Structural basis for both pro- and anti-inflammatory response induced by mannose-specific legume lectin from Cymbosema roseum

Legume lectins, despite high sequence homology, express diverse biological activities that vary in potency and efficacy. In studies reported here, the mannose-specific lectin from Cymbosema roseum (CRLI), which binds N-glycoproteins, shows both pro-inflammatory effects when administered by local injection and anti-inflammatory effects when by systemic injection. Protein sequencing was obtained by Tandem Mass Spectrometry and the crystal structure was solved by X-ray crystallography using a Synchrotron radiation source. Molecular replacement and refinement were performed using CCP4 and the carbohydrate binding properties were described by affinity assays and computational docking. Biological assays were performed in order to evaluate the lectin edematogenic activity. The crystal structure of CRLI was established to a 1.8 Å resolution in order to determine a structural basis for these differing activities. The structure of CRLI is closely homologous to those of other legume lectins at the monomer level and assembles into tetramers as do many of its homologues. The CRLI carbohydrate binding site was predicted by docking with a specific inhibitory trisaccharide. CRLI possesses a hydrophobic pocket for the binding of α-aminobutyric acid and that pocket is occupied in this structure as are the binding sites for calcium and manganese cations characteristic of legume lectins. CRLI route-dependent effects for acute inflammation are related to its carbohydrate binding domain (due to inhibition caused by the presence of α-methyl-mannoside), and are based on comparative analysis with ConA crystal structure. This may be due to carbohydrate binding site design, which differs at Tyr12 and Glu205 position. © 2011 Elsevier Masson SAS. All rights reserved

Purification, Characterization, and Preliminary X-Ray Diffraction Analysis of a Lactose-Specific Lectin from Cymbosema roseum Seeds

The unique carbohydrate-binding property of lectins makes them invaluable tools in biomedical research. Here, we report the purification, partial primary structure, carbohydrate affinity characterization, crystallization, and preliminary X-ray diffraction analysis of a lactose-specific lectin from Cymbosema roseum seeds (CRLII). Isolation and purification of CRLII was performed by a single step using a Sepharose-4B-lactose affinity chromatography column. The carbohydrate affinity characterization was carried using assays for hemagglutination activity and inhibition. CRLII showed hemagglutinating activity toward rabbit erythrocytes. O-glycoproteins from mucine mucopolysaccharides showed the most potent inhibition capacity at a minimum concentration of 1.2 A mu g mL(-1). Protein sequencing by mass spectrometry was obtained by the digestion of CRLII with trypsin, Glu-C, and AspN. CRLII partial protein sequence exhibits 46% similarity with the ConA-like alpha chain precursor. Suitable protein crystals were obtained with the hanging-drop vapor-diffusion method with 8% ethylene glycol, 0.1 M Tris-HCl pH 8.5, and 11% PEG 8,000. The monoclinic crystals belong to space group P2(1) with unit cell parameters a = 49.4, b = 89.6, and c = 100.8 A...

Pharmacological analysis of the neutrophil migration induced by D. rostrata lectin: Involvement of cytokines and nitric oxide

In the present study, we investigated the involvement of resident cell and inflammatory mediators in the neutrophil migration induced by chemotactic activity of a glucose/mannose-specific lectin isolated from Dioclea rostrata seeds (DrosL). Rats were injected i.p. with DrosL (125-1000 mu g/cavity), and at 2-96 h thereafter the leukocyte counts in peritoneal fluid were determined. DrosL-induced a dose-dependent neutrophil migration accumulation, which reached maximal response at 24 h after injection and declines thereafter. The carbohydrate ligand nearly abolished the neutrophil influx. Pre-treatment of peritoneal cavities with thioglycolate which increases peritoneal macrophage numbers, enhanced neutrophil migration induced by DrosL by 303%. However, the reduction of peritoneal mast cell numbers by treatment of the cavities with compound 48/80 did not modify DrosL-induced neutrophil migration. The injection into peritoneal cavities of supernatants from macrophage cultures stimulated with DrosL (125, 250 and 500 mu g/ml) induced neutrophil migration. In addition, DrosL treatment induced cytokines (TNF-alpha, IL-1 beta and CINC-1) and NO release into the peritoneal cavity of rats. Finally, neutrophil chemotaxis assay in vitro showed that the lectin (15 and 31 mu g/ml) induced neutrophil chemotaxis by even 180%. In conclusion, neutrophil migration induced by D. rostrata lectin occurs by way of the release of NO and cytokines such as IL-1 beta, TNF-alpha and CINC-1. (C) 2009 Elsevier Ltd. All rights reserved.FUNCAPCAPESCNPqAlencarAssreuyCavadaCunh

Crystallization and preliminary X-ray diffraction analysis of the lectin from Dioclea rostrata Benth seeds

D. rostrata lectin was crystallized by hanging-drop vapor diffusion. The crystal belongs to the orthorhombic space group I222 and diffracted to 1.87 Å resolution

High Primary Production Contrasts with Intense Carbon Emission in a Eutrophic Tropical Reservoir

Recent studies from temperate lakes indicate that eutrophic systems tend to emit less carbon dioxide (Co-2) and bury more organic carbon (OC) than oligotrophic ones, rendering them CO2 sinks in some cases. However, the scarcity of data from tropical systems is critical for a complete understanding of the interplay between eutrophication and aquatic carbon (C) fluxes in warm waters. We test the hypothesis that a warm eutrophic system is a source of both CO2 and CH4 to the atmosphere, and that atmospheric emissions are larger than the burial of OC in sediments. This hypothesis was based on the following assumptions: (i) OC mineralization rates are high in warm water systems, so that water column CO2 production overrides the high C uptake by primary producers, and (ii) increasing trophic status creates favorable conditions for CH4 production. We measured water-air and sediment-water CO2 fluxes, CH4 diffusion, ebullition and oxidation, net ecosystem production (NEP) and sediment OC burial during the dry season in a eutrophic reservoir in the semiarid northeastern Brazil. The reservoir was stratified during daytime and mixed during nighttime. In spite of the high rates of primary production (4858 +/- 934 mg C m(-2) d(-1)), net heterotrophy was prevalent due to high ecosystem respiration (5209 +/- 992 mg C m(-2) d(-1)). Consequently, the reservoir was a source of atmospheric CO2 (518 +/- 182 mg C m(-2) d(-1)). In addition, the reservoir was a source of ebullitive (17 +/- 10 mg C m(-2) d(-1)) and diffusive CH4 (11 +/- 6 mg C m(-2) d(-1)). OC sedimentation was high (1162 mg C m(-2) d(-1)), but our results suggest that the majority of it is mineralized to CO2 (722 +/- 182 mg C m(-2) d(-1)) rather than buried as OC (440 mg C m(-2) d(-1)). Although temporally resolved data would render our findings more conclusive, our results suggest that despite being a primary production and OC burial hotspot, the tropical eutrophic system studied here was a stronger CO2 and CH4 source than a C sink, mainly because of high rates of OC mineralization in the water column and sediments