Skip to main content

Advertisement

SpringerLink
Log in

Isolation, Functional Characterization and Proteomic Identification of CC2-PLA2 from Cerastes cerastes Venom: A Basic Platelet-Aggregation-Inhibiting Factor

  • Published:
The Protein Journal Aims and scope Submit manuscript

Abstract

Three-step chromatography and proteomic analysis have been used to purify and characterize a new basic phospholipase A2 named CC2-PLA2 from the venom of Cerastes cerastes. This phospholipase A2 has been isolated to an extent of about 50-folds and its molecular weight was estimated at 13,534 Da. For CC2-PLA2 identification and LC-MALDI-MS/MS analysis, the protein was reduced, alkylated and double hydrolyzed by lysine-C endopeptidase and trypsin. Tryptic fragments of LC–MS/MS analyzed CC2-PLA2 showed sequence similarities with other snake venom PLA2. This presents only 51 % (61/120 amino acid residues) sequence homology with the first PLA2 (gi |129506|) previously purified from the same venom. The isolated CC2-PLA2 displayed anti-aggregative effect on platelets and induced an inflammatory response characterized by leukocytosis in the peripheral blood. This inflammatory response is accompanied by a release of inflammatory mediators such as IL-6, eosinophil peroxidase and complement system. Obtained results indicate that CC2-PLA2 induced a release of high level of pro-inflammatory (IL-6) cytokine and no effect on the level of anti-inflammatory cytokine (IL-10) in blood sera. Furthermore, eosinophil peroxidase activity and hemolytic complement effect increased in peripheral blood. Mononuclear and neutrophil cells were found predominant in the induced leucocytosis following CC2-PLA2 administration into animals.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

Abbreviations

ACN:

Acetonitrile

CC2-PLA2 :

Second Cerastes cerastes phospholipase A2

CHCA:

Sinapinic acid

CID:

Collision ion dissociation

CM-Sephadex C-50:

Carboxymethyl-sephadex C-50

EDTA:

Ethylene diamine-tetra-acetic acid

EGTA:

Ethylene glycol tetraacetic acid

HPLC:

High performance liquid chromatography

IAM:

2-Iodoacetamide

TCEP:

Tris (2-carboxyethyl) phosphine hydrochloride

OPD:

O-Phenylene diamine dihydrochloride

PEG:

Polyethylene glycol

NaCl:

Sodium chloride

NIH:

Normal Institute of Health

SV-PLA2 :

Snake venom PLA2

TFA:

Tri fluoroacetic phosphate acid

References

  1. Andus T, Bauer J, Gerok W (1991) Effects of cytokines on the liver. Hepatology 13:364–369

    Article  CAS  Google Scholar 

  2. Bao Y, Bu P, Jin L, Hongxia W, Yang Q, An L (2005) Purification, characterization and gene cloning of a novel phospholipase A2 from the venom of Agkistrodon blomhoffii ussurensis. Int J Biochem Cell Biol 37:558–565

    Article  CAS  Google Scholar 

  3. Bazaa N, Marrakchi M, El Ayeb L, Sanz JJ, Calvete J (2005) Snake venomics: comparative analysis of the venom proteomes of the Tunisian snakes Cerastes cerastes, Cerastes vipera and Macrovipera lebetina. Proteomics 5:4223–4235

    Article  CAS  Google Scholar 

  4. Bertazzi DT, Assis-Pandochi AI, Azzolini AE, Talhaferro VL, Lazzarini M, Arantes EC (2003) Effect of Tityus serrulatus scorpion venom and its major toxin, TsTX-I, on the complement system in vivo. Toxicon 41:501–508

    Article  CAS  Google Scholar 

  5. Bertazzi DT, Assis-Pandochi AI, Talhaferro VL, Azzolini AECS, Crott LSP, Arantes EC (2005) Activation of complement system and leukocyte recruitment by Tityus serrulatus scorpion venom. Int Immunopharmacol 5:1077–1084

    Article  CAS  Google Scholar 

  6. Boukhalfa-Abib H, Meksem A, Laraba-Djebari F (2009) Purification and biochemical characterization of a novel hemorrhagic metalloproteinase from horned viper (Cerastes cerastes) venom. Comp Biochem Physiol Part C150:285–290

    Google Scholar 

  7. Braud S, Bon C, Wisner N (2000) Snake venom proteins acting on haemostasis. Biochimie 82:851–859

    Article  CAS  Google Scholar 

  8. Castro HC, Zingali RB, Albuquerque MG, Pujol-Luz M, Rodrigues CR (2004) Snake venom thrombin-like enzymes: from reptilase to now. Cell Mol Life Sci 61:843–856

    Article  CAS  Google Scholar 

  9. Castro HC, Rodriguez C (2006) Current status of snake venom thrombin-like enzymes. Toxin Rev 19:291–381

    Article  Google Scholar 

  10. Chaves F, Léon 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 snake Bothrops asper (terciopelo). Toxicon 36:1861–1869

    Article  CAS  Google Scholar 

  11. Chaves F, Teixeira CF, Gutierrez JM (2006) Role of nitric oxide in the local and systemic pathophysiological effects induced by Bothrops asper snake venom in mice. Inflamm Res 55:245–253

    Article  CAS  Google Scholar 

  12. Chérifi F, Laraba-Djebari F (2005) Effet de l’immunothérapie sur la biodistribution et les effets pulmonaires du venin de Cerastes cerastes. Rencontre en Toxinologie, «Toxines et Douleur» Lavoisier Editions TEC et DOC, pp 231–234

  13. Chérifi F, Laraba-Djebari F (2007) Purification et caractérisation d’une fraction anticoagulante et protéolytique du venin de Cerastes cerastes. «Toxines émergentes: nouveaux risques» Lavoisier, Editions TEC et DOC, pp 234–235

  14. Chérifi F, Laraba-Djebari F (2008) Mise en évidence et Caractérisation d’une Fraction Coagulante et Agrégante du venin de Cerastes cerastes. Toxines et fonctions cholinergiques neuronales et non neuronales, Editions de la SFET, pp 153–154

  15. Chérifi F, Laraba-Djebari F (2009) Réponse inflammatoire induite par la fraction coagulante C1 isolée du venin de Cerastes cerastes (editions de la SFET), Toxines et Signalisation, E-book RT17, 165–167

  16. Chérifi F, Rousselle JC, Namane A, Laraba-Djebari F (2010) CCSV-MPase, a Novel Procoagulant Metalloproteinase from Cerastes cerastes venom: purification, biochemical characterization and protein identification. Protein J 29:466–474

    Article  Google Scholar 

  17. Chérifi F, Rousselle JC, Namane A, Laraba-Djebari F (2011) Zn2+: a required ion for procoagulant metalloproteinase (CCSV-MPase) activities, isolated from Cerastes cerastes Venom. (SFET Editions) “Toxins and Ion transfers” E-Book RT19, pp 161–164

  18. Chérifi F, Laraba-Djebari F (2013) Pharmalocical interest of isolated biomolecules from Cerastes cerastes venom in haemostasis. J Venom Anim Toxins Includ Trop Dis 19:11

    Article  Google Scholar 

  19. Dennis EA (1994) Diversity of group types, regulation and function of phospholipases A2. J Biol Chem 269:13057–13060

    CAS  Google Scholar 

  20. D’Suze G, Monceda S, Gonzalez C, Sevcik C, Aguilar V, Alagon A (2003) Relationship between plasmatic levels of various cytokines, tumor necrosis factor, enzymes, glucose and venom concentrations following Tityus scorpion sting. Toxicon 41:367–375

    Article  Google Scholar 

  21. El-Asmar MF, Shaban E, Hagag M, Swelam N, Tu A (1986) Coagulant component in Cerastes cerastes (Egyptian sand viper) venom. Toxicon 24:1037–1044

    Article  CAS  Google Scholar 

  22. Faccioli LH, Souza GEP, Cunha FQS, Poole S, Ferreira SH (1990) Recombinant interleukin-1 and tumor necrosis factor induce neutrophil migration in vivo by indirect mechanisms. Agents Actions 30:344–349

    Article  CAS  Google Scholar 

  23. Faure G (2000) Natural inhibitors of phospholipases A2. Biochimie 82:833–840

    Article  CAS  Google Scholar 

  24. Faure G, Gowda VT, Maroun RC (2007) Characterization of a human coagulation factor Xa-binding site on Viperidae snake venom phospholipases A2 by affinity binding studies and molecular bioinformatics. BMC Struct Biol 7:82–89

    Article  Google Scholar 

  25. Farsky SHP, Costa-Cruz JWM, Cury Y, Teixeira CFP (1997) Leukocyte response induced by Bothrops jararaca crude venom. In vivo and in vitro studies. Toxicon 35:185–193

    Article  CAS  Google Scholar 

  26. Freitas MA, Geno PW, Summer LW, Cooke ME, Hudenberg SA, Ownby CL, Kaiser II, Odell GU (1992) Citrate is a major component of snake venoms. Toxicon 30:461–464

    Article  CAS  Google Scholar 

  27. Fuly AL, Calil-Elias S, Zingali RB, Guimaraes JA, Melo PA (2000) Myotoxic activity of acidic phospholipase A2 isolated from Lachesis muta (Bushmaster) snake venom. Toxicon 38(7):961–972

    Article  CAS  Google Scholar 

  28. Fukahara YDM, Reis ML, Dellalibera-Joviliani R, Cunha FQC, Donadi EA (2003) Increased levels of IL-1b, IL-6, IL-8, IL-10 and TNF-a in patients moderately or severely envenomed by Tityus serrulatus scorpion sting. Toxicon 41:49–55

    Article  Google Scholar 

  29. Grace SY, Shelat PB, Jensen MB, He Y, Sun AY, Simonyi A (2010) NeuroMol Med 12:133–148

    Article  Google Scholar 

  30. Hahn BS, Chang IM, Kim YS (1995) Purification and characterization of piscivorase I and II, the fibrinolytic enzymes from eastern cottonmouth moccasin venom (Agkistrodon piscivorus piscivorus). Toxicon 33:929–941

    Article  CAS  Google Scholar 

  31. Hamza L, Girardi T, Castelli S, Gargioli C, Cannata S, Patamia M, Luly P, Laraba-Djebari F, Petruzzelli R, Rufini S (2010) Isolation and characterization of a myotoxin from the venom of Macrovipera lebetina transmediterranea. Toxicon 56(3):381–390

    Article  CAS  Google Scholar 

  32. Hamza L, Gargioli C, Castelli S, Rufini S, Laraba-Djebari F (2010) Purification and characterization of a fibrinogenolytic and hemorrhagic metalloproteinase isolated from Vipera lebetina venom. Biochimie 92(7):797–805

    Article  CAS  Google Scholar 

  33. Hutton RA, Warrel DA (1993) Action of snake venom components on the haemostatic system. Blood Rev 7:840–855

    Article  Google Scholar 

  34. Kini RM (1997) Venom phospholipase A2 enzymes: structure, function and mechanism. Chichester, England, pp 1–511

    Google Scholar 

  35. Kishimoto T (1989) The biology of interleukin-6. Blood 74:1–10

    CAS  Google Scholar 

  36. Kornalik F (1990) Toxins affecting blood coagulation and fibrinolysis. In: Shier WT, Mebs D (eds) Handbook of toxicology. Marcel Dekker, New York, pp 697–709

    Google Scholar 

  37. Laraba-Djebari F, Martin-Eauclaire MF (1990) Purification and characterization of a phospholipase A2 from Cerastes cerastes (horn viper) snake venom. Toxicon 28:637–646

    Article  Google Scholar 

  38. Laraba-Djebari F, Martin-Eauclaire MF, Marchot PA (1992) A fibrinogen-clotting serine proteinase from Cerastes cerastes (Horned viper) with arginine–esterase an amidase activities, purification, characterization and kinetic parameter determination. Toxicon 30:1399–1410

    Article  CAS  Google Scholar 

  39. Laraba-Djebari F, Martin-Eauclaire MF, Mauco G, Matchot P (1995) Afaâcytin and α, β-fibrinogenase from Cerastes cerastes (Horned viper) venom, activates purified factor X and induces serotonin release from human blood platelet. Eur J Biochem 233:756–765

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  41. Leonardi A, Fox JW, Trampus-Bakija A, Krizaj I (2007) Ammodytase, a metalloproteinase from Vipera ammodytes ammodytes venom, possesses strong fibrinolytic activity. Toxicon 49:833–842

    Article  CAS  Google Scholar 

  42. Liu S, Sun MZ, Greenway FT (2006) A novel plasminogen activator from Agkistrodon blomhoffiussurensis venom (ABUSV-PA): purification and characterization. Bioch Biophys Res Commun 384:1279–1287

    Article  Google Scholar 

  43. Lizano S, Lomonte B, Fox JM, Gutiérrez JM (1997) Biochemical characterization and pharmacological properties of a phospholipase A2 myotoxin inhibitor from the plasma snake Bothrpos asper. Biochem 326:853–859

    Article  CAS  Google Scholar 

  44. Lomonte B, Tarkowski A, Hanson LA (1993) Host response to Bothrops asper snake. Analysis of edema formation, inflammatory cells and cytokine release in mouse model. Inflammation 17:93–105

    Article  CAS  Google Scholar 

  45. Marrakchi N, Barbouche R, Guermazi S, Bon C, El-Ayeb M (1997) Procoagulant and platelet-aggregating properties of Cerastocytin from Cerastes cerastes venom. Toxicon 35:261–272

    Article  CAS  Google Scholar 

  46. Marsh NA (1994) Snake venom affecting the haemostatic mechanism: a consideration of their mechanism, practical applications and biological significance. Blood Coagul Fibrinolysis 5:399–410

    CAS  Google Scholar 

  47. Martin MC, Gomez JJ, Esteban F, Saur R, Mourelle MI, Salgado A (1995) Cytokines and nitric oxide in streptococcal toxic shock syndrome. Med Clin 104:458–464

    CAS  Google Scholar 

  48. Meki ARMA, Mohey El-Dean ZM (1998) Serum interleukin 1b, interleukin-6, nitric oxide and a1-antitrypsin in scorpion envenomed children. Toxicon 36:1851–1859

    Article  CAS  Google Scholar 

  49. Mion G, Olive F, Hermandez E, Martin YN, Viellefosse S, Goyffon M (2002) Action des venins sur la coagulation sanguine: diagnostic des syndromes hémorragiques. Bull Soc Path Exot 95:132–138

    CAS  Google Scholar 

  50. Páramo L, Lomonte B, Pizarro-Cerda J, Bengoechea JA, Gorvel J, Moreno E (1998) Bactericidal activity of Lys49 and Asp49 myotoxic phospholipases A2 from Bothrops asper snake venom. Synthetic Lys49 myotoxin II-(115-129)-peptide identifies its bactericidal region. Eur J Biochem 253:452–461

    Article  Google Scholar 

  51. Oussedik-Oumehdi H, Laraba-Djebari F (2008) Irradiated Cerastes cerastes venom as a novel tool for immunotherapy. Immunopharmacol Immunotoxicol 30(1):37–52

    Article  CAS  Google Scholar 

  52. Quinton L, Girard E, Maiga A, Rekik M, Lluel P, Masuyer C, Ciolek J, Magnin T, Wagner R, Molgó J, Thai R, Fruchart-Gaillard C, Mourier G, Chamot-Rooke J, Ménez A, Palea S, Servent D, Gilles N (2010) Isolation and pharmacological characterization of AdTx1, a natural peptide displaying specific insurmountable antagonism of the alpha(1A)-adrenoceptor. Br J Pharmacol 159:316–325

    Article  CAS  Google Scholar 

  53. Smith JA (1994) Neutrophils, host defence, and inflammation: a double-edged sword. J Leukocyte Biol 56:672–686

    CAS  Google Scholar 

  54. Santamaría C, Larios S, Quiro′s S, Pizarro-Cerda J, Gorvel J (2004) Bactericidal and antiendotoxic properties of short cationic peptides derived from a snake venom Lys49 phospholipase A2. Antimicrob Agents Chemother 49(4):1340–1345

    Article  Google Scholar 

  55. Santamaría C, Larios S, Angulo Y, Pizarro-Cerda J, Gorvel J, Moreno E, Lomonte B (2005) Antimicrobial activity of myotoxic phospholipases A2 from crotalid snake venoms and synthetic peptide variants derived from their C-terminal region. Toxicon 45:807–815

    Article  Google Scholar 

  56. Soares AM, Guerra-Sa R, Borja-Oliveira C, Rodrigues VM, Rodrigues-Simioni L, Rodrigues V, Fontes M, Lomonte B (2000) Structural and functional characterization of BnSP-7, a Lys49 myotoxic phospholipase A2 homologue from Bothrops neuwiedi pauloensis venom. Arch Biochem Biophys 378:201–209

    Article  CAS  Google Scholar 

  57. Soares AM, Mancin AC, Cecchini AL, Arantes EC, França SC, Gutiérrez JM, Giglio JR (2001) Effects of chemical modifications of crotoxin B, the phospholipase A2 subunit of crotoxin from Crotalus durissus terrificus snake venom, on its enzymatic and pharmacological activities. Int J Biochem Cell Biol 33:877–888

    Article  CAS  Google Scholar 

  58. Swenson S, Markland FS (2005) Snake venom fibrin(ogen)olytic enzymes. Toxicon 45(8):1021–1039

    Article  CAS  Google Scholar 

  59. Teixeira S, Silveira LB, Da Silva FMN, Marchi-Salvador DP, Silva FP, Izidoro LFM, Fuly AL, Murakami CR (2011) Arch Toxicol 85(10):1219–1233

    Article  CAS  Google Scholar 

  60. Tsai IH, Tsai HY, Wang YM, Pe T, Warrel DA (2007) Venom phospholipases of Russell’s vipers from Myanmar and eastern India—cloning and phylogeographic analysis. Biochim Biophys Acta 1774:1020–1028

    Article  CAS  Google Scholar 

  61. Tsai YH, Wang YM, Tsai IH (2008) Cloning, characterization and phylogenetic analyses of members of three major venom families from a single specimen of Walterinnesia aegyptia. Toxicon 51:1245–1254

    Article  CAS  Google Scholar 

  62. Valentin E, Lambeau G (2000) What can venom phospholipases A2 tell us about the functional diversity of mammalian secreted phospholipases A2? Biochimie 82:815–831

    Article  CAS  Google Scholar 

  63. Valentin E, Lambeau G (2000) Increasing molecular diversity of secreted phospholipases A(2) and their receptors and binding proteins. Biochim Biophys Acta 1488:59–70

    Article  CAS  Google Scholar 

  64. Wei JF, Wei XL, Chen QY, Huang T, Qiao LY (2006) N49 phospholipase A2, a unique subgroup of snake venom group II phospholipase A2. Biochim Biophys Acta 1760:462–471

    Article  CAS  Google Scholar 

  65. Zuliani JP, Gutiérrez JM, Silva LLC, Sampaio SC, Lomonte B, CFP (2005) Activation of cellular functions in macrophages by venom secretory Asp-49 and Lys-49 phospholipases A2. Toxicon 46(5):523–532

    Article  CAS  Google Scholar 

  66. Zingali RB, Jandrot-Perrus M, Guillin MC, Bon C (1993) Bothrojaracin, a new thrombin inhibitor isolated from Bothrops jararaca venom: characterization and mechanism of thrombin inhibition. Biochemistry 32:10794–10802

    Article  CAS  Google Scholar 

  67. Zouari-Kessentini R, Luis-José L, Karry A, Kallech-Ziri O, Srairi-Abid N, Bazaa A, Loret E, Bezzine S, El-Ayeb M, Marrakchi N (2009) Two purified and characterized phospholipases A2 from Cerastes cerastes venom that inhibits cancerous cell adhesion and migration. Toxicon 53:444–453

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work was partially supported by ATRSS, Agence Thematique de la Recherche Scientifique en Santé (Thematic Agency of Scientific Resarech on Health, Oran, Algeria). The authors want to thank; Jean-Claude Rousselle and Benayad Tahar (Institut Pasteur, Plate-Forme de Protéomique, CNRS URA 2185, 75724 Paris cedex15, France and Laboratory of Food Security and the Environment, Scientific Police, Algiers, Algeria) for proteomic analysis and technical assistance.

Conflict of interest

Authors have no potential conflicts of interest.

Author information

Authors and Affiliations

  1. USTHB, Laboratory of Cellular and Molecular Biology, Faculty of Biological Sciences, USTHB, BP 32, El-Alia, Bab Ezzouar, Algiers, Algeria

    Fatah Chérifi & Fatima Laraba-Djebari

  2. Institut Pasteur Génétique des Interactions Macromoléculaires, 75724, Paris Cedex 15, France

    Abdelkader Namane

Authors
  1. Fatah Chérifi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Abdelkader Namane
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Fatima Laraba-Djebari
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Fatima Laraba-Djebari.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Chérifi, F., Namane, A. & Laraba-Djebari, F. Isolation, Functional Characterization and Proteomic Identification of CC2-PLA2 from Cerastes cerastes Venom: A Basic Platelet-Aggregation-Inhibiting Factor. Protein J 33, 61–74 (2014). https://doi.org/10.1007/s10930-013-9534-x

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10930-013-9534-x

Keywords

Search

Navigation