Biochemical and proteomic analyses of venom from a new pit viper, Protobothrops kelomohy

Abstract

Background: A 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. Methods: PKV was evaluated for its neutralizing capacity (ER 50 ), lethality (LD 50 ), 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. Results: Intravenous LD 50 of PKV was 0.67 µg/g. ER 50 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 A 2 (15.49%).

Phylogeny-Related Variations in Venomics: A Test in a Subset of Habu Snakes (Protobothrops)

“…These results are consistent with the previous investigations on the venom proteomes of three other habu snakes, which were also overwhelmingly dominated by SVMP, PLA 2 , and SVSP ( P. elegans 71.4%, P. flavoviridis 87.92%, and P. kelomohy 86.27% of the total abundance; Table 1 ) [ 31 , 33 , 36 ]; P. flavoviridis undoubtedly expressed the highest abundance of PLA 2 (55.14%) in its venom proteome among all adult habu snakes. However, the relative abundances of these three protein families were highly variable among these venoms, especially the abundance of SVSP in P. kelomohy which was 20.8-fold that of P. flavoviridis .…”

“…Statistical analyses further indicated the degrees of variation in the relative abundance of the 14 protein families in the venoms of eight adult habu snakes (three in previous studies [ 31 , 33 , 36 ] and five in the current study) ranged from 32.5 to 233.0 (coefficients of variations, CV), and the average CV of three highly abundant protein families (47.8 for SVMP, SVSP, and PLA 2 ) was much lower than that of trace protein families (206.6 for NGF, PLB, 5′NT, and QC). Similar trends were found in Agkistrodon snakes with an average CV of 19.5 estimated for highly abundant protein families (SVMP, SVSP, and PLA 2 ) and 71.3 for low abundance families (CRISP, disintegrin, CTL, LAAO, and NGF) [ 12 ], as well as in Sistrurus snakes with CV values of 23.9 and 125.4, respectively [ 10 ].…”

“…The venoms from adult habu snakes in the current study showed relatively high variability in their toxicity to ICR mice, among which the P. cornutus and P. mucrosquamatus venoms, respectively, produced the strongest (LD 50 = 0.61 μg/g) and the weakest (LD 50 = 3.82 μg/g) toxicities among these venoms, and the former was 6-fold more toxic than the latter ( Table 1 ). These venoms all seemed more toxic than P. elegans venom (LD 50 = 4.85 μg/g, [ 53 ]) and only the P. cornutus venom showed a strong toxicity similar to that of P. kelomohy venom (LD 50 = 0.67 μg/g, [ 36 ]). The other habu snake venoms investigated in a previous study [ 31 ] and the current study had moderate levels of toxicity.…”

“…However, most habu snakes are mainly endemic to small areas or lack enough detailed epidemiological and clinical data, and are classified as secondary medically important venomous snakes [ 23 ]. Except for P. elegans , P. kelomohy , and P. mangshanensis [ 33 , 36 , 37 ], the global venom composition of the remaining habu snakes has been less studied. Thus, the mystery of the venom profiles across the genus Protobothrops still needs to be comprehensively solved.…”

Challenges and Opportunities in Clinical Diagnostic Routine of Envenomation Using Blood Plasma Proteomics

“…Studies involving mass spectrometry in toxinology have focused on the study of proteins present in animal venoms, converging on (i) the identification of isolated and purified proteins from venoms [ 72 , 73 , 74 ], (ii) the description of the composition of venoms [ 75 , 76 , 77 , 78 , 79 , 80 , 81 ], and (iii) the identification of altered biological processes in tissues after tissue damage caused by snake venom [ 41 , 67 , 68 , 69 , 70 , 82 , 83 ]. MS can assist in the development and tracking of target toxins and improve the sensitivity and specificity of ELISA and Western blot assays by identifying toxins circulating in the blood of the victims.…”