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.
Highlights
The organic anion-transporting polypeptides (OATPs, gene symbol SLCO) are a family of transporters that mediate sodiumindependent transport of a wide spectrum of structurally independent compounds [1]
We treated plasma membrane proteins isolated from cells expressing OATP1B1 with N-glycosidase F, an enzyme that removes the Nlinked carbohydrate groups from glycoproteins
These results suggested that OATP1B1 is glycosylated in HEK293 cells and that glycosylation affects its transport function
Summary
The organic anion-transporting polypeptides (OATPs, gene symbol SLCO) are a family of transporters that mediate sodiumindependent transport of a wide spectrum of structurally independent compounds [1]. Substrates of OATPs are mainly amphipathic organic molecules including bile salts, hormones and their conjugates, toxins and different drugs. OATPs have been extensively recognized as key determinants of absorption, distribution, metabolism and excretion (ADME) of various drugs, xenobiotics and toxins because of their broad substrate specificity, wide tissue distribution and the involvement of drug-drug interaction [2,3]. There are 12 members of the human OATP family: OATP1A2, 1B1, 1B3, 1B7, 1C1, 2A1, 2B1, 3A1, 4A1, 4C1, 5A1 and 6A1 [4,5,6,7]. More and more drugs are identified as substrates of this OATP family member [9]
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