RESUMO
In this study, the amino acid sequence and anti-inflammatory effect of Bauhinia bauhinioides (BBL) lectin were evaluated. Tandem mass spectrometry revealed that BBL possesses 86 amino acid residues. BBL (1 mg/kg) intravenously injected in rats 30 min prior to inflammatory stimuli inhibited the cellular edema induced by carrageenan in only the second phase (21% - 3 h, 19% - 4 h) and did not alter the osmotic edema induced by dextran. BBL also inhibited carrageenan peritoneal neutrophil migration (51%), leukocyte rolling (58%) and adhesion (68%) and the neutrophil migration induced by TNF-α (64%). These effects were reversed by the association of BBL with galactose, demonstrating that the carbohydrate-binding domain is essential for lectin activity. In addition, BBL reduced myeloperoxidase activity (84%) and TNF-α (68%) and IL1-ß (47%) levels. In conclusion, the present investigation demonstrated that BBL contains highly homologous isolectins, resulting in a total of 86 amino acid residues, and exhibits anti-inflammatory activity by inhibiting neutrophil migration by reducing TNF-α and IL1-ß levels via the lectin domain.
Assuntos
Anti-Inflamatórios/farmacologia , Bauhinia/química , Galectinas/farmacologia , Neutrófilos/fisiologia , Extratos Vegetais/farmacologia , Lectinas de Plantas/farmacologia , Sequência de Aminoácidos , Animais , Anti-Inflamatórios/química , Adesão Celular , Citocinas/fisiologia , Avaliação Pré-Clínica de Medicamentos , Galectinas/química , Migração e Rolagem de Leucócitos , Dados de Sequência Molecular , Neutrófilos/efeitos dos fármacos , Peritonite/imunologia , Extratos Vegetais/química , Lectinas de Plantas/química , Ratos Wistar , Sementes/químicaRESUMO
Platymiscium floribundum lectin (PFL), a mannose/N-acetyl-D-glucosamine-specific lectin, was isolated from P. floribundum seeds using Sepharose-mannose affinity media chromatography. PFL is a glycoprotein that is a potent agglutinin for rabbit erythrocytes. In addition, PFL is highly stable because it is able to maintain its hemagglutinating activity after exposure to temperatures of up to 60 °C for 1 h and exposure to a wide pH range. The PFL purification process was monitored using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the results showed that the purified lectin consists of a single band with a molecular mass of approximately 29 kDa in either the presence or the absence of a reducing agent. The analysis of purified PFL by electrospray ionization-mass spectrometry showed that most ions had a molecular weight of 27,053 ± 2 Da, and other less abundant ions had similar molecular weights. Gel filtration shows that the lectin exists as a dimer in solution with mass at approximately 65 kDa. Sixteen peptides were sequenced, and as a result, a total of 130 amino acids were identified and resulted in a coverage of approximately 65% of the PFL sequence. The partial sequence of PFL was aligned with sequences of other lectins from evolutionarily related species, and PFL showed considerable similarity to the other lectins.
Assuntos
Acetilglucosamina/química , Fabaceae/química , Manose/química , Lectinas de Plantas/química , Lectinas de Plantas/isolamento & purificação , Sementes/químicaRESUMO
DwL, a lectin extracted from the seeds of Dioclea wilsonii, is a metalloprotein with strong agglutinating activity against rabbit and ABO erythrocytes, inhibited by glucose and mannose. DwL was purified by affinity chromatography on a Sephadex G-50 column and ion exchange chromatography on a HiTrap SP XL column. SDS-PAGE revealed three electrophoretic bands corresponding to the α (25,634 ± 2 Da), ß (12,873 ± 2 Da) and γ (12,779 ± 2 Da) chains. Protein sequencing was done by Tandem Mass Spectrometry. The primary sequence featured 237 amino acids and was highly homologous to other reported Diocleinae lectins. A complete X-ray dataset was collected at 2.0 Å for X-Man-complexed DWL crystals produced by the vapor diffusion method. The crystals were orthorhombic and belonged to the space group I222, with the unit-cell parameters a = 59.6, b = 67.9 and c = 109.0 Å. DWL differed in potency from other ConA-like lectins and was found to induce neutrophil migration in rats, making it particularly useful in structural/functional studies of this class of proteins.
Assuntos
Dioclea/química , Mediadores da Inflamação/química , Lectinas de Plantas/química , Sementes/química , Sequência de Aminoácidos , Animais , Movimento Celular/efeitos dos fármacos , Sequência Conservada , Cristalização , Eritrócitos/efeitos dos fármacos , Humanos , Mediadores da Inflamação/isolamento & purificação , Mediadores da Inflamação/farmacologia , Dados de Sequência Molecular , Neutrófilos/efeitos dos fármacos , Lectinas de Plantas/isolamento & purificação , Lectinas de Plantas/farmacologia , Estabilidade Proteica , Coelhos , Ratos , Ratos Wistar , Alinhamento de SequênciaRESUMO
This study aimed to evaluate the abilities of plant and algae lectins to inhibit planktonic growth and biofilm formation in bacteria and yeasts. Initially, ten lectins were tested on Staphylococcus epidermidis, Staphylococcus aureus, Klebsiella oxytoca, Pseudomonas aeruginosa, Candida albicans, and C. tropicalis at concentrations of 31.25 to 250 µ g/mL. The lectins from Cratylia floribunda (CFL), Vatairea macrocarpa (VML), Bauhinia bauhinioides (BBL), Bryothamnion seaforthii (BSL), and Hypnea musciformis (HML) showed activities against at least one microorganism. Biofilm formation in the presence of the lectins was also evaluated; after 24 h of incubation with the lectins, the biofilms were analyzed by quantifying the biomass (by crystal violet staining) and by enumerating the viable cells (colony-forming units). The lectins reduced the biofilm biomass and/or the number of viable cells to differing degrees depending on the microorganism tested, demonstrating the different characteristics of the lectins. These findings indicate that the lectins tested in this study may be natural alternative antimicrobial agents; however, further studies are required to better elucidate the functional use of these proteins.
Assuntos
Bactérias/crescimento & desenvolvimento , Biofilmes/crescimento & desenvolvimento , Eucariotos/química , Plâncton/crescimento & desenvolvimento , Plâncton/fisiologia , Lectinas de Plantas/farmacologia , Leveduras/crescimento & desenvolvimento , Bactérias/efeitos dos fármacos , Biofilmes/efeitos dos fármacos , Biomassa , Contagem de Colônia Microbiana , Eletroforese em Gel de Poliacrilamida , Viabilidade Microbiana/efeitos dos fármacos , Plâncton/efeitos dos fármacos , Lectinas de Plantas/isolamento & purificação , Leveduras/efeitos dos fármacosRESUMO
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.