N-glycosylation Dictates Proper Processing of Organic Anion Transporting Polypeptide 1B1

Abstract

Organic anion transporting polypeptides (OATPs) have been extensively recognized as key determinants of absorption, distribution, metabolism and excretion (ADME) of various drugs, xenobiotics and toxins. Putative N-glycosylation sites located in the extracellular loops 2 and 5 is considered a common feature of all OATPs and some members have been demonstrated to be glycosylated proteins. However, experimental evidence is still lacking on how such a post-translational modification affect the transport activity of OATPs and which of the putative glycosylation sites are utilized in these transporter proteins. In the present study, we substituted asparagine residues that are possibly involved in N-glycosylation with glutamine residues and identified three glycosylation sites (Asn134, Asn503 and Asn516) within the structure of OATP1B1, an OATP member that is mainly expressed in the human liver. Our results showed that Asn134 and Asn516 are used for glycosylation under normal conditions; however, when Asn134 was mutagenized, an additional asparagine at position 503 is involved in the glycosylation process. Simultaneously replacement of all three asparagines with glutamines led to significantly reduced protein level as well as loss of transport activity. Further studies revealed that glycosylation affected stability of the transporter protein and the unglycosylated mutant was retained within endoplasmic reticulum.

Expression Patterns of Organic Anion Transporting Polypeptides 1B1 and 1B3 Protein in Human Pediatric Liver

“…Posttranslational modification by glycosylation has been suggested to affect intracellular localization and the function of some transporters (Hardikar et al, 1995;Yao et al, 2012), including localization to the basolateral membrane and the transport function of OATP1B3 (Letschert et al, 2004;Schwarz et al, 2011). As the ratio of glycosylated to nonglycosylated protein changes, our data suggest that, in addition to protein expression, age-associated differences in post-translational modifications, such as glycosylation, may have an effect on the developmental pattern of OATP1B3 function.…”

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.…”

Molecular Characterization of Zebrafish Oatp1d1 (Slco1d1), a Novel Organic Anion-transporting Polypeptide

“…Cell Surface Biotinylation-Surface biotinylation of Oatp1d1 at the plasma membrane was performed as described previously (20) with some modifications. Transiently transfected cells (with pcDNA3.1/His vector alone or with pcDNA3.1/ His-Oatp1d1) were washed three times with PBS, and surface proteins were biotinylated with Sulfo-NHS-LC-LC-biotin (1 mg/ml; Pierce) in PBS for 2 h at 4°C.…”