Analysis of functional domains of Wilson's disease protein (ATP7B).

肝豆状核变性（Wilson's disease，Wilson's disease，Wilson's disease）患者存在ATP 7B基因缺陷，导致铜在肝内过度蓄积。我们最近揭示了ATP 7B蛋白肝铜代谢的功能重要性。基于氨基酸序列，推测ATP 7B蛋白在其分子中具有多个功能结构域，然而，这些结构域如何与ATP 7B功能的表现相关尚不清楚。为了解决这一问题，我们将各种突变，如点突变或缺失，引入到ATP 7B cDNA中，将这些突变体转染到缺失ATP 7B的酵母同源物CCC2基因（Δccc2）的酵母菌株中，并通过互补试验检测突变的ATP 7B蛋白的功能。仅含第六个铜结合结构域的突变体能挽救Δccc2的生长，表明第六个铜结合结构域足以使ATP 7B功能得以发挥。此外，发现在其C末端缺少88个氨基酸的ATP 7B蛋白丧失其功能。同样，位于ATP 7B蛋白铰链结构域的N1270S突变并不能挽救Δccc2突变，这意味着N1270S突变与Wilson病的发生高度相关。综上所述，第六个铜结合结构域，铰链结构域，和88个氨基酸的C端发挥重要作用的ATP 7B功能。

ATP7B gene, which encodes a putative copper transporting P-type ATPase (ATP7B protein), is defective in the patients with Wilson's disease, resulting in the excessive accumulation of copper in the liver. We have recently revealed the functional importance of ATP7B protein hepatic copper metabolism. Based on the amino acid sequence, ATP7B protein is assumed to have several functional domains in its molecule, however, how these domains correlate with the manifestation of ATP7B function is not known yet. To address this, we introduced various mutations, such as point mutation or deletion, into ATP7B cDNA, transfected these mutants into a yeast strain lacking the CCC2 gene (Δccc2), the yeast homologue of ATP7B, and examined the function of mutated ATP7B protein by the complementation test.Among the mutants bearing the deletion within the copper binding domains at the N terminus of ATP7B, the mutant containing only the sixth copper binding domain could rescue the growth of Δccc2, indicating the sixth copper binding domain is sufficient for the manifestation of ATP7B function. In addition, ATP7B protein lacking the 88 amino acids at its C terminus was found to lose its function. Similarly, the N1270S mutant, located in the hinge domain of ATP7B protein and found in patients showing the fulminant type of the disease, could not rescue the Δccc2 mutant, implying the N1270S mutation highly associates with the occurrence of Wilson's disease. In conclusion, the sixth copper binding domain, the hinge domain, and the 88 amino acids at the C terminus play important roles in ATP7B function.