title: Proteomic analysis and purification of an unusual germin-like protein with proteolytic activity in the latex of Thevetia peruviana.
abstract: 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.
authors.affiliation: Departamento de Bioquímica e Biologia Molecular da Universidade Federal do Ceará, Campus do Pici, Cx. Postal 6033, Fortaleza, CE, CEP 60451-970, Brazil. cleversondiniz@hotmail.com.
authors.forename: Cleverson D T
authors.initials: CD
authors.lastname: de Freitas
authors.affiliation: Departamento de Bioquímica e Biologia Molecular da Universidade Federal do Ceará, Campus do Pici, Cx. Postal 6033, Fortaleza, CE, CEP 60451-970, Brazil.
authors.forename: Wallace T
authors.initials: WT
authors.lastname: da Cruz
authors.affiliation: Departamento de Bioquímica e Biologia Molecular da Universidade Federal do Ceará, Campus do Pici, Cx. Postal 6033, Fortaleza, CE, CEP 60451-970, Brazil.
authors.forename: Maria Z R
authors.initials: MZ
authors.lastname: Silva
authors.affiliation: Departamento de Bioquímica e Biologia Molecular da Universidade Federal do Ceará, Campus do Pici, Cx. Postal 6033, Fortaleza, CE, CEP 60451-970, Brazil.
authors.forename: Ilka M
authors.initials: IM
authors.lastname: Vasconcelos
authors.affiliation: Centro de Ciências da Saúde da Universidade de Fortaleza, Fortaleza, CE, Brazil.
authors.forename: Frederico B M B
authors.initials: FB
authors.lastname: Moreno
authors.affiliation: Centro de Ciências da Saúde da Universidade de Fortaleza, Fortaleza, CE, Brazil.
authors.forename: Renato A
authors.initials: RA
authors.lastname: Moreira
authors.affiliation: Centro de Ciências da Saúde da Universidade de Fortaleza, Fortaleza, CE, Brazil.
authors.forename: Ana C O
authors.initials: AC
authors.lastname: Monteiro-Moreira
authors.affiliation: Departamento de Física da Universidade Federal do Ceará, Campus do Pici, Fortaleza, CE, Brazil.
authors.forename: Luciana M R
authors.initials: LM
authors.lastname: Alencar
authors.affiliation: Departamento de Física da Universidade Federal do Ceará, Campus do Pici, Fortaleza, CE, Brazil.
authors.forename: Jeanlex S
authors.initials: JS
authors.lastname: Sousa
authors.affiliation: Departamento de Bioquímica e Biologia Molecular da Universidade Federal do Ceará, Campus do Pici, Cx. Postal 6033, Fortaleza, CE, CEP 60451-970, Brazil.
authors.forename: Bruno A M
authors.initials: BA
authors.lastname: Rocha
authors.affiliation: Departamento de Bioquímica e Biologia Molecular da Universidade Federal do Ceará, Campus do Pici, Cx. Postal 6033, Fortaleza, CE, CEP 60451-970, Brazil.
authors.forename: Márcio V
authors.initials: MV
authors.lastname: Ramos
mesh_terms: D000935:Antifungal Agents / Q000494:pharmacology; D002364:Caseins / Q000378:metabolism; D057056:Cysteine Proteases / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D004353:Drug Evaluation, Preclinical / Q000379:methods; D007840:Latex / Q000737:chemistry* / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods; D010940:Plant Proteins / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology*; D040901:Proteomics / Q000379:methods; D031052:Thevetia / Q000737:chemistry*
chemical_list: D000935:Antifungal Agents; D002364:Caseins; D007840:Latex; D010940:Plant Proteins; C026018:azocasein; D057056:Cysteine Proteases
keywords: Apocynaceae; Atomic force microscopy; Peptidase; Proteome