Absence of N-linked glycosylation does not affect plasma membrane localization of breast cancer resistance protein (BCRP/ABCG2)
Absence of N-linked glycosylation does not affect plasma membrane localization of breast cancer resistance protein (BCRP/ABCG2)
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DOI：
10.1007/s00280-005-1004-5
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发表时间：
2005-10-01
影响因子：
3
通讯作者：
Schellens, JHM
中科院分区：
文献类型：
--
作者：
Mohrmann, K;van Eijndhoven, MAJ;Schellens, JHM
乳腺癌耐药蛋白(BCRP/ABCG2)是一种三磷酸腺苷结合盒(ABC)多药转运蛋白，对拓扑替康和米托蒽醌等多种抗癌药物具有耐药性。为了更深入地了解BCRP的细胞功能，我们研究了BCRP的磷酸化和N-连接糖基化。在上皮性Madin-Darby犬肾(MDCK)细胞系中，我们没有检测到BCRP的磷酸化，而MRP2则是磷酸化的。在卵巢癌细胞系T8中也未检测到磷酸化BCRP。由于BCRP在这两条线上都能有效地运输药物，看来药物运输并不需要BCRP的磷酸化(如果有的话)。我们进一步将BCRP三个N-连接糖基化基序中的天冬酰胺残基418、557和596突变为丙氨酸。用瞬时转染法在CHO9和MDCKII细胞中表达突变蛋白，并用Western印迹和免疫荧光分析对突变蛋白进行鉴定。我们发现，只有BCRP-N596A和一个三个天冬酰胺都突变的突变体(三个突变体)不再糖基化，这表明只有天冬酰胺596正常糖基化。天冬酰胺596(或418)突变对BCRP的亚细胞定位影响不大，说明N-连接的糖基化不是进入质膜所必需的。然而，BCRP-N557A和三个突变体主要定位于细胞内，可能位于内质网，提示该突变扰乱了BCRP的正常路径。
Breast cancer resistance protein (BCRP/ABCG2) is an ATP-binding cassette (ABC) multidrug transporter that confers resistance to various anticancer drugs like topotecan and mitoxantrone. To obtain more insight in its cellular functioning, we investigated phosphorylation and N-linked glycosylation of BCRP. In the epithelial Madin-Darby canine kidney (MDCK) cell line, we did not detect phosphorylation of BCRP, in contrast to MRP2, which was phosphorylated. In the ovarian carcinoma cell line T8 also no phosphorylated BCRP was detected. As BCRP in both lines effectively transports drugs, it appears that phosphorylation of BCRP (if it occurs at all) is not needed for drug transport. We further mutated the asparagine residues 418, 557 and 596 in three putative N-linked glycosylation motifs of BCRP to alanines. Mutant proteins were expressed in CHO9 and MDCKII cells by transient transfection and characterized by Western blot and immunofluorescence analysis. We found that only BCRP-N596A and a mutant with all three asparagines mutated (triple mutant) were not glycosylated anymore, indicating that only asparagine 596 is normally glycosylated. The mutation of asparagine 596 (or 418) had little effect on the subcellular localization of BCRP, indicating that N-linked glycosylation is not essential for routing to the plasma membrane. However, BCRP-N557A and the triple mutant were mainly localized intracellularly, probably in the endoplasmic reticulum, suggesting that this mutation disrupted proper routing of BCRP.