Deciphering the interactions of scopoletin and scopolin from Catunaregam nilotica roots against Naja nigricollis phospholipase A2 enzyme.

Catuneragam nilotica has been used in ethnomedicine to treat snakebite, inflammation, and diarrhea among others. The aim of this research is to isolate, and characterize potential potential phospholipase A2 (PLA2) inhibitors from the roots of C. nilotica. The plant material was collected, authenticated, and sequentially extracted using solvents of increasing polarity starting from n-hexane, ethyl acetate, and methanol. The extracts as reported in our previous work, were screened in vitro for their inhibitory activity against PLA2 enzyme from N. nigricollis venom using acidimetric assay. In line with the bio-activity guided isolation, methanol extract (being the most active) was subjected to chromatographic separation using silica gel and sephadex LH-20 which resulted in the isolation and characterization of scopoletin, and scopolin; the compounds were able to inhibit the hydrolytic actions of PLA2 enzyme with percentage inhibition ranging from 67.82 to 100.00 % and 65.76-93.15 %, respectively while the standard Antisnake Venom (ASV) had 74.96-85.04 % after 10 min incubation at 37 °C. The molecular docking of the compounds against PLA2 enzyme was performed using Auto Dock Vina while ADME-Tox analysis was evaluated using swissADME and ProTox-II online servers; The findings indicated that both compounds were able to bind to the active site of PLA2 enzyme with high affinity (-6.5 to -6.2 kcal/mol) and they exhibited favorable drug-likeness and pharmacokinetic properties, and according to toxicity predictions, scopolin was found to be non-toxic (LD50 of 5000 mg/kg) while scopoletin has a slight chance of being toxic (LD50 of 3800 mg/kg). In conclusion, the findings of the research revealed that the roots of C. nilotica contains phytoconstituents with anti-PLA2 enzyme activity and thus, validates the ethnomedicinal claim of the use of the plant as herbal therapy against N. nigricollis envenomation.

Heterologous Expression and Immunogenic Potential of the Most Abundant Phospholipase A2 from Coral Snake Micrurus dumerilii to Develop Antivenoms.

Micrurus dumerilii is a coral snake of clinic interest in Colombia. Its venom is mainly composed of phospholipases A2 being MdumPLA2 the most abundant protein. Nevertheless, Micrurus species produce a low quantity of venom, which makes it difficult to produce anticoral antivenoms. Therefore, in this work, we present the recombinant expression of MdumPLA2 to evaluate its biological activities and its immunogenic potential to produce antivenoms. For this, a genetic construct rMdumPLA2 was cloned into the pET28a vector and expressed heterologously in bacteria. His-rMdumPLA2 was extracted from inclusion bodies, refolded in vitro, and isolated using affinity and RP-HPLC chromatography. His-rMdumPLA2 was shown to have phospholipase A2 activity, a weak anticoagulant effect, and induced myonecrosis and edema. The anti-His-rMdumPLA2 antibodies produced in rabbits recognized native PLA2, the complete venom of M. dumerilii, and a phospholipase from another species of the Micrurus genus. Antibodies neutralized 100% of the in vitro phospholipase activity of the recombinant toxin and a moderate percentage of the myotoxic activity of M. dumerilii venom in mice. These results indicate that His-rMdumPLA2 could be used as an immunogen to improve anticoral antivenoms development. This work is the first report of an M. dumerilii functional recombinant PLA2.

Isolation and Characterization of A2-EPTX-Nsm1a, a Secretory Phospholipase A2 from Malaysian Spitting Cobra (Naja sumatrana) Venom.

Phospholipase A2 (PLA2) toxins are one of the main toxin families found in snake venom. PLA2 toxins are associated with various detrimental effects, including neurotoxicity, myotoxicity, hemostatic disturbances, nephrotoxicity, edema, and inflammation. Although Naja sumatrana venom contains substantial quantities of PLA2 components, there is limited information on the function and activities of PLA2 toxins from the venom. In this study, a secretory PLA2 from the venom of Malaysian N. sumatrana, subsequently named A2-EPTX-Nsm1a, was isolated, purified, and characterized. A2-EPTX-Nsm1a was purified using a mass spectrometry-guided approach and multiple chromatography steps. Based on LC-MSMS, A2-EPTX-Nsm1a was found to show high sequence similarity with PLA2 from venoms of other Naja species. The PLA2 activity of A2-EPTX-Nsm1 was inhibited by 4-BPB and EDTA. A2-EPTX-Nsm1a was significantly less cytotoxic in a neuroblastoma cell line (SH-SY5Y) compared to crude venom and did not show a concentration-dependent cytotoxic activity. To our knowledge, this is the first study that characterizes and investigates the cytotoxicity of an Asp49 PLA2 isolated from Malaysian N. sumatrana venom in a human neuroblastoma cell line.

Anti-ferroptotic mechanism of IL4i1-mediated amino acid metabolism.

Interleukin-4-induced-1 (IL4i1) is an amino acid oxidase secreted from immune cells. Recent observations have suggested that IL4i1 is pro-tumorigenic via unknown mechanisms. As IL4i1 has homologs in snake venoms (L-amino acid oxidases [LAAO]), we used comparative approaches to gain insight into the mechanistic basis of how conserved amino acid oxidases regulate cell fate and function. Using mammalian expressed recombinant proteins, we found that venom LAAO kills cells via hydrogen peroxide generation. By contrast, mammalian IL4i1 is non-cytotoxic and instead elicits a cell protective gene expression program inhibiting ferroptotic redox death by generating indole-3-pyruvate (I3P) from tryptophan. I3P suppresses ferroptosis by direct free radical scavenging and through the activation of an anti-oxidative gene expression program. Thus, the pro-tumor effects of IL4i1 are likely mediated by local anti-ferroptotic pathways via aromatic amino acid metabolism, arguing that an IL4i1 inhibitor may modulate tumor cell death pathways.

Convergent evolution of pain-inducing defensive venom components in spitting cobras.

Convergent evolution provides insights into the selective drivers underlying evolutionary change. Snake venoms, with a direct genetic basis and clearly defined functional phenotype, provide a model system for exploring the repeated evolution of adaptations. While snakes use venom primarily for predation, and venom composition often reflects diet specificity, three lineages of cobras have independently evolved the ability to spit venom at adversaries. Using gene, protein, and functional analyses, we show that the three spitting lineages possess venoms characterized by an up-regulation of phospholipase A2 (PLA2) toxins, which potentiate the action of preexisting venom cytotoxins to activate mammalian sensory neurons and cause enhanced pain. These repeated independent changes provide a fascinating example of convergent evolution across multiple phenotypic levels driven by selection for defense.

Proteomic characterization of Naja mandalayensis venom

Background Naja mandalayensis is a spitting cobra from Myanmar. To the best of our knowledge, no studies on this venom composition have been conducted so far. On the other hand, few envenomation descriptions state that it elicits mainly local inflammation in the victims' eyes, the preferred target of this spiting cobra. Symptoms would typically include burning and painful sensation, conjunctivitis, edema and temporary loss of vision. Methods We have performed a liquid-chromatography (C18-RP-HPLC) mass spectrometry (ESI-IT-TOF/MS) based approach in order to biochemically characterize N. mandalayensis venom. Results A wide variety of three-finger toxins (cardiotoxins) and metallopeptidases were detected. Less abundant, but still representative, were cysteine-rich secretory proteins, L-amino-acid oxidases, phospholipases A2, venom 5'-nucleotidase and a serine peptidase inhibitor. Other proteins were present, but were detected in a relatively small concentration. Conclusion The present study set the basis for a better comprehension of the envenomation from a molecular perspective and, by increasing the interest and information available for this species, allows future venom comparisons among cobras and their diverse venom proteins.(AU)

Enzymatic activity and brine shrimp lethality of venom from the large brown spitting cobra (Naja ashei) and its neutralization by antivenom.

OBJECTIVE: Naja ashei is a snake of medical importance in Kenya, Ethiopia, Somalia, Uganda, and Tanzania. Little is known about the enzymatic (snake venom phospholipases A2; svPLA2's) and toxic (lethal) activities of N. ashei venom and crucially, the safety and capacity of available antivenom to neutralize these effects. This study aimed to determine the enzymatic and toxic activities of N. ashei venom and the capacity of Indian and Mexican manufactured antivenoms to neutralize these effects. The protein content of the venom and the test antivenoms were also evaluated. A 12-point log concentration-response curve (0.5-22.5 µg/mL) was generated on an agarose-egg yolk model to predict the svPLA2 activity of the venom. The toxicity profiles of the venom and antivenoms were evaluated in the brine shrimp lethality assay. Lowry's method was used for protein estimation. RESULTS: Low and intermediate concentrations of the venom exhibited similar svPLA2 activities. The same was true for concentrations > 15 µg/mL. Intermediate and high doses of the venom exhibited similar mortalities in brine shrimp and test antivenoms were generally non-toxic but poorly neutralized svPLA2 activity. Mexican manufactured antivenom had lower protein content but neutralized venom-induced brine shrimp lethality much more effectively than Indian manufactured antivenom.

Atrase B, a novel metalloprotease with anti-complement and anti-coagulant activity, significantly delays discordant cardiac xenograft rejection.

BACKGROUND: Both the complement and the coagulation systems play important roles in the development of hyperacute or acute antibody-mediated xenograft rejection. Atrase B is a novel metalloproteinase isolated from the venom of Naja atra. In this study, we investigated the inhibitory effects of atrase B on complement activation and coagulation, as well as the effect on xenograft survival in a discordant xenotransplantation model. METHODS: The in vitro anti-complement activity of atrase B was evaluated using a normal human serum (NHS)-mediated complement-dependent cytotoxicity model with an immortalized porcine aortic endothelial cell line (iPEC) as the target. The in vivo inhibitory effects on complement activity and coagulation function were measured in rats after the administration of atrase B. Guinea pig hearts were transplanted heterotopically into Wistar rats with or without atrase B pre-treatment. RESULTS: Pre-treatment of the NHS with atrase B inhibited the cell lysis of iPECs in a dose-dependent manner. FACS analysis showed that atrase B potently suppressed the deposition of C5b-9, but not C3c and C4c, on iPECs. In vivo, atrase B-treated rats showed a significant reduction in serum complement activity; markedly prolonged PT, APTT, and TT; and a decreased plasma level of fibrinogen. When compared to PBS treatment evaluated at study endpoint, atrase B treatment significantly delayed xenograft rejection and attenuated pathologic damage, the formation of platelet microthrombi, and the deposition of fibrin and C5b-9. CONCLUSIONS: The dual activities of anti-complement and anti-coagulation make atrase B a potential adjuvant therapeutic drug for use in xenotransplantation.

Interspecific and intraspecific venom enzymatic variation among cobras (Naja sp. and Ophiophagus hannah).

The genera Ophiophagus and Naja comprise part of a clade of snakes referred to as cobras, dangerously venomous front-fanged snakes in the family Elapidae responsible for significant human mortality and morbidity throughout Asia and Africa. We evaluated venom enzyme variation for eleven cobra species and three N. kaouthia populations using SDS-PAGE venom fingerprinting and numerous enzyme assays. Acetylcholinesterase and PLA2 activities were the most variable between species, and PLA2 activity was significantly different between Malaysian and Thailand N. kaouthia populations. Venom metalloproteinase activity was low and significantly different among most species, but levels were identical for N. kaouthia populations; minor variation in venom L-amino acid oxidase and phosphodiesterase activities were seen between cobra species. Naja siamensis venom lacked the α-fibrinogenolytic activity common to other cobra venoms. In addition, venom from N. siamensis had no detectable metalloproteinase activity and exhibited an SDS-PAGE profile with reduced abundance of higher mass proteins. Venom profiles from spitting cobras (N. siamensis, N. pallida, and N. mossambica) exhibited similar reductions in higher mass proteins, suggesting the evolution of venoms of reduced complexity and decreased enzymatic activity among spitting cobras. Generally, the venom proteomes of cobras show highly abundant three-finger toxin diversity, followed by large quantities of PLA2s. However, PLA2 bands and activity were very reduced for N. haje, N. annulifera and N. nivea. Venom compositionalenzy analysis provides insight into the evolution, diversification and distribution of different venom phenotypes that complements venomic data, and this information is critical for the development of effective antivenoms and snakebite treatment.

Metabolites from the citrus extracts inhibit the activity of selected proteins in Indian Cobra (Naja naja) venom.

ETHNOPHARMACOLOGICAL RELEVANCE: Snakebite is a severe problem in many parts of the world, specifically in tropical and subtropical regions. A range of medicinal plant extracts are administered for treating snake bite. Of the many common plants, extracts of Citrus species have been documented to be used for treating snake bite and have been shown to decrease the snake venom toxicity. AIM: The aim of the current work is to evaluate the utility of citrus peel extracts (Citrus aurantium L. and Citrus reticulate Blanco) in the management of Indian cobra envenomation. MATERIALS AND METHODS: Peels of citrus species were evaluated for their phospholipase A2, protease and haemolytic inhibition properties. The phytochemicals present in the extract were inferred using GC-MS. In-vivo studies, using mice model, were done to confirm the inhibitory effect of the extracts. Molecular docking was used to understand the possible binding modes of selected phytochemicals to snake venom phospholipase. RESULTS: Citrus peel extracts are rich in polyphenols, flavonoids and tannins. The methanolic extract of Citrus aurantium L. and Citrus reticulate Blanco inhibits phospholipase (75%), protease (71%) and hemolysis (80%) activity of the venom. GC-MS analyses indicate the presence of ß-sitosterol, n-hexadecanoic acid, eicosanoic acid, and flavone in both the extracts. In addition, C. reticulate extract contains α-tocopherol and squalene. Molecular docking revealed that α-tocopherol, spiro [androst-5-ene-17,1'-cyclobutan]-2'-one,3-hydroxy-(3ß,17ß)- and ß-sitosterol acetate bind with moderate affinity to the catalytic site of phospholipase A2. CONCLUSION: The present study provides new molecular insight and scientific evidence on the utility of the methanolic extracts of citrus peels to neutralize the venom toxins of Naja naja.

Activity of two key toxin groups in Australian elapid venoms show a strong correlation to phylogeny but not to diet.

BACKGROUND: The relative influence of diet and phylogeny on snake venom activity is a poorly understood aspect of snake venom evolution. We measured the activity of two enzyme toxin groups - phospholipase A2 (PLA2), and L-amino acid oxidase (LAAO) - in the venom of 39 species of Australian elapids (40% of terrestrial species diversity) and used linear parsimony and BayesTraits to investigate any correlation between enzyme activity and phylogeny or diet. RESULTS: PLA2 activity ranged from 0 to 481 nmol/min/mg of venom, and LAAO activity ranged from 0 to 351 nmol/min/mg. Phylogenetic comparative methods, implemented in BayesTraits showed that enzyme activity was strongly correlated with phylogeny, more so for LAAO activity. For example, LAAO activity was absent in both the Vermicella and Pseudonaja/Oxyuranus clade, supporting previously proposed relationships among these disparate taxa. There was no association between broad dietary categories and either enzyme activity. There was strong evidence for faster initial rates of change over evolutionary time for LAAO (delta parameter mean 0.2), but no such pattern in PLA2 (delta parameter mean 0.64). There were some exceptions to the phylogenetic patterns of enzyme activity: different PLA2 activity in the ecologically similar sister-species Denisonia devisi and D. maculata; large inter-specific differences in PLA2 activity in Hoplocephalus and Austrelaps. CONCLUSIONS: We have shown that phylogeny is a stronger influence on venom enzyme activity than diet for two of the four major enzyme families present in snake venoms. PLA2 and LAAO activities had contrasting evolutionary dynamics with the higher delta value for PLA2 Some species/individuals lacked activity in one protein family suggesting that the loss of single protein family may not incur a significant fitness cost.

Enzymatic labelling of snake venom phospholipase A2 toxins.

Almost all animal venoms contain secretory phospholipases A2 (PLA2s), 14 kDa disulfide-rich enzymes that hydrolyze membrane phospholipids at the sn-2 position, releasing lysophospholipids and fatty acids. These proteins, depending on their sequence, show a wide variety of biochemical, toxic and pharmacological effects and deserve to be studied for their numerous possible applications, and to improve antivenom drugs. The cellular localization and activity of a protein can be studied by conjugating it with a tag. In this work, we applied an enzymatic labelling method, using Streptomyces mobaraense transglutaminase, on three snake venom PLA2s: a recombinant neuro- and myotoxic group I PLA2 from Notechis scutatus scutatus, and two myotoxic group II PLA2s from Bothrops asper - one of them a natural catalytically inactive variant. We demonstrate that TGase can be used to produce active mono- or bi-derivatives of these three PLA2s modified at specific Lys residues, and that all three of these proteins, conjugated with fluorescent peptides, are internalized in primary myotubes.

New insights into the phylogeographic distribution of the 3FTx/PLA2 venom dichotomy across genus Micrurus in South America.

Micrurus is a monophyletic genus of venomous coral snakes of the family Elapidae. The ~80 recognized species within this genus are endemic to the Americas, and are distributed from southeastern United States to northern Argentina. Although relatively few bites are recorded due to their reclusive nature, semi-fossorial habits, and their occurrence in sparsely populated areas, coral snakes possess powerful venoms that target the cholinergic system and, if early treatment is missed, can cause neuromuscular paralysis, respiratory failure, and death by asphyxiation within hours of envenoming. The to-date proteomically characterized 18 micrurine venoms exhibit a puzzling phenotypic dichotomy, characterized by the toxin arsenal being dominated either by pre-synaptically acting PLA2s or post-synaptic 3FTxs, and a general, but imperfect, distributional pattern of these venom phenotypes along the North-South axis of the American continent. The lack of perfect phylogenetic clustering suggests that phylogeny may not be the sole factor driving the evolution of the divergent venom phenotypes across Micrurus venoms. To shed new light on the origin and expression pattern of the 3FTx/PLA2 venom dichotomy, we have conducted a comparative proteomics analysis of venoms from the Brazilian ribbon coral snake, Micrurus lemniscatus carvalhoi, sourced from different localities in the Brazilian states of São Paulo; the Caatinga coral snake, M. ibiboboca, from central Bahia state (Brazil); two Micrurus specimens of uncertain taxonomy collected in the Brazilian states of Alagoas and Rio de Janeiro; and the Western ribbon coral snake, M. l. helleri, from Leticia, the southernmost town of the Colombian Department of Amazonas. Venoms from São Paulo and Rio de Janeiro showed 3FTx-predominant phenotypes, while in venoms from Leticia, Alagoas and Bahia PLA2s represented the major toxin family. Comparative venom proteomics suggests that both Micrurus venom phenotypes exhibit a high degree of toxin evolvability. Mapping the 3FTx/PLA2 dichotomy across the Americas points to a phylogeographic pattern for venom phenotypes consistent with, but more complex than, the North-South distribution hypothesis anticipated in previous investigations. BIOLOGICAL SIGNIFICANCE: New World coral snakes (Micrurus: Elapidae) produce potent venoms that target pre- and post-synaptically cholinergic nerve terminals resulting in neuromuscular paralysis, and in severe envenomings, may lead to death from asphyxiation by respiratory arrest. Presynaptic ß-neurotoxins of group IA PLA2 protein subfamily and postsynaptic α-neurotoxins with 3FTx fold are the major components (>80%) of coral snake venoms. Micrurine venoms exhibit a puzzling phenotypic venom dichotomy, characterized by the dominant expression of either α- or ß-neurotoxins. The distribution of these alternative compositional profiles has been fragmentarily studied both across Micrurus phylogeny and along the North-South axis of the genus radiation in the American continent, from southern United States to Northern Argentina. The unpredictability of the neurotoxin profile across the distribution range of the coral snakes represents a difficulty for applying the most appropriate treatment upon a coral snakebite. A deep knowledge of the phylogeographic distribution and the evolution of dichotomic Micrurus venoms would be useful for tracing the evolutionary path to their present day phenotypes, rationalizing the patchy cross-reactivity of current Micrurus antivenoms, and improving the efficacy of antivenoms to neutralize coral snake envenomings.

Binding of a Naja naja venom acidic phospholipase A2 cognate complex to membrane-bound vimentin of rat L6 cells: Implications in cobra venom-induced cytotoxicity.

An acidic phospholipase A2 enzyme (NnPLA2-I) interacts with three finger toxins (cytotoxin and neurotoxin) from Naja naja venom to form cognate complexes to enhance its cytotoxicity towards rat L6 myogenic cells. The cytotoxicity was further enhanced in presence of trace quantity of venom nerve growth factor. The purified rat myoblast cell membrane protein showing interaction with NnPLA2-I was identified as vimentin by LC-MS/MS analysis. The ELISA, immunoblot and spectrofluorometric analyses showed greater binding of NnPLA2-I cognate complex to vimentin as compared to the binding of individual NnPLA2-I. The immunofluorescence and confocal microscopy studies evidenced the internalization of NnPLA2-I to partially differentiated myoblasts post binding with vimentin in a time-dependent manner. Pre-incubation of polyvalent antivenom with NnPLA2-I cognate complex demonstrated better neutralization of cytotoxicity towards L6 cells as compared to exogenous addition of polyvalent antivenom 60-240 min post treatment of L6 cells with cognate complex suggesting clinical advantage of early antivenom treatment to prevent cobra venom-induced cytotoxicity. The in silico analysis showed that 19-22 residues, inclusive of Asp48 residue, of NnPLA2-I preferentially binds with the rod domain (99-189 and 261-335 regions) of vimentin with a predicted free binding energy (ΔG) and dissociation constant (KD) values of -12.86 kcal/mol and 3.67 × 10-10 M, respectively; however, NnPLA2-I cognate complex showed greater binding with the same regions of vimentin indicating the pathophysiological significance of cognate complex in cobra venom-induced cytotoxicity.

New insights into the proteomic characterization of the coral snake Micrurus pyrrhocryptus venom.

A proteomic analysis of the soluble venom of the coral snake Micrurus pyrrhocryptus is reported in this work. The whole soluble venom was separated by RP-HPLC and the molecular weights of its components (over 100) were determined by mass spectrometry. Three main sets of components were identified, corresponding to peptides with molecular masses from 5 to 8 kDa, proteins from 12 to 16 kDa and proteins from 20 to 30 kDa. Two components were fully sequenced: one α-neurotoxic peptide of 7210 Da with slight blocking activity of the nicotinic acetylcholine receptor (nAChR) and a phospholipase A2 (PLA2) with molecular weight 13517 Da and no effect on the nAChR. PLA2 activity was evaluated for all RP-HPLC components. In addition, N-terminal sequence was obtained for eleven components using Edman degradation. Among these, three were similar to known PLA2's, six to three-finger toxins (3FTx) and one to Kunitz-type serine protease inhibitors. Two-dimensional gel electrophoresis of the venom allowed the separation of about thirty spots with components of molecular weights from 25 to 70 kDa. Seventeen spots were recovered from the gel, digested with trypsin and the corresponding peptides (85) were sequenced by MS/MS allowing identification of amino acid sequences with similarities to snake venom metalloproteases (SVMP), PLA2's, L-amino acid oxidases (LAAO), acetylcholinesterases (AChE) and serine proteases (SP). In addition, LC-MS analysis of peptides obtained from tryptic digestion of whole soluble venom allowed the identification of 695 peptides, whose amino acid sequence could correspond to at least 355 components found in other snake venoms, where C-type lectins, vespryns, zinc finger proteins, and waprins were found, among others. These results show the complexity of the venom and provide important knowledge for future work on identification and activity determination of venom components from this coral snake.

Snake venoms from Angola: Intra-specific variations and immunogenicity.

Snakebite is a public health problem in many countries of world. These accidents are considered a Neglected Tropical Disease and are responsible for a high morbidity and mortality index in the South and Southeast Asia and Sub-Saharan Africa. Angolan snake venoms are poorly investigated and no specific antivenom against them is available in the country. Thus, the aim of this study was to evaluate biochemical and immunogenic properties of male and female venoms from Naja nigricollis, Bitis arietans and Bitis gabonica snakes. These animals were collected during an expedition covering 1350 km of Angola, including the Provinces of Cuanza Sul, Benguela, Huíla and Malanje. Results showed that Angolan snake venoms present distinctive immunogenic properties and large intra-specific variations, associated to the gender and the geographic origin of the animals. Thus, it is possible to suggest that for the preparation of a therapeutic antivenom, intra-species variability should be taken into account, in order to obtain an efficient serum to neutralize the toxic effects of the Angolan snake venoms.

Intravascular hemolysis induced by phospholipases A2 from the venom of the Eastern coral snake, Micrurus fulvius: Functional profiles of hemolytic and non-hemolytic isoforms.

A unique feature of the venom of Micrurus fulvius (Eastern coral snake) is its ability to induce severe intravascular hemolysis in particular species, such as dogs or mice. This effect was previously shown to be induced by distinct phospholipase A2 (PLA2) isoforms which cause direct hemolysis in vitro, an uncommon finding for such enzymes. The functional profiles of PLA2-17, a direct hemolytic enzyme, and PLA2-12, a co-existing venom isoform lacking such effect, were compared. The enzymes differed not only in their ability to cause intravascular hemolysis: PLA2-17 additionally displayed lethal, myotoxic, and anticoagulant actions, whereas PLA2-12 lacked these effects. PLA2-12 was much more active in hydrolyzing a monodisperse synthetic substrate than PLA2-17, but the catalytic activity of latter was notably higher on a micellar substrate, or towards pure phospholipid artificial monolayers under controlled lateral pressures. Interestingly, PLA2-17 could hydrolyze substrate at a pressure of 20 mN m-1, in contrast to PLA2-12 or the non-toxic pancreatic PLA2. This suggests important differences in the monolayer penetrating power, which could be related to differences in toxicity. Comparative examination of primary structures and predicted three-dimensional folding of PLA2-12 and PLA2-17, revealed that differences concentrate in their N-terminal and central regions, leading to variations of the surface properties at the membrane interacting interface. PLA2-17 presents a less basic interfacial surface than PLA2-12, but more bulky aromatic residues, which could be associated to its higher membrane-penetrating strength. Altogether, these structural and functional comparative observations suggest that the ability of PLA2s to penetrate substrate interfaces could be a major determinant of toxicity, perhaps more important than protein surface charge.

Proteomics and antivenomics of Echis carinatus carinatus venom: Correlation with pharmacological properties and pathophysiology of envenomation.

The proteome composition of Echis carinatus carinatus venom (ECV) from India was studied for the first time by tandem mass spectrometry analysis. A total of 90, 47, and 22 distinct enzymatic and non-enzymatic proteins belonging to 15, 10, and 6 snake venom protein families were identified in ECV by searching the ESI-LC-MS/MS data against non-redundant protein databases of Viperidae (taxid 8689), Echis (taxid 8699) and Echis carinatus (taxid 40353), respectively. However, analysis of MS/MS data against the Transcriptome Shotgun Assembly sequences (87 entries) of conger E. coloratus identified only 14 proteins in ECV. Snake venom metalloproteases and snaclecs, the most abundant enzymatic and non-enzymatic proteins, respectively in ECV account for defibrinogenation and the strong in vitro pro-coagulant activity. Further, glutaminyl cyclase, aspartic protease, aminopeptidase, phospholipase B, vascular endothelial growth factor, and nerve growth factor were reported for the first time in ECV. The proteome composition of ECV was well correlated with its biochemical and pharmacological properties and clinical manifestations observed in Echis envenomed patients. Neutralization of enzymes and pharmacological properties of ECV, and immuno-cross-reactivity studies unequivocally point to the poor recognition of <20 kDa ECV proteins, such as PLA2, subunits of snaclec, and disintegrin by commercial polyvalent antivenom.

Biological and molecular properties of yellow venom of the Amazonian coral snake Micrurus surinamensis.

INTRODUCTION:: The coral snake Micrurus surinamensis, which is widely distributed throughout Amazonia, has a neurotoxic venom. It is important to characterize the biological and molecular properties of this venom in order to develop effective antitoxins. METHODS:: Toxins from the venom of M. surinamensis were analyzed by two-dimensional polyacrylamide gel electrophoresis and their neurotoxic effects in vivo were evaluated. RESULTS AND CONCLUSIONS:: Most proteins in the venom had masses < 14kDa, low phospholipase A2 activity, and no proteolytic activity. The toxins inhibited the coagulation cascade. The venom had neurotoxic effects in mice, with a median lethal dose upon intravenous administration of 700 µg/kg. Immunogenic studies revealed abundant cross-reactivity of antielapidic serum with 14kDa toxins and limited cross-reactivity with toxins < 10kDa. These results indicate that antielapidic serum against M. surinamensis venom has weak potency (0.35mg/ml) in mice.

Biological and molecular properties of yellow venom of the Amazonian coral snake Micrurus surinamensis

Abstract INTRODUCTION: The coral snake Micrurus surinamensis, which is widely distributed throughout Amazonia, has a neurotoxic venom. It is important to characterize the biological and molecular properties of this venom in order to develop effective antitoxins. METHODS: Toxins from the venom of M. surinamensis were analyzed by two-dimensional polyacrylamide gel electrophoresis and their neurotoxic effects in vivo were evaluated. RESULTS AND CONCLUSIONS: Most proteins in the venom had masses < 14kDa, low phospholipase A2 activity, and no proteolytic activity. The toxins inhibited the coagulation cascade. The venom had neurotoxic effects in mice, with a median lethal dose upon intravenous administration of 700 µg/kg. Immunogenic studies revealed abundant cross-reactivity of antielapidic serum with 14kDa toxins and limited cross-reactivity with toxins < 10kDa. These results indicate that antielapidic serum against M. surinamensis venom has weak potency (0.35mg/ml) in mice.