Topological assessment of oatp1a1: a 12-transmembrane domain integral membrane protein with three N-linked carbohydrate chains
Topological assessment of oatp1a1: a 12-transmembrane domain integral membrane protein with three N-linked carbohydrate chains
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DOI：
10.1152/ajpgi.00584.2007
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发表时间：
2008-04-01
影响因子：
4.5
通讯作者：
Wolkoff, Allan W.
中科院分区：
文献类型：
--
作者：
Wang, Pijun;Hata, Soichiro;Wolkoff, Allan W.
有机阴离子转运蛋白1a 1（oatp 1a 1）是oatp家族高度同源转运蛋白的典型成员，表达于大鼠肝细胞基底外侧（窦状隙）表面。oatp 1a 1在质膜内的组织结构尚未得到很好的定义，基于计算机的模型预测了可能的12-和10-跨膜结构域结构。oatp 1a 1的四个潜在的N-连接糖基化位点实际上是糖基化的，它们对转运功能的影响尚未在哺乳动物系统中研究。在本研究中，拓扑结构的oatp 1a 1在大鼠肝细胞质膜的免疫荧光分析，使用表位特异性抗体，旨在区分10-从12-跨膜结构域模型。为了定位糖基化位点，将四个N-连接糖基化共有位点中的每一个处的天冬酰胺诱变为谷氨酰胺。在HeLa细胞中表达突变的oatp 1a 1构建体，并评估对蛋白质表达和转运活性的影响。这些研究表明，oatp 1a 1是一种12-跨膜结构域蛋白，其中第二和第五胞外环在天冬酰胺124、135和492处被糖基化，而天冬酰胺62处的潜在糖基化位点未被利用，这与其在跨膜结构域中的位置一致。消除一个以上糖基化位点的构建体的转运活性降低，但不一定降低转运蛋白表达。这与以下发现雅阁，即完全未糖基化的oatp 1a 1表达良好，但由于其细胞表面表达减少而位于细胞内，转运能力有限。
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.