Expression and functional contribution of hTHTR-2 in thiamin absorption in human intestine
Expression and functional contribution of hTHTR-2 in thiamin absorption in human intestine
复制标题
DOI：
10.1152/ajpgi.00361.2003
复制
发表时间：
2004-03-01
影响因子：
4.5
通讯作者：
Marchant, JS
中科院分区：
文献类型：
--
作者：
Said, HM;Balamurugan, K;Marchant, JS
本研究的目的是调查表达和相对贡献的人硫胺素转运蛋白（hTHTR）-2对整体载体介导的硫胺素摄取人肠上皮细胞。北方印迹分析显示hTHTR-2的信息沿着天然人胃肠道表达，其中最高表达在小肠的近端部分。通过Western印迹分析，发现hTHTR-2蛋白在天然人肠细胞的刷状缘膜（BBM）处表达，但不在基底外侧膜处表达。这种表达模式在使用hTHTR-2与增强型绿色荧光蛋白（hTHTR 2-EGFP）的融合蛋白的研究中得到证实，所述融合蛋白在生长于滤器上的活Caco-2细胞中表达。用hTHTR-2基因特异性小干扰RNA（siRNA）预处理Caco-2细胞（其也在RNA和蛋白水平表达hTHTR-2）导致载体介导的硫胺素摄取的显著（P < 0.01）和特异性抑制（48%）。类似地，用特异性靶向hTHTR-1（其在Caco-2细胞中表达）的siRNA预处理Caco-2细胞也显著（P < 0.01）和特异性抑制（56%）载体介导的硫胺素摄取。当用针对hTHTR-2和hTHTR-1基因的siRNA预处理Caco-2细胞时，观察到载体介导的硫胺素摄取几乎完全抑制。这些结果表明，hTHTR-2的信使沿着人胃肠道表达，并且其蛋白质在肠上皮中的表达主要定位于顶端BBM结构域。此外，研究结果表明，这种转运蛋白在载体介导的硫胺素在人体肠道的摄取中起着重要的作用。
The aim of this study was to investigate expression and relative contribution of human thiamin transporter (hTHTR)-2 toward overall carrier-mediated thiamin uptake by human intestinal epithelial cells. Northern blot analysis showed that the message of the hTHTR-2 is expressed along the native human gastrointestinal tract with highest expression being in the proximal part of small intestine. hTHTR-2 protein was found, by Western blot analysis, to be expressed at the brush-border membrane (BBM), but not at the basolateral membrane, of native human enterocytes. This pattern of expression was confirmed in studies using a fusion protein of hTHTR-2 with the enhanced green fluorescent protein (hTHTR2-EGFP) expressed in living Caco-2 cells grown on filter. Pretreating Caco-2 cells (which also express the hTHTR-2 at RNA and protein levels) with hTHTR-2 gene-specific small interfering RNA (siRNA) led to a significant (P < 0.01) and specific inhibition (48%) in carrier-mediated thiamin uptake. Similarly, pretreating Caco-2 cells with siRNA that specifically target hTHTR-1 (which is expressed in Caco-2 cells) also significantly (P < 0.01) and specifically inhibited (by 56%) carrier-mediated thiamin uptake. When Caco-2 cells were pretreated with siRNAs against both hTHTR-2 and hTHTR-1 genes, an almost complete inhibition in carrier-mediated thiamin uptake was observed. These results show that the message of hTHTR-2 is expressed along the human gastrointestinal tract and that expression of its protein in intestinal epithelia is mainly localized to the apical BBM domain. In addition, results show that this transporter plays a significant role in carrier-mediated thiamin uptake in human intestine.