Topological assessment of oatp1a1: a 12-transmembrane domain integral membrane protein with three N-linked carbohydrate chains

Abstract

Organic anion transport protein 1a1 (oatp1a1), a prototypical member of the oatp family of highly homologous transport proteins, is expressed on the basolateral (sinusoidal) surface of rat hepatocytes. The organization of oatp1a1 within the plasma membrane has not been well defined, and computer-based models have predicted possible 12- as well as 10-transmembrane domain structures. Which of oatp1a1's four potential N-linked glycosylation sites are actually glycosylated and their influence on transport function have not been investigated in a mammalian system. In the present study, topology of oatp1a1 in the rat hepatocyte plasma membrane was examined by immunofluorescence analysis using an epitope-specific antibody designed to differentiate a 10- from a 12-transmembrane domain model. To map glycosylation sites, the asparagines at the each of the four N-linked glycosylation consensus sites were mutagenized to glutamines. Mutagenized oatp1a1 constructs were expressed in HeLa cells, and effects on protein expression and transport activity were assessed. These studies revealed that oatp1a1 is a 12-transmembrane-domain protein in which the second and fifth extracellular loops are glycosylated at asparagines 124, 135, and 492, whereas the potential glycosylation site at asparagine 62 is not utilized, consistent with its position in a transmembrane domain. Constructs in which more than one glycosylation site were eliminated had reduced transport activity but not necessarily reduced transporter expression. This was in accord with the finding that fully unglycosylated oatp1a1 was well expressed but located intracellularly with limited transport ability as a consequence of its reduced cell surface expression.

Organic Anion Transporting Polypeptides

“…In 2008, the 12 TM domain structure of rOATP1A1 was confirmed using site-directed mutagenesis of putative N-glycosylation sites (P. Wang, Hata, Xiao, Murray, & Wolkoff, 2008). It was demonstrated that rOATP1A1 was N-glycosylated in the second and fifth extracellular loop, and that the unglycosylated protein was retained intracellularly and thus caused reduced transport function (P. Wang, et al, 2008). Similar results were recently published for hOATP1B1 (Yao et al, 2012).…”

“…Immunostaining was only observed in detergent permeabilized cells (Abe et al, 2001), confirming a topology with both termini on the cytoplasmic side of the membrane. In 2008, the 12 TM domain structure of rOATP1A1 was confirmed using site-directed mutagenesis of putative N-glycosylation sites (P. Wang, Hata, Xiao, Murray, & Wolkoff, 2008). It was demonstrated that rOATP1A1 was N-glycosylated in the second and fifth extracellular loop, and that the unglycosylated protein was retained intracellularly and thus caused reduced transport function (P. Wang, et al, 2008).…”

Oatp1a1 Requires PDZK1 to Traffic to the Plasma Membrane by Selective Recruitment of Microtubule-Based Motor Proteins

“…The superfamily is composed of six families based on 40% amino acid sequence identity divided into subfamilies that have 60% amino acid homology (Iusuf et al, 2012;Hagenbuch and Stieger, 2013). OATP family members are expressed in multiple organs, including the brain, heart, kidney, intestine, and liver, and they share structural similarities that include predicted 12 transmembrane domains (Wang et al, 2008;Hagenbuch and Stieger, 2013) and 3-4 N-linked glycosylation sites (Wang et al, 2008;Yao et al, 2012). Reduced transport activity of OATPs, accompanied by adverse drug reactions, have been described with Fig.…”

Xenobiotic, Bile Acid, and Cholesterol Transporters: Function and Regulation

“…Hypoglycosylation of Oatp1a1, using the glycosylation inhibitor tunicamycin, causes intracellular retention and diminished transport of taurocholate in X. laevis oocytes . Both membrane targeting and functional activity of Oatp1a1 are regulated by the extent of N-glycosylation Wang et al, 2008c). Similar to the post-translational regulation of uptake transporters, apically expressed efflux transporters Mrp2 , BCRP (Mohrmann et al, 2005), Bsep (Mochizuki et al, 2007), and MDR1 are regulated by glycosylation and cellular trafficking.…”