The functional evaluation of human peptide/histidine transporter 1 (hPHT1) in transiently transfected COS-7 cells
The functional evaluation of human peptide/histidine transporter 1 (hPHT1) in transiently transfected COS-7 cells
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DOI：
10.1016/j.ejps.2005.09.014
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发表时间：
2006-04-01
影响因子：
4.6
通讯作者：
Knipp, GT
中科院分区：
文献类型：
--
作者：
Bhardwaj, RK;Herrera-Ruiz, D;Knipp, GT
最近，人多肽/组氨酸转运体(hPHT1，SLC15A4)的mRNA在胃肠道和Caco-2细胞中有表达，提示其可能参与了多肽类药物的肠道吸收。本研究旨在阐明hPHT1(SLC15A4)在人小肠中的蛋白表达模式；(Ii)hPHT1全长序列的克隆；(Iii)hPHT1在瞬时转染的COS-7细胞中的功能鉴定。用Western印迹和免疫组织化学方法检测hPHT1的表达。从BeWo细胞中扩增出hPHT1全长序列，将其插入真核表达载体pcDNA3.1-V5/His Topo(R)中，瞬时转入COS-7细胞，研究[H-3]组氨酸和[H-3]卡莫辛的摄取动力学。在模拟(空载体)和hPHT1-COS-7细胞上进行了时间、pH和钠依赖摄取的研究。结果表明，hPHT1蛋白在不同肠区均有表达。在hPHT1-COS-7细胞中，组氨酸和棕榈碱摄取在15min内呈线性关系，且与pH呈正相关。与模型COS-7细胞相比，这些底物和万乃洛韦在pH为5.0时对hPHT1细胞的摄取显著增加，而对甘氨酰肌氨酸的摄取显著降低且不受pH的影响。其他二肽和三肽也表现出与hPHT1的亲和力。这项研究介绍了hPHT1转运蛋白的初步功能特征和蛋白表达，并为增加多肽和基于多肽的药物转运提供了一条潜在的不同途径。(C)2005 Elsevier B.V.保留所有权利。
Recently, the expression of the human peptide/histidine transporter (hPHT1, SLC15A4) mRNA was observed in the GI tract and in Caco-2 cells, suggesting that it may participate in the intestinal absorption of peptide-based agents. This study aims to elucidate the: (i) protein expression pattern of hPHT1 (SLC15A4) in human small intestine; (ii) cloning of the hPHT1 full-length sequence; (iii) functional characterization of hPHT1 in transiently transfected COS-7 cells. The expression of hPHT1 was measured using Western blot and immunohistochemical analysis. The hPHT1 full-sequence was amplified from BeWo cells, inserted into the pcDNA3.1-V5/His TOPO(R) plasmid and transiently transfected into COS-7 cells to investigate the uptake kinetics of [H-3]histidine and [H-3]camosine. Time, pH and sodium-dependent uptake studies were performed in mock (empty vector) and hPHT1-COS-7 cells. Results demonstrated hPHT1 protein expression in different intestinal regions. Histidine and camosine uptake was linear in hPHT1-COS-7 cells over 15 min and was found to be pH-dependent. These substrates and valacyclovir showed significantly higher uptake at pH 5.0 in the hPHT1 transients when contrasted to the mock COS-7 cells, whereas glycylsarcosine uptake was significantly lower and unaffected by pH. Other di- and tripeptides also showed affinity for hPHT1. This study presents the initial functional characterization, the protein expression of the hPHT1 transporter and provides insight into a potentially different route for increasing peptide and peptide-based drug transport. (c) 2005 Elsevier B.V. All rights reserved.