Interaction with the 5D3 monoclonal antibody is regulated by intramolecular rearrangements but not by covalent dimer formation of the human ABCG2 multidrug transporter
Interaction with the 5D3 monoclonal antibody is regulated by intramolecular rearrangements but not by covalent dimer formation of the human ABCG2 multidrug transporter
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DOI：
10.1074/jbc.m803230200
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发表时间：
2008-09-19
影响因子：
4.8
通讯作者：
Sarkadi, Balazs
中科院分区：
文献类型：
--
作者：
Ozvegy-Laczka, Csilla;Laczko, Rozalia;Sarkadi, Balazs
人ABCG 2是作为同源二聚体或同源寡聚体起作用的质膜糖蛋白。该蛋白在各种组织的保护/解毒中起重要作用，并且还可能负责癌细胞的多药耐药表型。在我们之前的研究中，我们发现5D3单克隆抗体对ABCG 2转运蛋白的细胞外表位显示出功能依赖性反应性。在当前的实验中，我们进一步表征了5D3-ABCG 2相互作用。深入研究了化学交联和细胞外S-S桥的调节对ABCG 2转运蛋白功能和5D3反应性的影响。我们发现，几种蛋白质交联剂大大增加了ABCG 2表达HEK细胞中的5D3标记;然而，共价二聚体形成、转运活性抑制和5D3结合增加之间没有相关性。二硫苏糖醇治疗，减少了细胞外的S-S桥形成的ABCG 2的半胱氨酸，没有影响运输功能，但导致5D3结合显着减少。当分析在细胞外环中携带Cys至Ala变化的ABCG 2突变体时，我们发现突变体C603 A（缺乏分子间S-S键）显示出与野生型ABCG 2相当的转运活性和5D3反应性。然而，在该环中的分子内S-S桥（在C592 A、C608 A或C592 A/C608 A突变体中）的破坏消除了5D3结合，而蛋白质的功能被保留。基于这些结果和从头折叠模拟，我们提出了一个模型的ABCG 2蛋白的大细胞外环。
Human ABCG2 is a plasma membrane glycoprotein working as a homodimer or homo-oligomer. The protein plays an important role in the protection/detoxification of various tissues and may also be responsible for the multidrug-resistant phenotype of cancer cells. In our previous study we found that the 5D3 monoclonal antibody shows a function-dependent reactivity to an extracellular epitope of the ABCG2 transporter. In the current experiments we have further characterized the 5D3-ABCG2 interaction. The effect of chemical cross-linking and the modulation of extracellular S-S bridges on the transporter function and 5D3 reactivity of ABCG2 were investigated in depth. We found that several protein cross-linkers greatly increased 5D3 labeling in ABCG2 expressing HEK cells; however, there was no correlation between covalent dimer formation, the inhibition of transport activity, and the increase in 5D3 binding. Dithiothreitol treatment, which reduced the extracellular S-S bridge-forming cysteines of ABCG2, had no effect on transport function but caused a significant decrease in 5D3 binding. When analyzing ABCG2 mutants carrying Cys-to-Ala changes in the extracellular loop, we found that the mutant C603A (lacking the intermolecular S-S bond) showed comparable transport activity and 5D3 reactivity to the wild-type ABCG2. However, disruption of the intramolecular S-S bridge (in C592A, C608A, or C592A/C608A mutants) in this loop abolished 5D3 binding, whereas the function of the protein was preserved. Based on these results and ab initio folding simulations, we propose a model for the large extracellular loop of the ABCG2 protein.