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1.
Int J Biol Macromol ; 117: 124-133, 2018 Oct 01.
Article in English | MEDLINE | ID: mdl-29802925

ABSTRACT

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.


Subject(s)
Fabaceae/chemistry , Molecular Docking Simulation , Molecular Dynamics Simulation , Molecular Structure , Plant Lectins/chemistry , Seeds/chemistry , Animals , Anti-Inflammatory Agents, Non-Steroidal/chemistry , Anti-Inflammatory Agents, Non-Steroidal/isolation & purification , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Binding Sites , Disease Models, Animal , Edema/drug therapy , Edema/etiology , Edema/pathology , Metals/chemistry , Mice , Plant Lectins/isolation & purification , Plant Lectins/pharmacology , Protein Binding , Protein Interaction Domains and Motifs , Structure-Activity Relationship
2.
Int J Biochem Cell Biol ; 92: 79-89, 2017 11.
Article in English | MEDLINE | ID: mdl-28939357

ABSTRACT

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.


Subject(s)
Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Dioclea/chemistry , Glioma/pathology , Plant Lectins/chemistry , Plant Lectins/pharmacology , Animals , Antineoplastic Agents/metabolism , Carbohydrate Metabolism , Cell Line, Tumor , Cell Survival/drug effects , Molecular Docking Simulation , Plant Lectins/metabolism , Protein Conformation , Rats , Seeds/chemistry
3.
Arch Biochem Biophys ; 630: 27-37, 2017 09 15.
Article in English | MEDLINE | ID: mdl-28754321

ABSTRACT

The Pisum arvense lectin (PAL), a legume protein belonging to the Vicieae tribe, is capable of specific recognition of mannose, glucose and its derivatives without altering its structure. In this work, the three-dimensional structure of PAL was determined by X-ray crystallography and studied in detail by a combination of molecular docking and molecular dynamics (MD). Crystals belonging to monoclinic space group P21 were grown by the vapor diffusion method at 293 K. The structure was solved at 2.16 Å and was similar to that of other Vicieae lectins. The structure presented Rfactor and Rfree of 17.04% and 22.08%, respectively, with all acceptable geometric parameters. Molecular docking was performed to analyze interactions of the lectin with monosaccharides, disaccharides and high-mannose N-glycans. PAL demonstrated different affinities on carbohydrates, depending on bond orientation and glycosidic linkage present in ligands. Furthermore, the lectin interacted with representative N-glycans in a manner consistent with the biological effects described for Vicieae lectins. Carbohydrate-recognition domain (CRD) in-depth analysis was performed by MD, describing the behavior of CRD residues in complex with ligand, stability, flexibility of the protein over time, CRD volume and topology. This is a first report of its kind for a lectin of the Vicieae tribe.


Subject(s)
Fabaceae/chemistry , Molecular Docking Simulation , Molecular Dynamics Simulation , Plant Lectins/chemistry , Polysaccharides/chemistry , Crystallography, X-Ray
4.
Int J Biol Macromol ; 102: 323-330, 2017 Sep.
Article in English | MEDLINE | ID: mdl-28380332

ABSTRACT

The lectin from Platypodium elegans seeds (PELa) was purified by affinity chromatography in a mannose-agarose column. The lectin agglutinated rabbit erythrocytes and the agglutinating effect was inhibited by previous incubation with the glycoprotein fetuin, along with N-acetyl-d-glucosamine, D-mannose and its derivatives. The lectin maintained complete activity in temperatures ranging from 40 to 60°C and pH values ranging from 9 to 10. As a glycoprotein, PELa has a carbohydrate content of 2.2%, and its activity requires divalent cations such as Ca2+ and Mn2+. Based on SDS-PAGE, PELa displays a profile similar to that of other Dalbergieae lectins with the main chain of molecular mass around 30kDa and two subunits of 19kDa and 10 kDa each. Two-dimensional (2D) electrophoresis revealed the presence of isoforms with different isoelectric points, and high-performance size exclusion chromatography (HPSEC) was performed to confirm the purity of the sample. The lectin was immobilized in CNBr-activated Sepharose 4B and successfully captured fetuin in solution, demonstrating that this lectin remains active and capable of binding carbohydrates. PELa showed effects different from those of its recombinant form in both pro- and anti-inflammatory tests.


Subject(s)
Edema/chemically induced , Fabaceae/chemistry , Immobilized Proteins/pharmacology , Plant Lectins/pharmacology , Recombinant Proteins/pharmacology , Seeds/chemistry , Sepharose/chemistry , Animals , Erythrocytes/drug effects , Erythrocytes/immunology , Hemagglutination/drug effects , Immobilized Proteins/chemistry , Male , Plant Lectins/chemistry , Rabbits , Rats , Recombinant Proteins/chemistry
5.
Biochimie ; 135: 126-136, 2017 Apr.
Article in English | MEDLINE | ID: mdl-28196677

ABSTRACT

Lectins are proteins, or glycoproteins, capable of reversibly binding to specific mono- or oligosaccharides via a noncatalytic domain. The Diocleinae subtribe presents lectins with high structural similarity, but different effects based on biological activity assays. This variability results from small structural differences. Therefore, in this context, the present study aimed to perform a structural analysis of the lectin from Dioclea lasiophylla Mart. ex Benth seeds (DlyL) and evaluate its inflammatory effect. To accomplish this, DlyL was purified in a single step by affinity chromatography on Sephadex® G-50 matrix. DlyL primary structure was determined through a combination of tandem mass spectrometry and DNA sequencing. DlyL showed high similarity with other species from the same genus. Its theoretical three-dimensional structure was predicted by homology modelling, and the protein was subjected to ligand screening with monosaccharides, oligosaccharides and complex N-glycans by molecular docking. Stability and binding of the lectin with α-methyl-d-mannoside were assessed by molecular dynamics. DlyL showed acute inflammatory response with hypernociceptive effect in the paw edema model, possibly by interaction with glycans present at the cell surface.


Subject(s)
Lectins/chemistry , Molecular Docking Simulation , Dioclea/chemistry , Molecular Dynamics Simulation , Monosaccharides/chemistry , Oligosaccharides/chemistry , Tandem Mass Spectrometry
6.
Int J Biol Macromol ; 98: 12-23, 2017 May.
Article in English | MEDLINE | ID: mdl-28130130

ABSTRACT

The three-dimensional structure of Dioclea reflexa seed lectin (DrfL) was studied in detail by a combination of X-ray crystallography, molecular docking and molecular dynamics. DrfL was purified by affinity chromatography using Sephadex G-50 matrix. Its primary structure was obtained by mass spectrometry, and crystals belonging to orthorhombic space group P212121 were grown by the vapor diffusion method at 293K. The crystal structure was solved at 1.765Å and was very similar to that of other lectins from the same subtribe. The structure presented Rfactor and Rfree of 21.69% and 24.89%, respectively, with no residues in nonallowed regions of Ramachandran plot. Similar to other Diocleinae lectins, DrfL was capable of relaxing aortic rings via NO induction, with CRD participation, albeit with low intensity (32%). In silico analysis results demonstrated that DrfL could strongly interact with complex N-glycans, components of blood vessel glycoconjugates. Despite the high similarity among Diocleinae lectins, it was also reported that each lectin has unique CRD properties that influence carbohydrate binding, resulting in different biological effects presented by these molecules.


Subject(s)
Dioclea/chemistry , Molecular Docking Simulation , Molecular Dynamics Simulation , Plant Lectins/chemistry , Plant Lectins/pharmacology , Seeds/chemistry , Amino Acid Sequence , Animals , Crystallography, X-Ray , Mannosides/chemistry , Mannosides/metabolism , Plant Lectins/metabolism , Polysaccharides/chemistry , Polysaccharides/metabolism , Protein Domains , Rats , Vasodilator Agents/chemistry , Vasodilator Agents/metabolism , Vasodilator Agents/pharmacology
7.
Int J Biol Macromol ; 94(Pt A): 271-282, 2017 Jan.
Article in English | MEDLINE | ID: mdl-27737777

ABSTRACT

A lectin from Canavalia virosa, Diocleinae subtribe, was purified by affinity chromatography with Sephadex G-50 matrix and named ConV. The primary structure of ConV was obtained by mass spectrometry and crystals were obtained by the vapor diffusion method at 293K and belonged to orthorhombic space group P21221 with two molecules in its asymmetric unit. The structure obtained presented Rfactor and Rfree of 18.91% and 24.92% respectively, with no residues in nonallowed regions of Ramachandran plot. The crystal structure was solved at 2.53Å and was demonstrated to be very similar to other lectins from the same subtribe. In inflammatory tests, ConV elicited paw edema, but incubation of lectin with glucose beforehand was able to reduce the edematogenic effect, indicating the involvement of the carbohydrate recognition domain in this process. The lectin also showed toxicity to rat C6 glioma cells, disrupting the mitochondrial membrane potential (ΔYm) and decreasing cell viability, indicating an anticancer potential for ConV. In silico studies confirmed that ConV interacts strongly with carbohydrates that comprise the N-glycans of glycoproteins. This finding corroborates the hypothesis which holds that the lectin domain interacts with glycans in molecular targets and that this contributes to the effects observed in biological activities.


Subject(s)
Anti-Inflammatory Agents/chemistry , Antineoplastic Agents, Phytogenic/chemistry , Plant Extracts/chemistry , Amino Acid Sequence , Animals , Anti-Inflammatory Agents/pharmacology , Antineoplastic Agents, Phytogenic/pharmacology , Binding Sites , Canavalia , Cell Line, Tumor , Cell Survival/drug effects , Conserved Sequence , Crystallography, X-Ray , Drug Screening Assays, Antitumor , Hydrogen Bonding , Male , Mannosides/chemistry , Mice , Molecular Docking Simulation , Plant Extracts/pharmacology , Plant Lectins/chemistry , Protein Binding , Protein Conformation, beta-Strand , Protein Structure, Quaternary , Rats , Seeds/chemistry
8.
Int J Biol Macromol ; 92: 194-201, 2016 Nov.
Article in English | MEDLINE | ID: mdl-27411295

ABSTRACT

The relation structure-activity of the Mimosoideae lectins of Parkia platycephala (PPL) and Parkia biglobosa (PBL) was analyzed in this study. PBL was solved by X-ray crystallography at a resolution of 2.1Å, and the crystal structure belonged to the C2221 space group. Structural organization and binding sites were also characterized. Specifically, PBL monomer consists of three ß-prism domains tandemly arranged with each one presenting a different carbohydrate recognition domain (CRD). PPL showed antinociceptive activity in the mouse model of acetic acid-induced writhes with maximal inhibitory effect by 74% at 1mg/mL. PPL also demonstrated anti-inflammatory effect causing inhibition of leukocyte migration induced by both direct and indirect chemoattractants. These PPL activities were compared to that of PBL described previously. Molecular docking of both PBL and PPL demonstrated some differences in carbohydrate-lectin interaction energy. Comparing structure and biological effects of the two lectins provided new data about their structure and the relation with its biological activities.


Subject(s)
Fabaceae/chemistry , Lectins/chemistry , Lectins/pharmacology , Amino Acid Sequence , Analgesics/pharmacology , Animals , Anti-Inflammatory Agents/pharmacology , Binding Sites , Cell Movement/drug effects , Leukocytes/cytology , Mice , Molecular Docking Simulation , Protein Domains , Protein Structure, Secondary , Sequence Alignment , Static Electricity
9.
Arch Biochem Biophys ; 596: 73-83, 2016 04 15.
Article in English | MEDLINE | ID: mdl-26946944

ABSTRACT

A glycosylated lectin (CTL) with specificity for mannose and glucose has been detected and purified from seeds of Centrolobium tomentosum, a legume plant from Dalbergieae tribe. It was isolated by mannose-sepharose affinity chromatography. The primary structure was determined by tandem mass spectrometry and consists of 245 amino acids, similar to other Dalbergieae lectins. CTL structures were solved from two crystal forms, a monoclinic and a tetragonal, diffracted at 2.25 and 1.9 Å, respectively. The carbohydrate recognition domain (CRD), metal-binding site and glycosylation site were characterized, and the structural basis for mannose/glucose-binding was elucidated. The lectin adopts the canonical dimeric organization of legume lectins. CTL showed acute inflammatory effect in paw edema model. The protein was subjected to ligand screening (dimannosides and trimannoside) by molecular docking, and interactions were compared with similar lectins possessing the same ligand specificity. This is the first crystal structure of mannose/glucose native seed lectin with proinflammatory activity isolated from the Centrolobium genus.


Subject(s)
Edema/chemically induced , Fabaceae/chemistry , Mannose-Binding Lectin , Molecular Docking Simulation , Plant Lectins , Seeds/chemistry , Amino Acid Sequence , Animals , Disease Models, Animal , Edema/pathology , Female , Glycosylation , Inflammation/chemically induced , Inflammation/pathology , Mannose-Binding Lectin/chemistry , Mannose-Binding Lectin/toxicity , Mass Spectrometry , Plant Lectins/chemistry , Plant Lectins/toxicity , Protein Footprinting , Rats , Rats, Wistar , Structure-Activity Relationship
10.
Arch Biochem Biophys ; 588: 33-40, 2015 Dec 15.
Article in English | MEDLINE | ID: mdl-26545483

ABSTRACT

A novel lectin from seeds of Clathrotropis nitida (CNA) was purified and characterized. CNA is a glycoprotein containing approximately 3.3% carbohydrates in its structure. CNA promoted intense agglutination of rabbit erythrocytes, which was inhibited by galactosides and porcine stomach mucin (PSM). The lectin maintained its hemagglutinating activity after incubation in a wide range of temperatures (30-60 °C) and pH (6.0-7.0), and its binding activity was dependent on divalent cations (Ca(+2) and Mg(+2)). SDS-PAGE showed an electrophoretic profile consisting of a single band of 28 kDa, as confirmed by electrospray ionization mass spectrometry, which indicated an average molecular mass of 27,406 ± 2 Da and the possible presence of isoforms and glycoforms. In addition, CNA exhibited no toxicity to Artemia sp. nauplii and elicited reversible and dose-dependent vasorelaxation in precontracted aortic rings. CNA was successfully immobilized on chitosan beads and was able to capture PSM in solution. This study demonstrated that CNA is a lectin that has potential as a biotechnological tool in glycomics and glycoproteomics applications.


Subject(s)
Fabaceae/chemistry , Plant Lectins/isolation & purification , Plant Lectins/pharmacology , Vasodilator Agents/isolation & purification , Vasodilator Agents/pharmacology , Amino Acid Sequence , Animals , Aorta, Thoracic/drug effects , Aorta, Thoracic/physiology , Artemia/drug effects , Chitosan , Fabaceae/genetics , Hemagglutination/drug effects , Humans , Immobilized Proteins/chemistry , In Vitro Techniques , Male , Molecular Sequence Data , Molecular Weight , Plant Lectins/genetics , Plants, Medicinal/chemistry , Plants, Medicinal/genetics , Rabbits , Rats , Rats, Wistar , Seeds/chemistry , Sequence Homology, Amino Acid , Vasodilator Agents/chemistry
11.
Molecules ; 18(9): 10857-69, 2013 Sep 04.
Article in English | MEDLINE | ID: mdl-24008245

ABSTRACT

Lectin from the seeds of Dioclea lasiophylla (DlyL) was purified in a single step by affinity chromatography on a Sephadex® G-50 column. DlyL strongly agglutinated rabbit erythrocytes and was inhibited by monosaccharides (D-mannose and α-methyl-D-mannoside) and glycoproteins (ovalbumin and fetuin). Similar to other Diocleinae lectins, DlyL has three chains, α, ß and γ, with mass of 25,569 ± 2, 12,998 ± 1 and 12,588 ± 1 Da, respectively, and has no disulfide bonds. The hemagglutinating activity of DlyL was optimal in pH 8.0, stable at a temperature of 70 °C and decreased in EDTA solution, indicating that lectin activity is dependent on divalent metals. DlyL exhibited low toxicity on Artemia sp. nauplii, but this effect was dependent on the concentration of lectin in solution. DlyL immobilized on cyanogen bromide-activated Sepharose® 4B bound 0.917 mg of ovalbumin per cycle, showing the ability to become a tool for glycoproteomics studies.


Subject(s)
Dioclea/chemistry , Hemagglutinins/pharmacology , Mannose-Binding Lectins/pharmacology , Plant Extracts/pharmacology , Seeds/chemistry , Animals , Artemia , Chelating Agents/chemistry , Chromatography, Affinity , Edetic Acid/chemistry , Erythrocytes/drug effects , Hemagglutination , Hemagglutinins/chemistry , Hemagglutinins/isolation & purification , Hydrogen-Ion Concentration , Lethal Dose 50 , Mannose-Binding Lectins/chemistry , Mannose-Binding Lectins/isolation & purification , Ovalbumin/chemistry , Plant Extracts/chemistry , Plant Extracts/isolation & purification , Protein Binding , Rabbits , Sepharose/chemistry
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