are three putative sites of N-glycosylation, one of which is situated in the small subunit region. The carbohydrate composition of this site was determined by mass spectrometry analysis and was found to contain three hexoses, two N-acetyl-hexoses and two deoxyhexoses. The protein has a calcium dependent phospholipase A(2) type of activity. It is lethal to arthropods (insects and isopods), but not toxic to mammals, using doses up to 20 microg per 20 g mouse body weight. For crickets, a dose of 5 microg per animal is lethal; however, when injected into mice it is capable of causing only muscular inflammation, without rupture of the basal membrane of cells. It has a direct hemolytic effect in human erythrocytes and retards the coagulation time of blood. It is an unusual phospholipase A(2), with only 36% and 50% amino acid sequence identities to the closest known phospholipases, imperatoxin I and phospholipin, respectively. Identities with bee and Heloderma venom phospholipase are only in the order of 28%.     

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.     

Two novel toxins from the Amazonian scorpion Tityus cambridgei that block Kv1.3 and Shaker B K(+)-channels with distinctly different affinities

Two novel toxic peptides (Tc30 and Tc32) were isolated and characterized from the venom of the Brazilian scorpion Tityus cambridgei. The first have 37 and the second 35 amino acid residues, with molecular masses of 3,871.8 and 3,521.5, respectively. Both contain three disulfide bridges but share only 27% identity. They are relatively potent inhibitors of K(+)-currents in human T lymphocytes with K(d) values of 10 nM for Tc32 and 16 nM for Tc30, but they are less potent or quite poor blockers of Shaker B K(+)-channels, with respective K(d) values of 74 nM and 4.7 microM. Tc30 has a lysine in position 27 and a tyrosine at position 36 identical to those of charybdotoxin. These two positions conform the dyad considered essential for activity. On the contrary, Tc32 has a serine in the position equivalent to lysine 27 of charybdotoxin and does not contain any aromatic amino acid. Due to its unique primary sequence and to its distinctive preference for K(+)-channels of T lymphocytes, it was classified as the first example of a new subfamily of K(+)-channel-specific peptides (alpha-KT x 18.1). Tc30 is a member of the Tityus toxin II-9 subfamily and was given the number alpha-KT x 4.4.     

A novel class of peptide found in scorpion venom with neurodepressant effects in peripheral and central nervous system of the rat

A novel toxin, named Cll9, was isolated from the venom of the scorpion Centruroides limpidus limpidus Karsch. It is composed of 63 amino acid residues closely packed by four disulfide bridges. It showed no apparent effect when injected to insects, crustaceans and i.p. to mice. However, when i.c.v. injected in the rat it immediately induced sleep, suggesting that it has a neurodepressant effect. We confirmed this by showing that it has a strong antiepileptic action, as assessed with the penicillin focus model. Its effectiveness in inhibiting Na(+) permeability in (cultured) rat peripheral ganglia further supports its neurodepressant actions. However, this peptide did not affect other Na(+) channels such as those from cerebellum granular cells in culture or the rSkM1 Na(+) channels expressed in HEK293. The cDNA and genomic regions encoding this peptide were cloned and sequenced. This peptide is synthesized as a precursor of 84 amino acid residues and processed by removing 19 amino acids (signal peptide) from the amino terminal region and a couple of lysine residues from the carboxyl end. The presence of an intron of 777 bases interrupting the region encoding the signal peptide was also revealed. A comparison of its primary sequence, with more than 100 scorpion toxins known, showed that together with toxin CsE9 they constitute a new subfamily of peptides considered to be one of the most divergent groups of scorpion toxin-like peptides discovered.     

Purification and primary structure of low molecular mass peptides from scorpion (Buthus sindicus) venom

The primary structures of four low molecular mass peptides (Bs 6, 8, 10 and 14) from scorpion Buthus sindicus were elucidated via combination of Edman degradation and matrix-assisted laser desorption ionization mass spectrometry. Bs 8 and 14 are cysteine-rich, thermostable peptides composed of 35–36 residues with molecular weights of 3.7 and 3.4 kDa, respectively. These peptides show close sequence homologies (55–78%) with other scorpion chlorotoxin-like short-chain neurotoxins (SCNs) containing four intramolecular disulfide bridges. Despite the sequence variation between these two peptides (37% heterogeneity) their general structural organization is very similar as shown by their clearly related circular dichroism spectra. Furthermore, Bs6 is a minor component, composed of 38 residues (4.1 kDa) containing six half-cystine residues and having close sequence identities (40–80%) with charybdotoxin-like SCNs containing three disulfide bridges. The non-cysteinic, bacic and thermolabile Bs10 is composed of 34 amino acid residues (3.7 kDa), and belongs to a new class of peptides, with no sequence resemblance to any other so far reported sequence isolated from scorpions. Surprisingly, Bs10 shows some limited sequence analogy with oocyte zinc finger proteins. Results of these studies are discussed with respect to their structural similarities within the scorpion LCNs, SCNs and other biologically active peptides.     

Purification and cDNA cloning of a novel neurotoxic peptide (Acra3) from the scorpion Androctonus crassicauda

Androctonus crassicauda is one of the Southeastern Anatolian scorpions of Turkey with ethno-medical and toxicological importance. Two toxic peptides (Acra1 and Acra2) were isolated and characterized from the venom of this scorpion. In this communication, the isolation of an additional toxin (Acra3) by chromatographic separations (HPLC and TSK-gel sulfopropyl) and its chemical and functional characterization is reported. Acra3 is a 7620Da molecular weight peptide, with 66 amino acid residues crosslinked by four disulfide bridges. The gene coding for this peptide was cloned and sequenced. Acra3 is anticipated to undergo post-translational modifications at the C-terminal region, having an amidated serine as last residue. Injection of Acra3 induces severe neurotoxic events in mice, such as: excitability and convulsions, leading to the death of the animals within a few minutes after injection. Electrophysiological assays conducted with pure Acra3, using cells that specifically expressed sodium channels (Nav1.1-Nav1.6) showed no clear effect. The exact molecular target of Acra3 remained undiscovered, similar to three other scorpion peptides that clustered very closely in the phylogenetic tree included here. The exact target of these four peptides is not very clear.     

Phaiodotoxin, a novel structural class of insect-toxin isolated from the venom of the Mexican scorpion Anuroctonus phaiodactylus.

A peptide called phaiodotoxin was isolated from the venom of the scorpion Anuroctonus phaiodactylus. It is lethal to crickets, but non toxic to mice at the doses assayed. It has 72 amino acid residues, with a molecular mass of 7971 atomic mass units. Its covalent structure was determined by Edman degradation and mass spectrometry; it contains four disulfide-bridges, of which one of the pairs is formed between cysteine-7 and cysteine-8 (positions Cys63-Cys71). The other three pairs are formed between Cys13-Cys38, Cys23-Cys50 and Cys27-Cys52. Comparative sequence analysis shows that phaiodotoxin belongs to the long-chain subfamily of scorpion peptides. Several genes coding for this peptide and similar ones were cloned by PCR, using cDNA prepared from the RNA of venomous glands of this scorpion. Electrophysiological assays conducted with this toxin in several mammalian cell lines (TE671, COS7, rat GH3 and cerebellum granular cells), showed no effect on Na+ currents. However, it shifts the voltage dependence of activation and inactivation of insect Na+ channels (para/tipE) to more negative and positive potentials, respectively. Therefore, the 'window' current is increased by 225%, which is thought to be the cause of its toxicity toward insects. Phaiodotoxin is the first toxic peptide ever purified from a scorpion of the family Iuridae.     

Isolation and molecular characterization of LVP1 lipolysis activating peptide from scorpion Buthus occitanus tunetanus

LVP1, a novel protein inducing lipolytic response in adipose cells, was purified from scorpion Buthus occitanus tunetanus venom. It represented 1% of crude venom proteins, with pHi approximately 6 and molecular mass of 16170 Da. In contrast to well-characterized scorpion toxins, reduction and alkylation of LVP1 revealed an heterodimeric structure. Isolated alpha and beta chains of LVP1 have a respective molecular mass of 8877 and 8807 Da as determined by mass spectrometry. The N-terminal and some internal peptide sequences of LVP1alpha and beta were determined by Edman degradation. The full amino acid sequences of both chains were deduced from nucleotide sequences of the corresponding cDNAs prepared based on peptide sequences and the 3' and 5' RACE methodologies. LVP1alpha and beta cDNAs encode a signal peptide of 22 residues and a mature peptide of 69 and 73 residues, respectively. Each mature peptide contains seven cysteines, which are compatible with an interchain disulfide bridge. The cDNA deduced protein structures share a high similarity with those of some Na+ channel scorpion toxins. LVP1 was not toxic to mice after intracerebro-ventricular injection. LVP1 stimulated lipolysis on freshly dissociated rat adipocytes in a dose-dependent manner with EC50 of approximately 1+0.5 microg/ml. LVP1 subunits did not display any lipolytic activity. As previously described for venom, beta adrenergic receptor (beta AR) antagonists interfere with LVP1 activity. Furthermore, it is shown that LVP1 competes with [3H]-CGP 12177 (beta1/beta2 antagonist) for binding to adipocyte plasma membrane with an IC50 of about 10(-7) M. These results demonstrate the existence of a new type of scorpion venom nontoxic peptides that are structurally related to Na+ channel toxins but can exert a distinct biological activity on adipocyte lipolysis through a beta-type adrenoreceptor pathway.     

OcyKTx2, a new K+-channel toxin characterized from the venom of the scorpion Opisthacanthus cayaporum

Opisthacanthus cayaporum belongs to the Liochelidae family, and the scorpions from this genus occur in southern Africa, Central America and South America and, therefore, can be considered a true Gondwana heritage. In this communication, the isolation, primary structure characterization, and K⁺-channel blocking activity of new peptide from this scorpion venom are reported. OcyKTx2 is a 34 amino acid long peptide with four disulfide bridges and molecular mass of 3807 Da. Electrophysiological assays conducted with pure OcyKTx2 showed that this toxin reversibly blocks Shaker B K⁺-channels with a Kd of 82 nM, and presents an even better affinity toward hKv1.3, blocking it with a Kd of ∼18 nM. OcyKTx2 shares high sequence identity with peptides belonging to subfamily 6 of α-KTxs that clustered very closely in the phylogenetic tree included here. Sequence comparison, chain length and number of disulfide bridges analysis classify OcyKTx2 into subfamily 6 of the α-KTx scorpion toxins (systematic name, α-KTx6.17).     

Molecular cloning and biochemical characterization of the first Na-channel α-type toxin peptide (Acra4) from Androctonus crassicauda scorpion venom

Due to the medical importance played in Turkey by stings of the scorpion Androctonus crassicauda, its venom has been studied with more attention. In this communication we report a new toxic peptide, named Acra4, because it is the fourth peptide completely characterized from venom of this scorpion. The peptide contains 64 amino acid residues stabilized by four disulfide bridges, with a molecular weight of 6937 Da. Purification of the lethal peptide was performed by three steps of high performance liquid chromatography (HPLC) separations, and the molecular weight was determined by mass spectrometry analysis and the full amino acid sequence was obtained by direct Edman degradation in conjunction with gene cloning. The LD₅₀ of Acra4 was 50.5 ng/20 g mouse body weight (95% confidence intervals from 48.8 to 52.2 ng/20 g mouse body weight). Additionally, from a sample of cDNA of A. crassicauda four genes were cloned displaying sequence similarities to known scorpion toxins, and are reported here as potentially toxic peptides, named Acra5 to Acra8. Electrophysiological studies of Acra4 were performed using Na⁺-channels expressed in F11 cell culture, by patch-clamp recordings. This is the first time that such peptide from A. crassicauda having a specific Na⁺-channel α-type effect is reported. Its affinity toward Na⁺-channels in F11 cell line is in the order of 1 μM concentration.     

Isolation and characterization of a novel type of neurotoxic peptide from the venom of the South African scorpion Parabuthus transvaalicus (Buthidae).

The venom of the South African scorpion Parabuthus transvaalicus was characterized using a combination of mass spectrometry and RP-HPLC separation and bioassays. The crude venom was initially separated into 10 fractions. A novel, moderately toxic but very high abundance peptide (birtoxin) of 58 amino-acid residues was isolated, identified and characterized. Each purification step was followed by bioassays and mass spectroscopy. First a C4 RP-HPLC column was used, then a C18 RP Microbore column purification resulted in > 95% purity in the case of birtoxin from a starting material of 230 microg of crude venom. About 12-14% of the D214 absorbance of the total venom as observed after the first chromatography step was composed of birtoxin. This peptide was lethal to mice at low microgram quantities and it induced serious symptoms including tremors, which lasted up to 24 h post injection, at submicrogram amounts. At least seven other fractions that showed different activities including one fraction with specificity against blowfly larvae were identified. Identification of potent components is an important step in designing and obtaining effective anti-venom. Antibodies raised against the critical toxic components have the potential to block the toxic effects and reduce the pain associated with the scorpion envenomation. The discovery of birtoxin, a bioactive long chain neurotoxin peptide with only three disulfide bridges, offers new insight into understanding the role of conserved disulfide bridges with respect to scorpion toxin structure and function.     

Purification and primary structure of low molecular mass peptides from scorpion (Buthus sindicus) venom

The primary structures of four low molecular mass peptides (Bs 6, 8, 10 and 14) from scorpion Buthus sindicus were elucidated via combination of Edman degradation and matrix-assisted laser desorption ionization mass spectrometry. Bs 8 and 14 are cysteine-rich, thermostable peptides composed of 35-36 residues with molecular weights of 3.7 and 3.4 kDa, respectively. These peptides show close sequence homologies (55-78%) with other scorpion chlorotoxin-like short-chain neurotoxins (SCNs) containing four intramolecular disulfide bridges. Despite the sequence variation between these two peptides (37% heterogeneity) their general structural organization is very similar as shown by their clearly related circular dichroism spectra. Furthermore, Bs6 is a minor component, composed of 38 residues (4.1 kDa) containing six half-cystine residues and having close sequence identities (40-80%) with charybdotoxin-like SCNs containing three disulfide bridges. The non-cysteinic, bacic and thermolabile Bs10 is composed of 34 amino acid residues (3.7 kDa), and belongs to a new class of peptides, with no sequence resemblance to any other so far reported sequence isolated from scorpions. Surprisingly, Bs10 shows some limited sequence analogy with oocyte zinc finger proteins. Results of these studies are discussed with respect to their structural similarities within the scorpion LCNs, SCNs and other biologically active peptides.