RESUMO
Plant lectins, especially those purified from species of the Leguminosae family, represent the best-studied group of carbohydrate-binding proteins. Lectins purified from seeds of the Diocleinae subtribe exhibit a high degree of sequence identity notwithstanding that they show very distinct biological activities. Two main factors have been related to this feature: variance in key residues influencing the carbohydrate-binding site geometry and differences in the pH-dependent oligomeric state profile. In this work, we have isolated a lectin from Canavalia boliviana (Cbol) and solved its x-ray crystal structure in the unbound form and in complex with the carbohydrates Man(α1-3)Man(α1-O)Me, Man(α1-4)Man(α1-O)Me and 5-bromo-4-chloro-3-indolyl-α-D-mannose. We evaluated its oligomerization profile at different pH values using Small Angle X-ray Scattering and compared it to that of Concanavalin A. Based on predicted pKa-shifts of amino acids in the subunit interfaces we devised a model for the dimer-tetramer equilibrium phenomena of these proteins. Additionally, we demonstrated Cbol anti-inflammatory properties and further characterized them using in vivo and in vitro models.
Assuntos
Anti-Inflamatórios/farmacologia , Canavalia/química , Edema/tratamento farmacológico , Manosídeos/química , Peritonite/tratamento farmacológico , Lectinas de Plantas/química , Lectinas de Plantas/farmacologia , Sementes/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Movimento Celular/efeitos dos fármacos , Quimiotaxia/efeitos dos fármacos , Cristalografia por Raios X , Edema/induzido quimicamente , Manosídeos/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Peritonite/induzido quimicamente , Conformação Proteica , Ratos , Ratos Wistar , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Lectins have been used as models for studies of the molecular basis of protein-carbohydrate interaction and specificity by deciphering codes present in the glycan structures. The purpose of the present study was to purify and solve the complete primary and crystal structure of the lectin of Camptosema pedicellatum (CPL) complexed with 5-bromo-4-chloro-3-indolyl-α-d-mannose (X-Man) using tandem mass spectrometry. CPL was purified by single-step affinity chromatography. Mass spectrometry findings revealed that purified CPL features a combination of chains weighing 25,298 ± 2 (α-chain), 12,835 ± 2 (ß-chain) and 12,481 ± 2 Da (γ-chain). The solved crystal structure of CPL features a conservative mutation in the hydrophobic subsite, a constituent of the carbohydrate recognition domain (CRD), indicating the relevance of hydrophobic interactions in the establishment of interactions with carbohydrates. The substitution and the analysis of the interactions with X-Man also revealed that the hydrophobic effect caused by a minor change in the hydrophobic subsite interferes in the formation of H-bonds due to the reorientation of the indolyl group in the CRD.
Assuntos
Lectinas de Plantas/química , Sequência de Aminoácidos , Cristalografia por Raios X , Fabaceae/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Indóis/química , Indóis/metabolismo , Manose/análogos & derivados , Manose/química , Manose/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Lectinas de Plantas/metabolismo , Espectrometria de Massas em TandemRESUMO
Diocleinae lectins are highly homologous in their primary structure which features metal binding sites and a carbohydrate recognition domain (CRD). Differences in the biological activity of legume lectins have been widely investigated using hemagglutination inhibition assays, isothermal titration microcalorimetry and co-crystallization with mono- and oligosaccharides. Here we report a new lectin crystal structure (ConBr) extracted from seeds of Canavalia brasiliensis, predict dimannoside binding by docking, identify the α-aminobutyric acid (Abu) binding pocket and compare the CRD of ConBr to that of homologous lectins. Based on the hypothesis that the carbohydrate affinity of lectins depends on CRD configuration, the relationship between tridimensional structure and endothelial NO synthase activation was used to clarify differences in biological activity. Our study established a correlation between the position of CRD amino acid side chains and the stimulation of NO release from endothelium.
Assuntos
Canavalia/metabolismo , Óxido Nítrico Sintase Tipo III/química , Lectinas de Plantas/química , Carboidratos , Cristalografia por Raios X , Ativação Enzimática , Conformação ProteicaRESUMO
This study investigated and compared vascular actions of leguminous lectins obtained from the Canavalia genus (Canavalia brasiliensis, Canavalia gladiata, and Canavalia maritima) in the rat models of paw edema and isolated aorta. Paw edema was induced by subcutaneous injection of lectins (0.01-1 mg/kg) in animals pre-treated or not with indomethacin or L-NAME. In isolated aorta, cumulative concentration curves of C. gladiata or C. brasiliensis (1-100 microg/ml) were performed at the contraction plateau induced by phenylephrine or at tissue basal tonus. The mechanism of the lectin relaxant action was investigated by previous addition of L-NAME, indomethacin, or tetraethylammonium. In both models, the lectin domain involvement was evaluated by incubation of lectins with their ligand and non-ligand sugars. The lectins induced paw edema paralleled by protein leakage. The edematogenic activity elicited by C. gladiata and C. brasiliensis involves prostaglandins and nitric oxide (NO), while that of C. maritima occurs without NO interference. C. gladiata and C. brasiliensis elicited aorta relaxation involving NO and prostacyclin, while that of C. gladiata included EDHF. All lectin effects were prevented by their binding sugars. The present study demonstrated important vasodilator effects of different degrees and mechanisms in vivo and in vitro of Canavalia lectins. In vivo, the edematogenic activity was paralleled by plasma exudation, and in vitro, aorta relaxation was strictly dependent on intact endothelium. All effects occurred via interaction with lectin domains and participation of NO and/or prostanoids.
Assuntos
Canavalia/química , Lectinas de Plantas/farmacologia , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologia , Animais , Aorta Torácica/efeitos dos fármacos , Aorta Torácica/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Edema/induzido quimicamente , Edema/fisiopatologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Masculino , Óxido Nítrico/metabolismo , Lectinas de Plantas/administração & dosagem , Lectinas de Plantas/isolamento & purificação , Prostaglandinas/metabolismo , Ratos , Ratos Wistar , Vasodilatadores/administração & dosagem , Vasodilatadores/isolamento & purificaçãoRESUMO
Plant lectins are the most studied group of carbohydrate-binding proteins. Despite the high similarity between the members of the Diocleinae subtribe (Leguminosae) group, they present differing biological activities. Canavalia boliviana lectin (Cbol) was purified using a Sephadex G-50 column and crystallized in the presence of X-Man by hanging-drop vapour diffusion at 293 K. After optimization, crystals suitable for diffraction were obtained under the condition 0.1 M HEPES pH 7.5 and 3.0 M sodium formate. The crystal belonged to the monoclinic space group C2, with unit-cell parameters a = 126.70, b = 66.64, c = 64.99 A, alpha = 90.0, beta = 120.8, gamma = 90.0 degrees . Assuming the presence of a dimer in the asymmetric unit, the solvent content was estimated to be about 46%. A complete data set was collected at 1.5 A resolution.