Department of Biomolecular Engineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Midori-ku, Yokohama, Japan.
FEBS J. 2009 Dec;276(24):7237-52. doi: 10.1111/j.1742-4658.2009.07423.x.
The human ATP-binding cassette (ABC) transporter, ABCG2 (BCRP/MXR/ABCP), is a plasma membrane protein containing intramolecular and intermolecular disulfide bonds and an N-linked glycan at Asn596. We have recently reported that the intramolecular disulfide bond is a critical checkpoint for determining the degradation fates of ABCG2. In the present study, we aimed to analyze quantitatively the impact of the N-linked glycan on the protein stability of ABCG2. For this purpose, we incorporated one single copy of ABCG2 cDNA into a designated site of genomic DNA in Flp-In-293 cells to stably express ABCG2 or its variant proteins. When ABCG2 wild type-expressing cells were incubated with various N-linked glycosylation inhibitors, tunicamycin profoundly suppressed the protein expression level of ABCG2 and, accordingly, reduced the ABCG2-mediated cellular resistance to the cancer chemotherapeutic SN-38. When Asn596 was converted to Gln596, the resulting variant protein was not glycosylated, and its protein level was about one-third of the wild type level in Flp-In-293 cells. Treatment with MG132, a proteasome inhibitor, increased the level of the variant protein. Immunoblotting with anti-ubiquitin IgG1k after immunoprecipitation of ABCG2 revealed that the N596Q protein was ubiquitinated at levels that were significantly enhanced by treatment with MG132. Immunofluorescence microscopy demonstrated that treatment with MG132 increased the level of ABCG2 N596Q protein both in intracellular compartments and in the plasma membrane. In conclusion, we propose that the N-linked glycan at Asn596 is important for stabilizing de novo-synthesized ABCG2 and that disruption of this linkage results in protein destabilization and enhanced ubiquitin-mediated proteasomal degradation.
人 ATP 结合盒（ABC）转运蛋白 ABCG2（BCRP/MXR/ABCP）是一种含有分子内和分子间二硫键以及 Asn596 处 N-连接聚糖的质膜蛋白。我们最近报道，分子内二硫键是决定 ABCG2 降解命运的关键检查点。在本研究中，我们旨在定量分析 N-连接聚糖对 ABCG2 蛋白稳定性的影响。为此，我们将 ABCG2 cDNA 的一个单拷贝整合到 Flp-In-293 细胞基因组 DNA 的指定位置，以稳定表达 ABCG2 或其变体蛋白。当 ABCG2 野生型表达细胞与各种 N-连接糖基化抑制剂孵育时，衣霉素显著抑制 ABCG2 的蛋白表达水平，从而降低 ABCG2 介导的细胞对癌症化疗药物 SN-38 的耐药性。当 Asn596 突变为 Gln596 时，产生的变体蛋白不发生糖基化，其蛋白水平约为 Flp-In-293 细胞中野生型的三分之一。蛋白酶体抑制剂 MG132 的处理增加了变体蛋白的水平。用抗泛素 IgG1k 进行免疫沉淀后进行免疫印迹分析，显示 N596Q 蛋白被泛素化，MG132 的处理显著增强了其水平。免疫荧光显微镜显示，MG132 的处理增加了 N596Q 蛋白在细胞内区室和质膜中的水平。总之，我们提出 Asn596 处的 N-连接聚糖对于稳定从头合成的 ABCG2 很重要，并且这种连接的破坏导致蛋白不稳定和增强的泛素介导的蛋白酶体降解。
