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Characterization of toxins from the broad-banded water snake Helicops angulatus (Linnaeus, 1758): isolation of a cysteine-rich secretory protein, Helicopsin

  • Molecular Toxicology
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Abstract

Helicops angulatus (broad-banded water snake) according to recent proposals is presently cited in the family Dipsadidae, subfamily Xenodontinae, forming the tribe Hydropsini along with the genera Hydrops and Pseudoeryx. The current work characterizes the proteolytic and neurotoxic activities of H. angulatus crude toxins from salivary excretion (SE) and describes the isolation and identification of a cysteine-rich secretory protein (CRISP) called helicopsin. The SE lethal dose (LD50) was 5.3 mg/kg; however, the SE did not contain hemorrhagic activity. Helicopsin was purified using activity-guided, Superose 12 10/300 GL molecular exclusion, Mono Q10 ion exchange, and Protein Pak 60 molecular exclusion. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) showed a highly purified band of approximately 20 kDa. The minimal lethal dose for helicopsin was 0.4 mg/kg. Liquid chromatography mass spectrometry (LC-MS/MS) analysis identified 2 unique peptides MEWYPEAAANAER and YTQIVWYK, representing a protein sequence (deleted homology) belonging to cysteine-rich secretory proteins, which are conserved in snake venoms (CRISPs). CRISPs are a large family of cysteine-rich secretory proteins found in various organisms and participate in diverse biological processes. Helicopsin exhibited robust neurotoxic activity as evidenced by immediate death (~8 min) due to respiratory paralysis in NIH mice. These observations for helicopsin purified from H. angulatus provide further evidence of the extensive distribution of highly potent neurotoxins in the Colubroidea superfamily of snakes than previously described.

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References

  • Anonymous (1985) Principles of laboratory animal care. National Institute of Health, Pub. 85 No 23, USA

  • AOAC INTERNATIONAL (1995) Official methods of analysis. In: Cunniff P (ed), Arlington, VA, sec. 991.31

  • Calvete JJ, Fasoli E, Sanz L, Boschetti E, Righetti PG (2009) Exploring the venom proteome of the Western diamondback rattlesnake, Crotalus atrox, via snake venomics and combinatorial peptide ligand library approaches. J Proteome Res 8:3055–3067

    Article  PubMed  CAS  Google Scholar 

  • Cameo MS, Blaquier JA (1976) Androgen-controlled specific proteins in rat epididymis. J Endocrinol 69:317–324

    Article  Google Scholar 

  • Dauplais M, Lecoq A, Song J, Cotton J, Jamin N, Gilquin B, Roumestand C, Vita C, de Medeiros CL, Rowan EG, Harvey AL, Ménez A (1997) On the convergent evolution of animal toxins. Conservation of a diad of functional residues in potassium channel-blocking toxins with unrelated structures. J Biol Chem 272:4302–4309

    Article  PubMed  CAS  Google Scholar 

  • Dixon JR, Soini P (1986) The reptiles of the upper Amazon Basin, Iquitos region, Peru. Milwaukee Public Museum, Wisconsin, pp 1–154

    Google Scholar 

  • Ellerman DA, Cohen DJ, Da Ros VG, Morgenfeld MM, Busso D, Cuasnicú PS (2006) Sperm protein “DE” mediates gamete fusion through an evolutionarily conserved site of the CRISP family. Dev Biol 297:228–237

    Article  PubMed  CAS  Google Scholar 

  • Ficarro SB, Zhang Y, Lu Y, Moghimi AR, Askenazi M, Hyatt E, Smith ED, Boyer L, Schlaeger TM, Luckey CJ, Marto JA (2009) Improved electrospray ionization efficiency compensates for diminished chromatographic resolution and enables proteomics analysis of tyrosine signaling in embryonic stem cells. Anal Chem 81:3440–3447

    Article  PubMed  CAS  Google Scholar 

  • Ford NB, Ford DF (2002) Notes on the ecology of the South American water snake Helicops angulatus (Squamata: Colubridae) in Nariva Swamp, Trinidad. Carib J Sci 38:129–131

    Google Scholar 

  • Franciosa G, Ferreira JL, Hatheway CL (1994) Detection of type A, B, and E botulism neurotoxin genes in Clostridium botulinum and other Clostridial species by PCR: evidence of unexpressed type B toxin genes in type A toxigenic organisms. J Clin Microbiol 32:1911–1917

    PubMed  CAS  Google Scholar 

  • Fry BG, Wüster W (2004) Assembling an arsenal: origin and evolution of the snake venom proteome inferred from phylogenetic analysis of toxin sequences. Mol Biol Evol 21:870–883

    Article  PubMed  CAS  Google Scholar 

  • Fry BG, Vidal N, Norman JA, Vonk FJ, Scheib H, Ramjan RF, Kurupp S, Fung K, Hedges SB, Richardson MK, Hodgson WC, Ignjatovic V, Summerhayes R, Kochva E (2006) Early evolution of the venom system in lizards and snakes. Nature 439:584–588

    Article  PubMed  CAS  Google Scholar 

  • Fry BG, Scheib H, van der Weerd L, Young B, McNaughtan J, Ramjan SF, Vidal N, Poelmann RE, Norman JA (2008) Evolution of an arsenal: structural and functional diversification of the venom system in the advanced snakes (Caenophidia). Mol Cell Proteomics 7:215–246

    PubMed  CAS  Google Scholar 

  • Galán JA, Guo M, Sánchez EE, Cantu E, Rodríguez-Acosta A, Perez JC, Tao WA (2008) Quantitative analysis of snake venoms using soluble polymer-based isotope labeling. Mol Cell Proteomics 4:785–799

    Google Scholar 

  • Gibbs GM, O’Bryan MK (2007) Cysteine rich secretory proteins in reproduction and venom. Soc Reprod Fertil Suppl 65:261–267

    PubMed  CAS  Google Scholar 

  • Gibbs GM, Roelants K, O’Bryan MK (2008) The CAP superfamily: cysteine-rich secretory proteins, antigen 5, and pathogenesis-related 1 proteins-roles in reproduction, cancer and immune defense. Endocrine Rev 29:865–897

    Article  CAS  Google Scholar 

  • Guo M, Teng M, Niu L, Liu Q, Huang Q, Hao Q (2005) Crystal structure of the cysteine-rich secretory protein stecrisp reveals that the cysteine-rich domain has a K+ channel inhibitor-like fold. J Biol Chem 280:12405–12412

    Article  PubMed  CAS  Google Scholar 

  • Gutiérrez JM, Gené JA, Rojas G, Cerdas L (1985) Neutralization of proteolytic and hemorrhagic activities of Costa Rican snake venoms by a polyvalent antivenom. Toxicon 23:887–893

    Article  PubMed  Google Scholar 

  • Hawgood BJ, Smith JW (1977) The mode of action at the mouse neuromuscular junction of the phospholipase A-crotapotin complex isolated from venom of the South American rattlesnake. Br J Pharmacol 61:597–606

    PubMed  CAS  Google Scholar 

  • Heading CE (2002) Conus peptides and neuroprotection. Curr Opin Investig Drugs 3:915–920

    PubMed  CAS  Google Scholar 

  • Heyborne WH, Mackessy SP (2009) Cysteine-rich proteins in reptile secretory venoms. In: Mackessy SP (ed) Handbook of venoms and toxins of reptiles. CRC Press, Taylor and Francis Group, USA

    Google Scholar 

  • Hill RE, Mackessy SP (2000) Characterization of venom (Duvernoy’s secretion) from twelve species of colubrid snakes and partial sequence of four venom proteins. Toxicon 38:1663–1687

    Article  PubMed  CAS  Google Scholar 

  • Huang SY, Perez JC (1980) Comparative study on hemorrhagic and proteolytic activities of snake venoms. Toxicon 18:421–426

    Article  PubMed  CAS  Google Scholar 

  • Kierszenbaum AL, Lea O, Petrusz P, French FS, Tres LL (1981) Isolation, culture, and immunocytochemical characterization of epididymal epithelial cells from pubertal and adult rats. Proc Natl Acad Sci USA 78:1675–1679

    Article  PubMed  CAS  Google Scholar 

  • Kraus F, Brown WM (1998) Phylogenetic relationships of colubroid snakes based on mitochondrial DNA sequences. Zool J Linn Soc 122:455–487

    Article  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  PubMed  CAS  Google Scholar 

  • Lemoine K, Rodríguez-Acosta A (2003) Hemorrhagic, proteolytic and neurotoxic activities produced by the false coral snake (Erythrolamprus Bizona Jan 1863) (Serpentes: Colubridae) Duvernoy’s gland secretion. Rev Cient FCV-LUZ 13:371–377

    Google Scholar 

  • Lemoine K, Salgueiro LM, Rodríguez-Acosta. A (2004a) Neurotoxic, haemorrhagic and proteolytic activities caused by Thamnodynastes strigilis (Serpentes: Colubridae) Duvernoy’s gland secretion. Vet Hum Toxicol 46:10–14

    PubMed  CAS  Google Scholar 

  • Lemoine K, Girón ME, Aguilar I, Navarrete LF, Rodríguez-Acosta A (2004b) Proteolytic, haemorrhagic and neurotoxic activities caused by Leptodeira annulata ashmeadii (Serpentes: Colubridae) Duvernoy’s gland secretion. J Wild Env Med 15:82–89

    Article  Google Scholar 

  • Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275

    PubMed  CAS  Google Scholar 

  • Mackessy SP (2002) Biochemistry and pharmacology of colubrid snake venoms. J Toxicol Toxin Rev 21:43–83

    CAS  Google Scholar 

  • Mackessy SP, Sixberry NM, Heyborne WH, Fritts T (2006) Venom of the Brown Treesnake, Boiga irregularis: ontogenetic shifts and taxa-specific toxicity. Toxicon 47:537–548

    Article  PubMed  CAS  Google Scholar 

  • Mochca-Morales J, Martin BM, Possani LD (1990) Isolation and characterization of helothermine, a novel toxin from Heloderma horridum horridum (Mexican beaded lizard) venom. Toxicon 28:299–309

    Article  PubMed  CAS  Google Scholar 

  • Morrissette J, Krätzschmar J, Haendler B, el-Hayek R, Mochca-Morales J, Martin BM, Patel JR, Moss RL, Schleuning WD, Coronado R, Possani L (1995) Primary structure and properties of helothermine, a peptide toxin that blocks ryanodine receptors. Biophys J 68:2280–2288

    Article  PubMed  CAS  Google Scholar 

  • Nobile M, Magnelli V, Lagostena L, Mochca-Morales J, Possani LD, Prestipino G (1994) The toxin helothermine affects potassium currents in newborn rat cerebellar granule cells. J Membr Biol 139:49–55

    PubMed  CAS  Google Scholar 

  • Ozkan O, Ciftci G, Pekmezci GZ, Kar S, Uysal H, Karaer KZ (2007) Proteins, lethality and in vivo effects of Iurus dufoureius asiaticus scorpion venom. Toxicon 50:394–399

    Article  PubMed  CAS  Google Scholar 

  • Rodríguez-Acosta A, Lemoine K, Navarrete LF, Girón ME, Aguilar I (2006) Experimental ophitoxemia produced by the opisthoglyphous Lora snake (Philodryas olfersii) (Serpentes: Colubridae) venom. Rev Soc Bras Med Trop 39:193–197

    Article  PubMed  Google Scholar 

  • Rossetto O, Montecucco C (2008) Presynaptic neurotoxins with enzymatic activities. Handb Exp Pharmacol 184:129–170

    Article  PubMed  CAS  Google Scholar 

  • Spearman-Karber R (1964) Alternative methods of analysis for quantal responses, 2nd ed. In: Finney D (ed) Statistical method in biological assay. Charles Griffin, London

    Google Scholar 

  • Tudor JE, Pallaghy PK, Pennington MW, Norton RS (1996) Solution structure of ShK toxin, a novel potassium channel inhibitor from a sea anemone. Nat Struct Biol 3:317–320

    Article  PubMed  CAS  Google Scholar 

  • Yamazaki Y, Morita T (2004) Structure and biology of snake venom cysteine-rich secretory proteins. Toxicon 44:227–231

    Article  PubMed  CAS  Google Scholar 

  • Yamazaki Y, Morita T (2007) Snake venom components affecting blood coagulation and the vascular system: structural similarities and marked diversity. Curr Pharm Des 13:2872–2886

    Article  PubMed  CAS  Google Scholar 

  • Yamazaki Y, Brown RL, Morita T (2002) Purification and cloning of toxins from elapid venoms that target cyclic nucleotide-gated ion channels. Biochemistry 41:11331–11337

    Article  PubMed  CAS  Google Scholar 

  • Zaher H, Gobbi-Grazziotin F, Cadle JE, Murphy RW, de Moura-Leite JC, Bonatto SL (2009) Molecular phylogeny of advanced snakes (Serpentes-Caenophidia) with an emphasis on South American Xenodontines: a revised classification and descriptions of new taxa. Pap Avulsos Zool 49:115–153

    Google Scholar 

Download references

Acknowledgments

This research was supported by a grant from the FONACIT (G-2005000400), Venezuela. We would like to thank the anonymous referee for the important comments that improved the manuscript.

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Correspondence to Alexis Rodríguez-Acosta.

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Amalid Estrella and Elda E. Sánchez contributed equally to this work.

Ethical statement: The persons behind the experiments declare that all the experimental events concerning the use of live animals were done by specialized personnel following the Venezuelan pertinent regulations as well as institutional guidelines, according to protocols approved by the Tropical Medicine Institute of the Universidad Central de Venezuela and the norms from the guide for the care and use of laboratory animals, published by the US National Institute of Health (Anonymous 1985).

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Estrella, A., Sánchez, E.E., Galán, J.A. et al. Characterization of toxins from the broad-banded water snake Helicops angulatus (Linnaeus, 1758): isolation of a cysteine-rich secretory protein, Helicopsin. Arch Toxicol 85, 305–313 (2011). https://doi.org/10.1007/s00204-010-0597-6

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