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Mimicking cell environment: carbohydrate-protein interactions under macromolecular crowding
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As molecular recognition players glycans mediate essential physiological events with high importance in Life. Galectin-3 (Gal-3) is a key regulator of the immune system and a potent effector in diverse cellular mechanisms inside the cell, at the cell surface and at extracellular matrix. Structurally Gal-3 FL is composed by a well-folded carbohydrate recognition domain (Gal-3 CRD) and a disorder tail. Gal-3 recognition events, including the dynamics and binding, are well characterized in diluted solutions. However, Gal-3 recognize galactose-unit in a complex biological medium that contains high concentrations of proteins, polysaccharides and metabolites. On this basis, the present project reports the consequences of distinct macromolecular crowders, two synthetic polymers (PEG 3350 and Ficoll 70) and two natural proteins (BSA and Lysozyme), into Gal-3/lactose recognition process combining NMR binding experiments from receptor to ligand viewpoint. 1H15N-HSQC experiments indicate no visible chemical shift perturbation of Gal-3 CRD signals after addition of crowders, noteworthy, the intensity of 1H15N-HSQC signals decrease for all crowders. Furthermore, the intensity of 1H15N-HSQC peaks of Gal-3 CRD recovered upon addition of a saturating amount of lactose. The solution viscosity accessed by water diffusion using diffusion experiments cannot explain the decrease of 1H15N-HSQC signals intensity in crowding conditions, as well as the raise of signals intensity upon addition of lactose. Hence, Gal-3 CRD/crowders interactions should take place in solution yielding an apparent large-sized complex that increases the global molecular tumbling of Gal-3 CRD with a dramatic broadening effect on 1H15N-HSQC Gal-3 CRD resonances. Preferential interactions occurred with the BSA, mainly due to its electrostatic protein surface and size. Crowding conditions reduce the diffusion coefficient of Gal-3 CRD in one order, accordingly to the literature data, and this value remains constant after addition of lactose. The dramatic line broadening effect precludes R2/R1 ratio determination of Gal-3 CRD in presence of BSA, noteworthy we were able to estimate R2/R1 ratio of Gal-3 CRD/lactose complex in presence of BSA. R2/R1 Gal-3 CRD/lactose values are thus different from that obtained in diluted solution mainly due to the raise in viscosity after addition of BSA. Gal-3 FL/crowder interactions were also exploited using BSA as the more physiological relevant crowder and ficoll as synthetic carbohydrate polymer. In contrast to Gal-3 CRD, no recovery of 1H15N-HSQC signal intensity was observed to Gal-3 FL after addition of lactose. Solution viscosity can prevent the detection of 1H15N-HSQC peaks in the case of larger Gal-3 FL by decreasing dramatically T2, or Gal-3 FL can self-associate in crowding conditions and potentiated by the presence of the unfolded tail. Noteworthy, STD-NMR experiment of lactose in presence of Gal-3 FL and BSA were performed indicating that lactose binding mode is conserved in crowding conditions. Finally, crowding conditions may have a biological implication contributing to co-localize Gal-3 at the cell surface favouring protein cell-adhesion and eventually promoting functional self-assembly of Gal-3.