Effects of N-glycosylation on the transport kinetics of organic anion transporting polypeptide (OATP) 1B1

Organic anion transporting polypeptide (OATP) 1B1 is crucial for hepatic drug uptake. Three N-glycosylation sites, Asn134, Asn503, and Asn516, have been identified, and substituting the amino acid at these sites with glutamine reduces the expression of the OATP1B1 protein on the plasma membrane. However, the effects of disrupted N-glycosylation on the transport kinetics of OATP1B1 molecule remained uninvestigated. This study quantitatively analyzed the impact of N-glycosylation modifications on the intrinsic transport activity of OATP1B1. We established artificial mutant OATP1B1-expressing HEK293 cell lines with substitution of asparagine at one or multiple N-glycosylation sites with glutamine. The change in molecular weight of OATP1B1 protein in the plasma membrane fraction was assessed by Western blotting. OATP1B1 transport activity was evaluated by measuring 2',7'-dichlorofluorescein (DCF) uptake to determine the Michaelis constant (K_m) and maximum transport rate (V_max). The V_max value per OATP1B1 molecule (V_max,c) was calculated by dividing V_max by the expression level of OATP1B1 in the plasma membrane fraction quantified by LC-MS/MS. Western blotting revealed that all mutant OATP1B1 proteins showed lower molecular weights than the wild-type. Among mutation sites, Asn134 was confirmed to be the primary glycosylation site, as reported previously. Regarding transport activity, the K_m value of the Asn134/503/516Gln mutant decreased to 0.3-fold that of the wild-type. However, its V_max,c value decreased to 0.2-fold that of the wild-type. Thus, N-glycosylation of OATP1B1 is not only important for the plasma membrane localization but also for transport activity.