Preservation of folate transport activity with a low-pH optimum in rat IEC-6 intestinal epithelial cell lines that lack reduced folate carrier function.
Preservation of folate transport activity with a low-pH optimum in rat IEC-6 intestinal epithelial cell lines that lack reduced folate carrier function.
复制标题
在缺乏叶酸载体功能的大鼠 IEC-6 肠上皮细胞系中，以低 pH 最佳值保存叶酸转运活性。
DOI：
10.1152/ajpcell.00307.2004
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发表时间：
2005
期刊：
影响因子：
--
通讯作者：
Zhao,Rongbao
中科院分区：
文献类型：
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作者：
Wang,Yanhua;Rajgopal,Arun;Goldman,IDavid;Zhao,Rongbao
肠道叶酸转运已得到很好的表征，大鼠小肠上皮（IEC-6）细胞已被用作在细胞水平上研究这一过程的模型系统。主要的肠道叶酸转运活性具有低pH最适值，并且目前的范例是该过程由还原叶酸载体（RFC）介导，尽管该载体在白血病细胞中具有中性pH最适值。目前的研究解决了IEC-6细胞中组成性低pH叶酸转运活性是否由RFC介导的问题。两个独立的IEC-6亚系，IEC-6/A4和IEC-6/PT 1，通过化学诱变，然后用抗叶酸剂选择性压力产生。在IEC-6/A4单元中，过早停止导致RFC在Gln 420处截断。与截短蛋白融合的绿色荧光蛋白（GFP）不稳定。在IEC-6/PT 1细胞中，Ser 135缺失，导致GFP融合蛋白在质膜上定位失败。在这两种细胞系中，与野生型IEC-6细胞相比，中性pH下的甲氨蝶呤（MTX）流入量明显减少，但在pH 5.5时MTX流入量没有受到抑制。GFP突变的RFC构建体瞬时转染到无RFC的HeLa细胞中证实了它们缺乏转运功能。这些结果表明，在IEC-6细胞中，在中性pH下的叶酸转运主要由RFC介导;然而，在pH 5.5下的叶酸转运活性是RFC独立的。因此，在这种肠细胞模型中，具有低pH最佳值的组成型叶酸转运活性是由完全不同于RFC的过程介导的。
Intestinal folate transport has been well characterized, and rat small intestinal epithelial (IEC-6) cells have been used as a model system for the study of this process on the cellular level. The major intestinal folate transport activity has a low-pH optimum, and the current paradigm is that this process is mediated by the reduced folate carrier (RFC), despite the fact that this carrier has a neutral pH optimum in leukemia cells. The current study addressed the question of whether constitutive low-pH folate transport activity in IEC-6 cells is mediated by RFC. Two independent IEC-6 sublines, IEC-6/A4 and IEC-6/PT1, were generated by chemical mutagenesis followed by selective pressure with antifolates. In IEC-6/A4 cells, a premature stop resulted in truncation of RFC at Gln420. A green fluorescent protein (GFP) fusion with the truncated protein was not stable. In IEC-6/PT1 cells, Ser135was deleted, and this alteration resulted in the failure of localization of the GFP fusion protein in the plasma membrane. In both cell lines, methotrexate (MTX) influx at neutral pH was markedly decreased compared with wild-type IEC-6 cells, but MTX influx at pH 5.5 was not depressed. Transient transfection of the GFP-mutated RFC constructs into RFC-null HeLa cells confirmed their lack of transport function. These results indicate that in IEC-6 cells, folate transport at neutral pH is mediated predominantly by RFC; however, the folate transport activity at pH 5.5 is RFC independent. Hence, constitutive folate transport activity with a low-pH optimum in this intestinal cell model is mediated by a process entirely distinct from that of RFC.