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Identification of proteins interacting with ammodytoxins in Vipera ammodytes ammodytes venom by immuno-affinity chromatography

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Abstract

In order to perform their function, proteins frequently interact with other proteins. Various methods are used to reveal protein interacting partners, and affinity chromatography is one of them. Snake venom is composed mostly of proteins, and various protein complexes in the venom have been found to exhibit higher toxicity levels than respective components separately. Complexes can modulate envenomation activity of a venom and/or potentiate its effect. Our previous data indicate that the most toxic components of the Vipera ammodytes ammodytes (Vaa) venom isolated so far—ammodytoxins (Atxs)—are contributing to the venom’s toxicity only moderately; therefore, we aimed to explore whether they have some interacting partner(s) potentiating toxicity. For screening of possible interactions, immuno-affinity chromatography combined with identification by mass spectrometry was used. Various chemistries (epoxy, carbonyldiimidazole, ethylenediamine) as well as protein G functionality were used to immobilize antibodies on monolith support, a Convective Interaction Media disk. Monoliths have been demonstrated to better suit the separation of large biomolecules. Using such approach, several proteins were indicated as potential Atx-binding proteins. Among these, the interaction of Atxs with a Kunitz-type inhibitor was confirmed by far-Western dot-blot and surface plasmon resonance measurement. It can be concluded that affinity chromatography on monolithic columns combined with mass spectrometry identification is a successful approach for screening of protein interactions and it resulted with detection of the interaction of Atx with Kunitz-type inhibitor in Vaa venom for the first time.

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References

  1. De Las Rivas J, Fontanillo C (2010) Protein–protein interactions essentials: key concepts to building and analyzing interactome networks. PLoS Comput Biol 6(e1000807):1–8

    Google Scholar 

  2. Koh GCKW, Porras P, Arranda B, Hermjakob H, Orchard SE (2012) Analyzing protein–protein interactions networks. J Prot Res 11:2014–2031

    Article  CAS  Google Scholar 

  3. Suter B, Kittanakom S, Stagljar I (2008) Two-hybrid technologies in proteomics research. Curr Opin Biotechnol 19:316–323

    Article  CAS  Google Scholar 

  4. Rigaut G, Sevchenko A, Rutz B, Wilm M, Mann M, Séraphine B (1999) A generic protein purification method for protein complex characterization and proteome exploration. Nat Biotechnol 17:1030–1032

    Article  CAS  Google Scholar 

  5. Puig O, Caspary F, Rigaut G, Rutz B, Bouveret E, Bragado-Nilsson E, Wilm M, Séraphine B (2001) The tandem affinity purification (TAP) method: a general procedure of protein complex purification. Methods 24:218–229

    Article  CAS  Google Scholar 

  6. Dziembowski A, Séraphine B (2004) Recent developments in the analysis of protein complexes. FEBS Lett 556:1–6

    Article  CAS  Google Scholar 

  7. Collins M, Choudhary J (2008) Mapping multiprotein complexes by affinity purification and mass spectrometry. Curr Opin Biotechnol 19:324–330

    Article  CAS  Google Scholar 

  8. Xu X, Song Y, Li Y, Chang J, Zhang H, An L (2010) The tandem affinity purification method: an efficient system for protein complex purification and protein interaction identification. Prot Exp Purif 72:149–156

    Article  CAS  Google Scholar 

  9. Beeckmans S (1999) Chromatographic methods to study protein–protein interactions. Methods 19:278–305

    Article  CAS  Google Scholar 

  10. Kuroda K, Kato M, Mima J, Ueda M (2006) Systems for the detection and analysis of protein–protein interactions. Applied Microbiol Biotechnol 71:127–136

    Article  CAS  Google Scholar 

  11. Lee W-C, Lee KH (2004) Applications of affinity chromatography in proteomics. Anal Biochem 324:1–10

    Article  CAS  Google Scholar 

  12. Azarkan M, Huet J, Baeyens-Volant D, Looze Y, Vandenbussche G (2007) Affinity chromatography: a useful tool in proteomics studies. J Chromatogr B 849:81–90

    Article  CAS  Google Scholar 

  13. Georgieva D, Risch M, Kardas A, Buck F, von Bergen M, Betzel C (2008) Comparative analysis of the venom proteomes of Vipera ammodytes ammodytes and Vipera ammodytes meridionalis. J Proteome Res 7:866–886

    Article  CAS  Google Scholar 

  14. Križaj I (2011) Ammodytoxin: a window into understanding presynaptic neurotoxicity of secreted phospholipases A2 and more. Toxicon 58:219–229

    Article  Google Scholar 

  15. Saul FA, Prijatelj-Znidarsic P, Vulliez-le Normand B, Villette B, Raynal B, Pungercar J, Krizaj I, Faure G (2009) Comparative structural studies of two natural isoforms of ammodytoxin, phospholipases A2 from Vipera ammodytes ammodytes which differ in neurotoxicity and anticoagulant activity. J Struct Biol 169:360–369

    Article  Google Scholar 

  16. Brgles M, Bertoša B, Winkler W, Kurtović T, Allmaier G, Marchetti-Deschmann M, Halassy B (2012) Chromatography, mass spectrometry, and molecular modeling studies on ammodytoxins. Anal Bioanal Chem 402:2737–2748

    Article  CAS  Google Scholar 

  17. Halassy B, Habjanec L, Brgles M, Lang Balija M, Leonardi A, Kovačić L, Prijatelj P, Tomašić J, Križaj I (2008) The role of antibodies specific for toxic sPLA2s and haemorrhagins in neutralizing potential of antisera raised against Vipera ammodytes ammodytes venom. Comp Biochem Physiol C Toxicol Pharmacol 148:178–183

    Article  Google Scholar 

  18. Prijatelj P, Vardjan N, Rowan EG, Križaj I, Pungerčar J (2006) Binding to the high-affinity M-type receptor for secreted phospholipases A(2) is not obligatory for the presynaptic neurotoxicity of ammodytoxin A. Biochimie 88:1425–1433

    Article  CAS  Google Scholar 

  19. Šribar J, Čopič A, Pariš A, Sherman NE, Gubenšek F, Fox JW, Križaj I (2001) A high affinity acceptor for phospholipase A2 with neurotoxic activity is a calmodulin. J Biol Chem 276:12493–12496

    Article  Google Scholar 

  20. Šribar J, Sherman NE, Prijatelj P, Faure G, Gubenšek F, Fox JW, Aitken A, Pungerčar J, Križaj I (2003) The neurotoxic phospholipase A2 associates, through a non-phosphorylated binding motif, with 14-3-3 protein γ and ε isoforms. Biochem Biophys Res Commun 302:691–696

    Article  Google Scholar 

  21. Doley R, Kini RM (2009) Protein complexes in snake venom. Cell Mol Life Sci 66:2851–2871

    Article  CAS  Google Scholar 

  22. Chu C-C, S-T CHU, Che S-W, Chen Y-H (1994) The non-phospholipase A2 subunit of beta-bungarotoxin plays an important role in the phospholipase A2-independent neurotoxic effect: characterization of three isotoxins with a common phospholipase A2 subunit. Biochem J 303:171–176

    CAS  Google Scholar 

  23. Aird SD, Kaiser II, Lewis RV, Kruggel WG (1985) Rattlesnake presynaptic neurotoxins: primary structure and evolutionary origin of the acidic subunit. Biochemistry 24:7054–7058

    Article  CAS  Google Scholar 

  24. Tchorbanov B, Grishin E, Aleksiev B, Ovchinnikov Y (1978) A neurotoxic complex from the venom of the Bulgarian viper (Vipera ammodytes ammodytes) and partial amino acid sequence of the toxic phospholipase A2. Toxicon 16:37–44

    Article  CAS  Google Scholar 

  25. Fohlman J, Eaker D, Karlsoon E, Thesleff S (1976) Taipoxin, an extremely potent presynaptic neurotoxin from the venom of the Australian snake taipan (Oxyuranus s. scutellatus): isolation, characterization, quaternary structure and pharmacological properties. Eur J Biochem 68:457–469

    Article  CAS  Google Scholar 

  26. Su MJ, Coulter AR, Sutherland SK, Chang CC (1983) The presynaptic neuromuscular blocking effect and phospholipase A2 activity of textilotoxin, a potent toxin isolated from the venom of the Australian brown snake Pseudonaja textiles. Toxicon 21:143–151

    Article  CAS  Google Scholar 

  27. Kumar JR, Basavarajappa BS, Arancio O, Aranha I, Gangadhara NS, Yajurvedi HN, Gowda TV (2008) Isolation and characterization of “Reprotoxin”, a novel protein complex from Daboia russelii snake venom. Biochimie 90:1545–1559

    Article  CAS  Google Scholar 

  28. Župunski V, Kordiš D, Gubenšek F (2003) Adaptive evolution in the snake venom Kunitz/BPTI protein family. FEBS Lett 547:131–136

    Article  Google Scholar 

  29. Bohlen CJ, Chesler AT, Sharif-Naeini R, Medzihradszky KF, Zhou S, King D, Sánchez EE, Burlingame AL, Basbaum AI, Julius D (2011) A heteromeric Texas coral snake toxin targets acid-sensing ion channels to produce pain. Nature 479:410–416

    Article  CAS  Google Scholar 

  30. Olivera BM, Teichert RW (2011) Chemical ecology of pain. Nature 479:306–307

    Article  CAS  Google Scholar 

  31. Kurtović T, Leonardi A, Lang Balija M, Brgles M, Habjanec L, Križaj I, Halassy B (2012) The standard mouse assay of anti-venom quality does not measure antibodies neutralising the haemorrhagic activity of Vipera ammodytes venom. Toxicon 59:709–717

    Article  Google Scholar 

  32. Jungbauer A, Hahn R (2008) Polymethacrylate monoliths for preparative and industrial separation of biomolecular assemblies. J Chromatogr A 1184:62–79

    Article  CAS  Google Scholar 

  33. Ehresmann B, Imbault P, Weil JH (1973) Spectrophotometric determination of protein concentration in cell extracts containing tRNA's and rRNA's. Anal Biochem 54:454–463

    Article  CAS  Google Scholar 

  34. Strohalm M, Kavan D, Novak P, Volny M, Havlicek V (2010) mMass 3: a cross-platform software environment for precise analysis of mass spectrometric dana. Anal Chem 82:4648–4651

    Article  CAS  Google Scholar 

  35. Theakston RDG, Reid HA (1983) Development of simple standard assay procedures for the characterization of snake venoms. Bull WHO 6:949–956

    Google Scholar 

  36. Lang Balija M, Vrdoljak A, Habjanec L, Dojnović B, Halassy B, Vranešić B, Tomašić J (2005) The variability of Vipera ammodytes ammodytes venoms from Croatia—biochemical properties and biological activity. Comp Biochem Physiol C 140:257–263

    CAS  Google Scholar 

  37. Davies D, Cohen GH (1996) Interactions of protein antigens with antibodies. PNAS 93:7–12

    Article  CAS  Google Scholar 

  38. Jones S, Thornton J (1996) Principles of protein–protein interactions. PNAS 93:13–20

    Article  CAS  Google Scholar 

  39. Leckband D (2000) Measuring the forces that control protein interactions. Annu Rev Biophys Biomol Struct 29:1–26

    Article  CAS  Google Scholar 

  40. Firer MA (2001) Efficient elution of functional proteins in affinity chromatography. J Biochem Biophys Methods 49:433–442

    Article  CAS  Google Scholar 

  41. Chan CS, Winstone TML, Turner RJ (2008) Investigating protein–protein interactions by far-Westerns. Adv Biochem Engin Biotechnol 110:195–214

    CAS  Google Scholar 

  42. Nicoli R, Gaud N, Stella C, Rudaz S, Veuthey J-L (2008) Trypsin immobilization on three monolithic disks for on-line protein digestion. J Pharm Biomed Anal 48:398–407

    Article  CAS  Google Scholar 

  43. Benčina K, Podgornik A, Štrancar A, Benčina M (2004) Enzyme immobilization on epoxy- and 1,1'-carbonyldiimidazole-activated methacrylate-based monoliths. J Sep Sci 27:811–818

    Article  Google Scholar 

Download references

Acknowledgments

This work was supported by the Bilateral Cooperation Grant Croatia–Austria (2010/2011 and 2012/2013) and the Croatian Ministry of Science, Education and Sports (grant 021-0212432-2033). CIM disks were kindly provided by BIA Separations.

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Authors and Affiliations

  1. Centre for Research and Knowledge Transfer in Biotechnology, University of Zagreb, Rockefellerova 10, 10000, Zagreb, Croatia

    Marija Brgles, Tihana Kurtović & Beata Halassy

  2. Department of Molecular and Biomedical Sciences, Jožef Stefan Institute, Jamova 39, 1000, Ljubljana, Slovenia

    Lidija Kovačič & Igor Križaj

  3. Department of Chemistry and Biochemistry, Faculty of Chemistry and Chemical Technology, University of Ljubljana, Aškerčeva 5, 1000, Ljubljana, Slovenia

    Igor Križaj

  4. Centre of Excellence for Integrated Approaches in Chemistry and Biology of Proteins, Jamova 39, 1000, Ljubljana, Slovenia

    Igor Križaj

  5. BIA Separations, Mirce 21, 5270, Ajdovščina, Slovenia

    Miloš Barut

  6. Institute of Immunology, Rockefellerova 2, 10000, Zagreb, Croatia

    Maja Lang Balija

  7. Institute of Chemical Technologies and Analytics, Vienna University of Technology, 1060, Vienna, Austria

    Günter Allmaier & Martina Marchetti-Deschmann

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  1. Marija Brgles
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Correspondence to Marija Brgles.

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Brgles, M., Kurtović, T., Kovačič, L. et al. Identification of proteins interacting with ammodytoxins in Vipera ammodytes ammodytes venom by immuno-affinity chromatography. Anal Bioanal Chem 406, 293–304 (2014). https://doi.org/10.1007/s00216-013-7453-5

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  • DOI: https://doi.org/10.1007/s00216-013-7453-5

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