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Proteomic analysis and purification of an unusual germin-like protein with proteolytic activity in the latex of Thevetia peruviana

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Abstract

Main Conclusion

The latex from Thevetia peruviana is rich in plant defense proteins, including a 120 kDa cysteine peptidase with structural characteristics similar to germin-like proteins.

More than 20,000 plant species produce latex, including Apocynaceae, Sapotaceae, Papaveraceae and Euphorbiaceae. To better understand the physiological role played by latex fluids, a proteomic analysis of Thevetia peruviana (Pers.) Schum latex was performed using two-dimensional gel electrophoresis and mass spectrometry. A total of 33 proteins (86 %) were identified, including storage proteins, a peptidase inhibitor, cysteine peptidases, peroxidases and osmotins. An unusual cysteine peptidase, termed peruvianin-I, was purified from the latex by a single chromatographic step involving gel filtration. The enzyme (glycoprotein) was inhibited by E-64 and iodoacetamide and exhibited high specific activity towards azocasein (K m 17.6 µM), with an optimal pH and temperature of 5.0–6.0 and 25–37 °C, respectively. Gel filtration chromatography, two-dimensional gel electrophoresis, and mass spectrometry revealed that peruvianin-I possesses 120 kDa, pI 4.0, and six subunits (20 kDa). A unique N-terminal amino acid sequence was obtained to oligomer and monomers of peruvianin-I (1ADPGPLQDFCLADLNSPLFINGYPCRNPALAISDDF36). High-resolution images from atomic force microscopy showed the homohexameric structure of peruvianin-I may be organized as a trimer of dimers that form a central channel similar to germin-like proteins. Peruvianin-I exhibited no oxalate oxidase and superoxide dismutase activity or antifungal effects. Peruvianin-I represents the first germin-like protein (GLP) with cysteine peptidase activity, an activity unknown in the GLP family so far.

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Acknowledgments

This study was supported by grants from the following Brazilian Agencies: Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). We thank Dr. Sandra Vairo Cavalli for critical reading of the manuscript.

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Authors and Affiliations

  1. Departamento de Bioquímica e Biologia Molecular da Universidade Federal do Ceará, Campus do Pici, Cx. Postal 6033, Fortaleza, CE, CEP 60451-970, Brazil

    Cleverson D. T. de Freitas, Wallace T. da Cruz, Maria Z. R. Silva, Ilka M. Vasconcelos, Bruno A. M. Rocha & Márcio V. Ramos

  2. Centro de Ciências da Saúde da Universidade de Fortaleza, Fortaleza, CE, Brazil

    Frederico B. M. B. Moreno, Renato A. Moreira & Ana C. O. Monteiro-Moreira

  3. Departamento de Física da Universidade Federal do Ceará, Campus do Pici, Fortaleza, CE, Brazil

    Luciana M. R. Alencar & Jeanlex S. Sousa

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  1. Cleverson D. T. de Freitas
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  2. Wallace T. da Cruz
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  3. Maria Z. R. Silva
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  4. Ilka M. Vasconcelos
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  5. Frederico B. M. B. Moreno
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  6. Renato A. Moreira
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  7. Ana C. O. Monteiro-Moreira
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  8. Luciana M. R. Alencar
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  9. Jeanlex S. Sousa
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  10. Bruno A. M. Rocha
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  11. Márcio V. Ramos
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Correspondence to Cleverson D. T. de Freitas.

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de Freitas, C.D.T., da Cruz, W.T., Silva, M.Z.R. et al. Proteomic analysis and purification of an unusual germin-like protein with proteolytic activity in the latex of Thevetia peruviana . Planta 243, 1115–1128 (2016). https://doi.org/10.1007/s00425-016-2468-8

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  • DOI: https://doi.org/10.1007/s00425-016-2468-8

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