RESUMO
We determined the specificity of BTL, a lectin from the red marine alga Bryothamnion triquetrum, toward fucosylated oligosaccharides. BTL showed a strict specificity for the core α1,6-fucosylation, which is an important marker for cancerogenesis and quality control of therapeutical antibodies. The double fucosylation α1,6 and α1,3 was also recognized, but the binding was totally abolished in the sole presence of the α1,3-fucosylation. A more detailed analysis of the specificity of BTL showed a preference for bi- and tri-antennary nonbisected N-glycans. Sialylation or fucosylation at the nonreducing end of N-glycans did not affect the recognition by the lectin. BTL displayed a strong affinity for a core α1,6-fucosylated octasaccharide with a Kd of 12 µM by titration microcalorimetry. The structural characterization of the interaction between BTL and the octasaccharide was obtained by STD-NMR. It demonstrated an extended epitope for recognition that includes the fucose residue, the distal GlcNAc and one mannose residue. Recombinant rBTL was obtained in Escherichia coli and characterized. Its binding properties for carbohydrates were studied using hemagglutination tests and glycan array analysis. rBTL was able to agglutinate rabbit erythrocytes with strong hemagglutination activity only after treatment with papain and trypsin, indicating that its ligands were not directly accessible at the cell surface. The hemagglutinating properties of rBTL confirm the correct folding and functional state of the protein. The results show BTL as a potent candidate for cancer diagnosis and as a reagent for the preparation and quality control of antibodies lacking core α1,6-fucosylated N-glycans.
Assuntos
Proteínas de Algas/química , Fucose/química , Lectinas/química , Polissacarídeos/química , Rodófitas/química , Proteínas de Algas/biossíntese , Proteínas de Algas/isolamento & purificação , Animais , Sítios de Ligação , Configuração de Carboidratos , Sequência de Carboidratos , Eritrócitos/metabolismo , Escherichia coli/química , Escherichia coli/metabolismo , Lectinas/biossíntese , Lectinas/isolamento & purificação , Dados de Sequência Molecular , Coelhos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Especificidade por SubstratoRESUMO
The aim of this work was to assess the effects of 1 week copper exposure (6.2, 108, 210 and 414microM) on Scenedesmus vacuolatus and Chlorella kessleri. The strains showed different susceptibility to copper. Copper content was determined in both strains by total X-ray reflection fluorescence analysis (TXRF). In S. vacuolatus, the increase of medium copper concentration induced an augmentation of protein and MDA content, and a significant decrease in the chlorophyll a/chlorophyll b ratio. S. vacuolatus showed a significant increase of catalase activity in 210 and 414microM of copper, and a significant increment of SOD activity and GSH content only in 414microM of copper. On the contrary, C. kessleri did not show significant differences in these parameters between 6.2 and 108microM of copper. Increased copper in the environment evokes oxidative stress and an increase in the antioxidant defenses of S. vacuolatus.