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
Knowledge of the different patterns of gene expression along the male reproductive tract can assist in understanding the physiological processes of species-specific reproduction in mammals. In the present work, expression profiles of buck spermadhesin (bodhesin) genes along the reproductive tract by qRT-PCR were investigated. Total RNA from the seminal vesicle, testis, epididymis, bulbourethral gland and ductus deferens were reverse transcribed and the cDNA produced was submitted to qRT-PCR. For each homologous bodhesin gene, namely Bdh-1, Bdh-2 and Bdh-3, sets of specific primers and recombinant plasmids were prepared for gene quantification. In buck seminal vesicles, Bdh-2 is the homologue predominantly expressed, with a copy number on the order of millions of times more than Bdh-1 and thousand times more than Bdh-3. The copy number of Bdh-3 mRNA is only 10-fold greater than that of Bdh-1. Bodhesin transcripts were detected in all tissues examined, except in ductus deferens. The quantitative analysis also demonstrated clearly the differential gene expression of spermadhesin in bulbourethral gland. The striking differences in bodhesin gene expression indicate that each isoform could have a specific biological function in the buck genital tract, which deserves further detailed studies.