Alternative splicing of the Menkes copper Atpase (Atp7a) transcript in the rat intestinal epithelium
Alternative splicing of the Menkes copper Atpase (Atp7a) transcript in the rat intestinal epithelium
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DOI：
10.1152/ajpgi.00203.2009
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发表时间：
2009-10-01
影响因子：
4.5
通讯作者：
Ranganathan, P. N.
中科院分区：
文献类型：
--
作者：
Collins, James F.;Hua, Ping;Ranganathan, P. N.
柯林斯 JF，华 P，陆 Y，Ranganathan PN。大鼠肠上皮中 Menkes 铜 Atpase (Atp7a) 转录物的选择性剪接。 Am J Physiol Gastrointest Liver Physiol 297：G695-G707，2009。首次发表于 2009 年 8 月 13 日； doi: 10.1152/ajpgi.00203.2009.-肠道 Menkes 铜 Atpase (Atp7a) 基因受到大鼠肠道缺铁的强烈诱导。我们试图通过在天然大鼠肠道和肠上皮 (IEC-6) 细胞中进行分子研究来开发体外模型来了解这种诱导的机制。 IEC-6 细胞表达 Atp7a，并且在缺铁时观察到诱导。 5' cDNA末端快速扩增和PCR实验揭示了大鼠肠和IEC-6细胞中的三种剪接变异体；所有变异均在缺铁期间强烈诱导（五到七倍）。剪接变体可能编码包含或不包含蛋白质末端 NH2 末端（包含铜结合结构域 1）的蛋白质。因此，我们假设存在不止一种版本的 Atp7a 蛋白。针对该蛋白质的 NH2 末端区域的抗体被开发出来（称为 N-term），并与之前报道的抗体（针对更多 COOH 末端区域，称为 54 10）一起使用来进行免疫印迹和免疫定位研究。 54-10 抗血清的结果揭示了一种类似于 190 kDa 的 Atp7a 蛋白变体，其定位于 IEC-6 细胞的跨高尔基体网络，并以铜负载运输到质膜。然而，使用 N 端抗血清，我们注意到类似于 97 和 64 kDa 的蛋白质。 97-kDa 蛋白是细胞质和细胞核特异性的，而 64-kDa 蛋白是细胞核特异性的。 N 端抗血清的免疫定位分析显示 IEC-6 和 Caco-2 细胞以及大鼠肠道中的细胞核染色强烈。我们得出的结论是，新型 Atp7a 蛋白变体可能存在于大鼠和人肠上皮细胞中，具有不同的细胞内位置和潜在不同的生理功能。
Collins JF, Hua P, Lu Y, Ranganathan PN. Alternative splicing of the Menkes copper Atpase (Atp7a) transcript in the rat intestinal epithelium. Am J Physiol Gastrointest Liver Physiol 297: G695-G707, 2009. First published August 13, 2009; doi: 10.1152/ajpgi.00203.2009.-The intestinal Menkes copper Atpase ( Atp7a) gene is strongly induced by iron deficiency in the rat intestine. We sought to develop an in vitro model to understand the mechanism of this induction by performing molecular studies in native rat intestine and in intestinal epithelial (IEC-6) cells. IEC-6 cells express Atp7a, and induction was noted with iron deprivation. 5' Rapid amplification of cDNA ends and PCR experiments revealed three splice variants in rat intestine and IEC-6 cells; all variants were strongly induced during iron deprivation (five- to sevenfold). The splice variants presumably encode proteins that would either contain the extreme NH2 terminus of the protein (containing copper binding domain 1) or not. We thus hypothesized that more than one version of Atp7a protein exists. Antibodies against this NH2-terminal region of the protein were developed (named N-term) and used along with previously reported antibodies (against more COOH-terminal regions, termed 54 10) to perform immunoblotting and immunolocalization studies. Results with the 54-10 antiserum revealed an Atp7a protein variant of similar to 190 kDa that localized to the trans-Golgi network of IEC-6 cells and trafficked to the plasma membrane with copper loading. Using the N-term antiserum, however, we noted protein of similar to 97 and 64 kDa. The 97-kDa protein was cytosolic and nuclear, whereas the 64-kDa protein was nuclear specific. Immunolocalization analyses with the N-term antiserum showed strong staining of nuclei in IEC-6 and Caco-2 cells and in rat intestine. We conclude that novel Atp7a protein variants may exist in rat and human intestinal epithelial cells, with different intracellular locations and potentially distinct physiological functions.