Substrate Transport Properties of the Human Peptide/Histidine Transporter PHT2 in Transfected MDCK Cells
Substrate Transport Properties of the Human Peptide/Histidine Transporter PHT2 in Transfected MDCK Cells
复制标题
转染 MDCK 细胞中人肽/组氨酸转运蛋白 PHT2 的底物转运特性
DOI：
10.1016/j.xphs.2019.06.016
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发表时间：
2019
影响因子：
3.8
通讯作者：
Jiang Huidi
中科院分区：
文献类型：
--
作者：
Wang Yuqing;Li Ping;Song Feifeng;Yang Xi;Weng Yayun;Ma Zhiyuan;Wang Lu;Jiang Huidi
PHT2是质子偶联寡肽转运蛋白家族的一员，参与小肽和组氨酸从溶酶体到胞浆的转运。它有助于维持细胞内肽的动态平衡。然而，由于PHT2定位于溶酶体膜上，其功能特性的阐明仍然是一个挑战。本研究的目的是将hPHT2突变体导入犬肾细胞，获得稳定表达的细胞膜蛋白，以探讨hPHT2的转运功能和底物性质。利用该细胞模型，我们发现hPHT2的转运活性在胞外pH为5.5时达到最大值。HPHT2对Gly-Sar亲和力较低，对d3-L组氨酸亲和力较高，Km值分别为428±88μM和66.9±5.7μM。几种典型的PEPT1和PEPT2的底物或抑制剂，包括万乃洛韦、甘氨酸-甘氨酸和头孢羟氨苄，但不是5-氨基乙酰丙酸或卡托普利，被证明是hPHT2的底物。然而，hPHT2对万乃洛韦的亲和力较低，Km值为5350±1234μM。因此，本研究建立了一种合适而有效的细胞模型来研究hPHT2在体外的功能，为研究hPHT2的转运活性和底物性质提供了重要的信息。
PHT2, a member of the proton-coupled oligopeptide transporter family, participates in the transportation of small peptides and histidine from lysosomes to the cytosol. It facilitates maintenance of intracellular peptide homeostasis. However, it remains a challenge to elucidate the functional properties of PHT2 due to its localization in the lysosomal membrane. The aim of this study was to explore the transport function and substrate properties of human PHT2 (hPHT2) by transfecting Madin-Darby canine kidney cells with hPHT2 mutants to obtain stably expressed protein in the cell membrane. Using this cell model, we found that the transport activity of hPHT2 reached a maximum capacity when the extracellular pH was 5.5. hPHT2 showed relatively low affinity for Gly-Sar and relatively high affinity for d3-L-histidine, withKmvalues of 428 ± 88 μM and 66.9 ± 5.7 μM, respectively. Several typical substrates or inhibitors of PEPT1 and PEPT2, including valacyclovir, Gly-Gly-Gly, and cefadroxil but not 5-aminolevulinic acid or captopril, were proven to be substrates of hPHT2. However, hPHT2 showed low affinity for valacyclovir with aKmvalue of 5350 ± 1234 μM. In conclusion, this study established a suitable and efficient cell model to explore the function of hPHT2in vitroand provided important information on the transport activity and substrate properties of hPHT2.