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

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, WNDcDNA 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.

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)(3)(4)(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)(12)(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 peptide is normal in the LEC rat (15). Since the incorporation of copper into CPN occurs through the secretory compartments in hepatocytes, the precise site for the incorporation is still controversial. Sato and Gitlin (16) suggested that the rough endoplasmic reticulum (rER) was the likely site for the incorporation, using the human hepatoma cell line HepG2. However, our previous report demonstrated that CPN-bound copper was present in the Golgi apparatus and not the rER isolated from rat liver and that this process was disturbed in the LEC rat (15). P-type ATPases, defined as those forming a covalent phosphorylated intermediate in their reaction cycle, transport a variety of cations across membranes (17). The general features of those include the TGEA/S motif (phosphatase domain), the DKTGT/S motif (phosphorylation domain), the TGDN motif (ATP-binding domain), and the sequence MXGDGXNDXP that connects the ATP-binding domain to the transmembrane segment. Among P-type ATPases, the product of WND or Atp7b is a member of a class of heavy metal transporting P-type AT-Pases that pump copper or cadmium (18,19). This class of ATPases contain several unique features including 6 -8 transmembrane segments, 1-6 metal binding motifs, GMTCXXC, at the N terminus of the molecule, and the CPX motif in an intramembranous region. ATP7B contains 6 metal binding motifs at the N terminus (5), whereas the rat homologue contains only 5 (9). Recent studies have reported that MNK protein, which is also classified as a heavy metal transporting P-type ATPase, and is defective in Menkes disease, localizes to the Golgi apparatus, and may function in transport of intracellular copper (20,21).
Recombinant adenoviruses have been used to deliver genes to several animal models of inherited disorders (22)(23)(24)(25)(26) as well as to human individuals (27,28). Recently, an efficient method for constructing recombinant adenoviral vectors has been established (29). The improved version of adenoviral vector bears the foreign gene under the transcriptional control of a CAG promoter (30) exhibited the efficient expression of the introduced gene (31).
In this study, we introduced WND cDNA to LEC rats using this adenoviral vector construct to investigate the in vivo function of the gene with respect to copper transport in the liver. Consequently, we observed transgene expression in the liver and the secretion of holo-CPN in the plasma of the treated LEC rats.

EXPERIMENTAL PROCEDURES
Construction of Full-length WND cDNA-Two partial cDNA clones, pCUN-C1 and pWD02, covering WND cDNA (GenBank accession number U11700) from nucleotide number 223 to 2031 and from 1489 to 5581, respectively, were kindly provided by Dr. T. C. Gilliam, Columbia University, NY. Since pCUN-C1 lacks the 5Ј-end region of WND cDNA, including the initiation codon, and pWD02 lacks exons 6, 7, 8, and 12, these regions were cloned by reverse transcription-polymerase chain reaction as follows. Primers used for the 5Ј-end region were 5Ј-CCGAC-CAGGTGACCTTTTGC-3Ј and 5Ј-CTGACTGTGCTGGTGGCCAC-3Ј and for exons 6, 7, 8, and 12 were 5Ј-TCAAGTATGACCCAGAGGTC-3Ј and 5Ј-CATCACAGTCTTTATCTTGT-3Ј. Total RNA was obtained from human liver tissue according to the method of Chomczynski (32), and poly(A) ϩ RNA was isolated using oligo-dT cellulose (Type III, Becton Dickinson Labware, Bedford, MA). First-strand cDNA synthesis utilized 1 g of poly(A) ϩ RNA primed with downstream antisense primers and reverse transcriptase (SuperScript™ II, Life Technologies, Inc., Gaithersburg, MD) following the protocol of the manufacturer. PCR was performed using both sense and antisense primers to amplify the desired regions of the cDNA. The sequences of PCR products were verified by using ABI PRISM™ dye terminator cycle sequencing ready reaction kit (Perkin Elmer, Foster City, CA). After ligating the PCR product of the 5Ј-end region with pCUN-C1 and inserting the other PCR product spanning exons 6, 7, 8, and 12 into pWD02, the entire WND cDNA was constructed by connecting the modified pCUN-C1 with pWD02, and the redundant sequence of 3Ј-untranslated region from nucleotide number 4730 to 5581 was removed. The resulting entire cDNA spans nucleotide numbers 141 to 4729 and was designated phWD-L.
Preparation of Recombinant Adenoviruses-The recombinant adenovirus containing WND cDNA was constructed according to the methods by Miyake et al. (29). Briefly, The fragment of WND cDNA was ligated into the cosmid cassette (pAxCAwt) at SwaI site, generating pAx-CAWD. pAxCAwt contains the CAG promoter consisting of the cytomegalovirus IE enhancer, chicken ␤-actin promoter, and rabbit ␤-globin polyadenylation signal (30). pAxCAWD and the DNA-terminal protein complex of Ad5-dlX (33) were cotransfected into human embryonic kidney 293 cells by the calcium phosphate method. The presence of phWD-L in the recombinant adenovirus, generated through homologous recombination in the 293 cells, was confirmed by restriction analysis and designated AxCAWD. Stocks of AxCAWD were expanded, purified twice by CsCl gradient centrifugation (34), and stored in PBS with 10% glycerol at Ϫ80°C.
Animals-Inbred LEC rats were bred under specific pathogen-free conditions in the Animal Facilities for Experimental Medicine, Akita University School of Medicine. Animals had free access to water and standard rat chow. Recombinant adenoviruses, 1 ϫ 10 10 plaque forming unit (pfu) in 0.5 ml of saline, were administered to 4-or 5-week-old LEC rats by tail vein injection. All experiments were performed in accordance with the animal guidelines of Akita University School of Medicine.
Antibody Production-The monoclonal antibody against the aminoterminal region of WND including 6 copper-binding domains (amino acid number from 21 to 623) was produced by Yang et al. (35). Polyclonal antibodies against rat CPN purified as described previously (36) were raised in rabbit. 2 Plasma Measurements-Blood was collected from the carotid vein at various intervals. Plasma was stored at Ϫ80°C until use. The ferroxidase activity of CPN was determined on 15 l of plasma using Deter-miner Cp Kit (Kyowa Medics, Tokyo). The copper concentration in plasma was determined by a polarized Zeeman atomic absorption spectrophotometer (Model 180 -80, Hitachi, Japan).
Preparation of Subcellular Fractions-The subcellular fractions were isolated from the LEC rat infused with recombinant adenoviruses using the method described previously (15,37) with modifications. The procedures described below were performed at 4°C. Five grams of fresh rat liver was minced and homogenized in 3 volumes of SH buffer (0.25 M sucrose, 10 mM HEPES-NaOH, pH 7.2) using a Potter-Elvehjem homogenizer with a Teflon pestle at 1,000 rpm for 5 strokes. The homogenate was centrifuged for 10 min at 600 ϫ g, and the resulting supernatant was saved as postnuclear supernatant. The postnuclear supernatant was centrifuged for 10 min at 5,000 ϫ g, and the supernatant was recentrifuged for 10 min at 25,000 ϫ g. The lysosomal fraction was prepared as described in Ref. 37. Briefly, the 25,000 ϫ g pellet was resuspended in 1.2 ml of SH buffer, mixed with 8.9 ml of 28% Percoll in SH buffer, and centrifuged for 1 h at 37,000 ϫ g in a swinging-bucket rotor. The resulting pellet was resuspended in 20 ml of SH buffer and centrifuged twice for 20 min at 15,000 ϫ g. The final pellet was suspended in SH buffer and homogenized with a Teflon pestle by hand. The Golgi and the rER fractions were obtained according to our previously described method (15) after the 25,000 ϫ g supernatant was adjusted to 1.28 M sucrose by adding 2.5 M sucrose as necessary. The pellets containing the enriched fractions were suspended in SH buffer and homogenized with a Teflon pestle by hand. For the cytosol fraction, the 25,000 ϫ g supernatant was centrifuged for 1 h at 140,000 ϫ g, and the resulting supernatant was saved as cytosol.
Enrichment of each fraction was confirmed by immunodetection using antibodies against proteins with known subcellular locations as follows: rabbit polyclonal IgG to human cathepsin D (Upstate Biotechnology, Lake Placid, NY) for lysosomes, mouse monoclonal antibody to rat mannosidase II for Golgi (Berkeley Antibody Co., Richmond, CA), and rabbit polyclonal IgG to rat protein disulfide isomerase (PDI) (38) for rER.
Western Blot Analysis-The protein samples were resolved by polyacrylamide gel electrophoresis (PAGE) under denaturing or non-denaturing conditions, and immunodetection was performed by Western blot analysis as described (15).
Indirect Immunofluorescent Study-Freshly isolated liver tissues were frozen gradually with OCT compound (Miles, Inc., Elkhart, IN) in liquid nitrogen, and 5 m sections were prepared by cryostat. The following procedures were performed at room temperature. Frozen sections were fixed with 4% paraformaldehyde in PBS for 2 min and permeabilized with 1% Nonidet P-40 in PBS for 5 min. The specimens were incubated with primary antibodies diluted to 1:2000 with 3% bovine serum albumin in PBS for 2 h, followed by washing with PBS, and then were incubated with secondary antibodies, rhodamine-conjugated antibodies to mouse IgG (Cappel, Organon Teknika Corp., West Chester, PA) diluted to 1:40. After further washing, the specimens were mounted in Gelmount (Biomedia Corp., Foster City, CA) and analyzed using a confocal laser scanning microscope (LSM 410 invert Laser Scan Microscope, Carl Zeiss, Germany).

RESULTS
To introduce human WND cDNA into the LEC rat liver, we constructed the recombinant adenovirus by using the COS-TPC method (29). The resulting viruses bearing WND cDNA were infused to the LEC rats by tail vein injection.

WND Protein Expression in LEC Rat Livers after Infusion of the Recombinant Adenoviruses
Bearing WND cDNA-To investigate the expression of WND protein introduced by the infusion of AxCAWD, the liver specimens were analyzed by the indirect immunofluorescence. Frozen sections of liver tissue were prepared from the rats sacrificed on days 1, 3, 7, and 10 after infusion of 1 ϫ 10 10 pfu of either AxCAWD or the control virus, AxCAwt. As shown in Fig. 1A, the WND protein was expressed on day 1 (Fig. 1A, a), and expression increased on day 3 (Fig. 1A, b) before WND protein levels diminishing to the same level as day 1 by day 10 (Fig. 1A, c and d). No fluorescent positive cells were detected in the liver specimens obtained from the rats infused with AxCAwt (data not shown). To verify this result, Western blot analysis was performed using the postnuclear supernatant of livers prepared from rats 3 days after AxCAWD infusion when the expression appeared to be maximal. Consistent with the above results, a band corresponding to WND was detected in the sample infused with AxCAWD but not with AxCAwt (Fig. 1B, lanes 3 and 4). The lower weight bands below WND protein (Fig. 1B, lane 4) seem to be degradation products since the protein sample was prepared from frozen liver tissue. Protein samples obtained from freshly isolated liver show no degradation products (describe below, Fig. 2). The Coomassie staining shows equivalent amounts of protein samples loaded (Fig. 1B, lanes 1 and 2).
Localization of WND Protein in Liver Cells-The above immunofluorescent study suggests that the WND protein is present in the cytoplasm of liver cells (Fig. 1A, e); however, its precise subcellular localization could not be determined. To this end, Golgi, rER, lysosomal, and cytosolic fractions of liver were freshly prepared from LEC rat 3 days after infusion of Ax-CAWD for use in immunoblot analyses. To assess the enrichment of each fraction, immunoblotting was performed using antibodies to marker proteins with known subcellular locations as described in "Experimental Procedures." Mannosidase II, PDI, and cathepsin D are detected mainly in the Golgi, the rER, and the lysosomal enriched fractions, respectively, suggesting the efficacy of subcellular fractionation study (Fig. 2, B-D). As shown in Fig. 2A, WND is detected mainly in the Golgi enriched fraction (lane 4), and not in the rER, lysosomal, nor  2) and Western blot analysis of WND protein (lanes 3 and 4) in equivalent amounts of protein samples (25 g) obtained from the postnuclear supernatants prepared from frozen livers of LEC rats 3 days after infusion with AxCAWD (lanes 2 and 4) or AxCAwt (lanes 1 and 3). The protein samples were subjected to 7% SDS-PAGE and transferred to a PVDF membrane. The blot was probed with anti-WND monoclonal antibody (1:100), and bound antibody was detected by chemiluminescence. Molecular mass markers shown are kDa ϫ 10 Ϫ3 .

FIG. 2. Detection of WND protein in the subcellular fractions of liver from LEC rat after infusion of the recombinant adenovirus.
Western blot analysis of equivalent amounts of protein samples (5 g) obtained from subcellular fractions prepared freshly from LEC rat liver 3 days after infusion with 1 ϫ 10 10 pfu of AxCAWD. A-D, the protein samples were subjected to 7% SDS-PAGE and transferred to a PVDF membrane. The blot was probed with anti-WND monoclonal antibody (1:100, panel A), anti-rat mannosidase II monoclonal antibody cytosol fractions, indicating that WND localizes to the Golgi fraction.
To confirm holo-CPN synthesis, PAGE was performed under non-denaturing conditions using the protein samples obtained from the subcellular fractions and plasma. Holo-CPN was detected in the Golgi enriched fraction (Fig. 2E, lane 4), where WND protein resides ( Fig. 2A), consistent with our previous report (15).

Appearance of Holoceruloplasmin in LEC Rat Plasma after Infusion of Recombinant Adenoviruses Bearing WND
cDNA-To investigate the in vivo function of WND cDNA, 4-week-old rats administered with 1 ϫ 10 10 pfu of either Ax-CAWD or AxCAwt were examined for the appearance of holo-CPN in blood samples collected before and on days 1, 3, 7, 10, and 14 after viral infusion. To detect holo-CPN in LEC rat plasma, the samples were analyzed by PAGE under non-denaturing conditions as previously reported (15). As shown in Fig.  3, the bands corresponding to holo-CPN were observed in plasma obtained from the LEC rats infused with AxCAWD but not with AxCAwt. While holo-CPN appeared already on day 1, the highest level of holo-CPN was found on day 3, and levels declined gradually by day 14. ApoCPN still appeared to be the major form of CPN in plasma. To confirm the existence of holo-CPN in plasma, the ferroxidase activities of CPN were determined. Ferroxidase activities increased in plasma from the LEC rats treated with AxCAWD were maximal at day 3 (Fig. 4), corresponding to the profile of holo-CPN expression obtained by PAGE (Fig. 3). Additionally, since more than 90% of copper in plasma is bound to CPN, we also measured the plasma copper concentration to verify the secretion of copper into plasma after viral infusion. The level of plasma copper was already elevated on day 1 after infusion of AxCAWD, reached maximal on day 3, and reduced gradually by day 14 (Fig. 5). Although neither the increase of the ferroxidase activities nor elevation of the copper concentration was observed in the rats administered the control virus, the maximal values of both the ferroxidase activities and the copper concentration in the rats with AxCAWD were lower than the lower limits of normal range for rats (11 milliunits/ml for the ferroxidase activity, 460 ng/ml for the plasma copper concentration) (7). The above results showing the secretion of holo-CPN to be partially corrected in the LEC rat indicate the introduction of a functional WND protein by adenoviral administration. DISCUSSION In this study, we demonstrate the appearance of holo-CPN, copper-bound and oxidase-active form, in plasma of the LEC rats after the introduction of human WND cDNA using the recombinant adenovirus. Additionally, we provide evidence that introduced WND protein localizes to the Golgi apparatus.
The gene transfer mediated by recombinant adenoviruses has been applied to a variety of metabolic diseases in liver (22)(23)(24)(25)(26)(27)(28)39). To introduce human WND cDNA into the LEC rat liver, we constructed the recombinant adenovirus by using the COS-TPC method (29) by which the desired recombinant adenovirus is efficiently obtained. Additionally, the high-level expression of the transgene can be achieved by using the CAG promoter. The major problem in the use of adenoviral vectors, so far, are host immune responses that result in transient expression of the transgene and the inability to readminister the same virus. Previous reports suggested that transient expression of the transgene was due to destructive immune responses to transduced cells and viral gene products (23,24,40,41). In addition, a recent report suggests that host immune responses directed against the non-self transgene product are the major determinants of the stability of the transgene expression (42). To minimize these problems, we used the recombinant adenovirus lacking regions of E1A, E1B, and E3 within the viral gene and employed the CAG promoter to achieve the efficient expression of the transgene to reduce the number of viral particles to be infused. Despite these attempts, the expression of WND protein lasted only 10 -14 days in the present study. We postulate that this transient expression is due to the induction of host immune responses against transduced cells expressing the exogenic transgene product efficiently, as previously suggested (24,42,43).
The export pathways for the hepatic copper consist of the secretion with CPN into plasma and excretion into bile. In the LEC rats, as well as in patients with Wilson's disease, both pathways are thought to be impaired, leading to the accumulation of copper in the liver (1,7,8,14,15,37,44). Normally, CPN is secreted as a copper-bound form; however, evidence from studies of the LEC rats suggest that the process of copper incorporation into CPN is disturbed in the secretory compartments of affected livers (14 -16). On the other hand, the reduced biliary excretion of copper is considered to be due to a defect in the entry of copper into lysosomes (37). These phenomena may be attributed to a defect in translocation of copper across the membrane of subcellular compartments in the secretory and excretory pathways. The WND protein is likely to play an important role in these processes, since the protein is believed to be a member of copper-transporting P-type ATPases from its deduced amino acid sequence and thus capable of transport copper into subcellular compartments. A recent report has indicated that MNK protein, which also encodes a putative copper-transporting P-type ATPase and shows 55% identity in amino acids to WND protein, localizes to the trans-Golgi network and may function in delivering copper to cuproproteins through the secretory pathway (20,21). Similarly, CCC2 protein, the copper transporting P-type ATPase in yeast, was found to deliver cytosolic copper into an extracytosolic compartment and give copper to a CPN-like oxidase, FET3 protein (45).
In this study, we introduced WND cDNA into LEC rat liver using the recombinant adenovirus, AxCAWD, to examine the function of WND protein with respect to the synthesis of holo-CPN as well as the intracellular localization of the protein. The results obtained by PAGE under non-denaturing conditions and the plasma measurement for the oxidase activity and the copper concentration reveal that the LEC rats infused with AxCAWD secrete holo-CPN into plasma. This indicates that the introduced WND protein functions in the copper transport and the incorporation of copper into CPN. Moreover, this last process was found to occur in the Golgi apparatus, suggesting that this is the likely site for the WND protein to manifest its function. Subcellular fractionation studies performed in this report and the recent reports of others (35,46) revealing the localization of WND protein to the Golgi apparatus support this notion. However, levels of circulating holo-CPN in the treated rats could not be restored to the normal level in the present study. This may be due to the insufficient transduction result-ing from a reduced number of infused viral particles as described above. It is also possible that the product of transgene derived from human is not able to manifest its complete function in the rodent model due to the differences in amino acid sequence (24). While the homology between human WND and rat Atp7b are 82% in amino acids and each functional domain is well conserved, there is the difference in the number of metal binding motifs, which is six in the human protein and five in the rat protein (5,9). These data suggest that the WND protein participates in the copper transport coupled with CPN synthesis; however, the association of WND protein with the biliary excretion pathway of copper still remains to be determined. FIG. 4. Ferroxidase activity of ceruloplasmin in plasma of LEC rats after infusion of the recombinant adenoviruses. Plasma samples were obtained from two 4-week-old LEC rats infused with 1 ϫ 10 10 pfu of either AxCAWD (-q-) or AxCAwt (-E-), the control virus. The samples were collected before and 1, 3, 7, 10, and 14 days after viral infusion. The enzyme activity was determined as described under "Experimental Procedures." The means Ϯ S.D. of the results obtained from two rats are shown. The lower limit of the normal range is 11 milliunits/ml (7).

FIG. 5. Copper concentration in plasma of LEC rats after infusion of the recombinant adenoviruses.
Plasma samples were obtained from two 4-week-old LEC rats infused with 1 ϫ 10 10 pfu of either AxCAWD (-q-) or Ax-CAwt (-E-), the control virus. The samples were collected before and 1, 3, 7, 10, and 14 days after viral infusion. The copper concentration was determined as described under "Experimental Procedures." The means Ϯ S.D. of the results obtained from two rats are shown. The lower limit of the normal range is 460 ng/ml (7).