Daboialipase, a phospholipase A2 from Vipera russelli russelli venom posesses anti-platelet, anti-thrombin and anti-cancer properties.

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 A2 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 PLA2 (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.

A Biochemical and Pharmacological Characterization of Phospholipase A2 and Metalloproteinase Fractions from Eastern Russell's Viper (Daboia siamensis) Venom: Two Major Components Associated with Acute Kidney Injury.

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 RvPLA2 and RvMP. Protein identification using LCMS/MS confirmed the identity of RvPLA2 to be snake venom phospholipase A2 (SVPLA2) 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. RvPLA2 (3-10 µg/mL) caused inhibition of direct twitches of the chick biventer cervicis muscle preparation. After administration of RvPLA2 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. RvPLA2 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 PLA2 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.

Synergism of in vitro plasmodicidal activity of phospholipase A2 isoforms isolated from panamanian Bothrops asper venom.

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 A2 (PLA2s) 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 PLA2 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 PLA2s, 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 PLA2s 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-PLA2-like. Acidic PLA2s catalyzed the degradation of all substrates evaluated; however, for the basic PLA2s, there was a preference for phosphatidylglycerol and phosphatidic acid. The antiparasitic potential of the toxins was evaluated, and the acidic PLA2s demonstrated action against the epimastigote forms of T. cruzi and promastigote forms of L. infantum, while the basic PLA2s 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-PLA2 and Lys49-PLA2 were associated with one another, denoting synergistic action between these PLA2 isoforms. These findings correspond to the first report of synergistic antiplasmodial action for svPLA2s, demonstrating that these molecules may be important targets in the search for new antiparasitic agents.

Bee Venom Components as Therapeutic Tools against Prostate Cancer.

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.

Bee Venom and Its Sub-Components: Characterization, Pharmacology, and Therapeutics.

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

Chikungunya virus entry is strongly inhibited by phospholipase A2 isolated from the venom of Crotalus durissus terrificus.

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 A2CB (PLA2CB), 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 PLA2CB on the CHIKV replicative cycle in BHK-21 cells using CHIKV-nanoluc, a marker virus carrying nanoluciferase reporter. The results demonstrated that PLA2CB possess a strong anti-CHIKV activity with a selectivity index of 128. We identified that PLA2CB treatment protected cells against CHIKV infection, strongly impairing virus entry by reducing adsorption and post-attachment stages. Moreover, PLA2CB presented a modest yet significant activity towards post-entry stages of CHIKV replicative cycle. Molecular docking calculations indicated that PLA2CB may interact with CHIKV glycoproteins, mainly with E1 through hydrophobic interactions. In addition, infrared spectroscopy measurements indicated interactions of PLA2CB and CHIKV glycoproteins, corroborating with data from in silico analyses. Collectively, this data demonstrated the multiple antiviral effects of PLA2CB on the CHIKV replicative cycle, and suggest that PLA2CB interacts with CHIKV glycoproteins and that this interaction blocks binding of CHIKV virions to the host cells.

Bee Venom Phospholipase A2 Ameliorates Atherosclerosis by Modulating Regulatory T Cells.

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-/-/Foxp3DTR 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.

Antibiofilm Activity of Acidic Phospholipase Isoform Isolated from Bothrops erythromelas Snake Venom.

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 A2 (PLA2), which have important antimicrobial activities. Bothropserythromelas 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 PLA2 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 (4N3OBA) substrate. In addition, hemolytic tests and antibacterial tests including action against Escherichiacoli, Staphylococcusaureus, and Acinetobacterbaumannii were carried out. Moreover, tests of antibiofilm action against A. baumannii were also performed. RESULTS: PLA2, 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 (IC50 = 30.2 µM) and antibiofilm activity against A. baumannii (IC50 = 1.1 µM) were observed. CONCLUSIONS: This is the first time that PLA2 purified from B. erythromelas venom has appeared as an alternative candidate in studies of new antibacterial medicines.

Expression, purification and biophysical characterization of recombinant Streptomyces violaceoruber phospholipase PLA2 overproduced in Pichia pastoris.

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. Ca2+ and Ba2+ ions significantly increased thermostability of the enzyme.

New approaches to old problems: Removal of phospholipase A2 results in highly active microsomal membranes from the honey bee, Apis mellifera.

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 A2 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.

Crotalus durissus ruruima Snake Venom and a Phospholipase A2 Isolated from This Venom Elicit Macrophages to Form Lipid Droplets and Synthesize Inflammatory Lipid Mediators.

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 A2 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 PGE2 biosynthesis was also assessed. Stimulation of murine macrophages with CdrV and CBr induced an increased number of LDs and release of prostanoids (PGE2, PGD2, and TXB2). 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, PGE2 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.

Venoms of Iranian Scorpions (Arachnida, Scorpiones) and Their Potential for Drug Discovery.

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.

Isolation, Biochemical Characterization and Antiparasitic Activity of BmatTX-IV, A Basic Lys49-Phospholipase A2 from the Venom of Bothrops mattogrossensis from Paraguay.

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.

The Anthelmintic Effect on Strongyloides venezuelensis Induced by BnSP- 6, a Lys49-phospholipase A2 Homologue from Bothrops pauloensis Venom.

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.

Comparison of the Protective Effects of Bee Venom Extracts with Varying PLA2 Compositions in a Mouse Model of Parkinson's Disease.

Bee venom contains a number of pharmacologically active components, including enzymes and polypeptides such as phospholipase A2 (PLA2) 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 PLA2 and melittin, among different compounds purified from honey bee venom, were evaluated against Parkinson's disease (PD). Notably, bee venom PLA2 (bvPLA2), 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 bvPLA2 treatment, depending on the PLA2 content. Further, bvPLA2 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 bvPLA2, but not melittin, protected against MPTP and alleviated inflammation in PD. Thus, bvPLA2 is a promising and effective therapeutic agent in Parkinson's disease.

Identification and characterization of phospholipases A2 from the skin secretion of Pithecopus azureus anuran.

The present work reports the isolation, characterization and the complete sequence of a phospholipase A2 (PLA2) 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 PLA2 activity was purified, assayed, sequenced and named Pa-PLA2. The Pithecopus azureus skin phospholipase A2 polypeptide chain is composed by 125 amino acid residues linked by seven disulfide bonds and two N-glycosylated sites (N67 and N108). The Pa-PLA2 enzymatic activity was qualitatively evaluated and compared to classical viperid PLA2 showing that both, native and deglycosylated Pa-PLA2 forms, are catalytically functional. The tridimensional molecular model of Pa-PLA2 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-PLA2 catalytic pocket and its substrates.

Venom Composition in a Phenotypically Variable Pit Viper ( Trimeresurus insularis) across the Lesser Sunda Archipelago.

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 A2 (PLA2), 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, PLA2 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.

Expression, purification and virucidal activity of two recombinant isoforms of phospholipase A2 from Crotalus durissus terrificus venom.

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 A2 (PLA2-CB). We showed previously the antiviral effect of PLA2-CB against dengue virus, yellow fever virus and other enveloped viruses. The aims of this study were to express two PLA2-CB isoforms in a prokaryotic system and to evaluate their virucidal effects. The sequences encoding the PLA2-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.

A prophylactic role of a secretory PLA2 of Spodoptera exigua against entomopathogens.

Phospholipase A2 (PLA2) hydrolyses phospholipids at sn-2 position to release free fatty acids and lysophospholipids. Secretory type of PLA2 (sPLA2) has been found in many different animals including insects. Insect sPLA2s have been divided into venomous and nonvenomous PLA2s. A non-venomous sPLA2 (Se-sPLA2) 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 sPLA2 activity in hemolymph remains unclear. To determine the physiological role of sPLA2 in hemolymph, a recombinant Se-sPLA2 (rSe-sPLA2) was expressed in a bacterial expression system and purified to test antimicrobial activity against various microbes. Purified rSe-sPLA2 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 sPLA2 also inhibited the enzyme activity of rSe-sPLA2 while other type PLA2 inhibitors did not. Furthermore, eight bacterial metabolites of Xenorhabdus and Photorhabdus known to be inhibitory against insect PLA2 significantly inhibited the enzyme activity of rSe-sPLA2. High concentrations of rSe-sPLA2 (above 0.5 mM) showed significant cytotoxicity to hemocytes of S. exigua. At concentrations without showing cytotoxicity, rSe-sPLA2 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-sPLA2 significantly lost such antimicrobial activities. However, the addition of rSe-sPLA2 to the hemolymph significantly rescued such antimicrobial activities. These results indicate that Se-sPLA2 possesses antimicrobial activity, suggesting that it might act as a prophylactic agent against microbial pathogens in the hemolymph of S. exigua.

A Novel Phospholipase A2 Isolated from Palythoa caribaeorum Possesses Neurotoxic Activity.

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 Ca2+-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.