RESUMO
The Tn antigen is an epitope containing N-acetyl-D-galactosamine present in the extracellular matrix of some carcinoma cells in humans, and it is often used as a biomarker. Lectins are proteins capable of binding to carbohydrates and can be used as a molecular tool to recognize antigens and to differentiate cancer cells from normal cells. In this context, the present work aimed to characterize the interaction of Vatairea guianensis seed lectin with N-acetyl-D-galactosamine and the Tn antigen by molecular dynamics and molecular mechanics/Poisson-Boltzmann solvent-accessible surface area analysis. This study revealed new interacting residues not previously identified in static analysis of the three-dimensional structures of Vatairea lectins, as well as the configuration taken by the carbohydrate recognition domain, as it interacts with each ligand. During the molecular dynamics simulations, Vatairea guianensis lectin was able to bind stably to Tn antigen, which, as seen previously for other lectins, enables its use in cancer research, diagnosis, and therapy. This work further demonstrates the efficiency of bioinformatics in lectinology.
Assuntos
Fabaceae/química , Simulação de Dinâmica Molecular , Lectinas de Plantas/química , Humanos , Neoplasias , Domínios ProteicosRESUMO
Lectins represent a class of proteins or glycoproteins capable of reversibly binding to carbohydrates. Seed lectins from the Dalbergieae tribe (Leguminosae) have structural variability, carbohydrate specificity, and biological effects, such as inflammation, vasorelaxation and cancer antigen binding. To comprehensively address these factors, the present work aimed to establish and characterize the three-dimensional structure of Centrolobium microchaete lectin (CML) by homology modeling, investigate protein-carbohydrate interactions and evaluate its inflammatory effect on mice. Molecular docking was performed to analyze interactions of the lectin with monosaccharides, disaccharides and N-glycans. Two dimannosides, methyl mannose-1,3-α-D-mannose (MDM) and mannose-1,3-α-D-mannose (M13), were used in molecular dynamics (MD) simulations to study the behavior of the carbohydrate-recognition domain (CRD) over time. Results showed an expanded domain within which hydrophobic interactions with the methyl group in the MDM molecule were established, thus revealing novel interactions for mannose-specific Dalbergieae lectins. To examine its biological activities, CML was purified in a single step by affinity chromatography on Sepharose-mannose matrix. The lectin demonstrated inflammatory response in the paw edema model and stimulated leukocyte migration to the animal peritoneal cavities, an effect elicited by CRD. For the first time, this work reports the molecular dynamics of a lectin from the Dalbergieae tribe.
Assuntos
Fabaceae/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Estrutura Molecular , Lectinas de Plantas/química , Sementes/química , Animais , Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/isolamento & purificação , Anti-Inflamatórios não Esteroides/farmacologia , Sítios de Ligação , Modelos Animais de Doenças , Edema/tratamento farmacológico , Edema/etiologia , Edema/patologia , Metais/química , Camundongos , Lectinas de Plantas/isolamento & purificação , Lectinas de Plantas/farmacologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Relação Estrutura-AtividadeRESUMO
Lectins are multidomain proteins that specifically recognize various carbohydrates. The structural characterization of these molecules is crucial in understanding their function and activity in systems and organisms. Most cancer cells exhibit changes in glycosylation patterns, and lectins may be able to recognize these changes. In this work, Dioclea lasiocarpa seed lectin (DLL) was structurally characterized. The lectin presented a high degree of similarity with other lectins isolated from legumes, presenting a jelly roll motif and a metal-binding site stabilizing the carbohydrate-recognition domain. DLL demonstrated differential interactions with carbohydrates, depending on type of glycosidic linkage present in ligands. As observed by the reduction of cell viability in C6 cells, DLL showed strong antiglioma activity by mechanisms involving activation of caspase 3.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Dioclea/química , Glioma/patologia , Lectinas de Plantas/química , Lectinas de Plantas/farmacologia , Animais , Antineoplásicos/metabolismo , Metabolismo dos Carboidratos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Simulação de Acoplamento Molecular , Lectinas de Plantas/metabolismo , Conformação Proteica , Ratos , Sementes/químicaRESUMO
The Pisum arvense lectin (PAL), a legume protein belonging to the Vicieae tribe, is capable of specific recognition of mannose, glucose and its derivatives without altering its structure. In this work, the three-dimensional structure of PAL was determined by X-ray crystallography and studied in detail by a combination of molecular docking and molecular dynamics (MD). Crystals belonging to monoclinic space group P21 were grown by the vapor diffusion method at 293 K. The structure was solved at 2.16 Å and was similar to that of other Vicieae lectins. The structure presented Rfactor and Rfree of 17.04% and 22.08%, respectively, with all acceptable geometric parameters. Molecular docking was performed to analyze interactions of the lectin with monosaccharides, disaccharides and high-mannose N-glycans. PAL demonstrated different affinities on carbohydrates, depending on bond orientation and glycosidic linkage present in ligands. Furthermore, the lectin interacted with representative N-glycans in a manner consistent with the biological effects described for Vicieae lectins. Carbohydrate-recognition domain (CRD) in-depth analysis was performed by MD, describing the behavior of CRD residues in complex with ligand, stability, flexibility of the protein over time, CRD volume and topology. This is a first report of its kind for a lectin of the Vicieae tribe.
Assuntos
Fabaceae/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Lectinas de Plantas/química , Polissacarídeos/química , Cristalografia por Raios XRESUMO
The lectin from Platypodium elegans seeds (PELa) was purified by affinity chromatography in a mannose-agarose column. The lectin agglutinated rabbit erythrocytes and the agglutinating effect was inhibited by previous incubation with the glycoprotein fetuin, along with N-acetyl-d-glucosamine, D-mannose and its derivatives. The lectin maintained complete activity in temperatures ranging from 40 to 60°C and pH values ranging from 9 to 10. As a glycoprotein, PELa has a carbohydrate content of 2.2%, and its activity requires divalent cations such as Ca2+ and Mn2+. Based on SDS-PAGE, PELa displays a profile similar to that of other Dalbergieae lectins with the main chain of molecular mass around 30kDa and two subunits of 19kDa and 10 kDa each. Two-dimensional (2D) electrophoresis revealed the presence of isoforms with different isoelectric points, and high-performance size exclusion chromatography (HPSEC) was performed to confirm the purity of the sample. The lectin was immobilized in CNBr-activated Sepharose 4B and successfully captured fetuin in solution, demonstrating that this lectin remains active and capable of binding carbohydrates. PELa showed effects different from those of its recombinant form in both pro- and anti-inflammatory tests.
Assuntos
Edema/induzido quimicamente , Fabaceae/química , Proteínas Imobilizadas/farmacologia , Lectinas de Plantas/farmacologia , Proteínas Recombinantes/farmacologia , Sementes/química , Sefarose/química , Animais , Eritrócitos/efeitos dos fármacos , Eritrócitos/imunologia , Hemaglutinação/efeitos dos fármacos , Proteínas Imobilizadas/química , Masculino , Lectinas de Plantas/química , Coelhos , Ratos , Proteínas Recombinantes/químicaRESUMO
Lectins are proteins, or glycoproteins, capable of reversibly binding to specific mono- or oligosaccharides via a noncatalytic domain. The Diocleinae subtribe presents lectins with high structural similarity, but different effects based on biological activity assays. This variability results from small structural differences. Therefore, in this context, the present study aimed to perform a structural analysis of the lectin from Dioclea lasiophylla Mart. ex Benth seeds (DlyL) and evaluate its inflammatory effect. To accomplish this, DlyL was purified in a single step by affinity chromatography on Sephadex® G-50 matrix. DlyL primary structure was determined through a combination of tandem mass spectrometry and DNA sequencing. DlyL showed high similarity with other species from the same genus. Its theoretical three-dimensional structure was predicted by homology modelling, and the protein was subjected to ligand screening with monosaccharides, oligosaccharides and complex N-glycans by molecular docking. Stability and binding of the lectin with α-methyl-d-mannoside were assessed by molecular dynamics. DlyL showed acute inflammatory response with hypernociceptive effect in the paw edema model, possibly by interaction with glycans present at the cell surface.
Assuntos
Lectinas/química , Simulação de Acoplamento Molecular , Dioclea/química , Simulação de Dinâmica Molecular , Monossacarídeos/química , Oligossacarídeos/química , Espectrometria de Massas em TandemRESUMO
A glycosylated lectin (CTL) with specificity for mannose and glucose has been detected and purified from seeds of Centrolobium tomentosum, a legume plant from Dalbergieae tribe. It was isolated by mannose-sepharose affinity chromatography. The primary structure was determined by tandem mass spectrometry and consists of 245 amino acids, similar to other Dalbergieae lectins. CTL structures were solved from two crystal forms, a monoclinic and a tetragonal, diffracted at 2.25 and 1.9 Å, respectively. The carbohydrate recognition domain (CRD), metal-binding site and glycosylation site were characterized, and the structural basis for mannose/glucose-binding was elucidated. The lectin adopts the canonical dimeric organization of legume lectins. CTL showed acute inflammatory effect in paw edema model. The protein was subjected to ligand screening (dimannosides and trimannoside) by molecular docking, and interactions were compared with similar lectins possessing the same ligand specificity. This is the first crystal structure of mannose/glucose native seed lectin with proinflammatory activity isolated from the Centrolobium genus.
Assuntos
Edema/induzido quimicamente , Fabaceae/química , Lectina de Ligação a Manose , Simulação de Acoplamento Molecular , Lectinas de Plantas , Sementes/química , Sequência de Aminoácidos , Animais , Modelos Animais de Doenças , Edema/patologia , Feminino , Glicosilação , Inflamação/induzido quimicamente , Inflamação/patologia , Lectina de Ligação a Manose/química , Lectina de Ligação a Manose/toxicidade , Espectrometria de Massas , Lectinas de Plantas/química , Lectinas de Plantas/toxicidade , Pegadas de Proteínas , Ratos , Ratos Wistar , Relação Estrutura-AtividadeRESUMO
A novel lectin from seeds of Clathrotropis nitida (CNA) was purified and characterized. CNA is a glycoprotein containing approximately 3.3% carbohydrates in its structure. CNA promoted intense agglutination of rabbit erythrocytes, which was inhibited by galactosides and porcine stomach mucin (PSM). The lectin maintained its hemagglutinating activity after incubation in a wide range of temperatures (30-60 °C) and pH (6.0-7.0), and its binding activity was dependent on divalent cations (Ca(+2) and Mg(+2)). SDS-PAGE showed an electrophoretic profile consisting of a single band of 28 kDa, as confirmed by electrospray ionization mass spectrometry, which indicated an average molecular mass of 27,406 ± 2 Da and the possible presence of isoforms and glycoforms. In addition, CNA exhibited no toxicity to Artemia sp. nauplii and elicited reversible and dose-dependent vasorelaxation in precontracted aortic rings. CNA was successfully immobilized on chitosan beads and was able to capture PSM in solution. This study demonstrated that CNA is a lectin that has potential as a biotechnological tool in glycomics and glycoproteomics applications.