Terada K, Nakako T, Yang X L, Iida M, Aiba N, Minamiya Y, Nakai M, Sakaki T, Miura N, Sugiyama T
Department of Biochemistry, Akita University School of Medicine, Japan.
J Biol Chem. 1998 Jan 16;273(3):1815-20. doi: 10.1074/jbc.273.3.1815.
Wilson's disease, an autosomal recessive disorder, is characterized by the excessive accumulation of copper in the liver. WND (ATP7B) gene, which encodes a putative copper transporting P-type ATPase, is defective in the patients. To investigate the in vivo function of WND protein as well as its intracellular localization, WND cDNA was introduced to the Long-Evans Cinnamon rat, known as a rodent model for Wilson's disease, by recombinant adenovirus-mediated gene delivery. An immunofluorescent study and a subcellular fractionation study revealed the transgene expression in liver and its localization to the Golgi apparatus. Moreover, since the synthesis of holoceruloplasmin is disturbed in the Long-Evans Cinnamon rat, the plasma level of holoceruloplasmin, oxidase-active and copper-bound form, was examined to evaluate the function of WND protein with respect to the copper transport. Consequently, the appearance of holoceruloplasmin in plasma was confirmed by Western blot analysis and plasma measurements for the oxidase activity and the copper content. These findings indicate that introduced WND protein may function in the copper transport coupled with the synthesis of ceruloplasmin and that the Golgi apparatus is the likely site for WND protein to manifest its function.
威尔逊氏病是一种常染色体隐性疾病，其特征是肝脏中铜过度积累。编码假定的铜转运P型ATP酶的WND（ATP7B）基因在患者体内存在缺陷。为了研究WND蛋白的体内功能及其细胞内定位，通过重组腺病毒介导的基因传递，将WND cDNA导入长 Evans 肉桂大鼠（一种已知的威尔逊氏病啮齿动物模型）。免疫荧光研究和亚细胞分级分离研究揭示了转基因在肝脏中的表达及其在高尔基体中的定位。此外，由于长 Evans 肉桂大鼠中全铜蓝蛋白的合成受到干扰，因此检测了氧化酶活性和铜结合形式的全铜蓝蛋白的血浆水平，以评估WND蛋白在铜转运方面的功能。结果，通过蛋白质免疫印迹分析以及对氧化酶活性和铜含量的血浆测量，证实了血浆中全铜蓝蛋白的出现。这些发现表明，导入的WND蛋白可能在与铜蓝蛋白合成相关的铜转运中发挥作用，并且高尔基体可能是WND蛋白发挥其功能的位点。
