Current Protein & Peptide ScienceCurrent Protein & Peptide ScienceCurrent Protein & Peptide Science1389-20371875-5550Bentham Science64565110.2174/0113892037277589231128103032Life SciencesResearch ArticleCTXP, The Major Cobra Toxin Peptide from Naja Naja Oxiana Venom; A Promising Target for Antivenom Agent DevelopmentChafiMohammadinfo@benthamscience.netEslamnezhad-NaminMohseninfo@benthamscience.netDastjerdehMansourehinfo@benthamscience.netZareinejadMohammadinfo@benthamscience.netOghalaieAkbarinfo@benthamscience.netBagheriKamraninfo@benthamscience.netKazemi-LomedashtFatemehinfo@benthamscience.netKarimiGholamrezainfo@benthamscience.netRazzaghi-AbyanehMehdiinfo@benthamscience.netSeyedjavadiSimainfo@benthamscience.netBehdaniMahdiinfo@benthamscience.netVenom and Biotherapeutics Molecules Laboratory, Medical Biotechnology Department, Biotechnology Research Center, Pasteur Institute of IranMedical Toxicology Research Center and Pharmacy School, Mashhad University of Medical SciencesDepartment of Mycology, Pasteur Institute of Iran0106202425646947911012025Copyright ©; 2024, Bentham Science Publishers2024Bentham Science Publishershttps://journals.eco-vector.com/1389-2037/article/view/645651

Background and Objective:Snakebite envenoming is a serious public health issue causing more than 135,000 annual deaths worldwide. Naja naja oxiana is one of the most clinically important venomous snakes in Iran and Central Asia. Conventional animal-derived polyclonal antibodies are the major treatment of snakebite envenoming. Characterization of venom components helps to pinpoint the toxic protein responsible for clinical manifestations in victims, which aids us in developing efficient antivenoms with minimal side effects. Therefore, the present study aimed to identify the major lethal protein of Naja naja oxiana by top-down proteomics.

Methods:Venom proteomic profiling was performed using gel filtration (GF), reversed-phase (RP) chromatography, and intact mass spectrometry. The toxicity of GF-, and RP-eluted fractions was analyzed in BALB/c mice. The rabbit polyclonal antisera were produced against crude venom, GF fraction V (FV), and RP peak 1 (CTXP) and applied in neutralization assays.

Results:Toxicity studies in BALB/c identified FV as the major toxic fraction of venom. Subsequently, RP separation of FV resulted in eight peaks, of which peak 1, referred to as "CTXP" (cobra toxin peptide), was identified as the major lethal protein. In vivo neutralization assays using rabbit antisera showed that polyclonal antibodies raised against FV and CTXP are capable of neutralizing at least 2-LD50s of crude venom, FV, and CTXP in all tested mice.

Conclusion:Surprisingly, the Anti-CTXP antibody could neutralize 8-LD50 of the CTXP peptide. These results identified CTXP (a 7 kDa peptide) as a potential target for the development of novel efficient antivenom agents.

Naja naja oxianavenomCTXP (Cobra toxin peptide)lethal proteinantivenomgel filtration.Gutiérrez, J.M.; Warrell, D.A.; Williams, D.J.; Jensen, S.; Brown, N.; Calvete, J.J.; Harrison, R.A. The need for full integration of snakebite envenoming within a global strategy to combat the neglected tropical diseases: The way forward. PLoS Negl. Trop. Dis., 2013, 7(6), e2162. doi: 10.1371/journal.pntd.0002162 PMID: 23785526Alirol, E.; Sharma, S.K.; Bawaskar, H.S.; Kuch, U.; Chappuis, F. Snake bite in South Asia: A review. PLoS Negl. Trop. Dis., 2010, 4(1), e603. doi: 10.1371/journal.pntd.0000603 PMID: 20126271Chippaux, J.P. Snake-bites: Appraisal of the global situation. Bull. World Health Organ., 1998, 76(5), 515-524. PMID: 9868843Kasturiratne, A.; Wickremasinghe, A.R.; de Silva, N.; Gunawardena, N.K.; Pathmeswaran, A.; Premaratna, R.; Savioli, L.; Lalloo, D.G.; de Silva, H.J. The global burden of snakebite: A literature analysis and modelling based on regional estimates of envenoming and deaths. PLoS Med., 2008, 5(11), e218. doi: 10.1371/journal.pmed.0050218 PMID: 18986210Dehghani, R; Dadpour, B; Mehrpour, O. Epidemiological profile of snakebite in Iran, 2009-2010 based on information of ministry of health and medical education. Int. J. Med. Toxicol. Forensic Med., 2014, 4(2), 33-41.Kohli, H.S.; Sakhuja, V. Snake bites and acute renal failure. Saudi J. Kidney Dis. Transpl., 2003, 14(2), 165-176. PMID: 18209442Pyron, R.A.; Burbrink, F.T.; Colli, G.R.; de Oca, A.N.M.; Vitt, L.J.; Kuczynski, C.A.; Wiens, J.J. The phylogeny of advanced snakes (Colubroidea), with discovery of a new subfamily and comparison of support methods for likelihood trees. Mol. Phylogenet. Evol., 2011, 58(2), 329-342. doi: 10.1016/j.ympev.2010.11.006 PMID: 21074626Hsiang, A.Y.; Field, D.J.; Webster, T.H.; Behlke, A.D.B.; Davis, M.B.; Racicot, R.A.; Gauthier, J.A. The origin of snakes: Revealing the ecology, behavior, and evolutionary history of early snakes using genomics, phenomics, and the fossil record. BMC Evol. Biol., 2015, 15(1), 87. doi: 10.1186/s12862-015-0358-5 PMID: 25989795Binh, D.V.; Thanh, T.T.; Chi, P.V. Proteomic characterization of the thermostable toxins from Naja naja venom. J. Venom. Anim. Toxins Incl. Trop. Dis., 2010, 16(4), 631-638. doi: 10.1590/S1678-91992010000400014Samianifard, M.; Nazari, A.; Tahoori, F.; Dounighi, M.N. Proteome analysis of toxic fractions of iranian cobra (naja naja oxiana) snake venom using two-dimensional electrophoresis and mass spectrometry. Arch. Razi Inst., 2021, 76(1), 127-138. PMID: 33818965Slagboom, J.; Kaal, C.; Arrahman, A.; Vonk, F.J.; Somsen, G.W.; Calvete, J.J.; Wüster, W.; Kool, J. Analytical strategies in venomics. Microchem. J., 2022, 175, 107187. doi: 10.1016/j.microc.2022.107187Lomonte, B.; Calvete, J.J. Strategies in ‘snake venomics’ aiming at an integrative view of compositional, functional, and immunological characteristics of venoms. J. Venom. Anim. Toxins Incl. Trop. Dis., 2017, 23(1), 26. doi: 10.1186/s40409-017-0117-8 PMID: 28465677Kakati, H; Patra, A.; Kalita, B.; Chanda, A.; Rapole, S.; Mukherjee, A.K. A comparison of two different analytical workflows to determine the venom proteome composition of Naja kaouthia from North-East India and immunological profiling of venom against commercial antivenoms. Int J Biol Macromol. , 2022, 208, 275-287.Valente, R.; AlvesNicolau, C.; Perales, J.; Costa Neves-Ferreira, A. Snake venom proteopeptidomics: What lies behind the curtain. Toxinology, 2014.Warrell, D.A.; Gutiérrez, J.M.; Calvete, J.J. New approaches & technologies of venomics to meet the challenge of human envenoming by snakebites in India. 2013, 138(1), 38.Wong, K.Y.; Tan, T.H. Venom and purified toxins of the spectacled cobra (Naja naja) from Pakistan: insights into toxicity and antivenom neutralization. 2016, 94(6), 1392.Melani, R.D.; Nogueira, F.C.S.; Domont, G.B. It is time for top-down venomics. J. Venom. Anim. Toxins Incl. Trop. Dis., 2017, 23(1), 44. doi: 10.1186/s40409-017-0135-6 PMID: 29075288Tare, T.G.; Sutar, N.K.; Renapurkar, D.M. A study of snake venom yield by different methods of venom extraction. Amphib.-Reptil., 1986, 7(2), 187-191. doi: 10.1163/156853886X00406Randhawa, M.A. Calculation of LD50 values from the method of Miller and Tainter, 1944. J. Ayub Med. Coll. Abbottabad, 2009, 21(3), 184-185. PMID: 20929045Hamilton, M.A.; Russo, R.C.; Thurston, R.V. Trimmed Spearman-Karber method for estimating median lethal concentrations in toxicity bioassays. Environ. Sci. Technol., 1977, 11(7), 714-719. doi: 10.1021/es60130a004Feola, A.; Marella, G.L.; Carfora, A.; Della Pietra, B.; Zangani, P.; Campobasso, C.P. Snakebite envenoming a challenging diagnosis for the forensic pathologist: A systematic review. Toxins (Basel), 2020, 12(11), 699. doi: 10.3390/toxins12110699 PMID: 33153179Bailon, H. Calderon1* VOYC, 2, Omar Alberto Cáceres Rey1, Elizabeth Gaby Colque Alave1, Walter Jhon Leiva Duran1, Carlos Padilla Rojas1, Harrison Montejo Arevalo1, David García Neyra1, Marco Galarza Pérez1, César Bonilla3, Benigno Tintaya3, Giulia Ricciardi4, Natalia Smiejkowska4, Ema Romão4, Cécile Vincke4, Juan Lévano1, Mary Celys1, Bruno Lomonte5 and Serge Muyldermans4. Development of Nanobodies Against Hemorrhagic and Myotoxic Components of Bothrops atrox Snake Venom. Front. Immunol., 2020, 11.Tan, N.H.; Wong, K.Y.; Tan, C.H. Venomics of Naja sputatrix, the Javan spitting cobra: A short neurotoxin-driven venom needing improved antivenom neutralization. 2017, 157, 18-32.Sintiprungrat, K.; Watcharatanyatip, K.; Senevirathne, W.; Chaisuriya, P.; Chokchaichamnankit, D.; Srisomsap, C. A comparative study of venomics of Naja naja from India and Sri Lanka, clinical manifestations and antivenomics of an Indian polyspecific antivenom. 2016, 132, 131-143. doi: 10.1016/j.jprot.2015.10.007Laustsen, A.H.; Gutiérrez, J.M.; Lohse, B.; Rasmussen, A.R.; Fernández, J.; Milbo, C. Snake venomics of monocled cobra (Naja kaouthia) and investigation of human IgG response against venom toxins Toxicon, 2015, 99, 23-35.Dutta, S.; Chanda, A.; Kalita, B.; Islam, T.; Patra, A.; Mukherjee, A.K. Proteomic analysis to unravel the complex venom proteome of eastern India Naja naja: Correlation of venom composition with its biochemical and pharmacological properties. Proteomics, 2017, 156, 29-39.Hashmi, S.U.; Alvi, A.; Munir, I.; Perveen, M.; Fazal, A.; Jackson, T.N. Functional venomics of the Big-4 snakes of Pakistan Toxicon, 2020, 179, 60-71. doi: 10.1016/j.toxicon.2020.03.001Chanda, A.; Mukherjee, A.K. Quantitative proteomics to reveal the composition of Southern India spectacled cobra (Naja naja) venom and its immunological cross-reactivity towards commercial antivenom. Int. J. Biol. Macromol., 2020, 160, 224-232.Khan, S.E.; Waheed, H.; Moin, S.F. Decomplexation of Venom Proteome of Pakistani Cobra (Naja naja). Proceedings of the Pakistan Academy of Sciences: B. Life Environ. Sci., 2023, 60(1), 125-131.Choudhury, M.; McCleary, R.J.; Kesherwani, M.; Kini, R.M.; Velmurugan, D.J.T. Comparison of proteomic profiles of the venoms of two of the ‘Big Four’snakes of India, the Indian cobra (Naja naja) and the common krait (Bungarus caeruleus), and analyses of their toxins Toxicon, 2017, 135, 33-42.Deka, A.; Gogoi, A.; Das, D.; Purkayastha, J. Doley RJJop. Proteomics of naja kaouthia venom from north east india and assessment of indian polyvalent antivenom by third generation antivenomics. Proteomics, 2019, 207, 103463.Manuwar, A.; Dreyer, B.; Böhmert, A.; Ullah, A.; Mughal, Z.; Akrem, A. Proteomic investigations of two Pakistani Naja snake venoms species unravel the venom complexity, posttranslational modifications, and presence of extracellular vesicles. Toxin, 2020, 12(11), 669. doi: 10.3390/toxins12110669Akbari, A.; Rabiei, H.; Hedayat, A.; Mohammadpour, N.; Zou, A.H.; Teymourzadeh, S. Production of effective antivenin to treat cobra snake (Naja naja oxiana) envenoming. 2010.Talebi, M.M.; Hajihosseini, R. Identification and isolation of immunodominant proteins of Naja naja (Oxiana) snake venom. Archives of Razi Institute, 2017, 72(2), 131-137.Asaadi, Y.; Jouneghani, F.F.; Janani, S.; Rahbarizadeh, F. A comprehensive comparison between camelid nanobodies and single chain variable fragments. Biomark. Res., 1998, 9(1), 1-20. doi: 10.1186/s40364-021-00332-6 PMID: 34863296Lauwereys, M.; Ghahroudi, M.A.; Desmyter, A.; Kinne, J.; Hölzer, W.; De Genst, E. Potent enzyme inhibitors derived from dromedary heavy-chain antibodies EMBO J., 1998, 17(13), 3512-3520. doi: 10.1093/emboj/17.13.3512Herrera, M.; León, G.; Segura, A.; Meneses, F.; Lomonte, B.; Chippaux, J.P. Factors associated with adverse reactions induced by caprylic acid-fractionated whole IgG preparations: comparison between horse, sheep and camel IgGs. Toxicon, 2005, 46(7), 775-781. doi: 10.1016/j.toxicon.2005.08.004Conrath, K.; Vincke, C.; Stijlemans, B.; Schymkowitz, J.; Decanniere, K.; Wyns, L. Antigen binding and solubility effects upon the veneering of a camel VHH in framework-2 to mimic a VH J. Mol. Biol., 2005, 350(1), 112-125. doi: 10.1016/j.jmb.2005.04.050Meddeb-Mouelhi, F.; Bouhaouala-Zahar, B.; Benlasfar, Z.; Hammadi, M.; Mejri, T.; Moslah, M. Immunized camel sera and derived immunoglobulin subclasses neutralizing Androctonus australis hector scorpion toxins. Toxicon, 2003, 42(7), 782-791. doi: 10.1016/j.toxicon.2003.10.021