Revealing molecular determinants governing mambalgin-3 pharmacology at acid-sensing ion channel 1 variants.

Acid-sensing ion channels (ASICs) are trimeric proton-gated cation channels that play a role in neurotransmission and pain sensation. The snake venom-derived peptides, mambalgins, exhibit potent analgesic effects in rodents by inhibiting central ASIC1a and peripheral ASIC1b. Despite their distinct species- and subtype-dependent pharmacology, previous structure-function studies have focussed on the mambalgin interaction with ASIC1a. Currently, the specific channel residues responsible for this pharmacological profile, and the mambalgin pharmacophore at ASIC1b remain unknown. Here we identify non-conserved residues at the ASIC1 subunit interface that drive differences in the mambalgin pharmacology from rat ASIC1a to ASIC1b, some of which likely do not make peptide binding interactions. Additionally, an amino acid variation below the core binding site explains potency differences between rat and human ASIC1. Two regions within the palm domain, which contribute to subtype-dependent effects for mambalgins, play key roles in ASIC gating, consistent with subtype-specific differences in the peptides mechanism. Lastly, there is a shared primary mambalgin pharmacophore for ASIC1a and ASIC1b activity, with certain peripheral peptide residues showing variant-specific significance for potency. Through our broad mutagenesis studies across various species and subtype variants, we gain a more comprehensive understanding of the pharmacophore and the intricate molecular interactions that underlie ligand specificity. These insights pave the way for the development of more potent and targeted peptide analogues required to advance our understating of human ASIC1 function and its role in disease.

Knowledge about Snake Venoms and Toxins from Colombia: A Systematic Review.

Colombia encompasses three mountain ranges that divide the country into five natural regions: Andes, Pacific, Caribbean, Amazon, and Orinoquia. These regions offer an impressive range of climates, altitudes, and landscapes, which lead to a high snake biodiversity. Of the almost 300 snake species reported in Colombia, nearly 50 are categorized as venomous. This high diversity of species contrasts with the small number of studies to characterize their venom compositions and natural history in the different ecoregions. This work reviews the available information about the venom composition, isolated toxins, and potential applications of snake species found in Colombia. Data compilation was conducted according to the PRISMA guidelines, and the systematic literature search was carried out in Pubmed/MEDLINE. Venom proteomes from nine Viperidae and three Elapidae species have been described using quantitative analytical strategies. In addition, venoms of three Colubridae species have been studied. Bioactivities reported for some of the venoms or isolated components-such as antibacterial, cytotoxicity on tumoral cell lines, and antiplasmodial properties-may be of interest to develop potential applications. Overall, this review indicates that, despite recent progress in the characterization of venoms from several Colombian snakes, it is necessary to perform further studies on the many species whose venoms remain essentially unexplored, especially those of the poorly known genus Micrurus.

Identification evidence unraveled by strict proteomics rules toward forensic samples.

Snake venom is a complex mixture of proteins and peptides secreted by venomous snakes from their poison glands. Although proteomics for snake venom composition, interspecific differences, and developmental evolution has been developed for a decade, current diagnosis or identification techniques of snake venom in clinical intoxication and forensic science applications are mainly dependent on morphological and immunoassay. It could be expected that the proteomics techniques directly offer great help. This work applied a bottom-up proteomics method to identify proteins' types and species attribution in suspected snake venom samples using ultrahigh-performance liquid chromatography-quadrupole-electrostatic field Orbitrap tandem mass spectrometric technique, and cytotoxicity assay was amended to provide a direct evidence of toxicity. Toward the suspicious samples seized in the security control, sample pretreatment (in-sol and in-gel digestion) and data acquisition (nontargeted and targeted screening) modes complemented and validated each other. We have implemented two consequent approaches in identifying the species source of proteins in the samples via the points of venom proteomics and strict forensic identification. First, we completed a workflow consisting of a proteomics database match toward an entire SWISS-PROT (date 2018-11-22) database and a result-directed specific taxonomy database. The latter was a helpful hint to compare master protein kinds and reveal the insufficiency of specific venom proteomics characterization rules. Second, we suggested strict rules for protein identification to meet the requirements of forensic science on improved identification correctness, that is, (1) peptide spectrum matches confidence, peptide confidence, and protein confidence were both high (with the false-discovery ratio less than 1%); (2) the number of unique peptides was more than or equal to two in one protein, and (3) within unique peptides, which at least 75% of the ∆m/z of the matched y and b ions were less than 5 ppm. We identified these samples as cobra venom containing 10 highly abundant proteins (P00597, P82463, P60770, Q9YGI4, P62375, P49123, P80245, P60302, P01442, and P60304) from two snake venom protein families (acid phospholipase A2 and three-finger toxins), and the most abundant proteins were cytotoxins.

Venom Variation of Neonate and Adult Chinese Cobras in Captivity Concerning Their Foraging Strategies.

The venom and transcriptome profile of the captive Chinese cobra (Naja atra) is not characterized until now. Here, LC-MS/MS and illumine technology were used to unveil the venom and trascriptome of neonates and adults N. atra specimens. In captive Chinese cobra, 98 co-existing transcripts for venom-related proteins was contained. A total of 127 proteins belong to 21 protein families were found in the profile of venom. The main components of snake venom were three finger toxins (3-FTx), snake venom metalloproteinase (SVMP), cysteine-rich secretory protein (CRISP), cobra venom factor (CVF), and phosphodiesterase (PDE). During the ontogenesis of captive Chinese cobra, the rearrangement of snake venom composition occurred and with obscure gender difference. CVF, 3-FTx, PDE, phospholipase A2 (PLA2) in adults were more abundant than neonates, while SVMP and CRISP in the neonates was richer than the adults. Ontogenetic changes in the proteome of Chinese cobra venom reveals different strategies for handling prey. The levels of different types of toxin families were dramatically altered in the wild and captive specimens. Therefore, we speculate that the captive process could reshape the snake venom composition vigorously. The clear comprehension of the composition of Chinese cobra venom facilitates the understanding of the mechanism of snakebite intoxication and guides the preparation and administration of traditional antivenom and next-generation drugs for snakebite.

Generation of Multivalent Nanobody-Based Proteins with Improved Neutralization of Long α-Neurotoxins from Elapid Snakes.

Recombinantly produced biotherapeutics hold promise for improving the current standard of care for snakebite envenoming over conventional serotherapy. Nanobodies have performed well in the clinic, and in the context of antivenom, they have shown the ability to neutralize long α-neurotoxins in vivo. Here, we showcase a protein engineering approach to increase the valence and hydrodynamic size of neutralizing nanobodies raised against a long α-neurotoxin (α-cobratoxin) from the venom of the monocled cobraNaja kaouthia. Based on the p53 tetramerization domain, a panel of anti-α-cobratoxin nanobody-p53 fusion proteins, termed Quads, were produced with different valences, inclusion or exclusion of Fc regions for endosomal recycling purposes, hydrodynamic sizes, and spatial arrangements, comprising up to 16 binding sites. Measurements of binding affinity and stoichiometry showed that the nanobody binding affinity was retained when incorporated into the Quad scaffold, and all nanobody domains were accessible for toxin binding, subsequently displaying increased blocking potency in vitro compared to the monomeric format. Moreover, functional assessment using automated patch-clamp assays demonstrated that the nanobody and Quads displayed neutralizing effects against long α-neurotoxins from both N. kaouthia and the forest cobra N. melanoleuca. This engineering approach offers a means of altering the valence, endosomal recyclability, and hydrodynamic size of existing nanobody-based therapeutics in a simple plug-and-play fashion and can thus serve as a technology for researchers tailoring therapeutic properties for improved neutralization of soluble targets such as snake toxins.

MT9, a natural peptide from black mamba venom antagonizes the muscarinic type 2 receptor and reverses the M2R-agonist-induced relaxation in rat and human arteries.

All five muscarinic receptors have important physiological roles. The endothelial M2 and M3 subtypes regulate arterial tone through direct coupling to Gq or Gi/o proteins. Yet, we lack selective pharmacological drugs to assess the respective contribution of muscarinic receptors to a given function. We used mamba snake venoms to identify a selective M2R ligand to investigate its contribution to arterial contractions. Using a bio-guided screening binding assay, we isolated MT9 from the black mamba venom, a three-finger toxin active on the M2R subtype. After sequencing and chemical synthesis of MT9, we characterized its structure by X-ray diffraction and determined its pharmacological characteristics by binding assays, functional tests, and ex vivo experiments on rat and human arteries. Although MT9 belongs to the three-finger fold toxins family, it is phylogenetically apart from the previously discovered muscarinic toxins, suggesting that two groups of peptides evolved independently and in a convergent way to target muscarinic receptors. The affinity of MT9 for the M2R is 100 times stronger than that for the four other muscarinic receptors. It also antagonizes the M2R/Gi pathways in cell-based assays. MT9 acts as a non-competitive antagonist against acetylcholine or arecaine, with low nM potency, for the activation of isolated rat mesenteric arteries. These results were confirmed on human internal mammary arteries. In conclusion, MT9 is the first fully characterized M2R-specific natural toxin. It should provide a tool for further understanding of the effect of M2R in various arteries and may position itself as a new drug candidate in cardio-vascular diseases.

Serum metabolomics of Bama miniature pigs bitten by Bungarus multicinctus.

Bungarus multicinctus is one of the top ten venomous snakes in China, and its bite causes acute and severe diseases, but its pathophysiology remains poorly elucidated. Thus, an animal model of Bungarus multicinctus bite was established by intramuscular injection of 30µg/kg of Bungarus multicinctus venom, and then the serum metabolites were subsequently screened, identified and validated by ultra-performance liquid chromatography-quadrupole-time of flight-mass spectrometry (UPLC-Q-TOF-MS) methods to explore the potential biomakers and possible metabolic pathways. Untargeted metabolomics analysis showed that 36 and 38 endogenous metabolites levels changed in ESI+ and ESI-, respectively, KEGG pathway analysis showed that 5 metabolic pathways, including mineral absorption, central carbon metabolism in cancer, protein digestion and absorption, aminoacyl-tRNA biosynthesis and ABC transporters might be closely related to Bungarus multicinctus bite. Targeted metabolomics analysis showed that there were significant differences in serum D-proline, L-leucine and L-glutamine after Bungarus multicinctus bite (P < 0.05). In addition, receiver operating characteristic (ROC) analysis showed that the diagnostic efficiency of L-Glutamine was superior to other potential biomarkers and the AUC value was 0.944. Moreover, we found evidence for differences in the pathophysiology of glutamine between Bungarus multicinctus bite group and normal group, specifically with the content of glutamine synthetase (GS) and glutaminase (GLS). Taken together, the current study has successfully established an animal model of Bungarus multicinctus bite, and further identified the links between the metabolic perturbations and the pathophysiology and the potential diagnostic biomakers of Bungarus multicinctus bite, which provided valuable insights for studying the mechanism of Bungarus multicinctus bite.

Novel Three-Finger Neurotoxins from Naja melanoleuca Cobra Venom Interact with GABAA and Nicotinic Acetylcholine Receptors.

Cobra venoms contain three-finger toxins (TFT) including α-neurotoxins efficiently binding nicotinic acetylcholine receptors (nAChRs). As shown recently, several TFTs block GABAA receptors (GABAARs) with different efficacy, an important role of the TFTs central loop in binding to these receptors being demonstrated. We supposed that the positive charge (Arg36) in this loop of α-cobratoxin may explain its high affinity to GABAAR and here studied α-neurotoxins from African cobra N. melanoleuca venom for their ability to interact with GABAARs and nAChRs. Three α-neurotoxins, close homologues of the known N. melanoleuca long neurotoxins 1 and 2, were isolated and sequenced. Their analysis on Torpedocalifornica and α7 nAChRs, as well as on acetylcholine binding proteins and on several subtypes of GABAARs, showed that all toxins interacted with the GABAAR much weaker than with the nAChR: one neurotoxin was almost as active as α-cobratoxin, while others manifested lower activity. The earlier hypothesis about the essential role of Arg36 as the determinant of high affinity to GABAAR was not confirmed, but the results obtained suggest that the toxin loop III may contribute to the efficient interaction of some long-chain neurotoxins with GABAAR. One of isolated toxins manifested different affinity to two binding sites on Torpedo nAChR.

Electric Blue: Molecular Evolution of Three-Finger Toxins in the Long-Glanded Coral Snake Species Calliophis bivirgatus.

The genus Calliophis is the most basal branch of the family Elapidae and several species in it have developed highly elongated venom glands. Recent research has shown that C. bivirgatus has evolved a seemingly unique toxin (calliotoxin) that produces spastic paralysis in their prey by acting on the voltage-gated sodium (NaV) channels. We assembled a transcriptome from C. bivirgatus to investigate the molecular characteristics of these toxins and the venom as a whole. We find strong confirmation that this genus produces the classic elapid eight-cysteine three-finger toxins, that δδ-elapitoxins (toxins that resemble calliotoxin) are responsible for a substantial portion of the venom composition, and that these toxins form a distinct clade within a larger, more diverse clade of C. bivirgatus three-finger toxins. This broader clade of C. bivirgatus toxins also contains the previously named maticotoxins and is somewhat closely related to cytotoxins from other elapids. However, the toxins from this clade that have been characterized are not themselves cytotoxic. No other toxins show clear relationships to toxins of known function from other species.

A unique snake venom neuritogenesis mechanism: A cornerstone in the treatment of neurodegenerative diseases?: An Editorial Highlight for "Transcriptomic, proteomic, and biochemical analyses reveal a novel neuritogenesis mechanism of Naja naja venom α-elapitoxin post binding to TrkA receptor of rat pheochromocytoma cells" on 612.

Neurodegenerative diseases are a worldwide health problem and are a major cause of death and disability. A progressive loss of defined neuronal populations is triggered by a diverse array of stimuli that converge in deficient neurotrophic signaling. Therefore, much effort has been placed in recent years in the characterization of the molecular mechanisms associated with the structure and function of neurotrophins, its receptors, signaling strategies, and their target genes. This Editorial highlights an impressive study by the group of Prof. Ashis K. Mukherjee, a renowned specialist in snake venoms, in which a component of the Indian Cobra N.naja venom with no significant similarity to nerve growth factor, is shown to induce sustained neuritogenesis. An elegant transcriptomic and functional analysis of this component, named Nn-α-elapitoxin, mapped novel domains in mammalian neurotrophic receptors that trigger both conventional and novel signal cascades that support neurite extension in the PC-12 neuronal model system. The authors discuss their findings in the context of the paradoxical neurite outgrowth properties of this toxin which originate in their unique receptor binding site. This study takes an important step towards a better understanding of the complexity of neuronal development and maintenance of the nervous system and provides a potential target to improve neurotrophic signaling, independent of endogenous growth factors, in the diseased brain.

Evolutionary Adaptations in Pseudonaja Textilis Venom Factor X Induce Zymogen Activity and Resistance to the Intrinsic Tenase Complex.

The venom of the Australian snake Pseudonaja textilis comprises powerful prothrombin activators consisting of factor X (v-ptFX)- and factor V-like proteins. While all vertebrate liver-expressed factor X (FX) homologs, including that of P. textilis, comprise an activation peptide of approximately 45 to 65 residues, the activation peptide of v-ptFX is significantly shortened to 27 residues. In this study, we demonstrate that exchanging the human FX activation peptide for the snake venom ortholog impedes proteolytic cleavage by the intrinsic factor VIIIa-factor IXa tenase complex. Furthermore, our findings indicate that the human FX activation peptide comprises an essential binding site for the intrinsic tenase complex. Conversely, incorporation of FX into the extrinsic tissue factor-factor VIIa tenase complex is completely dependent on exosite-mediated interactions. Remarkably, the shortened activation peptide allows for factor V-dependent prothrombin conversion while in the zymogen state. This indicates that the active site of FX molecules comprising the v-ptFX activation peptide partially matures upon assembly into a premature prothrombinase complex. Taken together, the shortened activation peptide is one of the remarkable characteristics of v-ptFX that has been modified from its original form, thereby transforming FX into a powerful procoagulant protein. Moreover, these results shed new light on the structural requirements for serine protease activation and indicate that catalytic activity can be obtained without formation of the characteristic Ile16-Asp194 salt bridge via modification of the activation peptide.

Molecular Mechanism by which Cobra Venom Cardiotoxins Interact with the Outer Mitochondrial Membrane.

Cardiotoxin CTII from Najaoxiana cobra venom translocates to the intermembrane space (IMS) of mitochondria to disrupt the structure and function of the inner mitochondrial membrane. At low concentrations, CTII facilitates ATP-synthase activity, presumably via the formation of non-bilayer, immobilized phospholipids that are critical in modulating ATP-synthase activity. In this study, we investigated the effects of another cardiotoxin CTI from Najaoxiana cobra venom on the structure of mitochondrial membranes and on mitochondrial-derived ATP synthesis. By employing robust biophysical methods including 31P-NMR and 1H-NMR spectroscopy, we analyzed the effects of CTI and CTII on phospholipid packing and dynamics in model phosphatidylcholine (PC) membranes enriched with 2.5 and 5.0 mol% of cardiolipin (CL), a phospholipid composition that mimics that in the outer mitochondrial membrane (OMM). These experiments revealed that CTII converted a higher percentage of bilayer phospholipids to a non-bilayer and immobilized state and both cardiotoxins utilized CL and PC molecules to form non-bilayer structures. Furthermore, in order to gain further understanding on how cardiotoxins bind to mitochondrial membranes, we employed molecular dynamics (MD) and molecular docking simulations to investigate the molecular mechanisms by which CTII and CTI interactively bind with an in silico phospholipid membrane that models the composition similar to the OMM. In brief, MD studies suggest that CTII utilized the N-terminal region to embed the phospholipid bilayer more avidly in a horizontal orientation with respect to the lipid bilayer and thereby penetrate at a faster rate compared with CTI. Molecular dynamics along with the Autodock studies identified critical amino acid residues on the molecular surfaces of CTII and CTI that facilitated the long-range and short-range interactions of cardiotoxins with CL and PC. Based on our compiled data and our published findings, we provide a conceptual model that explains a molecular mechanism by which snake venom cardiotoxins, including CTI and CTII, interact with mitochondrial membranes to alter the mitochondrial membrane structure to either upregulate ATP-synthase activity or disrupt mitochondrial function.

Transcriptomic, proteomic, and biochemical analyses reveal a novel neuritogenesis mechanism of Naja naja venom α-elapitoxin post binding to TrkA receptor of rat pheochromocytoma cells.

This is the first report showing unique neuritogenesis potency of Indian Cobra N. naja venom long-chain α-neurotoxin (Nn-α-elapitoxin-1) exhibiting no sequence similarity to conventional nerve growth factor, by high-affinity binding to its tyrosine kinase A (TrkA) receptor of rat pheochromocytoma (PC-12) cells without requiring low-affinity receptor p75NTR. The binding residues between Nn-α-elapitoxin-1 and mammalian TrkA receptor are predicted by in silico analysis. This binding results in a time-dependent internalization of TrkA receptor into the cytoplasm of PC-12 cells. The transcriptomic analysis has demonstrated the differential expression of 445 genes; 38 and 32 genes are up-regulated and down-regulated, respectively in the PC-12 cells post-treatment with Nn-α-elapitoxin-1. Global proteomic analysis in concurrence with transcriptomic data has also demonstrated that in addition to expression of a large number of common intracellular proteins in the control and Nn-α-elapitoxin-1-treated PC-12 cells, the latter cells also showed the expression of uniquely up-regulated and down-regulated intracellular proteins involved in diverse cellular functions. Altogether, the data from transcriptomics, proteomics, and inhibition of downstream signaling pathways by specific inhibitors, and the immunoblot analysis of major regulators of signaling pathways of neuritogenesis unambiguously demonstrate that, similar to mouse 2.5S-nerve growth factor, the activation of mitogen activated protein kinase/extracellular signal-regulated kinase is the major signaling pathway for neuritogenesis by Nn-α-elapitoxin-1. Nonetheless, fibroblast growth factor signaling and heterotrimeric G-protein signaling pathways were found to be uniquely expressed in Nn-α-elapitoxin-1-treated PC-12 cells and not in mouse 2.5S-nerve growth factor -treated cells. The TrkA binding region of Nn-α-elapitoxin-1 may be developed as a peptide-based drug prototype for the treatment of major central neurodegenerative diseases. Read the Editorial Highlight for this article on page 599.

Antibacterial activity of cardiotoxin-like basic polypeptide from cobra venom.

Antibacterial activity of the three-finger toxins from cobra venom, including cytotoxin 3 from N. kaouthia, cardiotoxin-like basic polypeptide A5 from N. naja (CLBP), and alpha-neurotoxin from N. oxiana venom, was investigated. All toxins failed to influence Gram-negative bacteria. The most pronounced activity against Bacillus subtilis was demonstrated by CLBP. The latter is ascribed to the presence of additional Lys-residues within the membrane-binding motif of this toxin.

Hydrostatin-SN10 Ameliorates Pancreatitis-Induced Lung Injury by Affecting IL-6-Induced JAK2/STAT3-Associated Inflammation and Oxidative Stress.

Hydrostatin-SN1 (peptide sequence, DEQHLETELHTLTSVLTANGFQ), a kind of peptides extracted from snake venom, has been reported to have anti-inflammatory effect, but its truncated mutant hydrostatin-SN10 (peptide sequence, DEQHLETELH) on pancreatitis-induced acute lung injury has not been well documented. Interleukin- (IL-) 6-induced Janus Kinase 2/Signal Transducer and Activator of Transcription 3 (JAK2/STAT3) pathway is involved with inflammatory and oxidative stress activities and may be associated with the pathogenesis of lung injury, and related molecules were measured. Taurocholate-induced pancreatitis associated with acute lung injury was established and treated with hydrostatin-SN10. Pancreatitis was confirmed by measuring the serum levels of amylase, lipase, and trypsinogen and urinary amylase. Lung injury was determined by histologically assessing acinar cell changes. The related molecules of IL-6-induced JAK2/STAT3-associated inflammation and oxidative stress were quantitated by real time-PCR, Western blot, and/or immunochemical assay. Hydrostatin-SN10 reduced the levels of serum amylase, lipase, and trypsinogen and urinary amylase when compared with the model group (p < 0.05). Hydrostatin-SN10 significantly inhibited the IL-6-stimulated JAK2/STAT3 pathway and reduced the number of apoptotic cells via the downregulation of caspase 3 and BAX (proapoptotic) and upregulation of Bcl2 (antiapoptotic) (p < 0.05). IL-6 induced the increase in the levels of JAK2 and STAT3, which was reversed by hydrostatin-SN10 treatment (p < 0.05). In addition, hydrostatin-SN10 reduced the expression of IL-6 and TNF- (tumor necrosis factor-) α and increased the level of IL-10 (p < 0.05). On the other hand, hydrostatin-SN10 treatment increased the levels of superoxide dismutase (SOD) and reduced glutathione (GSH) and the levels of malondialdehyde (MDA) and alanine aminotransferase (ALT) (p < 0.05). These results suggest that hydrostatin-SN10 may inhibit pancreatitis-induced acute lung injury by affecting IL-6-mediated JAK2/STAT3 pathway-associated inflammation and oxidative stress.

Variation in the Protein Composition and Biological Activity of King Cobra (Ophiophagus hannah) Venoms.

The biochemical properties and biological activities of the venom from three individual Ophiophagus hannah (King cobra) specimens was compared. The toxicity against mice, the cytotoxicity against five cell lines, and the antioxidant activity were measured. The KV2 venom showed a higher cytotoxicity than the KV6 and the non-cytotoxic KV9 venoms. Comparative analysis of the O. hannah venom proteins was performed after 2-dimensional (2-D) denaturing gel electrophoresis and reverse phase high performance liquid chromatography (RP-HPLC). 2-D analysis by isoelectric focusing (IEF) Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) resolution of the venoms revealed significant differences between all three venoms, with most spots being unique to that venom. Only 2 out of the 13-16 distinct spots were common to all three venoms, and four spots were common to KV6 and KV9. KV2 had the highest proportion of low molecular mass spots, and KV6 and KV9 appeared more related to each other than to KV9. From peptide mass mapping by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and MASCOT-based amino acid sequence database searching, the two venom proteins that were common to all three specimens are likely to be ophanin and acidic phospholipase A2 (PLA2), whilst the proteins unique to the cytotoxic KV2 venom, included three other PLA2 proteins. The RP-HPLC pattern of KV2 was different from the other two venoms with a higher protein concentration eluting in the 31-41% (v/v) acetonitrile (ACN) fraction than for the other two venoms.

Cytotoxicity of Nubein6.8 peptide isolated from the snake venom of Naja nubiae on melanoma and ovarian carcinoma cell lines.

This study was conducted to examine the cytotoxic effects of Nubein6.8 isolated from the venom of the Egyptian Spitting Cobra Naja nubiae on melanoma (A375) and ovarian carcinoma cell lines and to reveal its mode of action. The size of Nubein6.8 (6801.8 Da) and its N-terminal sequence are similar to cytotoxins purified from the venom of other spitting cobras. Nubein6.8 showed a high significant cytotoxic effect on A375 cell line and moderate effect on A2780. A clonogenic assay showed that Nubein6.8 has a significant long-term potency on A375 cell survival when compared to A2780. The molecular intracellular signaling pathways of Nubein6.8 have been investigated using Western blotting analysis, flow cytometry, and microscale protein labeling. This data revealed that Nubein6.8 has DNA damaging effects and the ability to activate apoptosis in both tumor cell lines. Cellular uptake recordings revealed that the labeled-Nubein6.8 was intracellularly present in A375 cells while A2780 displayed resistance against it. SEM examination showed that Nubein6.8 was found to have high accessibility to malignant melanoma cells. The apoptotic effect of Nubein6.8 was confirmed by TEM examination that revealed many evident characteristics for Nubein6.8 apoptotic efficacy on A375 cell sections. Also, TEM reflected many resistant characteristics that faced Nubein6.8 acquisition through ovarian carcinoma cell sections. Accordingly, the snake venom peptide of Nubein6.8 is a promising template for developing potential cytotoxic agents targeting human melanoma and ovarian carcinoma.

Venom gland transcriptomics and microRNA profiling of juvenile and adult yellow-bellied sea snake, Hydrophis platurus, from Playa del Coco (Guanacaste, Costa Rica).

With an extensive range in tropical and subtropical waters of Indo-Australian region and the Pacific coast of the American continent, the yellow-bellied sea snake, Hydrophis platurus, is the most broadly distributed snake species on our planet. We report a comparative analysis of the mRNA and microRNA transcriptional profiles of the venom glands of Costa Rican juvenile and adult yellow-bellied sea snakes, and correlate these datasets with the previously reported venom proteome gathered from an adult specimen of the same population of Playa del Coco. The transcriptomic profiles are qualitatively and quantitatively remarkably similar across both age classes, being overwhelmingly (>99%) dominated by only three toxin classes, three-finger toxins (3FTx, 88%), phospholipases A2 (PLA2, 10-11%), and cysteine-rich secretory proteins (CRISP, 1%). The only appreciable difference between the transcriptomes of juvenile and adult Costa Rican yellow-bellied sea snake lies in the relative abundance of short and long neurotoxic 3FTxs. Comparison of venom gland transcriptome and venom proteome datasets suggest that the venom arsenal of adult H. platurus from Playa del Coco (CR) is subjected to post-transcriptional modulation, involving repression of the translation of pelamitoxin a-encoding mRNAs and enhancement of the translational activity of PLA2 and CRISP mRNAs.

Proteomic and toxinological characterization of the venom of the South African Ringhals cobra Hemachatus haemachatus.

The protein composition and toxinological profile of the venom of the African spitting elapid Hemachatus haemachatus (Ringhals) were characterized by bottom-up proteomics and functional in vitro and in vivo assays. Venom is composed of abundant three-finger toxins (3FTxs; 63.3%), followed by phospholipases A2 (PLA2s; 22.8%), snake venom metalloproteinases (SVMPs; 7.1%), cysteine-rich secretory proteins (CRISPs; 4.1%) and Kunitz type protease inhibitors (KTPIs; 1.5%). 3FTxs are the main responsible for lethality and myotoxicity in mice and in vitro anticoagulant activity. In contrast to closely related spitting species, whose venom 3FTxs induces dermonecrosis, the 3FTxs of H. haemachatus did not induce dermonecrotic activity. The venom showed in vitro PLA2 activity, and most likely PLA2s contribute to some extent in venom lethality, as judged by partial reduction in toxicity after inhibition of their catalytic activity. Despite its relatively high content of SVMPs, compared to most elapids, the venom of H. haemachatus did not exert hemorrhagic effect, proteolytic activity on azocasein or defibrinogenating activity. Toxicovenomic characterization of H. haemachatus venom revealed that RP-HPLC fractions with higher abundance of 3FTxs presented lethal activity, while fractions with high content of PLA2s did not, underscoring the role of 3FTxs in the pathophysiology caused by this venom. BIOLOGICAL SIGNIFICANCE: The proteomic composition and toxinological profile of the venom of Ringhals snake, Hemachatus haemachatus, a cobra-like spitting snake endemic to southern Africa, were investigated. In vitro, Ringhals venom showed anticoagulant and phospholipase A2 activities, but was devoid of proteolytic activity on azocasein. In mice, venom induced lethality and myotoxicity, but no local hemorrhage or dermonecrosis. The lack of dermonecrotic activity is in sharp contrast to venoms of closely related spitting cobras which present a similar relative abundance of 3FTxs but are potently dermonecrotic. 3FTxs, the most abundant protein family in the venom, are predominantly responsible for toxic effects. PLA2 enzyme inactivation experiments suggest that H. haemachatus venom lethality is not dependent on PLA2s, but instead is more related to neurotoxic or cardiotoxic 3FTxs. The characterization of this venom, based on proteomic and toxicovenomic approaches, is useful for more in depth studies associated with biogeography, phylogeny, toxinology and antivenom efficacy towards the venom of this species, and its association with related elapids.

First Look at the Venom of Naja ashei.

Naja ashei is an African spitting cobra species closely related to N. mossambica and N. nigricollis. It is known that the venom of N. ashei, like that of other African spitting cobras, mainly has cytotoxic effects, however data about its specific protein composition are not yet available. Thus, an attempt was made to determine the venom proteome of N. ashei with the use of 2-D electrophoresis and MALDI ToF/ToF (Matrix-Assisted Laser Desorption/Ionization Time of Flight) mass spectrometry techniques. Our investigation revealed that the main components of analysed venom are 3FTxs (Three-Finger Toxins) and PLA2s (Phospholipases A2). Additionally the presence of cysteine-rich venom proteins, 5'-nucleotidase and metalloproteinases has also been confirmed. The most interesting fact derived from this study is that the venom of N. ashei includes proteins not described previously in other African spitting cobras-cobra venom factor and venom nerve growth factor. To our knowledge, there are currently no other reports concerning this venom composition and we believe that our results will significantly increase interest in research of this species.