Structural Analysis and Characterization of an Antiproliferative Lectin from Canavalia villosa Seeds.

Cells use glycans to encode information that modulates processes ranging from cell-cell recognition to programmed cell death. This information is encoded within a glycocode, and its decoding is performed by carbohydrate-binding proteins. Among these, lectins stand out due to their specific and reversible interaction with carbohydrates. Changes in glycosylation patterns are observed in several pathologies, including cancer, where abnormal glycans are found on the surfaces of affected tissues. Given the importance of the bioprospection of promising biomolecules, the current work aimed to determine the structural properties and anticancer potential of the mannose-specific lectin from seeds of Canavalia villosa (Cvill). Experimental elucidation of the primary and 3D structures of the lectin, along with glycan array and molecular docking, facilitated the determination of its fine carbohydrate-binding specificity. These structural insights, coupled with the lectin's specificity, have been combined to explain the antiproliferative effect of Cvill against cancer cell lines. This effect is dependent on the carbohydrate-binding activity of Cvill and its uptake in the cells, with concomitant activation of autophagic and apoptotic pathways.

Exploring the carbohydrate-binding ability of Canavalia bonariensis lectin in inflammation models.

Lectins are a group of proteins of non-immune origin recognized for their ability to bind reversibly to carbohydrates. Researchers have been intrigued by oligosaccharides and glycoconjugates for their involvement as mediators of complex cellular events and then many biotechnological applications of lectins are based on glycocode decoding and their activities. Here, we report a structural and biological study of a ConA-like mannose/glucose-specific lectin from Canavalia bonariensis seeds, CaBo. More specifically, we evaluate the binding of CaBo with α-methyl-D-mannoside (MMA) and mannose-1,3-α-D-mannose (M13) and the resultant in vivo effects on a rat model of acute inflammation. A virtual screening was also carried out to cover a larger number of possible bindings of CaBo. In silico analysis demonstrated the stability of CaBo interaction with mannose-type ligands, and the lectin was able to induce acute inflammation in rats with the participation of the carbohydrate recognition domain (CRD) and histamine release. These results confirm the ability of CaBo to interact with hybrid and high-mannose N-glycans, supporting the hypothesis that CaBo's biological activity occurs primarily through its interaction with cell surface glycosylated receptors.

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

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.

Purification, characterization and partial sequence of a pro-inflammatory lectin from seeds of Canavalia oxyphylla Standl. & L. O. Williams.

Recent studies have shown that lectins are promising tools for use in various biotechnological processes, as well as studies of various pathological mechanisms, isolation, and characterization of glycoconjugates and understanding the mechanisms underlying pathological mechanisms conditions, including the inflammatory response. This study aimed to purify, characterize physicochemically, and predict the biological activity of Canavalia oxyphylla lectin (CoxyL) in vitro and in vivo. CoxyL was purified by a single-step affinity chromatography in Sephadex® G-50 column. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that the pure lectin consists of a major band of 30 kDa (α-chain) and two minor components (ß-chain and γ-chain) of 16 and 13 kDa, respectively. These data were further confirmed by electrospray ionization mass spectrometry, suggesting that CoxyL is a typical ConA-like lectin. In comparison with the average molecular mass of α-chain, the partial amino acid sequence obtained corresponds to approximately 45% of the total CoxyL sequence. CoxyL presented hemagglutinating activity that was specifically inhibited by monosaccharides (D-glucose, D-mannose, and α-methyl-D-mannoside) and glycoproteins (ovalbumin and fetuin). Moreover, CoxyL was shown to be thermostable, exhibiting full hemagglutinating activity up to 60°C, and it was pH-sensitive for 1 h, exhibiting maximal activity at pH 7.0. CoxyL caused toxicity to Artemia nauplii and induced paw edema in rats. This biological activity highlights the importance of lectins as important tools to better understand the mechanisms underlying inflammatory responses.

Purification and characterization of a highly thermostable GlcNAc-binding lectin from Collaea speciosa seeds.

Lectins from plants of the Diocleinae subtribe often exhibit specificity towards mannose/glucose and derived sugars, with some plants also displaying a second lectin specific to lactose/GalNAc. Here, we present a novel lectin from Collaea speciosa, named CsL, that displays specificity for GlcNAc/glucose. The lectin was extracted from Collaea speciosa seeds and purified by a single chromatographic step on a Sephadex G-50 matrix. In solution, the lectin appears as a dimeric protein composed of 25 kDa monomers. The protein is stable at pH 7-8 and dependent on divalent cations. CsL maintained its agglutination activity after heating to 90 °C for 1 h. Glycan array studies revealed that CsL binds to N-glycans with terminal GlcNAc residues, chitobiose and chitotriose moieties. The partial amino acid sequence of the lectin is similar to that of some lactose-specific lectins from the same subtribe. In contrast to other ConA-like lectins, CsL is not toxic to Artemia. Because of its remarkably different properties and specificity, this lectin could be the first member of a new group inside the Diocleinae lectins.

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.

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.

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.

Lectin from Canavalia villosa seeds: A glucose/mannose-specific protein and a new tool for inflammation studies.

With important carbohydrate binding properties, lectins are proteins able to decipher the glycocode, and as such, they can be used in bioassays involving cell-cell communication, protein targeting, inflammation, and hypernociception, among others. In this study, a new glucose/mannose-specific lectin from Canavalia villosa seeds (Cvill) was isolated by a single affinity chromatography step in a Sephadex® G-50 column, with a purification yield of 19.35mg of lectin per gram of powdered seed. Analysis of intact protein by mass spectrometry showed the lectin is composed of three polypeptide chains, including a 25.6kDa α chain, 12.9KDa ß, and 12.6 KDa γ fragments, similar to the profile of ConA-like glucose/mannose-specific lectins. Partial sequence of the protein was obtained by MS-MALDI TOF/TOF covering 41.7% of its primary structure. Cvill presented sugar specificity to d-glucose, α-methyl-d-mannoside, d-mannose, and glycoproteins fetuin and ovoalbumin. The lectin characterization showed that Cvill presents high stability within a broad range of pH and temperature, also showing average toxicity against Artemia nauplii. The proinflammatory effect of Cvill was observed by induction of paw edema and hypernociception in mice, with the participation of the carbohydrate binding site, showing its potential to be used as tool in inflammation studies.

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.