Snake Phospholipase Research Articles

PLA2 Inhibitor Varespladib as an Alternative to the Antivenom Treatment for Bites from Nikolsky's Viper Vipera berus nikolskii.

Although envenoming by a small East European species of viper is rarely severe, and only exceptionally fatal, lack of specific antivenom stocks in a few areas within this region and possible severe side effects of antivenom application leave most bites to be treated only with antihistamines and supportive therapy. Varespladib is an effective inhibitor of snake phospholipase, and, as such, it could be considered as first-line therapy. The Nikolsky’s viper venom contains an extremely high concentration of phospholipase A2 (PLA2), responsible for the toxic effects of the venom, as well as minor amounts of other toxins. If Varespladib can successfully inhibit PLA2 activity, the Nikolsky’s viper could be one of the first venomous snakes having an antitoxin-specific treatment regimen. To assess that, Varespladib was administered alone subcutaneously to adult male CD-1 mice (8 mg/kg) and compared to mice exposed to Vipera berus nikolskii crude venom (8 mg/kg = 10 LD50) or a combination of Varespladib and the same amount of the venom. Experimental animals were monitored for the presence of envenoming symptoms and mortality for 48 h after injection. Eighty percent of mice receiving both Varespladib and venom survived, while 100% of the control group receiving venom alone died within 4 h. Experimental results are consistent with Varespladib acting as an effective antitoxin in the mouse model against Nikolsky’s viper venom. Further studies are needed under experimental conditions that more closely resemble natural envenoming (i.e., delayed administration).

Neurotoxicity of coral snake phospholipases A2 in cultured rat hippocampal neurons

The neurotoxicity of two secreted Phospholipases A2 from Brazilian coral snake venom in rat primary hippocampal cell culture was investigated. Following exposure to Mlx-8 or Mlx-9 toxins, an increase in free cytosolic Ca2+ and a reduction in mitochondrial transmembrane potential (ΔΨm) became evident and occurred prior to the morphological changes and cytotoxicity. Exposure of hippocampal neurons to Mlx-8 or Mlx-9 caused a decrease in the cell viability as assessed by MTT and LDH assays. Inspection using fluorescent images and ultrastructural analysis by scanning and transmission electron microscopy showed that multiphase injury is characterized by overlapping cell death phenotypes. Shrinkage, membrane blebbing, chromatin condensation, nucleosomal DNA fragmentation and the formation of apoptotic bodies were observed. The most striking alteration observed in the electron microscopy was the fragmentation and rarefaction of the neuron processes network. Degenerated terminal synapses, cell debris and apoptotic bodies were observed among the fragmented fibers. Numerous large vacuoles as well as swollen mitochondria and dilated Golgi were noted. Necrotic signs such as a large amount of cellular debris and membrane fragmentation were observed mainly when the cells were exposed to highest concentration of the PLA2-neurotoxins. PLA2s exposed cultures showed cytoplasmic vacuoles filled with cell debris, clusters of mitochondria presented mitophagy-like structures that are in accordance to patterns of programmed cell death by autophagy. Finally, we demonstrated that the sPLA2s, Mlx-8 and Mlx-9, isolated from the Micrurus lemniscatus snake venom induce a hybrid cell death with apoptotic, autophagic and necrotic features. Furthermore, this study suggests that the augment in free cytosolic Ca2+ and mitochondrial dysfunction are involved in the neurotoxicity of Elapid coral snake venom sPLA2s.

Alleviation of viper venom induced platelet apoptosis by crocin (Crocus sativus): implications for thrombocytopenia in viper bites

Viper envenomations are characterized by prominent local and systemic manifestations including hematological alterations. Snake venom metalloproteinases (SVMPs) and phospholipase A2 (PLA2) plays crucial role in the pathophysiology of hemorrhage by targeting/altering the platelets function which may result in thrombocytopenia. Platelets undergo the classic events of mitochondria-mediated apoptotic pathway due to augmented endogenous reactive oxygen species (ROS) levels. The observed anticoagulant effects during viper envenomations could be due to exacerbated platelet apoptosis and thrombocytopenia. Moreover, antivenin treatments are ineffective against the venom-induced oxidative stress; therefore, it necessitates an auxiliary therapy involving antioxidants which can effectively scavenge the endothelium-generated/endogenous ROS and protect the platelets. The present study explored the effects of viper venom on platelet apoptosis and its amelioration by a phytochemical crocin. The study evaluated the Vipera russelli venom-induced apoptotic events including endogenous ROS generation, intracellular Ca(2+) mobilization, mitochondrial membrane depolarization, cyt-c translocation, caspase activation and phosphatidylserine externalization which were effectively mitigated when the venom was pre-treated with crocin. The study highlights one of the less studied features of venom-induced secondary complications i.e. platelet apoptosis and sheds light on the underlying basis for venom-induced thrombocytopenia, systemic hemorrhage and in vivo anticoagulant effect.

202. Brazilian Coral Snake Phospholipases A2 Induce Neuronal Death in Primary Cultures of Hippocampal Neurons

202. Brazilian Coral Snake Phospholipases A2 Induce Neuronal Death in Primary Cultures of Hippocampal Neurons

Regional divergence of phospholipase A 2-like protein cDNAs between New Guinean and Australian Pseudechis australis

Snake Phospholipase A 2 (PLA 2) exhibits diverse pharmacological effects, such as hemolysis, myotoxicity, and neurotoxicity. In this study, we identified 10 novel PLA 2-like protein cDNAs, which we named Pr 1–10, from the venom gland cDNA library of Papuan pigmy mulga snake (New Guinean Pseudechis australis). The deduced amino acid sequence of Pr 1, which ortholog has not been reported in mulaga snake (Australian P. australis) yet, shows 78.8% identity with the ortholog in Australian tiger snake ( Notechis scutatus scutatus). The amino acid sequences of Pr 2–10 are 92.4–99.3% identical with their orthologs and paralogs in Australian P. australis.

Venom from the centipede Scolopendra viridis Say: Purification, gene cloning and phylogenetic analysis of a phospholipase A2

Venom components from the centipede Scolopendra viridis Say were studied, using both the soluble venom and a cDNA library prepared from mRNA of the venomous glands. Separation of the soluble venom by high performance liquid chromatography (HPLC) permitted to obtain at least 54 different fractions. The fraction eluting at 46.24 min showed phospholipase activity. The enzyme was purified to homogeneity and the first 25 amino acid residues were identified by Edman degradation. From the cDNA library several genes were cloned, one of which codes for a protein with identical amino acid sequence as the one experimentally determined. The cloned gene codes for a signal peptide of 28 amino acids and a mature peptide of 119 residues. The molecular weight of the enzyme was estimated by mass spectrometry and shown to be 13,752 Da, which matches exactly with the molecular mass expected from the deduced amino acid sequence of the gene. Phylogenetic analysis of this sequence, in comparison with other known from venomous animals, showed that it is more similar to snake phospholipases than to insect or arachnid sequences, suggesting that it has been submitted to convergent evolution. To the best of our knowledge this is the first time that a phospholipase from this species of animal is fully characterized. We have named it Scol/Pla.

A novel recombinant system for functional expression of myonecrotic snake phospholipase A2 in Escherichia coli using a new fusion affinity tag

A novel recombinant expression system in Escherichia coli was developed using conger eel galectin, namely, congerin II, as an affinity tag. This system was applied for the functional expression of myotoxic lysine-49-phospholipase A2 ([Lys49]PLA2), termed BPII and obtained from Protobothrops flavoviridis (Pf) venom. Recombinant Pf BPII fused with a congerin tag has been successfully expressed as a soluble fraction and showed better quantitative yield when folded correctly. The solubility of the recombinant congerin II-tagged BPII increased up to >90% in E. coli strain JM109 when coexpressed with the molecular chaperones GroEL, GroES, and trigger factor (Tf). The tag protein was cleaved by digestion with restriction protease, such as α-thrombin or Microbacterium liquefaciens protease (MLP), to obtain completely active recombinant BPII. Thus, the congerin-tagged fusion systems containing the cleavage recognition site for α-thrombin or MLP were demonstrated to be highly efficient and useful for producing proteins of desired solubility and activity.

Glycogen synthase kinase 3 activation is essential for the snake phospholipase A2 neurotoxin‐induced secretion in chromaffin cells

Neuroendocrine chromaffin cells were used to study the mechanism of the snake phospholipase A2 (PLA2) neurotoxin enhancement of exocytosis. Notexin, beta-bungarotoxin, taipoxin or textilotoxin enhanced the fast release of catecholamines elicited by flash photolysis of cytosolic caged calcium. Such an increase correlates with the capacity of these neurotoxins to cause fragmentation of the F-actin cortical barrier with subsequent accumulation of vesicles in the proximity of the plasma membrane. These PLA2 neurotoxins do not act via protein kinase C activation, which is known to promote F-actin fragmentation. Lithium, RO31-8220 and SB216763, three inhibitors of the glycogen synthase kinase 3, prevent both the alteration of the F-actin peripheral cortex and the enhancement of fast release elicited by these neurotoxins. In addition, glycogen synthase kinase 3 has been detected by immunolocalization in a membranous compartment of the chromaffin cell endoplasmic reticulum (ER). These results suggest that the activation of this enzyme plays a major role in the enhancement of exocytosis of the readily releasable granules caused by PLA2 neurotoxins in neuroendocrine chromaffin cells.

Divergence of genes encoding B chains of β-bungarotoxins

The structural organization of the genes encoding B2, B4, B5 and B6 chains of β-bungarotoxins are reported in this study. These genes shared virtually identical overall organization with three exons interrupted by two introns in similar positions. On the contrary, intron 1 of these genes had a similar size, a notable variation with the size of intron 2 was observed. It was found that two regions at the second intron of B1 and B2 chains were absent in that of B4, B5 and B6 chains. RT-PCR analyses indicated that Bungarus multicinctus venom gland, heart, liver and muscle expressed the RNA transcripts showing sequence similarity with the intronic segment being deleted in B4, B5 and B6 chain genes. This reflects that the ancestral gene of the intronic segment might insert in multiple loci of B. multicinctus genome. Comparative analyses of B chain genes showed that the protein-coding regions of the exons are more diverse than introns, except for in the signal peptide domain. These results suggest that intron insertions or deletions occur with the evolution of B chains, and that accelerated evolution may diversify the protein-coding sequence of B chain genes same as snake phospholipase A 2, neurotoxin and cardiotoxin genes.

Structural and functional analysis of BmjMIP, a phospholipase A 2 myotoxin inhibitor protein from Bothrops moojeni snake plasma

A protein, which neutralizes the enzymatic, toxic, and pharmacological activities of various basic and acidic phospholipases A 2 from the venoms of Bothrops moojeni, Bothrops pirajai, and Bothrops jararacussu, was isolated from B. moojeni snake plasma by affinity chromatography using immobilized myotoxins on Sepharose gel. Biochemical characterization of this myotoxin inhibitor protein (BmjMIP) showed it to be an oligomeric glycoprotein with a M r of 23,000–25,000 for the monomeric subunit. BmjMIP was stable in the pH range from 4.0 to 12.0, between 4 and 80 °C, even after deglycosylation. The role of the carbohydrate moiety was investigated and found not to affect the in vitro function of the inhibitor. The corresponding 500 bp cDNA obtained by RT-PCR from the liver of the snake encodes a mature protein of 166 amino acid residues including a 19 amino acid signal peptide. The primary structure of BmjMIP showed a high similarity with other snake phospholipase A 2 inhibitors (PLIs) in which the carbohydrate recognition domain (CRD) and the glycosylation site (Asn103) are conserved. Circular dichroism spectroscopy indicated that no significant alterations in the secondary structure of either the BmjMIP or the target protein occur upon their interaction. BmjMIP has a wide range of inhibitory properties against basic and acidic PLA 2s from Bothrops venoms (anti-enzymatic, anti-myotoxic, anti-edema inducing, anti-cytotoxic, anti-bactericidal, and anti-lethal). However, the inhibitor showed a reduced ability to neutralize the biological activities of crotoxin B (CB), the PLA 2 homologue associated with crotapotin in Crotalus durissus terrificus snake venom. Finally, the purified PLA 2 inhibitor was shown to protect in vivo against the toxic and pharmacological effects of a homologous PLA 2 enzyme, suggesting that PLIs or a corresponding derived peptide may prove useful in the treatment of snakebite victims or, more importantly, in the treatment of the many human diseases in which these enzymes have been implicated.

Soluble Expression of a Functional Recombinant Cytolytic Immunotoxin in Insect Cells

We have previously described the production of a recombinant melittin-based cytolytic immunotoxin (IT), scFv-mel-FLAG, in bacterial cells. While the IT exhibited specific cytotoxicity for a human lymphoblastoid cell line, HMy2, yields from expression were low. Here, we describe a baculovirus expression system for the overexpression and secretion of scFv-mel-FLAG. A novel snake phospholipase A2 inhibitor signal peptide was used to aid in the secretion of the immunotoxin. Sf21 insect cells infected with the recombinant virus secreted soluble scFv-mel-FLAG into the culture medium from which it was purified directly on an affinity column. The final yield of scFv-mel-FLAG was estimated at 3-5 mg/L, which was an improvement of 30-fold compared to expression in the prokaryotic system. The cell binding characteristics of the recombinant IT were assessed by flow cytometry using the antigen expressing cell line HMy2. ScFv-mel-FLAG bound specifically to HMy2 cells in direct binding assays and this binding was completely inhibited in the presence of an excess of soluble antigen. Significant cytotoxicity for HMy2 cells, measured by leakage of cytosolic LDH, was also observed for the IT at a concentration of 60 pmol/10(4) cells. Cytotoxicity was concentration dependent and was specific for antigen-positive cells. Thus the baculovirus expression system, under the control of a novel secretion signal, can be used for the production of soluble and functional recombinant cytolytic immunotoxins. To our knowledge, this is the first report of expression of a recombinant immunotoxin in the baculovirus expression vector system.

30-day intravenous administration of VRCTC-310-ONCO in rabbits

VRCTC-310-ONCO, an agent based on the snake phospholipase A 2 (crotoxin), is currently under clinical development. After phase I study in patients by intramuscular administration, the interest of intravenous (IV) dosing arose. To evaluate IV administration of VRCTC-310-ONCO in rabbits, ten animals were subjected to surgical implant of fixed jugular catheter, by which they received daily IV doses of 0.03 mg/kg body weight of VRCTC-310-ONCO for 30 days ( n=8) or saline ( n=2). The procedure was well tolerated in all rabbits. One of the animals died after the sixth dose of VRCTC-310-ONCO with CNS involvement; two additional rabbits required dose-reduction. All other rabbits achieved 30 days of treatment and were sacrificed. All rabbits (even controls) developed lymphocytosis and mild anaemia, without changes in blood neutrophils. No changes were found in serum transaminases (GOT and GPT), cholesterol, triglycerides, and γ-glutamyl transpeptidase. At necropsy, chronic granulation tissue was found surrounding the implant in all rabbits. VRCTC-310-ONCO-treated rabbits presented generalised and marked swelling of hepatocytes, with areas of cytoplasmic vacuolisation. No abnormalities were found in kidney, heart, lung, spleen, adrenal gland, uterus, testes and ovary. Additional studies with IV route for VRCTC-310-ONCO, including humans, are required to define its toxicity in the clinical setting.

Sequencing and two-dimensional structure prediction of a phospholipase A2 inhibitor from the serum of the common tiger snake (Notechis scutatus)

A phospholipase A2 inhibitor has been identified in the serum of the common tiger snake (Notechis scutatus). The inhibitor is composed of two chains, an α-chain and a β-chain, that form a non-covalently associated complex capable of inhibiting the enzymatic activity of all phospholipase A2 enzymes it was tested against. The α and β-chains have been purified to homogeneity, digested and sequenced. From the peptide sequence generated, degenerate PCR primers were designed and used to elucidate the complete cDNA sequence of the chains using 5′ and 3′ RACE PCR. A total of three α-chain isoforms were identified, only one isoform of the β-chain was detected. The two-dimensional structure of the three α-chains and one β-chain were predicted using five prediction programs (discrimination of secondary structure class; nearest neighbour secondary structure, profile network from Heidelberg; self-optimised prediction method from multiple alignment, SSPAL). For each protein chain a consensus prediction was generated. Results are discussed in relation to the function of the protein, and how they may influence the three-dimensional structure of the inhibitor. Additionally, the sequences of several snake phospholipase A2 inhibitors were used as the input for a motif prediction algorithm (MEME). The results are discussed in relation to the activity of these proteins.

In vivo effect of snake phospholipase A 2 (crotoxin+cardiotoxin) on serum IL-1α, TNF-α and IL-1ra level in humans

VRCTC-310-Onco (crotoxin, a secretory phospholipase A 2+cardiotoxin) is under development as an anti-neoplastic agent. Pro-inflammatory cytokines TNF-α and interleukin 1 α (IL-1α) and anti-inflammatory cytokine IL-1 receptor antagonist (IL-1ra) were measured with commercial ELISA kits in sera corresponding to 23 cycles with doses between 0.0025 and 0.023 μg/kg body weight, obtained during the phase I trial of VRCTC-310-Onco. Neither serum TNF-α nor IL-1α did change significantly after VRCTC-310-Onco. Basal IL-1ra was 794±97 pg/ml, by 3 h it was similar, 651±99 pg/ml and at 24 h p.i. it increased to 1197±122 pg/ml ( P<0.001). The increase was dose-dependent. The addition of dexamethasone (required to reduce pain with the highest doses) inhibited IL-1α and enhanced the induction of IL-1ra by VRCTC-310-Onco. Summing up, in vivo, in humans, in the dose range tested, VRCTC-310-Onco induces IL-1ra, and does not consistently modify IL-1α or TNF-α serum levels.

Functional Characteristics of a Phospholipase A2Inhibitor from Notechis ater Serum

A phospholipase A(2) inhibitor has been purified p6om the serum of Notechis ater using DEAE-Sephacel chromatography. The inhibitor was found to be composed of two protein subunits (alpha and beta) that form the intact complex of approximately 110 kDa. The alpha-chain is a 30-kDa glycoprotein and the beta-chain a nonglycosylated, 25-kDa protein. N-terminal sequence analysis reveals a high level of homology to other snake phospholipase A(2) inhibitors. The inhibitor was shown to be extremely pH and temperature stable. The inhibitor was tested against a wide variety of phospholipase A(2) enzymes and inhibited the enzymatic activity of all phospholipase A(2) enzymes tested, binding with micromole to nanomole affinity. Furthermore, the inhibitor was compared with the Eli-Lilly compound LY311727 and found to have a higher affinity for human secretory nonpancreatic phospholipase A(2) than this chemical inhibitor. The role of the carbohydrate moiety was investigated and found not to affect the in vitro function of the inhibitor.

Snake phospholipase A 2 introms as an excellent tool in resolving phylogenetic relationships

Snake phospholipase A 2 introms as an excellent tool in resolving phylogenetic relationships

Synergism between cytotoxin P4 from the snake venom of Naja nigricollis nigricollis and various phospholipases

The synergism between the lytic activity of cytotoxin P4 isolated from the venom of Naja nigricollis nigricollis and various phospholipases (PLA) is demonstrated. The highest synergistic activity was obtained with one of the basic phospholipases found in the same venom. It was also shown between cytotoxin P4 and heterologous elapid and viperid snake phospholipases. Moreover, remarkable synergism was also demonstrated between cytotoxin P4 and bee venom PLA 2 or porcine pancreatic PLA 2. However, the phospholipase fractions of Vipera palestinae and the bovine pancreas PLA 2 had no synergistic effect.

Conservative and variable regions of homologous snake phospholipases A2 sequences: Prediction of the taxonspecific peptides structure

Homologous amino acid sequences of phospholipases A2 (PLA2) of snakes belonging to the families Elapidae, Viperidae, and Colubridae were considered in order to study the conservative and variable regions location. The PLA2 sequences were divided into two groups (taxons) according to the phylogenetic tree reconstructed from the pair similarity matrix. Results of the intergroup comparison were plotted to facilitate the identification of significant conservative and variable regions. It was shown that the results of the comparison between two phylogenetic groups of snake PLA2 did not much depend on the number of each group representatives and did not markedly change if one of the groups was represented by the single sequence. The knowledge of the number and location of conservative and variable regions and their dependence on the phylogenetic relations between compared taxa may be used to predict a synthetic peptide structure to obtain specific antibodies against PLA2 of one of these taxons. Such prediction is possible if there is a specific region conservative for one taxon but variable for two of them.

Preliminary Crystallographic Study of Phospholipase A2 from the Venom of Trimeresurus flavoviridis (Habu Snake)1

Crystallization and a preliminary crystallographic study of Trimeresurus flavoviridis (habu snake) phospholipase A2 (PLA2) were carried out. Although crystals were obtained from various solutions, crystals suitable for X-ray analysis could be obtained from polyethylene glycol solutions only when a repeated seeding technique was applied starting from twinned crystals. The crystal is monoclinic with space group P21, with a = 44.1, b = 55.7, c = 48.8 A, and beta = 92.4 degrees. An asymmetric unit contains a dimer consisting of two identical subunits made of 122 amino acids. The crystal reflects X-rays beyond 2.5 A. A Pt derivative gave a good isomorphous crystal.

Immunological relationships of phospholipase A 2 neurotoxins from snake venoms

Polyclonal rabbit antisera were raised against ten snake phospholipase A 2 neurotoxins and one snake phospholipase A 2 cytotoxin. Immunological cross-reactivities between these toxins, two other snake phospholipase A 2 enzymes and pancreatic phospholipase A 2 were studied using ELISA technology. All snake phospholipase A 2 neurotoxins fell into two main antigenic classes. One antigenic class was composed of all the elapid toxins tested (textilotoxin, taipoxin, notexin, pseudexin and β-bungarotoxin), the cytotoxic phospholipase A 2 from Naja naja atra and pancreatic phospholipase A 2. β-Bungarotoxin seemed to be in an immunological subclass of its own compared to the rest of the elapid toxins. The second antigenic class was comprised of crotalid and viperid phospholipase A 2 neurotoxins (crotoxin, concolor toxin, Mojave toxin, vegrandis toxin, ammodytoxin and caudoxin). Our data indicated that the viperid toxins, caudoxin and ammodytoxin, were an immunological subclass apart from the crotalid toxins.