Ito L. Barroso-Neto

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.

Structural analysis of a Dioclea sclerocarpa lectin: Study on the vasorelaxant properties of Dioclea lectins

Lectins are proteins that show a variety of biological activities. However, they share in common at least one domain capable of recognizing specific carbohydrates reversibly without changing its structure. The legume lectins family is the most studied family of plant lectins, in particular the Diocleinae subtribe, which possesses high degree of structural similarity, but variable biological activities. This variability lies in small differences that can be analyzed in studies based on structures. In particular, Dioclea sclerocarpa seed lectin (DSL) presents low ability to relax endothelialized rat aorta in comparison with other Dioclea lectins such as Dioclea violacea (DVL), Dioclea virgata (DvirL) and Dioclea rostrata (DRL). The DSL relaxation mechanism relies on nitric oxide production and carbohydrate recognition domain (CRD). This feature can be explained by structural differences, since DSL has a carbohydrate recognition domain design less favorable. In addition, the presence of a glutamate residue at position 205 proved to be a decisive factor for the low relaxant effect of Dioclea lectins.

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.

Inactivation of ovine cyclooxygenase-1 by bromoaspirin and aspirin: a quantum chemistry description.

The resulting noncovalent bonding of the salicylic acid to ovine COX-1 after bromoaspirin and aspirin acetylation by Ser530 is investigated within the scope of density functional theory considering a 6.5 Å radius binding pocket. We have not only took full advantage of published X-ray structural data for the ovine COX-1 cocrystallized with bromoaspirin, but we also have improved that data through computation, finding good estimates for the hydrogen atom positions at the residues of the binding pocket, and repositioning the Ser530Ac[Br;H] lateral chain and salicylic acid by total energy minimization procedures employing LDA and GGA+D exchange-correlation functionals. Using bromoaspirin as a template, we have simulated the positioning of aspirin in the binding pocket, estimating its interaction energy with each of its neighbor COX-1 residues. We demonstrate that the binding energies of bromoaspirin and aspirin to COX-1 are very close when second-order quantum refinements of the structural data are performed, which points to an explanation on why the IC(50) values for the 126 μM COX-1 activity of both bromoaspirin and aspirin are practically the same. Attracting and repelling residues were identified, being shown that Arg120 is the most effective residue attracting the salicylic acid, followed by Ala527, Leu531, Leu359, and Ser353. On the other hand, Glu524 was found the most effective repulsive residue (strength interaction comparable to Arg120).

CRLI induces vascular smooth muscle relaxation and suggests a dual mechanism of eNOS activation by legume lectins via muscarinic receptors and shear stress

Lectins are proteins able to recognize carbohydrates, without modifying their structure, via the carbohydrate-recognition domain (CRD). Here, the three-dimensional structure of the mannose-binding lectin isolated from Cymbosema roseum (CRLI) was determined with X-man molecule modeled into the carbohydrate recognition domain. CRLI relaxant activity in thoracic rat aorta was also investigated, and based on the results, a molecular docking of CRLI with heparan sulfate was performed to investigate the possible interaction with mechanoreceptors involved in vasorelaxation. CRLI (IC₅₀=12.4 μg mL(-)(1)) elicited vasorelaxant response (96%) in endothelialized rat aorta contracted with phenylephrine. Endothelium-derived relaxant factors, extracellular calcium (Ca(2+)e) and muscarinic receptors were also evaluated as putative participants in the CRLI relaxant effect. CRLI relaxant effect was blocked by L-NAME, a nonselective inhibitor of nitric oxide synthase (NOS), and partially inhibited in a calcium-free solution (0Ca) and by atropine, but it remained unchanged in the presence of indomethacin and TEA. In summary, our data suggest interaction between CRLI and muscarinic receptors located in vascular endothelial cells leading to NOS activation triggered by a mechanism that involves Ca(2+)e along with the ability of CRLI to interact with heparan sulfate, a highly rated mechanoreceptor involved in eNOS activation.

An ab initio explanation of the activation and antagonism strength of an AMPA-sensitive glutamate receptor

We report quantum biochemistry calculations focusing on the binding pocket of the glutamate receptor co-crystallized with agonists (full and partial) and a antagonist. The calculated electronic binding energy follows the order AMPA > glutamate > kainate > DNQX, which explains published experimental data on GluR2 activation and antagonism strength.

Controlled Release of Nor-β-lapachone by PLGA Microparticles: A Strategy for Improving Cytotoxicity against Prostate Cancer Cells.

Prostate cancer is one of the most common malignant tumors in males and it has become a major worldwide public health problem. This study characterizes the encapsulation of Nor-β-lapachone (NβL) in poly(d,l-lactide-co-glycolide) (PLGA) microcapsules and evaluates the cytotoxicity of the resulting drug-loaded system against metastatic prostate cancer cells. The microcapsules presented appropriate morphological features and the presence of drug molecules in the microcapsules was confirmed by different methods. Spherical microcapsules with a size range of 1.03 ± 0.46 μm were produced with an encapsulation efficiency of approximately 19%. Classical molecular dynamics calculations provided an estimate of the typical adsorption energies of NβL on PLGA. Finally, the cytotoxic activity of NβL against PC3M human prostate cancer cells was demonstrated to be significantly enhanced when delivered by PLGA microcapsules in comparison with the free drug.

AIP1 is a novel Agenet/Tudor domain protein from Arabidopsis that interacts with regulators of DNA replication, transcription and chromatin remodeling

BackgroundDNA replication and transcription are dynamic processes regulating plant development that are dependent on the chromatin accessibility. Proteins belonging to the Agenet/Tudor domain family are known as histone modification “readers” and classified as chromatin remodeling proteins. Histone modifications and chromatin remodeling have profound effects on gene expression as well as on DNA replication, but how these processes are integrated has not been completely elucidated. It is clear that members of the Agenet/Tudor family are important regulators of development playing roles not well known in plants.MethodsBioinformatics and phylogenetic analyses of the Agenet/Tudor Family domain in the plant kingdom were carried out with sequences from available complete genomes databases. 3D structure predictions of Agenet/Tudor domains were calculated by I-TASSER server. Protein interactions were tested in two-hybrid, GST pulldown, semi-in vivo pulldown and Tandem Affinity Purification assays. Gene function was studied in a T-DNA insertion GABI-line.ResultsIn the present work we analyzed the family of Agenet/Tudor domain proteins in the plant kingdom and we mapped the organization of this family throughout plant evolution. Furthermore, we characterized a member from Arabidopsis thaliana named AIP1 that harbors Agenet/Tudor and DUF724 domains. AIP1 interacts with ABAP1, a plant regulator of DNA replication licensing and gene transcription, with a plant histone modification “reader” (LHP1) and with non modified histones. AIP1 is expressed in reproductive tissues and its down-regulation delays flower development timing. Also, expression of ABAP1 and LHP1 target genes were repressed in flower buds of plants with reduced levels of AIP1.ConclusionsAIP1 is a novel Agenet/Tudor domain protein in plants that could act as a link between DNA replication, transcription and chromatin remodeling during flower development.

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.