Döring F, Martini C, Walter J, Daniel H
Institute of Nutritional Science, Molecular Nutrition Unit, Technical University of Munich, Hochfeldweg 2, D-85350 Freising-Weihenstephan, Germany.
J Membr Biol. 2002 Mar 15;186(2):55-62. doi: 10.1007/s00232-001-0135-9.
The two closely related, proton-coupled, electrogenic mammalian peptide transporters PEPT1 and PEPT2 differ substantially in substrate affinity and mode of function. The intestinal carrier PEPT1 has a lower affinity for most substrates than the isoform PEPT2 that is expressed in kidney, lung, brain and other tissues. A previous analysis of PEPT1-PEPT2 chimeras has suggested that the N-terminal half of the carrier proteins is important for substrate affinity. We constructed and analyzed new PEPT1-PEPT2 chimeras for identifying smaller segments within the N-terminal region of the transporter proteins that contribute to the kinetic properties. The first 59 or 91 amino-acid residues of PEPT1 were used to replace the corresponding region in PEPT2 leading to the chimeras CH3 and CH4, which could be analyzed when expressed in Xenopus laevis oocytes. Substrate affinities of both chimeras for the zwitterionic substrate D-Phe-Ala ranged between those that are characteristic for either PEPT1 or PEPT2, but when charged dipeptide substrates were employed, both chimeras possessed PEPT1-like affinities. The chimera CH3 carrying the N-terminal 59 amino-acid residues of PEPT1 exhibited a PEPT2-like phenotype with respect to pHout-dependency as well as to the current-voltage relationship of inward currents. In the chimera CH4 possessing the 91 amino-terminal residues of PEPT1, a pronounced alteration in the pHout-dependence was observed, with highest transport rates occurring at pH values as low as pH 4.0. Based on this analysis, we propose that the two identified aminoterminal regions in mammalian peptide carriers play an important role in determining the substrate affinity and also other characteristic features of the two transporter subtypes.
两种密切相关的、质子偶联的、生电的哺乳动物肽转运体PEPT1和PEPT2在底物亲和力和功能模式上有很大差异。肠道载体PEPT1对大多数底物的亲和力低于在肾脏、肺、脑和其他组织中表达的同工型PEPT2。先前对PEPT1-PEPT2嵌合体的分析表明，载体蛋白的N端一半对底物亲和力很重要。我们构建并分析了新的PEPT1-PEPT2嵌合体，以确定转运蛋白N端区域内有助于动力学特性的较小片段。用PEPT1的前59个或91个氨基酸残基替换PEPT2中的相应区域，产生嵌合体CH3和CH4，当它们在非洲爪蟾卵母细胞中表达时可以进行分析。两种嵌合体对两性离子底物D-苯丙氨酸-丙氨酸的底物亲和力介于PEPT1或PEPT2的特征亲和力之间，但当使用带电荷的二肽底物时，两种嵌合体都具有类似PEPT1的亲和力。携带PEPT1的N端59个氨基酸残基的嵌合体CH3在pHout依赖性以及内向电流的电流-电压关系方面表现出类似PEPT2的表型。在具有PEPT1的91个N端残基的嵌合体CH4中，观察到pHout依赖性有明显改变，在低至pH 4.0的pH值下出现最高转运速率。基于此分析，我们提出哺乳动物肽载体中确定的两个N端区域在决定底物亲和力以及两种转运体亚型的其他特征方面发挥重要作用。
