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.     

Negative-shift activation, current reduction and resurgent currents induced by β-toxins from Centruroides scorpions in sodium channels

The β-toxins purified from the New World scorpion venoms of the Centruroides species affect several voltage-gated sodium channels (VGSCs) and thus are essential tools not only for the discrimination of different channel sub-types but also for studying the structure-function relationship between channels and toxins. This communication reports the results obtained with four different peptides purified from three species of Centruroides scorpions and assayed on seven distinct isoforms of VGSC (Na_v1.1-Na_v1.7) by specific functional analysis conducted through single cell electrophysiology. The toxins studied were CssII from Centruroides suffusus suffusus, Cll1 and Cll2 from Centruroides limpidus limpidus and a novel toxin from Centruroides noxius, which was characterized for the first time here. It has 67 amino acid residues and four disulfide bridges with a molecular mass of 7626 Da. Three different functional features were identified: current reduction of macroscopic conductance, left shift of the voltage-dependent activation and induction of resurgent currents at negative voltages following brief, strong depolarizations. The isoforms which revealed to be more affected resulted to be Na_v1.6 > 1.1 > 1.2 and, for the first time, a β-toxin is here shown to induce resurgent current also in isoforms different from Na_v1.6. Additionally, these results were analyzed with molecular modelling. In conclusion, although the four toxins have a high degree of identity, they display tri-modal function, each of which shows selectivity among the different sub-types of Na⁺-channels. Thus, they are invaluable as tools for structure-function studies of β-toxins and offer a basis for the design of novel ion channel-specific drugs.     

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.     

Isolation and molecular cloning of beta-neurotoxins from the venom of the scorpion Centruroides suffusus suffusus

This communication reports the identification and characterization of two new toxins from the venom of the scorpion Centruroides suffusus suffusus, named: CssVIII and CssIX, according to the original nomenclature of toxins previously described for this scorpion. The isolation was obtained by means of two chromatographic steps, and a cDNA library was used to fully identify their precursors. CssVIII and CssIX contain signal peptides of 19 and 17 amino acid residues, and mature peptides of 66 and 65 residues, respectively. Intracranial injections into mice of both purified toxins showed toxicity results similar to those found for toxins CssII and CssIV. Additionally, they compete with the parent toxin CssIV, in binding and displacement experiments, conducted with brain synaptosomes showing nanomolar affinities. These results strongly support the conclusion that they are new β-neurotoxins and certainly would be of the interest of researchers in the field of venomics for studying sodium channels.     

Antibacterial activity of six novel peptides from Tityus discrepans scorpion venom. A fluorescent probe study of microbial membrane Na permeability changes

Six novel peptides (named bactridines) were isolated from Tityus discrepans scorpion venom. From mass spectrometry molecular masses were 6916, 7362, 7226, 7011, 7101 and 7173 Da (bactridines 1–6). Bactridines 1 and 2 were sequenced by Edman degradation. The sequences and in silico analysis, indicated that they are positively charged polypeptides comprised of 61 and 64 amino acids (AA), respectively, bactridine 1 and bactridine 2 containing 4 disulfide bridges. Bactridine 1 was only toxic to cockroaches and crabs, and bactridine 2–6 were only toxic to mice. Bactridine 1 has a 78% sequence identity with ardiscretin. Ardisctretin is an insect specific sodium toxin which also produces a small depolarization and induces repetitive firing in squid axons resembling those of DDT [1,10(pchlorobenzyl) 2-trichloretane] in its ability to slow down action potential, to induce repetitive firing. Measured as the minimal inhibitory concentration, bactridines had high antibacterial activity against a wide range of Gram positive and Gram negative bacteria. Complete bacterial growth inhibition occurred at concentrations from 20 to 80 μM depending on the bacteria and peptide tested. Effects on membrane Na⁺ permeability induced by bactridines were observed on Yersinia enterocolitica loaded with 1 μM CoroNa™ Red. CoroNa™ Red fluorescence leakage from bacteria was observed after exposure to 0.3 μM of any bactridine tested, indicating that they modified Na⁺ membrane permeability. This effect was blocked by 10 μM amiloride and by 25 μM mibefradil drugs that affect Na⁺ and Ca²⁺ channels respectively. We found no evidence of changes of K⁺ or Ca²⁺ concentrations neither inside nor outside the bacteria in experiments using the fluorescent dyes Fluo 4AM (10 μM) and PBFI (20 μM).     

The Toxins Purified from Tityus Serrulatus (Lutz & Mello)Venom

Abstract

The toxicity of Tityus serrulatus venom is mainly due to a complex mixture of basic proteins of low molecular weight (MW< 8000 Da) which are active on the voltage-sensitive Na⁺ channel of excitable cells. One group of toxins, the α-toxins, delays inactivation of the Na⁺ channel. A second group, the β- toxins, produces a transient shift in the voltage-dependence of Na⁺ channel activation and increases the tendency of the cells to fire repetitively. The two groups bind specifically to two different binding sites, sites 3 and 4, of Na⁺ channels present in rat brain synaptosomes. The primary structure of the main toxins has been determined and consists of a single amino acid chain of 61 to 66 residues cross-linked by four disulfide bridges. Some secondary structural elements have also been determined. More recently, using molecular biological techniques, cDNAs encoding the precursors of α and β-toxins have been cloned from a cDNA library of Tityus serrulatus venom gland. The precursors contain a signal peptide of about 20 – 22 residues, the mature toxin and three additional Gly-Lys-Lys residues at the C-terminal that are not present in the mature toxins. The Lys residues are removed by a carboxypeptidase and the remaining Gly-extended peptides are converted into α-amidated C-terminal toxins. Toxins active at K⁺ channels have also been purified from Tityus serrulatus venom. At present, only two toxins have been characterized, namely a short polypeptide of 37 amino acid residues cross-linked by three disulfide bridges that competes for 125I-alpha DTX (dendrotoxin) binding sites in synaptic membranes, and a longer nonhomologous toxin of 8 016 Da, whose primary structure has only been determined at the level of its NH2 terminal.     

Biochemical and molecular characterization of the venom from the Cuban scorpion Rhopalurus junceus

This communication describes the first general biochemical, molecular and functional characterization of the venom from the Cuban blue scorpion Rhopalurus junceus, which is often used as a natural product for anti-cancer therapy in Cuba. The soluble venom of this arachnid is not toxic to mice, injected intraperitoneally at doses up to 200 μg/20 g body weight, but it is deadly to insects at doses of 10 μg per animal. The venom causes typical alpha and beta-effects on Na⁺ channels, when assayed using patch-clamp techniques in neuroblastoma cells in vitro. It also affects K⁺ currents conducted by ERG (ether-a-go-go related gene) channels. The soluble venom was shown to display phospholipase, hyaluronidase and anti-microbial activities. High performance liquid chromatography of the soluble venom can separate at least 50 components, among which are peptides lethal to crickets. Four such peptides were isolated to homogeneity and their molecular masses and N-terminal amino acid sequence were determined. The major component (RjAa12f) was fully sequenced by Edman degradation. It contains 64 amino acid residues and four disulfide bridges, similar to other known scorpion toxins. A cDNA library prepared from the venomous glands of one scorpion allowed cloning 18 genes that code for peptides of the venom, including RjA12f and eleven other closely related genes. Sequence analyses and phylogenetic reconstruction of the amino acid sequences deduced from the cloned genes showed that this scorpion contains sodium channel like toxin sequences clearly segregated into two monophyletic clusters. Considering the complex set of effects on Na⁺ currents verified here, this venom certainly warrant further investigation.     

Development of an in vitro drug screening system for Mycobacterium leprae based on the determination of the intrabacterial sodium to potassium ratio of individual bacterial organisms

In vitro drug effects on Mycobacterium leprae (M. leprae) in a cell-free system have been monitored by mass spectrometric determination of the ratio of the intrabacterial concentrations of the sodium and potassium ions (Na⁺, K⁺ ratio) of a limited number of individual bacteria per sample. From the drug-induced increase of the median values of the distributions of the Na⁺, K⁺ ratio, information on the concentration and time dependence of drug effects as well as on antagonistic or synergistic interactions of drugs has been obtained. Moreover, absolute values for the percentage of killed bacteria (% kill) have been derived from the distribution of the Na⁺, K⁺ ratios within a bacterial population. For this, the limiting value of the Na⁺, K⁺ ratio (up to which bacteria are viable) —which had been determined as 0.45 for cultivable bacteria — has been presumed to be valid also for M. leprae. Highest killing rates have been observed for fusidic acid and clarithromycin, followed by rifabutine, rifampin, and clofazimine. Minocycline and dapsone have shown only moderate killing effects and isoniazid and — probably due to the restricted metabolism of M. leprae in a cell-free medium — ofloxacin have been completely inactive. Strong ofloxacin effects, however, have been observed for cultivable mycobacteria and intracellular M. leprae phagocytized by a murine macrophage cell line.     

Purification, characterization and cDNA cloning of two natterin-like toxins from the skin secretion of oriental catfish Plotosus lineatus

The oriental catfish Plotosus lineatus is known to contain proteinaceous toxins in the skin secretion as well as in the venom gland. However, detailed properties and primary structures of the skin toxins have not been clarified. In this study, two proteinaceous toxins (toxins I and II) were purified from the skin secretion of oriental catfish by a combination of gel filtration, anion-exchange HPLC and hydroxyapatite HPLC. Toxins I and II are monomeric simple proteins with almost the same molecular mass (35 kDa for toxin I and 37 kDa for toxin II) and are distinguishable from each other in isoelectric point (6.5 for toxin I and 5.1 for toxin II). Both toxins display lethal, edema-forming and nociceptive activities, although toxin I is significantly more potent than toxin II. The primary structures of toxins I and II were elucidated by cloning experiments based on the determined partial amino acid sequences. Toxins I (317 amino acid residues) and II (315 amino acid residues) share as high as 86% sequence identity with each other and are also highly homologous (56–75% identities) with the known fish natterin-like proteins.     

Palytoxin binds to and inhibits kidney and erythrocyte Na+, K+-ATPase

Summary Hog kidney Na⁺, K⁺-ATPase, purified to the microsomal stage and activated with detergent, binds palytoxin, as shown by the nearly complete competition of the toxin with ³H-ouabain. The K _i-values of palytoxin, but not of ouabain, depend on the protein concentration; this indicates additional binding sites for the toxin on kidney membranes. — Palytoxin inhibits the enzymatic activity of the detergent-activated preparation nearly completely (IC₅₀ 8·10^–7 mol/l). Inhibition of ATPase activity and of ouabain binding are promoted by borate, a known activator of palytoxin. — Palytoxin also inhibits the Na⁺, K⁺-ATPase of erythrocyte ghosts in the same dose range.The data are discussed in context with the hypothesis (Chhatwal et al. 1983) that palytoxin raises the cellular permeability by altering the state of Na⁺, K⁺-ATPase or its environment.Part of the thesis (Dr. rer. nat.) of H. Böttinger     

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.     

Inhibition of Na+-pump enhances carbachol-induced influx of 45Ca2+ and secretion of catecholamines by elevation of cellular accumulation of 22Na+ in cultured bovine adrenal medullary cells

Summary In bovine adrenal medullary cells, we reported that ²²Na⁺ influx via nicotinic receptor-associated Na⁺ channels is involved in ⁴⁵Ca²⁺ influx, a requisite for initiating the secretion of catecholamines (Wada et al. 1984, 1985b).In the present study, we investigated whether the inhibition of Na⁺-pump modulates carbachol-induced ²²Na⁺ influx, ⁴⁵Ca²⁺ influx and catecholamine secretion in cultured bovine adrenal medullary cells. We also measured ⁸⁶Rb⁺ uptake by the cells to estimate the activity of Na⁺, K⁺-ATPase. (1) Ouabain and extracellular K⁺ deprivation remarkably potentiated carbachol-induced ²²Na⁺ influx, ⁴⁵Ca²⁺ influx and catecholamine secretion; this potentiation of carbachol-induced ⁴⁵Ca²⁺ influx and catecholamine secretion was not observed in Na⁺ free medium. (2) Carbachol increased the uptake of ⁸⁶Rb⁺; this increase was inhibited by hexamethonium and d-tubocurarine. In Na⁺ free medium, carbachol failed to increase ⁸⁶Rb⁺ uptake. (3) Ouabain inhibited carbachol-induced ⁸⁶Rb⁺ uptake in a concentration-dependent manner, as it increased the accumulation of cellular ²²Na⁺. These results suggest that Na⁺ influx via nicotinic receptor-associated Na⁺ channels increases the activity of Na⁺, K⁺-ATPase and the inhibition of Na⁺, K⁺-ATPase augmented carbachol-induced Ca²⁺ influx and catecholamine secretion by potentiating cellular accumulation of Na⁺. It seems that nicotinic receptor-associated Na⁺ channels and Na⁺, K⁺-ATPase, both modulate the influx of Ca²⁺ and secretion of catecholamines by accomodating cellular concentration of Na⁺.     

Inhibition of human colonic (Na++K+)-ATPase by arachidonic and linoleic acid

Summary The sodium pump, (Na⁺+K⁺)-ATPase, which is involved in the transport of cations and water movement by the colonic mucosa, may be decreased in various diarrhoeal states. In this study, we have measured ³H-ouabain binding and (Na⁺+K⁺)-ATPase activity in human colonic biopsy homogenates and the influence of various inflammatory and antiinflammatory compounds on these parameters. ³H-ouabain binds to one site of high affinity (K _D 1.9±0.2×10^–9 mol/l) with a maximal binding capacity of 7.5±0.8×10¹⁴ binding sites/g protein. Both arachidonic and linoleic acid inhibited (Na⁺+K⁺)-ATPase activity (IC₅₀ arachidonic acid: 7.5×10^–5 mol/l, linoleic acid: 6.5×10^–5 mol/l) and Mg²⁺-ATPase activity (IC₅₀ arachidonic acid: 9×10^–5 mol/l, linoleic acid: 4×10^–5 mol/l). Arachidonic acid inhibited ³H-ouabain binding, (IC₅₀ 3.2×10^–5 mol/l). The following antiinflammatory compounds, at concentrations up to 1×10^–3 mol/l, did not influence ATPase activity directly nor reverse the arachidonic acid-induced inhibition: indomethacin (cyclooxygenase inhibitor), nordihydroguaretic acid (lipoxygenase inhibitor), sulphasalazine and its metabolites: 5-aminosalicylic acid, N-acetylaminosalicylic acid and sulphapyridine.These results indicate that human colonic (Na⁺+K⁺)-ATPase is inhibited by the prostanoid precursors, arachidonic and linoleic acid. From a therapeutic point of view (effect on colonic (Na⁺+K⁺)-ATPase and perhaps diarrhoea), the suppression of the production of these prostanoid precursors by drugs may, therefore, be beneficial in the treatment of inflammatory bowel disease.Supported by DFG (Er65/4-4)     

Transport of NG-Nitro-L-Arginine Across Intestinal Brush Border Membranes by Na+-Dependent and Na+ Independent Amino Acid Transporters

Purpose. To clarify the transport mechanism of N^G-nitro-L-arginine (L-NNA), a potent NO-synthase inhibitor, across intestinal brush border membranes (BBM). Methods. Dog intestinal BBM vesicles were used. Results. The time course of L-NNA uptake showed a Na⁺-dependent overshoot phenomenon. Concentration-dependence curves of L-NNA initial uptake were saturable in the presence and absence of Na⁺, indicating participation of Na⁺-dependent and Na⁺-independent carrier-mediated transport systems. The calculated kinetic parameters of L-NNA initial uptake indicate that the former is a low-affinity high-capacity system and the latter is a high-affinity low-capacity one, similar to those in neutral amino acid transport. Neutral and basic amino acids showed cis-inhibitory and trans-stimulatory effects on L-NNA uptake in the presence or absence of Na⁺. N^G-Nitro-L-arginine methyl ester, another potent NO-synthase inhibitor, also had both effects, which were smaller than with amino acids. Conclusions. The present study clearly indicates that transport of L-NNA across the intestinal BBM occurs in the same manner as neutral amino acid transport. However, it is affected by both neutral and basic amino acids in the presence or absence of Na⁺ differently from that across plasma membranes of nonepithelial cells, because B^{0, +} and b^{0, +} amino acid transporters function partly in L-NNA transport across intestinal BBM.     

<Emphasis Type="Italic">N</Emphasis>-Ethylmaleimide differentially inhibits substrate uptake by and ligand binding to the noradrenaline transporter

Using transfected HEK293 cells that express the human (h) noradrenaline transporter (hNAT), we show differential inhibitory effects of the thiol reagent N-ethylmaleimide (NEM) on [³H]NA uptake and [³H]nisoxetine binding. Irreversible inhibition of uptake by NEM was complete, faster, and occurred at lower concentrations. Furthermore, hNAT ligands (substrates and inhibitors) prevented NEM-induced inhibition of binding but not that of uptake, indicating different underlying mechanisms of inhibition. NEM-induced uptake inhibition was not primarily due to inhibition of the Na⁺/K⁺-ATPase since ouabain caused only partial inhibition. For the first time, we show that NEM at low concentrations causes a rapid and complete depletion of cellular adenosine triphosphate (ATP) not only in HEK293 cells but also in several other eukaryotic cell lines. Thus, while high NEM concentrations alkylate the NAT protein in a ligand-protectable manner, low concentrations inhibit substrate uptake through a loss of the Na⁺ and K⁺ gradient as a driving force by depleting cellular ATP.     

The toxicity of H2O2 on the ionic homeostasis of airway epithelial cells in vitro

Oxygen species may be formed in the air spaces of the respiratory tract in response to environmental pollution such as particulate matter. The mechanisms and target molecules of these oxidants are still mainly unknown but may involve modifications of the ionic homeostasis in epithelial cells. Cytosolic concentrations of Ca²⁺ (Fura2) and Na⁺ (SBFI) and short-circuit current (Isc) were followed in primary cultures of human nasal epithelial cells and in the cell line 16HBE14o⁻ after exposure to H₂O₂ or ·OH (H₂O₂+Fe²⁺). Cells were grown on glass coverslips for ionic imaging or on permeable snapwell inserts for Isc studies. Exposure of the apical as well as the basal side of the cultures to H₂O₂ or ·OH induced a concentration-dependent transient increase in Isc which is due to a transient secretion of Cl⁻. Ca_i also increased transiently with approximately the same kinetics. The response was dependent on the release of calcium from intracellular stores. Na_i on the contrary increased steadily over more than an hour. When the apical membrane was permeabilized with gramicidin, ·OH inhibited the Na⁺ current (a measure of Na⁺-K⁺-ATPase activity in the baso-lateral membrane). The arrest of the pump was significant after 30 min exposure to oxidant. On the other hand no increase in the apical or baso-lateral sodium conductances could be detected. The progressive arrest of the Na⁺/K⁺-pump may contribute to the sustained elevation of Na_i. This strong modification in the cellular ionic homeostasis may participate in the stress response of the respiratory epithelium through alterations in signal transduction pathways.     

Steroid-like compounds in Chinese medicines promote blood circulation via inhibition of Na^＋/K^＋- ATPase

Aim:

To examine if steroid-like compounds found in many Chinese medicinal products conventionally used for the promotion of blood circulation may act as active components via the same molecular mechanism triggered by cardiac glycosides, such as ouabain.

Methods:

The inhibitory potency of ouabain and the identified steroid-like compounds on Na⁺/K⁺-ATPase activity was examined and compared. Molecular modeling was exhibited for the docking of these compounds to Na⁺/K⁺-ATPase.

Results:

All the examined steroid-like compounds displayed more or less inhibition on Na⁺/K⁺-ATPase, with bufalin (structurally almost equivalent to ouabain) exhibiting significantly higher inhibitory potency than the others. In the pentacyclic triterpenoids examined, ursolic acid and oleanolic acid were moderate inhibitors of Na⁺/K⁺-ATPase, and their inhibitory potency was comparable to that of ginsenoside Rh2. The relatively high inhibitory potency of ursolic acid or oleanolic acid was due to the formation of a hydrogen bond between its carboxyl group and the Ile322 residue in the deep cavity close to two K⁺ binding sites of Na⁺/K⁺-ATPase. Moreover, the drastic difference observed in the inhibitory potency of ouabain, bufalin, ginsenoside Rh2, and pentacyclic triterpenoids is ascribed mainly to the number of hydrogen bonds and partially to the strength of hydrophobic interaction between the compounds and residues around the deep cavity of Na⁺/K⁺-ATPase.

Conclusion:

Steroid-like compounds seem to contribute to therapeutic effects of many cardioactive Chinese medicinal products. Chinese herbs, such as Prunella vulgaris L, rich in ursolic acid, oleanolic acid and their glycoside derivatives may be adequate sources for cardiac therapy via effective inhibition on Na⁺/K⁺-ATPase.