Knütter I, Theis S, Hartrodt B, Born I, Brandsch M, Daniel H, Neubert K
Institute of Biochemistry, Department of Biochemistry/Biotechnology, and Biozentrum, Martin-Luther-University Halle-Wittenberg, Halle, Germany.
Biochemistry. 2001 Apr 10;40(14):4454-8. doi: 10.1021/bi0026371.
This study was initiated to develop inhibitors of the intestinal H(+)/peptide symporter. We provide evidence that the dipeptide derivative Lys[Z(NO(2))]-Pro is an effective competitive inhibitor of mammalian PEPT1 with an apparent binding affinity of 5-10 microM. Characterization of the interaction of Lys[Z(NO(2))]-Pro with the substrate binding domain of PEPT1 has been performed in (a) monolayer cultures of human Caco-2 cells expressing PEPT1, (b) transgenic Pichia pastoris cells expressing PEPT1, and (c) Xenopus laevis oocytes expressing PEPT1. By competitive uptake studies with radiolabeled dipeptides, HPLC analysis of Lys[Z(NO(2))]-Pro in cells, and electrophysiological techniques, we unequivocally show that Lys[Z(NO(2))]-Pro binds with high affinity to PEPT1, competes competitively with various dipeptides for uptake into cells, but is not transported itself. Lack of transport was substantiated by the absence of Lys[Z(NO(2))]-Pro in Caco-2 cell extracts as determined by HPLC analysis, and by the absence of any positive inward currents in oocytes when exposed to the inhibitor. The fact that Lys[Z(NO(2))]-Pro can bind to PEPT1 from the extracellular as well as the intracellular site was shown in the oocyte expression system by a strong inhibition of dipeptide-induced currents under voltage clamp conditions. Our findings serve as a starting point for the identification of the substrate binding domain in the PEPT1 protein as well as for studies on the physiological and pharmacological role of PEPT1.
本研究旨在开发肠道H(+)/肽同向转运体的抑制剂。我们提供的证据表明，二肽衍生物Lys[Z(NO(2))]-Pro是哺乳动物PEPT1的有效竞争性抑制剂，其表观结合亲和力为5-10 microM。已在以下体系中对Lys[Z(NO(2))]-Pro与PEPT1底物结合结构域的相互作用进行了表征：(a)表达PEPT1的人Caco-2细胞单层培养物；(b)表达PEPT1的转基因毕赤酵母细胞；(c)表达PEPT1的非洲爪蟾卵母细胞。通过用放射性标记二肽进行竞争性摄取研究、细胞中Lys[Z(NO(2))]-Pro的HPLC分析以及电生理技术，我们明确表明Lys[Z(NO(2))]-Pro以高亲和力与PEPT1结合，与各种二肽竞争性地摄取到细胞中，但自身不被转运。HPLC分析确定Caco-2细胞提取物中不存在Lys[Z(NO(2))]-Pro，以及卵母细胞暴露于抑制剂时不存在任何正向内向电流，证实了其缺乏转运。在卵母细胞表达系统中，通过电压钳制条件下对二肽诱导电流的强烈抑制表明，Lys[Z(NO(2))]-Pro可以从细胞外和细胞内位点与PEPT1结合。我们的研究结果为鉴定PEPT1蛋白中的底物结合结构域以及研究PEPT1的生理和药理作用提供了一个起点。
