Purification and biological effects of a C-type lectin isolated from Bothrops moojeni

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

A lectin from the lichenized Basidiomycete Dictyonema glabratum

A lectin with hemagglutinating activity has been isolated from an aqueous extract of the symbiotic phenotype of Dictyonema glabratum and its cyanobacterial photobiont Scytonema sp. The purified lectin had a pi of 6.8 and its molecular mass was investigated by electrospray ionization mass spectrometry, gel filtration and SDS-PAGE, which indicated its native conformation as a dimer formed by two identical subunits of 16540 Da. The lectin is a glycoprotein with a low degree of glycosylation, containing galactose, xylose, glucose and mannose as neutral monosaccharides, in addition to glucosamine, which could indicate both N- and O-linkages. Amino acid analysis showed the predominance of nonpolar residues such as phenylalanine. Agglutination of human erythrocytes required divalent cations, which is affected by addition of EDTA. The lectin was more stable at 30 degreesC or less for at least 1 h and between pH 5.0 and 7.0. Among the various compounds tested for hemagglutination inhibition, N-acetylgalactosamine was the most active. The potential role of this lectin in recognition of the compatible cyanobacterial photobiont is discussed.

Crystallization and preliminary x-ray analysis of Parkia pendula lectin

Lectins are proteins important in various biological processes such as infection, cell differentiation and metastasis. The Parkia pendula lectin has been crystallized using the hanging-drop vapour diffusion method. X-ray diffraction data were collected using a Rigaku RU300 rotating anode generator and R-AXIS IV diffractometer. The cell parameters for P. pendula lectin are a=93.7 Angstrom b=161.1 Angstrom, c=80.0 Angstrom and space group C222. The maximum resolution was of 2.98 Angstrom. These data showed a R-sym=12.8%.

Initial structural analysis of an alpha(4)beta(4) C-type lectin from the venom of Crotalus durissus terrificus

Convulxin, an alphabeta C-type lectin, is a potent platelet activator isolated from the venom of the South American rattlesnake Crotalus durissus terrificus. It is a 26.5 kDa alphabeta heterodimer consisting of two homologous disulfide-linked chains. The crystals belong to space group I4, with unit-cell parameters a = b = 131.61, c = 121.85 Angstrom, and diffraction data were collected to 2.7 Angstrom. The structure was solved by molecular replacement and the asymmetric unit contains two alphabeta heterodimers, each of which forms a disulfide-linked cyclic alpha(4)beta(4) tetramer in the unit cell. These alpha(4)beta(4) tetramers are stacked to form a large solvent channel.

Crotacetin, a novel snake venom C-type lectin homolog of convulxin, exhibits an unpredictable antimicrobial activity

Snake venom (sv) C-type lectins encompass a group of hemorrhagic toxins that are capable of interfering with blood stasis. A very well-studied svC-type lectin is the heterodimeric toxin, convulxin (CVX), from the venom of South American rattlesnake Crotalus durissus terrificus. CVX is able to activate platelets and induce their aggregation by acting via p62/GPVI collagen receptor. By using polymerase chain reaction homology screening, we have cloned several cDNA precursors of CVX subunit homologs. One of them, named crotacetin (CTC) beta-subunit, predicts a polypeptide with a topology very similar to the tridimensional conformations of other subunits of CVX-like snake toxins, as determined by computational analysis. Using gel permeation and reverse-phase high-performance liquid chromatography, CTC was purified from C. durissus venoms. CTC can be isolated from the venom of several C. durissus subspecies, but its quantitative predominance is in the venom of C. durissus cascavella. Functional analysis indicates that CTC induces platelet aggregation, and, importantly, exhibits an antimicrobial activity against Gram-positive and -negative bacteria, comparable with CVX.

Quartz Crystal Microbalance monitoring the real-time binding of lectin with carbohydrate with high and low molecular mass

Quartz Crystal Microbalance (QCM) was used to monitor the mass changes on a quartz crystal surface containing immobilized lectins that interacted with carbohydrates. The strategy for lectin immobilization was developed on the basis of a multilayer system composed of Au-cystamine-glutaraldehyde-lectin. Each step of the immobilization procedure was confirmed by FTIR analysis. The system was used to study the interactions of Concanavalin A (ConA) with maltose and Jacalin with Fetuin. The real-time binding of different concentrations of carbohydrate to the immobilized lectin was monitored by means of QCM measurements and the data obtained allowed for the construction of Langmuir isotherm curves. The association constants determined for the specific interactions analyzed here were (6.4 +/- 0.2) X 10(4) M-1 for Jacalin-Fetuin and (4.5 +/- 0.1) x 10(2) M-1 for ConA-maltose. These results indicate that the QCM constitutes a suitable method for the analysis of lectin-carbohydrate interactions, even when assaying low molecular mass ligands such as disaccharides. Published by Elsevier B.V.

Purification, partial characterization and preliminary X-ray diffraction analysis of a mannose-specific lectin from Cymbosema roseum seeds

A lectin from Cymbosema roseum seeds (CRL) was purified, characterized and crystallized. The best crystals grew in a month and were obtained by the vapour-diffusion method using a precipitant solution consisting of 0.1 M Tris-HCl pH 7.8, 8% (w/v) PEG 3350 and 0.2 M proline at a constant temperature of 293 K. A data set was collected to 1.77 angstrom resolution at a synchrotron-radiation source. CRL crystals are orthorhombic, belonging to space group P2(1)2(1)2(1). Crystallographic refinement and full amino-acid sequence determination are in progress.

Structure of a lectin from Canavalia gladiata seeds: new structural insights for old molecules

Background: Lectins are mainly described as simple carbohydrate- binding proteins. Previous studies have tried to identify other binding sites, which possible recognize plant hormones, secondary metabolites, and isolated amino acid residues. We report the crystal structure of a lectin isolated from Canavalia gladiata seeds ( CGL), describing a new binding pocket, which may be related to pathogen resistance activity in ConA- like lectins; a site where a non- protein amino- acid, aaminobutyric acid ( Abu), is bound.Results: the overall structure of native CGL and complexed with alpha- methyl- mannoside and Abu have been refined at 2.3 angstrom and 2.31 angstrom resolution, respectively. Analysis of the electron density maps of the CGL structure shows clearly the presence of Abu, which was confirmed by mass spectrometry.Conclusion: the presence of Abu in a plant lectin structure strongly indicates the ability of lectins on carrying secondary metabolites. Comparison of the amino acids composing the site with other legume lectins revealed that this site is conserved, providing an evidence of the biological relevance of this site. This new action of lectins strengthens their role in defense mechanisms in plants.

Crystal structure of a lectin from Canavalia maritima (ConM) in complex with trehalose and maltose reveals relevant. mutation in ConA-like lectins

The crystal structure of Canavalia maritima lectin (ConM) complexed with trehalose and maltose revealed relevant point mutations in ConA-like lectins. ConM with the disaccharides and other ConA-like lectins complexed with carbohydrates demonstrated significant differences in the position of H-bonds. The main difference in the ConM structure is the replacement of Pro202 by Ser202, a residue that promotes the approximation of Tyr12 to the carbohydrate-binding site. The O-6' of the second glucose ring in maltose interacts with Tyr12, while in trehalose the interaction is established by the O-2' and Tyr12, explaining the higher affinity of ConM for disaccharides compared to monosaccharides. (c) 2006 Elsevier B.V. All rights reserved.

Crystallization and preliminary X-ray diffraction analysis of an anti-H(O) lectin from Lotus tetragonolobus seeds

The seed lectin from Lotus tetragonolobus (LTA) has been crystallized. The best crystals grew over several days and were obtained using the vapour-diffusion method at a constant temperature of 293 K. A complete structural data set was collected at 2.00 angstrom resolution using a synchrotron-radiation source. LTA crystals were found to be monoclinic, belonging to space group P2(1), with unit-cell parameters a = 68.89, b = 65.83, c = 102.53 angstrom, alpha = gamma = 90, beta = 92 degrees. Molecular replacement yielded a solution with a correlation coefficient and R factor of 34.4 and 51.6%, respectively. Preliminary analysis of the molecular-replacement solution indicates a new quaternary association in the LTA structure. Crystallographic refinement is under way.

Genotypes of the mannan-binding lectin gene and susceptibility to visceral leishmaniasis and clinical complications

Background. Visceral leishmaniasis (VL) is almost always lethal if not treated, but most infections with the causative agents are clinically silent. Mannan-binding lectin (MBL), an opsonin, is a candidate molecule for modifying progression to VL because it may enhance infection with intracellular pathogens. Mutations in the MBL2 gene decrease levels of MBL and may protect against development of VL. This case-control study examines genotypes of MBL2 and levels of MBL in individuals presenting with different outcomes of infection with Leishmania chagasi.Methods. Genotypes for MBL2 and levels of serum MBL were determined in uninfected control subjects (n=76) and in individuals presenting with asymptomatic infection (n=90) or VL (n=69).Results. Genotypes resulting in high levels of MBL were more frequent (odds ratio [OR], 2.5 [95% confidence interval {CI}, 1.3-5.0]; P=.006) among individuals with VL than among those with asymptomatic infections and were even more frequent (OR, 3.97 [95% CI, 1.10-14.38];P=.043) among cases of VL presenting with clinical complications than among those with uneventful courses. Serum levels of MBL were higher (P=.011) in individuals with VL than in asymptomatic infections.Conclusions. Genotypes of the MBL2 gene predict the risk for developing VL and clinical complications in infections with L. chagasi.

Native crystal structure of a nitric oxide-releasing lectin from the seeds of Canavalia maritima

Here, we report the crystallographic study of a lectin from Canavalia maritima seeds (ConM) and its relaxant activity on vascular smooth muscle, to provide new insights into the understanding of structure/function relationships of this class of proteins. ConM was crystallized and its structure determined by standard molecular replacement techniques. The amino acid residues, previously suggested incorrectly by manual sequencing, have now been determined as I17, I53, S129, S134, G144, S164, P165, S187, V190, S169, T196, and S202. Analysis of the structure indicated a dimer in the asymmetric unit, two metal binding sites per monomer, and loops involved in the molecular oligomerization. These confer 98% similarity between ConM and other previously described lectins, derived from Canavalia ensiformis and Canavalia brasiliensis. Our functional data indicate that ConM exerts a concentration-dependent relaxant action on isolated aortic rings that probably occurs via an interaction with a specific lectin-binding site on the endothelium, resulting in a release of nitric oxide. (C) 2005 Elsevier B.V. All rights reserved.