The lectin isolated from Lonchocarpus araripensis seed elicits endothelium-dependent vasorelaxation / A lectina isolada de sementes de Lonchocarpus araripensis promove efeito vasorelaxante dependente de endotélio

Background: The vasorelaxant effect of lectins from leguminous plants (Diocleinae subtribe) is well described. However, this effect has been little explored for lectins isolated from Dalbergieae tribe, except for that of Vatairea guianensis, that induces vasorelaxation involving nitric oxide and the lectin domain. Objective: To evaluate the vasorelaxant effect of a lectin isolated from Lonchocarpus araripensis (LAL), Dalbergieae tribe, and the involvement of the lectin domain and endothelium derived relaxing factors. Methods: Aortic rings of Wistar rats (250 - 300 g) were mounted in organ bath and mantained in physiological conditions (CEUA No. 10130208-8/40). LAL (0.1­100 µg/ml) was added to phenylephrine (0.1 µM)-contracted tissues with either endothelium intact or denuded. In order to investigate the mechanisms of LAL relaxation, inhibitors of NOS (L-NAME: 100 µM), cyclooxygenase (indomethacin: 10 µM), or potassium channels (TEA: 5 mM) were added to endothelized tissues 30 min before contraction. The involvement of lectin domain was assessed by previous incubation of LAL (30 µg/ml) with GlcNAc (0.1 M). Results: LAL (0.1-100 µg/ml) induced relaxation only in endothelized aorta, being maximal at 100 µg/ml (62.57 ± 7.8%). The relaxant effect induced by LAL at 30 µg/ml (52.49 ± 10.32%) was abolished by previous incubation with GlcNAc. LAL relaxant effect (IC50 9.75 ± 7.1) was partially reversed by indomethacin (IC50 LAL + indomethacin: 30.47 ± 10.93) and was abolished by L-NAME or TEA. Conclusion: LAL exhibits vasorelaxant activity in contracted endothelized aorta of rats, involving the lectin domain, muscarinic receptor of acetylcholine and endothelial derived relaxing factors. (AU)

Introdução: O efeito vasorrelaxante de lectinas de plantas leguminosas (Subtribo Diocleinae) já é bem descrito, embora pouco explorado para lectinas isoladas da tribo Dalbergieae, com exceção da lectina de Vatairea guianensis, que induz relaxamento com envolvimento de óxido nítrico e do domínio lectínico. Objetivo: Avaliar o efeito vasorrelaxante da lectina isolada de Lonchocarpus araripensis (LAL), tribo Dalbergieae, e o envolvimento do domínio lectínico e de fatores relaxantes derivados do endotélio (EDRF). Métodos: Anéis de aorta de ratos Wistar (250-300 g) foram montados em banho de órgãos em condições fisiológicas (Tyrode, 37 ° C, 95% de O2 e 5% de CO2, pH = 7,4) (CEUA No. 10130208-8/40). LAL (0,1-100 µg/ml) foi adicionada a tecidos pré-contraídos com fenilefrina (0,1 µM) com ou sem endotélio. Para investigar os mecanismos de relaxamento, foram adicionados inibidores de NOS (L-NAME: 100 µM), guanilato ciclase (ODQ: 10 µM), receptor muscarínico (atropina: 1 µM), ciclooxigenase (indometacina: 10 µM) ou canais de potássio (TEA: 5 mM) aos tecidos endotelizados 30 minutos antes da contração. O envolvimento do domínio lectínico foi avaliado por incubação prévia da LAL (30 µg/ml) com GlcNAc (0,1 M). Resultados: LAL (0,1-100 µg/ml) relaxou apenas anéis de aorta endotelizadas, com efeito máximo na dose de 100 µg/ml (62,57 ± 7,8%). O efeito relaxante da LAL a 30 µg/ml (52,49 ± 10,32%) foi abolido por incubação prévia com GlcNAc, atropina ou ODQ. O relaxamento da LAL (IC50 9,75 ± 7,1) a 10, 30 e 100 µg/ml foi parcialmente revertido por indometacina (IC50 LAL + indometacina: 30,47 ± 10,93) e abolido por L-NAME e TEA. Conclusão: A LAL exibe atividade vasorrelaxante em aorta endotelizada de ratos, no estado contraído, envolvendo o domínio lectínico, receptor muscarínico e fatores relaxantes derivados do endotélio. (AU)

Detection, purification and characterization of a lectin from freshwater green algae Spirogyra spp

ABSTRACT Freshwater algae are rich sources of structurally biologically active metabolites, such as fatty acids, steroids, carotenoids and polysaccharides. Among these metabolites, lectins stand out. Lectins are proteins or glycoproteins of non-immune origin which bind to carbohydrates or glycoconjugates, without changing ligand structure. Many studies have reported on the use of Spirogyra spp. as effective bioindicators of heavy metals; however, reports on Spirogyra molecular bioprospecting are quite limited. Therefore, this study aimed to detect, isolate, purify and characterize a lectin present in the freshwater green algae Spirogyra. Presence of the lectin protein in the extract was detected by hemagglutination assays. Subsequently, the protein extract was subjected to a sugar inhibition assay to identify the lectin-specific carbohydrate. Following this, the extract was applied to a guar gum column to afford the pure lectin. The lectin was inhibited by N-acetyl-glucosamine and N-acetyl-beta-D-mannose, but more strongly by D-galactose. The apparent molecular mass of the purified lectin was evaluated by Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE). Electrophoretic analysis revealed a single protein band with an apparent molecular mass of 56 kDa. Thus, it could be concluded that a lectin was purified from Spirogyra spp.

The effect of Cratylia floribunda lectin on renal hemodynamics and ion transport

Lectins have been described as glycoproteins that reversibly and specifically bind to carbohydrates. Legume lectins isolated from the subtribe Diocleinae (Canavalia, Dioclea andCratylia) are structurally homologous with respect to their primary structures. The Diocleinae lectins of Canavalia brasiliensis, Dioclea guianensis andCanavalia ensiformis have been shown to distinctly alter physiological parameters in isolated rat kidneys. Thus, the aim of this study was to investigate the effect of Cratylia floribunda lectin (CFL) on renal hemodynamics and ion transport in rats. In isolated perfused kidneys, CFL (10 mg/mL, n=5) increased RPP, RVR and decreased %TK+, but did not change urinary flow, glomerular filtration rate, sodium or chloride tubular transport. In isolated perfused mesenteric bed, CFL (3 and 10 mg/mL/min; n=4) did not alter tissue basal tonus or tissue contraction by phenylephrine (1 mM/mL/min). In conclusion, the seed lectin of Cratylia floribunda increased renal hemodynamic parameters showing a kaliuretic effect. This effect could be of tubular origin, rather than a result from haemodynamic alterations.

As lectinas são descritas como (glico)proteínas que se ligam, especificamente e reversivelmente, a carboidratos. Lectinas de leguminosas isoladas da subtribo Diocleinae (Canavalia, Dioclea eCratylia) são estruturalmente homólogas em relação às suas estruturas primárias. Demonstrou-se que as lectinas de DiocleinaeCanavalia brasiliensis, Dioclea guianensis eCanavalia ensiformis alteram diferentemente parâmetros fisiológicos em rins isolados de ratos. Dessa maneira, o objetivo deste estudo foi investigar o papel da lectina de Cratylia floribunda (CFL) na hemodinâmica renal e no transporte de íons em ratos. Em rins isolados perfundidos, CFL (10 mg/mL, n=5) aumentou a pressão de perfusão renal, a resistência vascular renal e reduziu o percentual do transporte tubular de K+, mas não alterou o fluxo urinário, a taxa de filtração glomerular e o percentual de transporte tubular dos íons sódio e cloreto. No leito mesentérico isolado perfundido, CFL (3 e 10 mg/mL/min, n=4) não alterou o tônus basal ou a contração do tecido induzida por fenilefrina (1 mM/mL/min). Em conclusão, a lectina de sementes de Cratylia floribunda altera parâmetros hemodinâmicos renais, provavelmente de origem tubular, e não por alterações hemodinâmicas.

Antifungal activity of lectins against yeast of vaginal secretion

Lectins are carbohydrate-binding proteins of non-imune origin. This group of proteins is distributed widely in nature and they have been found in viruses, microorganisms, plants and animals. Lectins of plants have been isolated and characterized according to their chemical, physical-chemical, structural and biological properties. Among their biological activities, we can stress its fungicidal action. It has been previously described the effect of the lectins Dviol, DRL, ConBr and LSL obtained from the seeds of leguminous plants on the growth of yeasts isolated from vaginal secretions. In the present work the experiments were carried out in microtiter plates and the results interpreted by both methods: visual observations and a microplate reader at 530nm. The lectin concentrations varied from 0.5 to 256µg/mL, and the inoculum was established between 65-70% of trammitance. All yeast samples isolated from vaginal secretion were evaluated taxonomically, where were observed macroscopic and microscopic characteristics to each species. The LSL lectin did not demonstrate any antifungal activity to any isolate studied. The other lectins DRL, ConBr and DvioL, showed antifungal potential against yeast isolated from vaginal secretion. These findings offering offer a promising field of investigation to develop new therapeutic strategies against vaginal yeast infections, collaborating to improve women's health.

Perfil de proteases de lesões cutâneas experimentais em camundongos tratadas com a lectina isolada das sementes de Canavalia brasiliensis / Proteases profile of skin wounds treated with lectin from Canavalia brasiliensis seeds

O objetivo deste estudo foi determinar o perfil de proteases em lesões cutâneas experimentais tratadas com a lectina isolada das sementes da Canavalia brasiliensis (ConBr) livre e conjugada com o seu açúcar específico. Lesões cirúrgicas foram produzidas assepticamente na região dorsal de camundongos (n=120), divididos de acordo com o tratamento empregado: Grupo NaCl (NaCl 150mM), Grupo manose (manose 100mM), Grupo ConBr (ConBr 100µg mL-1) e Grupo ConBr/manose (solução contendo ConBr 100µg mL-1 preparada em manose 100mM). Amostras da área lesada foram coletadas para determinação do perfil de proteases e atividade colagenolítica no 2°, no 7° e no 12° dia de pós-operatório. O perfil das proteínas realizado através de eletroforese SDS-PAGE demonstrou a presença de proteínas com massa molecular de 67kDa em todos os grupos. O Grupo ConBr/manose apresentou a maior atividade colagenolítica no 12° dia de pós-operatório. A lectina isolada das sementes da Canavalia brasiliensis influenciou a expressão de proteases com atividade colagenolítica podendo assim interferir no processo cicatricial das lesões cutâneas em camundongos.

The objective of the present study was determining the proteases profile of cutaneous healings treated with free and conjugated lectin of Canavalia brasiliensis (ConBr) and their specific sugar. An aseptic wound was produced in the thoracic area of the mice (n=120), divided according to the employed treatment: NaCl Group (150mM NaCl), manose Group (100mM manose), ConBr Group (100µg mL-1 ConBr) and ConBr/manose Group (solution containing 100µg mL-1 ConBr prepared in 100mM manose). Samples of the injured area were collected for determination of proteases profile and collagenolytic activity on 2nd, 7th e 12th days after the surgery. Electrophoresis SDS-PAGE demonstrated proteins with molecular mass of 67kDa in all groups. Group IV presented the highest collagenolytic activity on the 12th day post surgery. ConBr lectin influenced proteases expression with collagenolytic activity thus being able to intervene on skin wound healing in mice.

Purification and molecular cloning of a new galactose-specific lectin from Bauhinia variegata seeds.

A new galactose-specific lectin was purified from seeds of a Caesalpinoideae plant, Bauhinia variegata, by affinity chromatography on lactose-agarose. Protein extracts haemagglutinated rabbit and human erythrocytes (native and treated with proteolytic enzymes), showing preference for rabbit blood treated with papain and trypsin. Among various carbohydrates tested, the lectin was best inhibited by D-galactose and its derivatives, especially lactose. SDS-PAGE showed that the lectin, named BVL, has a pattern similar to other lectins isolated from the same genus, Bauhinia purpurea agglutinin (BPA). The molecular mass of BVL subunit is 32 871 Da, determined by MALDI-TOF spectrometry. DNA extracted from B.variegata young leaves and primers designed according to the B. purpurea lectin were used to generate specific fragments which were cloned and sequenced, revealing two distinct isoforms. The bvl gene sequence comprised an open reading frame of 876 base pairs which encodes a protein of 291 amino acids. The protein carried a putative signal peptide. The mature protein was predicted to have 263 amino acid residues and 28 963 Da in size.

Caracterizaçao química parcial e bioquímica de sementes de Bauhinia forficata link / Biochemical and chemical partial characterization of Bauhinia forficata link seeds

Sementes quiescentes de Bauhinia forficata foram submetidas à caracterizaçao bioquímica por medio de análise de ácidos graxos, (fraccionamiento de proteínas e atividade hemaglutinante específica. A análise elementar da semente mostrou grande quantidade de proteína total e de lipídeos com 21,24 por cento e 19.45 por cento, respectivamente. Na fraçao lipídica, o ácido linolêico foi o mais abundante com 46,47 por cento. Com exceçao das prolaminas, as diferentes françoes protéicas (albuminas, globulinas, glutelinas ácidas e básicas) apresentaram actividade hemaglutinante contra hemácias de coelho tratadas e nao tratadas com enzimas proteolíticas. A maior actividade hemaglutinante específica foi evidenciada na fraçao glutelinas ácidas (1.072,25 U.H./mgP) contra sangue de coelho tratado com tripsina. Os aminoácidos presentes em maior teor foram glutamina (16,20 por cento) e valina (11,07 por cento). Assim, por apresentarem alto valor energético as sementes de Bauhinia forficata sao uma possível fonte apcional na alimentaçao

Interaction of diocleinae lectins with glycoproteins based in surface plasmon resonance

Interaction of glucose/mannose-binding lectins in solution with immobilized glycoproteins was followed in real time using surface plasmon resonance technology. The lectins which share many biochemical and structural features could be clearly differentiated in terms of their specificity for complex glycoconjugates. The most prominent interaction of the lectins with PHA-E comparing with soybean agglutinin, both glycoproteins exhibiting high mannose oligosaccharides, suggests that the whole structure of the glycoproteins themselves, may interfere in affinity. These findings also support the hypothesis that minor amino acid replacements in the primary sequence of the lectins might be responsible for their divergence in fine specificity and biological activities. This is the first report using surface plasmon resonance technology that evidences differences of Diocleinae lectins in respect their fine glycan-specificity

In vivo lymphocyte activation and apoptosis by lectins of the Diocleinae subtribe

This paper reports the overall effects of three lectins, extracted from Canavalia brasiliensis, Dioclea violacea, and D. grandiflora, on BALB/c mice popliteal draining lymph nodes. These lectins have presented high stimulatory capacity on lymph node T cells. Additionally, they were able to induce apoptosis and inflammation (frequently associated with high endothelial venule necrosis). The data presented here suggest that the Diocleinae lectins studied can stimulate in vivo T cell activation and apoptosis, as well as present important side effects

Carbohydrate/glycan-binding specificity of legume lectins in respect to their proposed biological functions

The lectins, proteins which specifically recognize carbohydrate moieties, have been extensively studied in many biochemical and structural aspects in order to establish the molecular basis of this non-catalytic event. On the other hand, their clinical and agricultural potentials have been growing fast. Although lectins, mainly those from legume plants, had been investigated for biological properties, studies about the physiological functions of lectins are scarce in literature. Therefore, despite the accumulated data on lectins (as proteins), the role played by these signalizing molecules is poorly discussed. In the light of our accumulated results on legume lectins, specially those obtained from plants belonging to the Diocleinae sub-tribe and available data in literature, we discuss here the main hypothesis of their functions according to their carbohydrate/glycan-binding specificity.

As lectinas, proteinas que especificamente reconhecem estruturas que contém carboidratos, têm sido extensivamente estudadas em muitos aspectos bioquímicos e estruturais, objetivando estabelecer as bases moleculares deste evento não-catalítico. Por outro lado, os potenciais clínicos e agriculturais destas proteínas têm crescido rapidamente. Embora as lectinas, principalmente aquelas de legumes tenham sido bastante investigadas em suas propriedades biológicas, estudos sobre as funcões fisiológicas de lectinas são escassos na literatura. Além disto, a despeito da quantidade de dados acumulados sobre lectinas (como proteínas), o papel desempenhado por estas moléculas de sinalização é pobremente discutido. Valendo-se de nossos estudos sobre lectinas de leguminosas, principalmente da sub-tribo Diocleinae, e outros dados presentes na literatura, discutimos aqui, as principais hipóteses de suas funções com base na especificidade por carboidratos e glicanos complexos.

The carbohydrate-binding specificity and molecular modelling of Canavalia maritima and Dioclea grandiflora lectins

The carbohydrate-binding specificity of lectins from the seeds of Canavalia maritima and Dioclea grandiflora was studied by hapten-inhibition of haemagglutination using various sugars and sugar derivatives as inhibitors, including N-acetylneuraminic acid and N-acetylneuraminic acid and N-acetylmuramic acid. Despite some discrepancies, both lectins exhibited a very similiar carbohydrate-binding specificity as previously reported for other lectins from Diocleinae (tribe Phaseoleae, sub-tribe Diocleinae). Accordingly, both lectins exhibited almost identical hydropathic profiles and their three-dimensional models built up from the atomic coordinates of ConsA looked very similar. However, doking experiments of glucose and mannose in their monosaccharide-binding sites, by comparison with the ConA-mannose complex used as a model, reveled conformational changes in side chains of the animo acid residues invlved in the binding of monosaccharides. These results fully agree with crystallographic data showing that binding of specific ligands to ConsA requires conformational chances of this monosaccharide-binding site.

Plant lectins, chemical and biological aspects

Lectins, carbohydrate-binding proteins of non-immune origin, that agglutinate cells or precipitate polysaccharides and glycoconjugates, are well distributed in nature, mainly in the Plant Kingdom. The great majority of the plante lectins are present in seed cotyledons where they are found in the cytoplasm or int he protein bodies, although they have also been found in roots, stems and leaves. Due to their peculiar properties, the lectins are used as a tool both for analytical and preparative purposes in biochemistry, cellular biology, immunology and related areas. In agriculture and medicine the use of lectins greatly improved in the last few years. The lextins, with few exceptions, are glycoproteins, need divalent cations to display full activity and are, in general, oligomers with variable molecular weight. Although the studies on lectins have completed a century, their role in nature is yet ynknown . Several hypotheses on their physiological functions have been suggested. Thus, lectins could play important roles in defense against pathogens, plant-microorganism symbiosis, cell organization, embryo morphogenesis, phagocytosis, cell wall elongation, pollen recognition and as reserve proteins. A brief review on the general properties and roles of the lectins is given.