Bot IT2: a new scorpion toxin to study receptor site on insect sodium channels

AbstractThe insect-specific Bothus occitanus tunetanus IT2 toxin is distinguishable from other scorpion toxins by its amino acid sequence and effects on sodium conductance. The present study reveals that Bot IT2 possesses in cockroach neuronal membranes a single class of high affinity (Kd=0.3±0.1 nM) and low capacity (Bmax=2.4±0.5 pmol/mg) binding sites. Competitive binding experiments with several known sodium channel neurotoxins reveal that the Bot IT2 binding site is in close proximity to the other toxins.© 1997 Federation of European Biochemical Societies

Purification and Characterization of two novel hemiscorpius Lepturus Scorpion venom-Derived Phospolipases A(2) which inhibit angiogenesis un vitro

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Hemilipin, a novel Hemiscorpius lepturus venom heterodimeric phospholipase A2, which inhibits angiogenesis in vitro and in vivo

International audiencePhospholipases A(2) (PLA(2)) are enzymes which specifically hydrolyze the sn-2 acyl ester bond of phospholipids producing free fatty acids and lysophospholipids. The secreted PLA(2) (sPLA(2)) are the most common types of PLA(2) purified from the snake venom, mammalian pancreatic juice and other sources. They display a variety of toxic actions and biological activities, including antitumoral and antiangiogenic effects. In this study, we report the isolation, characterization and the antiangiogenic activity of Hemilipin, a novel sPLA(2) extracted from Hemiscomius lepturus venom, the most dangerous scorpion in Iran. Hemilipin was purified by HPLC and analyzed by MALDI TOF/MS. The primary structure was determined by EDMAN degradation method and the PLA(2) activity by titration of fatty acids released from the egg yolk phospholipids. Its antiangiogenic activity was studied in vitro by evaluating effects on apoptosis, Matrigel angiogenesis, migration and adhesion of human umbilical vein endothelial cells (HUVECs) and human pulmonary artery endothelial cells (HPAECs) and in vivo by the chorioallantoic membrane (CAM) assay. Mass spectrometry profile showed that Hemilipin is heterodimeric and the PLA(2) test demonstrated its strong hydrolytic activity. N-terminal aminoacid sequence highlighted a significant homology of Hemilipin's small and large subunits with other sPLA(2) group III. Hemilipin had no effect on apoptosis, but strongly impacted angiogenesis both in vitro and in vivo. Our results demonstrate that this novel non toxic sPLA(2) could be a new tool to disrupt at different steps human angiogenesis. (C) 2015 Elsevier Ltd. All rights reserved

Hemitoxin, the first potassium channel toxin from the venom of the Iranian scorpion Hemiscorpius lepturus.

International audienceHemitoxin (HTX) is a new K+ channel blocker isolated from the venom of the Iranian scorpion Hemiscorpius lepturus. It represents only 0.1% of the venom proteins, and displaces [125 I]alpha-dendrotoxin from its site on rat brain synaptosomes with an IC50 value of 16 nm. The amino acid sequence of HTX shows that it is a 35-mer basic peptide with eight cysteine residues, sharing 29-69% sequence identity with other K+ channel toxins, especially with those of the alphaKTX6 family. A homology-based molecular model generated for HTX shows the characteristic alpha/beta-scaffold of scorpion toxins. The pairing of its disulfide bridges, deduced from MS of trypsin-digested peptide, is similar to that of classical four disulfide bridged scorpion toxins (Cys1-Cys5, Cys2-Cys6, Cys3-Cys7 and Cys4-Cys8). Although it shows the highest sequence similarity with maurotoxin, HTX displays different affinities for Kv1 channel subtypes. It blocks rat Kv1.1, Kv1.2 and Kv1.3 channels expressed in Xenopus oocytes with IC50 values of 13, 16 and 2 nM, respectively. As previous studies have shown the critical role played by the beta-sheet in Kv1.3 blockers, we suggest that Arg231 is also important for Kv1.3 versus Kv1.2 HTX positive discrimination. This article gives information on the structure-function relationships of Kv1.2 and Kv1.3 inhibitors targeting developing peptidic inhibitors for the rational design of new toxins targeting given K+ channels with high selectivity

BotIT6: a potent depressant insect toxin from Buthus occitanus tunetanus venom.

International audienceA new depressant insect toxin Buthus occitanus tunetanus insect-toxin 6 (BotIT6) was purified by high-performance liquid chromatography from Buthus occitanus tunetanus (Bot) venom. BotIT6 is very active against Blatella germanica (LD50=10ng/100mg body mass) thus being one of the most potent anti-insect toxin so far characterised. When compared to other insect toxin sequences, BotIT6 present high similarities with depressant insect toxins with an additional arginine residue at the C-terminus and a methionine at position 27. The calculated net charge of BotIT6 is positive (+3) whereas it is negative for classical depressant toxins: this might be associated with its high toxicity. Voltage current clump studies show that BotIT6 is not a very potent depressant insect toxin despite its high toxicity in vivo. BotIT6 is able to fully inhibit the specific binding of 125I AaHIT and 125I-BotIT2 on Periplaneta americana synaptosomal membrane vesicles with high affinities. Despite its higher toxicity BotIT6 is a weaker competitor with 125I AaHIT and 125I BotIT2 as compared to the other beta toxins.Altogether, these results may suggest that BotIT6 probably defines a novel sub-group of depressant anti-insect toxins for which the receptor site can be overlapping, but not identical to that for classical depressant insect toxins

The small subunit of Hemilipin2, a new heterodimeric phospholipase A2 from Hemiscorpius lepturus scorpion venom, mediates the antiangiogenic effect of the whole protein

International audienceIn a previous study, we reported the identification of Hemilipin, the first secreted heterodimeric phospholipase A2 (sPLA2) from Hemiscorpius lepturus scorpion venom and demonstrated its effective inhibition of all angiogenesis key steps in vitro and in vivo. Here, we aimed to characterize a second sPLA2, Hemilipin2, from the same venom and to elucidate its antiangiogenic effect. The protein was purified by chromatography separation and analyzed by MALDI/TOF mass spectrometry. Its N terminal amino acid sequence was determined by Edman degradation method and the enzymatic activity by fatty acids release assay. Hemilipin2 antiangiogenic activity was investigated by studying its effect in vitro on adhesion, migration and capillary like tube formation of Human Umbilical Vein Endothelial Cells (HUVECs) and Human Pulmonary Artery Endothelial Cells (HPAECs); and in vivo on the chick embryo chorioallantoic membrane (CAM) assay. Data to be presented show that Hemilipin2 is heterodimeric composed by two subunits: the large one has a molecular weight of 12,866 and the small one of 2461 a.m.u. It has a strong calcium-dependent PLA2 activity and impacts angiogenesis in vitro and in vivo without showing any cytotoxic or apoptotic signs. Its chemical modification with p-Bromophenacyl Bromide abolishes the enzymatic activity without affecting the antiangiogenic effect. Furthermore, it has been proved that Hemilipin2 small subunit was able to inhibit blood vessel formation both in vitro and in vivo. These findings may serve as a starting point for the designing of a new generation of specific inhibitor of human angiogenesis at different steps. (C) 2016 Elsevier Ltd. All rights reserved

Heminecrolysin, the first hemolytic dermonecrotic toxin purified from scorpion venom.

Envenomation caused by Hemiscorpius (H.) lepturus from Liochlidae family presents clinical features that have not been previously described for the Buthidae family scorpions. The most significant manifestations of H. lepturus envenomation are hemolysis and dermonecrosis which could lead in severe cases to renal, cardio-respiratory failure, and death. In this study, we aimed to identify and characterize the protein(s) causing these effects. We have purified a 33 kDa protein from the venom of H. lepturus and named it Heminecrolysin. Tryptic digestion and MS/MS analysis of obtained peptides showed homology with previously described brown spider sphingomyelinases D. Functional characterization of Heminecrolysin indicated a sphingomyelinase D, a complement-dependent hemolysis properties and a dermonecrosis activity. Heminecrolysin displayed higher hemolytic activity to human erythrocytes (ED50 of 0.025 μg/ml), a stronger inflammatory and dermonecrotic effects when injected intra-dermally to rabbit skins, while its efficiency to hydrolyze sphingomyelin seems weaker than other known spider dermonecrotic SMasesD (149 ± 32.5 nmol/mg). Step of sensitization of human erythrocytes by Heminecrolysin was shown to be Mg(2+) and Ca(2+)-independent while hemolysis step in the presence of complement required both bivalent ions. Heminecrolysin is the first hemolytic dermonecrotic toxin identified in venom other than spiders. Except in spider Loxosceles genus and some pathogenic strains of Corynebacteria, sphingomyelinase D activity is unknown in the animal kingdom

Histopathological changes induced by Hemiscorpius lepturus scorpion venom in mice.

International audienceEnvenomation by Hemiscorpius lepturus (H. lepturus) is associated with local necrosis, followed by systemic manifestations. In this work the LD₅₀ of H. lepturus venom were determined by subcutaneous (SC) injection in white Balb/c mice (5 mg/kg). Histopathological alterations in organs such as kidney, heart, liver, lungs, stomach and intestine were determined in 3, 6, 12 and 24 h following experimental (SC) envenoming injection of one LD ₅₀ of the venom in Balb/c mice. Histological studies showed degenerative changes in the kidney with disorganized glomeruli and necrotic tubular in 3 h and reached to its climax in 6 h. Myocardium showed massive myocytolysis with interstitial necrosis in 3 h and reached to its peak after 6 h past envenoming. Bowels showed edema of lamina propria and slight villous necrosis. The enzymatic activities of creatine kinase (CK) and lactate dehydrogenase (LDH) were significantly increased in the serum in 9 h. No necrotic lesion observed in lungs and liver. The results indicate that the venom of H. lepturus is a highly cytotoxic, and induces massive tissue damages in specific organs, starting from the heart and kidney as the first target in 3 h and ends to the bowels in 6 h post envenomation

Antigenic and Substrate Preference Differences between Scorpion and Spider Dermonecrotic Toxins, a Comparative Investigation

The Hemiscorpius lepturus scorpion and brown spider Loxosceles intermedia represent a public health problem in Asia and America, respectively. Although distinct, these organisms contain similar toxins responsible for the principal clinical signs of envenomation. To better understand the properties of these toxins, we designed a study to compare recombinant Heminecrolysin (rHNC) and rLiD1, the major phospholipase D toxins of scorpion and spider venom, respectively. Using a competitive ELISA and a hemolytic inhibition test, we come to spot a cross reaction between scorpion and spider venoms along with an epitopic similarity between rHNC and rLiD1 associated with neutralizing antibodies. Results show that the ability of the rHNC to hydrolyze lysophosphatidylcholine (LPC) is equivalent to that of rLiD1 to hydrolyze sphingomyelin and vice-versa. rHNC exclusively catalyze transphosphatidylation of LPC producing cyclic phosphatidic acid (cPA). The in-silico analysis of hydrogen bonds between LPC and toxins provides a possible explanation for the higher transphosphatidylase activity of rHNC. Interestingly, for the first time, we reveal that lysophosphatidic acid (LPA) can be a substrate for both enzymes using cellular and enzymatic assays. The finding of the usage of LPA as a substrate as well as the formation of cPA as an end product could shed more light on the molecular basis of Hemiscorpius lepturus envenomation as well as on loxoscelism

Hemicalcin, a new toxin from the Iranian scorpion Hemiscorpius lepturus which is active on ryanodine-sensitive Ca2+ channels

In the present work, we purified and characterized a novel toxin named hemicalcin from the venom of the Iranian chactoid scorpion Hemiscorpius lepturus where it represents 0.6% of the total protein content. It is a 33-mer basic peptide reticulated by three disulfide bridges, and that shares between 85 and 91% sequence identity with four other toxins, all known or supposed to be active on ryanodine-sensitive calcium channels. Hemicalcin differs from these other toxins by seven amino acids at positions 9 (leucine/arginine), 12 (alanine/glutamic acid), 13 (aspartic acid/asparagine), 14 (lysine/asparagine), 18 (serine/glycine), 26 (threonine/alanine) and 28 (proline/isoleucine/alanine). In spite of these differences, hemicalcin remains active on ryanodine-sensitive Ca2+ channels, since it increases [3H]ryanodine binding on RyR1 (ryanodine receptor type 1) and triggers Ca2+ release from sarcoplasmic vesicles. Bilayer lipid membrane experiments, in which the RyR1 channel is reconstituted and its gating properties are analysed, indicate that hemicalcin promotes an increase in the opening probability at intermediate concentration and induces a long-lasting subconductance level of 38% of the original amplitude at higher concentrations. Mice intracerebroventricular inoculation of 300 ng of hemicalcin induces neurotoxic symptoms in vivo, followed by death. Overall, these data identify a new biologically active toxin that belongs to a family of peptides active on the ryanodine-sensitive channel