Identification of the histidyl residue obligatory for the catalytic activity of the human H+/peptide cotransporters PEPT1 and PEPT2
Identification of the histidyl residue obligatory for the catalytic activity of the human H+/peptide cotransporters PEPT1 and PEPT2
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DOI：
10.1021/bi962058p
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发表时间：
1997-01-14
期刊：
影响因子：
2.9
通讯作者：
Leibach, FH
中科院分区：
文献类型：
--
作者：
Fei, YJ;Liu, W;Leibach, FH
组氨酸残基对于肠道和肾脏表达的H+偶联多肽转运体的催化功能是必不可少的，很可能参与了HF的结合和转位。三个组氨酸残基在不同动物的肠肽和肾肽转运蛋白(分别为PEPT1和PEPT2)中保守，在hPEPT1中，这些残基是His-57、His-121和His-260。HPEPT2中相应的残基是His-87、His-142和His-278。我们分别突变了hPEPT1和hPEPT2中的每一个组氨酸残基，并通过在非洲爪哇卵母细胞和HeLa细胞中表达这些转运蛋白来比较突变体和它们各自的野生型转运蛋白的催化功能，发现hPEPT1中的His-57和hPEPT2中的His-87对于催化活性是绝对必要的，因为相应的突变体没有检测到的肽转运活性。HPEPT1中的His-121不是必需的，因为这个残基的突变不会损害运输功能，hPEPT2中的His-142在运输功能的维持中起着重要的作用，但没有发现是必需的，因为突变体具有明显的运输活性。必需的组氨酸残基(hPEPT1中的His-57和hPEPT2中的His-87)位于两个转运蛋白中几乎相同的拓扑位置，靠近第二个假定的跨膜结构域的细胞外表面。第二个保守的组氨酸残基位于hPEPT1和hPEPT2的第四个跨膜区。第三个保守的组氨酸残基存在于跨膜区6和7之间的细胞质环中，不太可能在H+的结合和转位中发挥任何重要作用，这得到了hPEPT1中这个组氨酸残基的突变不会干扰转运功能的研究结果的支持。当hPEPT1中的His-57和hPEPT2中的His-87发生突变时，hPEPT1和hPEPT2的转运功能的丧失并不是由于蛋白质表达的变化，因为这些突变体的表达水平与免疫印迹分析显示的HeLa细胞中各自野生型转运蛋白的表达水平相似。对表达hPEPT1野生型和3个组氨酸突变体的乳支睾吸虫卵母细胞的免疫荧光共聚焦分析表明，hPEPT1转运蛋白仅在质膜上表达，野生型和3个突变体的表达水平相当。这些定点突变研究清楚地表明，hPEPT1中的His-57和hPEPT2中的His-87是这些转运蛋白催化功能所必需的最关键的组氨酸残基。
Histidyl residues are known to be essential for the catalytic function of the H+-coupled peptide transporters expressed in the intestine and the kidney, most likely participating in the binding and translocation of Hf. Three histidyl residues are conserved among the intestinal and renal peptide transporters (PEPT1 and PEPT2, respectively) from different animal species, In hPEPT1, these residues are His-57, His-121, and His-260. The corresponding residues in hPEPT2 are His-87, His-142, and His-278. We have individually mutated each of these histidyl residues in hPEPT1 and in hPEPT2 and compared the catalytic function of the mutants with that of their respective wild type transporters by expressing the transporters in Xenopus laevis oocytes and also in HeLa cells, His-57 in hPEPT1 and His-87 in hPEPT2 were found to be absolutely essential for catalytic activity because the corresponding mutants had no detectable peptide transport activity. His-121 in hPEPT1 is not essential since mutation of this residue did not impair transport function, His-142 in hPEPT2 was found to play a significant role in the maintenance of transport function but was not found to be obligatory because the mutant had appreciable transport activity. The obligatory histidyl residue (His-57 in hPEPT1 and His-87 in hPEPT2) is located in an almost identical topological position in both transporters, near the extracellular surface of the second putative transmembrane domain. The second conserved histidyl residue is located in the fourth putative transmembrane domain in hPEPT1 as well as in hPEPT2. The third conserved histidyl residue is present in the cytoplasmic loop between the transmembrane domains 6 and 7 and is unlikely to play any significant role in the binding and translocation of H+ and this was supported by the findings that mutation of this histidyl residue in hPEPT1 did not interfere with transport function. The loss of transport function of hPEPT1 and hPEPT2, when His-57 in hPEPT1 and His-87 in hPEPT2 were mutated, was not due to alterations in protein expression because the expression levels of these mutants were similar to those of the respective wild type transporters in HeLa cells as assessed by immunoblot analysis. Confocal analysis of immunofluorescence in X. laevis oocytes expressing the wild type and the three histidine mutants of hPEPT1 showed that the transporter protein is expressed exclusively in the plasma membrane and that the level of expression is comparable among the wild type and the three mutants. These site-directed mutagenesis studies clearly show that His-57 in hPEPT1 and His-87 in hPEPT2 an the most critical histidyl residues necessary for the catalytic function of these transporters.