ß-keto amyrin isolated from Cryptostegia grandiflora R. br. inhibits inflammation caused by Daboia russellii viper venom: Direct binding of ß-keto amyrin to phospholipase A2.

The search for mechanism-based anti-inflammatory therapies is of fundamental importance to avoid undesired off-target effects. Phospholipase A2 (PLA2) activity is a potential molecular target for anti-inflammatory drugs because it fuels arachidonic acid needed to synthesize inflammation mediators, such as prostaglandins. Herein, we aim to investigate the molecular mechanism by which ß-keto amyrin isolated from a methanolic extract of Cryptostegia grandiflora R. Br. Leaves can inhibit inflammation caused by Daboia russellii viper (DR) venom that mainly contains PLA2. We found that ß-keto amyrin neutralizes DR venom-induced paw-edema in a mouse model. Molecular docking of PLA2 with ß-keto amyrin complex resulted in a higher binding energy score of -8.86 kcal/mol and an inhibition constant of 611.7 nM. Diclofenac had a binding energy of -7.04 kcal/mol and an IC50 value of 620 nM, which predicts a poorer binding interaction than ß-keto amyrin. The higher conformational stability of ß-keto amyrin interaction compared to diclofenac is confirmed by molecular dynamics simulation. ß-keto amyrin isolated from C. grandiflora inhibits the PLA2 activity contained in Daboia russellii viper venom. The anti-inflammatory property of ß-keto amyrin is due to its direct binding into the active site of PLA2, thus inhibiting its enzyme activity.

Viper Venom Phospholipase A2 Database: The Structural and Functional Anatomy of a Primary Toxin in Envenomation.

Viper venom phospholipase A2 enzymes (vvPLA2s) and phospholipase A2-like (PLA2-like) proteins are two of the principal toxins in viper venom that are responsible for the severe myotoxic and neurotoxic effects caused by snakebite envenoming, among other pathologies. As snakebite envenoming is the deadliest neglected tropical disease, a complete understanding of these proteins' properties and their mechanisms of action is urgently needed. Therefore, we created a database comprising information on the holo-form, cofactor-bound 3D structure of 217 vvPLA2 and PLA2-like proteins in their physiologic environment, as well as 79 membrane-bound viper species from 24 genera, which we have made available to the scientific community to accelerate the development of new anti-snakebite drugs. In addition, the analysis of the sequenced, 3D structure of the database proteins reveals essential aspects of the anatomy of the proteins, their toxicity mechanisms, and the conserved binding site areas that may anchor universal interspecific inhibitors. Moreover, it pinpoints hypotheses for the molecular origin of the myotoxicity of the PLA2-like proteins. Altogether, this study provides an understanding of the diversity of these toxins and how they are conserved, and it indicates how to develop broad, interspecies, efficient small-molecule inhibitors to target the toxin's many mechanisms of action.

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

Snake venoms are known to contain toxins capable of interfering with normal physiological processes of victims. Specificity of toxins from snake venoms give scope to identify new molecules with therapeutic action and/or help to understand different cellular mechanisms. Russell's viper venom (RVV) is a mixture of many bioactive molecules with enzymatic and non-enzymatic proteins. The present article describes Daboialipase (DLP), an enzymatic phospholipase A2 with molecular mass of 14.3 kDa isolated from RVV. DLP was obtained after cation exchange chromatography followed by size-exclusion high performance liquid chromatography (SE-HPLC). The isolated DLP presented strong inhibition of adenosine di-phosphate (ADP) and collagen induced platelet aggregation. It also showed anti-thrombin properties by significantly extending thrombin time in human blood samples. Trypan blue and resazurin cell viability assays confirmed time-dependent cytotoxic and cytostatic activities of DLP on MCF7 breast cancer cells, in vitro. DLP caused morphological changes and nuclear damage in MCF7 cells. However, DLP did not cause cytotoxic effects on non-cancer HaCaT cells. Peptide sequences of DLP obtained by O-HRLCMS analysis showed similarity with a previously reported PLA2 (Uniprot ID: PA2B_DABRR/PDB ID: 1VIP_A). An active Asp at 49th position, calcium ion binding site and anticoagulant activity sites were identified in 1 VIP_A. These findings are expected to contribute to designing new anti-platelet, anticoagulant and anti-cancer molecules.

Proteome analysis of Daboia russelii venom, a medically important snake from the Indian sub-continent.

Daboia russelii is a category-I medically important snake throughout the Indian sub-continent contributing to majority of snakebite incidences in this part of the world. As such, extensive studies on its venom composition and search of efficient and appropriate interventions for its treatment become crucial. In this study, the proteome of Daboia russelii venom from Tanore, Rajshahi, Bangladesh was profiled using a combination of chromatographic and mass spectrometric techniques. A total of 37 different proteins belonging to 11 different snake venom protein families were detected. Proteomics analysis revealed the presence of major phospholipase A2 toxins. Daboiatoxin (both A and B subunits), the main lethal PLA2 toxin in the venom of Daboia siamensis (Myanmar viper) which is neurotoxic, myotoxic and cytotoxic was detected. Presence of Daboxin P, which is a major protein in the venom of Indian Daboia russelii with strong anticoagulant activity, was also observed. Inconsistent distribution of such lethal toxins in the venom of same species calls for more investigations of snake venoms from lesser explored regions and formulation of better alternatives to the current antivenom therapy for efficient treatment.

Composition characterization of various viperidae snake venoms using MS-based proteomics N-glycoproteomics and N-glycomics.

Viperidae snake species is widely abundant and responsible for most envenomation cases in Turkey. The structural and compositional profiles of snake venom have been investigated to study the venom component variation across different species and to profile the venom biological activity variation against prey. In this context, we used proteomics, glycoproteomics and glycomics strategies to characterize the protein, glycoproteins and glycan structural and compositional profiles of various snake venoms in the Viperidae family. Moreover, we compared these profiles using the downstream bioinformatics and machine learning classification modules. The overall mass spectrometry profiles identified 144 different proteins, 36 glycoproteins and 78 distinct N-glycan structures varying in composition across the five venoms. A high amount of the characterized proteins belongs to the glycosylated protein family Trypsin-like serine protease (Tryp_SPc), Disintegrin (DISIN), and ADAM Cysteine-Rich (ACR). Most identified N-glycans have a complex chain carrying galactosylated N-glycans abundantly. The glycan composition data obtained from glycoproteomics aligns consistently with the findings from glycomics. The clustering and principal component analyses (PCA) illustrated the composition-based similarities and differences between each snake venom species' proteome, glycoproteome and glycan profiles. Specifically, the N-glycan profiles of M. xanthina (Mx) and V. a. ammodytes (Vaa) venoms were identical and difficult to differentiate; in contrast, their proteome profiles were distinct. Interestingly, the variety of the proteins across the species highlighted the impact of glycosylation on the diversity of the glycosylated protein families. This proposed high throughput approach provides accurate and comprehensive profiles of the composition and function of various Viperidae snake venoms.

Functional Characterization and Anti-Tumor Effect of a Novel Group II Secreted Phospholipase A2 from Snake Venom of Saudi Cerastes cerates gasperetti.

Secreted phospholipases A2 are snake-venom proteins with many biological activities, notably anti-tumor activity. Phospholipases from the same snake type but different geographical locations have shown similar biochemical and biological activities with minor differences in protein sequences. Thus, the discovery of a new phospholipase A2 with unique characteristics identified in a previously studied venom could suggest the origins of these differences. Here, a new Group II secreted phospholipase A2 (Cc-PLA2-II) from the snake venom of Saudi Cerastes cerastes gasperetti was isolated and characterized. The purified enzyme had a molecular weight of 13.945 kDa and showed high specific activity on emulsified phosphatidylcholine of 1560 U/mg at pH 9.5 and 50 °C with strict calcium dependence. Interestingly, stability in extreme pH and high temperatures was observed after enzyme incubation at several pH levels and temperatures. Moreover, a significant dose-dependent cytotoxic anti-tumor effect against six human cancer cell lines was observed with concentrations of Cc-PLA2 ranging from 2.5 to 8 µM. No cytotoxic effect on normal human umbilical-vein endothelial cells was noted. These results suggest that Cc-PLA2-II potentially has angiogenic activity of besides cytotoxicity as part of its anti-tumor mechanism. This study justifies the inclusion of this enzyme in many applications for anticancer drug development.

Therapeutic outcome of quercetin nanoparticles on Cerastes cerastes venom-induced hepatorenal toxicity: a preclinical study.

Aim: The objective of this study was to investigate the therapeutic potential of quercetin (QT) and QT-loaded poly(lactic-co-glycolic acid) nanoparticles (QT-NPs) on Cerastes cerastes venom-mediated inflammation, redox imbalance, hepatorenal tissue damage and local hemorrhage. Methods: The developed QT-NPs were first submitted to physicochemical characterization and then evaluated in the 'challenge then treat' and 'preincubation' models of envenoming. Results: QT-NPs efficiently alleviated hepatorenal toxicity, inflammation and redox imbalance and significantly attenuated venom-induced local hemorrhage. Interestingly, QT-NPs were significantly more efficient than free QT at 24 h postenvenoming, pointing to the efficacy of this drug-delivery system. Conclusion: These findings highlight the therapeutic potential of QT-NPs on venom-induced toxicity and open up the avenue for their use in the management of snakebite envenoming.

Type I-like metalloproteinase in the venom of the West African saw-scaled carpet viper (Echis ocellatus) has anti-trypanosomal activity against African trypanosomes.

African trypanosomiasis is an infectious disease caused by hemoparasites of the genus Trypanosoma and remains a major health problem in Africa - killing around 4000 people and animals worth an estimated $5 billion, annually. The absence of a vaccine and satisfactory drug against African trypanosomiasis (AT) necessitates the continued search for new chemotherapy options. Owing to the rich biochemical diversity in snake venom, it has recently become a source of therapeutic peptides that are being explored for the development of novel drug candidates for diverse ailments such as cancers and infectious diseases. To explore this, Echis ocellatus venom (EOV) was investigated for the presence of an anti-Trypanosoma factor, with the subsequent aim to isolate and identify it. Crude EOV was collected and tested in vitro on the bloodstream form (BSF) i.e. long and slender morphological form of Trypanosoma brucei and T. congolense. This initial testing was followed by a sequential anti-trypanosomal assay guided purification of EOV using ethanol precipitation, distillation, and ion exchange (IEX) chromatography to obtain the active trypanocidal component. The purified anti-Trypanosoma factor, estimated to be a 52-kDa protein on SDS-PAGE, was subjected to in-gel trypsin digestion and 2D RP HPLC-MS/MS to identify the protein. The anti-Trypanosoma factor was revealed to be a zinc-dependent metalloproteinase that contains the HEXXHXXGXXH adamalysin motif. This protein may provide a conceptual framework for the possible design of a safe and effective anti-trypanosomal peptide for the treatment of AT.

Exploring Toxin Genes of Myanmar Russell's Viper, Daboia siamensis, through De Novo Venom Gland Transcriptomics.

The Russell's viper (Daboia siamensis) is a medically important venomous snake in Myanmar. Next-generation sequencing (NGS) shows potential to investigate the venom complexity, giving deeper insights into snakebite pathogenesis and possible drug discoveries. mRNA from venom gland tissue was extracted and sequenced on the Illumina HiSeq platform and de novo assembled by Trinity. The candidate toxin genes were identified via the Venomix pipeline. Protein sequences of identified toxin candidates were compared with the previously described venom proteins using Clustal Omega to assess the positional homology among candidates. Candidate venom transcripts were classified into 23 toxin gene families including 53 unique full-length transcripts. C-type lectins (CTLs) were the most highly expressed, followed by Kunitz-type serine protease inhibitors, disintegrins and Bradykinin potentiating peptide/C-type natriuretic peptide (BPP-CNP) precursors. Phospholipase A2, snake venom serine proteases, metalloproteinases, vascular endothelial growth factors, L-amino acid oxidases and cysteine-rich secretory proteins were under-represented within the transcriptomes. Several isoforms of transcripts which had not been previously reported in this species were discovered and described. Myanmar Russell's viper venom glands displayed unique sex-specific transcriptome profiles which were correlated with clinical manifestation of envenoming. Our results show that NGS is a useful tool to comprehensively examine understudied venomous snakes.

De Novo Venom Gland Transcriptome Assembly and Characterization for Calloselasma rhodostoma (Kuhl, 1824), the Malayan Pit Viper from Malaysia: Unravelling Toxin Gene Diversity in a Medically Important Basal Crotaline.

In Southeast Asia, the Malayan Pit Viper (Calloselasma rhodostoma) is a venomous snake species of medical importance and bioprospecting potential. To unveil the diversity of its toxin genes, this study de novo assembled and analyzed the venom gland transcriptome of C. rhodostoma from Malaysia. The expression of toxin genes dominates the gland transcriptome by 53.78% of total transcript abundance (based on overall FPKM, Fragments Per Kilobase Million), in which 92 non-redundant transcripts belonging to 16 toxin families were identified. Snake venom metalloproteinase (SVMP, PI > PII > PIII) is the most dominant family (37.84% of all toxin FPKM), followed by phospholipase A2 (29.02%), bradykinin/angiotensin-converting enzyme inhibitor-C-type natriuretic peptide (16.30%), C-type lectin (CTL, 10.01%), snake venom serine protease (SVSP, 2.81%), L-amino acid oxidase (2.25%), and others (1.78%). The expressions of SVMP, CTL, and SVSP correlate with hemorrhagic, anti-platelet, and coagulopathic effects in envenoming. The SVMP metalloproteinase domains encode hemorrhagins (kistomin and rhodostoxin), while disintegrin (rhodostomin from P-II) acts by inhibiting platelet aggregation. CTL gene homologues uncovered include rhodocytin (platelet aggregators) and rhodocetin (platelet inhibitors), which contribute to thrombocytopenia and platelet dysfunction. The major SVSP is a thrombin-like enzyme (an ancrod homolog) responsible for defibrination in consumptive coagulopathy. The findings provide insight into the venom complexity of C. rhodostoma and the pathophysiology of envenoming.

Unraveling the Reaction Mechanism of Russell's Viper Venom Factor X Activator: A Paradigm for the Reactivity of Zinc Metalloproteinases?

Snake venom metalloproteinases (SVMPs) are important drug targets against snakebite envenoming, the neglected tropical disease with the highest mortality worldwide. Here, we focus on Russell's viper (Daboia russelii), one of the "big four" snakes of the Indian subcontinent that, together, are responsible for ca. 50,000 fatalities annually. The "Russell's viper venom factor X activator" (RVV-X), a highly toxic metalloproteinase, activates the blood coagulation factor X (FX), leading to the prey's abnormal blood clotting and death. Given its tremendous public health impact, the WHO recognized an urgent need to develop efficient, heat-stable, and affordable-for-all small-molecule inhibitors, for which a deep understanding of the mechanisms of action of snake's principal toxins is fundamental. In this study, we determine the catalytic mechanism of RVV-X by using a density functional theory/molecular mechanics (DFT:MM) methodology to calculate its free energy profile. The results showed that the catalytic process takes place via two steps. The first step involves a nucleophilic attack by an in situ generated hydroxide ion on the substrate carbonyl, yielding an activation barrier of 17.7 kcal·mol-1, while the second step corresponds to protonation of the peptide nitrogen and peptide bond cleavage with an energy barrier of 23.1 kcal·mol-1. Our study shows a unique role played by Zn2+ in catalysis by lowering the pKa of the Zn2+-bound water molecule, enough to permit the swift formation of the hydroxide nucleophile through barrierless deprotonation by the formally much less basic Glu140. Without the Zn2+ cofactor, this step would be rate-limiting.

Computational and in vitro analyses to identify the anticoagulant regions of Echicetin, a snake venom anticoagulant C-type lectin (snaclec): possibility to develop anticoagulant peptide therapeutics?

Snake venom C-type lectins (Snaclecs) display anticoagulant and platelet-modulating activities; however, their interaction with the critical components of blood coagulation factors was unknown. Computational analysis revealed that Echicetin (Snaclec from Echis carinatus venom) interacted with heavy chain of thrombin, and heavy and light chains of factor Xa (FXa). Based on FXa and thrombin binding regions of Echicetin, the two synthetic peptides (1A and 1B) were designed. The in silico binding studies of the peptides with thrombin and FXa showed that peptide 1B interacted with both heavy and light chains of thrombin and, peptide 1A interacted with only heavy chain of thrombin. Similarly, peptide 1B interacted with both heavy and light chains of FXa; however, peptide 1A interacted only with heavy chain of FXa. Alanine screening predicted the hot-spots residues for peptide 1A (Aspartic acid6, Valine8, Valine9, and Tyrosine17 with FXa, and Isoleucine14, Lysine15 with thrombin) and peptide 1B (Valine16 with FXa). Spectrofluorometric interaction study showed a lower Kd value for peptide 1B binding with both FXa and thrombin than peptide 1A, indicating higher binding strength of the former peptide. The circular dichroism spectroscopy also established the interaction between thrombin and the custom peptides. The in vitro study demonstrated higher anticoagulant activity of peptide 1B than peptide 1A due to higher inhibition of thrombin and FXa. Inhibition of anticoagulant activity of the peptides by respective anti-peptide antibodies corroborates our hypothesis that peptides 1A and 1B represent the anticoagulant regions of Echicetin and may be developed as antithrombotic peptide drug prototypes.Communicated by Ramaswamy H. Sarma.

SPAD-1, a serine proteinase associated disintegrin from Russell's viper venom disrupts adhesion of MCF7 human breast cancer cells.

Serine Proteinase Associated Disintegrin-1 (SPAD-1) is a low molecular mass (26 kDa) positively charged protein purified from Russell's viper venom (RVV) possessing cytotoxic activity on MCF7, human breast cancer cells. Primary sequence analysis of the protein confirms that it is a novel Snake Venom Serine Proteinase (SVSP) and a member of the trypsin family. SPAD-1 contains a conserved triad of Histidine (H), Aspartic acid(D) and Serine(S) residues at its active site for proteinase activity and also an adjacent histidine-glycine-aspartic acid (HGD) disintegrin-like motif. The serine proteinase and disintegrin parts are functionally active and independent. SPAD-1 showed proteolytic digestion of fibrinogen and fibronectin, but laminin digestion was below the detectable limit. Proteolytically inactivated SPAD-1 inhibited collagen and ADP-induced platelet aggregation. This study proposes considering Serine Proteinase Associated Disintegrin (SPAD) as a new group of snake venom proteins. Members of this group contain a serine proteinase catalytic triad and a disintegrin-like motif. SPAD-1 caused visible morphological changes in MCF7 cells, including a reduction of the cell-to-cell attachments, rounding of cell shape and death, in vitro. SPAD-1 also showed a dose-dependent significant decrease in the invasive potency of breast cancer cells. Confocal microscopic analysis revealed the breakage of nuclei of the SPAD-1-treated cells. SPAD-1 also increased cell detachment from the poly L-lysine-coated, laminin-coated and fibronectin-coated culture plate matrices, confirming the disintegrin activity. This study concludes that SPAD-1 may be a good candidate for anti-tumour drug design in the future.

How snake venom disintegrins affect platelet aggregation and cancer proliferation.

Disintegrins are small peptides possessing a tripeptide motif capable of binding to integrins. These were first isolated from viper venoms and are now also found in many other hematophagous organisms. Many integrins have been studied for their role in the onset of disease and the interaction of disintegrins with these receptors makes them potential therapeutic molecules. Disintegrins are also used as molecular scaffolds to design effective drugs for cardiovascular diseases and cancer. Even the gene and protein sequencing data of disintegrins have provided insights into understanding the molecular complexity of disintegrins. In this review, we try to summarize the structural and functional importance of disintegrins in identifying the biological targets and triggering various signaling pathways involved in platelet aggregation and cancer. Also, we have tried to elucidate a possible molecular mechanism behind the action of disintegrins on platelet aggregation and cancer. This understanding will help us to design and to explore more of these integrin-binding molecules.

The Middle Eastern Cousin: Comparative Venomics of Daboia palaestinae and Daboia russelii.

Among the medically most important snakes in the world, the species belonging to the genus Daboia have been attributed to the highest number of human envenomings, deaths and disabilities. Given their significant clinical relevance, the venoms of Russell's vipers (D. russelii and D. siamensis) have been the primary focus of research. In contrast, the composition, activity, ecology and evolution of venom of its congener, the Palestine viper (D. palaestinae), have remained largely understudied. Therefore, to unravel the factors responsible for the enhanced medical relevance of D. russelii in comparison to D. palaestinae, we comparatively evaluated their venom proteomes, biochemical activities, and mortality and morbidity inflicting potentials. Furthermore, the synthesis and regulation of venom in snakes have also remained underinvestigated, and the relative contribution of each venom gland remains unclear. We address this knowledge gap by sequencing the tissue transcriptomes of both venom glands of D. palaestinae, and comparatively evaluating their contribution to the secreted venom concoction. Our findings highlight the disparity in the venom composition, function and toxicities of the two Daboia species. We also show that toxin production is not partitioned between the two venom glands of D. palaestinae.

In Silico Study of the Mechanisms Underlying the Action of the Snake Natriuretic-Like Peptide Lebetin 2 during Cardiac Ischemia.

Lebetin 2 (L2), a natriuretic-like peptide (NP), exerts potent cardioprotection in myocardial infarction (MI), with stronger effects than B-type natriuretic peptide (BNP). To determine the molecular mechanisms underlying its cardioprotection effect, we used molecular modeling, molecular docking and molecular dynamics (MD) simulation to describe the binding mode, key interaction residues as well as mechanistic insights into L2 interaction with NP receptors (NPRs). L2 binding affinity was determined for human, rat, mouse and chicken NPRs, and the stability of receptor-ligand complexes ascertained during 100 ns-long MD simulations. We found that L2 exhibited higher affinity for all human NPRs compared to BNP, with a rank preference for NPR-A > NPR-C > NPR-B. Moreover, L2 affinity for human NPR-A and NPR-C was higher in other species. Both docking and MD studies revealed that the NPR-C-L2 interaction was stronger in all species compared to BNP. Due to its higher affinity to human receptors, L2 could be used as a therapeutic approach in MI patients. Moreover, the stronger interaction of L2 with NPR-C could highlight a new L2 signaling pathway that would explain its additional effects during cardiac ischemia. Thus, L2 is a promising candidate for drug design toward novel compounds with high potency, affinity and stability.

Comparative Venom Proteomics of Iranian, Macrovipera lebetina cernovi, and Cypriot, Macrovipera lebetina lebetina, Giant Vipers.

Envenoming by Macrovipera lebetina subspecies causes severe life-threatening difficulties for people living in North Africa and the Middle East. To better understand the pathophysiology of envenoming and improve patient management, knowledge about the venom components of the subspecies is essential. Here, the venom proteomes of Macrovipera lebetina lebetina from Cyprus and Macrovipera lebetina cernovi from Iran were characterized using RP-HPLC separation of the crude venom proteins, SDS-PAGE of fractionated proteins, and LC-MS/MS of peptides obtained from in-gel tryptic digestion of protein bands. Moreover, we also used high-resolution shot-gun proteomics to gain more reliable identification, where the whole venom proteomes were subjected directly to in-solution digestion before LC-HR-MS/MS. The data revealed that both venoms consisted of at least 18 protein families, of which snake venom Zn2+-dependent metalloprotease (SVMP), serine protease, disintegrin, phospholipase A2, C-type lectin-like, and L-amino acid oxidase, together accounted for more than 80% of the venoms' protein contents. Although the two viper venoms shared mostly similar protein classes, the relative occurrences of these toxins were different in each snake subspecies. For instance, P-I class of SVMP toxins were found to be more abundant than P-III class in the venoms of M. l. cernovi compared to M. l. lebetina, which gives hints at a more potent myonecrotic effect and minor systemic hemorrhage following envenoming by M. l. cernovi than M. l. lebetina. Moreover, single-shot proteomics also revealed many proteins with low abundance (<1%) within the venoms, such as aminopeptidase, hyaluronidase, glutaminyl-peptide cyclotransferase, cystatin, phospholipase B, and vascular endothelial growth factor. Our study extends the in-depth understanding of the venom complexity of M. lebetina subspecies, particularly regarding toxin families associated with envenoming pathogenesis and those hard-detected protein classes expressed in trace amounts.

Diverse and Dynamic Alpha-Neurotoxicity Within Venoms from the Palearctic Viperid Snake Clade of Daboia, Macrovipera, Montivipera, and Vipera.

The targeting of specific prey by snake venom toxins is a fascinating aspect of molecular and ecological evolution. Neurotoxic targeting by elapid snakes dominates the literature in this regard; however, recent studies have revealed viper toxins also induce neurotoxic effect. While this effect is thought to primarily be driven by prey selectivity, no study has quantified the taxonomically specific neurotoxicity of the viper clade consisting of Daboia, Macrovipera, Montivipera, and Vipera genera. Here, we tested venom toxin binding from 28 species of vipers from the four genera on the alpha 1 neuronal nicotinic acetylcholine receptors (nAChRs) orthosteric sites of amphibian, avian, lizard, rodent, and human mimotopes (synthetic peptides) using the Octet HTX biolayer interferometry platform. Daboia siamensis and D. russelii had broad binding affinity towards all mimotopes, while D. palestinae had selectivity toward lizard. Macrovipera species, on the other hand, were observed to have a higher affinity for amphibian mimotopes except for M. schweizeri, which inclined more toward lizard mimotopes. All Montivipera and most Vipera species also had higher affinity toward lizard mimotopes. Vipera a. montandoni, V. latastei, V. nikolski, and V. transcaucasina had the least binding to any of the mimotopes of the study. While a wide range of affinity binding towards various mimotopes were observed within the clade, the lowest affinity occurred towards the human target. Daboia siamensis and Macrovipera lebetina exhibited the greatest affinity toward the human mimotope, albeit still the least targeted of the mimotopes within those species. Overlaying this toxin-targeting trait over phylogeny of this clade revealed multiple cases of amplification of this trait and several cases of secondary loss. Overall, our results reveal dynamic variation, amplification, and some secondary loss of the prey targeting trait by alpha-neurotoxins within the venoms of this clade, indicating evolutionary selection pressure shaping the basic biochemistry of these venoms. Our work illustrates the successful use of this biophysical assay to further research snake venom neurotoxins and emphasizes the risk of generalizing venom effects observed on laboratory animals to have similar effects on humans.

Differences between Two Groups of Burmese Vipers (Viperidae: Azemiops) in the Proteomic Profiles, Immunoreactivity and Biochemical Functions of Their Venoms.

Two recently revised Azemiops snakes with apparent differences in their external appearances and skeletal morphologies but unclear genetic boundaries have been proposed. Some researchers have refrained from using the newly proposed taxonomy because these two "species" might be two clades corresponding to different geographical populations of Azemiops feae. To improve the understanding of the kinship of these two Burmese viper groups, more of their characteristics should be explored in depth. We performed a comparative analysis of the proteomic profiles and biochemical activities of snake venoms from these two groups (Sichuan A. feae and Zhejiang A. feae) and evaluated the immunorecognition capacity of commercial antivenoms toward them. Eight protein families were identified in venoms from these two groups, while phospholipase B was only detected in venom from Sichuan A. feae. These protein families displayed varying degrees of differences in relative abundance between venoms, and phospholipase A2 (Sichuan A. feae: 57.15%; Zhejiang A. feae: 65.94%) was the predominated component. Gloydius brevicaudus antivenom exhibited the strongest capacity to immunologically recognize these two venoms, but this was mainly limited to components with high molecular masses, some of which differed between venoms. Additionally, Zhejiang A. feae venom was more toxic than Sichuan A. feae venom, and the venoms expressed remarkable differences in enzymatic activities, probably resulting from the variation in the relative abundance of specific protein families. Our findings unveil differences between the two Burmese viper groups in terms of proteomic profiles, immunoreactivity, and the biochemical functions of their venoms. This information will facilitate the management of snakebites caused by these snakes.

The Phospholipase Activity of Ammodytoxin, a Prototype Snake Venom ß-Neurotoxin, Is Not Obligatory for Cell Internalisation and Translocation to Mitochondria.

ß-Neurotoxins are secreted phospholipase A2 molecules that inhibit transmission in neuromuscular synapses by poisoning the motor neurons. These toxins specifically and rapidly internalise into the nerve endings of motor neurons. Ammodytoxin (Atx) is a prototype ß-neurotoxin from the venom of the nose-horned viper (Vipera ammodytes ammodytes). Here, we studied the relevance of the enzymatic activity of Atx in cell internalisation and subsequent intracellular movement using transmission electron microscopy (TEM). We prepared a recombinant, enzymatically inactive mutant of Atx, Atx(D49S), labelled with gold nanoparticles (GNP), and incubated this with PC12 cells, to analyse its localisation by TEM. Atx(D49S)-GNP internalised into the cells. Inside the cells, Atx(D49S)-GNP was detected in different vesicle-like structures, cytosol, endoplasmic reticulum and mitochondria, where it was spotted in the intermembrane space and matrix. Co-localization of fluorescently labelled Atx(D49S) with mitochondria in PC12 cells by confocal fluorescence microscopy confirmed the reliability of results generated using Atx(D49S)-GNP and TEM and allowed us to conclude that the phospholipase activity of Atx is not obligatory for its cell internalisation and translocation into the mitochondrial intermembrane space and matrix.