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首页> 外文期刊>CNS neuroscience & therapeutics. >EGFP Tags Affect Cellular Localization of ATP7B Mutants
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EGFP Tags Affect Cellular Localization of ATP7B Mutants

机译：EGFP标签影响ATP7B突变体的细胞定位。

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Summary Aims Wilson's disease is an autosomal recessive disorder of copper metabolism due to mutations within ATP7B gene. Clinical investigations indicate that ATP7B truncations are associated with an early age of onset when compared to its missense mutations. In vitro studies show that mislocalization of ATP7B mutants is involved in disease‐causing mechanisms. Enhanced green fluorescent protein (EGFP) tags are commonly used in in vitro studies of cellular localization of ATP7B mutants. However, there is still much unknown about cellular localization of ATP7B truncations. Methods Here, we subcloned full‐length human wild‐type, a missense mutation (T935M), and four truncating mutants (E332X, Q511X, Q547X, Q819X) of ATP7B into pEGFP‐C1 , pEGFP‐N2 and pCMV–myc , and transfected Chinese hamster ovary (CHO) and SH ‐ SY5Y cells with them, respectively. Results ATP7B truncations all showed a diffuse and homogenous distribution pattern within the cytosol of CHO and SH ‐ SY5Y cells, whereas its wild‐type proteins and T935M mutation were clustered in the Golgi apparatus. Furthermore, we found that EGFP tags at N‐ or C‐terminal would severely affect cellular localization of ATP7B truncations, and EGFP tags at N‐terminal also have an influence on T935M localization. Conclusion EGFP tags may not be suitable for the detection of cellular localization of ATP7B mutants.
机译：概述目的威尔逊氏病是由于ATP7B基因内的突变导致的铜代谢的常染色体隐性遗传疾病。临床研究表明，与它的错义突变相比，ATP7B截短与发病年龄早有关。体外研究表明，ATP7B突变体的错误定位与致病机制有关。增强型绿色荧光蛋白（EGFP）标签通常用于ATP7B突变体的细胞定位的体外研究。然而，关于ATP7B截短的细胞定位仍然未知。方法在此，我们将人野生型，一个错义突变（T935M）和ATP7B的四个截断突变体（E332X，Q511X，Q547X，Q819X）亚克隆到pEGFP-C1，pEGFP-N2和pCMV-myc中，并进行了转染。中国仓鼠卵巢（CHO）和SH SY5Y细胞分别与它们在一起。结果ATP7B截短均在CHO和SH SY5Y细胞的胞质溶胶中显示出弥散且均匀的分布模式，而其野生型蛋白和T935M突变则聚集在高尔基体中。此外，我们发现在N或C末端的EGFP标签会严重影响ATP7B截短的细胞定位，而在N末端的EGFP标签也对T935M定位产生影响。结论EGFP标签可能不适合检测ATP7B突变体的细胞定位。

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