Tanaka Kunihiko, Xu Wen, Zhou Fanfan, You Guofeng
Department of Pharmaceutics, Rutgers, The State University of New Jersey, Piscataway, New Jersey 08854, USA.
J Biol Chem. 2004 Apr 9;279(15):14961-6. doi: 10.1074/jbc.M400197200. Epub 2004 Jan 28.
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.
有机阴离子转运体（OAT）在临床上重要的阴离子药物（包括抗病毒药物、抗肿瘤药物、抗生素、抗高血压药物和抗炎药物）的体内处置过程中发挥着重要作用。我们之前报道过（Kuze, K., Graves, P., Leahy, A., Wilson, P., Stuhlmann, H., and You, G. (1999) J. Biol. Chem. 274, 1519 - 1524），天冬酰胺连接的糖基化抑制剂衣霉素显著抑制了表达小鼠有机阴离子转运体（mOAT1）的COS - 7细胞中的有机阴离子转运，这表明糖基化在mOAT1功能中起重要作用。在本研究中，我们通过将天冬酰胺突变为谷氨酰胺来研究破坏mOAT1及其人类对应物hOAT1中假定的糖基化位点的影响，并在HeLa细胞中评估突变的转运体。我们发现mOAT1中假定的糖基化位点Asp - 39未被糖基化，但hOAT1中的相应位点（Asp - 39）被糖基化。破坏Asp - 39导致mOAT1和hOAT1的转运活性完全丧失，而不影响它们在细胞表面的表达，这表明功能丧失不是因为Asp - 39本身的去糖基化，而是可能因为这个关键参与底物结合的重要氨基酸的改变。在其他位点单个替换天冬酰胺对转运活性没有影响，这表明单个位点的糖基化对OAT功能不是必需的。相反，同时替换mOAT1和hOAT1中的所有天冬酰胺会损害转运体向质膜的运输。总之，我们提供的证据表明：1）Asp - 39关键参与OAT1的底物识别；2）OAT1功能不需要单个位点的糖基化；3）糖基化在OAT1靶向质膜过程中起重要作用。本研究是首次对OAT1糖基化进行分子鉴定和表征，可能为有机阴离子转运体家族的结构 - 功能关系提供重要见解。
