Search

Snake venoms are known to contain toxins capable of interfering with normal physiological processes of victims. Specificity of toxins from snake venoms give scope to identify new molecules with therapeutic action and/or help to understand different cellular mechanisms. Russell's viper venom (RVV) is a mixture of many bioactive molecules with enzymatic and non-enzymatic proteins. The present article describes Daboialipase (DLP), an enzymatic phospholipase A 2 with molecular mass of 14.3 kDa isolated from RVV. DLP was obtained after cation exchange chromatography followed by size-exclusion high performance liquid chromatography (SE-HPLC). The isolated DLP presented strong inhibition of adenosine di-phosphate (ADP) and collagen induced platelet aggregation. It also showed anti-thrombin properties by significantly extending thrombin time in human blood samples. Trypan blue and resazurin cell viability assays confirmed time-dependent cytotoxic and cytostatic activities of DLP on MCF7 breast cancer cells, in vitro. DLP caused morphological changes and nuclear damage in MCF7 cells. However, DLP did not cause cytotoxic effects on non-cancer HaCaT cells. Peptide sequences of DLP obtained by O-HRLCMS analysis showed similarity with a previously reported PLA 2 (Uniprot ID: PA2B_DABRR/PDB ID: 1VIP_A). An active Asp at 49th position, calcium ion binding site and anticoagulant activity sites were identified in 1 VIP_A. These findings are expected to contribute to designing new anti-platelet, anticoagulant and anti-cancer molecules., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Bothrops asper is one of the most important snake species in Central America, mainly because of its medical importance in countries like Ecuador, Panama and Costa Rica, where this species causes a high number of snakebite accidents. Several basic phospholipases A 2 (PLA 2 s) have been previously characterized from B. asper venom, but few studies have been carried out with its acidic isoforms. In addition, since snake venom is a rich source of bioactive substances, it is necessary to investigate the biotechnological potential of its components. In this context, this study aimed to carry out the biochemical characterization of PLA 2 isoforms isolated from B. asper venom and to evaluate the antiparasitic potential of these toxins. The venom and key fractions were subjected to different chromatographic steps, obtaining nine PLA 2 s, four acidic ones (BaspAc-I, BaspAc-II, BaspAc-III and BaspAc-IV) and five basic ones (BaspB-I, BaspB-II, BaspB-III, BaspB-IV and BaspB-V). The isoelectric points of the acidic PLA 2 s were also determined, which presented values ranging between 4.5 and 5. The findings indicated the isolation of five unpublished isoforms, four Asp49-PLA, corresponding to the group of acidic isoforms, and one Lys49-PLA 2 -like. Acidic PLA 2 s catalyzed the degradation of all substrates evaluated; however, for the basic PLA 2 s, there was a preference for phosphatidylglycerol and phosphatidic acid. The antiparasitic potential of the toxins was evaluated, and the acidic PLA 2 s demonstrated action against the epimastigote forms of T. cruzi and promastigote forms of L. infantum, while the basic PLA 2 s BaspB-II and BaspB-IV showed activity against P. falciparum. The results indicated an increase of up to 10 times in antiplasmodial activity, when the Asp49-PLA 2 and Lys49-PLA 2 were associated with one another, denoting synergistic action between these PLA 2 isoforms. These findings correspond to the first report of synergistic antiplasmodial action for svPLA 2 s, demonstrating that these molecules may be important targets in the search for new antiparasitic agents., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Acute kidney injury (AKI) following Eastern Russell's viper ( Daboia siamensis ) envenoming is a significant symptom in systemically envenomed victims. A number of venom components have been identified as causing the nephrotoxicity which leads to AKI. However, the precise mechanism of nephrotoxicity caused by these toxins is still unclear. In the present study, we purified two proteins from D. siamensis venom, namely RvPLA 2 and RvMP. Protein identification using LCMS/MS confirmed the identity of RvPLA 2 to be snake venom phospholipase A 2 (SVPLA 2 ) from Thai D. siamensis venom, whereas RvMP exhibited the presence of a factor X activator with two subunits. In vitro and in vivo pharmacological studies demonstrated myotoxicity and histopathological changes of kidney, heart, and spleen. RvPLA 2 (3-10 µg/mL) caused inhibition of direct twitches of the chick biventer cervicis muscle preparation. After administration of RvPLA 2 or RvMP (300 µg/kg, i.p.) for 24 h, diffuse glomerular congestion and tubular injury with minor loss of brush border were detected in envenomed mice. RvPLA 2 and RvMP (300 µg/kg; i.p.) also induced congestion and tissue inflammation of heart muscle as well as diffuse congestion of mouse spleen. This study showed the significant roles of PLA 2 and SVMP in snake bite envenoming caused by Thai D. siamensis and their similarities with observed clinical manifestations in envenomed victims. This study also indicated that there is a need to reevaluate the current treatment strategies for Thai D. siamensis envenoming, given the potential for irreversible nephrotoxicity.

Prostate cancer is one of the most common cancers in men. Despite the development of a variety of therapeutic agents to treat either metastatic hormone-sensitive prostate cancer, advanced prostate cancer, or nonmetastatic/metastatic castration-resistant prostate cancer, the progression or spread of the disease often cannot be avoided. Additionally, the development of resistance of prostate cancer cells to available therapeutic agents is a well-known problem. Despite extensive and cost-intensive research over decades, curative therapy for metastatic prostate cancer is still not available. Therefore, additional therapeutic agents are still needed. The animal kingdom offers a valuable source of natural substances used for the treatment of a variety of diseases. Bee venom of the honeybee is a mixture of many components. It contains proteins acting as enzymes such as phospholipase A2, smaller proteins and peptides such as melittin and apamin, phospholipids, and physiologically active amines such as histamine, dopamine, and noradrenaline. Melittin has been shown to induce apoptosis in different cancer cell lines, including prostate cancer cell lines. It also influences cell proliferation, angiogenesis, and necrosis as well as motility, migration, metastasis, and invasion of tumour cells. Hence, it represents an interesting anticancer agent. In this review article, studies about the effect of bee venom components on prostate cancer cells are discussed. An electronic literature research was performed utilising PubMed in February 2021. All scientific publications, which examine this interesting subject, are discussed. Furthermore, the different types of application of these promising substances are outlined. The studies clearly indicate that bee venom or melittin exhibited anticancer effects in various prostate cancer cell lines and in xenografts. In most of the studies, a combination of bee venom or the modified melittin with another molecule was utilised in order to avoid side effects and, additionally, to target selectively the prostate cancer cells or the surrounding tissue. The studies showed that systemic side effects and unwanted damage to healthy tissue and organs could be minimised when the anticancer drug was not activated until binding to the cancer cells or the surrounding tissue. Different targets were used, such as the matrix metalloproteinase 2, hormone receptors expressed by prostate cancer cells, the extracellular domain of PSMA, and the fibroblast activation protein occurring in the stroma of prostate cancer cells. Another approach used loaded phosphate micelles, which were cleaved by the enzyme secretory phospholipase A2 produced by prostate cancer cells. In a totally different approach, targeted nanoparticles containing the melittin gene were used for prostate cancer gene therapy. By the targeted nonviral gene delivery, the gene encoding melittin was delivered to the prostate cancer cells without systemic side effects. This review of the scientific literature reveals totally different approaches using bee venom, melittin, modified melittin, or protoxin as anticancer agents. The toxic agents acted through several different mechanisms to produce their anti-prostate cancer effects. These mechanisms are not fully understood yet and more experimental studies are necessary to reveal the complete mode of action. Nevertheless, the researchers have conducted pioneering work. Based on these results, further experimental and clinical studies about melittin and modifications of this interesting agent deriving from nature are necessary and could possibly lead to a complementary treatment option for prostate cancer.

Chikungunya virus (CHIKV) is the etiologic agent of Chikungunya fever, a globally spreading mosquito-borne disease. There is no approved antiviral or vaccine against CHIKV, highlighting an urgent need for novel therapies. In this context, snake venom proteins have demonstrated antiviral activity against several viruses, including arboviruses which are relevant to public health. In particular, the phospholipase A2 CB (PLA2 CB ), a protein isolated from the venom of Crotalus durissus terrificus was previously shown to possess anti-inflammatory, antiparasitic, antibacterial and antiviral activities. In this study, we investigated the multiple effects of PLA2 CB on the CHIKV replicative cycle in BHK-21 cells using CHIKV-nanoluc, a marker virus carrying nanoluciferase reporter. The results demonstrated that PLA2 CB possess a strong anti-CHIKV activity with a selectivity index of 128. We identified that PLA2 CB treatment protected cells against CHIKV infection, strongly impairing virus entry by reducing adsorption and post-attachment stages. Moreover, PLA2 CB presented a modest yet significant activity towards post-entry stages of CHIKV replicative cycle. Molecular docking calculations indicated that PLA2 CB may interact with CHIKV glycoproteins, mainly with E1 through hydrophobic interactions. In addition, infrared spectroscopy measurements indicated interactions of PLA2 CB and CHIKV glycoproteins, corroborating with data from in silico analyses. Collectively, this data demonstrated the multiple antiviral effects of PLA2 CB on the CHIKV replicative cycle, and suggest that PLA2 CB interacts with CHIKV glycoproteins and that this interaction blocks binding of CHIKV virions to the host cells.

Bee venom, which is a complex substance produced by Apis mellifera , is widely used to treat various diseases, such as pain [...].

Atherosclerosis is a chronic inflammatory disease caused by lipids and calcareous accumulations in the vascular wall due to an inflammatory reaction. Recent reports have demonstrated that regulatory T (Treg) cells have an important role as a new treatment for atherosclerosis. This study suggests that bee venom phospholipase A2 (bvPLA2) may be a potential therapeutic agent in atherosclerosis by inducing Treg cells. We examined the effects of bvPLA2 on atherosclerosis using ApoE -/- and ApoE -/- /Foxp3 DTR mice. In this study, bvPLA2 increased Treg cells, followed by a decrease in lipid accumulation in the aorta and aortic valve and the formation of foam cells. Importantly, the effect of bvPLA2 was found to depend on Treg cells. This study suggests that bvPLA2 can be a potential therapeutic agent for atherosclerosis.

Introduction: Bacterial resistance is a worldwide public health problem, requiring new therapeutic options. An alternative approach to this problem is the use of animal toxins isolated from snake venom, such as phospholipases A 2 (PLA 2 ), which have important antimicrobial activities. Bothrops erythromelas is one of the snake species in the northeast of Brazil that attracts great medical-scientific interest. Here, we aimed to purify and characterize a PLA 2 from B. erythromelas , searching for heterologous activities against bacterial biofilms., Methods: Venom extraction and quantification were followed by reverse-phase high-performance liquid chromatography (RP-HPLC) in C18 column, matrix-assisted ionization time-of-flight (MALDI-ToF) mass spectrometry, and sequencing by Edman degradation. All experiments were monitored by specific activity using a 4-nitro-3-(octanoyloxy) benzoic acid (4N 3 OBA) substrate. In addition, hemolytic tests and antibacterial tests including action against Escherichia coli , Staphylococcus aureus, and Acinetobacter baumannii were carried out. Moreover, tests of antibiofilm action against A. baumannii were also performed., Results: PLA 2 , after one purification step, presented 31 N -terminal amino acid residues and a molecular weight of 13.6564 Da, with enzymatic activity confirmed in 0.06 µM concentration. Antibacterial activity against S. aureus (IC 50 = 30.2 µM) and antibiofilm activity against A. baumannii (IC 50 = 1.1 µM) were observed., Conclusions: This is the first time that PLA 2 purified from B. erythromelas venom has appeared as an alternative candidate in studies of new antibacterial medicines.

Aim: The main purpose of this work was to develop new protocols for high yield purification of secretory phospholipase A2 (PLA2) and to investigate its biophysical properties. Materials and methods: We have used a Pichia pastoris expression system for PLA2 expression and two-stage chromatography for its purification. The biophysical properties of PLA2 were investigated by circular dichroism. Results: A scalable method for high yield purification of recombinant Streptomyces violaceruber PLA2 was developed. The PLA2 from S. violaceruber was expressed in the methylotrophic yeast P. pastoris . Functional active phospholipase A2 with specific activity 73 U/mg was purified with a concentration of at least 3 mg/mL. The role of different divalent ions in PLA2 thermostability were evaluated. Ca 2+ and Ba 2+ ions significantly increased thermostability of the enzyme.

Viper snake Crotalus durissus ruruima (Cdr) is a subspecies found in northern area of Brazil. Among the snakes of Crotalus genus subspecies, the venom of Cdr presents highest level of crotoxin, which is the major component of Crotalus snake venoms, formed by two subunits (crotapotin and a phospholipase A 2 named CBr) and presents potent neurotoxic activity. Curiously, the venom of C. d. ruruima (CdrV) is better neutralized by antibothropic than by anticrotalic serum, strongly suggesting that this venom has similarities with venom of Bothrops genus snakes with regard to the ability to induce inflammation. Macrophages are cells with a central role in inflammatory and immunological responses. Upon inflammatory stimuli, these cells exhibit increased numbers of lipid droplets, which are key organelles in the synthesis and release of inflammatory mediators. However, the effects of CdrV and CBr in macrophage functions are unknown. We herein investigated the ability of CdrV and CBr to activate macrophages with focus on the formation of lipid droplets (LDs), synthesis of lipid mediators, and mechanisms involved in these effects. The involvement of LDs in PGE 2 biosynthesis was also assessed. Stimulation of murine macrophages with CdrV and CBr induced an increased number of LDs and release of prostanoids (PGE 2 , PGD 2 , and TXB 2 ). Neither CdrV nor CBr induced the expression of COX-1 and COX-2 by macrophages. LDs induced by both CdrV and CBr are associated to PLIN2 recruitment and expression and were shown to be dependent on COX-1, but not COX-2 activity. Moreover, PGE 2 colocalized to CdrV- and CBr-induced LDs, revealing the role of these organelles as sites for the synthesis of prostanoids. These results evidence, for the first time, the ability of a whole snake venom to induce formation of LDs and the potential role of these organelles for the production of inflammatory mediators during envenomation by Crotalus snakes., Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this paper., (Copyright © 2019 Ana Eduarda Zulim de Carvalho et al.)

Over the last 50 years numerous studies were published by insect toxicologists using native microsomal membrane preparations in order to investigate in vitro cytochrome P450-(P450) mediated oxidative metabolism of xenobiotics, including insecticides. Whereas the preparation of active microsomal membranes from many pest insect species is straightforward, their isolation from honey bees, Apis mellifera (Hymenoptera: Apidae) remained difficult, if not impossible, due to the presence of a yet unidentified endogenous inhibitory factor released during abdominal gut membrane isolation. Thus hampering in vitro toxicological studies on microsomal oxidative phase 1 metabolism of xenobiotics, including compounds of ecotoxicological concern. The use of microsomal membranes rather than individually expressed P450s offers advantages and allows to develop a better understanding of phase 1 driven metabolic fate of foreign compounds. Here we biochemically investigated the problems associated with the isolation of active honey bee microsomes and developed a method resulting in highly active native microsomal preparations from adult female worker abdomens. This was achieved by removal of the abdominal venom gland sting complex prior to microsomal membrane preparation. Molecular sieve chromatography of the venom sac content leads to the identification of phospholipase A 2 as the enzyme responsible for the immediate inhibition of cytochrome P450 activity in microsomal preparations. The substrate specificity of functional honey bee microsomes was investigated with different fluorogenic substrates, and revealed a strong preference for coumarin over resorufin derivatives. Furthermore we were able to demonstrate the metabolism of insecticides by honey bee microsomes using an approach coupled to LC-MS/MS analysis of hydroxylated metabolites. Our work provides access to a new and simple in vitro tool to study honey bee phase 1 metabolism of xenobiotics utilising the entire range of microsomal cytochrome P450s., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The present work reports the isolation, characterization and the complete sequence of a phospholipase A 2 (PLA 2 ) present in the skin secretion of Pithecopus azureus. Among several peptides and small proteins previously described by our group from some species belonging to this amphibian genus (formerly named Phyllomedusa), a 15 kDa N-glycosylated protein showing PLA 2 activity was purified, assayed, sequenced and named Pa-PLA 2 . The Pithecopus azureus skin phospholipase A 2 polypeptide chain is composed by 125 amino acid residues linked by seven disulfide bonds and two N-glycosylated sites (N67 and N108). The Pa-PLA 2 enzymatic activity was qualitatively evaluated and compared to classical viperid PLA 2 showing that both, native and deglycosylated Pa-PLA 2 forms, are catalytically functional. The tridimensional molecular model of Pa-PLA 2 indicates that the observed glycan moieties are suggestively placed far from the active site of that enzyme and therefore having little or no significant role on the direct interaction of the Pa-PLA 2 catalytic pocket and its substrates., (Copyright © 2019. Published by Elsevier Ltd.)

Scorpions, a characteristic group of arthropods, are among the earliest diverging arachnids, dating back almost 440 million years. One of the many interesting aspects of scorpions is that they have venom arsenals for capturing prey and defending against predators, which may play a critical role in their evolutionary success. Unfortunately, however, scorpion envenomation represents a serious health problem in several countries, including Iran. Iran is acknowledged as an area with a high richness of scorpion species and families. The diversity of the scorpion fauna in Iran is the subject of this review, in which we report a total of 78 species and subspecies in 19 genera and four families. We also list some of the toxins or genes studied from five species, including Androctonus crassicauda , Hottentotta zagrosensis, Mesobuthus phillipsi, Odontobuthus doriae, and Hemiscorpius lepturus , in the Buthidae and Hemiscorpiidae families. Lastly, we review the diverse functions of typical toxins from the Iranian scorpion species, including their medical applications.

Bee venom contains a number of pharmacologically active components, including enzymes and polypeptides such as phospholipase A 2 (PLA 2 ) and melittin, which have been shown to exhibit therapeutic benefits, mainly via attenuation of inflammation, neurotoxicity, and nociception. The individual components of bee venom may manifest distinct biological actions and therapeutic potential. In this study, the potential mechanisms of action of PLA 2 and melittin, among different compounds purified from honey bee venom, were evaluated against Parkinson's disease (PD). Notably, bee venom PLA 2 (bvPLA 2 ), but not melittin, exhibited neuroprotective activity against PD in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD. MPTP-induced behavioral deficits were also abolished after bvPLA 2 treatment, depending on the PLA 2 content. Further, bvPLA 2 administration activated regulatory T cells (Tregs) while inhibiting inflammatory T helper (Th) 1 and Th17 cells in the MPTP mouse model of PD. These results indicate that bvPLA 2 , but not melittin, protected against MPTP and alleviated inflammation in PD. Thus, bvPLA 2 is a promising and effective therapeutic agent in Parkinson's disease.

Phospholipase A 2 (PLA 2 ) hydrolyses phospholipids at sn-2 position to release free fatty acids and lysophospholipids. Secretory type of PLA 2 (sPLA 2 ) has been found in many different animals including insects. Insect sPLA 2 s have been divided into venomous and nonvenomous PLA 2 s. A non-venomous sPLA 2 (Se-sPLA 2 ) has been identified in beet armyworm, Spodoptera exigua. Its high enzyme activity is detected in hemolymph of naïve larvae. However, the physiological role of high sPLA 2 activity in hemolymph remains unclear. To determine the physiological role of sPLA 2 in hemolymph, a recombinant Se-sPLA 2 (rSe-sPLA 2 ) was expressed in a bacterial expression system and purified to test antimicrobial activity against various microbes. Purified rSe-sPLA 2 exhibited typical enzyme kinetic properties, including becoming saturated at high substrate concentrations, exhibiting optimal activity at pH 7-9, and being inactivated at high temperatures. However, a reducing agent (dithiothreitol) or calcium chelator treatment inhibited the catalytic activity. A specific inhibitor to sPLA 2 also inhibited the enzyme activity of rSe-sPLA 2 while other type PLA 2 inhibitors did not. Furthermore, eight bacterial metabolites of Xenorhabdus and Photorhabdus known to be inhibitory against insect PLA 2 significantly inhibited the enzyme activity of rSe-sPLA 2 . High concentrations of rSe-sPLA 2 (above 0.5 mM) showed significant cytotoxicity to hemocytes of S. exigua. At concentrations without showing cytotoxicity, rSe-sPLA 2 possessed significant antimicrobial activities against entomopathogenic bacteria (Serratia marscens and Entercoccus mondtii) and fungi (Beauveria bassiana and Metarhyzium rileyi). Hemolymph obtained from larvae treated with RNA interference specific to Se-sPLA 2 significantly lost such antimicrobial activities. However, the addition of rSe-sPLA 2 to the hemolymph significantly rescued such antimicrobial activities. These results indicate that Se-sPLA 2 possesses antimicrobial activity, suggesting that it might act as a prophylactic agent against microbial pathogens in the hemolymph of S. exigua., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The genus Trimeresurus comprises a group of venomous pitvipers endemic to Southeast Asia and the Pacific Islands. Of these, Trimeresurus insularis, the White-lipped Island Pitviper, is a nocturnal, arboreal species that occurs on nearly every major island of the Lesser Sunda archipelago. In the current study, venom phenotypic characteristics of T. insularis sampled from eight Lesser Sunda Islands (Flores, Lembata, Lombok, Pantar, Sumba, Sumbawa, Timor, and Wetar) were evaluated via SDS-PAGE, enzymatic activity assays, fibrinogenolytic assays, gelatin zymography, and RP-HPLC, and the Sumbawa sample was characterized by venomic analysis. For additional comparative analyses, venoms were also examined from several species in the Trimeresurus complex, including T. borneensis, T. gramineus, T. puniceus, T. purpureomaculatus, T. stejnegeri, and Protobothrops flavoviridis. Despite the geographical isolation, T. insularis venoms from all eight islands demonstrated remarkable similarities in gel electrophoretic profiles and RP-HPLC patterns, and all populations had protein bands in the mass ranges of phosphodiesterases (PDE), l-amino acid oxidases (LAAO), P-III snake venom metalloproteinases (SVMP), serine proteases, cysteine-rich secretory proteins (CRISP), phospholipases A 2 (PLA 2 ), and C-type lectins. An exception was observed in the Lombok sample, which lacked protein bands in the mass range of serine protease and CRISP. Venomic analysis of the Sumbawa venom also identified these protein families, in addition to several proteins of lesser abundance (<1%), including glutaminyl cyclase, aminopeptidase, PLA 2 inhibitor, phospholipase B, cobra venom factor, 5'-nucleotidase, vascular endothelial growth factor, and hyaluronidase. All T. insularis venoms exhibited similarities in thrombin-like and PDE activities, while significant differences were observed for LAAO, SVMP, and kallikrein-like activities, though these differences were only observed for a few islands. Slight but noticeable differences were also observed with fibrinogen and gelatin digestion activities. Trimeresurus insularis venoms exhibited overall similarity to the other Trimeresurus complex species examined, with the exception of P. flavoviridis venom, which showed the greatest overall differentiation. Western blot analysis revealed that all major T. insularis venom proteins were recognized by Green Pitviper ( T. albolabris) antivenom, and reactivity was also seen with most venom proteins of the other Trimeresurus species, but incomplete antivenom-venom recognition was observed against P. flavoviridis venom proteins. These results demonstrate significant conservation in the venom composition of T. insularis across the Lesser Sunda archipelago relative to the other Trimeresurus species examined.

The global emergence and re-emergence of arthropod-borne viruses (arboviruses) over the past four decades have become a public health crisis of international concern, especially in tropical and subtropical countries. A limited number of vaccines against arboviruses are available for use in humans; therefore, there is an urgent need to develop antiviral compounds. Snake venoms are rich sources of bioactive compounds with potential for antiviral prospection. The major component of Crotalus durissus terrificus venom is a heterodimeric complex called crotoxin, which is constituted by an inactive peptide (crotapotin) and a phospholipase A 2 (PLA 2 -CB). We showed previously the antiviral effect of PLA 2 -CB against dengue virus, yellow fever virus and other enveloped viruses. The aims of this study were to express two PLA 2 -CB isoforms in a prokaryotic system and to evaluate their virucidal effects. The sequences encoding the PLA 2 -CB isoforms were optimized and cloned into a plasmid vector (pG21a) for recombinant protein expression. The recombinant proteins were expressed in the E. coli BL21(DE3) strain as insoluble inclusion bodies; therefore, the purification was performed under denaturing conditions, using urea for protein solubilization. The solubilized proteins were applied to a nickel affinity chromatography matrix for binding. The immobilized recombinant proteins were subjected to an innovative protein refolding step, which consisted of the application of a decreasing linear gradient of urea and dithiothreitol (DTT) concentrations in combination with the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate (CHAPS) as a protein stabilizer. The refolded recombinant proteins showed phospholipase activity and virucidal effects against chikungunya virus, dengue virus, yellow fever virus and Zika virus.

Zoanthids of the genus Palythoa are distributed worldwide in shallow waters around coral reefs. Like all cnidarians, they possess nematocysts that contain a large diversity of toxins that paralyze their prey. This work was aimed at isolating and functionally characterizing a cnidarian neurotoxic phospholipase named A2-PLTX-Pcb1a for the first time. This phospholipase was isolated from the venomous extract of the zoanthid Palythoa caribaeorum . This enzyme, which is Ca 2+ -dependent, is a 149 amino acid residue protein. The analysis of the A2-PLTX-Pcb1a sequence showed neurotoxic domain similitude with other neurotoxic sPLA₂´s, but a different catalytic histidine domain. This is remarkable, since A2-PLTX-Pcb1a displays properties like those of other known PLA₂ enzymes.

Phospholipase A 2 (PLA 2 ) enzyme could be acted as a unique biomarker for forecasting and diagnosing certain diseases. Therefore, it is important to monitor PLA 2 activity in biological and clinical samples. In this work, a simple electrochemical assay for PLA 2 activity was developed based on a screen-printed carbon electrode (SPCE) with 3D graphene-like surface. When the PLA 2 -containing sample was mixed with the nanoprobes, i.e. the electroactive marker methylene blue (MB) encapsulated within nanometer-sized phospholipid liposomes, MB was released and adsorbed/enriched in site onto the surface of SPCE in a micro-cell. The encapsulation and enzymatic release of MB were evaluated using UV-Vis and fluorescence. The peak current due to oxidation of the adsorbed MB on the SPCE was measured by square-wave voltammetry (SWV). The current was directly linear to the PLA 2 activity from 5 U/L to 200 U/L with a detection limit of 3 U/L. The same method can also be used for screening PLA 2 inhibitors. Thus, the enrichment strategy developed in this work could be a promising signal amplification method for the sensitive and selective detection of PLA 2 in biological or clinical samples., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The emergence of antibiotic resistance drives an essential race against time to reveal new molecular structures capable of addressing this alarming global health problem. Snake venoms are natural catalogs of multifunctional toxins and privileged frameworks, which serve as potential templates for the inspiration of novel treatment strategies for combating antibiotic resistant bacteria. Phospholipases A 2 (PLA 2 s) are one of the main classes of antibacterial biomolecules, with recognized therapeutic value, found in these valuable secretions. Recently, a number of biomimetic oligopeptides based on small fragments of primary structure from PLA 2 toxins has emerged as a meaningful opportunity to overcome multidrug-resistant clinical isolates. Thus, this review will highlight the biochemical and structural properties of antibacterial PLA 2 s and peptides thereof, as well as their possible molecular mechanisms of action and key roles in development of effective therapeutic strategies. Chemical strategies possibly useful to convert antibacterial peptides from PLA 2 s to efficient drugs will be equally addressed., (© 2018 Wiley Periodicals, Inc.)

A mRNA transcript that codes for a phospholipase (PLA 2 ) was isolated from a single venom gland of the Bothrops ammodytoides viper. The PLA 2 transcript was cloned onto a pCR ® 2.1-TOPO vector and subsequently expressed heterologously in the E. coli strain M15, using the pQE30 vector. The recombinant phospholipase was named rBamPLA2&#95;1, and is composed of an N-terminal fusion protein of 16 residues, along with 122 residues from the mature protein that includes 14 cysteines that form 7 disulfide bonds. Following bacterial expression, rBamPLA2&#95;1 was obtained from inclusion bodies and extracted using a chaotropic agent. rBamPLA2&#95;1 had an experimental molecular mass of 15,692.5 Da that concurred with its theoretical molecular mass. rBamPLA2&#95;1 was refolded in in vitro conditions and after refolding, three main protein fractions with similar molecular masses, were identified. Although, the three fractions were considered to represent different oxidized cystine isoforms, their secondary structures were comparable. All three recombinant isoforms were active on egg-yolk phospholipid and recognized similar cell membrane phospholipids to be native PLA 2 s, isolated from B. ammodytoides venom. A mixture of the three rBamPLA2&#95;1 cystine isoforms was used to immunize a horse in order to produce serum antibodies (anti-rBamPLA2&#95;1), which partially inhibited the indirect hemolytic activity of B. ammodytoides venom. Although, anti-rBamPLA2&#95;1 antibodies were not able to recognize crotoxin, a PLA 2 from the venom of a related but different viper genus, Crotalus durissus terrificus, they recognized PLA 2 s in other venoms from regional species of Bothrops., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: Functional and structural diversity of proteins of snake venoms is coupled with a wide repertoire of pharmacological effects. Snake venoms are targets of studies linked to searching molecules with biotechnological potential., Methods: A homologue phospholipase A2 (BmatTX-IV) was obtained using two chromatographic techniques. Mass spectrometry and two-dimensional gel electrophoresis were used to determine the molecular mass and isoelectric point, respectively. By means of Edman degradation chemistry, it was possible to obtain the partial sequence of amino acids that comprise the isolated toxin. Trypanocidal, leishmanicidal and cytoxic activity against Trypanosoma cruzi, Leishmania infantum and murine fibrobasts was determinated., Results: Combination of both chromatographic steps used in this study demonstrated efficacy to obtain the PLA2-Lys49. BmatTX-IV showed molecular mass and isoelectric point of 13.55 kDa and 9.3, respectively. Amino acid sequence of N-terminal region (51 residues) shows the presence of Lys49 residue at position 49, a distinctive trait of enzymatically inactive PLA2. Bothrops mattogrossensis snake venom showed IC50 values of 11.9 μg/mL against Leishmania infantum promastigotes and of 13.8 μg/mL against Trypanosoma cruzi epimastigotes, respectively. On the other hand, the venom showed a high cytotoxic activity (IC50 value of 16.7 μg/mL) against murine fibroblasts, whereas the BmatTX-IV showed IC50 value of 81.2 μg/mL., Conclusion: Physicochemical and biological characterization of snake venoms components is critically important, since these complex mixtures provide a source of molecules with antiparasitic potential, making further studies necessary to identify and characterize components with higher efficacy and selectivity., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Background: Phospholipases A2 (PLA2) from snake venoms have a broad potential as pharmacological tools on medicine. In this context, strongyloidiasis is a neglected parasitic disease caused by helminths of the genus Strongyloides. Currently, ivermectin is the drug of choice for treatment, however, besides its notable toxicity, therapeutic failures and cases of drug resistance have been reported. BnSP-6, from Bothorps pauloensis snake venom, is a PLA2 with depth biochemical characterization, reporting effects against tumor cells and bacteria., Objective: The aim of this study is to demonstrate for the first time the action of the PLA2 on Strongyloides venezuelensis., Methods: After 72 hours of treatment with BnSP-6 mortality of the infective larvae was assessed by motility assay. Cell and parasite viability was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Furthermore, autophagic vacuoles were labeled with Monodansylcadaverine (MDC) and nuclei of apoptotic cells were labeled with Propidium Iodide (PI). Tissue degeneration of the parasite was highlighted by Transmission Electron Microscopy (TEM)., Results: The mortality index demonstrated that BnSP-6 abolishes the motility of the parasite. In addition, the MTT assay attested the cytotoxicity of BnSP-6 at lower concentrations when compared with ivermectin, while autophagic and apoptosis processes were confirmed. Moreover, the anthelmintic effect was demonstrated by tissue degeneration observed by TEM. Furthermore, we report that BnSP-6 showed low cytotoxicity on human intestinal cells (Caco-2)., Conclusion: Altogether, our results shed light on the potential of BNSP-6 as an anthelmintic agent, which can lead to further investigations as a tool for pharmaceutical discoveries., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Crotoxin (CTX), a heterodimeric phospholipase present in venom of snakes of the genus Crotalus, has demonstrated a broad spectrum of pharmacological properties, such as antimicrobial, hemostatic, and antitumoral. However, the precise mechanism of its cytotoxicity and antitumoral properties remains to be determined. Therefore, in the present study, we isolated crotoxin (F1 CTX) through two steps DEAE-Sepharose and Heparin-Sepharose FF chromatography. The C-terminal sequence of the A- and B-chain protein fragment was determined by LC-MS/MS mass spectrometry, which showed 100% identity to crotoxin structure. In order to investigate its cytotoxic effects, we demonstrated that the F1 CTX fraction at 0-30 μg/mL concentrations for 72 h presented a heterogeneous response profile on nine human cancer-derived cell lines from four tumor types (pancreatic, esophagus, cervical cancer, and glioma). The glioma (GAMG and HCB151) and pancreatic (PSN-1 and PANC-1) cancer cells showed a higher sensitivity with IC 50 of <0.5, 4.1, 0.7 and < 0.5 μg/mL, respectively. Conversely, F1 CTX does not reduce the viability of normal cells. On the other hand, cervical (SiHa) and esophagus (KYSE270) cancer cell lines presented higher resistance, with IC 50 higher than 30.2 and 8.7 μg/mL, respectively. Moreover, F1 CTX did not affect cell cycle distribution under the conditions evaluated and seems to be more cytotoxic than cytostatic. The pro-apoptotic effect of F1 CTX treatment was demonstrated in glioma (HCB151) cell line. In addition, crotoxin revealed a potential to initiate cell responses such as DNA damage in glioma (HCB151) and pancreatic cancer by H2AX activity induction. Conversely, F1 CTX does not reduce the viability of normal cells. Importantly, the comparison of F1 CTX effect with standard chemotherapeutic agents demonstrated a greater cytotoxic potential in the majority of tumor types (glioma, pancreatic, and cervical cancer). On the other hand, F1 CTX was less cytotoxic in esophageal cell lines compared to the gemcitabine agent used in clinical practice. Therefore, this work showed that F1 CTX has a cytotoxic activity and pro-apoptotic potential, contributing to the knowledge about the F1 crotoxin properties as well as its possible use in cancer research, particularly in glioma and pancreatic cancer cell lines., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

This study aimed to elucidate anticoagulant/antiplatelet mechanisms of two previously purified PLA 2 s from Cerastes cerastes venom, here, termed Cc 1 -PLA 2 and Cc 2 -PLA 2 . Both PLA 2 s present close molecular weights of 13,534 and 13,430 Da and Isoectric pH (pI) 7.38 and 7.86 respectively, for Cc 1 -PLA 2 and Cc 2 -PLA 2 . These Ca 2+ -dependent enzymes showed a high catalytic activity upon phospholipids, inducing indirect hemolysis, since they conserve the catalytic domain of PLA 2 s 26 CYCGWGGKG 34 . They exhibited dual inhibition of platelet aggregation by targeting P 2 Y 12 and TPα receptors preventing Adenosine diphosphate/arachidonate binding and blood clotting. These effects are due to the interaction of Cc 1 -PLA 2 s/Cc 2 -PLA 2 s with factor FXa through a noncatalytic PL-independent mechanism leading to nonreleased thrombin. Both proteins consist of 120 amino acid residues and share similar three-dimensional structures close to other SV-PLA 2 s. Structural data of PLA 2 s allowed the relevant residues involved in binding to FXa and platelet receptors. These findings may lead to the design of novel noncompetitive FXa inhibitors., (© 2018 Wiley Periodicals, Inc.)

Jellyfish are a type of poisonous cnidarian invertebrate that secrete lethal venom for predation or defense. Human beings often become victims of jellyfish stings accidentally while swimming or fishing and suffer severe pain, itching, swelling, inflammation, shock, and even death. Jellyfish venom is composed of various toxins, and the lethal toxin is the most toxic and hazardous component of the venom, which is responsible for deaths caused by jellyfish stings and envenomation. Our previous study revealed many toxins in jellyfish venom, including phospholipase A2, metalloproteinase, and protease inhibitors. However, it is still unknown which type of toxin is lethal and how it works. Herein a combined toxicology analysis, proteome strategy, and purification approach was employed to investigate the lethality of the venom of the jellyfish Cyanea nozakii. Toxicity analysis revealed that cardiotoxicity including acute myocardial infarction and a significant decrease in both heart rate and blood pressure is the primary cause of death. Purified lethal toxin containing a fraction of jellyfish venom was subsequently subjected to proteome analysis and bioinformation analysis. A total of 316 and 374 homologous proteins were identified, including phospholipase A2-like toxins and metalloprotease-like toxins. Furthermore, we confirmed that the lethality of the jellyfish venom is related to metalloproteinase activity but without any phospholipase A2 activity or hemolytic activity. Altogether, this study not only provides a comprehensive understanding of the lethal mechanism of jellyfish venom but also provides very useful information for the therapeutic or rescue strategy for severe jellyfish stings.

Ophidic accidents are among the problems of public health in Brazil. The components from bothropic venom are responsible for many systemic clinical complications resulting from envenomation. The present work aimed to analyse the systemic changes induced in mice after intraperitoneal administration of BmooTX-I, a myotoxic acidic phospholipase A 2 isolated from Bothrops moojeni venom. Urinalysis was performed and the following plasma biochemical markers were documented: urea, creatinine and uric acid (renal function); glucose and amylase (pancreatic function); alanine aminotransferase, alkaline phosphatase and gamma-GT (intra- and extrahepatic function); creatine kinase and enzymatic lactate (muscle function). Our results showed that after the intraperitoneal injection of BmooTX-I the urine of these animals showed glycosuria, proteinuria, haematuria, bacteriuria, bilirubinuria, polyuria and nitrite. The plasma biochemical analysis showed alterations in levels of urea, creatinine and uric acid. Amylase concentration was not altered significantly, but the plasma glucose increased significantly compared to controls. The plasma levels of alanine aminotransferase and alkaline phosphatase decreased and increased, respectively, in these same animals. On the other hand, the plasma γGT concentration did not undergo significant modification compared to the control group. The plasma concentration of CK increased, while the enzymatic lactate concentration decreased after the injection of the BmooTX-I. Therefore, in mice BmooTX-I is capable of causing systemic alterations which manifest as renal, muscular, hepatic and pancreatic impairment., (© 2018 The Authors. International Journal of Experimental Pathology © 2018 International Journal of Experimental Pathology.)

The Asian world is home to a multitude of venomous and dangerous snakes, which are used to induce various medical effects in the preparation of traditional snake tinctures and alcoholics, like the Japanese snake wine, named Habushu. The aim of this work was to perform the first quantitative proteomic analysis of the Protobothrops flavoviridis pit viper venom. Accordingly, the venom was analyzed by complimentary bottom-up and top-down mass spectrometry techniques. The mass spectrometry-based snake venomics approach revealed that more than half of the venom is composed of different phospholipases A2 (PLA₂). The combination of this approach and an intact mass profiling led to the identification of the three main Habu PLA₂s. Furthermore, nearly one-third of the total venom consists of snake venom metalloproteinases and disintegrins, and several minor represented toxin families were detected: C-type lectin-like proteins (CTL), cysteine-rich secretory proteins (CRISP), snake venom serine proteases (svSP), l-amino acid oxidases (LAAO), phosphodiesterase (PDE) and 5'-nucleotidase. Finally, the venom of P. flavoviridis contains certain bradykinin-potentiating peptides and related peptides, like the svMP inhibitors, pEKW, pEQW, pEEW and pENW. In preliminary MTT cytotoxicity assays, the highest cancerous-cytotoxicity of crude venom was measured against human neuroblastoma SH-SY5Y cells and shows disintegrin-like effects in some fractions.

Bothrops cotiara is a pitviper found in Southeastern Brazil and, scarcely, in the Misiones province of Argentina. In contrast to considerable information available on the venom of the Brazilian snake population, that of Misiones has received little attention. While exploring the chromatographic venom profile of Argentinean B. cotiara, a major protein peak was found which, according to a previous study, is not present in the venom of Brazilian origin. The corresponding protein was isolated by RP-HPLC, and characterized by electrophoresis, mass spectrometry, phospholipase A 2 (PLA 2 ) assay, and myotoxic activities. Representing nearly 15% of B. cotiara venom from Misiones, this protein was identified as a Lys49 PLA 2 homologue. In accordance with the characteristics of this toxin family, the protein induced myotoxicity in mice and was devoid of PLA 2 activity. Since previous work reported that no PLA 2 or PLA 2 -homologues occur in B. cotiara venom of Brazilian origin, the presence of an abundant Lys49 PLA 2 homologue in the venom from Misiones highlights a striking phenotypic variation in toxin expression within two populations of a single snake species inhabiting different geographic areas. The considerable proportion of B. cotiara Lys49 PLA 2 homologue myotoxin in the venom alerts that skeletal muscle necrosis might be a potentially relevant consequence of eventual envenomings by this species in Misiones., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Snake venom phospholipases A 2 (PLA 2 s) are responsible for numerous pathophysiological effects in snakebites; however, their biochemical properties favour antimicrobial actions against different pathogens, thus constituting a true source of potential microbicidal agents. This study describes the isolation of a Lys49 PLA 2 homologue from Lachesis muta muta venom using two chromatographic steps: size exclusion and reverse phase. The protein showed a molecular mass of 13,889 Da and was devoid of phospholipase activity on an artificial substrate. The primary structure made it possible to identify an unpublished protein from L. m. muta venom, named LmutTX, that presented high identity with other Lys49 PLA 2 s from bothropic venoms. Synthetic peptides designed from LmutTX were evaluated for their cytotoxic and antimicrobial activities. LmutTX was cytotoxic against C2C12 myotubes at concentrations of at least 200 μg/mL, whereas the peptides showed a low cytolytic effect. LmutTX showed antibacterial activity against Gram-positive and Gram-negative bacteria; however, S. aureusATCC 29213 and MRSA strains were more sensitive to the toxin's action. Synthetic peptides were tested on S. aureus, MRSA and P. aeruginosaATCC 27853 strains, showing promising results. This study describes for the first time the isolation of a Lys49 PLA 2 from Lachesis snake venom and shows that peptides from specific regions of the sequence may constitute new sources of molecules with biotechnological potential., (© 2017 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).)

Aggregation of recombinant proteins into inclusion bodies (IBs) is the major drawback of heterologous expression in Escherichia coli. Here, we evaluated the effects of a pH shift after expression induction on recombinant phospholipase A2 production and its aggregation in IBs in E. coli Origami™, as compared to cultures with pH maintained at 7.5 or uncontrolled pH. Cultures shifted from 7.5 to pH 6.5 or 8.5 produced ∼15-25% less biomass as compared with those kept at 7.5 or without pH control. The cultures shifted to pH 8.5 showed a ∼50% higher yield of acetate per biomass, and the rPLA2 yield was improved 2.4-fold. Purified IBs formed at pH 8.5 containing ∼50% of rPLA2, were more susceptible to proteinase-K cleavage and bound less thioflavin-T, indicating lower amyloid content, with the concomitant enrichment of α-helical and random-coil secondary structures, as demonstrated by FTIR. Moreover, only one IB per cell was formed at pH 8.5; instead, more than two were observed under the other culture pH conditions. Nevertheless, under uncontrolled pH conditions, ∼300nm larger IBs were observed. Our work presents evidence of the usefulness of recombinant protein expression cultivated at pH 8.5 allowing the reduction of amyloid content in IBs., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Phospholipases A 2 (PLA 2 s) are important enzymes present in snake venoms and are related to a wide spectrum of pharmacological effects, however the toxic potential and therapeutic effects of acidic isoforms have not been fully explored and understood. Due to this, the present study describes the isolation and biochemical characterization of two new acidic Asp49-PLA 2 s from Bothrops brazili snake venom, named Braziliase-I and Braziliase-II. The venom was fractionated in three chromatographic steps: ion exchange, hydrophobic interaction and reversed phase. The isoelectric point (pI) of the isolated PLA 2 s was determined by two-dimensional electrophoresis, and 5.2 and 5.3 pIs for Braziliase-I and II were observed, respectively. The molecular mass was determined with values ​​of 13,894 and 13,869Da for Braziliase-I and II, respectively. Amino acid sequence by Edman degradation and mass spectrometry completed 87% and 74% of the sequences, respectively for Braziliase-I and II. Molecular modeling of isolated PLA 2 s using acid PLA 2 BthA-I-PLA 2 from B. jararacussu template showed high quality. Both acidic PLA 2 s showed no significant myotoxic activity, however they induced significant oedematogenic activity. Braziliase-I and II (100μg/mL) showed 31.5% and 33.2% of cytotoxicity on Trypanosoma cruzi and 26.2% and 19.2% on Leishmania infantum, respectively. Braziliase-I and II (10μg) inhibited 96.98% and 87.98% of platelet aggregation induced by ADP and 66.94% and 49% induced by collagen, respectively. The acidic PLA 2 s biochemical and structural characterization can lead to a better understanding of its pharmacological effects and functional roles in snakebites pathophysiology, as well as its possible biotechnological applications as research probes and drug leads., (Copyright © 2017 Elsevier B.V. All rights reserved.)

In this study, we have produced for the first time a fish phospholipase (PLA 2 ) in heterologous system (E. coli). The Diplodus annularis PLA 2 (DaPLA 2 ) was then refolded from inclusion bodies and purified by Ni-affinity chromatography. We used the pH-stat method (with emulsified phosphatidylcholine as substrate) and the monomolecular film technique (using various glycerophospholipids substrates spread in the form of monomolecular films at the air-water interface) to access the biochemical and kinetic properties of the recombinant DaPLA 2 . The DaPLA 2 was found to be active and stable at higher temperatures (37-50 °C) than expected. Interestingly, DaPLA 2 hydrolyzes efficiently both purified phosphatidylglycerol and phosphatidylethanolamine at 20 mN/m. These analytical results corroborate with the fact that the catalytic activity of DaPLA 2 , measured with the pH-stat using egg yolk as substrate, is mainly due to the hydrolysis of the PE fraction present in egg yolk, whereas the phosphatidylglycerol is a hallmark substrate for the most secreted PLA 2 -IB.

Phospholipase A 2 -like (PLA 2 -like) proteins contribute to the development of muscle necrosis in Viperidae snake bites and are not efficiently neutralized by current antivenom treatments. The toxic mechanisms of PLA 2 -like proteins are devoid of catalytic activity and not yet fully understood, although structural and functional experiments suggest a dimeric assembly and that the C-terminal residues are essential to myotoxicity. Herein, we characterized the functional mechanism of bothropic PLA 2 -like structures related to global and local measurements using the available models in the Protein Data Bank and normal mode molecular dynamics (NM-MD). Those measurements include: (i) new geometric descriptions between their monomers, based on Euler angles; (ii) characterizations of canonical and non-canonical conformations of the C-terminal residues; (iii) accessibility of the hydrophobic channel; (iv) inspection of ligands; and (v) distance of clustered residues to toxin interface of interaction. Thus, we described the allosteric activation of PLA 2 -like proteins and hypothesized that the natural movement between monomers, calculated from NM-MD, is related to their membrane disruption mechanism, which is important for future studies of the inhibition process. These methods and strategies can be applied to other proteins to help understand their mechanisms of action.

Myotoxic phospholipases A₂ (PLA₂) are responsible for many clinical manifestations in envenomation by Bothrops snakes. A new myotoxic acidic Asp49 PLA₂ (BaCol PLA₂) was isolated from Colombian Bothrops asper venom using reverse-phase high performance liquid chromatography (RP-HPLC). BaCol PLA₂ had a molecular mass of 14,180.69 Da (by mass spectrometry) and an isoelectric point of 4.4. The complete amino acid sequence was obtained by cDNA cloning (GenBank accession No. MF319968) and revealed a mature product of 124 amino acids with Asp at position 49. BaCol PLA₂ showed structural homology with other acidic PLA₂ isolated from Bothrops venoms, including a non-myotoxic PLA₂ from Costa Rican B. asper . In vitro studies showed cell membrane damage without exposure of phosphatidylserine, an early apoptosis hallmark. BaCol PLA₂ had high indirect hemolytic activity and moderate anticoagulant action. In mice, BaCol PLA₂ caused marked edema and myotoxicity, the latter seen as an increase in plasma creatine kinase and histological damage to gastrocnemius muscle fibers that included vacuolization and hyalinization necrosis of the sarcoplasm., Competing Interests: The authors declare no potential conflicts of interest regarding this manuscript. The founding sponsors had no role in the design of the study, in the collection, analysis or interpretation of the data, in the writing of the manuscript, or in the decision to publish the results.

Two phospholipase A 2 s (PLA 2 s), Asp49 (D49) and Lys49 (K49), were purified by one-step reverse-phase high performance liquid chromatography (RP-HPLC) from the venom of each of the three subspecies of cottonmouth snake, Western cottonmouth (Agkistrodon piscivorus leucostoma; Apl), Eastern cottonmouth (Agkistrodon piscivorus piscivorus; App) and Florida cottonmouth (Agkistrodon piscivorus conanti; Apc). Venom protein profiles and PLA 2 s elution pattern of the three cottonmouth snakes were remarkably similar displaying four similar sharp and two wide peaks; in all cases K49 PLA 2 eluted first followed by D49 PLA 2 . The yields of K49 and D49 PLA 2 s were, respectively, 13.2 and 17.5 mg/g venom from the Western cottonmouth, 16.8 and 19.2 mg/g from the Eastern cottonmouth, and 17.3 and 22.7 mg/g from the Florida cottonmouth. Biochemical and enzymatic techniques were used to characterize the purified PLA 2 . The amino acid sequences of all three K49PLA 2 s were identical; App-D49 and Apc-D49 were also identical but displayed a single amino acid difference with Apl-D49. To our knowledge, this is the first report on the amino acid sequence and molecular mass of Apc-D49 and Apc-K49 PLA 2 s from Florida cottonmouth venom. As expected, PLA 2 enzymatic analysis revealed that D49 PLA 2 s from all three venoms hydrolyze phospholipids to a similar extent, whereas no phospholipid hydrolysis was detectable by any of the K49 PLA 2 s purified. Cottonmouth snake venoms contain abundant PLA 2 isoforms, thus the identification of PLA 2 s in these venoms is of interest to facilitate the purification of specific PLA 2 from rich sources of subspecies venom for future biological and biomedical research. Results of this study also contribute towards the understanding of venom protein profiles, variation, and evolution in subspecies of venomous snakes., (Published by Elsevier Ltd.)

Bothrops fonsecai (B. fonsecai), a pitviper endemic to southeastern Brazil, has a venom mainly composed by snake venom phospholipases (PLA 2 ) and metalloproteases, compounds that could interfere with neuromuscular junction in vitro. In this work, we investigated the role of PLA 2 in the myotoxicity and neuromuscular blockade caused by B. fonsecai venom using different procedures frequently associated with PLA 2 activity inhibition: 24 °C bath temperature, Ca 2+ - Sr 2+ replacement and chemical modification with p-bromophenacyl bromide (p-BPB). Mice extensor digitorum longus preparations (EDL) were incubated with usual or modified Tyrode solution (prepared with Ca 2+ or Sr 2+ respectively) at 24 °C or 37 °C (as controls) and in addition of B. fonsecai venom (100 μg/mL) alone or after its incubation with buffer (24 h, 23 °C) on the absence (alkylation control) and presence of p-BPB; all muscle were processed for histological analysis. The PLA 2 , proteolytic and amidolytic activities under the same conditions (24 °C or 37 °C, Ca 2+ - Sr 2+ replacement, absence or presence p-BPB) were also assessed. The B. fonsecai venom caused total neuromuscular blockade after 100 min of incubation, in Ca 2+ Tyrode solution at 37 °C (usual conditions); on Sr 2+ Tyrode solution (37 °C) the twitch height were 31.7 ± 7.4% of basal, and at 24 °C (Ca 2+ Tyrode solution) were 53.6 ± 7.0% of basal. The alkylation of PLA 2 with p-BPB promoted a great blockade decrease at 100 min of incubation (88.7 ± 5.7% of basal), but it was also observed on alkylation control preparations (66.2 ± 6.6%). The venom produced 50% of blockade at 40.5 ± 5.9 min, in Ca 2+ Tyrode solution at 37 °C. The protocols delayed the time for 50% blockade: 105.7 ± 7.1 min (at 24 °C, in Ca 2+ Tyrode solution) and 71.1 ± 9.0 min (at 37 °C, in Sr 2+ Tyrode solution). Regarding p-BPB incubation and alkylation control preparations, 50% of blockade was not reached during the 120 min of venom incubation. Regarding to enzymatic activities, the 24 °C protocol reduced not only PLA 2 (to 62.3%) but also proteolytic (52.3%) and amidolytic (73.4%) activities, as well as observed on p-BPB alkylation protocol which markedly inhibited all enzymes (<10%). The alkylation control promoted the same proteolytic and amidolytic inhibition but no reduction of PLA 2 activity; Ca 2+ - Sr 2+ replacement reduced only the PLA 2 activity (to 15.3%). We observed a strict relation between the inhibition of PLA 2 activity and the myotoxicity. On the other hand, this relation was not observed with neuromuscular blockade, suggesting that blockade and muscle damage may not be strictly related. It suggests that the neuromuscular blockade may be induced by non-catalytic PLA 2 or other venom components, such as metalloproteinases., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Novel phospholipase (PLA 2 ) genes from the Sparidae family were cloned. The sequenced PLA 2 revealed an identity with pancreatic PLA 2 group IB. To better understand the structure/function relationships of these enzymes and their evolution, the Diplodus annularis PLA 2 (DaPLA 2 ) was overexpressed in E. coli. The refolded enzyme was purified by Ni-affinity chromatography and has a molecular mass of 15 kDa as determined by MALDI-TOF spectrometry. Interestingly, unlike the pancreatic type, the DaPLA 2 was active and stable at higher temperatures, which suggests its great potential in biotechnological applications. The 3D structure of DaPLA 2 was constructed to gain insights into the functional properties of sparidae PLA 2 . Molecular docking and dynamic simulations were performed to explain the higher thermal stability and the substrate specificity of DaPLA 2 . Using the monolayer technique, the purified DaPLA 2 was found to be active on various phospholipids ranging from 10 to 20 mN·m -1 , which explained the absence of the hemolytic activity for DaPLA 2 .

Tropidolaemus wagleri (temple pit viper) is a medically important snake in Southeast Asia. It displays distinct sexual dimorphism and prey specificity, however its venomics and inter-sex venom variation have not been thoroughly investigated. Applying reverse-phase HPLC, we demonstrated that the venom profiles were not significantly affected by sex and geographical locality (Peninsular Malaya, insular Penang, insular Sumatra) of the snakes. Essentially, venoms of both sexes share comparable intravenous median lethal dose (LD 50 ) (0.56-0.63 μg/g) and cause neurotoxic envenomation in mice. LCMS/MS identified six waglerin forms as the predominant lethal principles, comprising 38.2% of total venom proteins. Fourteen other toxin-protein families identified include phospholipase A 2 , serine proteinase, snaclec and metalloproteinase. In mice, HPLC fractions containing these proteins showed insignificant contribution to the overall venom lethality. Besides, the unique elution pattern of approximately 34.5% of non-lethal, low molecular mass proteins (3-5 kDa) on HPLC could be potential biomarker for this primitive crotalid species. Together, the study unveiled the venom proteome of T. wagleri that is atypical among many pit vipers as it comprises abundant neurotoxic peptides (waglerins) but little hemotoxic proteinases. The findings also revealed that the venom is relatively well conserved intraspecifically despite the drastic morphological differences between sexes.

Venoms are integrated phenotypes that evolved independently in, and are used for predatory and defensive purposes by, a wide phylogenetic range of organisms. The same principles that contribute to the evolutionary success of venoms, contribute to making the study of venoms of great interest in such diverse fields as evolutionary ecology and biotechnology. Evolution is profoundly contingent, and nature also reinvents itself continuosly. Changes in a complex phenotypic trait, such as venom, reflect the influences of prior evolutionary history, chance events, and selection. Reconstructing the natural history of venoms, particularly those of snakes, which will be dealt with in more detail in this review, requires the integration of different levels of knowledge into a meaningful and comprehensive evolutionary framework for separating stochastic changes from adaptive evolution. The application of omics technologies and other disciplines have contributed to a qualitative and quantitative advance in the road map towards this goal. In this review we will make a foray into the world of animal venoms, discuss synergies and complementarities of the different approaches used in their study, and identify current bottlenecks that prevent inferring the evolutionary mechanisms and ecological constraints that molded snake venoms to their present-day variability landscape., (© 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.)

Atopic dermatitis (AD) is a biphasic inflammatory skin disease that is provoked by epidermal barrier defects, immune dysregulation, and increased skin infections. Previously, we have demonstrated that bvPLA2 evoked immune tolerance by inducing regulatory T cells (Treg), and thus alleviated Th2 dominant allergic asthma in mice. Here, we would like to determine whether treatment with bvPLA2 exacerbates the AD-like allergic inflammations induced by house dust mite extract (DFE) in a murine model. Epidermal thickness, immune cell infiltration, serum immunoglobulin, and cytokines were measured. Ear swelling, skin lesions, and the levels of total serum IgE and Th1/Th2 cytokines were elevated in DFE/DNCB-induced AD mice. Topical application of bvPLA2 elicited significant suppression of the increased AD symptoms, including ear thickness, serum IgE concentration, inflammatory cytokines, and histological changes. Furthermore, bvPLA2 treatment inhibited mast cell infiltration into the ear. On the other hand, Treg cell depletion abolished the anti-atopic effects of bvPLA2, suggesting that the effects of bvPLA2 depend on the existence of Tregs. Taken together, the results revealed that topical exposure to bvPLA2 aggravated atopic skin inflammation, suggesting that bvPLA2 might be a candidate for the treatment of AD.

The proteome composition of western India (WI) Russell's viper venom (RVV) was correlated with pharmacological properties and pathological manifestations of RV envenomation. Proteins in the 5-19 and 100-110 kDa mass ranges were the most predominate (∼35.1%) and least abundant (∼3.4%) components, respectively, of WI RVV. Non-reduced SDS-PAGE indicated the occurrence of multiple subunits, non-covalent oligomers, self-aggregation, and/or interactions among the RVV proteins. A total of 55 proteins belonging to 13 distinct snake venom families were unambiguously identified by ESI-LC-MS/MS analysis. Phospholipase A 2 (32.5%) and Kunitz-type serine protease inhibitors (12.5%) represented the most abundant enzymatic and non-enzymatic proteins, respectively. However, ATPase, ADPase, and hyaluronidase, detected by enzyme assays, were not identified by proteomic analysis owing to limitations in protein database deposition. Several biochemical and pharmacological properties of WI RVV were also investigated. Neurological symptoms exhibited by some RV-bite patients in WI may be correlated to the presence of neurotoxic phospholipase A 2 enzymes and Kunitz-type serine protease inhibitor complex in this venom. Monovalent antivenom was found to be better than polyvalent antivenom in immuno-recognition and neutralization of the tested pharmacological properties and enzyme activities of WI RVV; nevertheless, both antivenoms demonstrated poor cross-reactivity and neutralization of pharmacological activities shown by low-molecular-mass proteins (<18 kDa) of this venom.

In 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 sPLA 2 , 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., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Jellyfish envenomations are emerging as an important public health concern occurred worldwide. In China, the situation is getting worse with numerous people stung by jellyfish Nemopilema nomurai (N. nomurai) and Cyanea nozakii (C. nozakii) in the summer. However, the proteinaceous mixtures in nematocysts responsible for the symptoms of jellyfish stings were scarcely characterized and understood in view of enzymatic constituents and toxicity. In the present study, enzymatic properties of jellyfish N. nomurai and C. nozakii nematocyst venom were analyzed biochemically and kinetically. The current data revealed that N. nomurai and C. nozakii nematocyst venom exhibited various enzymatic activities, of which metalloproteinases activity and PLA 2 s-like activity were predominant. Moreover, the catalytic activities of metalloproteinases and PLA 2 s-like were dependent on different physiochemical conditions such as temperature, pH and divalent ions. Kinetic profiling revealed their catalytic behaviors fitted the Michaelis-Menten equation under specific conditions. Findings suggested jellyfish nematocyst venom possessed diverse enzymatic constituents, which may underlie the extensively characterized bioactivities of jellyfish venom and human envenomations. Hence, our study will contribute to understanding the enzymatic constituents and toxicity of jellyfish nematocyst venom and may afford potential therapeutic targets for developing drugs for jellyfish stings., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Snake venom is a rich source of peptides and proteins with a wide range of actions. Many of the venom components are currently being tested for their usefulness in the treatment of many diseases ranging from neurological and cardiovascular to cancer. It is also important to constantly search for new proteins and peptides with properties not yet described. The venom of Vipera berus berus has hemolytic, proteolytic and cytotoxic properties, but its exact composition and the factors responsible for these properties are not known. Therefore, an attempt was made to identify proteins and peptides derived from this species venom by using high resolution two-dimensional electrophoresis and MALDI ToF/ToF mass spectrometry. A total of 11 protein classes have been identified mainly proteases but also l-amino acid oxidases, C-type lectin like proteins, cysteine-rich venom proteins and phospholipases A₂ and 4 peptides of molecular weight less than 1500 Da. Most of the identified proteins are responsible for the highly hemotoxic properties of the venom. Presence of venom phospholipases A₂ and l-amino acid oxidases cause moderate neuro-, myo- and cytotoxicity. All successfully identified peptides belong to the bradykinin-potentiating peptides family. The mass spectrometry data are available via ProteomeXchange with identifier PXD004958.

The venom of Micrurus lemniscatus, a coral snake of wide geographical distribution in South America, was fractionated by reverse-phase HPLC and the fractions screened for phospholipase A2 (PLA2) activity. The major component of the venom, a PLA2, here referred to as 'Lemnitoxin', was isolated and characterized biochemically and toxicologically. It induces myotoxicity upon intramuscular or intravenous injection into mice. The amino acid residues Arg15, Ala100, Asn108, and a hydrophobic residue at position 109, which are characteristic of myotoxic class I phospholipases A2, are present in Lemnitoxin. This PLA2 is antigenically related to M. nigrocinctus nigroxin, Notechis scutatus notexin, Pseudechis australis mulgotoxin, and Pseudonaja textilis textilotoxin, as demonstrated with monoclonal and polyclonal antibodies. Lemnitoxin is highly selective in its targeting of cells, being cytotoxic for differentiated myotubes in vitro and muscle fibers in vivo, but not for undifferentiated myoblasts or endothelial cells. Lemnitoxin is not lethal after intravenous injection at doses up to 2μg/g in mice, evidencing its lack of significant neurotoxicity. Lemnitoxin displays anticoagulant effect on human plasma and proinflammatory activity also, as it induces paw edema and mast cell degranulation. Thus, the results of this work demonstrate that Lemnitoxin is a potent myotoxic and proinflammatory class I PLA2., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

The present study investigated the kinetic and interfacial properties of two secreted phospholipases isolated from Tunisian vipers'venoms: Cerastes cerastes (CC-PLA2) and Macrovipera lebetina transmediterranea (MVL-PLA2). Results show that these enzymes have great different abilities to bind and hydrolyse phospholipids. Using egg-yolk emulsions as substrate at pH 8, we found that MVL-PLA2 has a specific activity of 1473U/mg at 37°C in presence of 1mM CaCl2. Furthermore the interfacial kinetic and binding data indicate that MVL-PLA2 has a preference to the zwitterionic phosphatidylcholine monolayers (PC). Conversely, CC-PLA2 was found to be able to hydrolyse preferentially negatively charged head group phospholipids (PG and PS) and exhibits a specific activity 9 times more important (13333U/mg at 60°C in presence of 3mM CaCl2). Molecular models of both CC-PLA2 and MVL-PLA2 3D structures have been built and their electrostatic potentials surfaces have been calculated. A marked anisotropy of the overall electrostatic charge distribution leads to a significantly difference in the dipole moment intensity between the two enzymes explaining the great differences in catalytic and binding properties, which seems to be governed by the electrostatic and hydrophobic forces operative at the surface of the two phospholipases., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Phospholipase A2 (PLA2) and protease (P) are enzymes responsible of myotoxic, edematogenic and hemostasis disorder effects observed in the envenomation by Bothrops alternatus pitviper. Their partitioning coefficient (Kp) in different polyethyleneglycol/potassium phosphate aqueous two-phase systems (ATPSs) was determined in order to both achieve a better understanding of the partitioning mechanism and define optimal conditions for toxin isolation. Polyethyleneglycols (PEGs) of molecular weights 1000; 3350; 6000 and 8000; different temperatures (5, 20 and 37 °C) and phase volume ratios of 0.5; 1 and 2 were assayed. PLA2 partitioned preferentially to the top phase while P mainly distributed to the bottom phase. Either entropically- or enthalpically-driven mechanisms were involved in each case (PLA2 and P). The aqueous two-phase system formed by PEG of MW 3350 (12.20% wt/wt) and KPi pH 7.0 (11.82% wt/wt) with a volume ratio of one and a load of 1.25 mg of venom/g of system showed to be the most efficient to recover both enzymes. It allowed obtaining the 72% of PLA2 in the top phase with a purification factor of 2 and the 82% of P at the bottom phase simultaneously. A further adsorption batch step with DEAE-cellulose was used to remove satisfactorily the PEG from the top phase and recover the active PLA2. The proposed methodology is simple, inexpensive, and only requires professionals trained in handling basic laboratory equipment. It could be easily adoptable by developing countries in which the snakebite accidents cause considerable morbidity and mortality., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Commonly, phospholipases A2 (PLA2s) play key roles in the pathogenesis of the local tissue damage characteristic of crotaline and viperine snake envenomations. Crotalus oreganus lutosus snake venom has not been extensively studied; therefore, the characterization of its components represents a valuable biotechnological tool for studying pathophysiological processes of envenoming and for gaining a deeper understanding of its biological effects. In this study, for the first time, a basic PLA2 myotoxin, ColTx-I, was purified from C. o. lutosus through two chromatographic steps. ColTx-I is monomeric with calculated molecular mass weight (Mw) of 14,145 Da and a primary structure closely related to basic PLA2s from viperid venoms. The pure enzyme has a specific activity of 15.87 ± 0.65 nmol/min/mg at optimal conditions (pH 8.0 and 37 °C). ColTx-I activity was found to be dependent on Ca(2+), as its substitution by other ionic species as well as the addition of chelating agents significantly reduced its phospholipase activity. In vivo, ColTx-I triggered dose-dependent inflammatory responses, measured using the paw edema model, with an increase in IL-6 levels, systemic and local myotoxicity, characterized by elevated plasma creatine kinase activity. ColTx-I induced a complex series of degenerative events associated with edema, inflammatory infiltrate and skeletal muscle necrosis. These biochemical and functional results suggest that ColTx-I, a myotoxic and inflammatory mediator, plays a relevant role in C. o. lutosus envenomation. Thus, detailed studies on its mechanism of action, such as evaluating the synergism between ColTx-I and other venom components may reveal targets for the development of more specific and effective therapies., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

We showed that bee venom phospholipase A2 can be used as a membrane-binding vector to anchor to the surface of cells a soluble protein fused to its C-terminus. ZZ, a two-domain derivative of staphylococcal protein A capable of binding constant regions of antibodies was fused to the C-terminus of the phospholipase or to a mutant devoid of enzymatic activity. The fusion proteins bound to the surface of cells and could themselves bind IgGs. Their fate depended on the cell type to which they bound. On the A431 carcinoma cell line the proteins remained exposed on the cell surface. In contrast, on human dendritic cells the proteins were internalized into early endosomes., (Copyright © 2015 Elsevier Ltd. All rights reserved.)