The Effects of N-Glycosylation on the Expression and Transport Activity of OATP1A2 and OATP2B1.

• This study revealed that Asn124, Asn135 and Asn492 in OATP1A2 and Asn176 and Asn538 in OATP2B1 are N -glycosylation sites. • The effect of deletion of N -glycans on the transport activity of OATPs was evaluated based on the kinetics parameters per single molecules using the expression level of OATPs on the plasma membrane quantified by LC-MS/MS. • Among the three N -glycans of OATP1A2, those at Asn124 and Asn135 are especially important for the expression on the plasma membrane and transport activity. • Deletion of each N -glycan of OATP2B1 reduces the expression on the plasma membrane and enhances substrate affinity, and these effects becomes stronger when both N -glycans are disrupted. Organic anion transporting polypeptide (OATP)1A2 and OATP2B1 have potential N -glycosylation sites, but their influence remains unclear. This study aimed to identify the N -glycosylation sites of OATP1A2/2B1 and investigate their impact on the expression and function of OATP1A2/2B1. Human embryonic kidney cells expressing OATP1A2 or OATP2B1 (HEK293-OATP1A2/2B1) were exposed to tunicamycin, an N -glycosylation inhibitor, and a plasma membrane fraction (PMF) Western blot assay and an estrone 3-sulfate (E3S) uptake study were conducted. HEK293-OATP1A2/OATP2B1 cell lines with mutation(s) at potential N -glycosylation sites were established, and the Western blotting and uptake study were repeated. Tunicamycin reduced the PMF levels and E3S uptake of OATP1A2/OATP2B1. The Asn124Gln, Asn135Gln, and Asn492Gln mutations in OATP1A2 and Asn176Gln and Asn538Gln mutations in OATP2B1 reduced the molecular weights of the OATP molecules and their PMF levels. The PMF levels of OATP1A2 Asn124/135Gln, OATP1A2 Asn124/135/492Gln, and OATP2B1 Asn176/538Gln were further reduced. The maximum transport velocities of OATP1A2 Asn124Gln, OATP1A2 Asn135Gln, and OATP2B1 Asn176/538Gln were markedly reduced to 10 %, 4 %, and 10 % of the wild-type level, respectively. In conclusion, the N -glycans at Asn124 and Asn135 of OATP1A2 and those at Asn176 and Asn538 of OATP2B1 are essential for the plasma membrane expression of these molecules and also affect their transport function. [ABSTRACT FROM AUTHOR]