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

Molecules ◽  
2018 ◽  
Vol 23 (8) ◽  
pp. 1893 ◽  
Author(s):  
Maik Damm ◽  
Benjamin-Florian Hempel ◽  
Ayse Nalbantsoy ◽  
Roderich Süssmuth
Keyword(s):  
Mass Spectrometry ◽  
Snake Venom ◽  
Serine Proteases ◽  
Secretory Proteins ◽  
Crude Venom ◽  
Human Neuroblastoma ◽  
Phospholipases A2 ◽  
Cytotoxicity Assays ◽  
Intact Mass ◽  
Snake Venomics

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.

scholarly journals Comprehensive snake venomics of the Okinawa Habu pit viper, Protobothrops flavoviridis, by complementary mass spectrometry-guided approaches

2018 ◽  
Author(s):  
Maik Damm ◽  
Benjamin-Florian Hempel ◽  
Ayse Nalbantsoy ◽  
Roderich D. Süssmuth
Keyword(s):  
Mass Spectrometry ◽  
Snake Venom ◽  
Proteomic Analysis ◽  
Top Down ◽  
Crude Venom ◽  
Human Neuroblastoma ◽  
Phospholipases A2 ◽  
Cytotoxicity Assays ◽  
Intact Mass ◽  
Snake Venomics

The Asian world is home to a multitude of venomous and dangerous snakes, which are attributed to various medical effects used 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 with 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 toxins families were detected: CTL, CRISP, svSP, LAAO, 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 the crude venom was measured against human neuroblastoma SH-SY5Y cells and shows in some fractions disintegrin-like effects.

scholarly journals Venomics of Trimeresurus (Popeia) nebularis, the Cameron Highlands Pit Viper from Malaysia: Insights into Venom Proteome, Toxicity and Neutralization of Antivenom

Toxins ◽  
2019 ◽  
Vol 11 (2) ◽  
pp. 95 ◽  
Author(s):  
Choo Tan ◽  
Kae Tan ◽  
Tzu Ng ◽  
Evan Quah ◽  
Ahmad Ismail ◽  
...  
Keyword(s):  
Snake Venom ◽  
Serine Proteases ◽  
Cross Reactivity ◽  
Secretory Proteins ◽  
Proteomic Profiling ◽  
Phospholipases A2 ◽  
Central Highland ◽  
Liquid Chromatography Tandem Mass ◽  
Cameron Highlands ◽  
Venom Proteins

Trimeresurus nebularis is a montane pit viper that causes bites and envenomation to various communities in the central highland region of Malaysia, in particular Cameron’s Highlands. To unravel the venom composition of this species, the venom proteins were digested by trypsin and subjected to nano-liquid chromatography-tandem mass spectrometry (LC-MS/MS) for proteomic profiling. Snake venom metalloproteinases (SVMP) dominated the venom proteome by 48.42% of total venom proteins, with a characteristic distribution of P-III: P-II classes in a ratio of 2:1, while P-I class was undetected. Snaclecs constituted the second most venomous protein family (19.43%), followed by snake venom serine proteases (SVSP, 14.27%), phospholipases A2 (5.40%), disintegrins (5.26%) and minor proteins including cysteine-rich secretory proteins, L-amino acid oxidases, phosphodiesterases, 5′-nucleotidases. The venomic profile correlates with local (painful progressive edema) and systemic (hemorrhage, coagulopathy, thrombocytopenia) manifestation of T. nebularis envenoming. As specific antivenom is unavailable for T. nebularis, the hetero-specific Thai Green Pit viper Monovalent Antivenom (GPVAV) was examined for immunological cross-reactivity. GPVAV exhibited good immunoreactivity to T. nebularis venom and the antivenom effectively cross-neutralized the hemotoxic and lethal effects of T. nebularis (lethality neutralizing potency = 1.6 mg venom per mL antivenom). The findings supported GPVAV use in treating T. nebularis envenoming.

scholarly journals Snake venomics of Bothrops punctatus, a semi-arboreal pitviper species from Antioquia, Colombia

2013 ◽  
Author(s):  
Maritza Fernández Culma ◽  
Jaime A Pereañez ◽  
Vitelbina Núñez Rangel ◽  
Bruno Lomonte
Keyword(s):  
Amino Acid ◽  
Protein Composition ◽  
Secretory Proteins ◽  
Acid Oxidase ◽  
Crude Venom ◽  
Amino Acid Oxidase ◽  
Analytical Strategy ◽  
Phospholipases A2 ◽  
Snake Venomics

Bothrops punctatus is an endangered, semi-arboreal pitviper species distributed in Panamá, Colombia, and Ecuador, whose venom is poorly characterized. In the present work, the protein composition of this venom was profiled using the 'snake venomics' analytical strategy. Decomplexation of the crude venom by RP-HPLC and SDS-PAGE, followed by tandem mass spectrometry of tryptic digests, showed that it consists of proteins assigned to at least nine snake toxin families. Metalloproteinases are predominant in this secretion (41.5% of the total proteins), followed by C-type lectin/lectin-like proteins (16.7%), bradykinin-potentiating peptides (10.7%), phospholipases A2 (9.3%), serine proteinases (5.4%), disintegrins (3.8%), L-amino acid oxidases (3.1%), vascular endothelial growth factors (1.7%), and cysteine-rich secretory proteins (1.2%). Altogether, 6.6% of the proteins were not identified. In vitro, the venom exhibited proteolytic, phospholipase A2, and L-amino acid oxidase activities, as well as angiotensin-converting enzyme (ACE)-inhibitory activity, in agreement with the obtained proteomic profile. Cytotoxic activity on murine C2C12 myoblasts was negative, suggesting that the majority of venom phospholipases A2 likely belong to the acidic type, which often lack major toxic effects. The protein composition of B. punctatus venom shows a good correlation with toxic activities here and previously reported, and adds further data in support of the wide diversity of strategies that have evolved in snake venoms to subdue prey, as increasingly being revealed by proteomic analyses.

scholarly journals Proteomics and immunocharacterization of Asian mountain pit viper (Ovophis monticola) venom

PLoS ONE ◽  
2021 ◽  
Vol 16 (12) ◽  
pp. e0260496
Author(s):  
Siravit Sitprija ◽  
Lawan Chanhome ◽  
Onrapak Reamtong ◽  
Tipparat Thiangtrongjit ◽  
Taksa Vasaruchapong ◽  
...  
Keyword(s):  
Snake Venom ◽  
Serine Proteases ◽  
Indirect Elisa ◽  
Secretory Proteins ◽  
Acid Oxidase ◽  
Two Dimensional ◽  
Crude Venom ◽  
Amino Acid Oxidase ◽  
Specific Antigens ◽  
Phospholipases A

The venomic profile of Asian mountain pit viper Ovophis monticola is clarified in the present study. Using mass spectrometry-based proteomics, 247 different proteins were identified in crude venom of O. monticola found in Thailand. The most abundant proteins were snake venom metalloproteases (SVMP) (36.8%), snake venom serine proteases (SVSP) (31.1%), and phospholipases A2 (PLA2) (12.1%). Less abundant proteins included L-amino acid oxidase (LAAO) (5.7%), venom nerve growth factor (3.6%), nucleic acid degrading enzymes (3.2%), C-type lectins (CTL) (1.6%), cysteine-rich secretory proteins (CRISP) (1.2%) and disintegrin (1.2%). The immunoreactivity of this viper’s venom to a monovalent antivenom against green pit viper Trimeresurus albolabris, or to a polyvalent antivenom against hemotoxic venom was investigated by indirect ELISA and two-dimensional (2D) immunoblotting. Polyvalent antivenom showed substantially greater reactivity levels than monovalent antivenom. A titer for the monovalent antivenom was over 1:1.28x107 dilution while that of polyvalent antivenom was 1:5.12x107. Of a total of 89 spots comprising 173 proteins, 40 spots of predominantly SVMP, SVSP and PLA2 were specific antigens for antivenoms. The 49 unrecognized spots containing 72 proteins were characterized as non-reactive proteins, and included certain types of CTLs and CRISPs. These neglected venom constituents could limit the effectiveness of antivenom-based therapy currently available for victims of pit viper envenomation.

scholarly journals Snake venomics of Bothrops punctatus, a semi-arboreal pitviper species from Antioquia, Colombia

2013 ◽  
Author(s):  
Maritza Fernández Culma ◽  
Jaime A Pereañez ◽  
Vitelbina Núñez Rangel ◽  
Bruno Lomonte
Keyword(s):  
Amino Acid ◽  
Protein Composition ◽  
Secretory Proteins ◽  
Acid Oxidase ◽  
Crude Venom ◽  
Amino Acid Oxidase ◽  
Analytical Strategy ◽  
Phospholipases A2 ◽  
Snake Venomics

Bothrops punctatus is an endangered, semi-arboreal pitviper species distributed in Panamá, Colombia, and Ecuador, whose venom is poorly characterized. In the present work, the protein composition of this venom was profiled using the 'snake venomics' analytical strategy. Decomplexation of the crude venom by RP-HPLC and SDS-PAGE, followed by tandem mass spectrometry of tryptic digests, showed that it consists of proteins assigned to at least nine snake toxin families. Metalloproteinases are predominant in this secretion (41.5% of the total proteins), followed by C-type lectin/lectin-like proteins (16.7%), bradykinin-potentiating peptides (10.7%), phospholipases A2 (9.3%), serine proteinases (5.4%), disintegrins (3.8%), L-amino acid oxidases (3.1%), vascular endothelial growth factors (1.7%), and cysteine-rich secretory proteins (1.2%). Altogether, 6.6% of the proteins were not identified. In vitro, the venom exhibited proteolytic, phospholipase A2, and L-amino acid oxidase activities, as well as angiotensin-converting enzyme (ACE)-inhibitory activity, in agreement with the obtained proteomic profile. Cytotoxic activity on murine C2C12 myoblasts was negative, suggesting that the majority of venom phospholipases A2 likely belong to the acidic type, which often lack major toxic effects. The protein composition of B. punctatus venom shows a good correlation with toxic activities here and previously reported, and adds further data in support of the wide diversity of strategies that have evolved in snake venoms to subdue prey, as increasingly being revealed by proteomic analyses.

scholarly journals New Insights on Moojase, a Thrombin-Like Serine Protease from Bothrops moojeni Snake Venom

Toxins ◽  
2018 ◽  
Vol 10 (12) ◽  
pp. 500 ◽  
Author(s):  
Fernanda Amorim ◽  
Danilo Menaldo ◽  
Sante Carone ◽  
Thiago Silva ◽  
Marco Sartim ◽  
...  
Keyword(s):  
Snake Venom ◽  
Serine Proteases ◽  
New Drugs ◽  
Coagulation Cascade ◽  
Dependent Manner ◽  
Crude Venom ◽  
Human Fibrinogen ◽  
Factor Xia ◽  
Bothrops Moojeni

Snake venom serine proteases (SVSPs) are enzymes that are capable of interfering in various parts of the blood coagulation cascade, which makes them interesting candidates for the development of new therapeutic drugs. Herein, we isolated and characterized Moojase, a potent coagulant enzyme from Bothrops moojeni snake venom. The toxin was isolated from the crude venom using a two-step chromatographic procedure. Moojase is a glycoprotein with N-linked glycans, molecular mass of 30.3 kDa and acidic character (pI 5.80–6.88). Sequencing of Moojase indicated that it is an isoform of Batroxobin. Moojase was able to clot platelet-poor plasma and fibrinogen solutions in a dose-dependent manner, indicating thrombin-like properties. Moojase also rapidly induced the proteolysis of the Aα chains of human fibrinogen, followed by the degradation of the Bβ chains after extended periods of incubation, and these effects were inhibited by PMSF, SDS and DTT, but not by benzamidine or EDTA. RP-HPLC analysis of its fibrinogenolysis confirmed the main generation of fibrinopeptide A. Moojase also induced the fibrinolysis of fibrin clots formed in vitro, and the aggregation of washed platelets, as well as significant amidolytic activity on substrates for thrombin, plasma kallikrein, factor Xia, and factor XIIa. Furthermore, thermofluor analyses and the esterase activity of Moojase demonstrated its very high stability at different pH buffers and temperatures. Thus, studies such as this for Moojase should increase knowledge on SVSPs, allowing their bioprospection as valuable prototypes in the development of new drugs, or as biotechnological tools.

scholarly journals Snake Venom Proteomics, Immunoreactivity and Toxicity Neutralization Studies for the Asiatic Mountain Pit Vipers, Ovophis convictus, Ovophis tonkinensis, and Hime Habu, Ovophis okinavensis

Toxins ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 514
Author(s):  
Choo Hock Tan ◽  
Praneetha Palasuberniam ◽  
Kae Yi Tan
Keyword(s):  
Snake Venom ◽  
Serine Proteases ◽  
Southern China ◽  
Snake Venoms ◽  
Phospholipases A2 ◽  
Limited Efficacy ◽  
Chromatography Tandem Mass Spectrometry ◽  
Liquid Chromatography Tandem Mass ◽  
Species Specific ◽  
Venom Proteins

Snakebite envenomation is a serious neglected tropical disease, and its management is often complicated by the diversity of snake venoms. In Asia, pit vipers of the Ovophis species complex are medically important venomous snakes whose venom properties have not been investigated in depth. This study characterized the venom proteomes of Ovophis convictus (West Malaysia), Ovophis tonkinensis (northern Vietnam, southern China), and Ovophis okinavensis (Okinawa, Japan) by applying liquid chromatography-tandem mass spectrometry, which detected a high abundance of snake venom serine proteases (SVSP, constituting 40–60% of total venom proteins), followed by phospholipases A2, snake venom metalloproteinases of mainly P-III class, L-amino acid oxidases, and toxins from other protein families which were less abundant. The venoms exhibited different procoagulant activities in human plasma, with potency decreasing from O. tonkinensis > O. okinavensis > O. convictus. The procoagulant nature of venom confirms that consumptive coagulopathy underlies the pathophysiology of Ovophis pit viper envenomation. The hetero-specific antivenoms Gloydius brevicaudus monovalent antivenom (GbMAV) and Trimeresurus albolabris monovalent antivenom (TaMAV) were immunoreactive toward the venoms, and cross-neutralized their procoagulant activities, albeit at variably limited efficacy. In the absence of species-specific antivenom, these hetero-specific antivenoms may be useful in treating coagulotoxic envenomation caused by the different snakes in their respective regions.

scholarly journals Bleeding and Thrombosis: Insights into Pathophysiology of Bothrops Venom-Related Hemostasis Disorders

2021 ◽  
Vol 22 (17) ◽  
pp. 9643
Author(s):  
Sébastien Larréché ◽  
Jean-Philippe Chippaux ◽  
Lucie Chevillard ◽  
Simon Mathé ◽  
Dabor Résière ◽  
...  
Keyword(s):  
Acute Kidney Injury ◽  
Snake Venom ◽  
Thrombotic Microangiopathy ◽  
Serine Proteases ◽  
Kidney Injury ◽  
Coagulation Factors ◽  
Cardiovascular Collapse ◽  
Cell Integrity ◽  
Consumption Coagulopathy ◽  
Phospholipases A2

Toxins from Bothrops venoms targeting hemostasis are responsible for a broad range of clinical and biological syndromes including local and systemic bleeding, incoagulability, thrombotic microangiopathy and macrothrombosis. Beyond hemostais disorders, toxins are also involved in the pathogenesis of edema and in most complications such as hypovolemia, cardiovascular collapse, acute kidney injury, myonecrosis, compartmental syndrome and superinfection. These toxins can be classified as enzymatic proteins (snake venom metalloproteinases, snake venom serine proteases, phospholipases A2 and L-amino acid oxidases) and non-enzymatic proteins (desintegrins and C-type lectin proteins). Bleeding is due to a multifocal toxicity targeting vessels, platelets and coagulation factors. Vessel damage due to the degradation of basement membrane and the subsequent disruption of endothelial cell integrity under hydrostatic pressure and tangential shear stress is primarily responsible for bleeding. Hemorrhage is promoted by thrombocytopenia, platelet hypoaggregation, consumption coagulopathy and fibrin(ogen)olysis. Onset of thrombotic microangiopathy is probably due to the switch of endothelium to a prothrombotic phenotype with overexpression of tissue factor and other pro-aggregating biomarkers in association with activation of platelets and coagulation. Thrombosis involving large-caliber vessels in B. lanceolatus envenomation remains a unique entity, which exact pathophysiology remains poorly understood.

scholarly journals Contribution of metalloproteases, serine proteases and phospholipases A2 to the inflammatory reaction induced by Bothrops jararaca crude venom in mice

Toxicon ◽  
2010 ◽  
Vol 55 (2-3) ◽  
pp. 227-234 ◽  
Author(s):  
Bianca Cestari Zychar ◽  
Camila Squazoni Dale ◽  
Denise Soares Demarchi ◽  
Luis Roberto C. Gonçalves
Keyword(s):  
Inflammatory Reaction ◽  
Serine Proteases ◽  
Crude Venom ◽  
Phospholipases A2 ◽  
Bothrops Jararaca

scholarly journals Use of MALDI-TOF Mass Spectrometry for Specificity Studies of Biomedically Important Proteases

2002 ◽  
Vol 16 (3-4) ◽  
pp. 161-170 ◽  
Author(s):  
Jüri Siigur ◽  
Katrin Trummal ◽  
Külli TÕnismägi ◽  
Mari Samel ◽  
Ene Siigur ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Snake Venom ◽  
Serine Proteases ◽  
Factor Ix ◽  
Diagnostic Tools ◽  
Factor V ◽  
Factor X ◽  
Maldi Tof Mass Spectrometry ◽  
Maldi Tof ◽  
Vipera Lebetina

Proteases play crucial role starting from fertilization until to cell death. Our studies of the two Viperidae venoms (Levantine viperVipera lebetina, Common viperVipera berus) have demonstrated the existence of biomedically important proteases, both coagulants and anticoagulants that may be useful as diagnostic tools or potential therapeutics. We showed that venoms of both snakes contain: (i) metalloproteases and serine proteases that degrade fibrinogen, but not fibrin; (ii) factor X activators (VLFXA, VBFXAE); (iii) bradykinin-releasing serine proteases. AdditionallyVipera lebetinasnake venom contains thrombolytic fibrin degrading metalloenzyme (lebetase), HUVEC cell apoptosis inducing metalloprotease (VLAIP), factor V activator (VLFVA), thermostable β-fibrinogenase and α-fibrinogenase which has no homolog among known serine proteases. We examined the activity of snake venom proteases against bradykinin, substance P, insulin B-chain and 6–10 amino acid residues containing peptides synthesized according to potential cleavage regions of fibrinogen, factor X, factor IX, factor V, α2-macroglobulin bait region and pregnancy zone protein (PZP). We used MALDI TOF mass spectrometry technique for the discovery and identification of peptides released by protease hydrolysis. The sensitive and quick MALDI-TOF mass spectrometry methodology allows us to obtain the primary information about the substrate specificity of different proteases against various peptides and proteins.

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