Crystal structure of Pisum arvense seed lectin (PAL) and characterization of its interaction with carbohydrates by molecular docking and dynamics.

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.

Structural analysis of Centrolobium tomentosum seed lectin with inflammatory activity.

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.

Vasorelaxant activity of Canavalia grandiflora seed lectin: A structural analysis.

Lectins are comprised of a large family of proteins capable of the specific and reversible recognition of carbohydrates. Legume lectins, the most studied plant lectins, show high structural similarity, but with modifications that imply a variation in the intensity of some biological activities. In this work, the primary and tertiary structures of Canavalia grandiflora (ConGF) were determined. ConGF, a lectin isolated from C. grandiflora seeds, is able to induce relaxant activity in rat aortic rings. The complete sequence of ConGF comprises 237 amino acids. This particular protein has primary sequence variations commonly found in lectins from Dioclea and Canavalia genera. The protein structure was solved at 2.3 Å resolution by X-ray crystallography. An X-Man molecule was modeled into the carbohydrate recognition domain. Still, ConGF (30 and 100 µg mL(-1)) elicited 25% of vasorelaxation (IC50=34.48 ± 5.07 µg mL(-1)) in endothelialized aortic rings. A nonselective inhibitor of nitric oxide blocked ConGF relaxant effect, showing mediation by nitric oxide. Key distances between ConGF carbohydrate recognition domain residues were determined in order to explain this effect, in turn revealing some structural aspects that could differentiate lectins from the Canavalia genera with respect to different efficacy in vasorelaxant effect.

Galectin-9 is a target for the treatment of cancer: A patent review.

Galectins, which correspond to a group of proteins capable of recognizing and reversibly binding to ß-galactoside carbohydrates, have been the subject of innovation and development of technological products. Galectins play biological roles, such as cell proliferation and apoptosis, and some studies showed differences in the concentrations of galectins dispersed in serum of patients with cancer. For this reason, different studies have evaluated the biotechnological potential of these proteins as biomarkers for the prognosis and/or diagnosis of physiological disorders. Thus, this review discusses recent technological advancements in targeting galectins for the treatment of cancer and using galectins for cancer prognosis and diagnosis. Data mining was performed using the search descriptors "Galectin 9* and cancer*" and the ESPACENET and Cortellis Drug Discovery Intelligence (CDDI) databases. PRISMA guidelines were followed as a basis for literature review which aimed to conduct a systematic study of galectin-9 patents related to cancer prognosis, diagnosis and treatment. Results showed the importance of galectin-9 protein patents in furthering biomedical advancements in the global fight against cancer.

Kinetics characterization of a low immunogenic recombinant l-asparaginase from Phaseolus vulgaris with cytotoxic activity against leukemia cells.

l-asparaginases play a crucial role in the treatment of acute lymphoblastic leukemia (ALL), a type of cancer that mostly affects children and teenagers. However, it is common for these molecules to cause adverse reactions during treatment. These downsides ignite the search for novel asparaginases to mitigate these problems. Thus, this work aimed to produce and characterize a recombinant asparaginase from Phaseolus vulgaris (Asp-P). In this study, Asp-P was expressed in Escherichia coli with high yields and optimum activity at 40 °C, pH 9.0. The enzyme Km and Vmax values were 7.05 mM and 1027 U/mg, respectively. Asp-P is specific for l-asparagine, showing no activity against l-glutamine and other amino acids. The enzyme showed a higher cytotoxic effect against Raji than K562 cell lines, but only at high concentrations. In silico analysis indicated that Asp-P has lower immunogenicity than a commercial enzyme. Asp-P induced biofilm formation by Candida sp. due to sublethal dose, showing an underexplored potential of asparaginases. The absence of glutaminase activity, lower immunogenicity and optimal activity similar to physiological temperature conditions are characteristics that indicate Asp-P as a potential new commercial enzyme in the treatment of ALL and its underexplored application in the treatment of other diseases.

Halilectin 1 (H-1) and Halilectin 2 (H-2): two new lectins isolated from the marine sponge Haliclona caerulea.

Two new lectins named Halilectin 1 (H-1) and Halilectin 2 (H-2) were isolated from the marine sponge Haliclona caerulea using a combination of affinity chromatography on stroma fixed onto Sephadex G-25 and cation and anion exchange chromatography. H-1 is a monomeric protein with a molecular mass of 40 kDa estimated using sodium dodecyl sulfate polyacrylamide gel electrophoresis and 15 kDa estimated using a TSK gel. Conversely, H-2 is a homodimeric protein with 15 kDa monomers linked via weak interactions. H-1 more effectively agglutinates trypsinized rabbit erythrocytes, whereas H-2 more effectively agglutinates native rabbit erythrocytes. The hemagglutinating activity of H-1 could be not inhibited by any tested sugars, but H-2 was inhibited by orosomucoid and porcine stomach mucin. Neither lectin was dependent on divalent ions. H-1 was stable at basic pH range and temperatures up to 50 °C, whereas H-2 was stable at acid pH range and temperatures up to 80 °C. The H. caerulea lectins exhibited dose-dependent toxicity against Artemia nauplii. Additionally, 76% of the primary structure of H-2 was determined using tandem mass spectrometry to contain a unique amino acid sequence with no similarity to any members of the animal lectin family.

Structural evolution of an amphibian-specific globin: A computational evolutionary biochemistry approach.

Studies on the globin family are continuously revealing insights into the mechanisms of gene and protein evolution. The rise of a new globin gene type in Pelobatoidea and Neobatrachia (Amphibia:Anura) from an α-globin precursor provides the opportunity to investigate the genetic and physical mechanisms underlying the origin of new protein structural and functional properties. This amphibian-specific globin (globin A/GbA) discovered in the heart of Rana catesbeiana is a monomer. As the ancestral oligomeric state of α-globins is a homodimer, we inferred that the ancestral state was lost somewhere in the GbA lineage. Here, we combined computational molecular evolution with structural bioinformatics to determine the extent to which the loss of the homodimeric state is pervasive in the GbA clade. We also characterized the loci of GbA genes in Bufo bufo. We found two GbA clades in Neobatrachia. One was deleted in Ranidae, but retained and expanded to yield a new globin cluster in Bufonidae species. Loss of the ancestral oligomeric state seems to be pervasive in the GbA clade. However, a taxonomic sampling that includes more Pelobatoidea, as well as early Neobatrachia, lineages would be necessary to determine the oligomeric state of the last common ancestor of all GbA. The evidence presented here points out a possible loss of oligomerization in Pelobatoidea GbA as a result of amino acid substitutions that weaken the homodimeric state. In contrast, the loss of oligomerization in both Neobatrachia GbA clades was linked to independent deletions that disrupted many packing contacts at the homodimer interface.

An innovative insecticidal approach based on plant protease inhibitor and Bt protoxins inhibits trypsin-like activity in zebrafish.

The Leucaena leucocephala trypsin inhibitor (LTI) + Bacillus thuringiensis (Bt) protoxins mix has been proposed as a novel larvicide agent in order to control the vector mosquito of dengue virus, Aedes aegypti, in their aquatic breeding sites. However, use of this insecticide formulation has raised concerns about its impacts on aquatic biota. In this context, this work aimed to assess the effects of LTI and Bt protoxins, separately or in combination, in zebrafish, in regard to the evaluation of toxicity at early life stages and to the presence of LTI inhibitory effects on intestinal proteases of this fish. Results showed that LTI and Bt concentrations (250 mg/L, and 0.13 mg/L, respectively), and LTI + Bt mix (250 mg/L + 0.13 mg/L) - 10 times superior to those with insecticidal action - did not cause death nor did it induce morphological changes during embryonic and larval development (3 to 144 h post-fertilization) of zebrafish. Molecular docking analyses highlighted a possible interaction between LTI and zebrafish trypsin, especially through hydrophobic interactions. In concentrations near to those with larvicidal action, LTI (0.1 mg/mL) was able to inhibit in vitro intestinal extracts of trypsin in female and male fish by 83 % and 85 %, respectively, while LTI + Bt mix promoted trypsin inhibition of 69 % in female and 65 % in male ones. These data show that the larvicidal mix can potentially promote deleterious effects to nutrition and survival in non-target aquatic organisms, especially those with trypsin-like dependent protein digestion.

Structural study and antimicrobial and wound healing effects of lectin from Solieria filiformis (Kützing) P.W.Gabrielson.

The SfL-1 isoform from the marine red algae Solieria filiformis was produced in recombinant form (rSfL-1) and showed hemagglutinating activity and inhibition similar to native SfL. The analysis of circular dichroism revealed the predominance of ß-strands structures with spectra of ßI-proteins for both lectins, which had Melting Temperature (Tm) between 41 °C and 53 °C. The three-dimensional structure of the rSfL-1 was determined by X-ray crystallography, revealing that it is composed of two ß-barrel domains formed by five antiparallel ß chains linked by a short peptide between the ß-barrels. SfL and rSfL-1 were able to agglutinate strains of Escherichia coli and Staphylococcus aureus and did not show antibacterial activity. However, SfL induced a reduction in E. coli biomass at concentrations from 250 to 125 µg mL-1, whereas rSfL-1 induced reduction in all concentrations tested. Additionally, rSfL-1 at concentrations from 250 to 62.5 µg mL-1, showed a statistically significant reduction in the number of colony-forming units, which was not noticed for SfL. Wound healing assay showed that the treatments with SfL and rSfL-1 act in reducing the inflammatory response and in the activation and proliferation of fibroblasts by a larger and fast deposition of collagen.

Purification and characterization of a mannose/N-acetyl-D-glucosamine-specific lectin from the seeds of Platymiscium floribundum Vogel.

Platymiscium floribundum lectin (PFL), a mannose/N-acetyl-D-glucosamine-specific lectin, was isolated from P. floribundum seeds using Sepharose-mannose affinity media chromatography. PFL is a glycoprotein that is a potent agglutinin for rabbit erythrocytes. In addition, PFL is highly stable because it is able to maintain its hemagglutinating activity after exposure to temperatures of up to 60 °C for 1 h and exposure to a wide pH range. The PFL purification process was monitored using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the results showed that the purified lectin consists of a single band with a molecular mass of approximately 29 kDa in either the presence or the absence of a reducing agent. The analysis of purified PFL by electrospray ionization-mass spectrometry showed that most ions had a molecular weight of 27,053 ± 2 Da, and other less abundant ions had similar molecular weights. Gel filtration shows that the lectin exists as a dimer in solution with mass at approximately 65 kDa. Sixteen peptides were sequenced, and as a result, a total of 130 amino acids were identified and resulted in a coverage of approximately 65% of the PFL sequence. The partial sequence of PFL was aligned with sequences of other lectins from evolutionarily related species, and PFL showed considerable similarity to the other lectins.

Protein crystal content analysis by mass spectrometry and preliminary X-ray diffraction of a lectin from Canavalia grandiflora seeds with modulatory role in inflammation.

RATIONALE: Lectins are a family of proteins capable of deciphering the glycan code. Several authors have published works about crystallization and mass spectrometry analyses of ConA-like lectins. However, mass spectrometry has never been used to characterize lectin crystal content. In this study, Canavalia grandiflora lectin (ConGF), a ConA-like lectin, was crystallized, part of its primary structure sequenced and the pro-inflammatory activity evaluated. In addition, the crystal content was analyzed by mass spectrometry. METHODS: ConGF was crystallized in the presence of X-Man by hanging-drop vapor diffusion at 293 K and the protein crystal content was analyzed by electrospray ionization in a SYNAPT HDMS mass spectrometer. Partial sequence was obtained by protein digestion with several proteolytic enzymes and the peptides sequenced by liquid chromatography/tandem mass spectrometry (LC/MS/MS). The pro-inflammatory potential of ConGF was also evaluated in the model of rat paw edema. RESULTS: The protein crystals consist of mature α chain and ß and γ fragments measuring 25 612 ± 2 Da, 12 962 ± 2 Da and 12 667 ± 2 Da, respectively. The crystal belongs to the orthorhombic space group I222 (unit cell parameters: a = 67.70, b = 55.90, c = 107.46 Å), assuming a monomer in the asymmetric unit. The solvent content was calculated as 43.50% and the protein content as 2.5 µg. Furthermore, a significant part of the primary structure (65.8%) was determined by mass spectrometry. CONCLUSIONS: As far as we know this is the first report of lectin crystal content characterized by mass spectrometry. Like other ConA-like lectins, GonGF induced paw edema however differing in potency and duration. The observed pro-inflammatory activity suggests that ConGF might be a useful tool in the study of inflammation processes and structure/function relationships.

Lectin from Canavalia brasiliensis seeds (ConBr) is a valuable biotechnological tool to stimulate the growth of Rhizobium tropici in vitro.

To study the interactions between a Rhizobium tropici strain and lectins isolated from the seeds of Canavalia ensiformis (ConA) and Canavalia brasiliensis (ConBr), a lectin fluorescence assay was performed. In addition, an experiment was designed to evaluate the effect of the two lectins on bacterial growth. Both lectins were found to bind to R. tropici cells, but the interactions were inhibited by D-mannose. Interestingly, only ConBr stimulated bacterial growth in proportion to the concentrations used (15.6-500 µg/mL), and the bacterial growth stimulation was inhibited by D-mannose as well. Structure/Function analyses by bioinformatics were carried out to evaluate the volume and carbohydrate recognition domain (CRD) configuration of ConA and ConBr. The difference of spatial arrangement and volume of CRD may indicate the variation between biological activities of both lectins. The results suggest that ConBr could be a promising tool for studies focusing on the interactions between rhizobia and host plants.

Codium isthmocladum lectin 1 (CiL-1): Interaction with N-glycans explains antinociceptive and anti-inflammatory activities in adult zebrafish (Danio rerio).

Inflammation and oxidative stress are processes associated with different human diseases. They are treated using drugs that have several side effects. Seaweed are sources of potentially relevant natural compounds for use as treatment of these disorders. Lectins are able to reversibly interact with complex carbohydrates and modulate cell membrane glycosylated receptors through this interaction. This study aimed to determine the antinociceptive and anti-inflammatory potential of CiL-1 in adult zebrafish by modulation of TRPA1 through lectin-glycan binding. Possible neuromodulation by TRPA1 channel was also evaluated by camphor pretreatment. CiL-1 was efficacious at all tested doses, revealing anti-nociceptive and anti-inflammatory effects in adult zebrafish. This galactose-binding lectin was also able to reduce the content of ROS in brain and liver. In silico analyses showed CiL-1 interactions with both ligands tested. LacNac2 presents the most favorable binding energy with the protein. The interaction occurs at 4 subsites as an extended conformation at the site. LacNac2-Sia had a less favorable curved-shape interaction energy. Based on the predictions made for the oligosaccharides, a tetra-antenate putative glycan was schematically constructed, illustrating an interaction between TRPA1 N-glycan and CiL-1. This binding seems to be related to CiL-1 anti-inflammatory activity as result of receptor modulation.

Structural analysis of ConBr reveals molecular correlation between the carbohydrate recognition domain and endothelial NO synthase activation.

Diocleinae lectins are highly homologous in their primary structure which features metal binding sites and a carbohydrate recognition domain (CRD). Differences in the biological activity of legume lectins have been widely investigated using hemagglutination inhibition assays, isothermal titration microcalorimetry and co-crystallization with mono- and oligosaccharides. Here we report a new lectin crystal structure (ConBr) extracted from seeds of Canavalia brasiliensis, predict dimannoside binding by docking, identify the α-aminobutyric acid (Abu) binding pocket and compare the CRD of ConBr to that of homologous lectins. Based on the hypothesis that the carbohydrate affinity of lectins depends on CRD configuration, the relationship between tridimensional structure and endothelial NO synthase activation was used to clarify differences in biological activity. Our study established a correlation between the position of CRD amino acid side chains and the stimulation of NO release from endothelium.

Molecular characterization and tandem mass spectrometry of the lectin extracted from the seeds of Dioclea sclerocarpa Ducke.

Lectin from the seeds of Dioclea sclerocarpa (DSL) was purified in a single step by affinity chromatography on a Sephadex G-50 column. The primary sequence, as determined by tandem mass spectrometry, revealed a protein with 237 amino acids and 81% of identity with ConA. DSL has a molecular mass of 25,606 Da. The ß and γ chains weigh 12,873 Da and 12,752 Da, respectively. DSL hemagglutinated rabbit erythrocytes (both native and treated with proteolytic enzymes), showing stability even after one hour of exposure to a specific pH range. The hemagglutinating activity of DSL was optimal between pH 6.0 and 8.0, but was inhibited after incubation with D-galactose and D-glucose. The pure protein possesses a molecular mass of 25 kDa by SDS-PAGE and 25,606 Da by mass spectrometry. The secondary structure content was estimated using the software SELCON3. The results indicate that b-sheet secondary structures are predominant in DSL (approximately 42.3% antiparallel b-sheet and 6.7% parallel b-sheet). In addition to the b-sheet, the predicted secondary structure of DSL features 4.1% a-helices, 15.8% turns and 31.3% other contributions. Upon thermal denaturation, evaluated by measuring changes in ellipticity at 218 nm induced by a temperature increase from 20 °C to 98 °C, DSL displayed cooperative sigmoidal behavior with transition midpoint at 84 °C and permitted the observation of two-state model (native and denatured).

Crystallization and characterization of an inflammatory lectin purified from the seeds of Dioclea wilsonii.

DwL, a lectin extracted from the seeds of Dioclea wilsonii, is a metalloprotein with strong agglutinating activity against rabbit and ABO erythrocytes, inhibited by glucose and mannose. DwL was purified by affinity chromatography on a Sephadex G-50 column and ion exchange chromatography on a HiTrap SP XL column. SDS-PAGE revealed three electrophoretic bands corresponding to the α (25,634 ± 2 Da), ß (12,873 ± 2 Da) and γ (12,779 ± 2 Da) chains. Protein sequencing was done by Tandem Mass Spectrometry. The primary sequence featured 237 amino acids and was highly homologous to other reported Diocleinae lectins. A complete X-ray dataset was collected at 2.0 Å for X-Man-complexed DWL crystals produced by the vapor diffusion method. The crystals were orthorhombic and belonged to the space group I222, with the unit-cell parameters a = 59.6, b = 67.9 and c = 109.0 Å. DWL differed in potency from other ConA-like lectins and was found to induce neutrophil migration in rats, making it particularly useful in structural/functional studies of this class of proteins.

The rise and fall of globins in the amphibia.

The globin gene repertoire of gnathostome vertebrates is dictated by differential retention and loss of nine paralogous genes: androglobin, neuroglobin, globin X, cytoglobin, globin Y, myoglobin, globin E, and the α- and ß-globins. We report the globin gene repertoire of three orders of modern amphibians: Anura, Caudata, and Gymnophiona. Combining phylogenetic and conserved synteny analysis, we show that myoglobin and globin E were lost only in the Batrachia clade, but retained in Gymnophiona. The major amphibian groups also retained different paralogous copies of globin X. None of the amphibian presented αD-globin gene. Nevertheless, two clades of ß-globins are present in all amphibians, indicating that the amphibian ancestor possessed two paralogous proto ß-globins. We also show that orthologs of the gene coding for the monomeric hemoglobin found in the heart of Rana catesbeiana are present in Neobatrachia and Pelobatoidea species we analyzed. We suggest that these genes might perform myoglobin- and globin E-related functions. We conclude that the repertoire of globin genes in amphibians is dictated by both retention and loss of the paralogous genes cited above and the rise of a new globin gene through co-option of an α-globin, possibly facilitated by a prior event of transposition.

Costus spiralis (Jacq.) Roscoe leaves fractions have potential to reduce effects of inflammatory diseases.

ETHNOPHARMACOLOGICAL RELEVANCE: Since drugs currently used to manage pain and inflammatory conditions present several side effects, the investigation of new anti-inflammatory and antinociceptive agents from folk-medicine plants is an important approach. Costus spiralis (Costaceae) has been used in Brazilian medicinal teas to treat urinary infection, cough, inflammation, arthritis, among others. AIM OF THE STUDY: The current study focused on investigating anti-inflammatory and antinociceptive effects of fractions from C. spiralis leaves using animal models. MATERIALS AND METHODS: Adults Swiss mice were used in the following experimental models: acetic acid-induced abdominal writhing, formalin-induced nociception, hot plate, zymosan-induced peritonitis, and arthritis induced by complete Freund's adjuvant. RESULTS: The presence of steroids was confirmed in all fractions. Flavonoids, condensed tannins and saponins were observed in EFL. In methanolic fraction leaves (MFL), the presence of flavonoids and pentacyclic triterpenoids was confirmed. Orally administered leaf fractions significantly reduced abdominal writhing. Fractions were ineffective in the neurogenic stage of the formalin test, but in the inflammatory stage, ethyl acetate fraction levaes (AcFL), ethanolic fraction leaves (EFL), and MFL significantly reduced paw licking time by 69.6 ± 11.9%, 58.2 ± 9.4%, and 79.6 ± 8.3%, respectively. In the hot plate test, the reaction latency was similar for treated animals and controls. However, in the peritonitis test, cell migration was significantly reduced in animals treated with chloroform fractions leaves ClFL (61.8 ± 11.4%), AcFL (58.7 ± 8.3%), EFL (39.2 ± 5.0%), and MFL (64.8 ± 4.4%). This was similar to the result observed in the chronic inflammation model, this time only the chloroform fraction was able to reduce paw edema. CONCLUSION: Our results show that leaf fractions of Costus spiralis are capable of modulating peripheral nociceptive and inflammatory responses without effects on central nervous system being potential substrates for phytochemical purification, structural and mechanistic studies.

Crystallization and preliminary X-ray diffraction analysis of the lectin from Canavalia boliviana Piper seeds.

Plant lectins are the most studied group of carbohydrate-binding proteins. Despite the high similarity between the members of the Diocleinae subtribe (Leguminosae) group, they present differing biological activities. Canavalia boliviana lectin (Cbol) was purified using a Sephadex G-50 column and crystallized in the presence of X-Man by hanging-drop vapour diffusion at 293 K. After optimization, crystals suitable for diffraction were obtained under the condition 0.1 M HEPES pH 7.5 and 3.0 M sodium formate. The crystal belonged to the monoclinic space group C2, with unit-cell parameters a = 126.70, b = 66.64, c = 64.99 A, alpha = 90.0, beta = 120.8, gamma = 90.0 degrees . Assuming the presence of a dimer in the asymmetric unit, the solvent content was estimated to be about 46%. A complete data set was collected at 1.5 A resolution.

Structural studies of a vasorelaxant lectin from Dioclea reflexa Hook seeds: Crystal structure, molecular docking and dynamics.

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.