Explaining Echis: Proteotranscriptomic Profiling of Echis carinatus carinatus Venom
Abstract
1. Introduction
2. Results and Discussion
2.1. Comparative Venom Gland Transcriptomics
2.2. Venom Proteome
2.3. Biochemical Activities of Venom
2.3.1. Phospholipase Activity of Venoms
2.3.2. LAAO Activity of Venoms
2.3.3. Protease Activity of Venoms
2.4. The Preclinical Efficacy of Indian Antivenom Against E. c. carinatus Venom
2.4.1. In Vitro Binding Potential
2.4.2. In Vivo Neutralisation Potential
3. Conclusions
4. Materials and Methods
4.1. Venoms and Antivenoms
4.2. Ethical Statement
4.3. Comparative Tissue Transcriptomics
4.3.1. RNA Isolation, Library Preparation, and Sequencing
4.3.2. Transcriptome Assembly, Annotation, and Quantification
4.4. Venom Proteomics
4.4.1. Protein Estimation and One-Dimensional Gel Electrophoresis
4.4.2. Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC)
4.4.3. In-Gel and In-Solution Digestion and Liquid Chromatography—Tandem Mass Spectrometry (LC-MS/MS)
4.4.4. Relative Abundance of Toxin Families
4.5. Biochemical Characterisation
4.5.1. Phospholipase Assay
4.5.2. Protease Assay
4.5.3. L-Amino Acid Oxidase (LAAO) Assay
4.6. In Vitro Binding Assay
4.7. The Median Lethal Dose (LD50)
4.8. The Median Effective Dose (ED50)
4.9. Statistics
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
- Arnold, N.; Robinson, M.; Carranza, S. A Preliminary Analysis of Phylogenetic Relationships and Biogeography of the Dangerously Venomous Carpet Vipers, Echis (Squamata, Serpentes, Viperidae) Based on Mitochondrial DNA Sequences. Amphib-Reptil 2009, 30, 273–282. [Google Scholar] [CrossRef]
- Campbell, J.A.; Lamar, W.W. The Venomous Reptiles of the Western Hemisphere; Comstock Books in Herpetology; Comstock Publishing Associates: Ithaca, NY, USA, 2004; ISBN 9780801441417. [Google Scholar]
- Pook, C.E.; Joger, U.; Stümpel, N.; Wüster, W. When Continents Collide: Phylogeny, Historical Biogeography and Systematics of the Medically Important Viper Genus Echis (Squamata: Serpentes: Viperidae). Mol. Phylogenet. Evol. 2009, 53, 792–807. [Google Scholar] [CrossRef] [PubMed]
- Suraweera, W.; Warrell, D.; Whitaker, R.; Menon, G.; Rodrigues, R.; Fu, S.H.; Begum, R.; Sati, P.; Piyasena, K.; Bhatia, M.; et al. Trends in Snakebite Deaths in India from 2000 to 2019 in a Nationally Representative Mortality Study. Elife 2020, 9, e54076. [Google Scholar] [CrossRef]
- Warrell, D.A.; Davidson, N.M.; Greenwood, B.M.; Ormerod, L.D.; Pope, H.M.; Watkins, B.J.; Prentice, C.R. Poisoning by Bites of the Saw-Scaled or Carpet Viper (Echis Carinatus) in Nigeria. Q. J. Med. 1977, 46, 33–62. [Google Scholar] [PubMed]
- Sarkar, S.; Sinha, R.; Chaudhury, A.R.; Maduwage, K.; Abeyagunawardena, A.; Bose, N.; Pradhan, S.; Bresolin, N.L.; Garcia, B.A.; McCulloch, M. Snake Bite Associated with Acute Kidney Injury. Pediatr. Nephrol. 2021, 36, 3829–3840. [Google Scholar] [CrossRef]
- Bhatia, S.; Vasudevan, K. Comparative Proteomics of Geographically Distinct Saw-Scaled Viper (Echis Carinatus) Venoms from India. Toxicon X 2020, 7, 100048. [Google Scholar] [CrossRef] [PubMed]
- Patra, A.; Kalita, B.; Chanda, A.; Mukherjee, A.K. Proteomics and Antivenomics of Echis Carinatus Carinatus Venom: Correlation with Pharmacological Properties and Pathophysiology of Envenomation. Sci. Rep. 2017, 7, 17119. [Google Scholar] [CrossRef]
- Patra, A.; Mukherjee, A.K. Proteomic Analysis of Sri Lanka Echis Carinatus Venom: Immunological Cross-Reactivity and Enzyme Neutralization Potency of Indian Polyantivenom. J. Proteome Res. 2020, 19, 3022–3032. [Google Scholar] [CrossRef]
- Senji Laxme, R.R.; Khochare, S.; de Souza, H.F.; Ahuja, B.; Suranse, V.; Martin, G.; Whitaker, R.; Sunagar, K. Beyond the “Big Four”: Venom Profiling of the Medically Important yet Neglected Indian Snakes Reveals Disturbing Antivenom Deficiencies. PLoS Negl. Trop. Dis. 2019, 13, e0007899. [Google Scholar] [CrossRef]
- Attarde, S.; Iyer, A.; Khochare, S.; Shaligram, U.; Vikharankar, M.; Sunagar, K. The Preclinical Evaluation of a Second-Generation Antivenom for Treating Snake Envenoming in India. Toxins 2022, 14, 168. [Google Scholar] [CrossRef]
- Casewell, N.R.; Harrison, R.A.; Wüster, W.; Wagstaff, S.C. Comparative Venom Gland Transcriptome Surveys of the Saw-Scaled Vipers (Viperidae: Echis) Reveal Substantial Intra-Family Gene Diversity and Novel Venom Transcripts. BMC Genom. 2009, 10, 564. [Google Scholar] [CrossRef]
- Wagstaff, S.C.; Harrison, R.A. Venom Gland EST Analysis of the Saw-Scaled Viper, Echis Ocellatus, Reveals Novel Alpha9beta1 Integrin-Binding Motifs in Venom Metalloproteinases and a New Group of Putative Toxins, Renin-like Aspartic Proteases. Gene 2006, 377, 21–32. [Google Scholar] [CrossRef] [PubMed]
- Tasoulis, T.; Isbister, G.K. A Review and Database of Snake Venom Proteomes. Toxins 2017, 9, 290. [Google Scholar] [CrossRef] [PubMed]
- Bittenbinder, M.A.; van Thiel, J.; Cardoso, F.C.; Casewell, N.R.; Gutiérrez, J.-M.; Kool, J.; Vonk, F.J. Tissue Damaging Toxins in Snake Venoms: Mechanisms of Action, Pathophysiology and Treatment Strategies. Commun. Biol. 2024, 7, 358. [Google Scholar] [CrossRef] [PubMed]
- Cedro, R.C.A.; Menaldo, D.L.; Costa, T.R.; Zoccal, K.F.; Sartim, M.A.; Santos-Filho, N.A.; Faccioli, L.H.; Sampaio, S.V. Cytotoxic and Inflammatory Potential of a Phospholipase A2 from Bothrops Jararaca Snake Venom. J. Venom. Anim. Toxins Incl. Trop. Dis. 2018, 24, 33. [Google Scholar] [CrossRef]
- Guo, C.; Liu, S.; Yao, Y.; Zhang, Q.; Sun, M.-Z. Past Decade Study of Snake Venom L-Amino Acid Oxidase. Toxicon 2012, 60, 302–311. [Google Scholar] [CrossRef]
- Du, X.-Y.; Clemetson, K.J. Snake Venom L-Amino Acid Oxidases. Toxicon 2002, 40, 659–665. [Google Scholar] [CrossRef]
- Costa, T.R.; Burin, S.M.; Menaldo, D.L.; de Castro, F.A.; Sampaio, S.V. Snake Venom L-Amino Acid Oxidases: An Overview on Their Antitumor Effects. J. Venom. Anim. Toxins Incl. Trop. Dis. 2014, 20, 23. [Google Scholar] [CrossRef]
- Matsui, T.; Fujimura, Y.; Titani, K. Snake Venom Proteases Affecting Hemostasis and Thrombosis. Biochim. Biophys. Acta 2000, 1477, 146–156. [Google Scholar] [CrossRef]
- Bhatia, S.; Blotra, A.; Vasudevan, K. Evaluating Antivenom Efficacy against Echis Carinatus Venoms-Screening for in Vitro Alternatives. Toxins 2022, 14, 481. [Google Scholar] [CrossRef]
- Bolger, A.M.; Lohse, M.; Usadel, B. Trimmomatic: A Flexible Trimmer for Illumina Sequence Data. Bioinformatics 2014, 30, 2114–2120. [Google Scholar] [CrossRef]
- Andrews, S. FastQC: A quality control tool for high throughput sequence data. In Babraham Bioinformatics; Babraham Institute: Cambridge, UK, 2010. [Google Scholar]
- Grabherr, M.G.; Haas, B.J.; Yassour, M.; Levin, J.Z.; Thompson, D.A.; Amit, I.; Adiconis, X.; Fan, L.; Raychowdhury, R.; Zeng, Q.; et al. Full-Length Transcriptome Assembly from RNA-Seq Data without a Reference Genome. Nat. Biotechnol. 2011, 29, 644–652. [Google Scholar] [CrossRef] [PubMed]
- Simão, F.A.; Waterhouse, R.M.; Ioannidis, P.; Kriventseva, E.V.; Zdobnov, E.M. BUSCO: Assessing Genome Assembly and Annotation Completeness with Single-Copy Orthologs. Bioinformatics 2015, 31, 3210–3212. [Google Scholar] [CrossRef] [PubMed]
- Langmead, B.; Salzberg, S.L. Fast Gapped-Read Alignment with Bowtie 2. Nat. Methods 2012, 9, 357–359. [Google Scholar] [CrossRef] [PubMed]
- Haas, B.J.; Papanicolaou, A.; Yassour, M.; Grabherr, M.; Blood, P.D.; Bowden, J.; Couger, M.B.; Eccles, D.; Li, B.; Lieber, M.; et al. De Novo Transcript Sequence Reconstruction from RNA-Seq Using the Trinity Platform for Reference Generation and Analysis. Nat. Protoc. 2013, 8, 1494–1512. [Google Scholar] [CrossRef]
- Altschul, S.F.; Gish, W.; Miller, W.; Myers, E.W.; Lipman, D.J. Basic Local Alignment Search Tool. J. Mol. Biol. 1990, 215, 403–410. [Google Scholar] [CrossRef]
- Li, B.; Dewey, C.N. RSEM: Accurate Transcript Quantification from RNA-Seq Data with or without a Reference Genome. BMC Bioinformatics 2011, 12, 323. [Google Scholar] [CrossRef]
- Tarazona, S.; Furió-Tarí, P.; Turrà, D.; Pietro, A.D.; Nueda, M.J.; Ferrer, A.; Conesa, A. Data Quality Aware Analysis of Differential Expression in RNA-Seq with NOISeq R/Bioc Package. Nucleic Acids Res. 2015, 43, e140. [Google Scholar] [CrossRef]
- Bradford, M.M. A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding. Anal. Biochem. 1976, 72, 248–254. [Google Scholar] [CrossRef]
- Smith, B.J. Quantification of Proteins on Polyacrylamide Gels (Nonradioactive). Methods Mol. Biol. 1984, 1, 119–125. [Google Scholar]
- Senji Laxme, R.R.; Attarde, S.; Khochare, S.; Suranse, V.; Martin, G.; Casewell, N.R.; Whitaker, R.; Sunagar, K. Biogeographical Venom Variation in the Indian Spectacled Cobra (Naja Naja) Underscores the Pressing Need for Pan-India Efficacious Snakebite Therapy. PLoS Negl. Trop. Dis. 2021, 15, e0009150. [Google Scholar] [CrossRef]
- Calvete, J.J.; Lomonte, B.; Saviola, A.J.; Calderón Celis, F.; Ruiz Encinar, J. Quantification of Snake Venom Proteomes by Mass Spectrometry-Considerations and Perspectives. Mass Spectrom. Rev. 2024, 43, 977–997. [Google Scholar] [CrossRef]
- Finney, D.J. Probit Analysis, 2nd ed.; Cambridge University Press: Cambridge, UK, 1971; ISBN 9780521080415. [Google Scholar]
Name of Sample | Challenge Dose 5× LD50 (μg/mouse) | Amount of Antivenom Injected in the Venom-Antivenom Mixture (µL) | ED50 (μL) | Potency of Antivenom (mg/mL) | |||
---|---|---|---|---|---|---|---|
EcCaKA (pooled) | 75 | 39.1 | 45 | 51.75 | 68.4 | 43.07 39.7–46.72 | 1.39 1.28–1.51 |
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Javed, S.; Gond, P.G.; Samanta, A.; Unawane, A.; Mudavath, M.N.; Jaglan, A.; Sunagar, K. Explaining Echis: Proteotranscriptomic Profiling of Echis carinatus carinatus Venom. Toxins 2025, 17, 353. https://doi.org/10.3390/toxins17070353
Javed S, Gond PG, Samanta A, Unawane A, Mudavath MN, Jaglan A, Sunagar K. Explaining Echis: Proteotranscriptomic Profiling of Echis carinatus carinatus Venom. Toxins. 2025; 17(7):353. https://doi.org/10.3390/toxins17070353
Chicago/Turabian StyleJaved, Salil, Prasad Gopalkrishna Gond, Arpan Samanta, Ajinkya Unawane, Muralidhar Nayak Mudavath, Anurag Jaglan, and Kartik Sunagar. 2025. "Explaining Echis: Proteotranscriptomic Profiling of Echis carinatus carinatus Venom" Toxins 17, no. 7: 353. https://doi.org/10.3390/toxins17070353
APA StyleJaved, S., Gond, P. G., Samanta, A., Unawane, A., Mudavath, M. N., Jaglan, A., & Sunagar, K. (2025). Explaining Echis: Proteotranscriptomic Profiling of Echis carinatus carinatus Venom. Toxins, 17(7), 353. https://doi.org/10.3390/toxins17070353