N-glycans, not the GPI anchor, mediate the apical targeting of a naturally glycosylated, GPI-anchored protein in polarised epithelial cells
N-glycans, not the GPI anchor, mediate the apical targeting of a naturally glycosylated, GPI-anchored protein in polarised epithelial cells
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DOI：
10.1242/jcs.01386
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发表时间：
2004-10-01
影响因子：
4
通讯作者：
Hooper, NM
中科院分区：
文献类型：
--
作者：
Pang, S;Urquhart, P;Hooper, NM
糖基-磷脂酰肌醇（GPI）锚通过与脂筏的相互作用介导极化上皮细胞中蛋白质的顶端分选。在这里，我们研究了通过选择性点突变去除GPI锚添加信号或N-连接糖基化位点或两者的天然N-糖基化和GPI锚定的膜二肽酶的顶端靶向所需的信号。活性测定、免疫印迹和免疫荧光显微镜显示，缺乏GPI锚的构建体从Madin-Darby犬肾（MDCK）细胞分泌，而保留GPI锚的构建体附着在细胞表面，与糖基化状态无关。野生型膜二肽酶优先表达于NH CK和CaCo-2细胞的顶端表面。相比之下，缺乏N-聚糖的GPI锚定构建体优先靶向两种细胞类型的基底外侧表面。在缺乏GPI锚的构建体中，N-聚糖也将蛋白质靶向顶端表面。顶部靶向的糖基化和基底侧靶向的非糖基化GPI锚定形式的蛋白质都位于洗涤剂不溶性脂筏中。这些数据表明，是N-聚糖，而不是GPI锚与脂筏的结合，决定了极化上皮细胞中内源性N-糖基化的GPI锚定蛋白的顶端靶向。
The glycosyl-phosphatidylinositol (GPI) anchor mediates the apical sorting of proteins in polarised epithelial cells through its interaction with lipid rafts. Here we investigated the signals required for the apical targeting of the naturally N-glycosylated and GPI-anchored membrane dipeptidase by selective point mutation to remove the GPI anchor addition signal or the sites for N-linked glycosylation, or both. Activity assays, immunoblotting and immunofluorescence microscopy revealed that the constructs lacking the GPI anchor were secreted from Madin-Darby canine kidney (MDCK) cells, whereas those retaining the GPI anchor were attached at the cell surface, irrespective of the glycosylation status. Wild-type membrane dipeptidase was expressed preferentially on the apical surface of both NH)CK and CaCo-2 cells. By contrast, the GPI-anchored construct lacking the N-glycans was targeted preferentially to the basolateral surface of both cell types. In constructs lacking the GPI anchor, the N-glycans also targeted the protein to the apical surface. Both the apically targeted, glycosylated and the basolaterally targeted, unglycosylated GPI-anchored forms of the protein were located in detergent-insoluble lipid rafts. These data indicate that it is the N-glycans, not the association of the GPI anchor with lipid rafts, which determine apical targeting of an endogenously N-glycosylated, GPI-anchored protein in polarised epithelia] cells.