Mayara Torquato Lima Silva

Purification of a thermostable antinociceptive lectin isolated from Andira anthelmia.

Andira anthelmia (tribe Dalbergieae), a plant from Brazilian Amazon, possesses a seed lectin that was purified by affinity chromatography in sepharose–mannose. This novel Dalbergieae lectin, named AAL, agglutinated rabbit erythrocytes treated with trypsin. The hemagglutinating activity of AAL was maintained after incubation at a wide range of temperature (40 to 70 °C) and pH, was shown to be dependent on divalent cations, and was inhibited by d‐mannose and d‐sucrose. AAL showed an electrophoretic profile in sodium dodecyl sulfate–polyacrylamide gel electrophoresis similar to other lectins of the tribe Dalbergieae, presenting a double band of molecular weight with approximately 20 kDa and other minor bands of 17, 15, and 13 kDa, being the smaller fragment glycosylated. AAL injected by intravenous route in mice showed antinociceptive activity in two behavioral tests (writhing and formalin). In the writhing test induced by acetic acid, AAL showed inhibitory effect at 0.01 mg/kg (68%), 0.1 mg/kg (46%) and 1 mg/kg (74%). In the formalin test, AAL (0.1 mg/kg) inhibited by 48% the licking time in the inflammatory phase, an effect that was recovered by the lectin association with mannose. In conclusion, AAL presents analgesic effect involving the lectin domain via peripheral mechanisms of inflammatory nociception. This activity highlights the importance of lectins as tools to be used for understanding the interaction of protein–carbohydrate in processes associated to inflammatory pain. Copyright

Purification and partial characterization of a new mannose/glucose‐specific lectin from Dialium guineense Willd seeds that exhibits toxic effect

A new mannose/glucose‐specific lectin, named DigL, was purified from seeds of Dialium guineense by a single step using a Sepharose 4b‐Mannose affinity chromatography column. DigL strongly agglutinated rabbit erythrocytes and was inhibited by d‐mannose, d‐glucose, and derived sugars, especially α‐methyl‐d‐mannopyranoside and N‐acetyl‐d‐glucosamine. DigL has been shown to be a stable protein, maintaining its hemagglutinating activity after incubation at a wide range of temperature and pH values and after incubation with EDTA. DigL is a glycoprotein composite by approximately 2.9% of carbohydrates by weight. By sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis, the purified DigL exhibited an electrophoretic profile consisting of a broad band of 28–30 kDa. Analysis using electrospray ionization mass spectrometry indicated that purified DigL possesses a molecular average mass of 28 452 ± 2 Da and shows the presence of possible glycoforms. In addition, DigL exhibited an intermediary toxic effect on Artemia sp. nauplii, and this effect was both dependent on native structure and mediated by a carbohydrate‐binding site. Copyright

Canavalia bonariensis lectin: Molecular bases of glycoconjugates interaction and antiglioma potential

CaBo is a mannose/glucose-specific lectin purified from seeds of Canavalia bonariensis. In the present work, we report the CaBo crystal structure determined to atomic resolution in the presence of X-man, a specific ligand. Similar to the structural characteristics of other legume lectins, CaBo presented the jellyroll motif, a metal binding site occupied by calcium and manganese ions close to the carbohydrate-recognition domain (CRD). In vitro test of CaBo cytotoxicity against glioma cells demonstrated its ability to decrease the cellular viability and migration by induction of autophagy and cell death. Molecular docking simulations corroborate previous data indicating that the lectins biological activities occur mostly through interactions with glycoproteins since the lectin interacted favorably with several N-glycans, especially those of the high-mannose type. Together, these results suggest that CaBo interacts with glycosylated cell targets and elicits a remarkable antiglioma activity.

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.

Structural analysis, molecular docking and molecular dynamics of an edematogenic lectin from Centrolobium microchaete seeds

Lectins represent a class of proteins or glycoproteins capable of reversibly binding to carbohydrates. Seed lectins from the Dalbergieae tribe (Leguminosae) have structural variability, carbohydrate specificity, and biological effects, such as inflammation, vasorelaxation and cancer antigen binding. To comprehensively address these factors, the present work aimed to establish and characterize the three-dimensional structure of Centrolobium microchaete lectin (CML) by homology modeling, investigate protein-carbohydrate interactions and evaluate its inflammatory effect on mice. Molecular docking was performed to analyze interactions of the lectin with monosaccharides, disaccharides and N-glycans. Two dimannosides, methyl mannose-1,3-α-D-mannose (MDM) and mannose-1,3-α-D-mannose (M13), were used in molecular dynamics (MD) simulations to study the behavior of the carbohydrate-recognition domain (CRD) over time. Results showed an expanded domain within which hydrophobic interactions with the methyl group in the MDM molecule were established, thus revealing novel interactions for mannose-specific Dalbergieae lectins. To examine its biological activities, CML was purified in a single step by affinity chromatography on Sepharose-mannose matrix. The lectin demonstrated inflammatory response in the paw edema model and stimulated leukocyte migration to the animal peritoneal cavities, an effect elicited by CRD. For the first time, this work reports the molecular dynamics of a lectin from the Dalbergieae tribe.

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 β, 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.

Isolation, purification and partial physicochemical characterization of a lectin in Andira pisonis Mart seed.

Lectins are ubiquitous proteins in nature, with non-immune origin, which have at least one non-catalytic domain that binds carbohydrates specifically and reversibly. They can be found in vegetables leaves, stems and seeds. The Dalbergieae tribe has lectins which have specificity for different carbohydrates and also have several biological activities such as induction of rat paw edema, release of chemotactic mediators by macrophages, vasorelaxant effect in rat aortas, among others. This study aimed to isolate, purify and physiochemically characterize a lectin found in seeds of Andira pisonis Mart (Dalbergieae). Andira pisonis Mart seeds were ground into a fine powder and subjected to total protein extraction in 1 M ammonium sulfate. Soluble proteins were subjected to hemagglutination activity quantification by the Bradford method and essays of hemagglutination inibition activity by sugar. The lectin from Andira pisonis Mart (APL) was purified by affinity chromatography on Sepharose- Mannose matrix eluted in 0.1 M glycine buffer pH 2.6 with 0.15 M NaCl. The eluted fraction was dialyzed against distilled water, lyophilized and subjected to ion exchange chromatography on HiTrap SP XL 01. APL was eluted on 20 mM sodium acetate buffer pH 4.5 gradient of 0-1M NaCl. APL hemagglutinated rabbit erythrocytes (enzymatically treated) and other lectins from the tribe Dalbergieae and showed specificity for mannose (25 mM). SDS-PAGE analysis showed that APL is composed of a major 34 kDa double band and a minor 8 and 9 kDa double band. APL showed thermostability at 60° C. Further studies are still needed in order to better physicochemically characterize this protein and study its biotechnological potential on the referred conditions of vasorelaxant effect and chemotatic mediator.

Physico-chemical characterization and partial sequence of a lectin from Canavalia bonariensis Lindl seeds

Lectins are (glycol) proteins that bind specifically and reversibly to carbohydrates. These proteins, in particular those from plant, are important tools in glycobiochemistry and glycobiology. Canavalia bonariensis Lindl is a species of Leguminosae family, Papilionoideae subfamily, tribe Phaseoleae, subtribe Diocleinae, native of the southern region of the country. The objective of this work was to purify a lectin from C. bonariensis (CaBo) seeds through affinity chromatographic. The process of purification of CaBo (Canavalia bonariensis Lectin) was monitored by SDS-PAGE and hemagglutinating activity and showed that the purified lectin is characterized by an electrophoretic profile consists of a higher band with approximately 26 kDa, and two bottom bands with apparent molecular mass of 14 and 12 kDa. The analysis by mass spectrometry indicated that CaBo has a chain with molecular mass of 25,512 kDa and and two subunits (β and γ chains) with molecular mass of 12,999 Da and 12,537 Da, respectively. CaBo also had its primary sequence partially determined by tandem mass spectrometry, obtaining 61% of the total sequence of the protein. CaBo was tested for the thermostability of their hemagglutinating activity after incubation for one hour at different temperatures (40° to 80° C), losing activity only at 80° C after one hour. Regarding its stability at different pH (4.0 to 10.0), CaBo was stable in a pH range between 7.0 and 9.0. The CaBo activity was also affected after serial dilution in the presence of the chelating agent EDTA and it was recovered significantly after addition of CaCl2 and MnCl2 0,005 mol/L, proving to be dependent of divalent metal cations.

Isolation and characterization of a lectin from Andira anthelmia seeds

Andira anthelmia seeds, a species belonging to the Leguminosae family, Papilionoideae subfamily, Dalbergieae tribe, have a glucose/mannose specific lectin that agglutinates rabbit erythrocytes treated with trypsin. The Dalbergieae tribe has lectins which have specificity for different carbohydrates including mannose and also have several biological activities such as induction of rat paw edema, release of chemotactic mediators by macrophages, vasorelaxant effect in rat aortas, termiticide activity, potential fungicide action, among others. This study aimed to isolate, purify and physicochemically characterize a lectin found in seeds of Andira anthelmia. The lectin from Andira anthelmia seeds was purified by affinity chromatography on Mannose-Sepharose matrix followed by ion exchange chromatography on DEAE-Sephacel matrix. This procedure resulted in a purified lectin, named AAL. AAL purification process was monitored by specific hemagglutinating activity and SDS-PAGE, in which it was observed that this lectin has a molecular weight of approximately 20 kDa and four others subunits of approximately 15 and 14 kDa. This lectin is a glycoprotein with approximately 1.89% of carbohydrates on its composition and shows high stability, being able to maintain their hemagglutinating activity in a wide pH range and after exposure to temperatures of 70 °C for one hour. After dialysis against the chelating agent EDTA, AAL lost its hemagglutinating activity, but recovered its action after the addition of metals, being, therefore, dependent on divalent metal cations. In this study a new lectin from Dalbergieae was purified and characterized. Further analyzes are needed in order to best evaluate their biotechnological applications.