Snake Venom Serine Proteinases Research Articles

Comprehensive Snake Venomics of the Okinawa Habu Pit Viper, Protobothrops flavoviridis, by Complementary Mass Spectrometry-Guided Approaches.

The Asian world is home to a multitude of venomous and dangerous snakes, which are used to induce various medical effects in the preparation of traditional snake tinctures and alcoholics, like the Japanese snake wine, named Habushu. The aim of this work was to perform the first quantitative proteomic analysis of the Protobothrops flavoviridis pit viper venom. Accordingly, the venom was analyzed by complimentary bottom-up and top-down mass spectrometry techniques. The mass spectrometry-based snake venomics approach revealed that more than half of the venom is composed of different phospholipases A2 (PLA2). The combination of this approach and an intact mass profiling led to the identification of the three main Habu PLA2s. Furthermore, nearly one-third of the total venom consists of snake venom metalloproteinases and disintegrins, and several minor represented toxin families were detected: C-type lectin-like proteins (CTL), cysteine-rich secretory proteins (CRISP), snake venom serine proteases (svSP), l-amino acid oxidases (LAAO), phosphodiesterase (PDE) and 5′-nucleotidase. Finally, the venom of P. flavoviridis contains certain bradykinin-potentiating peptides and related peptides, like the svMP inhibitors, pEKW, pEQW, pEEW and pENW. In preliminary MTT cytotoxicity assays, the highest cancerous-cytotoxicity of crude venom was measured against human neuroblastoma SH-SY5Y cells and shows disintegrin-like effects in some fractions.

Proteomic profile, biological activities and antigenic analysis of the venom from Bothriopsis bilineata smaragdina (“loro machaco”), a pitviper snake from Peru

In order to determine Bothriopsis bilineata smaragdina venom (BbsV) composition, proteomic approaches were performed. Venom components were analyzed by RP-HPLC, SDS- PAGE and nano LC on line with LTQ Orbitrap XL. Results showed a total of 189 identified proteins, grouped into 11 different subgroups, which include snake venom metalloproteinases (SVMPs, 54.67%), snake C-type lectins (Snaclecs, 15.78%), snake venom serine proteinases (SVSPs, 14.69%), cystein-rich secretory proteins (CRISP, 2.61%), phospholipases A2 (PLA2, 1.14%), phosphodiesterase (PDE, 1.17%), venom endothelial growth factor (VEGF, 1.06%) 5’nucleotidases (0.33%), L-amino acid oxidases (LAAOs, 0.28%) and other proteins. In vitro enzymatic activities (SVMP, SVSP, LAAO, Hyal and PLA2) of BbsV were also analyzed. BbsV showed high SVSP activity but low PLA2 activity, when compared to other Bothrops venoms. In vivo, BbsV induced hemorrhage and edema in mice and showed intraperitoneal median lethal dose (LD50) of 92.74 (± 0.15) μg/20 g of mice. Furthermore, BbsV reduced cell viability when incubated with VERO cells. Peruvian and Brazilian bothropic antivenoms recognize BbsV proteins, as detected by ELISA and Western Blotting. Both antivenoms were able to neutralize in vivo edema and hemorrhage. SignificanceIn Peru, snakebite is a public health problem, especially in the rain forest, as a result of progressive colonization of this geographical area. This country is the second in Latin America, after Brazil, to exhibit the largest variety of venomous snakes. B. atrox and B. b. smaragdina snakes are sympatric species in Peruvian Amazon region and are responsible for approximately 95% of the envenomings reported in this region. B. b. smaragdina may cause a smaller share (3 to 38%) of those accidents, due to its arboreal habits, that make human encounters with these snakes less likely to happen. Despite B. b. smaragdina recognized medical importance, its venom composition and biological activities have been poorly studied. Furthermore, BbsV is not a component of the antigenic pool used to produce the corresponding Peruvian bothropic antivenom (P-BAV). Our results not only provide new insights on BbsV composition and biological activity, but also demonstrate that both P-BAV and B-BAV polyvalent antivenoms have a considerable recognition of proteins from BbsV and, more importantly, neutralized hemorrhage and edema, the main local effects of bothropic envenomation.

Proteomic characterization of six Taiwanese snake venoms: Identification of species-specific proteins and development of a SISCAPA-MRM assay for cobra venom factors

Deinagkistrodon acutus, Trimeresurus stejnegeri, Protobothrops mucrosquamatus, Daboia russelii siamensis, Bungarus multicinctus and Naja atra are the six medically important venomous snake species in Taiwan. In this study, we characterized and compared their venom protein profiles using proteomic approaches. The major snake venom proteins were identified by GeLC-MS/MS and the total venom proteome was characterized by in-solution digestion coupled with LC-MS/MS. A total of 27–52 proteins, categorized into 23 protein families, were identified in each snake's venom. The major venom components found in Viperidae species (D. acutus, T. stejnegeri, P. mucrosquamatus and D. russelii) were C-type lectin, snake venom serine proteinase, venom metalloproteinase and phospholipase A2, whereas three-finger toxin and phospholipase A2 were the major components detected in the venom of Elapidae snakes (B. multicinctus and N. atra). This study also provided the first demonstration of some low-abundance proteins in these six snake venoms, including 5′-nucleotidase, glutaminyl-peptide cyclotransferase and phosphodiesterase, among others. Furthermore, we found that cobra venom factor (CVF) is a cobra-specific protein. We produced anti-peptide antibodies against CVF and used it to develop a highly sensitive SISCAPA-MRM assay for quantifying CVF. The limit of detection and lower limit of quantification were 3.2 and 9.6 attomoles, respectively. This assay was used to precisely quantify CVF in 1 μg crude venom proteins from three Naja species and king cobra. The amount of CVF varied from 0.9 to 54.36 femtomoles (equivalent to 0.16–10.03 mg/g of venom protein). Biological significanceThere are six medically significant venomous snakes in Taiwan. The venoms of the four Viperidae species (Deinagkistrodon acutus, Trimeresurus stejnegeri, Protobothrops mucrosquamatus and Daboia russelii siamensis) cause local tissue swelling; this symptom is also seen in N. atra envenomation in humans, potentially complicating the differential diagnosis of envenomation by N. atra and Viperidae species. Thus, characterization of the venom proteomes of the six Taiwanese snakes, including the relative abundance of the major components and species-specific protein(s) in each venom type, could be useful for future venom research, including the development of new assay(s) for detecting snake species-specific venom protein(s) and new type(s) of antivenom.

MOLECULAR DOCKING STUDIES OF SNAKE VENOM SERINE PROTEASE OF SHARP-NOSED PIT VIPER WITH HESPERETIN

Objective: The management of snake bite envenomation is a global challenge affecting millions of people. Immunotherapy is still regarded as the treatment of choice; however, their subsequent adverse effects restrict their potential use for therapy against snake venom poisoning. In recent years, more attention has been given to the exploration of indigenous medicinal plants for their outstanding benefits for the treatment of several diseases and disorders, including snake venom poisoning. Hesperetin is a naturally occurring compound derived from a flavanone glycoside, hesperidin and is obtained from various citrus fruits. It is known to possess significant inhibitory activity against snake venom serine proteases. The aim of our present study was to investigate the significant inhibitory action of hesperetin on thrombin-like serine protease from sharp-nosed pit viper (Deinagkistrodon acutus) snake venom.Methods: We have employed molecular docking analysis by implementing the state-of-the-art docking program to validate the hypothesis of the prospective inhibitory properties of hesperetin on thrombin-like serine proteases of snake venom. AutoDock 4.2, InterProSurf, MGLTools, and MTiAutoDock were utilized for the molecular docking analysis of thrombin-like serine protease obtained from the snake venom of sharp-nosed pit viper with the natural compound hesperetin.Results: The results generated from in silico based approach reveals the significant inhibitory role of hesperetin against thrombin-like snake venom proteases, which might lead to the drug designing of the inhibitors of snake venom serine proteases.Conclusion: The implementation of molecular docking analysis by the employment of state-of-the-art docking program supports the potential of inhibitory activity of naturally obtained hesperetin compound on thrombin-like serine proteases of snake venom. The generated in silico results suggests that the novel structure hesperetin - flavanone might act as a potent inhibitor of thrombin-like snake venom proteases, and unlocks the possibilities for designing drugs of the inhibitors of snake venom serine proteases. Moreover, the investigation of the novel compound obtained from natural sources for their inhibitory activity against snake venom serine proteases would lead to the discovery of newer inhibitory compound from a highly uninvestigated research arena.

Proteomics, functional characterization and antivenom neutralization of the venom of Pakistani Russell's viper (Daboia russelii) from the wild

The venom proteome of wild Pakistani Russell's viper (Daboia russelii) was investigated through nano-ESI-LCMS/MS of the reverse-phase HPLC fractions. A total of 54 venom proteins were identified and clustered into 11 protein families. Phospholipase A2 (PLA2, 63.8%) and Kunitz-type serine protease inhibitor (KSPI, 16.0%) were most abundant, followed by snake venom serine protease (SVSP, 5.5%, mainly Factor V activating enzyme), vascular endothelial growth factor (VEGF, 4.3%), snake venom metalloproteinase (SVMP, 2.5%, mainly Factor X activating enzyme) and phosphodiesterase (PDE, 2.5%). Other minor proteins include cysteine-rich secretory protein (CRiSP), snake venom C-type lectin/lectin-like protein (snaclec), nerve growth factor, L-amino acid oxidase and 5′-nucleotidase. PLA2, KSPI, SVSP, snaclec and SVMP are hemotoxic proteins in the venom. The study indicated substantial venom variation in D. russelii venoms of different locales, including 3 Pakistani specimens kept in the USA. The venom exhibited potent procoagulant activity on human plasma (minimum clotting dose = 14.5 ng/ml) and high lethality (rodent LD50 = 0.19 μg/g) but lacked hemorrhagic effect locally. The Indian VINS Polyvalent Antivenom bound the venom immunologically in a concentration-dependent manner. It moderately neutralized the venom procoagulant and lethal effects (normalized potency against lethality = 2.7 mg venom neutralized per g antivenom). Biological significanceComprehensive venom proteomes of D. russelii from different locales will facilitate better understanding of the geographical variability of the venom in both qualitative and quantitative terms. This is essential to provide scientific basis for the interpretation of differences in the clinical presentation of Russell's viper envenomation. The study revealed a unique venom proteome of the Pakistani D. russelii from the wild (Indus Delta), in which PLA2 predominated (~60% of total venom proteins). The finding unveiled remarkable differences in the venom compositions between the wild (present study) and the captive specimens reported previously. The integration of toxicity tests enabled the correlation of the venom proteome with the envenoming pathophysiology, where the venom showed potent lethality mediated through coagulopathic activity. The Indian VINS Polyvalent Antivenom (VPAV) showed binding activity toward the venom protein antigens; however the immunorecognition of small proteins and PLA2-dominating fractions was low to moderate. Consistently, the antivenom neutralized the toxicity of the wild Pakistani Russell's viper venom at moderate efficacies. Our results suggest that it may be possible to enhance the Indian antivenom potency against the Pakistani viper venom by the inclusion of venoms from a wider geographical range including that from Pakistan into the immunogen formulation.

Citrus bioflavonoid, hesperetin, as inhibitor of two thrombin-like snake venom serine proteases isolated from Crotalus simus

Around 5.5 million people suffer from snakebites per year, with about 400,000 cases with some type of sequelae, such as amputation, and 20,000 to 125,000 cases with the fatal end. Usually, the victim outcome depends on correct, agile and many times in situ intervention based on the proper identification of the snake venom type and its potential effects, among other factors. Therefore, knowledge on the snake venom composition and a research on inhibitors of snake venom target components might ameliorate envenoming dangerous outcome. Herein, two thrombin-like serine proteases from the Crotalus simus snake venom - SVSP1 and SVSP2 - were isolated in two chromatographic steps, using gel filtration and then RP-HPLC. They showed molecular masses of around 31.3 and 24.6 kDa, respectively, and mostly β-sheet secondary structure features. The SVSP1 and SVSP2 were sequenced using tandem mass spectrometry (Q-TOF). Using the known serine protease structure (PDB entry: 4e7n), which was evaluated as homologous to the two target proteins, in silico docking results showed that hesperetin is its excellent inhibitor. Using in vitro tests with the commercial hesperetin, kinetic parameters were obtained for SVSPs against the synthetic substrate BApNA. Obtained results pointed that hesperetin might act as an uncompetitive (SVSP1) or mixed (SVSP2) inhibitor. Also, the fluorescence quenching upon inhibition was observed, as well as, red shift in maximums of around 20 nm, which indicate that the tryptophan residues in the target enzymes suffered conformational changes caused by hesperetin binding. Thus, a naturally occurring flavone that can easily be extracted from oranges might serve as low-cost inhibitor of the investigated snake venom proteases.

A Single Mutation Unlocks Cascading Exaptations in the Origin of a Potent Pitviper Neurotoxin.

Evolutionary innovations and complex phenotypes seemingly require an improbable amount of genetic change to evolve. Rattlesnakes display two dramatically different venom phenotypes. Type I venoms are hemorrhagic with low systemic toxicity and high expression of tissue-destroying snake venom metalloproteinases. Type II venoms are highly neurotoxic and lack snake venom metalloproteinase expression and associated hemorrhagic activity. This dichotomy hinges on Mojave toxin (MTx), a phospholipase A2 (PLA2) based β-neurotoxin expressed in Type II venoms. MTx is comprised of a nontoxic acidic subunit that undergoes extensive proteolytic processing and allosterically regulates activity of a neurotoxic basic subunit. Evolution of the acidic subunit presents an evolutionary challenge because the need for high expression of a nontoxic venom component and the proteolytic machinery required for processing suggests genetic changes of seemingly little immediate benefit to fitness. We showed that MTx evolved through a cascading series of exaptations unlocked by a single nucleotide change. The evolution of one new cleavage site in the acidic subunit unmasked buried cleavage sites already present in ancestral PLA2s, enabling proteolytic processing. Snake venom serine proteases, already present in the venom to disrupt prey hemostasis, possess the requisite specificities for MTx acidic subunit proteolysis. The dimerization interface between MTx subunits evolved by exploiting a latent, but masked, hydrophobic interaction between ancestral PLA2s. The evolution of MTx through exaptation of existing functional and structural features suggests complex phenotypes that depend on evolutionary innovations can arise from minimal genetic change enabled by prior evolution.

Identification of B cell recognized linear epitopes in a snake venom serine proteinase from the central American bushmaster Lachesis stenophrys

Snake venom serine proteinases are toxins that perturb hemostasis acting on proteins from the blood coagulation cascade, the fibrinolytic or the kallikrein–kinin system. Despite the relevance of these enzymes in envenomations by viper bites, the characterization of the antibody response to these toxins at the molecular level has not been previously addressed. In this work surface-located B cell recognized linear epitopes from a Lachesis stenophrys venom serine proteinase (UniProt accession number Q072L7) were predicted using an artificial neuronal network at the ABCpred server, the corresponding peptides were synthesized and their immunoreactivity was analyzed against a panel of experimental and therapeutic antivenoms. A molecular model of the L. stenophrys enzyme was built using as a template the structure of the D. acutus Dav-PA serine proteinase (Q9I8X1), which displays the highest degree of sequence similarity to the L. stenophrys enzyme among proteins of known 3D structure, and the surface-located epitopes were identified in the protein model using iCn3D. A total of 13 peptides corresponding to the surface exposed predicted epitopes from L. stenophrys serine proteinase were synthesized and, their reactivity with a rabbit antiserum against the recombinant enzyme and a panel of antivenoms was evaluated by a capture ELISA. Some of the epitopes recognized by monospecific and polyspecific antivenoms comprise sequences overlapping motifs conserved in viper venom serine proteinases. The identification and characterization of relevant epitopes recognized by B cells in snake venom toxins may provide valuable information for the preparation of immunogens that help in the production of improved therapeutic antivenoms.

Cross-recognition of a pit viper (Crotalinae) polyspecific antivenom explored through high-density peptide microarray epitope mapping.

Snakebite antivenom is a 120 years old invention based on polyclonal mixtures of antibodies purified from the blood of hyper-immunized animals. Knowledge on antibody recognition sites (epitopes) on snake venom proteins is limited, but may be used to provide molecular level explanations for antivenom cross-reactivity. In turn, this may help guide antivenom development by elucidating immunological biases in existing antivenoms. In this study, we have identified and characterized linear elements of B-cell epitopes from 870 pit viper venom protein sequences by employing a high-throughput methodology based on custom designed high-density peptide microarrays. By combining data on antibody-peptide interactions with multiple sequence alignments of homologous toxin sequences and protein modelling, we have determined linear elements of antibody binding sites for snake venom metalloproteases (SVMPs), phospholipases A2s (PLA2s), and snake venom serine proteases (SVSPs). The studied antivenom antibodies were found to recognize linear elements in each of the three enzymatic toxin families. In contrast to a similar study of elapid (non-enzymatic) neurotoxins, these enzymatic toxins were generally not recognized at the catalytic active site responsible for toxicity, but instead at other sites, of which some are known for allosteric inhibition or for interaction with the tissue target. Antibody recognition was found to be preserved for several minor variations in the protein sequences, although the antibody-toxin interactions could often be eliminated completely by substitution of a single residue. This finding is likely to have large implications for the cross-reactivity of the antivenom and indicate that multiple different antibodies are likely to be needed for targeting an entire group of toxins in these recognized sites.

Combined venom profiling and cytotoxicity screening of the Radde's mountain viper (Montivipera raddei) and Mount Bulgar Viper (Montivipera bulgardaghica) with potent cytotoxicity against human A549 lung carcinoma cells

Here we report the first characterization of the endemic Mount Bulgar Viper (Montivipera bulgardaghica) and Radde's mountain viper (Montivipera raddei) venom by a combined approach using intact mass profiling and bottom-up proteomics. The cytotoxicity screening of crude venom as well as isolated serine proteases revealed a high activity against A549 human lung carcinoma cells. By means of intact mass profiling of native and reduced venom we observed basic and acidic phospholipases type A2. Moreover, the analysis revealed snake venom metalloproteases, cysteine-rich secretory proteins, disintegrins, snake venom serine proteases, C-type lectins, a vascular endothelial growth factor and an L-amino acid oxidase.

The genetics of venom ontogeny in the eastern diamondback rattlesnake (Crotalus adamanteus).

The same selective forces that give rise to rapid inter- and intraspecific divergence in snake venoms can also favor differences in venoms across life-history stages. Ontogenetic changes in venom composition are well known and widespread in snakes but have not been investigated to the level of unambiguously identifying the specific loci involved. The eastern diamondback rattlesnake was previously shown to undergo an ontogenetic shift in venom composition at sexual maturity, and this shift accounted for more venom variation than geography. To characterize the genetics underlying the ontogenetic venom compositional change in C. adamanteus, we sequenced adult/juvenile pairs of venom-gland transcriptomes from five populations previously shown to have different adult venom compositions. We identified a total of 59 putative toxin transcripts for C. adamanteus, and 12 of these were involved in the ontogenetic change. Three toxins were downregulated, and nine were upregulated in adults relative to juveniles. Adults and juveniles expressed similar total levels of snake-venom metalloproteinases but differed substantially in their featured paralogs, and adults expressed higher levels of Bradykinin-potentiating and C-type natriuretic peptides, nerve growth factor, and specific paralogs of phospholipases A2 and snake venom serine proteinases. Juvenile venom was more toxic to mice, indicating that the expression differences resulted in a phenotypically, and therefore potentially ecologically, significant difference in venom function. We also showed that adult and juvenile venom-gland transcriptomes for a species with known ontogenetic venom variation were equally effective at individually providing a full characterization of the venom genes of a species but that any particular individual was likely to lack several toxins in their transcriptome. A full characterization of a species’ venom-gene complement therefore requires sequencing more than one individual, although the ages of the individuals are unimportant.

Comparative venom gland transcriptomics ofNaja kaouthia(monocled cobra) from Malaysia and Thailand: elucidating geographical venom variation and insights into sequence novelty

BackgroundThe monocled cobra (Naja kaouthia) is a medically important venomous snake in Southeast Asia. Its venom has been shown to vary geographically in relation to venom composition and neurotoxic activity, indicating vast diversity of the toxin genes within the species. To investigate the polygenic trait of the venom and its locale-specific variation, we profiled and compared the venom gland transcriptomes of N. kaouthia from Malaysia (NK-M) and Thailand (NK-T) applying next-generation sequencing (NGS) technology.MethodsThe transcriptomes were sequenced on the Illumina HiSeq platform, assembled and followed by transcript clustering and annotations for gene expression and function. Pairwise or multiple sequence alignments were conducted on the toxin genes expressed. Substitution rates were studied for the major toxins co-expressed in NK-M and NK-T.Results and discussionThe toxin transcripts showed high redundancy (41–82% of the total mRNA expression) and comprised 23 gene families expressed in NK-M and NK-T, respectively (22 gene families were co-expressed). Among the venom genes, three-finger toxins (3FTxs) predominated in the expression, with multiple sequences noted. Comparative analysis and selection study revealed that 3FTxs are genetically conserved between the geographical specimens whilst demonstrating distinct differential expression patterns, implying gene up-regulation for selected principal toxins, or alternatively, enhanced transcript degradation or lack of transcription of certain traits. One of the striking features that elucidates the inter-geographical venom variation is the up-regulation of α-neurotoxins (constitutes ∼80.0% of toxin’s fragments per kilobase of exon model per million mapped reads (FPKM)), particularly the long-chain α-elapitoxin-Nk2a (48.3%) in NK-T but only 1.7% was noted in NK-M. Instead, short neurotoxin isoforms were up-regulated in NK-M (46.4%). Another distinct transcriptional pattern observed is the exclusively and abundantly expressed cytotoxin CTX-3 in NK-T. The findings suggested correlation with the geographical variation in proteome and toxicity of the venom, and support the call for optimising antivenom production and use in the region. Besides, the current study uncovered full and partial sequences of numerous toxin genes from N. kaouthia which have not been reported hitherto; these include N. kaouthia-specific l-amino acid oxidase (LAAO), snake venom serine protease (SVSP), cystatin, acetylcholinesterase (AChE), hyaluronidase (HYA), waprin, phospholipase B (PLB), aminopeptidase (AP), neprilysin, etc. Taken together, the findings further enrich the snake toxin database and provide deeper insights into the genetic diversity of cobra venom toxins.

What killed Karl Patterson Schmidt? Combined venom gland transcriptomic, venomic and antivenomic analysis of the South African green tree snake (the boomslang), Dispholidus typus

BackgroundNon-front-fanged colubroid snakes comprise about two-thirds of extant ophidian species. The medical significance of the majority of these snakes is unknown, but at least five species have caused life-threatening or fatal human envenomings. However, the venoms of only a small number of species have been explored. MethodsA combined venomic and venom gland transcriptomic approach was employed to characterise of venom of Dispholidus typus (boomslang), the snake that caused the tragic death of Professor Karl Patterson Schmidt. The ability of CroFab™ antivenom to immunocapture boomslang venom proteins was investigated using antivenomics. ResultsTranscriptomic-assisted proteomic analysis identified venom proteins belonging to seven protein families: three-finger toxin (3FTx); phospholipase A2 (PLA2); cysteine-rich secretory proteins (CRISP); snake venom (SV) serine proteinase (SP); C-type lectin-like (CTL); SV metalloproteinases (SVMPs); and disintegrin-like/cysteine-rich (DC) proteolytic fragments. CroFab™ antivenom efficiently immunodepleted some boomslang SVMPs. ConclusionsThe present work is the first to address the overall proteomic profile of D. typus venom. This study allowed us to correlate the toxin composition with the toxic activities of the venom. The antivenomic analysis suggested that the antivenom available at the time of the unfortunate accident could have exhibited at least some immunoreactivity against the boomslang SVMPs responsible for the disseminated intravascular coagulation syndrome that caused K.P. Schmidt's fatal outcome. General significanceThis study may stimulate further research on other non-front-fanged colubroid snake venoms capable of causing life-threatening envenomings to humans, which in turn should contribute to prevent fatal human accidents, such as that unfortunately suffered by K.P. Schmidt.

Proteomic analysis to unravel the complex venom proteome of eastern India Naja naja: Correlation of venom composition with its biochemical and pharmacological properties

The complex venom proteome of the eastern India (EI) spectacled cobra (Naja naja) was analyzed using tandem mass spectrometry of cation-exchange venom fractions. About 75% of EI N. naja venom proteins were <18kDa and cationic at physiological pH of blood. SDS-PAGE (non-reduced) analysis indicated that in the native state venom proteins either interacted with each-other or self-aggregated resulting in the formation of higher molecular mass complexes. Proteomic analysis revealed that 43 enzymatic and non-enzymatic proteins in EI N. naja venom with a percent composition of about 28.4% and 71.6% respectively were distributed over 15 venom protein families. The three finger toxins (63.8%) and phospholipase A2s (11.4%) were the most abundant families of non-enzymatic and enzymatic proteins, respectively. nanoLC-ESI-MS/MS analysis demonstrated the occurrence of acetylcholinesterase, phosphodiesterase, cholinesterase and snake venom serine proteases in N. naja venom previously not detected by proteomic analysis. ATPase, ADPase, hyaluronidase, TAME, and BAEE-esterase activities were detected by biochemical analysis; however, due to a limitation in the protein database depository they were not identified in EI N. naja venom by proteomic analysis. The proteome composition of EI N. naja venom was well correlated with its in vitro and in vivo pharmacological properties in experimental animals and envenomed human. Biological significanceProteomic analysis reveals the complex and diverse protein profile of EI N. naja venom which collectively contributes to the severe pathophysiological manifestation upon cobra envenomation. The study has also aided in comprehending the compositional variation in venom proteins of N. naja within the Indian sub-continent. In addition, this study has also identified several enzymes in EI N. naja venom which were previously uncharacterized by proteomic analysis of Naja venom.

Structure Insights and Fibrinogen Peptides Molecular Recognition by Bothrombin, A Snake Venom Serine Protease from Bothropus jararaca

Bothrombin is a snake venom serine protease from Bothropus jararaca, which aggregates platelets in the presence of fibrinogen. In this context, the 3D model of Bothrombin was design for molecular modeling studies by modeller9v5 using as template the protein C Activator (PDB ID 2AIP). Energy minimized Bothrombin model was validated by methods such as PROCHECK, PROSA, ERRAT, WHAT-IF and VERITY 3D. After confirming all suggested evidences, such as geometric quality of the backbone conformation, residue interaction, energy profile etc., the interaction study based on docking was further calculated with ClusPro server. This protocol was designed in order to assess the dependence of bothrombin model from its template and how it compares to thrombin. Crystal structure of fibrinopeptides of PDB ID: 1DM4 and 1YCP were selected for docking studies. These fibrinopeptide also docked with thrombin and protein C Activator. All docked complexes were then further analysed by means of interaction energy between subunits and their corresponding binding energy estimates. The present study gain insight into protein structure-function relationships among serine proteinases, and its importance in biomedical applications.

Identification of snake venom allergens by two-dimensional electrophoresis followed by immunoblotting

This allergic reaction to snake venom was described to occur in patients after recurrent exposure through bites in amateur and professional snake handlers, which might be underestimated and contribute to fatal snakebites in victim, independently from the toxicity of the venom itself. Few allergens were identified from snake venoms by normal SDS-PAGE, which cannot separate the snake venom completely. In the present study, we identified nine potential allergens by two-dimensional (2D) electrophoresis followed by immunoblotting (named as allergenomics) in Protobothrops mucrosquamatus venom. By multidimensional liquid chromatography-ion trap mass spectrometry (MDLC-ESI-LTQ-MS/MS) analysis, six allergens showed sequence similarity to snake venom serine proteinases. Other allergens showed sequence similarity to snake venom metalloproteinase. These allergic reactions to snake venom allergens might contribute to fatal snakebites in victim, independently.

Venomics of Calloselasma rhodostoma, the Malayan pit viper: A complex toxin arsenal unraveled

The venom of Malayan pit viper (Calloselasma rhodostoma) is highly toxic but also valuable in drug discovery. However, a comprehensive proteome of the venom that details its toxin composition and abundance is lacking. This study aimed to unravel the venom complexity through a multi-step venomic approach. At least 96 distinct proteins (29 basic, 67 acidic) in 11 families were identified from the venom. The venom consists of mainly snake venom metalloproteinases (SVMP, 41.17% of total venom proteins), within which the P-I (kistomin, 20.4%) and P-II (rhodostoxin, 19.8%) classes predominate. This is followed by C-type lectins (snaclec, 26.3%), snake venom serine protease (SVSP, 14.9%), L-amino acid oxidase (7.0%), phospholipase A2 (4.4%), cysteine-rich secretory protein (2.5%), and five minor toxins (nerve growth factor, neurotrophin, phospholipase B, 5′ nucleotidase and phosphodiesterase, totaling 2.6%) not reported in the proteome hitherto. Importantly, all principal hemotoxins unveiled correlate with the syndrome: SVSP ancrod causes venom-induced consumptive coagulopathy, aggravated by thrombocytopenia caused by snaclec rhodocytin, a platelet aggregation inducer, while P-II rhodostoxin mediates hemorrhage, exacerbated by P-I kistomin and snaclec rhodocetin that inhibit platelet plug formation. These toxins exist in multiple isoforms and/or complex subunits, deserving further characterization for the development of an effective, polyspecific regional antivenom. Biological significanceAdvents in proteomics and bioinformatics have vigorously propelled the scientific discoveries of toxins from various lineages of venomous snakes. The Malayan pit viper, Calloselasma rhodostoma, is a medically important species in Southeast Asia as its bite can cause envenomation, while the venom is also a source of bioactive compounds for drug discovery. Detailed profiling of the venom, however, is inadequate possibly due to the complex nature of the venom and technical limitation in separating the constituents into details. Integrating a multi-step fractionation method, this study successfully revealed a comprehensive and quantitative profile of the composition of the venom of this medically important venomous snake. The relative abundance of the various venom proteins is determined in a global profile, providing useful information for understanding the pathogenic roles of the different toxins in C. rhodostoma envenomation. Notably, the principal hemotoxins were identified in great details, including the variety of toxin subunits and isoforms. The findings indicate that these toxins are the principal targets for effective antivenom neutralization, and should be addressed in the production of a pan-regional polyspecific antivenom. In addition, minor toxin components not reported previously in the venom were also detected in this study, enriching the current toxin database for the venomous snakes.

Revisiting Notechis scutatus venom: on shotgun proteomics and neutralization by the “bivalent” Sea Snake Antivenom

Recent advances in proteomics enable deep profiling of the compositional details of snake venoms for improved understanding on envenomation pathophysiology and immunological neutralization. In this study, the venom of Australian tiger snake (Notechis scutatus) was trypsin-digested in solution and subjected to nano-ESI-LCMS/MS. Applying a relative quantitative proteomic approach, the findings revealed a proteome comprising 42 toxin subtypes clustered into 12 protein families. Phospholipases A2 constitute the most abundant toxins (74.5% of total venom proteins) followed by Kunitz serine protease inhibitors (6.9%), snake venom serine proteases (5.9%), alpha-neurotoxins (5.6%) and several toxins of lower abundance. The proteome correlates with N. scutatus envenoming effects including pre-synaptic and post-synaptic neurotoxicity and consumptive coagulopathy. The venom is highly lethal in mice (intravenous median lethal dose=0.09μg/g). BioCSL Sea Snake Antivenom, raised against the venoms of beaked sea snake (Hydrophis schistosus) and N. scutatus (added for enhanced immunogenicity), neutralized the lethal effect of N. scutatus venom (potency=2.95mg/ml) much more effectively than the targeted H.schistosus venom (potency=0.48mg/ml). The combined venom immunogen may have improved the neutralization against phospholipases A2 which are abundant in both venoms, but not short-neurotoxins which are predominant only in H. schistosus venom. SignificanceA shotgun proteomic approach adopted in this study revealed the compositional details of the venom of common tiger snake from Australia, Notechis scutatus. The proteomic findings provided additional information on the relative abundances of toxins and the detection of proteins of minor expression unreported previously. The potent lethal effect of the venom was neutralized by bioCSL Sea Snake Antivenom, an anticipated finding due to the fact that the Sea Snake Antivenom is actually bivalent in nature, being raised against a mix of venoms of the beaked sea snake (Hydrophis schistosus) and N. scutatus. However, it is surprising to note that bioCSL Sea Snake Antivenom neutralized N. scutatus venom much more effectively compared to the targeted sea snake venom by a marked difference in potency of approximately 6-fold. This phenomenon may be explained by the main difference in the proteomes of the two venoms, where H. schistosus venom is dominated by short-neurotoxins in high abundance – this is a poorly immunogenic toxin group that has been increasingly recognized in the venoms of a few cobras. Further investigations should be directed toward strategies to improve the neutralization of short-neurotoxins, in line with the envisioned production of an effective pan-regional elapid antivenom.

Expression and partial biochemical characterization of a recombinant serine protease from Bothrops pauloensis snake venom

Snake venom serine proteases (SVSPs) are enzymes capable of interfering at several points of hemostasis. Some serine proteases present thrombin-like activity, which makes them targets for the development of therapeutics agents in the treatment of many hemostatic disorders. In this study, a recombinant thrombin-like serine protease, denominated rBpSP-II, was obtained from cDNA of the Bothrops pauloensis venom gland and was characterized enzymatically and biochemically. The enzyme rBpSP-II showed clotting activity on bovine plasma and proteolytic activity on fibrinogen, cleaving exclusively the Aα chain. The evaluation of rBpSP-II activity on chromogenic substrates demonstrated thrombin-like activity of the enzyme due to its capacity to hydrolyze the thrombin substrate. These characteristics make rBpSP-II an attractive molecule for additional studies. Further research is needed to verify whether rBpSP-II can serve as a template for the synthesis of therapeutic agents to treat hemostatic disorders.

BbrzSP-32, the first serine protease isolated from Bothrops brazili venom: Purification and characterization

Snake venom toxins are related not only in detention, death and the promotion of initial digestion of prey but also due to their different biochemical, structural and pharmacological effects they can result in new drugs. Among these toxins snake venom serine proteases (SVSPs) should be highlighted because they are responsible for inducing changes in physiological functions such as blood coagulation, fibrinolysis, and platelet aggregation. This article presents the first serine protease (SP) isolated from Bothrops brazili: BbrzSP-32. The new SP showed 36kDa of relative molecular mass and its absolute mass was confirmed by mass spectrometry as 32,520Da. It presents 79.48% identity when compared to other SVSPs and was able to degrade the α-chain of fibrinogen, in in vitro models, because of this it is considered a SVTLE-A. It showed dose-dependent activity in the process of degradation of fibrin networks demonstrating greater specificity for this activity when compared to its thrombolytic action. BbrzSP-32 demonstrated proteolytic activity on gelatin and chromogenic substrates for serine proteases and thrombin-like enzymes (S-2288 and S-2238 respectively), besides having coagulant activity on human plasma. After pre-incubation with PMSF and benzamidine the coagulant and proteolytic activities on the S-2288 and S-2238 substrates were reduced. BbrzSP-32 shows stability against pH and temperature variations, demonstrating optimum activity between 30 and 40°C and in the pH range 7.5 to 8.5. A new SP with potential biotechnological application was isolated.