An overview of some enzymes from buthid scorpion venoms from Colombia: Centruroides margaritatus, Tityus pachyurus, and Tityus n. sp. aff. metuendus

Background: In Colombia, several species of Buthidae scorpions belonging to the genera Centruroides and Tityus coexist, and their stings are considered life-threatening to humans because of their venom neurotoxins. Despite previous studies focusing on neurotoxins from these scorpion genera, little is known about the enzymes present in their venoms and their relationship with whole venom toxicity. Methods: Here, using proteomic and biochemical protocols the enzymatic activities of the venoms of three Colombian scorpion species, C. margaritatus, T. pachyurus, and T. n. sp. aff. metuendus, were compared to establish the presence and absence of enzymes such as phospholipases, hyaluronidases, and proteases that could be related to venom toxicity. Results: C. margaritatus was positive for hyaluronidases, T. n. sp. aff. metuendus for proteases, and T. pachyurus exhibited activity for all three mentioned enzymes. Conclusion: This information provides valuable insights into the specific enzyme diversity of each species' venom and their potential role in venom toxicity, which could contribute to the development of better treatments and prevention strategies for scorpion 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.

Venom characterization of the bark scorpion Centruroides edwardsii (Gervais 1843): Composition, biochemical activities and in vivo toxicity for potential prey.

In this study, we characterize the venom of Centruroides edwardsii, one of the most abundant scorpions in urban and rural areas of Costa Rica, in terms of its biochemical constituents and their biological activities. C. edwardsii venom is rich in peptides but also contains some higher molecular weight protein components. No phospholipase A2, hemolytic or fibrinogenolytic activities were found, but the presence of proteolytic and hyaluronidase enzymes was evidenced by zymography. Venom proteomic analysis indicates the presence of a hyaluronidase, several cysteine-rich secretory proteins, metalloproteinases and a peptidylglycine α-hydroxylating monooxygenase like-enzyme. It also includes peptides similar to the K+-channel blocker margatoxin, a dominant toxin in the venom of the related scorpion C. margaritatus. MS and N-terminal sequencing analysis also reveals the presence of Na+-channel-modulating peptides with sequence similarity to orthologs present in other scorpion species of the genera Centruroides and Tityus. We purified the hyaluronidase (which co-eluted with an allergen 5-like CRiSP) and sequenced ~60% of this enzyme. We also sequenced some venom gland transcripts that include other cysteine-containing peptides and a Non-Disulfide Bridged Peptide (NDBP). Our in vivo experiments characterizing the effects on potential predators and prey show that C. edwardsii venom induces paralysis in several species of arthropods and geckos; crickets being the most sensitive and cockroaches and scorpions the most resistant organisms tested. Envenomation signs were also observed in mice, but no lethality was reached by intraperitoneal administration of this venom up to 120 µg/g body weight.

Purification and Biochemical Characterization of TsMS 3 and TsMS 4: Neuropeptide-Degrading Metallopeptidases in the Tityus serrulatus Venom.

Although omics studies have indicated presence of proteases on the Tityus serrulatus venom (TsV), little is known about the function of these molecules. The TsV contains metalloproteases that cleave a series of human neuropeptides, including the dynorphin A (1-13) and the members of neuropeptide Y family. Aiming to isolate the proteases responsible for this activity, the metalloserrulase 3 and 4 (TsMS 3 and TsMS 4) were purified after two chromatographic steps and identified by mass spectrometry analysis. The biochemical parameters (pH, temperature and cation effects) were determined for both proteases, and the catalytic parameters (Km, kcat, cleavage sites) of TsMS 4 over fluorescent substrate were obtained. The metalloserrulases have a high preference for cleaving neuropeptides but presented different primary specificities. For example, the Leu-enkephalin released from dynorphin A (1-13) hydrolysis was exclusively performed by TsMS 3. Neutralization assays using Butantan Institute antivenoms show that both metalloserrulases were well blocked. Although TsMS 3 and TsMS 4 were previously described through cDNA library studies using the venom gland, this is the first time that both these toxins were purified. Thus, this study represents a step further in understanding the mechanism of scorpion venom metalloproteases, which may act as possible neuropeptidases in the envenomation process.

Developing recombinant phospholipase D1 (rPLD1) toxoid from Iranian Hemiscorpius lepturus scorpion and its protective effects in BALB/c mice.

Hemiscorpius lepturus (H. lepturus) is one of the most dangerous scorpions and the most medically important scorpion in Iran. The clinical signs of H. lepturus envenomation, including dermonecrosis, hematuria, renal failure and early death, are attributed to phospholipase D activity. This study was conducted to develop a novel recombinant phospholipase D1 (rPLD1) toxoid and investigate its immunogenicity and protective effects against the lethality of H. lepturus venom. The lethal protein recombinant phospholipase D1 was expressed from PLD H. lepturus venom gland. The rPLD1 toxin was converted into toxoid (the first toxoid of H. lepturus PLD) with a 0.25% concentration of formalin and stored for ten days at room temperature. In the toxicity test, the lethal activity of recombinant phospholipase D1 was fully inhibited. When it reached up to 3 times higher than the maximal effective concentration of the purified toxin (11.1 µg), rPLD1 toxoid was used. The sphingomyelinase activity was inhibited when up to 5.4 times of the LD100 of the purified toxin (20 µg), toxoid was used. It was then used to produce an antibody in BALB/c as an antigen and the mice were then challenged with rPLD1 toxin and the whole venom. The immunogenicity of rPLD1 toxoid was evaluated and the maximum titer of the raised antibodies was determined by ELISA assay. The optimum titer for anti-rPLD1 toxoid sera was obtained at the third intraperitoneal injection of rPLD1 toxoid, and a high titer was reached at the fourth injection in the mice. This toxoid increased the amount of antibodies and produced a protective antiserum against the whole venom of H. lepturus and rPLD1 toxin. The in-vivo test results showed that the mice were completely resistant against 200 times the LD100 of recombinant phospholipase D1 and the whole venom of H. lepturus. To conclude, rPLD1 can be used in toxoid form as an immunogen in the production of a new generation of neutralizing antibodies against the lethality and toxicity of H. lepturus whole venom.

Integrin-mediated human glioblastoma cells adhesion, migration and invasion by native and recombinant phospholipases of Scorpio maurus venom glands.

Integrins are a large family of cell surface receptors mediating the interaction of cells with their microenvironment and they play an important role in glioma biology. In the present work, we reported the anti-tumor effect of Sm-PLGV a phospholipase A2 from Tunisian scorpion venom glands-as well as its recombinant forms expressed in Escherichia coli-through interference with integrin receptor function in malignant glioma cells U87. These phospholipases inhibited in a dose dependent manner the adhesion, migration and invasion onto fibrinogen and fibronectin without any cytotoxicity. We showed that Sm-PLGV and its recombinant constructs blocked U87 migration by reducing their velocity and directional persistence. The inhibitory effect was related to a blockage of the integrins αvß3 and α5ß1 function. Inactivation of the enzymatic activity of Sm-PLGV by chemical modification with p-bromophenacyl bromide did not affect its anti-tumor properties, suggesting the presence of 'pharmacological sites' distinct from the catalytic site in scorpion venom phospholipases A2.

Anti-angiogenic effect of phospholipases A2 from Scorpio maurus venom glands on Human Umbilical Vein Endothelial Cells.

In a previous study, we purified Sm-PLGV an heterodimeric phospholipase A2, from the venom glands of the Tunisian scorpion Scorpio maurus. This enzyme contains a Long chain, a penta-peptide insertion, which is cut out during the maturation process, followed by a short chain. A disulfide bridge links the two chains. Three recombinant forms of this enzyme were produced in Escherichia coli: rPLA2(+5) with a penta-peptide insert, rPLA2(-5) without the penta-peptide, and the Long chain alone without the short one. In the present study, we showed that Sm-PLGV, rPLA2(+5) and rPLA2(-5) displayed more potent anti-angiogenic activity in vitro than the recombinant Long chain and the short one obtained by chemical synthesis. These phospholipases A2 inhibited in a dose-dependent manner adhesion, migration and invasion of Human Umbilical Vein Endothelial Cells. Using Matrigel™, we demonstrated that Sm-PLGV, rPLA2(+5) and rPLA2(-5) significantly inhibited tubulogensesis. We also showed a clear dissociation between the anti-angiogenic effect of Sm-PLGV and its catalytic activity.

Heterologous expression of rTsHyal-1: the first recombinant hyaluronidase of scorpion venom produced in Pichia pastoris system.

In general, hyaluronidases have a broad potential application on medicine and esthetics fields. Hyaluronidases from animal venoms cleave hyaluronan present in the extracellular matrix, acting as spreading factors of toxins into the tissues of the victim. However, the in-depth characterization of hyaluronidase from animal venoms has been neglected due to its instability and low concentration in the venom, which hamper its isolation. Thus, heterologous expression of hyaluronidase acts as a biotechnological tool in the obtainment of enough amounts of the enzyme for structural and functional studies. Therefore, this study produced a recombinant hyaluronidase from Tityus serrulatus scorpion venom, designated as rTsHyal-1, in the Pichia pastoris system. Thus, a gene for TsHyal-1 (gb|KF623285.1) was synthesized and cloned into the pPICZαA vector (GenScript Corporation) for heterologous expression in P. pastoris. rTsHyal-1 was expressed in laboratorial scale in a buffered minimal medium containing methanol (BMM) for 96 h with daily addition of methanol. Expression of rTsHyal-1 resulted in a total protein yield of 0.266 mg/mL. rTsHyal-1 partially purified through cation exchange chromatography presented a specific activity of 1097 TRU/mg, against 838 TRU/mg for the final expressed material, representing a 1.31-fold purification. rTsHyal-1 has molecular mass of 49.5 kDa, and treatment with PNGase F and analysis by mass spectrometry (MALDI-TOF) indicated a potential N-glycosylation of 4.5 kDa. Additionally, de novo sequencing of rTsHyal-1, performed in MALDI-TOF and Q Exactive Orbitrap MS, resulted in 46.8% of protein sequence coverage. rTsHyal-1 presents the highest substrate specificity to hyaluronan followed by chondroitin-6-sulfate, chondroitin-4-sulfate, and dermatan sulfate and showed an optimum activity at pH 6.0 and 40 °C. These results validate the biotechnological process for the heterologous expression of rTsHyal-1. This is the first recombinant hyaluronidase from scorpion venoms expressed in the P. pastoris system with preserved enzyme activity.

Venom gland transcriptomic and venom proteomic analyses of the scorpion Megacormus gertschi Díaz-Najera, 1966 (Scorpiones: Euscorpiidae: Megacorminae).

The soluble venom from the Mexican scorpion Megacormus gertschi of the family Euscorpiidae was obtained and its biological effects were tested in several animal models. This venom is not toxic to mice at doses of 100 µg per 20 g of mouse weight, while being lethal to arthropods (insects and crustaceans), at doses of 20 µg (for crickets) and 100 µg (for shrimps) per animal. Samples of the venom were separated by high performance liquid chromatography and circa 80 distinct chromatographic fractions were obtained from which 67 components have had their molecular weights determined by mass spectrometry analysis. The N-terminal amino acid sequence of seven protein/peptides were obtained by Edman degradation and are reported. Among the high molecular weight components there are enzymes with experimentally-confirmed phospholipase activity. A pair of telsons from this scorpion species was dissected, from which total RNA was extracted and used for cDNA library construction. Massive sequencing by the Illumina protocol, followed by de novo assembly, resulted in a total of 110,528 transcripts. From those, we were able to annotate 182, which putatively code for peptides/proteins with sequence similarity to previously-reported venom components available from different protein databases. Transcripts seemingly coding for enzymes showed the richest diversity, with 52 sequences putatively coding for proteases, 20 for phospholipases, 8 for lipases and 5 for hyaluronidases. The number of different transcripts potentially coding for peptides with sequence similarity to those that affect ion channels was 19, for putative antimicrobial peptides 19, and for protease inhibitor-like peptides, 18. Transcripts seemingly coding for other venom components were identified and described. The LC/MS analysis of a trypsin-digested venom aliquot resulted in 23 matches with the translated transcriptome database, which validates the transcriptome. The proteomic and transcriptomic analyses reported here constitute the first approach to study the venom components from a scorpion species belonging to the family Euscorpiidae. The data certainly show that this venom is different from all the ones described thus far in the literature.

Neuropeptide Y family-degrading metallopeptidases in the Tityus serrulatus venom partially blocked by commercial antivenoms.

Accidents caused by scorpions represent a relevant public health issue in Brazil, being more recurring than incidents with snakes and spiders. The main species responsible for this situation is the yellow scorpion, Tityus serrulatus, due especially to the great frequency with which accidents occur and the potential of its venom to induce severe clinical manifestations, even death, mainly among children. Although neurotoxins are well characterized, little information is known about other components of scorpion venoms, such as peptidases, and their effect on envenomation. Previous results from our group showed that the metallopeptidases present in this venom are capable of hydrolyzing the neuropeptide dynorphin 1-13 in vitro, releasing Leu-enkephalin, which may interact with ion channels and promote indirect neurotoxicity. Thus, this study aims to get more information about the effect of toxic peptidase activity present in the venom on biologically active peptides, and to evaluate the in vitro neutralizing potential of commercial antivenoms produced by the Butantan Institute. A set of human bioactive peptides were studied as substrates for the peptidases, and the members of the neuropeptide Y family were found to be the most susceptible ones. All new substrate hydrolyses were totally inhibited by ethylenediaminetetracetic and not blocked by phenylmethanesulfonylfluoride, indicating that metallopeptidases were responsible for the peptidase activity. Also, peptidase activities were only partially inhibited by therapeutic Brazilian scorpion antivenom (SAV) and arachnid antivenom (AAV). The dose-response inhibition by both antivenoms indicates that AAV neutralizes better than SAV at the used doses. These characterizations, unpublished until now, can contribute to the improvement of our knowledge about the venom and envenomation processes by T. serrulatus.

Molecular and functional characterization of metalloserrulases, new metalloproteases from the Tityus serrulatus venom gland.

Tityus serrulatus is a Brazilian scorpion species with great medical significance. While the effects of neurotoxins have been extensively studied, little is known about the proteases expressed in the venom gland of this arthropod. In this study, clones from a T. serrulatus (Ts) venom gland cDNA library were selected according to homology to proteases. The sequences were aligned in the database and classified by homology. Similarity and identity analyses of the sequences were carried out, and a phylogenetic tree was constructed with the sequences of other proteases. These cDNA sequences correspond to ten different metalloproteases, named metalloserrulases (TsMS). TsMS 1-9 belong to the metzincin family, which has three domains: signal peptide, propeptide, and metalloprotease domain; while TsMS 10 belongs to the gluzincin family. The proteolytic activity of the venom was inferred from the cleavage of fibrinogen, and the residues recognized by the proteases were determined by cleavage of a tripeptide library using a fluorescence resonance energy transfer assay. The Ts venom showed proteolytic activity on fibrinogen and preferential cleavage close to the basic residues K and R. Its activity could be inhibited by EDTA, indicating that the venom from this scorpion predominantly consists of metalloproteases.

Molecular, immunological, and biological characterization of Tityus serrulatus venom hyaluronidase: new insights into its role in envenomation.

BACKGROUND: Scorpionism is a public health problem in Brazil, and Tityus serrulatus (Ts) is primarily responsible for severe accidents. The main toxic components of Ts venom are low-molecular-weight neurotoxins; however, the venom also contains poorly characterized high-molecular-weight enzymes. Hyaluronidase is one such enzyme that has been poorly characterized. METHODS AND PRINCIPAL FINDINGS: We examined clones from a cDNA library of the Ts venom gland and described two novel isoforms of hyaluronidase, TsHyal-1 and TsHyal-2. The isoforms are 83% identical, and alignment of their predicted amino acid sequences with other hyaluronidases showed conserved residues between evolutionarily distant organisms. We performed gel filtration followed by reversed-phase chromatography to purify native hyaluronidase from Ts venom. Purified native Ts hyaluronidase was used to produce anti-hyaluronidase serum in rabbits. As little as 0.94 µl of anti-hyaluronidase serum neutralized 1 LD50 (13.2 µg) of Ts venom hyaluronidase activity in vitro. In vivo neutralization assays showed that 121.6 µl of anti-hyaluronidase serum inhibited mouse death 100%, whereas 60.8 µl and 15.2 µl of serum delayed mouse death. Inhibition of death was also achieved by using the hyaluronidase pharmacological inhibitor aristolochic acid. Addition of native Ts hyaluronidase (0.418 µg) to pre-neutralized Ts venom (13.2 µg venom+0.94 µl anti-hyaluronidase serum) reversed mouse survival. We used the SPOT method to map TsHyal-1 and TsHyal-2 epitopes. More peptides were recognized by anti-hyaluronidase serum in TsHyal-1 than in TsHyal-2. Epitopes common to both isoforms included active site residues. CONCLUSIONS: Hyaluronidase inhibition and immunoneutralization reduced the toxic effects of Ts venom. Our results have implications in scorpionism therapy and challenge the notion that only neurotoxins are important to the envenoming process.

Fibrin(ogen)olytic enzymes in scorpion (Tityus discrepans) venom.

Several fibrin(ogen)olytic enzymes from Tityus discrepans (Buthidae, Buthoidea) venom (TdV) were partially purified on a Sephadex G-50 column, by affinity and molecular exclusion high-performance chromatography. Fractions SB1-I and SB1-II had fibrinolytic, fibrinogenolytic (Aα-chains degradation) and tissue plasminogen activator (t-PA)-like activities. SB1-III was only fibrinogenolytic (fast degradation of Aα-chains and slower degradation of fibrinogen Bß-chains). These results showed the presence of α-fibrinogenases in TdV. The fibrino(geno)lytic activity in these fractions was abolished by metalloprotease inhibitors (MPI). Fractions SB3-I and SB3-II contain fibrinogenolytic (Aα-chains degradation) and fibronectinolytic activities. Also fraction SB3-I had a t-PA-like activity. Activities in SB3-I and SB3-II were abolished by serine protease inhibitors (SPI). None of the fractions degraded fibrinogen γ-chains. Fibrinogen degradation by active fractions is associated with an anticoagulant effect supported by a reduced coagulant activity. The overall outcome suggests that metalloproteases and serine proteases in TdV are responsible for fibrin(ogen)olytic activity because MPI and SPI inhibited these activities.

Antarease-like Zn-metalloproteases are ubiquitous in the venom of different scorpion genera.

BACKGROUND: The venoms of several scorpion species have long been associated with pancreatitis in animal models and humans. Antarease, a Zn-metalloprotease from Tityus serrulatus, is able to penetrate intact pancreatic tissue and disrupts the normal vesicular traffic necessary for secretion, so it could play a relevant role in the onset of acute pancreatitis. METHODS: The cDNA libraries from five different scorpion species were screened for antarease homologs with specific primers. The amplified PCR products were cloned and sequenced. A structural model was constructed to assess the functionality of the putative metalloproteases. A phylogenetic analysis was performed to identify clustering patterns of these venom components. RESULTS: Antarease-like sequences were amplified from all the screened cDNA libraries. The complete sequence of the antarease from T. serrulatus was obtained. The structural model of the putative antarease from Tityus trivittatus shows that it may adopt a catalytically active conformation, sharing relevant structural elements with previously reported metalloproteases of the ADAM family. The phylogenetic analysis reveals that the reported sequences cluster in groups that correlate with the geographical localization of the respective species. CONCLUSIONS: Antareases are ubiquitous to a broad range of scorpion species, where they could be catalytically active enzymes. These molecules can be used to describe the evolution of scorpion venoms under different ecogeographic constrains. GENERAL SIGNIFICANCE: For the first time the complete sequence of the antareases is reported. It is demonstrated that antareases are common in the venom of different scorpion species. They are now proposed as targets for antivenom therapies.

A thermoactive secreted phospholipase A2 purified from the venom glands of Scorpio maurus: relation between the kinetic properties and the hemolytic activity.

A lipolytic activity was located in the scorpion venom glands (telsons), from which a phospholipase A2 (Sm-PLVG) was purified. Like known phospholipases A2 from scorpion venom, which are 14-18 kDa proteins, the purified Scorpio maurus-Phospholipase from Venom Glands (Sm-PLVG) has a molecular mass of 17 kDa containing long and short chains linked by disulfide bridge. It has a specific activity of 5500 U/mg measured at 47 °C and pH 8.5 using phosphatidylcholine as a substrate in presence of 8 mM NaTDC and 12 mM CaCl2. The NH2-terminal amino acid sequences of the purified Sm-PLVG showed similarities with those of long and short chains of some previously purified phospholipases from venom scorpions. Moreover, the Sm-PLVG exhibits hemolytic activity toward human, rabbit or rat erythrocytes. This hemolytic activity was related to its ability to interact with phospholipids' monolayer at high surface pressure. These properties are similar to those of phospholipases isolated from snake venoms.

Anticoagulant and factor Xa-like activities of Tityus discrepans scorpion venom

Tityus discrepans venom (TdV) produces a variety of haemostatic manifestations including alveoli fbrin deposition and/ or prothrombin and partial thromboplastin time (PT, PTT) alterations in mammals. In vitro studies have demonstrated that TdV contains tissue plasminogen activator-like (t-PA), fbrinolytic and plasmin inhibitory compounds and produces platelets activation through GPVI and a novel Src-dependent signalling pathway. The aim of this study is to describe the initial characterization of procoagulant and anticoagulant components from TdV. This venom was fractionated by exclusion molecular chromatography on a Sephadex G-50 column. The eluted material was collected as fve fractions called S1 to S5. These fractions and the whole venom were used to evaluate factor Xa- and thrombin-like activities, fbrinogen degradation, furthermore thrombin- and factor Xa-inhibitory activities. The results demonstrated that TdV contain components with factor Xa-like activity (procoagulants) as well fbrinogenolytic compounds present in the fraction S1 and components with factor Xa inhibitory activity in the fractions S4 and S5 (anticoagulants).

El veneno de Tityus discrepans (TdV) produce en mamíferos una variedad de manifestaciones hemostáticas tales como depósitos de fbrina en alveolos y/o alteración en los tiempos de protrombina y tromboplastina parcial (PT, PTT). Estudios in vitro han demostrado que el TdV contiene componentes semejantes al activador del plasminógeno tipo tisular (t-PA), fbrino-líticos, compuestos que inhiben la actividad de plasmina y además componentes que promueven la activación de plaquetas a través del receptor GPVI y por una nueva vía de señalización dependiente de las Src kinasas. El objetivo de este estudio es describir la caracterización inicial de componentes procoagulantes y anticoagulantes a partir del TdV. Este veneno fue fraccionado por cromatografía de exclusión molecular sobre una columna Sephadex G-50. El material eluido fue colectado en cinco fracciones denominadas S1 a S5. Estas fracciones y el veneno completo fueron usados para evaluar actividades semejantes a factor Xa y trombina, degradación de fbrinógeno, como también la inhibición de la actividad del factor Xa y de la trombina. Los resultados demostraron que TdV contiene componentes con actividad semejante al factor Xa (procoagulantes) y compuestos fbrinogenolíticos presentes en la fracción S1, además de componentes con actividad inhibitoria del factor Xa presentes en la fracción S4 y S5 (anticoagulantes).

Heteromtoxin (HmTx), a novel heterodimeric phospholipase A(2) from Heterometrus laoticus scorpion venom.

Heteromtoxin (HmTx) is a group III phospholipase A(2) produced in Heterometrus laoticus, in Thailand. In this study, HmTx was purified from venom by separation chromatography, and the PLA(2) activity of the fractions was determined by lecithin agar assay. The enzyme is an acidic protein with a pI of 5.6 and an apparent molecular weight of 14018.4 Da. The nucleotide sequence of HmTx contains 649 bp, and the mature protein is predicted to have 131 amino acid residues-104 of which make up the large subunit, and 27 of which make up the small subunit. The subunit structure of HmTx is highly similar to that of the other toxin, Pandinus imperator imperatoxin I (IpTx(i)) and to Mesobuthus tamulus phospholipase A(2) (MtPLA(2)). The 3D-structure of HmTx consists of three conserved alpha-helices: h1 (Lys24-His34), h2 (Cys59-Asp71), and h3 (Ala80-Phe89). The beta-sheet consisted of a single stranded anti-parallel beta-sheet (b1.1 at Glu43-Lys45 and b1.2 at Lys48-Asn50) that was highly similar to the conserved sequences (-CGXG-, -CCXXHDXC- and CXCEXXXXXC-) of Apis mellifera (bee) phospholipases.

An in vitro comparative study upon the toxic properties of the venoms from Hemiscorpius lepturus, Androctonus crassicauda and Mesobuthus eupeus scorpions.

The aim of the present study was to compare the toxic effects of the venoms from Hemiscorpius lepturus (H. lepturus), Androctonus crassicauda (A. crassicauda) and Mesobuthus eupeus (M. eupeus). For this purpose, three in vitro models were employed to compare the toxic effects of various concentrations of the venoms from these three scorpions, namely: hemolytic potential using human RBCs, phospholipase activity using Saubouraud's dextrose agar (SDA) supplemented with 2% egg yolk and lactate dehydrogenase (LDH) enzyme releasing effect using K562 leukemia cell line. In addition, the neutralizing effectiveness of the antivenom against these toxic properties was assessed. The results showed that, unlike the venoms from A. crassicauda and M. eupeus, the venom from H. lepturus produced dose-dependent lysis of human RBCs and showed phospholipase activity. However, all the tested venoms showed variable degrees of LDH releasing properties. The venom from H. lepturus had highest and the venom from M. eupeus had the lowest LDH releasing effect. The antivenom effectively inhibited all the tested toxicities. In conclusion, these results suggest that the venoms from the studied scorpions have variable toxic properties, which may explain the underlying reason for the differences in their clinical manifestations. In addition, the antivenom was effective in neutralizing all the tested toxic effects.

Enzymatic analysis of Hemiscorpius lepturus scorpion venom using zymography and venom-specific antivenin.

Hemiscorpius lepturus envenomation exhibits various pathological changes in the affected tissues, including skin, blood cells, cardiovascular and central nervous systems. The enzymatic activity and protein component of the venom have not been described previously. In the present study, the electrophoretic profile of H. lepturus venom was determined by SDS-PAGE (12 and 15%), resulting in major protein bands at 3.5-5, 30-35 and 50-60 kDa. The enzymatic activities of the venom was, for the first time, investigated using various zymography techniques, which showed the gelatinolytic, caseinolytic, and hyaluronidase activities mainly at around 50-60 kDa, 30-40 kDa, and 40-50 kDa, respectively. Among these, the proteolytic activities was almost completely disappeared in the presence of a matrix metalloproteinase inhibitor, 1, 10-phenanthroline. Antigen-antibody interactions between the venom and its Iranian antivenin was observed by Western blotting, and it showed several antigenic proteins in the range of 30-160 kDa. This strong antigen-antibody reaction was also demonstrated through an enzyme-linked immunosorbent assay (ELISA). The gelatinase activity of the venom was suppressed by Razi institute polyvalent antivenin, suggesting the inhibitory effect of the antivenin against H. lepturus venom protease activities. Prudently, more extensive clinical studies are necessary for validation of its use in envenomed patients.

Vesicle-associated membrane protein (VAMP) cleavage by a new metalloprotease from the Brazilian scorpion Tityus serrulatus.

We present evidence that venom from the Brazilian scorpion Tityus serrulatus and a purified fraction selectively cleave essential SNARE proteins within exocrine pancreatic tissue. Western blotting for vesicle-associated membrane protein type v-SNARE proteins (or synaptobrevins) reveals characteristic alterations to venom-treated excised pancreatic lobules in vitro. Immunocytochemistry by electron microscopy confirms both the SNARE identity as VAMP2 and the proteolysis of VAMP2 as a marked decrease in secondary antibody-conjugated colloidal gold particles that are predominantly associated with mature zymogen granules. Studies with recombinant SNARE proteins were used to determine the specific cleavage site in VAMP2 and the susceptibility of VAMP8 (endobrevin). The VAMP2 cleavage site is between the transmembrane anchor and the SNARE motif that assembles into the ternary SNARE complex. Inclusion of divalent chelating agents (EDTA) with fraction nu, an otherwise active purified component from venom, eliminates SNARE proteolysis, suggesting the active protein is a metalloprotease. The unique cleavages of VAMP2 and VAMP8 may be linked to pancreatitis that develops following scorpion envenomation as both of these v-SNARE proteins are associated with zymogen granule membranes in pancreatic acinar cells. We have isolated antarease, a metalloprotease from fraction nu that cleaves VAMP2, and report its amino acid sequence.