resumo
Carbohydrate microarrays are a powerful, sensitive and high-throughput method to study carbohydrate-protein interactions, enabling the elucidation of several physiological functions and pathological mechanisms. Galactomannans (GMs) are a polysaccharide family which specific structure can have several particularities upon the source and/or undergo several changes upon the application of different extraction/purification methods. In this study, 2 lots of GMs derived from locust bean gum (LBG), were purified to ≈100% of carbohydrates, lot 1 and 2, presenting distinctive mannose/galactose (M/G) ratios, 2.4 and 3.3, respectively. Due to a closer structural simillarity reported for LBG with lot 2, this sample was selected for further treatment: dissolved and purified by diafiltration achieving reproductive mass yields of 63-67%. The purified samples were submitted to a partial acid hydrolysis treatment assisted by microwaves (MW), yielding 45% of mass and allowing a viscosity reduction, and depolymerisation effect occuring in minutes time-length. The aplication of an ultrafiltration separation process allowed a large portfolio of GMs with different molecular weights (Mw) fractions (6-21% of mass in each range of cut-off). Structural analysis, both neutral sugars and methylation, were used to characterise the structure of the carbohydrates obtained, and the main glycosidic bonds identified were 4-Manp (43-49%), 4,6-Manp (23-31%) and T-Gal (19-24%) in accordance with GMs composition. The purified LBG lot 1 and 2, and higher Mw retentates were non-convalently immobilized onto nitrocellulose-coated glass slides, with other well characterized saccharides as controls, constructing a microarray. The array was probed for recognition with different carbohydrate specific proteins, such as monoclonal antibodies (mAbs), and plant and mammalian lectins. Microarray analysis showed that, on one hand, the mannan recognizing mAbs 400-4 and LM21, confirmed the presence of β1,4-mannose backbone in the LBG-derived fractions. On the other hand, anti-galactomannan CCRC-M70 demonstrated binding correlation, associating recognition to the M/G ratio differences instead of the source, and beeing less tolerant to lower Mw. Microarray analysis of the two plant lectins used, RCA120 and ConA revelead that RCA120 binding to the saccharides presented, was unable to demonstrate a different identification between GMs and arabinogalactans, while ConA non significant binding confirms that only β-Man is present in the samples. Microarray analysis of the immune related lectins, DC-SIGN and SIGNR1, showed no to negligible binding, showing a potencial application of LBG fractions obtained during this study to clinical applications not involving an immune recognition.
autores
Bernardo A. C. Ferreira
nossos autores
Bernardo Ferreira
Estudante de doutoramentoorientadores
Lisete Machado Silva, Cláudia Pereira e Passos