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

ABSTRACT: 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.

SUBMITTER: Sitprija S

PROVIDER: S-EPMC8635378 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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

Sitprija Siravit S Chanhome Lawan L Reamtong Onrapak O Thiangtrongjit Tipparat T Vasaruchapong Taksa T Khow Orawan O Noiphrom Jureeporn J Laoungbua Panithi P Tubtimyoy Arissara A Chaiyabutr Narongsak N Kumkate Supeecha S

PloS one 20211201 12

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%), nuclei ...[more]

PMID: 34851989

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Project description:BackgroundA new pit viper, Protobothrops kelomohy, has been recently discovered in northern and northwestern Thailand. Envenoming by the other Protobothrops species across several Asian countries has been a serious health problem since their venom is highly hematotoxic. However, the management of P. kelomohy bites is required as no specific antivenom is available. This study aimed to investigate the biochemical properties and proteomes of P. kelomohy venom (PKV), including the cross-neutralization to its lethality with antivenoms available in Thailand.MethodsPKV was evaluated for its neutralizing capacity (ER50), lethality (LD50), procoagulant and hemorrhagic effects with three monovalent antivenoms (TAAV, DSAV, and CRAV) and one polyvalent (HPAV) hematotoxic antivenom. The enzymatic activities were examined in comparison with venoms of Trimeresurus albolabris (TAV), Daboia siamensis (DSV), Calloselasma rhodostoma (CRV). Molecular mass was separated on SDS-PAGE, then the specific proteins were determined by western blotting. The venom protein classification was analyzed using mass spectrometry-based proteomics.ResultsIntravenous LD50 of PKV was 0.67 µg/g. ER50 of HPAV, DSAV and TAAV neutralize PKV at 1.02, 0.36 and 0.12 mg/mL, respectively. PKV exhibited procoagulant effect with a minimal coagulation dose of 12.5 ± 0.016 µg/mL and hemorrhagic effect with a minimal hemorrhagic dose of 1.20 ± 0.71 µg/mouse. HPAV was significantly effective in neutralizing procoagulant and hemorrhagic effects of PKV than those of TAAV, DSAV and CRAV. All enzymatic activities among four venoms exhibited significant differences. PKV proteome revealed eleven classes of putative snake venom proteins, predominantly metalloproteinase (40.85%), serine protease (29.93%), and phospholipase A2 (15.49%).ConclusionsEnzymatic activities of PKV are similarly related to other viperid venoms in this study by quantitatively hematotoxic properties. Three major venom toxins were responsible for coagulopathy in PKV envenomation. The antivenom HPAV was considered effective in neutralizing the lethality, procoagulant and hemorrhagic effects of PKV.

Project description:Snake venoms are complex mixtures of proteins with toxic activities, with many distinct isoforms, affecting different physiological targets, comprised in a few protein families. It is currently accepted that this diversity in venom composition is an adaptive advantage for venom efficacy on a wide range of prey. However, on the other side, variability on isoforms expression has implications in the clinics of human victims of snakebites and in the efficacy of antivenoms. B. atrox snakes are responsible for most of the human accidents in Brazilian Amazon and the type and abundance of protein families on their venoms present individual variability. Thus, in this study we attempted to correlate the individual venom proteome of the snake brought to the hospital by the patient seeking for medical assistance with the clinical signs observed in the same patient. Individual variability was confirmed in venoms of the 14 snakes selected for the study. The abundance of each protein family was quite similar among the venom samples, while the isoforms composition was highly variable. Considering the protein families, the SVMP group presented the best correlation with bleeding disorders and edema. Considering individual isoforms, some isoforms of venom metalloproteinase (SVMP), C-type lectin-like toxins (CTL) and snake venom serine proteinases (SVSP) presented expression levels that with statistically significant positive correlation to signs and symptoms presented by the patients as bleeding disorders, edema, ecchymosis and blister formation. However, some unexpected data were also observed as the correlation between a CTL, CRISP or LAAO isoforms with blister formation, still to be confirmed with a larger number of samples. Although this is still a small number of patient samples, we were able to indicate that venom composition modulates clinical manifestations of snakebites, to confirm at the bedside the prominent role of SVMPs and to include new possible toxin candidates for the development of toxin inhibitors or to improve antivenom selectiveness, important actions for the next generation treatments of snakebites.

Project description:The Malayan pit viper (Calloselasma rhodostoma) is a hemotoxic snake widely found in Southeast Asia and is responsible for the majority of poisoning cases in this region, including Thailand. However, a comprehensive knowledge of the venom protein profile and classification, as well as novel venom proteins, of this viper is still limited. Recently, the detailed composition of several snake venoms has been discovered through the use of transcriptome analysis. Therefore, the aim of this study was to employ a next-generation sequencing platform and bioinformatics analysis to undertake venom-gland de novo transcriptomics of Malayan pit vipers. Furthermore, 21,272 functional coding genes were identified from 36,577 transcripts, of which 314 transcripts were identified as toxin proteins, accounting for 61.41% of total FPKM, which can be categorized into 22 toxin gene families. The most abundant are snake venom metalloproteinase kistomin (P0CB14) and zinc metalloproteinase/disintegrin (P30403), which account for 60.47% of total toxin FPKM and belong to the SVMP toxin family, followed by snake venom serine protease 1 (O13059) and Snaclec rhodocetin subunit beta (P81398), which account for 6.84% and 5.50% of total toxin FPKM and belong to the snake venom serine protease (SVSP) and Snaclec toxin family, respectively. Amino acid sequences of the aforementioned toxins were compared with those identified in other important medical hemotoxic snakes from Southeast Asia, including the Siamese Russell's viper (Daboia siamensis) and green pit viper (Trimeresurus albolabris), in order to analyze their protein homology. The results demonstrated that ranges of 58%-62%, 31%-60%, and 48%-59% identity was observed among the SVMP, Snaclec, and SVSP toxin families, respectively. Understanding the venom protein profile and classification is essential in interpreting clinical symptoms during human envenomation and developing potential therapeutic applications. Moreover, the variability of toxin families and amino acid sequences among related hemotoxic snakes found in this study suggests the use and development of universal antivenom for the treatment of envenomating patients is still challenging.

Project description:Envenomation by the Hypnale hypnale in the Western Ghats of India (particularly in the Malabar region of Kerala) and the subcontinent island nation of Sri Lanka is known to inflict devastating mortality and morbidity. Currently, H. hypnale bites in India are devoid of anti-venom regimens. A detailed characterization of the venom is essential to stress the need for therapeutic anti-venom. Notably, the deleterious effects of this venom on human blood cells have largely remained less explored. Therefore, in continuation of our previous study, in the present study, we envisioned investigating the effect of venom on the morphological and physiological properties of red blood cells (RBCs). The venom readily induced deleterious morphological changes and, finally, the aggregation of washed RBCs. The aggregation process was independent of the ROS and the intracellular Ca2+ ion concentration. Confocal and scanning electron microscopy (SEM) images revealed the loss of biconcave morphology and massive cytoskeletal disarray. Crenation or serrated plasma membrane projections were evenly distributed on the surface of the RBCs. The venom did not cause the formation of methemoglobin in washed RBCs but was significantly induced in whole blood. Venom did not affect glucose uptake and Na+/K+ -ATPase activity but inhibited glucose 6 phosphate dehydrogenase activity and decreased the fluidity of the plasma membrane. Venom-induced RBC aggregates exhibited pro-coagulant activity but without affecting platelet aggregation. In pre-incubation or co-treatment studies, none of the bioactive compounds, such as melatonin, curcumin, fisetin, berberine, and quercetin, sugars such as mannose and galactose, and therapeutic polyvalent anti-venoms (Bharat and VINS) were inhibited, whereas only N-acetylcysteine and H. hypnale monovalent anti-venom could inhibit venom-induced deleterious morphological changes and aggregation of RBCs. In post-treatment studies, paradoxically, none of the bioactives and anti-venoms, including N-acetylcysteine and H. hypnale monovalent anti-venom, reversed the venom-induced RBC aggregates.

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

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

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