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Biochemical characterization of VQ-VII, a cysteine peptidase with broad specificity, isolated from Vasconcellea quercifolia latex

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Abstract

The latex from Vasconcellea quercifolia (“oak leaved papaya”), a member of the Caricaceae family, contains at least seven cysteine endopeptidases with high proteolytic activity, which helps to protect these plants against injury. In this study, we isolated and characterized the most basic of these cysteine endopeptidases, named VQ-VII. This new purified enzyme was homogeneous by bidimensional electrophoresis and MALDI-TOF mass spectrometry, and exhibited a molecular mass of 23,984 Da and an isoelectric point >11. The enzymatic activity of VQ-VII was completely inhibited by E-64 and iodoacetic acid, confirming that it belongs to the catalytic group of cysteine endopeptidases. By investigating the cleavage of the oxidized insulin B-chain to establish the hydrolytic specificity of VQ-VII, we found 13 cleavage sites on the substrate, revealing that it is a broad-specificity peptidase. The pH profiles toward p-Glu-Phe-Leu-p-nitroanilide (PFLNA) and casein showed that the optimum pH is about 6.8 for both substrates, and that in casein, it is active over a wide pH range (activity higher than 80 % between pH 6 and 9.5). Kinetic enzymatic assays were performed with the thiol peptidase substrate PFLNA (K m = 0.454 ± 0.046 mM, k cat = 1.57 ± 0.07 s−1, k cat/K m = 3.46 × 103 ± 14 s−1 M−1). The N-terminal sequence (21 amino acids) of VQ-VII showed an identity >70 % with 11 plant cysteine peptidases and the presence of highly conserved residues and motifs shared with the “papain-like” family of peptidases. VQ-VII proved to be a new latex enzyme of broad specificity, which can degrade extensively proteins of different nature in a wide pH range.

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Abbreviations

AMPSO:

3-[(1,1-Dimethyl-2-hydroxyethyl)amino]-2-hydroxy-propanesulfonic acid

BLAST:

Basic local alignment search tool

CAPS:

3-(Cyclohexylamino)-1-propanesulfonic acid

CHAPS:

3-[(3-Cholamidopropyl)-dimethylammonio]-1-propane sulfonate

2D-PAGE:

Two dimensional-polyacrylamide gel electrophoresis

DTT:

Dithiothreitol

E-64:

Trans-epoxysuccinyl-l-leucylamido-(4-guanidino)butane

HCCA:

α-Cyano-4-hydroxy-cinnamic acid

MALDI-TOF MS:

Matrix-assisted laser desorption/ionization time of flight mass spectrometry

MES:

2-Morpholinoethanesulfonic acid

MOPS:

3-(N-morpholino) propanesulfonic acid

PFLNA:

p-Glu-Phe-Leu-p-nitroanilide

PMF:

Peptide mass fingerprinting

SDS:

Sodium dodecyl sulfate

SDS-PAGE:

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

SP:

Sulfopropyl

TAPS:

N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid

TFA:

Trifluoroacetic acid

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Acknowledgments

The present work was supported by grants from CONICET (PIP 0477), UNLP and CIC (Argentina).  C.L. Natalucci is member of the CICPBA Researcher Career, L.M.I. López is member of the CONICET Researcher Career. The MALDI-TOF MS experiments were carried out in the proteomics facility (SePBioEs) from IBB-UAB, a member of ProteoRed-ISCIII.

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Authors and Affiliations

  1. Departamento de Ciencias Básicas y Experimentales, Universidad Nacional del Noroeste de la Provincia de Buenos Aires, Junín, Argentina

    María José Torres

  2. Servei de Proteòmica del Institut de Biotecnologia i de Biomedicina (IBB), Universitat Autònoma de Barcelona, Campus Universitari, Bellaterra, Cerdanyola del Vallès, 08193, Barcelona, Spain

    Sebastián Alejandro Trejo

  3. Laboratorio de Investigación de Proteínas Vegetales (LIProVe), Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata, Argentina

    Claudia Luisa Natalucci & Laura María Isabel López

Authors
  1. María José Torres
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  2. Sebastián Alejandro Trejo
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  3. Claudia Luisa Natalucci
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  4. Laura María Isabel López
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Corresponding author

Correspondence to Laura María Isabel López.

Additional information

C. L. Natalucci and L. M. I. López contributed equally to this work.

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Torres, M.J., Trejo, S.A., Natalucci, C.L. et al. Biochemical characterization of VQ-VII, a cysteine peptidase with broad specificity, isolated from Vasconcellea quercifolia latex. Planta 237, 1651–1659 (2013). https://doi.org/10.1007/s00425-013-1872-6

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