The reconstituted P-glycoprotein multidrug transporter is a flippase for glucosylceramide and other simple glycosphingolipids
The reconstituted P-glycoprotein multidrug transporter is a flippase for glucosylceramide and other simple glycosphingolipids
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DOI：
10.1042/bj20050047
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发表时间：
2005-07-15
影响因子：
4.1
通讯作者：
Sharom, FJ
中科院分区：
文献类型：
--
作者：
Eckford, PDW;Sharom, FJ
Pgp（P-糖蛋白）多药转运蛋白与人类癌症的多药耐药性有关，其功能是非极性药物的外排泵，由其核苷酸结合结构域的ATP水解提供动力。Pgp的药物结合位点似乎位于膜双层的细胞质小叶内，这表明Pgp可能作为疏水化合物的“翻转酶”发挥作用。Pgp已被证明可以移位荧光磷脂，并且已经表明它也可以与GlcCer（葡糖神经酰胺）相互作用。在这里，我们使用连二亚硫酸盐荧光淬灭技术，以显示重建的Pgp可以翻转几个NBD（硝基苯并-2-氧杂-1，3-二唑）标记的简单鞘糖脂，包括NBD-GlcCer，从一个小叶的双层到其他的ATP依赖性，钒酸盐敏感的方式。NBD-GlcCer翻转的速率与NBD标记的PC（磷脂酰胆碱）观察到的速率相似。NBD-GlcCer翻转被各种Pgp底物和调节剂以浓度依赖性、可饱和的方式抑制，并且抑制与蛋白结合的Kd良好相关。与NBD-PC相比，向糖脂的头基添加第二种糖以形成NBD-乳糖神经酰胺显著降低了翻转速率，这可能是因为额外的糖残基增加了尺寸和极性。我们得出结论，Pgp功能作为一个广泛的特异性外向翻转酶简单的鞘糖脂和膜磷脂。
The Pgp (P-glycoprotein) multidrug transporter, which is linked to multidrug resistance in human cancers, functions as an efflux pump for non-polar drugs, powered by the hydrolysis of ATP at its nucleotide binding domains. The drug binding sites of Pgp appear to be located within the cytoplasmic leaflet of the membrane bilayer, suggesting that Pgp may function as a 'flippase' for hydrophobic compounds. Pgp has been shown to translocate fluorescent phospholipids, and it has been suggested that it may also interact with GlcCer (glucosylceramide). Here we use a dithionite fluorescence quenching technique to show that reconstituted Pgp can flip several NBD (nitrobenzo-2-oxa- 1,3-diazole)-labelled simple glycosphingolipids, including NBD-GlcCer, from one leaflet of the bilayer to the other in an ATP-dependent, vanadate-sensitive fashion. The rate of NBD-GlcCer flipping was similar to that observed for NBD-labelled PC (phosphatidylcholine). NBD-GlcCer flipping was inhibited in a concentration-dependent, saturable fashion by various Pgp substrates and modulators, and inhibition correlated well with the K-d for binding to the protein. The addition of a second sugar to the headgroup of the glycolipid to form NBD-lactosylceramide drastically reduced the rate of flipping compared with NBD-PC, probably because of the increased size and polarity contributed by the additional sugar residue. We conclude that Pgp functions as a broad-specificity outwardly-directed flippase for simple glycosphingolipids and, membrane phospholipids.