Francisco Nascimento Pereira-Junior

Purification and partial characterization of a new pro-inflammatory lectin from Bauhinia bauhinioides Mart (Caesalpinoideae) seeds.

A new galactose-specific lectin, named BBL, was purified from seeds of Bauhinia bauhinioides by precipitation with ammonium sulfate, followed by two steps of ion exchange chromatography. BBL haemagglutinated rabbit erythrocytes (native and treated with proteolytic enzymes) showing stability even after exposure to 60 °C for an hour. The lectin haemagglutinating activity was optimum between pH 8.0 and 9.0 and inhibited after incubation with D-galactose and its derivatives, especially α-methyl-D-galactopyranoside. The pure protein possessed a molecular mass of 31 kDa by SDS-PAGE and 28.310 Da by mass spectrometry. The lectin pro-inflammatory activity was also evaluated. The s.c. injection of BBL into rats induced a dose-dependent paw edema, an effect that occurred via carbohydrate site interaction and was significantly reduced by L-NAME, suggesting an important participation of nitric oxide in the late phase of the edema. These findings indicate that BBL can be used as a tool to better understand the mechanisms involved in inflammatory responses.

Purification, partial characterization and immobilization of a mannose-specific lectin from seeds of Dioclea lasiophylla mart.

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, β 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.

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 (β‐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. Copyright

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.

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 β 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.

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. Copyright

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.

Purification and Partial Characterization of a New Mannose/Glucose-Specific Lectin from Centrolobium tomentosum Guill. ex Benth Seeds Exhibiting Low Toxicity on Artemia sp.

Centrolobium tomentosum seeds possess a mannose/glucose-specific lectin, named CTL, which was purified using a Sepharose-4B-mannose affinity chromatography column. CTL agglutinated rabbit erythrocytes and was inhibited by D-mannose, D-glucose, N-acetyl-D-glucosamine and α-methyl-D-mannopyranoside. CTL maintained its hemagglutinating activity after incubation at a wide range of temperature, pH values and EDTA. CTL is a glycoprotein with 1.7% of carbohydrates by weight. By SDS-PAGE, CTL exhibited a profile consisting of a single band of approximately 27 kDa. Electrospray ionization mass spectrometry indicated that CTL has a molecular average mass of 27,452±2 Da. CTL partial sequence was determined by tandem mass spectrometry and showed identity with other mannosebinding lectins from Dalbergieae tribe. In addition, CTL exhibited low toxicity on Artemia sp. nauplii and appears to be mediated by interaction of the carbohydrate-binding site with the digestive tract of the nauplii, suggesting a potential avenue for exploring the exact mechanism by which lectins exert their toxicity.

Purification and molecular characterization of a novel mannose‐specific lectin from Dioclea reflexa hook seeds with inflammatory activity

A novel lectin present in Dioclea reflexa seeds (DrfL) was discovered and described in this study. DrfL was purified in a single step by affinity chromatography in a Sephadex G‐50 column. The lectin strongly agglutinated rabbit erythrocytes and was inhibited by α‐methyl‐d‐mannoside, d‐mannose, and d‐glucose. The hemagglutinating activity of DrfL is optimum at pH 5.0–7.0, stable up to 50 °C, and dependent on divalent cations. Similar to other lectins of the subtribe Diocleinae, the analysis by mass spectrometry indicated that DrfL has three chains (α, β, and γ) with masses of 25 562, 12 874, and 12 706 Da, respectively, with no disulfide bonds or glycosylation. DrfL showed inflammatory activity in the paw edema model and exhibited low cytotoxicity against Artemia sp. Copyright

Toxicity and Binding Profile of Lectins from the Genus Canavalia on Brine Shrimp

Lectins are sugar-binding proteins widely distributed in nature with many biological functions. Although many lectins have a remarkable biotechnological potential, some of them can be cytotoxic. Thus, the aim of this study was to assess the toxicity of five lectins, purified from seeds of different species of Canavalia genus. In order to determine the toxicity, assays with Artemia nauplii were performed. In addition, a fluorescence assay was carried out to evaluate the binding of lectins to Artemia nauplii. In order to verify the relationship between the structure of lectins and their cytotoxic effect, structural analysis was carried out to evaluate the volume of the carbohydrate recognition domain (CRD) of each lectin. The results showed that all lectins exhibited different toxicities and bound to a similar area in the digestive tract of Artemia nauplii. Concerning the structural analysis, differences in spatial arrangement and volume of CRD may explain the variation of the toxicity exhibited by each lectin. To this date, this is the first study that establishes a link between toxicity and structure of CRD from Diocleinae lectins.