The Protein Journal

, Volume 29, Issue 2, pp 103–113 | Cite as

Neurotoxic, Myotoxic and Cytolytic Activities of the New Basic PLA2 Isoforms BmjeTX-I and BmjeTX-II Isolated from the Bothrops marajoensis (Marajó Lancehead) Snake Venom

  • L. A. Ponce-SotoEmail author
  • D. Martins-de-Souza
  • S. Marangoni


The BmjeTX-I and BmjeTX-II isoforms of PLA2 were purified from Bothrops marajoensis venom by ion-exchange chromatography and reverse phase HPLC. Both isoforms showed a molecular mass of 13808.89 Da (BmjeTX-I) and 13863.97 Da (BmjeTX-II) determined by based on the determined primary structures and SDS–PAGE and confirmed experimentally by MALDI-TOF mass spectrometry. Multiple alignment of BmjeTX-I and BmjeTX-II isoforms of PLA2 show high degree of homology with basic PLA2 myotoxins from other Bothrops venoms. Ex vivo, both isoforms caused a blockade of the neuromuscular transmission in young chick biventer cervicis preparations in a similar way to other Bothrops species. In chick preparations, contractures to exogenous acetylcholine (55 and 110 μM) or KCl (13.4 mM) were unaltered after complete blockade for the both isoforms BmjeTX-I and BmjeTX-II of PLA2. These results, which strongly suggested a presynaptic mechanism of action for these toxins. In mice, both isoforms induced myonecrosis and a systemic interleukin-6 response upon intramuscular injection. Both isoforms BmjeTX-I and BmjeTX-II of PLA2 also induced moderate marked paw edema, evidencing the local increase in vascular permeability. Since both isoforms of PLA2 exert a strong proinflammatory effect, the enzymatic hydrolysis of phospholipids might be relevant for this phenomenon and produced cytotoxicity in murine skeletal muscle C2C12 myoblasts and myotubes.


Phospholipase A2 Snake venom Bothrops marajoensis Myotoxin Neurotoxin Cytotoxitic C2C12 Isoforms 



The authors thank Paulo A. Baldasso for general technical help. This work was supported by FAPESP process 06/51566-0 and it is part of Luis Alberto Ponce-Soto’ Post-Doctorate.


  1. 1.
    Avila-Aguero ML, Parýs MM, Hu S, Peterson PK, Gutiérrez JM, Lomonte B, Faingezicht I (2001) Systemic cytokine response in children bitten by snakes in Costa Rica. Ped Emerg Care 17:425–429CrossRefGoogle Scholar
  2. 2.
    Barraviera B, Lomonte B, Tarkowski A, Hanson LA, Meira DA (1995) Acute-phase reactions, including cytokines, in patients bitten by Bothrops and Crotalus snakes in Brazil. Toxicon 33:283CrossRefGoogle Scholar
  3. 3.
    Bon C, Changeux JP, Jeng TW, Fraenkel-Conrat H (1979) Postsynaptic effects of crotoxin and of its isolated subunits. Eur J Biochem 99(3):471–481CrossRefGoogle Scholar
  4. 4.
    Brusés JL, Capaso J, Katz E, Pilar G (1993) Specific in vitro biological activity of snake venom myotoxins. J Neurochem 60(3):1030–1042CrossRefGoogle Scholar
  5. 5.
    Calgarotto AK, Damico DC, Ponce-Soto LA, Baldasso PA, Da Silva SL, Souza GH, Eberlin MN, Marangoni S (2008) Biological and biochemical characterization of new basic phospholipase A2 BmTX-I isolated from Bothrops moojeni snake venom. Toxicon 51(8):1509–1519CrossRefGoogle Scholar
  6. 6.
    Chaves F, León G, Alvarado VH, Gutiérrez JM (1998) Pharmacological modulation of edema induced by Lys-49 and Asp-49 myotoxic phospholipases A2 isolated from the venom of the snake Bothrops asper (terciopelo). Toxicon 36:1861–1869CrossRefGoogle Scholar
  7. 7.
    Chijiwa T, Hamai S, Tsubouchi S, Ogawa T, Deshimaru M, Oda-Ueda N, Hattori S, Kihara H, Tsunasawa S, Ohno M (2003) Interisland mutation of a novel phospholipase A2 from Trimeresurus flavoviridis venom and evolution of Crotalinae group II phospholipases A2. J Mol Evol 57:546–554CrossRefGoogle Scholar
  8. 8.
    Condrea E, Yang CC, Rosenberg P (1981) Lack of correlation between anticoagulant activity and phospholipid hydrolysis by snake venom phospholipases A2. Thromb Haemost 45:82–85Google Scholar
  9. 9.
    Díaz C, León G, Rucavado A, Rojas N, Schroit AJ, Gutiérrez JM (2001) Modulation of the susceptibility of human erythrocytes to snake venom myotoxic phospholipases A2: role of negatively charged phospholipids as potential membrane binding sites. Arch Biochem Biophys 391(1):56–64CrossRefGoogle Scholar
  10. 10.
    Fletcher JE, Jiang MS (1998) Lys49 phospholipase A2 myotoxins lyse cell cultures by two distinct mechanisms. Toxicon 36:1549–1555CrossRefGoogle Scholar
  11. 11.
    Fuly AL, de Miranda ALP, Zingali RB, Guimaraes JA (2002) Purification and characterization of a phospholipase A2 isoenzyme isolated from Lachesis muta snake venom. Biochem Pharmacol 63:1589–1597CrossRefGoogle Scholar
  12. 12.
    Goldsby RA, Kindt TJ, Osborne BA (2000) Kuby immunology. W.H. Freeman, New YorkGoogle Scholar
  13. 13.
    Gutiérrez JM, Ponce-Soto LA, Marangoni S, Lomonte B (2008) Systemic and local myotoxicity induced by snake venom group II phospholipases A2: comparison between crotoxin, crotoxin B and a Lys49 PLA2 homologue. Toxicon 51(1):80–92CrossRefGoogle Scholar
  14. 14.
    Gutierrez JM, Ownby CL (2003) Skeletal muscle degeneration induced by venom phospholipases A2: insights into the mechanisms of local and systemic myotoxicity. Toxicon 42:915–931 ReviewCrossRefGoogle Scholar
  15. 15.
    Gutiérrez JM, Rojas G, Lomonte B, Gené JA, Cerdas L (1986) Comparative study of the edema-forming activity of Costa Rican snake venoms and its neutralization by a polyvalent antivenom. Comp Biochem Physiol 85:171–175Google Scholar
  16. 16.
    Gutierrez JM, Lomonte B (1995) Phospholipase A2 myotoxins from Bothrops snake venoms. Toxicon 33:1405–1424CrossRefGoogle Scholar
  17. 17.
    Hanasaki K, Arita H (1999) Biological and pathological functions of phospholipase A2 receptor. Arch Biochem Biophys 372:215–223CrossRefGoogle Scholar
  18. 18.
    Holzer M, Mackessy SP (1996) An aqueous endpoint assay of snake venom phospholipase A2. Toxicon 34:1149–1155CrossRefGoogle Scholar
  19. 19.
    Ketelhut DF, de Mello MH, Veronese EL, Esmeraldino LE, Murakami MT, Arni RK, Giglio JR, Cintra AC, Sampaio SV (2003) Isolation, characterization and biological activity of acidic phospholipase A2 isoforms from Bothrops jararacussu snake venom. Biochimie 85(10):983–991CrossRefGoogle Scholar
  20. 20.
    Kini RM (1997) Venom phospholipase A2 enzymes: structure, function and mechanism. Wiley, Chichester, pp 1–511Google Scholar
  21. 21.
    Kini RM (2003) Excitement ahead: structure, function and mechanism of snake venom phospholipase A2 enzymes. Toxicon 42:827–840CrossRefGoogle Scholar
  22. 22.
    Krizaj I, Gubensek F (2000) Neuronal receptors for phospholipases A2 and beta-neurotoxicity. Biochimie 82(9–10):807–814CrossRefGoogle Scholar
  23. 23.
    Krizaj I, Dolly JO, Gubensek F (1994) Identification of the neuronal acceptor in bovine cortex for ammodytoxin C, a presynaptically neurotoxic phospholipase A2. Biochemistry 33:13938–13945CrossRefGoogle Scholar
  24. 24.
    Krizaj I, Faure G, Gubensek F, Bon C (1997) Neurotoxic phospholipases A2 ammodytoxin and crotoxin bind to distinct high-affinity protein acceptors in Torpedo marmorata electric organ. Biochemistry 36:2779–2787CrossRefGoogle Scholar
  25. 25.
    Krizaj I, Rowan EG, Gubensek F (1995) Ammodytoxin A acceptor in bovine brain synaptic membranes. Toxicon 33:437–449CrossRefGoogle Scholar
  26. 26.
    Kudo I, Murakami M (2002) Phospholipase A2 enzymes. Prostaglandins Other Lipid Mediat 68–69:3–58CrossRefGoogle Scholar
  27. 27.
    Lambeau G, Barhanin J, Schweitz H, Qar J, Lazdunski M (1989) Identification and properties of very high affinity brain membrane-binding sites for a neurotoxic phospholipase from the taipan venom. J Biol Chem 264(19):11503–11510Google Scholar
  28. 28.
    Lambeau G, Lazdunski M (1999) Receptors for a growing family of secreted phospholipases A2. Trends Pharmacol Sci 20:162–170CrossRefGoogle Scholar
  29. 29.
    Landucci ECT, de Castro RC, Pereira MF, Cintra ACO, Giglio JR, Marangoni S, Oliveira B, Cirino G, Antunes E, de Nucci G (1998) Mast cell degranulation induced by two phospholipase A2 homologues: dissociation between enzymatic and biological activities. Eur J Pharmacol 343:257–263CrossRefGoogle Scholar
  30. 30.
    Lloret S, Moreno JJ (1993) Oedema formation and degranulation of mast cells by phospholipase A2 purified from porcine pancreas and snake venoms. Toxicon 31(8):949–956CrossRefGoogle Scholar
  31. 31.
    Lomonte B, Tarkowski A, Hanson LA (1994) Broad cytolytic specificity of myotoxin II, a lysine-49 phospholipase A2 of Bothrops asper snake venom. Toxicon 32(11):1359–1369CrossRefGoogle Scholar
  32. 32.
    Lomonte B, Angulo Y, Rufini S, Cho W, Giglio JR, Ohno M, Daniele JJ, Geoghegan P, Gutiérrez JM (1999) Comparative study of the cytolytic activity of myotoxic phospholipases A2 on mouse endothelial (tEnd) and skeletal muscle (C2C12) cells in vitro. Toxicon 37:145–158CrossRefGoogle Scholar
  33. 33.
    Lomonte B, Tarkowski A, Hanson LA (1993) Host response to Bothrops asper snake venom. Analysis of edema formation, inflammatory cells, and cytokine release in a mouse model. Inflammation 17:93–105CrossRefGoogle Scholar
  34. 34.
    Mebs D, Ownby CL (1990) Myotoxic components of snake venoms: their biochemical and biological activities. Pharmacol Ther 48:223–236CrossRefGoogle Scholar
  35. 35.
    Moura-da-Silva AM, Desmond H, Laing G, Theakston RDG (1991) Isolation and comparison of myotoxins isolated from venoms of different species of Bothrops snakes. Toxicon 29:713–723CrossRefGoogle Scholar
  36. 36.
    Ponce-Soto LA, Bonfim VL, Rodrigues-Simioni L, Novello JC, Marangoni S (2006) Determination of primary structure of two isoforms 6-1 and 6-2 PLA2 D49 from Bothrops jararacussu snake venom and neurotoxic characterization using in vitro neuromuscular preparation. Protein J 25:147–155CrossRefGoogle Scholar
  37. 37.
    Ponce-Soto LA, Lomonte B, Gutiérrez JM, Rodrigues-Simioni L, Novello JC, Marangoni S (2007) Structural and functional properties of BaTX, a new Lys49 phospholipase A2 homologue isolated from the venom of the snake Bothrops alternatus. Biochim Biophys Acta 1770:585–593Google Scholar
  38. 38.
    Ponce-Soto LA, Toyama MH, Hyslop S, Novello JC, Marangoni S (2002) Isolation and preliminary enzymatic characterization of a novel PLA2 from Crotalus durissus collilineatus venom. J Protein Chem 21:131–136CrossRefGoogle Scholar
  39. 39.
    Pungercar J, Krizaj I (2007) Understanding the molecular mechanism underlying the presynaptic toxicity of secreted phospholipases A2. Toxicon 50(7):871–892Google Scholar
  40. 40.
    Randazzo-Moura P, Ponce-Soto LA, Rodrigues-Simioni L, Marangoni S (2008) Structural characterization and neuromuscular activity of a new Lys49 phospholipase A2 homologous (Bp-12) isolated from Bothrops pauloensis snake venom. Protein J. doi: 10.1007/s10930-008-9144-1
  41. 41.
    Rodrigues RS, Izidoro LF, Teixeira SS, Silveira LB, Hamaguchi A, Homsi-Brandeburgo MI, Selistre-de-Araújo HS, Giglio JR, Fuly AL, Soares AM, Rodrigues VM (2007) Isolation and functional characterization of a new myotoxic acidic phospholipase A2 from Bothrops pauloensis snake venom. Toxicon 50(1):153–165CrossRefGoogle Scholar
  42. 42.
    Rouault M, Bollinger JG, Lazdunski M, Gelb MH, Lambeau G (2003) Novel mammalian group XII secreted phospholipase A2 lacking enzymatic activity. Biochemistry 42:11494–11503CrossRefGoogle Scholar
  43. 43.
    Sai-Ngam A, Phongtananant S, Nuchprayoon I (2008) Phospholipase A2 genes and their expressions in Thai Russell’s viper venom glands. Toxicon 52:395–399CrossRefGoogle Scholar
  44. 44.
    Schagger HA, Von Jagow G (1987) Comassie blue-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for direct visualization of polypeptides during electrophoresis. Anal Biochem 166:368–379CrossRefGoogle Scholar
  45. 45.
    Toyama MH, Costa PD, Novello JC, de Oliveira B, Giglio JR, da Cruz Hofling MA, Marangoni S (1999) Purification and amino acid sequence of MP-III 4R D49 phospholipase A2 from Bothrops pirajai snake venom, a toxin with moderate PLA2 and anticoagulant activities and high myotoxic activity. J Protein Chem 18:371–378CrossRefGoogle Scholar
  46. 46.
    Valentin E, Lambeau G (2000) Increasing molecular diversity of secreted phospholipases A2 and their receptors and binding proteins. Biochim Biophys Acta 1488:59–70Google Scholar
  47. 47.
    Vishwanath BS, Kini RM, Gowda TV (1987) Characterization of three edema-inducing phospholipase A2 enzymes from habu (Trimeresurus flavoviridis) venom and their interaction with the alkaloid aristolochic acid. Toxicon 25(5):501–515CrossRefGoogle Scholar
  48. 48.
    Wang YM, Wang JH, Tsai IH (1996) Molecular cloning and deduced primary structures of acidic and basic phospholipases A2 from the venom of Deinagkistrodon acutus. Toxicon 34:1191–1196CrossRefGoogle Scholar
  49. 49.
    Zuliani JP, Fernandes CM, Zamuner SR, Gutiérrez JM, Teixeira CFP (2005) Inflammatory events induced by Lys-49 and Asp-49 phospholipases A2 isolated from Bothrops asper snake venom: role of catalytic activity. Toxicon 45:335–346CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • L. A. Ponce-Soto
    • 1
    • 2
    Email author
  • D. Martins-de-Souza
    • 1
    • 3
  • S. Marangoni
    • 1
  1. 1.Department of Biochemistry, Institute of Biology (IB)State University of Campinas (UNICAMP)CampinasBrazil
  2. 2.Department of Pharmacology, Faculty of Medical Science (FCM)State University of Campinas (UNICAMP)CampinasBrazil
  3. 3.Max Planck Institute of PsychiatryMunichGermany

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