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Crystal Structure of Crataeva Tapia Bark Protein (Cratabl) and Its Effect in Human Prostate Cancer Cell Lines

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Crystal Structure of Crataeva Tapia Bark Protein (Cratabl) and Its Effect in Human Prostate Cancer Cell Lines Crystal Structure of Crataeva tapia Bark Protein (CrataBL) and Its Effect in Human Prostate Cancer Cell Lines Rodrigo da Silva Ferreira1., Dongwen Zhou2., Joana Gasperazzo Ferreira1, Mariana Cristina Cabral Silva1, Rosemeire Aparecida Silva-Lucca3, Reinhard Mentele4,5, Edgar Julian Paredes-Gamero1, Thiago Carlos Bertolin6, Maria Tereza dos Santos Correia7, Patrı´cia Maria Guedes Paiva7, Alla Gustchina2, Alexander Wlodawer2*, Maria Luiza Vilela Oliva1* 1 Departamento de Bioquı´mica, Universidade Federal de Sa˜o Paulo, Sa˜o Paulo, Sa˜o Paulo, Brazil, 2 Macromolecular Crystallography Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, Maryland, United States of America, 3 Centro de Engenharias e Cieˆncias Exatas, Universidade Estadual do Oeste do Parana´, Toledo, Parana´, Brazil, 4 Institute of Clinical Neuroimmunology LMU, Max-Planck-Institute for Biochemistry, Martinsried, Munich, Germany, 5 Department for Protein Analytics, Max-Planck-Institute for Biochemistry, Martinsried, Munich, Germany, 6 Departamento de Biofı´sica, Universidade Federal de Sa˜o Paulo, Sa˜o Paulo, Sa˜o Paulo, Brazil, 7 Departamento de Bioquı´mica, Universidade Federal de Pernambuco, Recife, Pernambuco, Brazil Abstract A protein isolated from the bark of Crataeva tapia (CrataBL) is both a Kunitz-type plant protease inhibitor and a lectin. We have determined the amino acid sequence and three-dimensional structure of CrataBL, as well as characterized its selected biochemical and biological properties. We found two different isoforms of CrataBL isolated from the original source, differing in positions 31 (Pro/Leu); 92 (Ser/Leu); 93 (Ile/Thr); 95 (Arg/Gly) and 97 (Leu/Ser). CrataBL showed relatively weak inhibitory activity against trypsin (Kiapp =43 mM) and was more potent against Factor Xa (Kiapp = 8.6 mM), but was not active against a number of other proteases. We have confirmed that CrataBL contains two glycosylation sites and forms a dimer at high concentration. The high-resolution crystal structures of two different crystal forms of isoform II verified the b-trefoil fold of CrataBL and have shown the presence of dimers consisting of two almost identical molecules making extensive contacts (,645 A˚ 2). The structure differs from those of the most closely related proteins by the lack of the N-terminal b-hairpin. In experiments aimed at investigating the biological properties of CrataBL, we have shown that addition of 40 mM of the protein for 48 h caused maximum growth inhibition in MTT assay (47% of DU145 cells and 43% of PC3 cells). The apoptosis of DU145 and PC3 cell lines was confirmed by flow cytometry using Annexin V/FITC and propidium iodide staining. Treatment with CrataBL resulted in the release of mitochondrial cytochrome c and in the activation of caspase-3 in DU145 and PC3 cells. Citation: Ferreira RdS, Zhou D, Ferreira JG, Silva MCC, Silva-Lucca RA, et al. (2013) Crystal Structure of Crataeva tapia Bark Protein (CrataBL) and Its Effect in Human Prostate Cancer Cell Lines. PLoS ONE 8(6): e64426. doi:10.1371/journal.pone.0064426 Editor: Enrique Pe´rez-Paya´, Centro de Investigacion Principe Felipe and IBV-CSIC, Spain Received December 17, 2012; Accepted April 15, 2013; Published June 18, 2013 Copyright: ß 2013 Ferreira et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Use of the APS was supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences under Contract No. W-31-109-Eng- 38. The authors are grateful to CAPES, Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq) and Sa˜o Paulo Research Foundation (FAPESP) (Process 09/53766-5) for providing financial support. This project was also supported in part by the Intramural Research Program of the National Institutes of Health (NIH), National Cancer Institute, Center for Cancer Research. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] (MLVO); [email protected] (AW) . These authors contributed equally to this work. Introduction modification (miraculin) [6], binding to cytokine receptors (IL- 1b) [7], ribosome poisoning (ricin) [8] or carbohydrate binding, A large number of proteins are characterized by their b-trefoil exemplified by the Clitocybe nebularis lectin, CNL [9]. fold [1]. Members of this superfamily share structural features but Plant lectins are proteins that possess at least one non-catalytic not necessarily other properties and their biological role may be domain that binds reversibly and specifically mono- or oligosac- widely different [2]. Prominent among them are plant protease charides [10,11]. Owing to these properties, such proteins are inhibitors of the Kunitz type, called Kunitz-P or Kunitz-STI being utilized in characterization of glycoconjugates and in cell- inhibitors [2,3]. These proteins primarily inhibit serine proteases, surface or cell-cell architecture studies [12,13]. A few protease although some also inhibit cysteine and aspartic proteases [4]. inhibitors also show lectin activity, an example provided by the Kunitz-P inhibitors are characterized by molecular mass of about inhibitor isolated from the epidermis of the eel, named Eel-CPI-1 20,000 Da (for the whole protein or a domain), low content of [14]. Troncoso et al. [15] isolated an inhibitor with lectin cysteine residues, and one or two reactive sites that are the basis of properties from Peltophorum dubium seeds which decreases the their inhibitory activity. However, some structurally related b- viability of the Nb2 rat lymphoma cells. trefoil proteins are not protease inhibitors at all, but exhibit varied Crataeva tapia (also known as Crateva tapia) belongs to the other properties, for example chlorophyll binding [5], taste Capparaceae family that is found in northeastern Brazil. A protein PLOS ONE | www.plosone.org 1 June 2013 | Volume 8 | Issue 6 | e64426 Crataeva tapia Lectin Characterization from the Crataeva tapia bark has been purified using reversed Primary structure of CrataBL micelles in isooctane [16] and through chromatographic processes The sequencing of the native protein was performed by Edman [17]. Named CrataBL (Crataeva tapia Bark Lectin), this protein was degradation [28]. The sample was reduced, alkylated, and then shown to possess some specificity for binding glucose and galactose submitted to digestion with trypsin, chymotrypsin, Asp-N, and [17]. A number of its biological properties have been character- Lys-C endopeptidases (sequencing grade, Roche, Germany). ized, including delay in clot formation by impairing the intrinsic Fragments resulting from the treatment with endopeptidases were pathway of coagulation cascade [18]. In addition, CrataBL was purified on a 16150 mm Jupiter Proteo 90A column (Phenom- shown to exhibit anti-inflammatory, analgesic [19], insecticidal enex, Aschaffenburg, Germany) at a flow rate of 0.1 mL/min, [17], and anti-tumor [19] activities. using an acetonitrile gradient (0–60%) with 0.1% (v/v) trifluor- Prostate cancer is the most common cancer in men [20], with oacetic acid for 40 min. Sequencing was performed on an DU145 and PC3 being the most extensively studied prostate automatic gas-phase sequencer (492cLC; Applied Biosystems, cancer cell lines that serve as in vitro models for cancer treatment Foster City, USA). The glycosylated peptides were analyzed by studies [21,22,23]. Prostate cancer is associated with a high level of mass spectrometry to determine the molecular mass of the expression of proteases [24] and glycoproteins [25], making carbohydrate moieties. Sequence similarities were assessed with CrataBL a potentially useful tool for studies involving prostate the program BLAST against the NCBI protein database [29]. The cancer cell lines, due to its combination of properties that include sequences were aligned using the program MULTALIN [30]. both protease inhibition and carbohydrate binding. In this work, we determined the amino acid sequence and the Enzyme inhibition assays three-dimensional structure of CrataBL and also investigated a The inhibitory activity of CrataBL against proteases was range of biochemical activities, comparing them to the properties measured using specific chromogenic or fluorogenic substrates in of other members of this superfamily. Subsequently, we charac- 96-well microtiter plates at 37uC. The enzymes (human Factor terized the effect of CrataBL on growth and stability of human Xa, bovine trypsin, bovine chymotrypsin, human plasma kallikrein prostate cancer cell lines. (HuPK), porcine pancreatic kallikrein (PoPK), human neutrophil elastase (HNE), human plasmin, and papain) were pre-incubated Materials and Methods with either CrataBL or with solvent for 10 min. The reactions Isolation of CrataBL were initiated by the addition of the substrates, and the color or Isolation of the protein was performed according to the fluorescence were monitored continuously at 405 nm using a procedure of Arau´jo et al. [17]. Briefly, 10% (w/v) extracts from spectrophotometer (Packard, SpectraCount), or at 380/460 nm Crataeva tapia bark were fractionated with 30–60% ammonium using a Hitachi F-2000 spectrofluorimeter (Tokyo, Japan), sulfate. The fraction containing the protein
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