Role of Glycosylation in the Organic Anion Transporter OAT1

Abstract

Organic anion transporters (OAT) play essential roles in the body disposition of clinically important anionic drugs, including antiviral drugs, antitumor drugs, antibiotics, antihypertensives, and anti-inflammatories. We reported previously (Kuze, K., Graves, P., Leahy, A., Wilson, P., Stuhlmann, H., and You, G. (1999) J. Biol. Chem. 274, 1519 -1524) that tunicamycin, an inhibitor of asparagine-linked glycosylation, significantly inhibited organic anion transport in COS-7 cells expressing a mouse organic anion transporter (mOAT1), suggesting an important role of glycosylation in mOAT1 function. In the present study, we investigated the effect of disrupting putative glycosylation sites in mOAT1 as well as its human counterpart, hOAT1, by mutating asparagine to glutamine and assessing mutant transporters in HeLa cells. We showed that the putative glycosylation site Asp-39 in mOAT1 was not glycosylated but the corresponding site (Asp-39) in hOAT1 was glycosylated. Disrupting Asp-39 resulted in a complete loss of transport activity in both mOAT1 and hOAT1 without affecting their cell surface expression, suggesting that the loss of function is not because of deglycosylation of Asp-39 per se but rather is likely because of the change of this important amino acid critically involved in the substrate binding. Single replacement of asparagines at other sites had no effect on transport activity indicating that glycosylation at individual sites is not essential for OAT function. In contrast, a simultaneous replacement of all asparagines in both mOAT1 and hOAT1 impaired the trafficking of the transporters to the plasma membrane. In summary, we provided the evidence that 1) Asp-39 is crucially involved in substrate recognition of OAT1, 2) glycosylation at individual sites is not required for OAT1 function, and 3) glycosylation plays an important role in the targeting of OAT1 onto the plasma membrane. This study is the first molecular identification and characterization of glycosylation of OAT1 and may provide important insights into the structure-function relationships of the organic anion transporter family. Organic anion transporters (OAT)1 play essential roles in the body disposition of clinically important anionic drugs including anti-human immunodeficiency virus therapeutics, antitumor drugs, antibiotics, antihypertensives, and anti-inflammatories (1). Several OAT isoforms have been identified by us and others (2). OAT1 and -3 are predominantly expressed in the kidney and brain. In the kidney, these transporters utilize a tertiary transport mechanism to move organic anions across the basolateral membrane into the proximal tubule cells for subsequent exit/elimination across the apical membrane into urine. Through this tertiary transport mechanism, Na ϩ -K ϩ -ATPase maintains an inwardly directed (blood to cell) Na ϩ gradient. The Na ϩ gradient then drives a Na ϩ -dicarboxylate cotransporter, sustaining an outwardly directed dicarboxylate gradient that is utilized by a dicarboxylate/organic anion (OA) exchanger to move th...

A Three-dimensional Model of Human Organic Anion Transporter 1

“…These deviations likely result from slight differences in alignment of the long extracellular loop and differences in the energy minimization procedures. The rbOCT2 model also takes into account that residue Asn 39 is a glycosylation site in loop 1-2 and not present in domain 1 as based on the GlpT template (35,37). In the hOAT1 model, the Asn 39 was left in domain 1 as it did not affect the remaining alignment.…”

Organic Anion Transporter OAT1 Undergoes Constitutive and Protein Kinase C-regulated Trafficking through a Dynamin- and Clathrin-dependent Pathway

“…Recent investigation from our laboratory on the structure-function relationship of OATs revealed that glycosylation is necessary for the targeting of these transporters to the plasma membrane (17,18).…”

Transport Mechanisms of Nucleosides and Nucleoside Analogues ReverseTranscriptase Inhibitors in the Brain

“…After exiting the ER, hENT1 was translocated to the Golgi and glycosylated. Glycosylation probably plays a significant role in trafficking and function for a number of transporters including ENTs (Cai et al, 2005, Hendriks et al, 2004, Tanaka et al, 2004. hENT1 was then trafficked to the plasma membrane in association with the microtubule network in a variety of vesicles; in the plasma membrane hENT1 was co-localized with actin, which suggested that this transporter was anchored in the membrane by the actin cytoskeleton (Nivillac et al, 2011).…”