ABSTRACT
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.
Subject(s)
Dioclea/chemistry , Plant Lectins/chemistry , Animals , Aorta/drug effects , Cell Line, Tumor , Chromatography, Affinity , Escherichia coli/genetics , Escherichia coli/metabolism , Glioma/metabolism , Hemagglutination , Mannose/chemistry , Plant Lectins/metabolism , Protein Structure, Secondary , Rats , Recombinant Proteins/analysis , Recombinant Proteins/biosynthesis , Recombinant Proteins/isolation & purification , Recombinant Proteins/pharmacology , Seeds/chemistry , Vasodilator Agents/chemistryABSTRACT
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.
Subject(s)
Autophagy/drug effects , Dioclea/chemistry , Gene Expression/drug effects , Neuroglia/drug effects , Plant Lectins/pharmacology , Animals , Apoptosis/drug effects , Apoptosis/genetics , Autophagy/genetics , Canavalia/chemistry , Caspase 3/genetics , Caspase 3/metabolism , Cell Cycle/drug effects , Cell Cycle/genetics , Cell Line, Tumor , Cell Membrane/drug effects , Cell Membrane/metabolism , Cell Membrane/ultrastructure , Cell Movement/drug effects , Cell Proliferation/drug effects , Concanavalin A/isolation & purification , Concanavalin A/pharmacology , L-Lactate Dehydrogenase/metabolism , Membrane Potential, Mitochondrial/drug effects , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolism , Mitochondria/drug effects , Mitochondria/metabolism , Neuroglia/metabolism , Neuroglia/pathology , Plant Lectins/isolation & purification , RatsABSTRACT
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.