Identification of bioactive peptides released from in vitro gastrointestinal digestion of yam proteins (Dioscorea cayennensis).

Bioactive peptides have been broadly studied for their contribution to human health. This study aimed to identify bioactive peptides generated by in vitro gastrointestinal digestion of yam proteins. Yam protein concentrate (YPC) was submitted to simulated digestion. Gastric phase hydrolysate (GPH) and total gastrointestinal phase hydrolysate (GIPH) had their peptides identified by nanoLC-ESI-MS/MS. Peptide sequences were subjected to a database-driven (BIOPEP) bioactivity search. In vitro tests included: Antioxidant activity, DNA damage protection, ACE-inhibitory activity and antibacterial activity against the bacteria Escherichia coli, Salmonella sp. and Lysteria monocytogenes. Simulated digestion generated small peptides (mostly MW < 3500 Da), several of them with potential bioactive sequences predicted in silico. In both GPH and GIPH biological activities were detected, although GIPH displayed stronger DNA damage protection and antibacterial activity against Escherichia coli. The digestion of yam proteins releases promising biologically active peptides which can contribute to the prevention of bacterial infection and chronic degenerative diseases, with beneficial effects to human health.

Antiulcer and Antioxidant Activity of a Lectin from Mucuna pruriens Seeds on Ethanol- induced Gastropathy: Involvement of Alpha-2 Adrenoceptors and Prostaglandins.

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.

Αlpha-2 Adrenergic and Opioids Receptors Participation in Mice Gastroprotection of Abelmoschus esculentus Lectin.

Lectins are a heterogeneous group of proteins and glycoproteins with potential role as therapeutic and diagnostic tools to combat various diseases, besides some functions on human organism. Abelmoschus esculentus (Okra), a horticultural plant of African origin, is cultivated in northeastern Brazil, and used for different medicinal purposes. This work is aimed to elucidate the action mechanisms of Abelmoschus esculentus lectin (AEL) gastro protective effect on gastropathy induced by ethanol. Fasted mice treated with Ethanol 99.9% (0.2 ml/animal, p.o.) received previously AEL (0.01, 0.1, 1.0, 10 or 50 mg/kg, i.v.), saline (5 ml/kg; i.v.) or ranitidine (80 mg/kg, p.o.) in four experimental series, in which pharmacological tools (yohimbine, naloxone, L-NAME or indomethacin), were administered with the purpose of make clear possible molecular action mechanisms. Mice were euthanized 30 min after ethanol challenge to verify the stomach damages. Establishment of gastric oxidative stress, tissue hemoglobin (Hb) content and microscopic features (H&E) were taken in order to characterize the AEL gastro protective effect. AEL (1 mg/kg) was capable of protect mucosa against ethanol damages in presence of two (L-NAME and indomethacin) of four antagonists/inhibitors used. The AEL effect was reversed by naloxone and yohimbine, showing the involvement of opioids and Αlpha-2 adrenergic receptors on gastric protective effect of this lectin. Evaluation of microscopic features, oxidative stress, and Hb levels pointed the protective effects of AEL. This activity seems to be mediated by alpha-2 adrenergic and opioid receptors activation. Nitric oxide or prostaglandins were not involved. AEL simultaneously showed antioxidant effect that is probably implicated in its intricate defensive mechanism of action.

Antibacterial and hemolytic activity of a new lectin purified from the seeds of Sterculia foetida L.

The aim of this study was to isolate, characterize, and verify possible antibacterial and hemolytic activity for a lectin found in the seeds of Sterculia foetida L. Purification of the lectin from S. foetida (SFL) was realized with ion exchange chromatography DEAE-Sephacel coupled to HPLC. The purity and the molecular weight was determined by SDS-PAGE. The isolated SFL was characterized as to its glycoprotein nature, and sugar specificity, as well as resistance to pH, temperature, denaturing agents, reduction, oxidation, and chelation. A microdilution method was used to determine antibacterial activity, and hemolytic activity was observed in human erythrocytes. The SFL has a molecular weight of 17 kDa, and a carbohydrate content of 53 µg/mL, specific for arabinose and xylose, and is resistant to treatment with urea, sensitive to treatment with sodium metaperiodate and ß-mercaptoethanol, and in the presence of EDTA lost its hemagglutinating activity (HA). However, in the presence of divalent cations (Ca(2 +) and Mn(2 +)) the HA was increased. The SFL remained active even after incubation at 80 °C, and, within pH values of between 5 and 11. The SFL inhibited the bacterial growth of all the tested strains and caused little hemolysis in human erythrocytes when compared to the positive control Triton X-100.

Marine sponge lectins: actual status on properties and biological activities.

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.

Comparative protein composition analysis of goat milk produced by the Alpine and Saanen breeds in northeastern Brazil and related antibacterial activities.

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.

Lectin of Abelmoschus esculentus (okra) promotes selective antitumor effects in human breast cancer cells.

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.

Interactions between indole-3-acetic acid (IAA) with a lectin from Canavalia maritima seeds reveal a new function for lectins in plant physiology.

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.

Molecular modeling of lectin-like protein from Acacia farnesiana reveals a possible anti-inflammatory mechanism in Carrageenan-induced inflammation.

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.

Assessment of plant lectin antifungal potential against yeasts of major importance in medical mycology.

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.

Involvement of ABCB1 and ABCC1 transporters in sea urchin Echinometra lucunter fertilization.

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.

Purification and biological activities of Abelmoschus esculentus seed lectin.

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.

Antinociceptive and anti-inflammatory effects of a lectin-like substance from Clitoria fairchildiana R. Howard seeds.

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.

Structure of Dioclea virgata lectin: Relations between carbohydrate binding site and nitric oxide production.

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.

Mass spectrometry and X-ray diffraction analysis of two crystal types of Dioclea virgata lectin: an antinociceptive protein candidate to structure/function analysis.

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.

Central action of Araucaria angustifolia seed lectin in mice.

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.

Structure of a lectin from Canavalia gladiata seeds: new structural insights for old molecules.

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.

Purification and biological effects of Araucaria angustifolia (Araucariaceae) seed lectin.

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.

Modulation of acute inflammation by a chitin-binding lectin from Araucaria angustifolia seeds via mast cells.

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.

Crystal structure of a lectin from Canavalia maritima (ConM) in complex with trehalose and maltose reveals relevant mutation in ConA-like lectins.

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.