Restoration of Holoceruloplasmin Synthesis in LEC Rat after Infusion of Recombinant Adenovirus Bearing WND cDNA

Abstract

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 LongEvans 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.Wilson's disease, an autosomal recessive disorder, is characterized by the excessive accumulation of copper in the liver (1). This phenomenon is thought to be due to reduced biliary excretion of copper and disturbed incorporation of copper into ceruloplasmin (CPN).1 Hepatic copper at toxic levels causes liver cirrhosis, and extrahepatic copper toxicity occurs especially in the brain due to the released copper from the damaged liver. WND (officially designated ATP7B), identified as the gene responsible for this disease, encodes a putative copper transporting P-type ATPase (2-5). The observations of single base changes or small deletions within WND of Wilson's disease patients have already been reported (3, 6).The Long-Evans Cinnamon (LEC) rat, known as an animal model for Wilson's disease, shows some of the clinical features similar to Wilson's disease, including hepatic copper accumulation, reduced biliary copper excretion, reduced copper in plasma, and a remarkable decrease of serum CPN activity (7,8). Atp7b, the rat gene homologous to WND, has been cloned, and a partial deletion at the 3Ј end in this gene is reported in the LEC rat (9). It is also known that the expression of Atp7b mRNA is absent in the LEC rat (10).CPN, a blue copper oxidase in plasma, contains 90 -95% of plasma copper. This protein is synthesized mainly in hepatocytes and secreted into plasma with 6 atoms of copper per molecule as the oxidase active holoprotein (11-13). The reduced levels of oxidase activity of CPN in the circulation of Wilson's disease patients and LEC rats is due to the secretion of apoceruloplasmin, copper-free and oxidase-inactive form, resulting from the disturbed incorporation of copper atoms into the protein (1, 14), while the intracellular synthesis of CPN...

Biochemical basis of regulation of human copper-transporting ATPases

“…The Cu-ATPases use the energy of ATP hydrolysis to transport copper from the cytosol into the secretory pathway and thus supply the metal for subsequent biosynthetic incorporation into various copper-dependent enzymes. ATP7A is required for formation of functional tyrosinase (1), peptidyl-α-monooxygenase (2), lysyl oxidase (3), and possibly some other enzymes (4), while ATP7B is essential for the biosynthesis of holo-ceruloplasmin, a copper-dependent ferroxidase (5). In addition to their biosynthetic role, human Cu-ATPases participate in the export of excess copper from the cells.…”

Binding of Copper(I) by the Wilson Disease Protein and Its Copper Chaperone

“…localized in the trans-Golgi network of hepatocytes where it delivers copper to key metalloenzymes, including ceruloplasmin, a multicopper oxidase involved in high affinity iron uptake (4,5). This process is dependent on interaction with Atox1, a copper chaperone that delivers Cu(I) ions to WND (6,7).…”

Structure of human Wilson protein domains 5 and 6 and their interplay with domain 4 and the copper chaperone HAH1 in copper uptake

“…WLNP acquires copper from the cytosolic metallochaperone HAH1 (commonly also called Atox1) (6), and then, after passage of copper into the Golgi, copper is inserted into the multicopper oxidase ceruloplasmin (7,8). WLNP also translocates from Golgi membrane to the cytosolic membrane for copper detoxification (9).…”