Glucose-Binding Dioclea bicolor Lectin (DBL): Purification, Characterization, Structural Analysis, and Antibacterial Properties.

In this study, we purified a lectin isolated from the seeds of Dioclea bicolor (DBL) via affinity purification. Electrophoresis analysis revealed that DBL had three bands, α, ß, and γ chains, with molecular masses of approximately 29, 14, and 12 kDa, respectively. Gel filtration chromatography revealed that the native form of DBL had a molecular mass of approximately 100 kDa, indicating that it is a tetramer. Interestingly, DBL-induced hemagglutination was inhibited by several glucosides, mannosides, ampicillin, and tetracycline with minimum inhibitory concentration (MIC) values of 1.56-50 mM. Analysis of the complete amino acid sequence of DBL revealed the presence of 237 amino acids with high similarity to other Diocleinae lectins. Circular dichroism showed the prominent ß-sheet secondary structure of DBL. Furthermore, DBL structure prediction revealed a Discrete Optimized Protein Energy (DOPE) score of -26,642.69141/Normalized DOPE score of -1.84041. The DBL monomer was found to consist a ß-sandwich based on its 3D structure. Molecular docking showed the interactions between DBL and α-D-glucose, N-acetyl-D-glucosamine, α-D-mannose, α-methyl-D-mannoside, ampicillin, and tetracycline. In addition, DBL showed antimicrobial activity with an MIC of 125 µg/mL and exerted synergistic effects in combination with ampicillin and tetracycline (fractional inhibitory concentration index ≤ 0.5). Additionally, DBL significantly inhibited biofilm formation and showed no toxicity in murine fibroblasts (p < 0.05). These results suggest that DBL exhibits antimicrobial activity and works synergistically with antibiotics.

Dioclea violacea lectin inhibits tumorigenesis and tumor angiogenesis in vivo.

Dioclea violacea seed mannose-binding lectin (DvL) has attracted considerable attention because of its interesting biological activities, including antitumor, antioxidant, and anti-inflammatory activities. This study evaluated the cytotoxic effect of DvL on tumor and normal cells using the mitochondrial activity reduction (MTT) assay, the carcinogenic and anti-carcinogenic activity by the epithelial tumor test (ETT) in Drosophila melanogaster, and the anti-angiogenic effect by the chick embryo chorioallantoic membrane (CAM) assay. Data demonstrated that DvL promoted strong selective cytotoxicity against tumor cell lines, especially A549 and S180 cells, whereas normal cell lines were weakly affected. Furthermore, DvL did not promote carcinogenesis in D. melanogaster at any concentration tested, but modulated DXR-induced carcinogenesis at the highest concentrations tested. In the CAM and immunohistochemical assays, DvL inhibited sarcoma 180-induced angiogenesis and promoted the reduction of VEGF and TGF-ß levels at all concentrations tested. Therefore, our results demonstrated that DvL is a potent anticancer, anti-angiogenic, and selective cytotoxic agent for tumor cells, suggesting its potential application as a prototype molecule for the development of new drugs with chemoprotective and/or antitumor effects.

DVL, lectin from Dioclea violacea seeds, has multiples mechanisms of action against Candida spp via carbohydrate recognition domain.

Lectins are proteins of non-immunological origin with the ability to bind to carbohydrates reversibly. They emerge as an alternative to conventional antifungals, given the ability to interact with carbohydrates in the fungal cell wall inhibiting fungal growth. The lectin from D. violacea (DVL) already has its activity described as anti-candida in some species. Here, we observed the anti-candida effect of DVL on C. albicans, C. krusei and C. parapsilosis and its multiple mechanisms of action toward the yeasts. Additionally, it was observed that DVL induces membrane and cell wall damage and ROS overproduction. DVL was also able to cause an imbalance in the redox system of the cells, interact with ergosterol, inhibit ergosterol biosynthesis, and induce cytochrome c release from the mitochondrial membrane. These results endorse the potential application of DVL in developing a new antifungal drug to fight back against fungal resistance.

Dioclea violacea lectin increases the effect of neomycin against multidrug-resistant strains and promotes the purification of the antibiotic in immobilized lectin column.

DVL is a Man/Glc-binding lectin from Dioclea violacea seeds that has the ability to interact with the antibiotic gentamicin. The present work aimed to evaluate whether the DVL has the ability to interact with neomycin via CRD and to examine the ability of this lectin to modulate the antibiotic effect of neomycin against multidrug-resistant strains (MDR). The hemagglutinating activity test revealed that neomycin inhibited the hemagglutinating activity of DVL with a minimum inhibitory concentration of 50 mM, indicating that the antibiotic interacts with DVL via the carbohydrate recognition domain (CRD). DVL immobilized on cyanogen bromide-activated Sepharose® 4B bound 41 % of the total neomycin applied to the column, indicating that the DVL-neomycin interaction is efficient for purification processes. Furthermore, the minimum inhibitory concentrations (MIC) obtained for DVL against all strains studied were not clinically relevant. However, when DVL was combined with neomycin, a significant increase in antibiotic activity was observed against S. aureus and P. aeruginosa. These results demonstrate the first report of lectin-neomycin interaction, indicating that immobilized DVL has the potential to isolate neomycin by affinity chromatography. Moreover, DVL increased the antibiotic activity of neomycin against MDR, suggesting that it is a potent adjuvant in the treatment of infectious diseases.

The lectin DrfL inhibits cell migration, adhesion and triggers autophagy-dependent cell death in glioma cells.

Glioblastoma multiforme (GBM) is the most aggressive type of glioma, displaying atypical glycosylation pattern that may modulate signaling pathways involved in tumorigenesis. Lectins are glycan binding proteins with antitumor properties. The present study was designed to evaluate the antitumor capacity of the Dioclea reflexa lectin (DrfL) on glioma cell cultures. Our results demonstrated that DrfL induced morphological changes and cytotoxic effects in glioma cell cultures of C6, U-87MG and GBM1 cell lines. The action of DrfL was dependent upon interaction with glycans, and required a carbohydrate recognition domain (CRD), and the cytotoxic effect was apparently selective for tumor cells, not altering viability and morphology of primary astrocytes. DrfL inhibited tumor cell migration, adhesion, proliferation and survival, and these effects were accompanied by activation of p38MAPK and JNK (p46/54), along with inhibition of Akt and ERK1/2. DrfL also upregulated pro-apoptotic (BNIP3 and PUMA) and autophagic proteins (Atg5 and LC3 cleavage) in GBM cells. Noteworthy, inhibition of autophagy and caspase-8 were both able to attenuate cell death in GBM cells treated with DrfL. Our results indicate that DrfL cytotoxicity against GBM involves modulation of cell pathways, including MAPKs and Akt, which are associated with autophagy and caspase-8 dependent cell death.

Dioclea Altissima Seed Lectin (DAL) Prevents Anxiety-like Behavioral Responses in Adult Zebrafish (Danio Rerio): Involvement of GABAergic and 5-HT Systems.

BACKGROUND: Plant lectins have shown promising neuropharmacological activities in animal models. OBJECTIVE: This study evaluated the effect of Dioclea altissima seed lectin (DAL) on adult zebrafish behavior. METHOD: Zebrafish (n=6/group) were treated (i.p.; 20 µL) with DAL (0.025; 0.05 or 0.1 mg/mL), vehicle or diazepam (DZP) and submitted to several tests (open field, light/dark preference or novel tank). Flumazenil, pizotifen or granisetron were administered 15 min before DAL (0.05 mg/mL), and the animals were evaluated on light/dark preference test. It was also verified whether the DAL effect depended on its structural integrity and ability to interact with carbohydrates. RESULTS: DAL decreased the locomotor activity of adult zebrafish (0.025; 0.05 or 0.1 mg/mL), increased the time spent in the upper region of the aquarium (0.025 mg/mL), and decreased the latency time of adult zebrafish to enter the upper region on the novel tank test. DAL (0.05 mg/mL) also increased their permanence in the light zone of the light/dark preference test. The effect of DAL was dependent on carbohydrate interaction and protein structure integrity and was prevented by pizotifen, granizetron and flumazenil. CONCLUSION: DAL was found to have an anxiolytic-like effect mediated by the 5-HT and GABAergic receptors.

In depth analysis on the carbohydrate-binding properties of a vasorelaxant lectin from Dioclea lasiophylla Mart Ex. Benth seeds.

Lectins are a class of proteins or glycoproteins capable of recognizing and interacting with carbohydrates in a specific and reversible manner. Owing to this property, these proteins can interact with glycoconjugates present on the cell surface, making it possible to decipher the glycocode, as well as elicit biological effects, such as inflammation and vasorelaxation. Here, we report a structural and biological study of the mannose/glucose-specific lectin from Dioclea lasiophylla seeds, DlyL. The study aimed to evaluate in detail the interaction of DlyL with Xman and high-mannose N-glycans (MAN3, MAN5 and MAN9) by molecular dynamics (MD) and the resultant in vitro effect on vasorelaxation using rat aortic rings. In silico analysis of molecular docking was performed to obtain the initial coordinates of the DlyL complexes with the carbohydrates to apply as inputs in MD simulations. The MD trajectories demonstrated the stability of DlyL over time as well as different profiles of interaction with Xman and N-glycans. Furthermore, aortic rings assays demonstrated that the lectin could relax pre-contracted aortic rings with the participation of the carbohydrate recognition domain (CRD) and nitric oxide (NO) when endothelial tissue is preserved. These results confirm the ability of DlyL to interact with high-mannose N-glycans with its expanded CRD, supporting the hypothesis that DlyL vasorelaxant activity occurs primarily through its interaction with cell surface glycosylated receptors.Communicated by Ramaswamy H. Sarma.

Differential vasodilator effect of Dioclea rostrata lectin in conductance and resistance arteries: Mechanisms and glycoconjugate binding relationships.

Lectins are proteins that recognize specific carbohydrates, and the vasorelaxant effect of legume lectins has been previously reported, for example the Dioclea rostrata lectin (DRL). This study evaluated major pathways of DRL-induced relaxation in different artery segments and the possible molecular interactions involved. Rat thoracic aorta, coronary and mesenteric resistance arteries were tested "in vitro" with concentration-response curves to DRL (0.01-100 µg/mL). L-NAME, indomethacin and high KCl were used to evaluate nitric oxide, cyclooxygenase and hyperpolarization-dependent effects. DRL promoted relaxation of all vessels throughout different mechanisms. L-NAME blunted DRL-induced effects only in the aorta and mesenteric resistance artery. By the use of depolarizing KCl solution, vasodilation was reduced in all arteries, while incubation with indomethacin indicated a role of cyclooxygenase-derived factors for DRL effects in mesenteric and coronary arteries, but not in the aorta. Molecular docking results suggested interactions between DRL and heparan sulphate, CD31 and other glycans present on the membrane surface. These data indicate that the mechanisms involved in DRL-mediated vasodilation vary between conductance and resistance arteries of different origins, and these effects may be related to the capacity of DRL to bind a diversity of glycans, especially heparan sulphate, a proposed mechanoreceptor for nitric oxide synthase and cyclooxygenase activation.

Cognitive impairment by non-insulin-dependent diabetes mellitus was attenuated by dietary supplements of marble vine (Dioclea reflexa) and plantain (Musa paradisiaca) dough meals in albino rats.

This study investigates the protective effect of formulated marble vine/plantain dough meals on cognitive impairment in diabetic rats. Wistar rats were divided into eight groups (n = 6) and fed with HFD for 14 days and a single dose of streptozotocin intraperitoneally on the 14th day (except control rats). Diabetic rats were treated with formulated diets and metformin. The ameliorative effect of the formulated doughs on cerebral damage in diabetic rats with respect to weight gain/loss, glucose and insulin levels, oxidative damage, neurological dysfunction, and histological alterations were assessed. The formulated diet had high protein and fiber content values ranged from 13.00 to 25.04 g/100 g and from 5.23 to 6.20 g/100 g, respectively compared to the control. Blood glucose level was observed, thereby mitigating the cerebral oxidative damage. The diet significantly ameliorated the neurological dysfunction as adjudged by increased dopamine concentration and lowered acetylcholinesterase activity; results were also supported by the outcomes from brain histopathological study. PRACTICAL APPLICATIONS: Underutilized leguminous seeds such as marble vine seeds are known for their nutraceutical potentials due to their numerous biochemical components. The study provides preliminary information on the potential of marble vine/plantain functional dough meals in the management of neurological complications resulting from type 2 diabetes mellitus in albino rats. Generally, the formulated doughs possess neuroprotective potentials in preventing neurological complications arising from diabetes. However, the effect of marble vine-plantain dough meal in managing the brain damage should be further investigated through the clinical trials before development for pharmaceutical applications.

Heterologous production of α-chain of Dioclea sclerocarpa lectin: Enhancing the biological effects of a wild-type lectin.

Lectins from Diocleinae subtribe species (family Leguminosae) are of special interest since they present a wide spectrum of biological activities, despite their high structural similarity. During their synthesis in plant cells, these proteins undergo post-translational processing resulting in the formation of three chains (α, ß, γ), which constitute the lectins' subunits. Furthermore, such wild-type proteins are presented as isolectins or with different combinations of these chains, which undermine their biotechnological potential. Thus, the present study aimed to produce a recombinant form of the lectin from Dioclea sclerocarpa seeds (DSL), exclusively constituted by α-chain. The recombinant DSL (rDSL) was successfully expressed in E. coli BL21 (DE3) and purified by affinity chromatography (Sephadex G-50), showing a final yield of 74 mg of protein per liter of culture medium and specificity for D-mannose, α-methyl-mannoside and melibiose, unlike the wild-type protein. rDSL presented an effective vasorelaxant effect in rat aortas up to 100% and also interacted with glioma cells C6 and U87. Our results demonstrated an efficient recombinant production of rDSL in a bacterial system that retained some biochemical properties of the wild-type protein, showing wider versatility in sugar specificities and better efficacy in its activity in the biological models evaluated in this work.

Neuropharmacological Characterization of Dioclea altissima Seed Lectin (DAL) in Mice: Evidence of Anxiolytic-like Effect Mediated by Serotonergic, GABAergic Receptors and NO Pathway.

BACKGROUND: Plant lectins have shown promising biological activities in the central nervous system (CNS). OBJECTIVE: This study evaluated the effect of DAL, a lectin isolated from the seeds of the Dioclea altissima species, having binding affinity to D-glucose or D-mannose residues, on mice behavior. METHODS: Mice (n=6/group) were treated (i.p.) with DAL (0.25, 0.5 or 1 mg/kg) or vehicle and subjected to several tests (open field/OFT, marble-burying/MBT, hole-board/HBT, elevated plus maze/PMT, tail suspension/ TST, forced swimming/FST or rotarod/RRT). Pizotifen, cyproheptadine, flumazenil, L-NAME, 7-NI, Larginine or yohimbine were administered 15 min before DAL (0.5 mg/kg) and the animals were evaluated on PMT. It was also verified whether the DAL effect depended on its structural integrity and ability to interact with carbohydrates. RESULTS: The results showed there were no neurobehavioral changes in the mice at the RRT, FST and locomotion in the OFT. DAL (0.25, 0.5 or 1 mg/kg) increased the behavior of grooming and rearing in the OFT, head dips in the HBT, pedalling in the TST and decreased the number of marbles hidden in the MBT. In the PMT, DAL (0.25, 0.5 and 1 mg/kg) and Diazepam increased the frequency of entries in the open arms and the time of permanence in the open arms without affecting the locomotor activity. The effect of DAL was dependent on carbohydrate interaction and protein structure integrity and it prevented by pizotifen, cyproheptadine, flumazenil, L-NAME and 7-NI, but not by L-arginine or yohimbine. CONCLUSION: DAL was found to have an anxiolytic-like effect mediated by the 5-HT and GABAergic receptors and NO pathway.

Dioclea violacea lectin modulates the gentamicin activity against multi-resistant strains and induces nefroprotection during antibiotic exposure.

Gentamicin is an aminoglycoside antibiotic used to treat infections of various origins. In the last few decades, the constant use of gentamicin has resulted in increased bacterial resistance and nephrotoxicity in some cases. In this study, we examined the ability of Dioclea violacea lectin (DVL) in modulate the antimicrobial activity of gentamicin and reduce the nephrotoxicity induced by this drug. The minimum inhibitory concentration (MIC) obtained for DVL against all strains studied was not clinically relevant (MIC ≥ 1024 µg/mL). However, when DVL was combined with gentamicin, a significant increase in antibiotic action was observed against Staphylococcus aureus and Escherichia coli. DVL also reduced antibiotic tolerance in S. aureus during 10 days of continuous treatment. In addition, DVL presented a nephroprotective effect, reducing sodium excretion, N-Gal expression and urinary protein, that are important markers of glomerular and tubular injuries. Taken together, studies of inhibition of hemagglutinating activity, fluorescence spectroscopy and molecular docking revealed that gentamicin can interact with DVL via the carbohydrate recognition domain (CRD), suggesting that the results obtained in this study may be directly related to the interaction of DVL-gentamicin and with the ability of the lectin to interact with glycans present in the cells of the peritoneum.

Lectin from Dioclea violacea induces autophagy in U87 glioma cells.

The antitumor activity of DVL, a lectin purified from Dioclea violacea seeds, on the U87 human glioma cell line was evaluated and compared with Canavalia ensiformis lectin (ConA). Treatment with DVL (10-100 µg/mL; 24-96 h) induced alterations in cell morphology, decreased cell numbers and clonogenic survival in a time- and concentration-dependent manner. DVL caused significant decreases in cell viability and impaired cell migration. Mechanistically, DVL treatment (12 h) disrupted mitochondrial electrochemical gradient, without ROS accumulation or caspase activation. In the absence of apoptosis, DVL (30-100 µg/mL), instead, induced autophagy, as detected by acridine orange staining and cleavage of LC3I. Inhibition of autophagy with 3-Methyladenine (3-MA) and Chloroquine partially abrogated DVL, but not ConA, cytotoxicity. The modulation of signaling pathways that orchestrate autophagic and cell survival processes were analyzed. DVL (30-100 µg/mL) decreased Akt, mTORC1 and ERK1/2 phosphorylation and augmented JNK(p54) and p38MAPK phosphorylation. DVL was more potent than ConA for most parameters analyzed. Even though both lectins showed cytotoxicity to glioma cells, they spared primary astrocyte cultures. The results suggest a selective antiglioma activity of DVL by inhibiting U87 glioma cell migration and proliferation and inducing cell death, partially associated with autophagy, and likely involving Akt and mTORC1 dephosphorylation.

Electrochemical Response of Saccharomyces cerevisiae Corresponds to Cell Viability upon Exposure to Dioclea reflexa Seed Extracts and Antifungal Drugs.

Dioclea reflexa bioactive compounds have been shown to contain antioxidant properties. The extracts from the same plant are used in traditional medical practices to treat various diseases with impressive outcomes. In this study, ionic mobility in Saccharomyces cerevisiae cells in the presence of D. reflexa seed extracts was monitored using electrochemical detection methods to link cell death to ionic imbalance. Cells treated with ethanol, methanol, and water extracts were studied using cyclic voltammetry and cell counting to correlate electrochemical behavior and cell viability, respectively. The results were compared with cells treated with pore-forming Amphotericin b (Amp b), as well as Fluconazole (Flu) and the antimicrobial drug Rifampicin (Rif). The D. reflexa seed water extract (SWE) revealed higher anodic peak current with 58% cell death. Seed methanol extract (SME) and seed ethanol extract (SEE) recorded 31% and 22% cell death, respectively. Among the three control drugs, Flu revealed the highest cell death of about 64%, whereas Amp b and Rif exhibited cell deaths of 35% and 16%, respectively, after 8 h of cell growth. It was observed that similar to SWE, there was an increase in the anodic peak current in the presence of different concentrations of Amp b, which also correlated with enhanced cell death. It was concluded from this observation that Amp b and SWE might follow similar mechanisms to inhibit cell growth. Thus, the individual bioactive compounds from the water extracts of D. reflexa seeds could further be purified and tested to validate their potential therapeutic application. The strategy to link electrochemical behavior to biochemical responses could be a simple, fast, and robust screening technique for new drug targets and to understand the mechanism of action of such drugs against disease models.

Dioclea violacea lectin ameliorates inflammation in the temporomandibular joint of rats by suppressing intercellular adhesion molecule-1 expression.

Inflammation of temporomandibular joint (TMJ) tissues are the most common cause of pain conditions associated with temporomandibular disorders (TMDs). After a tissue and/or neural damage, the inflammatory response is characterized by plasma extravasation and leukocytes infiltration in the TMJ tissues, which in turn, release inflammatory cytokines cascades responsible for inflammatory pain. Lectins are glycoproteins widely distributed in nature that may exhibit anti-inflammatory properties. This study demonstrated by molecular docking and MM/PBSA that the lectin from Dioclea violacea (DVL) interacts favorably with α-methyl-D-mannoside, N-acetyl-D-glucosamine, and core1-sialyl-Lewis X which are associated with leukocytes migration during an inflammatory response. Wistar rats pretreated with intravenously injection of DVL demonstrated a significant inhibition of plasma extravasation induced by carrageenan (a non-neurogenic inflammatory inductor) and mustard oil (a neurogenic inflammatory inductor) in the TMJ periarticular tissues (p < 0.05; ANOVA, Tukey's test). In addition, DVL significantly reduced carrageenan-induced leukocyte migration in the TMJ periarticular tissues mediated by down-regulation of ICAM-1 expression. These results suggest a potential anti-inflammatory effect of DVL in inflammatory conditions of TMJ.

Anti-glioma properties of DVL, a lectin purified from Dioclea violacea.

Plant lectins have been studied owing to their structural properties and biological effects that include agglutinating activity, antidepressant-like effect and antitumor property. The results from this work showed the effects of the lectin extracted from the Dioclea violacea plant (DVL) on the C6 rat glioma cell line. DVL treatment was able to induce caspase-3 activation, apoptotic cell death and cellular membrane damage. Furthermore, DVL decreased mitochondrial membrane potential and increased the number of acidic vesicles and cleavage of LC3, indicating activation of autophagic processes. DVL also significantly inhibited cell migration. Compared to ConA, a well-studied lectin extracted from Canavalia ensiformes seeds, some effects of DVL were more potent, including decreasing C6 glioma cell viability and migration ability. Taken together, the results suggest that DVL can induce glioma cell death, autophagy and inhibition of cell migration, displaying potential anti-glioma activity.

Differential recognition of natural and remodeled glycotopes by three Diocleae lectins.

The carbohydrate specificities of Dioclea grandiflora lectins DGL-I1 and DGL-II, and Galactia lindenii lectin II (GLL-II) were explored by use of remodeled glycoproteins as well as by the lectin hemagglutinating activity against erythrocytes from various species with different glycomic profiles. The three lectins exhibited differences in glycan binding specificity but also showed overlapping recognition of some glycotopes (i.e. Tα glycotope for the three lectins; IIß glycotope for DGL-II and GLL-II lectins); in many cases the interaction with distinct glycotopes was influenced by the structural context, i.e., by the neighbouring sugar residues. Our data complement and expand the existing knowledge about the binding specificity of these three Diocleae lectins, and taken together with results of previous studies, allow us to suggest a functional map of the carbohydrate recognition which illustrate the impact of modification of basic glycotopes enhancing, permiting, or inhibiting their recognition by each lectin.

Dioclins A and B, new antioxidant flavonoids from Dioclea reflexa.

Dioclins A (1) and B (2), the new flavonoids, have been isolated from the ethyl acetate soluble fraction of the roots of Dioclea reflexa along with 3,5-dihydroxy-4 methoxybenzoic acid (3), lupeol (4) and the rare dipeptide, auratiamide acetate (5). Their structures have been elucidated by spectroscopic techniques. The compounds 1 and 2 showed a significant antioxidant activity in DPPH radical scavenging assay.

Structural analysis of Dioclea lasiocarpa lectin: A C6 cells apoptosis-inducing protein.

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.