RESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Mucuna pruriens (Mp) belongs to Leguminosae family, it is native of tropical regions and used to treat several maladies such as urinary, neurological, and menstruation disorders, constipation, edema, fever, tuberculosis, ulcers, diabetes, arthritis, dysentery, and cardiovascular diseases. Mp seeds are rich in bioactive compounds, for instance, lectins, a heterogeneous group of proteins and glycoproteins with a potential role as therapeutic tools for several conditions, including gastric disorders. This study investigated the acute toxicity, gastroprotective, and antioxidant activities of a lectin from Mucuna pruriens seeds (MpLec) on ethanol-induced gastropathy model in mice. MATERIAL AND METHODS: Mice received MpLec (5 or 10 mg/kg; i.v.) and were observed for acute toxicity signs; in another experimental series, mice were pre-treated with MpLec (0.001; 0.01 or 0.1 mg/kg, i.v.), ranitidine (80 mg/kg, p.o.), or saline (0.3 mL/30g, i.v.) before ethanol 99.9% (0.2 mL/animal, p.o.), and euthanized 30 min after ethanol challenge. Macroscopic and microscopic gastric aspects, biochemical parameters (tissue hemoglobin levels, iron-induced lipid peroxidation, GSH content, SOD activity, and gastric mucosal PGE2) were measured. Additionally, pharmacological tools (yohimbine, indomethacin, naloxone, L-NAME) were opportunely used to clarify MpLec gastroprotective mechanisms of action. RESULTS: No toxicity signs nor death were observed at acute toxicity tests. MpLec reduced ethanol-induced gastric damage, edema, and hemorrhagic patches formation, as well as decreased lipid peroxidation, SOD activity, and increased GSH content. Yohimbine and indomethacin prevented MpLec effects, suggesting the involvement of alpha-2 adrenoceptors and prostaglandins in the MpLec-mediated effects. CONCLUSION: MpLec does not present toxicity signs and shows gastroprotective and antioxidant activities via alpha-2 adrenoceptors and prostaglandins in the ethanol-induced gastropathy model.
Assuntos
Antioxidantes/farmacologia , Mucosa Gástrica/efeitos dos fármacos , Lectinas/farmacologia , Mucuna/química , Prostaglandinas/metabolismo , Receptores Adrenérgicos/metabolismo , Úlcera Gástrica/terapia , Animais , Etanol/efeitos adversos , Peroxidação de Lipídeos , Camundongos , Fitoterapia , Extratos Vegetais/uso terapêutico , Sementes/química , Úlcera Gástrica/induzido quimicamente , Testes de Toxicidade AgudaRESUMO
Marine sponges are primitive metazoans that produce a wide variety of molecules that protect them against predators. In studies that search for bioactive molecules, these marine invertebrates stand out as promising sources of new biologically-active molecules, many of which are still unknown or little studied; thus being an unexplored biotechnological resource of high added value. Among these molecules, lectins are proteins that reversibly bind to carbohydrates without modifying them. In this review, various structural features and biological activities of lectins derived from marine sponges so far described in the scientific literature are discussed. From the results found in the literature, it could be concluded that lectins derived from marine sponges are structurally diverse proteins with great potential for application in the production of biopharmaceuticals, especially as antibacterial and antitumor agents.
Assuntos
Lectinas/química , Lectinas/farmacologia , Biologia Marinha , Poríferos/química , Animais , BiotecnologiaRESUMO
The protein composition of goat milk differs between goat breeds and could present regional trends. The aim of this study was to comparatively analyze the protein composition of goat milk produced by the Alpine and Saanen breeds in northeastern Brazil and to evaluate the antibacterial activity of its protein fractions. SDS-PAGE, 2-DE electrophoresis and RP-HPLC analyses revealed the absence of αs1-casein in the milk of both breeds and no differences between the αs2-casein, ß-casein, ß-lactoglobulin and α-lactalbumin profiles. The amounts of soluble proteins and ß-casein hydrolysis residues were higher in Saanen milk. Only the protein fraction containing the largest amounts of casein (F60-90%) inhibited bacterial growth, with MIC values between 50 and 100 mg/mL. This study describe for the first time three important points about the goat milk protein of two Brazilian goat breeders: absence of α-s1 casein in the protein profile, differences between the milk protein composition produced by goats of Alpine and Saanen breeders and antibacterial activity of unbroken proteins (casein-rich fraction) present in these milk.
Assuntos
Antibacterianos/farmacologia , Caseínas/farmacologia , Lactalbumina/farmacologia , Leite/química , Animais , Antibacterianos/química , Antibacterianos/isolamento & purificação , Bacillus subtilis/efeitos dos fármacos , Bacillus subtilis/crescimento & desenvolvimento , Brasil , Cruzamento , Caseínas/química , Caseínas/isolamento & purificação , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Feminino , Cabras , Hidrólise , Lactalbumina/química , Lactalbumina/isolamento & purificação , Masculino , Testes de Sensibilidade Microbiana , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/crescimento & desenvolvimento , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimentoRESUMO
The anti-tumor effects of a newly-discovered lectin, isolated from okra, Abelmoschus esculentus (AEL), were investigated in human breast cancer (MCF7) and skin fibroblast (CCD-1059 sk) cells. AEL induced significant cell growth inhibition (63 %) in MCF7 cells. The expression of pro-apoptotic caspase-3, caspase-9, and p21 genes was increased in MCF7 cells treated with AEL, compared to those treated with controls. In addition, AEL treatment increased the Bax/Bcl-2 ratio in MCF7 cells. Flow cytometry also indicated that cell death (72 %) predominantly occurred through apoptosis. Thus, AEL in its native form promotes selective antitumor effects in human breast cancer cells and may represent a potential therapeutic to combat human breast cancer.
Assuntos
Abelmoschus/química , Antineoplásicos/farmacologia , Apoptose , Células Epiteliais/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Lectinas/farmacologia , Antineoplásicos/isolamento & purificação , Caspases/análise , Linhagem Celular Tumoral , Células Epiteliais/fisiologia , Fibroblastos/fisiologia , Humanos , Lectinas/isolamento & purificaçãoRESUMO
Indole-3-acetic acid (IAA) bound is considered a storage molecule and is inactive. However, some studies have proposed an additional possible regulatory mechanism based on the ability of lectins to form complexes with IAA. We report the first crystal structure of ConM in complex with IAA at 2.15 Å resolution. Based on a tetrameric model of the complex, we hypothesize how the lectin controls the availability of IAA during the early seedling stages, indicating a possible new physiological role for these proteins. A free indole group is also bound to the protein. The ConM interaction with different forms of IAA is a strategy to render the phytohormone unavailable to the cell. Thus, this new physiological role proposed for legume lectins might be a novel mechanism by which IAA levels are decreased in addition to the destruction and formation of new complexes in the later stages of seed germination.
Assuntos
Canavalia/fisiologia , Ácidos Indolacéticos/metabolismo , Lectinas de Plantas/metabolismo , Sementes/metabolismo , Animais , Canavalia/metabolismo , Hemaglutinação/efeitos dos fármacos , Simulação de Acoplamento Molecular , Lectinas de Plantas/química , Lectinas de Plantas/farmacologia , Ligação Proteica , Conformação Proteica , CoelhosRESUMO
The search for new compounds with antifungal activity is accelerating due to rising yeast and fungal resistance to commonly prescribed drugs. Among the molecules being investigated, plant lectins can be highlighted. The present work shows the potential of six plant lectins which were tested in vitro against yeasts of medical importance, Candida albicans, Candida tropicalis, Candida parapsilosis, Cryptococcus gattii, Cryptococcus neoformans, Malassezia pachydermatis, Rhodotorula sp. and Trichosporon sp. Broth microdilution susceptibility testing was performed in accordance with standard protocols to evaluate antifungal activity. Minimum inhibitory concentration (MIC) was determined at 80% yeast growth inhibition, whereas the minimum fungicidal concentration (MFC) was evaluated after making the subcultures of each dilution. Only C. parapsilosis growth was inhibited by the lectins tested. Abelmoschus esculentus lectin showed the highest MIC (0.97 µg ml(-1)). Lectins from Canavalia brasiliensis, Mucuna pruriens and Clitoria fairchildiana presented the highest MFC at (3.90 µg ml(-1)). These results encourage further studies with wider yeast strain selections, and open new perspectives for the development of pharmacological molecules.
Assuntos
Antifúngicos/farmacologia , Fungos/efeitos dos fármacos , Lectinas/farmacologia , Plantas/química , Antifúngicos/isolamento & purificação , Lectinas/isolamento & purificação , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacosRESUMO
Acacia farnesiana lectin-like protein (AFAL) is a chitin-binding protein and has been classified as phytohaemagglutinin from Phaseolus vulgaris (PHA). Legume lectins are examples for structural studies, and this family of proteins shows a remarkable conservation in primary, secondary, and tertiary structures. Lectins have ability to reduce the effects of inflammation caused by phlogistic agents, such as carrageenan (CGN). This paper explains the anti-inflammatory activity of AFAL through structural comparison with anti-inflammatory legume lectins. The AFAL model was obtained by molecular modeling and molecular docking with glycan and carrageenan were performed to explain the AFAL structural behavior and biological activity. Pisum sativum lectin was the best template for molecular modeling. The AFAL structure model is folded as a ß sandwich. The model differs from template in loop regions, number of ß strands and carbohydrate-binding site. Carrageenan and glycan bind to different sites on AFAL. The ability of AFAL binding to carrageenan can be explained by absence of the sixth ß -strand (posterior ß sheets) and two ß strands in frontal region. AFAL can inhibit pathway inflammatory process by carrageenan injection by connecting to it and preventing its entry into the cell and triggers the reaction.
Assuntos
Anti-Inflamatórios/química , Inflamação/tratamento farmacológico , Modelos Moleculares , Lectinas de Plantas/química , Acacia , Animais , Anti-Inflamatórios/metabolismo , Carragenina/toxicidade , Quitina/química , Cristalografia por Raios X , Inflamação/induzido quimicamente , Inflamação/patologia , Camundongos , Simulação de Acoplamento Molecular , Fito-Hemaglutininas/química , Fito-Hemaglutininas/metabolismo , Lectinas de Plantas/administração & dosagem , Lectinas de Plantas/isolamento & purificação , Lectinas de Plantas/metabolismo , Ligação ProteicaRESUMO
Fertilization is an ordered sequence of cellular interactions that promotes gamete fusion to form a new individual. Since the pioneering work of Oskar Hertwig conducted on sea urchins, echinoderms have contributed to the understanding of cellular and molecular aspects of the fertilization processes. Studies on sea urchin spermatozoa reported the involvement of a plasma membrane protein that belongs to the ABC proteins superfamily in the acrosome reaction. ABC transporters are expressed in membranes of eukaryotic and prokaryotic cells, and are associated with the transport of several compounds or ions across biomembranes. We aimed to investigate ABCB1 and ABCC1 transporter activity in sea urchin spermatozoa and their involvement in fertilization. Our results indicate that Echinometra lucunter spermatozoa exhibit a low intracellular calcein accumulation (18.5% stained cells); however, the ABC blockers reversin205, verapamil, and MK571 increased dye accumulation (93.0-96.6% stained cells). We also demonstrated that pharmacologically blocking ABCB1 and ABCC1 decreased spermatozoa fertilizing capacity (70% inhibition), and this phenotype was independent of extracellular calcium. These data suggest that functional spermatozoa ABCB1 and ABCC1 transporters are crucial for a successful fertilization. Additional studies must be performed to investigate the involvement of membrane lipid homeostasis in the fertilization process.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Fertilização/efeitos dos fármacos , Proteínas de Membrana Transportadoras/metabolismo , Transportadores de Ânions Orgânicos/metabolismo , Ouriços-do-Mar/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Acrossomo/metabolismo , Reação Acrossômica , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Fluoresceínas/metabolismo , Antagonistas de Leucotrienos/farmacologia , Masculino , Proteínas Associadas à Resistência a Múltiplos Medicamentos/antagonistas & inibidores , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Oligopeptídeos/farmacologia , Transportadores de Ânions Orgânicos/antagonistas & inibidores , Propionatos/farmacologia , Quinolinas/farmacologia , Ouriços-do-Mar/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Espermatozoides/metabolismo , Verapamil/farmacologiaRESUMO
The Abelmoschus esculentus (Malvaceae) plant originated in Africa and has spread across a number of tropic countries, including northeastern Brazil. The plant has been used to treat various disorders, such as cancer, microbial infections, hypoglycemia, constipation, urine retention and inflammation. The lectin of A. esculentus (AEL) was isolated by precipitation with ammonium sulfate at a saturation level of 30/60 and purified by ion exchange chromatography (Sephacel-DEAE). The electrophoresis (SDS-PAGE) profile of the AEL showed two protein bands of apparent molecular mass of approximately 15.0 and 21.0 kDa. The homogenity of the protein was confirmed by electrospray mass spectrometry (ESI-MS), which revealed the presence of a 10.29-kDa monomer and a 20.58-kDa dimer. The AEL exhibits agglutinating activity against rabbit (74.41 UH/mP) and human type ABO erythrocytes (21.00 UH/mP). This activity does not require the presence of divalent cations and is specifically inhibited by lactose, fructose and mannose. The intravenous treatment with 0.01, 0.1 and 1 mg/kg of AEL inhibited the paw edema elicited by carrageenan by approximately 15, 22 and 44 %, respectively, but not that induced by dextran. In addition, treatment with 0.1, 1 and 10 mg/kg of AEL also inhibited the abdominal writhing induced by acetic acid by approximately 52, 57 and 69 %, respectively. In conclusion, AEL is a new lectin with a molecular mass of 20.0 kDa, which is -composed of a 10.291-Da monomer and a 20.582-kDa dimer, that exhibits anti-inflammatory, antinociceptive and hemagglutinating activities. In addition, the lectin hemagglutinating property is both metallo-independent and associated with the lectin domain.
Assuntos
Abelmoschus/química , Analgésicos/isolamento & purificação , Analgésicos/farmacologia , Anti-Inflamatórios/isolamento & purificação , Anti-Inflamatórios/farmacologia , Lectinas de Plantas/isolamento & purificação , Lectinas de Plantas/farmacologia , Ácido Acético , Analgésicos/química , Análise de Variância , Animais , Anti-Inflamatórios/química , Metabolismo dos Carboidratos , Carragenina/efeitos adversos , Edema/induzido quimicamente , Agregação Eritrocítica/efeitos dos fármacos , Feminino , Testes de Hemaglutinação , Humanos , Inflamação/induzido quimicamente , Masculino , Camundongos , Medição da Dor/efeitos dos fármacos , Lectinas de Plantas/química , Coelhos , Ratos , Ratos Wistar , Sementes/químicaRESUMO
Lectins are proteins that have the ability to bind specifically and reversibly to carbohydrates and glycoconjugates, without altering the structure of the glycosyl ligand. They are found in organisms such as viruses, plants and humans, and they have been shown to possess important biological activities. The objective of this study was to purify and characterize lectins in the seeds of Clitoria fairchildiana, as well as to verify their biological activities. The results indicated the presence of a lectin (CFAL) in the glutelin acid protein fraction, which agglutinated native rabbit erythrocytes. CFAL was purified by column chromatography ion-exchange, DEAE-Sephacel, which was obtained from a peak of protein retained in the matrix by applying 0.5 M NaCl using the step-wise method. Electrophoretic analysis of this lectin in SDS-PAGE indicated a two band pattern protein molecular mass of approximately 100 and 116 kDa. CFAL proved to be unspecific to all carbohydrates/glycoconjugates in common use for the sugar inhibition test. This lectin showed no significant cytotoxicity to human red blood cells. It was observed that CFAL has anti-inflammatory activity in the paw edema induced by carrageenan model, in which a 64% diminution in edema was observed. Antinociceptive effects were observed for CFAL in the abdominal writhing test (induced by acetic acid), in which increasing doses of the lectin caused reduction in the number of contortions by up to 72%. It was concluded that the purified and characterized lectin from the seeds of Clitoria fairchildiana has anti-inflammatory and antinociceptive activity, and is not cytotoxic to human erythrocytes.
Assuntos
Analgésicos/farmacologia , Anti-Inflamatórios/farmacologia , Clitoria/química , Extratos Vegetais/farmacologia , Lectinas de Plantas/farmacologia , Sementes/química , Analgésicos/isolamento & purificação , Animais , Anti-Inflamatórios/isolamento & purificação , Carragenina , Eritrócitos/efeitos dos fármacos , Hemaglutinação , Humanos , Inflamação/induzido quimicamente , Inflamação/tratamento farmacológico , Camundongos , Nociceptividade/efeitos dos fármacos , Extratos Vegetais/isolamento & purificação , Lectinas de Plantas/isolamento & purificação , Coelhos , Ratos WistarRESUMO
The lectin of Dioclea virgata (DvirL), both native and complexed with X-man, was submitted to X-ray diffraction analysis and the crystal structure was compared to that of other Diocleinae lectins in order to better understand differences in biological properties, especially with regard to the ability of lectins to induce nitric oxide (NO) production. An association was observed between the volume of the carbohydrate recognition domain (CRD), the ability to induce NO production and the relative positions of Tyr12, Arg228 and Leu99. Thus, differences in biological activity induced by Diocleinae lectins are related to the configuration of amino acid residues in the carbohydrate binding site and to the structural conformation of subsequent regions capable of influencing site-ligand interactions. In conclusion, the ability of Diocleinae lectins to induce NO production depends on CRD configuration.
Assuntos
Carboidratos/química , Dioclea/química , Óxido Nítrico/metabolismo , Lectinas de Plantas/química , Lectinas de Plantas/metabolismo , Animais , Aorta/efeitos dos fármacos , Sítios de Ligação , Masculino , Lectinas de Plantas/farmacologia , Ligação Proteica , RatosRESUMO
The lectin from seeds of Dioclea virgata (DvirL) was purified in a single step affinity chromatography, sequenced by tandem mass spectrometry and submitted to crystallization and biological experiments. DvirL has a molecular mass of 25,412 ± 2 Da and the chains ß and γ has 12,817 Da ± 2 and 12,612 Da ± 2, respectively. Primary sequence determination was assigned by tandem mass spectrometry and revealed a protein with 237 amino acids and 87% of identify with ConA. The protein crystals were obtained native and complexed with X-Man using vapor-diffusion method at a constant temperature of 293 K. A complete X-ray dataset was collected at 1.8 Å resolution. DvirL crystals were found to be orthorhombic, belonging to the space group I222, with a unit cell parameters a = 647.5 Å, b = 86.6 Å, c = 90.2 Å. Molecular replacement search found a solution with a correlation coefficient of 77.1% and an R(factor) of 44.6%. The present study also demonstrated that D. virgata lectin presents edematogenic and antinociceptive activities in rodents electing this protein as a candidate to structure/function analysis.
Assuntos
Analgésicos/química , Dioclea/química , Lectinas de Plantas/química , Sequência de Aminoácidos , Analgésicos/isolamento & purificação , Analgésicos/farmacologia , Animais , Cristalização , Edema/tratamento farmacológico , Humanos , Masculino , Espectrometria de Massas , Camundongos , Dados de Sequência Molecular , Mapeamento de Peptídeos , Lectinas de Plantas/isolamento & purificação , Lectinas de Plantas/farmacologia , Sementes/química , Alinhamento de Sequência , Difração de Raios XRESUMO
Possible central nervous system effects of the gymnosperm lectin from Araucaria angustifolia seeds were studied in seizure and open field tests. Male Swiss mice were administered saline (control), lectin (0.1, 1, and 10 mg/kg), flumazenil (1 mg/kg), or diazepam (1 mg/kg) intraperitoneally. Lectin at the highest dose increased time to death in the pentylenetetrazole- and strychnine-induced seizure models as compared with control, but not in the pilocarpine model. In the open field test, lectin reduced locomotor activity at all doses tested, as did diazepam, when compared with control. These locomotor effects were reversed by flumazenil pretreatment. In conclusion, A. angustifolia lectin had a protective effect in the pentylenetetrazole- and strychnine-induced seizure models, suggestive of activity in the GABAergic and glycinergic systems, respectively, and also caused a reduction in animal movements, which was reversed by flumazenil, pointing to a depressant action mediated by a GABAergic mechanism.
Assuntos
Lectinas/farmacologia , Lectinas/uso terapêutico , Sementes/química , Convulsões/tratamento farmacológico , Análise de Variância , Animais , Anticonvulsivantes/farmacologia , Anticonvulsivantes/uso terapêutico , Diazepam/farmacologia , Diazepam/uso terapêutico , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Comportamento Exploratório/efeitos dos fármacos , Flumazenil/farmacologia , Flumazenil/uso terapêutico , Locomoção/efeitos dos fármacos , Masculino , Camundongos , Pentilenotetrazol , Fitoterapia/métodos , Extratos Vegetais/uso terapêutico , Tempo de Reação/efeitos dos fármacos , Convulsões/induzido quimicamente , EstricninaRESUMO
Talinum fruticosum Juss (L.) is an unconventional plant belonging to the family Portulacaceae; it is a herbaceous perennial plant that grows in tropical regions as a foliaceous vegetable. T. fruticosum leaves area source of fiber and minerals and they contribute to assemble the micronutrients for daily nutritional requirements. These plants supply the essential nutrients to the human organism; however, these plants contain antinutritinal factors such as lectins and tannins which may compromise their bioavailability. In the present study, the T. fruticosum leaves samples were investigated on proximate composition and antinutritional factors. Samples were exposed to heat treatment for washing and for cooking for 10 min. These procedures neither significantly changed the carbohydrates and lipids contents, nor eliminated the hemagglutinating activity which is inactivated at 70 C, although no effective reduction of tannin from the leaves was observed.
Talinum fruticosum Juss (L.) é uma planta não convencional da família Portulacaceae e trata-se de planta herbácea perene que cresce em regiões tropicais como vegetais folhosos. As plantas fornecem nutrientes essenciais ao organismo, no entanto, a biodisponibilidade pode ser comprometida pela presença de fatores antinutricionais tais como lectinas e taninos. Em virtude das folhas de T. fruticosum serem fonte de fibra e de minerais e por contribuem para reunir os micronutrientes necessários para a nutrição diária, no presente estudo as amostras de folhas dessa planta foram investigadas quanto à composição centesimal, bem como os fatores antinutricionais. As amostras foram submetidas ao tratamento térmico nos procedimentos de lavagem e de cozimento durante 10 min. Estes processos não foram capazes de alterar significativamente os teores de carboidratos e lipídeos, tampouco a supressão da atividade hemaglutinante que é inativada a 70C. Contudo não houve eficácia na redução dos teores de tanino presentes nas folhas estudadas.
RESUMO
BACKGROUND: Lectins are mainly described as simple carbohydrate-binding proteins. Previous studies have tried to identify other binding sites, which possible recognize plant hormones, secondary metabolites, and isolated amino acid residues. We report the crystal structure of a lectin isolated from Canavalia gladiata seeds (CGL), describing a new binding pocket, which may be related to pathogen resistance activity in ConA-like lectins; a site where a non-protein amino-acid, alpha-aminobutyric acid (Abu), is bound. RESULTS: The overall structure of native CGL and complexed with alpha-methyl-mannoside and Abu have been refined at 2.3 A and 2.31 A resolution, respectively. Analysis of the electron density maps of the CGL structure shows clearly the presence of Abu, which was confirmed by mass spectrometry. CONCLUSION: The presence of Abu in a plant lectin structure strongly indicates the ability of lectins on carrying secondary metabolites. Comparison of the amino acids composing the site with other legume lectins revealed that this site is conserved, providing an evidence of the biological relevance of this site. This new action of lectins strengthens their role in defense mechanisms in plants.
Assuntos
Canavalia/química , Lectinas de Plantas/química , Sementes/química , Aminobutiratos/química , Aminobutiratos/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Lectinas de Plantas/metabolismo , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
This paper describes the purification and characterization of a new N-acetyl-d-glucosamine-specific lectin from Araucaria angustifolia (AaL) seeds (Araucariaceae) and its anti-inflammatory and antibacterial activities. AaL was purified using a combination of affinity chromatography on a chitin column and ion exchange chromatography on Sephacel-DEAE. The pure protein has 8.0kDa (SDS-PAGE) and specifically agglutinates rabbit erythrocytes, effect that was independent of the presence of divalent cations and was inhibited after incubation with glucose and N-acetyl-d-glucosamine. AaL showed antibacterial activity against Gram-negative and Gram-positive strains, shown by scanning electron microscopy. AaL, intravenously injected into rats, showed anti-inflammatory effect, via carbohydrate site interaction, in the models of paw edema and peritonitis. This lectin can be used as a tool for studying bacterial infections and inflammatory processes.
Assuntos
Bactérias/citologia , Bactérias/efeitos dos fármacos , Cycadopsida/metabolismo , Inflamação/tratamento farmacológico , Lectinas de Plantas/administração & dosagem , Sementes/química , Animais , Relação Dose-Resposta a Droga , Extratos Vegetais/administração & dosagem , Extratos Vegetais/isolamento & purificação , Lectinas de Plantas/isolamento & purificação , RatosRESUMO
The effects of a lectin (AaL) from seeds of Araucaria angustifolia were investigated in the model of rat paw edema. In vivo anti-and pro-inflammatory activities, role of sugar residues, inflammatory mediators and systemic toxicity were assessed. Intravenous injection of AaL (0.1-1 mg/kg) dose-dependently inhibited the dextran-induced increase in edema and vascular permeability, which were prevented by association of the lectin with its binding sugar N-acetyl-glucosamine (Glyc-Nac). AaL also significantly inhibited edema induced by serotonin (18%) and compound 48/80 (33%), but not edema induced by histamine. In contrast, when applied by the s.c. route, AaL evoked a paw edema that peaked 1 h later and was partially prevented by association with Glyc-Nac (59%) or by prior i.v. administration of the lectin itself (38.8%). This AaL edematogenic activity was significantly inhibited by pentoxifylline (44.4%) or dexamethasone (51%) and also by depletion of rat paw mast cells (45.6%), but not by L-N-nitro-arginine methyl ester or indomethacin, excluding involvement of nitric oxide and prostaglandins. Treatment of animals with a single anti-inflammatory dose of AaL (1 mg/kg, i.v.) for 7 days did not affect rat corporal mass, liver, kidney, spleen or stomach wet weight, blood leukocyte count, and urea, creatinine or serum transaminase activity. Systemic toxicity was apparent only at much higher doses (LD50=88.3 mg/kg) than those required for the anti-inflammatory effect. Summarizing, AaL exerts anti-and pro-edematogenic actions via interaction with its specific lectin domain. These actions may share a common pathway involving either activation or inhibition of inflammatory mediators from resident mast cells.
Assuntos
Anti-Inflamatórios/farmacologia , Quitina/metabolismo , Mastócitos/fisiologia , Lectinas de Plantas/farmacologia , Sementes/química , Traqueófitas/química , Doença Aguda , Animais , Permeabilidade Capilar/efeitos dos fármacos , Relação Dose-Resposta a Droga , Edema/prevenção & controle , Histamina/farmacologia , Masculino , Pentoxifilina/farmacologia , Ratos , Ratos Wistar , Serotonina/farmacologia , p-Metoxi-N-metilfenetilamina/farmacologiaRESUMO
The crystal structure of Canavalia maritima lectin (ConM) complexed with trehalose and maltose revealed relevant point mutations in ConA-like lectins. ConM with the disaccharides and other ConA-like lectins complexed with carbohydrates demonstrated significant differences in the position of H-bonds. The main difference in the ConM structure is the replacement of Pro202 by Ser202, a residue that promotes the approximation of Tyr12 to the carbohydrate-binding site. The O-6' of the second glucose ring in maltose interacts with Tyr12, while in trehalose the interaction is established by the O-2' and Tyr12, explaining the higher affinity of ConM for disaccharides compared to monosaccharides.
Assuntos
Canavalia/metabolismo , Concanavalina A/química , Cristalografia por Raios X/métodos , Lectinas/química , Maltose/química , Trealose/química , Sítios de Ligação , Carboidratos/química , Elétrons , Ligação de Hidrogênio , Conformação Molecular , Mutação , Conformação Proteica , SoftwareRESUMO
Here, we report the crystallographic study of a lectin from Canavalia maritima seeds (ConM) and its relaxant activity on vascular smooth muscle, to provide new insights into the understanding of structure/function relationships of this class of proteins. ConM was crystallized and its structure determined by standard molecular replacement techniques. The amino acid residues, previously suggested incorrectly by manual sequencing, have now been determined as I17, I53, S129, S134, G144, S164, P165, S187, V190, S169, T196, and S202. Analysis of the structure indicated a dimer in the asymmetric unit, two metal binding sites per monomer, and loops involved in the molecular oligomerization. These confer 98% similarity between ConM and other previously described lectins, derived from Canavalia ensiformis and Canavalia brasiliensis. Our functional data indicate that ConM exerts a concentration-dependent relaxant action on isolated aortic rings that probably occurs via an interaction with a specific lectin-binding site on the endothelium, resulting in a release of nitric oxide.
Assuntos
Canavalia/química , Óxido Nítrico/metabolismo , Lectinas de Plantas/química , Sementes/química , Sequência de Aminoácidos , Animais , Aorta Torácica/efeitos dos fármacos , Aorta Torácica/fisiologia , Sítios de Ligação , Canavalia/genética , Concanavalina A/genética , Concanavalina A/farmacologia , Cristalografia por Raios X , Endotélio Vascular/fisiologia , Inibidores Enzimáticos/farmacologia , Técnicas In Vitro , Masculino , Modelos Moleculares , Dados de Sequência Molecular , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/metabolismo , Fenilefrina/farmacologia , Lectinas de Plantas/genética , Lectinas de Plantas/farmacologia , Conformação Proteica , Estrutura Quaternária de Proteína , Ratos , Ratos Wistar , Homologia de Sequência de Aminoácidos , Eletricidade Estática , Vasodilatação/efeitos dos fármacosRESUMO
A lectin from Canavalia maritima seeds (ConM) was purified and submitted to crystallization experiments. The best crystals were obtained using the vapour-diffusion method at a constant temperature of 293 K and grew in 7 d. A complete structural data set was collected to 2.1 A resolution using a synchrotron-radiation source. The ConM crystal belongs to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 67.15, b = 70.90, c = 97.37 A. A molecular-replacement search found a solution with a correlation coefficient of 69.2% and an R factor of 42.5%. Crystallographic refinement is under way.