The complex processes of cellular vitamin A uptake and delivery: insights by NMR and other biophysical techniques

Vitamin A has diverse biological functions and is essential for human survival. It circulates in blood bound to serum retinol binding protein (RBP) and is transported into cells by a membrane receptor termed STRA6. The cellular trafficking and metabolism of vitamin A are regulated primarily by specific high-affinity carriers called CRBP-I and CRBP-II. Both proteins deliver retinol to membrane-bound enzymes, either for esterification with fatty acids or for oxidation to retinaldehyde. Until now it was not clear whether CRBPs may couple directly to STRA6 and to the enzymes, or the biomembranes modulate ligand uptake and delivery. To gain insights into these complex processes, we have investigated the interactions of CRBPs (in the apo and holo forms) with biomembrane mimetic systems. NMR experiments were performed at different protein:vesicles molar ratios and by varying the composition of the phospholipid liposomes and the ionic strength. Chemical shifts perturbations and line shape analysis provided information on the interacting residues and proteins conformational dynamics. As the signals were broadened beyond detection at latest steps of the titrations, the NMR data have been complemented by other biophysical measurements. The results revealed striking differences between CRBP-I and CRBP-II, despite they exhibit the same fold (a beta-barrel with two short alpha-helices) and identical retinol-binding motifs. Moreover, the interactions of the two homologs with the lipid bilayers depend upon the phospholipid composition and ionic strength. The new evidences complement our previous studies which suggested that the two primary cellular retinol carriers adopt different mechanisms for ligand uptake [1, 2]. These differences may account for their distinct functional roles in the modulation of retinoid metabolism. [1] T. Mittag, L. Franzoni, D. Cavazzini, B. Schaffhausen, G.L. Rossi, U.L. Günther J. Am. Chem. Soc. 128, 9844-9848 (2006). [2] L. Franzoni, D. Cavazzini, G.L. Rossi, C. Lücke J. Lipid Res., 51, 1332-1343 (2010).