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Planta

, Volume 243, Issue 5, pp 1115–1128 | Cite as

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

  • Cleverson D. T. de FreitasEmail author
  • Wallace T. da Cruz
  • Maria Z. R. Silva
  • Ilka M. Vasconcelos
  • Frederico B. M. B. Moreno
  • Renato A. Moreira
  • Ana C. O. Monteiro-Moreira
  • Luciana M. R. Alencar
  • Jeanlex S. Sousa
  • Bruno A. M. Rocha
  • Márcio V. Ramos
Original Article

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.

Keywords

Atomic force microscopy Apocynaceae Peptidase Proteome 

Notes

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.

Supplementary material

425_2016_2468_MOESM1_ESM.doc (32 kb)
Supplementary material 1 (DOC 32 kb)
425_2016_2468_MOESM2_ESM.doc (178 kb)
Supplementary material 2 (DOC 178 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Cleverson D. T. de Freitas
    • 1
    Email author
  • Wallace T. da Cruz
    • 1
  • Maria Z. R. Silva
    • 1
  • Ilka M. Vasconcelos
    • 1
  • Frederico B. M. B. Moreno
    • 2
  • Renato A. Moreira
    • 2
  • Ana C. O. Monteiro-Moreira
    • 2
  • Luciana M. R. Alencar
    • 3
  • Jeanlex S. Sousa
    • 3
  • Bruno A. M. Rocha
    • 1
  • Márcio V. Ramos
    • 1
  1. 1.Departamento de Bioquímica e Biologia Molecular da Universidade Federal do CearáCampus do PiciFortalezaBrazil
  2. 2.Centro de Ciências da Saúde da Universidade de FortalezaFortalezaBrazil
  3. 3.Departamento de Física da Universidade Federal do CearáCampus do PiciFortalezaBrazil

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