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[en] Scorpionism is responsible for most accidents involving venomous animals in Brazil, which leads to severe symptoms that can evolve to death. Scorpion venoms consist of complexes cocktails, including peptides, proteins, and non-protein compounds, making separation and purification procedures extremely difficult and time-consuming. Scorpion toxins target different biological systems and can be used in basic science, for clinical, and biotechnological applications. This study is the first to explore the venom content of the unexplored scorpion species Rhopalurus crassicauda, which inhabits exclusively the northernmost state of Brazil, named Roraima, and southern region of Guyana. Here, we pioneer the fractionation of the R. crassicauda venom and isolated and characterized a novel scorpion beta-neurotoxin, designated Rc1, and a monomeric hyaluronidase. R. crassicauda venom and Rc1 (6,882 Da) demonstrated pro-inflammatory activities in vitro and a nociceptive response in vivo. Moreover, Rc1 toxin showed specificity for activating Na(v)1.4, Na(v)1.6, and BgNa(v)1 voltage-gated ion channels. This study also represents a new perspective for the treatment of envenomings in Roraima, since the Brazilian scorpion and arachnid antivenoms were not able to recognize R. crassicauda venom and its fractions (with exception of hyaluronidase). Our work provides useful insights for the first understanding of the painful sting and pro-inflammatory effects associated with R. crassicauda envenomings.
Disciplines :
Chemistry
Author, co-author :
Abreu, Caio B.
Bordon, Karla C. F.
Cerni, Felipe A.
Oliveira, Isadora S.
Balenzuela, Carla
Alexandre-Silva, Gabriel M.
Zoccal, Karina F.
Reis, Mouzarllem B.
Wiezel, Gisele A.
Peigneur, Steve
Pinheiro-Júnior, Ernesto L.
Tytgat, Jan
Cunha, Tiago M.
Quinton, Loïc ; Université de Liège - ULiège > Département de chimie (sciences) > Chimie biologique
Nelsen DR, Nisani Z, Cooper AM, Fox GA, Gren ECK, Corbit AG, et al. Poisons, toxungens, and venoms: redefining and classifying toxic biological secretions and the organisms that employ them. Biol Rev Camb Philos Soc. (2014) 89:450–65. 10.1111/brv.12062 24102715
Chippaux JP, Goyffon M., Venomous and poisonous animals – I. Overview. Med Trop. (2006) 66:215–20.
Souza WMP, Alexandre-Silva G, Cerni FA, Oliveira IS, Zottich U, Bassoli BK, et al. Envenomings caused by venomous animals in Roraima: a neglected health problem in the Brazil’s Northernmost state. TCR. (2019) 3:1–8. 10.24966/TCR-3735/100011
Cupo P., Bites and stings from venomous animals: a neglected Brazilian tropical disease. Rev Soc Bras Med Trop. (2015) 48:639–41. 10.1590/0037-8682-0387-2015 26676486
Pucca MB, Cerni FA, Pinheiro Junior EL, Bordon KCF, Amorim FG, Cordeiro FA, et al. Tityus serrulatus venom – a lethal cocktail. Toxicon. (2015) 108:272–84. 10.1016/j.toxicon.2015.10.015 26522893
Pucca MB, Oliveira FN, Schwartz EF, Arantes EC, Lira-da-Silva RM., Scorpionism and dangerous species of Brazil. In: Gopalakrishnakone P, editor. Toxinology: Scorpion Venoms. Dordrecht: Springer Netherlands. (2014). p. 1–24. 10.1007/978-94-007-6647-1_20-1
Torrez PPQ, Dourado FS, Bertani R, Cupo P, França FOS., Scorpionism in Brazil: exponential growth of accidents and deaths from scorpion stings. Revista da Sociedade Brasileira de Medicina. Tropical. (2019) 52:e20180350. 10.1590/0037-8682-0350-2018 31141047
di Caporiacco L., Diagnosi preliminari di specie nuove di Aracnidi della Guiana britannica. Monitore zoologico Italiano. (1947) 56: 20–34.
Lenarducci ÂRIP, Pinto-da-Rocha R, Lucas SM., Descrição de uma nova espécie de Rhopalurus thorell, 1876 (scorpiones: Buthidae) do nordeste brasileiro. Biota Neotropica. (2005) 5:173–80. 10.1590/S1676-06032005000200015
Lourenço WR, Pinto-da-Rocha R., A reappraisal of the geographic distribution of the genus Rhopalurus Thorell (Scorpiones, Buthidae) and description of two new species. Biogeographica. (1997) 73:181–91.
Teruel R, Tietz AK., The true identity of Rhopalurus pintoi Mello-Leitão, 1932, with notes on the status and distribution of rhopalurus crassicauda caporiacco, 1947 (Scorpiones: Buthidae). Euscorpius. (2008) 70: 1–4.
Esposito LA, Yamaguti HY, Souza CA, Pinto-Da-Rocha R, Prendini L., Systematic revision of the neotropical club-tailed scorpions, Physoctonus, Rhopalurus, and Troglorhopalurus, revalidation of Heteroctenus, and descriptions of two new genera and three new species (Buthidae: Rhopalurusinae). Bull Am Museum Nat His. (2017) 415:1–136. 10.1206/0003-0090-415.1.1
Ministério da Saúde do Brasil DATASUS – Doenças e Agravos de Notificação. (2020). Available online at: https://datasus.saude.gov.br/acesso-a-informacao/doencas-e-agravos-de-notificacao-de-2007-em-diante-sinan/ (accessed July 6, 2020).
Chippaux J-P., Emerging options for the management of scorpion stings. Drug Des Devel Ther. (2012) 6:165–73. 10.2147/DDDT.S24754
Cupo P, Cupo P., Clinical update on scorpion envenoming. Rev Soc Bras Med Trop. (2015) 48:642–9. 10.1590/0037-8682-0237-2015 26676487
Pucca MB, Zoccal KF, Roncolato EC, Bertolini TB, Campos LB, Cologna CT, et al. Serrumab: a human monoclonal antibody that counters the biochemical and immunological effects of Tityus serrulatus venom. J Immunotoxicol. (2012) 9:173–83. 10.3109/1547691X.2011.649220 22424317
Laustsen AH, Gutiérrez JM, Knudsen C, Johansen KH, Bermúdez-Méndez E, Cerni FA, et al. Pros and cons of different therapeutic antibody formats for recombinant antivenom development. Toxicon. (2018) 146:151–75. 10.1016/j.toxicon.2018.03.004 29534892
Utkin YN., Animal venom studies: current benefits and future developments. World J Biol Chem. (2015) 6:28–33. 10.4331/wjbc.v6.i2.28 26009701
Pucca MB, Cerni FA, Janke R, Bermúdez-Méndez E, Ledsgaard L, Barbosa JE, et al. History of envenoming therapy and current perspectives. Front Immunol. (2019) 10:598. 10.3389/fimmu.2019.01598 31354735
Williams HF, Layfield HJ, Vallance T, Patel K, Bicknell AB, Trim SA, et al. The urgent need to develop novel strategies for the diagnosis and treatment of snakebites. Toxins. (2019) 11:363. 10.3390/toxins11060363 31226842
de Oliveira GH, Cerni FA, Cardoso IA, Arantes EC, Pucca MB., Tityus serrulatus envenoming in non-obese diabetic mice: a risk factor for severity. J Venom Anim Toxins Incl Trop Dis. (2016) 22:26. 10.1186/s40409-016-0081-8 27660634
Schwartz EF, Diego-Garcia E, Rodríguez de la Vega RC, Possani LD., Transcriptome analysis of the venom gland of the Mexican scorpion Hadrurus gertschi (Arachnida: Scorpiones). BMC Genomics. (2007) 8:119. 10.1186/1471-2164-8-119 17506894
Verano-Braga T, Dutra AAA, León IR, Melo-Braga MN, Roepstorff P, Pimenta AMC, et al. Moving pieces in a venomic puzzle: unveiling post-translationally modified toxins from Tityus serrulatus. J Proteome Res. (2013) 12:3460–70. 10.1021/pr4003068 23731212
Fox JW, Serrano SMT., Insights into and speculations about snake venom metalloproteinase (SVMP) synthesis, folding and disulfide bond formation and their contribution to venom complexity. FEBS J. (2008) 275:3016–30. 10.1111/j.1742-4658.2008.06466.x 18479462
Cerni FA, Pucca MB, Amorim FG, de Castro Figueiredo Bordon K, Echterbille J, Quinton L, et al. Isolation and characterization of Ts19 Fragment II, a new long-chain potassium channel toxin from Tityus serrulatus venom. Peptides. (2016) 80:9–17. 10.1016/j.peptides.2015.06.004 26116782
Cologna CT, Rodrigues RS, Santos J, de Pauw E, Arantes EC, Quinton L., Peptidomic investigation of Neoponera villosa venom by high-resolution mass spectrometry: seasonal and nesting habitat variations. J Venom Anim Toxins Incl Trop Dis. (2018) 24:6. 10.1186/s40409-018-0141-3 29467797
Bordon KCF, Wiezel GA, Amorim FG, Arantes EC., Arthropod venom Hyaluronidases: biochemical properties and potential applications in medicine and biotechnology. J Venom Anim Toxins Incl Trop Dis. (2015) 21:43. 10.1186/s40409-015-0042-7 26500679
Oliveira-Mendes BBR, de, Miranda SEM, Sales-Medina DF, Magalhães BF, Kalapothakis Y, Souza RP, et al. Inhibition of Tityus serrulatus venom hyaluronidase affects venom biodistribution. PLoS Negl Trop Dis. (2019) 13:e0007048. 10.1371/journal.pntd.0007048 31002673
Ramanaiah M, Parthasarathy PR, Venkaiah B., Isolation and characterization of hyaluronidase from scorpion (Heterometrus fulvipes) venom. Biochem Int. (1990) 20:301–10.
Batista CVF, Román-González SA, Salas-Castillo SP, Zamudio FZ, Gómez-Lagunas F, Possani LD., Proteomic analysis of the venom from the scorpion Tityus stigmurus: biochemical and physiological comparison with other Tityus species. Comp Biochem Physiol C Toxicol Pharmacol. (2007) 146:147–57. 10.1016/j.cbpc.2006.12.004 17270501
Lourenço WR., The evolution and distribution of noxious species of scorpions (Arachnida: Scorpiones). J Venom Anim Toxins Includ Trop Dis. (2018) 24:1. 10.1186/s40409-017-0138-3 29308066
Uzawa S., Über die phosphomonoesterase und die phosphodiesterase. J Biochem. (1932) 15:1–10. 10.1093/oxfordjournals.jbchem.a125167
Dhananjaya BL, D’souza CJM., An overview on nucleases (DNase, RNase, and phosphodiesterase) in snake venoms. Biochem Moscow. (2010) 75:1–6. 10.1134/S0006297910010013 20331418
Fox JW., A brief review of the scientific history of several lesser-known snake venom proteins: l-amino acid oxidases, hyaluronidases and phosphodiesterases. Toxicon. (2013) 62:75–82. 10.1016/j.toxicon.2012.09.009 23010165
Boldrini-França J, Cologna CT, Pucca MB, Bordon KCF, Amorim FG, Anjolette FAP, et al. Minor snake venom proteins: Structure, function and potential applications. Biochim Biophys Acta Gen Subj. (2017) 1861:824–38. 10.1016/j.bbagen.2016.12.022 28012742
Díaz-García A, Ruiz-Fuentes JL, Yglesias-Rivera A, Rodríguez-Sánchez H, Riquenes Garlobo Y, Fleitas Martinez O, et al. Enzymatic analysis of venom from cuban scorpion Rhopalurus junceus. J Venom Res. (2015) 6:11–8.
Rodríguez-Ravelo R, Coronas FIV, Zamudio FZ, González-Morales L, López GE, Urquiola AR, et al. The Cuban scorpion Rhopalurus junceus (Scorpiones, Buthidae): component variations in venom samples collected in different geographical areas. J Venom Anim Toxins Incl Trop Dis. (2013) 19:13. 10.1186/1678-9199-19-13 23849540
Zamudio FZ, Conde R, Arévalo C, Becerril B, Martin BM, Valdivia HH, et al. The mechanism of inhibition of ryanodine receptor channels by imperatoxin I, a heterodimeric protein from the scorpion Pandinus imperator. J Biol Chem. (1997) 272:11886–94. 10.1074/jbc.272.18.11886 9115249
Conde R, Zamudio FZ, Becerril B, Possani LD., Phospholipin, a novel heterodimeric phospholipase A2 from Pandinus imperator scorpion venom. FEBS Lett. (1999) 460:447–50. 10.1016/S0014-5793(99)01392-7
Valdez−Cruz NA, Batista CVF, Possani LD., Phaiodactylipin, a glycosylated heterodimeric phospholipase A2 from the venom of the scorpion Anuroctonus phaiodactylus. Eur J Biochem. (2004) 271:1453–64. 10.1111/j.1432-1033.2004.04047.x 15066171
Louati H, Krayem N, Fendri A, Aissa I, Sellami M, Bezzine S, et al. A thermoactive secreted phospholipase A2 purified from the venom glands of Scorpio maurus: relation between the kinetic properties and the hemolytic activity. Toxicon. (2013) 72:133–42. 10.1016/j.toxicon.2013.06.017 23831286
Schwartz EF, Camargos TS, Zamudio FZ, Silva LP, Bloch C, Caixeta F, et al. Mass spectrometry analysis, amino acid sequence and biological activity of venom components from the Brazilian scorpion Opisthacanthus cayaporum. Toxicon. (2008) 51:1499–508. 10.1016/j.toxicon.2008.03.029 18502464
Ramanaiah M, Parthasarathy PR, Venkaiah B., Purification and properties of phospholipase A2 from the venom of scorpion, (Heterometrus fulvipes). Biochem Int. (1990) 20:931–40.
Incamnoi P, Patramanon R, Thammasirirak S, Chaveerach A, Uawonggul N, Sukprasert S, et al. Heteromtoxin (HmTx), a novel heterodimeric phospholipase A(2) from Heterometrus laoticus scorpion venom. Toxicon. (2013) 61:62–71. 10.1016/j.toxicon.2012.10.012 23142507
Bordon KCF, Cologna CT, Arantes EC., Scorpion venom research around the world: Tityus serrulatus. In: Gopalakrishnakone P, Possani LD, Schwartz EF, Rodríguez de la Vega RC, editors. Scorpion Venoms. Dordrecht: Springer Netherlands. (2014). p. 411–37. 10.1007/978-94-007-6404-0_7
Polikarpov I, Junior MS, Marangoni S, Toyama MH, Teplyakov A., Crystal structure of neurotoxin Ts1 from Tityus serrulatus provides insights into the specificity and toxicity of scorpion toxins. J Mol Biol. (1999) 290:175–84. 10.1006/jmbi.1999.2868 10388565
Pucca MB, Cerni FA, Peigneur S, Bordon KCF, Tytgat J, Arantes EC., Revealing the function and the structural model of Ts4: Insights into the “Non-Toxic” toxin from Tityus serrulatus venom. Toxins. (2015) 7:2534–50. 10.3390/toxins7072534 26153865
Tanaka T, Narazaki M, Kishimoto T., IL-6 in inflammation, immunity, and disease. Cold Spring Harb Perspect Biol. (2014) 6:a016295. 10.1101/cshperspect.a016295 25190079
Abbas AK, Lichtman AH, Pillai S, Baker DL, Baker A., Cellular and Molecular Immunology. 9th ed. Philadelphia, PA: Elsevier. (2018).
Fukuhara YDM, Reis ML, Dellalibera-Joviliano R, Cunha FQC, Donadi EA., Increased plasma levels of IL-1β, IL-6, IL-8, IL-10 and TNF-α in patients moderately or severely envenomed by Tityus serrulatus scorpion sting. Toxicon. (2003) 41:49–55. 10.1016/S0041-0101(02)00208-8
Zoccal KF, Bitencourt CS, Sorgi CA, Bordon KCF, Sampaio SV, Arantes EC, et al. Ts6 and Ts2 from Tityus serrulatus venom induce inflammation by mechanisms dependent on lipid mediators and cytokine production. Toxicon. (2013) 61:1–10. 10.1016/j.toxicon.2012.10.002 23085190
Reis MB, Zoccal KF, Gardinassi LG, Faccioli LH., Scorpion envenomation and inflammation: beyond neurotoxic effects. Toxicon. (2019) 167:174–9. 10.1016/j.toxicon.2019.06.219 31228480
Zoccal KF, Bitencourt CS, Paula-Silva FWG, Sorgi CA, de Castro Figueiredo Bordon K, Arantes EC, et al. TLR2, TLR4 and CD14 recognize venom-associated molecular patterns from Tityus serrulatus to induce macrophage-derived inflammatory mediators. PLoS One. (2014) 9:e88174. 10.1371/journal.pone.0088174 24516606
Zoccal KF, Bitencourt CS, Secatto A, Sorgi CA, Bordon KCF, Sampaio SV, et al. Tityus serrulatus venom and toxins Ts1, Ts2 and Ts6 induce macrophage activation and production of immune mediators. Toxicon. (2011) 57:1101–8. 10.1016/j.toxicon.2011.04.017 21549737
Zoccal KF, Sorgi CA, Hori JI, Paula-Silva FWG, Arantes EC, Serezani CH, et al. Opposing roles of LTB4 and PGE2 in regulating the inflammasome-dependent scorpion venom-induced mortality. Nat Commun. (2016) 7:10760. 10.1038/ncomms10760 26907476
Pucca MB, Peigneur S, Cologna CT, Cerni FA, Zoccal KF, Bordon KCF, et al. Electrophysiological characterization of the first Tityus serrulatus alpha-like toxin, Ts5: Evidence of a pro-inflammatory toxin on macrophages. Biochimie. (2015) 115:8–16. 10.1016/j.biochi.2015.04.010 25906692
Shih VF-S, Tsui R, Caldwell A, Hoffmann A., A single NFκB system for both canonical and non-canonical signaling. Cell Res. (2011) 21:86–102. 10.1038/cr.2010.161 21102550
Kendellen MF, Bradford JW, Lawrence CL, Clark KS, Baldwin AS., Canonical and non-canonical NF-κB signaling promotes breast cancer tumor-initiating cells. Oncogene. (2014) 33:1297–305. 10.1038/onc.2013.64 23474754
Díaz-García A, Ruiz-Fuentes JL, Frión-Herrera Y, Yglesias-Rivera A, Riquenez Garlobo Y, Rodríguez Sánchez H, et al. Rhopalurus junceus scorpion venom induces antitumor effect in vitro and in vivo against a murine mammary adenocarcinoma model. Iran J Basic Med Sci. (2019) 22:759–65. 10.22038/ijbms.2019.33308.7956 32373297
Mikaelian AG, Traboulay E, Zhang XM, Yeritsyan E, Pedersen PL, Ko YH, et al. Pleiotropic anticancer properties of scorpion venom peptides: Rhopalurus princeps venom as an anticancer agent. DDDT. (2020) 14:881–93. 10.2147/DDDT.S231008 32161447
Pucca MB, Cerni FA, Cordeiro FA, Peigneur S, Cunha TM, Tytgat J, et al. Ts8 scorpion toxin inhibits the Kv4.2 channel and produces nociception in vivo. Toxicon. (2016) 119:244–52. 10.1016/j.toxicon.2016.06.014 27346450
Isbister GK, Volschenk ES, Balit CR, Harvey MS., Australian scorpion stings: a prospective study of definite stings. Toxicon. (2003) 41:877–83. 10.1016/S0041-0101(03)00065-5
Pucca MB, Cerni FA, Oliveira IS, Timothy Jenkins TP, Argemí LM, Sørensen CV, et al. Bee updated: current knowledge on bee venom and bee envenoming therapy. Front Immunol. (2019) 10:2090. 10.3389/fimmu.2019.02090 31552038
Dib-Hajj SD, Binshtok AM, Cummins TR, Jarvis MF, Samad T, Zimmermann K., Voltage-gated sodium channels in pain states: Role in pathophysiology and targets for treatment. Brain Res Rev. (2009) 60:65–83. 10.1016/j.brainresrev.2008.12.005 19150627
Cardoso FC, Lewis RJ., Sodium channels and pain: from toxins to therapies. Br J Pharmacol. (2018) 175:2138–57. 10.1111/bph.13962 28749537
Pucca MB, Amorim FG, Cerni FA, Bordon KCF, Cardoso IA, Anjolette FAP, et al. Influence of post-starvation extraction time and prey-specific diet in Tityus serrulatus scorpion venom composition and hyaluronidase activity. Toxicon. (2014) 90:326–36. 10.1016/j.toxicon.2014.08.064 25199494
Smith JJ, Herzig V, King GF, Alewood PF., The insecticidal potential of venom peptides. Cell Mol Life Sci. (2013) 70:3665–93. 10.1007/s00018-013-1315-3 23525661
García-Gómez BI, Coronas FIV, Restano-Cassulini R, Rodríguez RR, Possani LD., Biochemical and molecular characterization of the venom from the Cuban scorpion Rhopalurus junceus. Toxicon. (2011) 58:18–27. 10.1016/j.toxicon.2011.04.011 21605585
Cerni FA, Pucca MB, Peigneur S, Cremonez CM, Bordon KCF, Tytgat J, et al. Electrophysiological characterization of Ts6 and Ts7, K+ channel toxins isolated through an improved Tityus serrulatus venom purification procedure. Toxins. (2014) 6:892–913. 10.3390/toxins6030892 24590385
Rochat H, Rochat C, Kupeyan C, Miranda F, Lissitzky S, Edman P., Scorpion neurotoxins: a family of homologous proteins. FEBS Lett. (1970) 10:349–51. 10.1016/0014-5793(70)80470-7
Oliveira-Mendes BBR, Miranda ‘, Sales-Medina DF, Magalhães B, de F, Kalapothakis Y, et al. Hyaluronidase: the spreading factor of Tityus serrulatus venom. bioRxiv [Preprint]. (2018). 10.1101/487298 bioRxiv:487298,
Schägger H, von Jagow G., Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem. (1987) 166:368–79. 10.1016/0003-2697(87)90587-2
Cevallos MA, Navarro-Duque C, Varela-Julia M, Alagon AC., Molecular mass determination and assay of venom hyaluronidases by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Toxicon. (1992) 30:925–30. 10.1016/0041-0101(92)90392-i
Bjork W., Purification of phosphodiesterase from Bothrops atrox venom, with special consideration of the elimination of monophosphatases. J Biol Chem. (1963) 238:2487–90.
Valério AA, Corradini AC, Panunto PC, Mello SM, Hyslop S., Purification and characterization of a phosphodiesterase from Bothrops alternatus snake venom. J Protein Chem. (2002) 21:495–503.
Habermann E, Hardt KL., A sensitive and specific plate test for the quantitation of phospholipases. Anal Biochem. (1972) 50:163–73.
Mosmann T., Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. (1983) 65:55–63. 10.1016/0022-1759(83)90303-4
Peigneur S, Cheneval O, Maiti M, Leipold E, Heinemann SH, Lescrinier E, et al. Where cone snails and spiders meet: design of small cyclic sodium-channel inhibitors. FASEB J. (2019) 33:3693–703. 10.1096/fj.201801909R 30509130
