Skip to main content
SpringerLink
Log in

Cloning and Identification of a Complete cDNA Coding for a Bactericidal and Antitumoral Acidic Phospholipase A2 from Bothrops jararacussu Venom

  • Published:
The Protein Journal Aims and scope Submit manuscript

Abstract

In order to better understand the function of acidic phospholipases A2 (PLA2s) from snake venoms, expressed sequence tags (ESTs) that code for acidic PLA2s were isolated from a cDNA library prepared from the poly(A)+ RNA of venomous glands of Bothrops jararacussu. The complete nucleotide sequence (366 bp), named BOJU-III, encodes the BthA-I-PLA2 precursor, which includes a signal peptide and the mature protein with 16 and 122 amino acid residues, respectively. Multiple comparison of both the nucleotide and respective deduced amino acid sequence with EST and protein sequences from databases revealed that the full-length cDNA identified (BOJU III – AY145836) is related to an acidic PLA2 sharing similarity, within the range 55–81%, with acidic phospholipases from snake venoms. Moreover, phylogenetic analysis of amino acid sequences of acidic PLA2s from several pit viper genera showed close evolutionary relationships among acidic PLA2s from Bothrops, Crotalus, and Trimeresurus. The molecular modeling showed structural similarity with other dimeric class II PLA2s from snake venoms. The native protein BthA-I-PLA2, a nontoxic acidic PLA2 directly isolated from Bothrops jararacussu snake venom, was purified and submitted to various bioassays. BthA-I-PLA2 displayed high catalytic activity and induced Ca2+-dependent liposome disruption. Edema induced by this PLA2 was inhibited by indomethacin and dexamethasone, thus suggesting involvement of the cyclo-oxygenase pathway. BthA-I-PLA2 showed anticoagulant activity upon human plasma and inhibited phospholipid-dependent platelet aggregation induced by collagen or ADP. In addition, it displayed bactericidal activity against Escherichia coli and Staphylococcus aureus and antitumoral effect upon breast adrenocarcinoma as well as upon human leukemia T and Erlich ascitic tumor. Following chemical modification with p-bromophenacyl bromide, total loss of the enzymatic and pharmacological activities were observed. This is the first report on the isolation and identification of a cDNA encoding a complete acidic PLA2 from Bothrops venom, exhibiting bactericidal and antitumoral effects.

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

Similar content being viewed by others

References

  • Alvarado, J., and Gutiérrez, J. M. (1988). Rev. Biol. Trop. 36: 563-565.

    Google Scholar 

  • Andrião-Escarso, S. H., Soares, A. M., Rodrigues, V. M., Angulo, Y., Díaz-Oreiro, C., Lomonte, B., et al. (2000). Biochimie 82: 755-763.

    Google Scholar 

  • Andrião-Escarso, S. H., Soares, A. M., Fontes, M. R. M., Fuly, A. L., Correa, F. M. A., Rosa, J. C., et al. (2002). Biochem. Pharmacol. 64: 723-732.

    Google Scholar 

  • Balsinde, J., Balboa, M. A., Insel, P. A., and Dennis, E. A. (1999). Annu. Rev. Pharmacol. Toxicol. 39: 175-189.

    Google Scholar 

  • Butron, E., Thelestam, M., and Gutiérrez, J. M. (1993). Biochim. Biophys. Acta 1179: 253-259.

    Google Scholar 

  • Carson, M. (1997). Methods Enzymol. 277: 493-505.

    Google Scholar 

  • Chaves, F., Léon, G., Alvarado, V. H., and Gutiérrez, J. M. (1998). Toxicon 36: 1861-1869.

    Google Scholar 

  • Chwetzoff, S., Tsunasawa, S., Sakiyama, F., and Menez, A. (1989). J. Biol. Chem. 264: 13289-13297.

    Google Scholar 

  • Corin, R. E., Viskatis, L. J., Vidal, J. C., and Etcheverry, M. A. (1993). Invest New Drugs 11: 11-15.

    Google Scholar 

  • Cura, J. E., Blanzaco, D. P., Brisson, C., Cura, M. A., Cabrol, R., Larrateguy, L., et al. (2002). Clin. Cancer Res. 8: 1033-1041.

    Google Scholar 

  • Cummings, B. S., McHowat, J. G., and Schnellmann, R. G. (2000). J. Pharmacol. Exp. Ther. 294: 793-799.

    Google Scholar 

  • Díaz, C., Gutiérrez, J. M., Lomonte, B., and Gené, J. A. (1991). Biochim. Biophys. Acta 1070: 455-460.

    Google Scholar 

  • Felsenstein, J. (1997). PHYLIP: The Phylogeny Inference Package, Version 3.5, Computer program distributed by the Department of Genetics, University of Washington, Seattle, WA.

    Google Scholar 

  • Fuly, A. L., de Miranda, A. L., Zingali, R. B., and Guimarães, J. A. (2002). Biochem. Pharmacol. 63: 1589-1597.

    Google Scholar 

  • Gutiérrez, J. M., Avila, C., Rojas, E., and Cerdas, L. (1988). Toxicon 26: 411-413.

    Google Scholar 

  • Gutiérrez, J. M., and Lomonte, B. (1997). In: Kini, R. M. (ed.), Venom Phospholipase A2 Enzymes: Structure, Function and Mechanism, John Wiley and Sons, Chichester, pp. 321-352.

    Google Scholar 

  • de Haas, G. H., Postema, N. M., Nieuwenhuizen, W., and van Deenen, L. L. M. (1968). Biochem. Biophys. Acta 159: 103-110.

    Google Scholar 

  • Homsi-Brandeburgo, M. I., Queiroz, L. S., Santo-Neto, H., Rodrigues-Simioni, L., and Giglio, J. R. (1988). Toxicon 26: 615-627.

    Google Scholar 

  • Itzhaki, R. F., and Gill, D. M. (1964). Anal. Biochem. 9: 401-410.

    Google Scholar 

  • Junqueira-de-Azevedo, I. L., and Ho, P. L. (2002). Gene 299: 279-291.

    Google Scholar 

  • Kashima, S., Soares, A. M., Roberto, P. G., Pereira, J. O., Astolfi-Filho, S., Cintra, A. C. O., et al. (2002). Biochimie 84: 675-680.

    Google Scholar 

  • Kashima, S., Roberto, P. G., Soares, A. M., Astolfi-Filho, S., Pereira, J. O., Giuliatti, S., et al. (2004). Biochimie 86 (in press).

  • Kimura, M. (1980). J. Mol. Evol. 16: 111-120.

    Google Scholar 

  • Kini, R. M., and Chan, Y. M. (1999). J. Mol. Evol. 48: 127-132.

    Google Scholar 

  • Lomonte, B., and Carmona, E. (1992). Comp. Biochem. Physiol. 102B: 325-329.

    Google Scholar 

  • Lu, Q. M., Jin, Y., Wei, J. F., Li, D. S., Zhu, S. W., Wang, W. Y., et al. (2002). Toxicon 40: 1313-1319.

    Google Scholar 

  • Mosmann, T. (1983). J. Immunol. Methods 65: 55-63.

    Google Scholar 

  • Moura-da-Silva, A. M., Paine, M. J. I., Diniz, M. R. V., Theakston, R. D. G., and Crampton, J. M. (1995). J. Mol. Evol. 41: 174-179.

    Google Scholar 

  • Ownby, C. L., Selistre-de-Araújo, H. S., White, S. P., and Fletcher, J. E. (1999). Toxicon 37: 411-445.

    Google Scholar 

  • Pan, H., Liu, X. L., Ou-Yang, L. L., Yang, G. Z., Zhou, Y. C., Li, Z. P., et al. (1998). Toxicon 36: 1155-1163.

    Google Scholar 

  • Panini, S. R., Yang, L., Rusinol, A. E., Sinensky, M. S., Bonventre, J. V., and Leslie, C. C. (2001). J. Lipid Res. 42: 1678-1686.

    Google Scholar 

  • Rodrigues, V. M., Soares, A. M., Mancin, A. C., Fontes, M. R. M., Homsi-Brandeburgo, M. I., and Giglio, J. R. (1998). Comp. Biochem. Physiol. 121A: 215-222.

    Google Scholar 

  • Rudrammaji, L. M., and Gowda, T. V. (1998). Toxicon 36: 921-932.

    Google Scholar 

  • Serrano, S. M. T., Reichl, A. P., Mentele, R., Auerswald, E. A., Santoro, M. L., Sampaio, C. A. M., et al. (1999). Arch. Biochem. Biophys. 367: 26-32.

    Google Scholar 

  • Six, D. A., and Dennis, E. A. (2000). Biochim. Biophys. Acta 1488: 1-19.

    Google Scholar 

  • Soares, A. M., Anzaloni-Pedrosa, L. H., Fontes, M. R. M., da Silva, R. J., and Giglio, J. R. (1998a). J. Venom. Anim. Toxins 4: 137-142.

    Google Scholar 

  • Soares, A. M., Rodrigues, V. M., Homsi-Brandeburgo, M. I., Toyama, M. H., Lombardi, F. R., Arni, R. K., et al. (1998b). Toxicon 36: 503-514.

    Google Scholar 

  • Soares, A. M., Guerra-Sá, R., Borja-Oliveira, C., Rodrigues, V., Rodrigues-Simioni, L., Rodrigues, V., et al. (2000). Arch. Biochem. Biophys. 378: 201-209.

    Google Scholar 

  • Soares, A. M., Andriao-Escarso, S. H., Bortoleto, R. K., Rodrigues-Simioni, L., Arni, R. K., Ward, R. J., et al. (2001). Arch. Biochem. Biophys. 387: 188-196.

    Google Scholar 

  • Soares, A. M., and Giglio, J. R. (2003). Toxicon 42: 855-868.

    Google Scholar 

  • Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F., and Higgins, D. G. (1997). Nucl. Acids Res. 24: 4876-4882.

    Google Scholar 

  • Tsai, I. H., Lu, P. J., Wang, Y. M., Ho, C. L., and Liaw, L. L. (1995). Biochem. J. 311: 895-900.

    Google Scholar 

  • Tsai, I. H., Wang, Y. M., Au, L. C., Ko, T. P., Chen, Y. H., and Chu, Y. F. et al. (2000). Eur. J. Biochem. 267: 6684-6691.

    Google Scholar 

  • Tsai, I. H., Wang, Y. M., Chen, Y. H., and Tu, A. T. (2003). Arch. Biochem. Biophys. 411: 289-296.

    Google Scholar 

  • Valiente, C., Moreno, E., Sittenfeld, A., Lomonte, B., and Gutiérrez, J. M. (1992). Toxicon 30: 815-823.

    Google Scholar 

  • Vesterberg, O. (1972). Biochim. Biophys. Acta 257: 11-30.

    Google Scholar 

  • Wang, Y. M., Wang, J. H., and Tsai, I. H. (1996). Toxicon 34: 1191-1196.

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Unidade de Biotecnologia, Universidade de Ribeirão Preto, UNAERP, Ribeirão Preto-SP, Brazil

    Patrícia G. Roberto, Simone Kashima, Silvana Marcussi, Andreimar M. Soares & Suzelei C. França

  2. Faculdade de Ciências Agrárias, Universidade Federal do Amazonas, Manaus-AM, Brazil

    José O. Pereira & Spartaco Astolfi-Filho

  3. Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, FCFRP, Universidade de São Paulo, Ribeirão Preto-SP, Brazil

    Auro Nomizo

  4. Departamento de Bioquímica e Imunologia, FMRP, Universidade de São Paulo, Ribeirão Preto-SP, Brazil

    José R. Giglio

  5. Departamento de Física e Biofísica, IB, Universidade Estadual Paulista, Botucatu-SP, Brazil

    Marcos R.M. Fontes

Authors
  1. Patrícia G. Roberto
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Simone Kashima
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Silvana Marcussi
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. José O. Pereira
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Spartaco Astolfi-Filho
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Auro Nomizo
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. José R. Giglio
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Marcos R.M. Fontes
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Andreimar M. Soares
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Suzelei C. França
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Andreimar M. Soares.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Roberto, P.G., Kashima, S., Marcussi, S. et al. Cloning and Identification of a Complete cDNA Coding for a Bactericidal and Antitumoral Acidic Phospholipase A2 from Bothrops jararacussu Venom. J Protein Chem 23, 273–285 (2004). https://doi.org/10.1023/B:JOPC.0000027852.92208.60

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1023/B:JOPC.0000027852.92208.60

Search

Navigation