Department of Neurology and Institute of Neurology, Huashan Hospital, Institutes of Brain Science and State Key Laboratory of Medical Neurobiology, Shanghai Medical College, Fudan University, Shanghai, China.
CNS Neurosci Ther. 2013 May;19(5):346-51. doi: 10.1111/cns.12091.
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.
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.
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.
EGFP tags may not be suitable for the detection of cellular localization of ATP7B mutants.
威尔逊病是一种常染色体隐性遗传性铜代谢疾病，由 ATP7B 基因突变引起。临床研究表明，与错义突变相比，ATP7B 截断突变与发病年龄较早有关。体外研究表明，ATP7B 突变体的定位异常与致病机制有关。增强型绿色荧光蛋白（EGFP）标签常用于 ATP7B 突变体的细胞定位体外研究。然而，关于 ATP7B 截断突变体的细胞定位仍有许多未知。
在这里，我们将全长人野生型、错义突变（T935M）和四个截断突变体（E332X、Q511X、Q547X、Q819X）的 ATP7B 亚克隆到 pEGFP-C1、pEGFP-N2 和 pCMV-myc 中，并分别转染中国仓鼠卵巢（CHO）和 SH-SY5Y 细胞。
ATP7B 截断突变体在 CHO 和 SH-SY5Y 细胞的细胞质中均显示出弥散和均匀的分布模式，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 EGFP 标签位于 N 端或 C 端会严重影响 ATP7B 截断突变体的细胞定位，而 EGFP 标签位于 N 端也会影响 T935M 的定位。
EGFP 标签可能不适合检测 ATP7B 突变体的细胞定位。
