Inhibition of the copper incorporation into ceruloplasmin leads to the ...

Vol. 268, No.12, Issue of April 25, pp. 8965-8971.1993 Printed in U.S.A.

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc.

Inhibition of the Copper Incorporation into Ceruloplasmin Leads to the Deficiency in SerumCeruloplasmin Activity in Long-Evans Cinnamon Mutant Rat* (Received for publication, August 3, 1992)

Takahisa YamadaSQ, Takashi Ami$$$, Yasuo Suzukill, Mitsuru Sat011,and Kozo Matsumoto$** From the $Institute for Animal Experimentation and the YDepartment of Hygiene, University of Tokushima School of Medicine, Kuramoto 3, Tokushima 770, Japan and IlDepartment of Anatomy, Akita University School of Medicine, Akita 010, Japan

Although ceruloplasmin is known to be a coppertransporting protein, little is known about the biochemical mechanisms of copper incorporation into ceruloplasmin during the biosynthesis. We have examined various levels of ceruloplasmin biosynthesis in the Long-Evans Cinnamon (LEC) rat, which possesses a mutation causing the deficiency in serum ceruloplasmin activity associated with excess hepatic copper accumulation. Southern and Northern blot analyses revealed that the gene and mRNA encoding ceruloplasmin resided normally in LEC rat liver. Western blot analysis showed a normal level of ceruloplasmin in LEC rat serum. Following metabolic labeling of hepatocytes with “Cu, no radioactive copper was detected in the ceruloplasmin fraction in LEC rat hepatocytes using Sephadex G-75 column chromatography, indicating that copper incorporation into ceruloplasmin is deficient in the LEC rat. Furthermore, LEC rat hepatocytes incubated with 64Cualso showed a reduction in the efficiency of copper transport from cytosolic to noncytosolic fractionsanda reduced copper efflux from the hepatocytes, indicating that LEC rat hepatocytes possess an abnormality in copper metabolism. These results suggest that anabnormality of the copper delivery mechanism causes an inhibition of copper incorporationintothe ceruloplasmin molecule in the liver, leading to the deficiency in serumceruloplasmin activity in the LEC rat. In addition, this abnormality also seems to cause an inhibition of biliary copper excretion. The blockingof these twocopper exclusion pathways is thought to lead to excess hepatic copper accumulation in theLEC rat. Thus, theLEC rat should be a good model for studying the biochemical process responsible for copper delivery.

Ceruloplasmin is an important protein that circulates in * This work was supported in part by a research grant from the Ministry of Education, Science, and Culture and by a grant-in-aid for cancer research from the Ministry of Health and Welfare of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must thereforebe hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Supported by fellowships from the Japan Society for the Promotion of Science for Japanese Junior Scientists. **To whom correspondence should be addressed. Institute for Animal Experimentation, University of Tokushima School of Medicine, Kuramoto 3, Tokushima770, Japan. Tel.: 81-886-31-3111; Fax: 81-886-32-9963. $3 Present address: Institute for Experimental Animal Science, School of Medicine, Nagoya City University, Mizuho-ku, Nagoya 467, Japan.

plasmaas amajor serum copper transporterproteinand contains greater than 95% of the copper found in serum (1, 2). An apoceruloplasmin is synthesized in liver as a single polypeptide chain and secreted into plasma as a holoceruloplasmin associated with six atoms of copper/molecule (3). The functions of ceruloplasmin are copper transport, iron metabolism, antioxidantdefense, tissue angiogenesis, and coagulation (4-11). Serum ceruloplasmin activity increases during inflammation, infection, and injury, suggesting that serum ceruloplasmin actspossibly as an antioxidant and as an acute phase protein (10-13). Copper is an integral enzyme cofactoressential for a variety of processes in homeostasis such as electron transport, amino acid metabolism, connective tissue biosynthesis, pigment formation, and neurotransmitter and hormone production (14). Ceruloplasmin provides an attractive model for studying the mechanisms of copper protein biosynthesis. The complete amino acid and nucleotide sequences of ceruloplasmin have been determined in both the human and the rat (3, 13, 15, 16), and the cis-acting element in the 5”flanking region of the ceruloplasmin gene has been recently characterized (17). Copper is incorporated into newly synthesized ceruloplasmin within hepatocytes (18), and turnover data indicate that very little copper exchanges from the protein in the circulation (19, 20). About 10% of circulating ceruloplasmin occurs as apoprotein, presumably synthesized and secreted from the liver without copper incorporation (21). The biosynthesis and secretion of apo- and holoceruloplasmin from a human cell line occur at identical rates (18). However, little is known about the molecular structure essential for copper incorporation into ceruloplasmin during biosynthesis and the biochemistry involved in this process. IthasbeenreportedthattheLong-EvansCinnamon (LEC)’ mutant rat spontaneously develops a necrotizing hepatic injury and liver cancer (22, 23). Recently, Li et al. (24) reported that the LEC rat exhibits an abnormal accumulation of hepatic copper anda marked decrease in serum ceruloplasmin activity, and they proposed the hypothesis that the cytotoxicity of excessively accumulated hepatic copper is likely to cause necrotizing hepatic injury. However, the molecular mechanisms of the deficiency in ceruloplasmin activityassociated with hepatic copper accumulation in the LEC rat have not been clarified. In the present study, we have examined various levels of ceruloplasmin biosynthesis in LEC rat liver. Our results suggest that an abnormality of the copper delivery mechanism inhibits the incorporation of copper atoms into the ceruloplasmin molecule in the liver, and asa result, the ceruloplasThe abbreviations used are: LEC, Long-Evans Cinnamon; PBS, phosphate-buffered saline; kb, kilobase pair(s).

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min without copper atoms cannot exertits enzymatic activity in the serum of the LEC rat. In addition, the abnormality probably causes the inhibition of biliary copper excretion. This inhibition together with the inhibition of copper exclusion by ceruloplasmin seem to lead to copper accumulation in LEC rat liver. Our present report suggests that the copper incorporation into ceruloplasmin and the biliary copper excretion require an identical intracellular biochemical process responsible for the copper delivery. The LEC rat should be an interesting animalwhich is primarily deficient in aprocess responsible for the copper deliveries into a site of copper incorporation into the ceruloplasmin and biliary copper excretion pathway. Thus, the LEC rat is a good and unique animal model for studying the specific biochemical process responsible for copper delivery. MATERIALS ANDMETHODS

Animals-Inbred strains of LEC, F344, and WKAH rats are bred under specific pathogen-free conditions in the Institute for Animal Experimentation, University of Tokushima School of Medicine, which is coded as Tj (Tokushima Japan). F344/Tj and WKAH/Tj rats were used as a control. Measurement of Copper Content and CeruloplasminActivity-Liver or fractionseluted from column chromatography were treated with a mixture of nitric, perchloric, and sulfuric acids. Copper concentrations were determined with an inductively coupled argon plasma emission spectrophotometer, model ICAP-750N (Nippon Jarrell-Ash Co., Kyoto, Japan). The ceruloplasmin activities were measured as an oxidase activity by the method of Schosinsky et al. (25). Serum or fractions eluted from column chromatography (50 pl) were incubated in 1 mlof 0.1 M acetate buffer (pH 5.0) containing 7.88 mM odianisidine &hydrochloride for 5 and15 min at 30 "C, andthe absorption at 540 nm was measured. The oxidase activity was obtained by subtracting the absorbance a t 5 min from that of 15 min. Probe-The probe used in this study was the cDNA clone, phCpl, which contains DNA encoding amino acid residues 202-1046 of the ceruloplasmin (16). This DNAwas kindly provided by Dr. MacGillivray (University of British Columbia, Vancouver, Canada). The clone, phCpl, was labeled with [w3'P]dCTP (Amersham International, Amersham, U. K.) using a nick translation kit (Amersham International). The specific activity of the probe was 1.5 X 10' dpm/

a.

Southern BlotAnalysis-High molecular weightDNA was extracted from rat liver and digested with restriction enzymes BgnI, EcoRI, EcoRV, and Hind111 (Toyobo, Kyoto, Japan). Digested DNAs (IO pg) were electrophoresed, transferred to Biodyne A nylon membranes (Pall, Glen Cove, NY), and hybridized according to the supplier's instructions (Pall). Filters were washed twice in 2 X SSC (1X SSC is 0.15 M NaCl, 0.015 M sodium citrate (pH 7.4)) containing 0.1% SDS at room temperature for 30 min and 0.5 X SSC containing 0.1% SDS at 65 "C for 30 min and were exposed to XAR-5 x-ray film (Eastman Kodak) with intensifying screens at -70 "C overnight. Northern Blot Analysis-RNA was prepared from the guanidine thiocyanate (5.5 M) extracts by cesium chloride (5 M ) density gradient centrifugation as described previously (26) with slight modifications. An aliquot (10 rg) of RNA was treated with formaldehyde, subjected to 1% agarose gel electrophoresis, and then transferred to the nylon membrane. The conditions of hybridization, washing, and exposure were the same as theprocedure described for Southern blot analysis. To determine the ratio of the steady-state levels of ceruloplasmin to that of &actin mRNAs, signal intensities were examined with a laser densitometer (Ultrascan XL, Pharmacia, Uppsala, Sweden). Western Blot Analysis-Sera were collected from LEC and normal rats. An aliquot of sera was subjected to SDS-polyacrylamide (7%) gel electrophoresis as described previously (27). After transferring to the nitrocellulose membrane, the blot was blocked with phosphatebuffered saline (PBS) (pH7.0) containing 2% BSA and 0.05% Tween 80 and incubated with rabbit anti-rat ceruloplasmin (18).After the blot was washed three times with PBS containing 0.05% Tween 80, immune complexes on the blot were detected with a Vectastain ABC kit (Vector Laboratories Inc., Burlingame, CA). For treatment of sera with endoglycosidase H, 5-pl aliquots of sera were treated with 50 milliunits of endoglycosidase H in 25 mM sodium acetate (pH 5.5) for 16 h at 37 "C. For endoglycosidase F treatment, 5-pl aliquots of sera were treated with 0.5 unit of endoglycosidase F in 50 mM sodium

into Ceruloplasmin acetate (pH 5.0),20mM EDTA, 0.1% SDS, 0.75% Nonidet P-40, and 1%(v/v) 2-mercaptoethanol for 16 h at 37 "C. Following enzymatic digests the aliquots were analyzed as described above. Fractionation of Serum Proteins on a Sepharose CL-4B ColumnSera were collected and pooled from four male rats at1month of age. Nine ml of the serum was applied to a Sepharose CL-4B column equilibrated with PBS. The column was eluted at 20 ml/h by using a peristatic pump (model P-1, Pharmacia), and 5-ml fractions were collected. The fractions were assayed for copper content and ceruloplasmin oxidase activity. Hepatocyte Isolation-Male rats at 6 weeks of age, weighing 150200 g, were used for hepatocyte isolation. Hepatocytes were isolated as reported previously (28). Briefly, the livers were perfused with a washing solution containing 0.5 mM EGTA and thenwith a collagenase-containing buffer. The isolated cells were washed with Eagle's minimum essential medium to remove nonparenchymal cells, suspended in Dulbecco's modified Eagle's medium containing 5% fetal calf serum and 30 mg/liter of kanamycin, and then approximately 2 X lo6 cells were placed in collagen-coated dishes (15 X 60 mm). After culture for 3 h, loosely attached and dead cells were removed, and fresh medium was added. The cells were incubated in a CO, incubator overnight and used on the following day. This method gave a yield of 80-95% viable cells by the trypanblue exclusion criterion. "CU Accumulation in Hepatocytes-Cultured hepatocytes were washed three times with Hanks' balanced salt solution, and Earle's balanced salt solution containing 10% fetal calf serum was added. The accumulation of copper by hepatocytes was initiated by the addition of 5 pCi of "Cu to each plate. Cells were incubated at 37 "C for various periods up to 16 h. After incubation, the cells were washed three times with PBS containing 10 mM EDTA. Cells were then scraped off the dish surface with a rubber policeman following the addition of 1 mlof ice-cold PBS. They were disrupted by freezing and thawing several times, and a 100-pl aliquot of the lysedcell suspension wasremoved for protein analysis. To measure copper accumulation in cytosolic and noncytosolic fractions, a 900-pl aliquot of the suspension was centrifuged at 100,000 X g for 10 min, and the supernatant and pellet were counted with a y-counter. The "CU as Cu(CH3C00), in 5% CH3COOH wasobtained from the JapanAtomic Energy Research Institute (Ibaragi, Japan)atan initial specific activity of 200 mCi/mg and was used within 2 days. y-Counting was performed with a y-counter (Aloka model ARC-361, Tokyo, Japan) with automatic decay correction and background subtraction. Protein content was assayed by BCA protein assay kit (Pierce Chemical Co.). "CU Efflux from Hepatocytes-Hepatocytes were loaded with "Cu for 6 h as described above.At the end of incubation, radioactive medium was removed, and the cells were washed three times with Hanks' balanced salt solution. Fresh, nonradioactive mediumwas then added to each plate, and theincubation was continued at 37 "C for an additional 5, 15, and 60 min and 16 h. The cells were assayed for remaining intracellular '%u. Fractionation of Hepatocyte Lysates on a Sephadex G-75 ColumnFor column chromatography, the hepatocytes were removed from the plates after exposure to "CU overnight. The cells pooled from five plates were resuspended in 1.5 ml of isolation buffer consisting of 100 mM KC1,20 mM Hepes, 1 mM dithiothreitol, and 0.1% mebutamate (pH 7.3). They were frozen and thawed several times and centrifuged at 5,000 X g for 10 min to get the cytoplasmic fraction. The supernatant was applied to a Sephadex G-75 column equilibrated with the isolation buffer. The column was eluted at 10 ml/h by using a peristatic pump (model P-1, Pharmacia) at 4 "C, and 0.8 ml of each fraction was collected. Blue dextran (2,000 kDa) and cytochrome c (12.4 kDa) were used as molecular mass markers. RESULTS

Levels of Hepatic Copper Content and Serum Ceruloplasmin Activity in the LEC Rat-Table I shows the mean f S.E. of copper concentrations in liver and the levels of serum ceruloplasmin activity in six male animals of WKAH, (LEC x WKAH)FI, and LEC rats. The copper concentrations were approximately 40-fold higher in the LEC rat than in the normal rat. (LEC X WKAH)Fl rats showed a normal level of hepatic copper concentration. The difference between LEC and WKAH or (LEC X WKAH)F1rats was statistically significant. Since the difference between WKAH and (LEC X WKAH)Fl rats was not found, it was indicated that the

Inhibition of Copper Incorporation Ceruloplasmin into TABLE I Copper concentrations in the liver and serum level of ceruloplasmin activity in WKAH, (LEC X WKAH)F,, and LEC rats The copper concentration was determined by atomic absorption spectrophotometry. The serum ceruloplasmin level was determined as a serum oxidase activity. Values are expressed as mean f S.E. of six animals. Strain and age

Copper concentration

Ceruloplasmin activity

g / g wet weight

nrnol/rnin/rnl

WKAH 1 month 3.9 f 0.37" 152.7 f 16.12" 5 months 4.2 f 0.85" 259.6 f 8.15' (LEC X WKAHIF, . . 5 months 119.4 f 7.91' 4.8 f 0.85" LEC 1 month 147 2 48.70" 0.6 f 1.51" 5 months 7.3 f 5.61b 205 f 52.05" " p < 0.005 between LEC and WKAH or (LEC X WKAH)F, rats (Student's t test). b p < 0.005 between LEC and WKAH rats;p < 0.01 between (LEC X WKAH)FI and LEC or WKAH rats (Student's t test). 1

2

3

4

5

6

7

8

-2.3

FIG.1. Southern blot analysis of the rat ceruloplasmin gene. Genomic DNA was digested with four kindsof restriction endonucleases and electrophoresed in 0.7% agarose gel. After being transferred to nylon membranes, the blot was hybridized with 32P-labeled ceruloplasmin cDNA probe. Lanes 1 , 3 , 5 , and 7, normal rat; lanes 2,4,6, and 8, LEC rat; lanes 1 and 2, genomic DNA digested with BglII; lanes 3 and 4, genomic DNA digested with EcoRV; lanes 5 and 6, genomic DNA digested with HindIII; lanes 7 and 8, genomic DNA digested with EcoRI. The length (in kb)of the DNA is shown on the right side of each panel. The result shown is from a representative experiment repeated for a t least three separate DNAs prepared from different animals in both normal and LEC rats.

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EcoRI, 6.0 and 9.4 kb for EcoRV, and 2.4 and 9.0 kb for HindIII. The resultsof Southern blot analysis indicated that the ceruloplasmin gene exists normally on the LEC rat genome, excluding the possibility of deletion of the ceruloplasmin gene because of a deficiency in serum ceruloplasmin activity. Among 15 restriction enzymes tested, a restriction fragment length polymorphism between LEC and F344 was detected by using KpnI (29). Detection of Ceruloplasmin mRNA in the LEG Rat by Northern Blot Analysis-RNAs prepared from liver of LEC and normalrats at 1 and 5 months of age were subjected to Northern blot analysis. The autoradiographof Northern blot hybridization is shown in Fig. 2. There were no qualitative differences in the ceruloplasmin mRNA between LEC and normalrats.The molecular size of the mRNA was 4,800 nucleotides in the LEC rat, consistent with that of the normal rat. No quantitative difference between LEC and normal rats was observed. The ratio of the steady-state levels of ceruloplasmin to thatof P-actin mRNAs in LEC rats at1 month of age (0.397 f 0.002 in three separate RNA preparations from three individual rats) was indistinguishable from that of agematched normal rats (0.411 f 0.016 in three separate RNA preparations from three individual rats) by Student's t test. Moreover, the ratio of the steady-statelevels at 5 months of age was also indistinguishable between normal and LEC rats by Student's t test (0.110 f 0.008 and 0.093 f 0.015 in three separate RNA preparations of normal and LEC rats, respectively). Detection of normal levels of ceruloplasmin mRNA in the LEC rat indicated that the expression process from the ceruloplasmin gene to the mRNA for ceruloplasmin occurs normally. Detection of Serum Ceruloplasmin in the LEC Ratby Western Blot Analysis-Sera taken from LEC and normal rats at 1and 5 months of age were subjected to Westernblot analysis (Fig. 3). Both 135-kDa fragmentand 115-kDaproteolytic fragment were identified with rabbit anti-rat ceruloplasmin in LEC as well as in normal rats, consistent with the results reported previously (18).The resultsof Western blot analysis indicated that the expressionprocess from the mRNA to single peptide for ceruloplasmin occurs normally in LEC rat Ceruloplasmin 1

2

3

4

-

-28s

abnormal copper accumulation in the LEC rat liver is inherited as a recessive trait. The levels of serum ceruloplasmin activity were about 40-fold lower in the LEC rat than in the normal rat. The levels of serum ceruloplasmin activity in (LEC x WKAH)FI ratsshowed an almost intermediatevalue between those of LEC and normal rats. differences The among the three strains of rats were statistically significant. Since the value of the serum ceruloplasmin activity level in the F1 rat was intermediate between those of WKAH and LEC rats, it was suggested that the deficiency in serum ceruloplasmin activity in the LEC rat is inherited aascodominant trait. Southern Blot Analysis of the Ceruloplasmin Gene-The deficiency inceruloplasminactivity could beexplained by either a deletion of gene, a defectivetranscription, a defective translation, or a defect in a post-translational modification mechanism. To test theirpossibilities, first theceruloplasmin gene in LEC and normal ratswas analyzed by Southern blot hybridization. The hybridization pattern of the genomic DNA digested by restriction endonucleases with a probe for ceruloplasmin is shown in Fig. 1. In both strains, thesizes of the fragments were 5.2 and 9.2 kb for BglII, 2.3 and 3.9 kb for

beta-actin 1

2

3

4

FIG. 2. Northern blot analysis of the rat ceruloplasminspecific mRNA. RNA was preparedand electrophoresed in 1% agarose gel. After being transferred tonylon membranes, the blot was first hybridized with "P-labeled ceruloplasmin cDNA probe and then rehybridized with 32P-labeled p-actin cDNA probe. Lane 1, normal rat at theage of 1 month; lane 2, LEC rat a t the age of 1 month; lane 3, normal rat a t the age of 5 months; lane 4, LEC rat at theage of 5 months. Thearrow indicates the mRNA for ceruloplasmin. The result shown is from a representative experimentrepeated for three separate RNAs prepared from different animalsin both normal and LEC rats.

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liver and that the produced ceruloplasminis normally secreted into LEC rat serum. Furthermore, the treatment of sera with endoglycosidase H or F resulted in no change in themobility in the electrophoresis, indicating that the serum ceruloplasmin of both LEC and normal rats was resistant to these by endoglycosidase endoglycosidases (Fig. 3). In the treatment H or F, no difference in the glycosylation was a t least found between LEC and normal rat serum ceruloplasmin. Copper atoms bound on the ceruloplasmin molecule appear to be essentialfor the oxidase activity of serum ceruloplasmin. Therefore, we propose the mechanism that the ceruloplasmin in LEC rat serumincludes no copper and, as a result, cannot exert oxidase activity. Identification of Serum Ceruloplasmin in the LEC Rat as Apoceruloplasmin-To depict that the ceruloplasmin in the LEC rat serum includes no copper, the serum was chromatographed on a Sepharose CL-4B column (Fig. 4). One peak containing ceruloplasminoxidase activity was detectedin normal rat and coeluted with copper atoms, indicating that the peak contains holoceruloplasmin (copper atoms-ceruloplasmin molecule complex). In contrast, in the LEC rat, no 1 2 3 4 5 6 7 8 I

I-

205

FIG.3. Western blot analysis of the serum ceruloplasmin. Sera were electrophoresed in SDS-polyacrylamide (7%) gel. After being transferred to the nitrocellulose membrane, the blotwas incubated with rabbit anti-rat ceruloplasmin serum. Immune complexes were detected with a Vectastain ABC kit. Lanes I , 5, and 7, normal rat at theage of 1 month; lanes 2,6, and 8, LEC rat at the age of 1 month; lane 3, normal rat at theage of 5 months; lane 4, LEC rat at the age of 5 months; lanes 1-4, untreated serum;lanes 5 and 6, serum treated with endoglycosidase H; lanes 7 and 8, serum treated with endoglycosidase F. The result shown is from a representative experiment repeated for a t least three separate sera prepared from different animals in both normal and LEC rats.

t

peak showing ceruloplasmin activity was detected, and only a very small amount of copper atoms was detected in fractions corresponding to thepeak containing ceruloplasmin in normal rat. This result indicates that the ceruloplasmin in the LEC rat serum exists apoceruloplasmin as (ceruloplasmin molecule including no copper atoms), which, therefore, cannot exert the oxidase activity. We thought that theoccurrence of apoceruloplasmin in LEC rat serum was caused by a defect in either the transportation of holoceruloplasmin from liver to serum or formation of acopper-ceruloplasmin complex in liver. Therefore, we examined copper binding to ceruloplasmin by fractionating copper-binding proteins in 64Cu-loaded hepatocytes. Distribution of -Cu among Hepatic Copper-binding Proteins-Rat hepatocytes were incubated with 64Cu overnight and chromatographed on a Sephadex G-75 column. Two major peaks (I and 11) containing 64Cu activity were detectedin normal rat hepatocytes (Fig. 5). Peaks I and I1 were eluted at the void volume and near 10-kDa position, respectively. From the molecular mass,peaks I and I1 were thought tocorrespond to major copper-binding proteins, ceruloplasmin and metallothionein, respectively. Peak I was confirmed to be ceruloplasmin by Western blot analysis with anti-rat ceruloplasmin hepatocytes, LEC rat (Fig. 5, inset). In contrast to normal rat hepatocytes showed no @Cu activityin the ceruloplasmin fraction. Since the total cytoplasmic fraction of LEC rat liver contains a ceruloplasmin level equal to that of control rats when analyzed with Western blot (data not shown), it was indicated that thedeficiency in serum ceruloplasmin activity was caused by the nonformationof the copper-ceruloplasmin complex, namely the inhibition of copper incorporation into ceruloplasmin in LEC rathepatocytes. The lack of formation of the copper-ceruloplasmin complex could be explained by either an abnormality of copper delivery into ceruloplasmin or a mutation in the ceruloplasmin molecule in LECrat hepatocytes. However, the possibility of a mutation in the ceruloplasmin molecule was excluded by the data in genetic analysis (see “Discussion”).A question is raised as to whether the abnormal accumulation of hepatic copper (Table I) could be explained only by the blocking of the copper exclusion by

a

b

%

-0-

F344

U

F344

0.30

0.20

0.10

0.00 t

Fraction No.

Fraction No.

FIG. 4. Identification of LEC rat serum ceruloplasmin as apoceruloplasmin. Sera (9 ml) were chromatographed on a Sepharose CL-4B column, and 5 ml of each fraction was collected. Panel a, ceruloplasmin oxidase activity was examined in each fraction of normal (open circles) and LEC (closed circles) rat serum. Panel b, the fractions were assayed for copper content in normal (open circles) and LEC (closed circles) rats. The experiments were repeated for three separate sera prepared from different animals in both normal and LEC rats, and this is representativeof the three similar results. In panel b, the copper content value in a mixture of fractions 70-85 was significantly different between normal and LEC rats(166.0& 5.1 and 15.3 & 0.5 ng/ml in the three preparationsof normal and LEC rats,respectively) ( p < 0.05 by Student’s t test).

Inhibition of Copper Incorporation Ceruloplasmin into 30 Peak I1

Peak I

v

v

8969 I

la

I

30

F344

I

20

la

Y

C

25

Time (min)

15

Fraction No.

FIG. 5. Distribution of "Cu within hepatic copper-binding proteins in hepatocytes. Hepatocytes were incubated with 5 pCi of W u overnight.Thecells were suspendedin isolation buffer and disrupted as described under "Materials and Methods." The cytoplasmic fraction of normal (open circles)and LEC (closed circles) rat hepatocytes was applied to a Sephadex G-75 column and eluted with the isolation buffer at a flow rate of 10 ml/h. 0.8 ml of each fraction was collected and counted for T u . Western blot analysis showed the presence of ceruloplasmin in fractions 8-10. The arrow indicates the position of cytochrome c. The arrowheads indicate the positions of peaks I and 11. The experiments wererepeatedfor three separate hepatocytes prepared from different animals in both normaland LEC rats, and this is representative of the three similar results. Total "2u content value in a mixture of fractions 8-10 was significantlydifferent between normal and LEC rats (12.76 k 5.04 and 0.41 & 0.15 ng in the three preparations of normal and LEC rats, respectively) ( p < 0.05 by Student's t test).

ceruloplasmin. We thought thata putative defect in LEC rat liver might affect the other copper metabolism pathways. Therefore, we examined the copper metabolism, namely uptake, compartmentalization, and efflux in LEC rat hepatocytes, by loading them with 64Cu. @CuAccumulation by Cytosolic and Noncytosolic Fractions in LEC RatHepatocytes-When the total copper contents in isolated hepatocytes were measured, they were about 170 and 330 pmol/mg of protein in cytosolic and noncytosolic fractions, respectively. On the contrary, the totalcopper content in LEC rat hepatocytesshowed about 6,500 and 2,100 pmol/ mg of protein in the cytosolic and noncytosolic fractions, respectively. The ratio of the cytosolic copper content to the noncytosolic copper content in the LEC ratwas 3:1, whereas that in the normal ratwas 1:2. The uptake of copper into the cytosol largely precedes the copper uptake into other noncytosolic compartments (30), and in hepatocytes loaded with excess coppermost of the excess copper is accumulated in the noncytosolic compartment, which includes nuclear, mitchondrial,and microsomal fractions (31, 32). Therefore, we thought of the possibility that in LEC rat hepatocytes the copper cannot be transported from the cytosol into noncytosolic compartments. To assess this possibility, the accumulation of radioactive copper in cytosolic and noncytosolic fractions of hepatocytes was examined using primary-cultured hepatocytes. 64Cu accumulation in the noncytosolic fraction of LEC rat hepatocytes was about 2-fold lower than that of normal rat hepatocytes,whereas W u accumulation in the cytosolic fraction of LEC rat hepatocytes was indistinguishable from that of normal rat hepatocytes(Fig. 6). Student's t test confirmed that at the loading time of 16 h, the value of noncytosolic T u accumulated was significantly different be-

b 200

-0- F344 L K :

100

-

Time (min)

FIG.6. '%u accumulation in cytosolic and noncytosolic fractions of hepatocytes. Hepatocytes were incubated with 5 pCi of %u in Earle's balanced salt solution plus 10% fetal calf serum at 37 "C. A t the indicated times, the cells were washed and processed as described under "Materials and Methods" followed by the determination of MCu accumulation in cytosolic (panel a ) and noncytosolic (panel b) fractions of normal (open circles) and LEC (closed circles) rat hepatocytes. Each point and bar represents the mean & S.E. of triplicate observations. The experiments were repeated for two and three separate hepatocytes prepared from different animalsin normal andLEC rats, respectively,and this is representative of the two similar results for control rats and three for LEC rats. tween normal and the LEC rats (182.2 f 13.6 and 69.2 f 13.6 pmol/mgincombined data from two andthree separate experiments using different hepatocyte preparations of normal and LEC rats, respectively), whereasthe value of cytosolic 64Cuaccumulated was indistinguishable (17.8 f 0.6 and 20.8 f 3.0 pmol/mg in combined data from two and threeseparate experiments using different hepatocyte preparations of normal andLECrats, respectively).Therefore, these results suggest that theefficiency of copper transport from the cytosol to noncytosolic compartments is reduced in the LEC rat compared with the normal rat and that as a result, a large part of theabnormally accumulated copper existsin the cytosolic fraction in LEC rat hepatocytes. Efflux of 64Cu from Preloaded Hepatocytes-Hepatocytes were preloaded with 64Cu for 6 h, and then the cells were recultured in 64Cu-freemedia. As shown in Fig. 7,65% of the preloaded '%u was released from normal rat hepatocytes a t

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present data in 64Cudistribution among copper-binding proteins together with the result of the genetic study, we have suggested that theinhibition of the copper incorporation into ceruloplasmin is because of an abnormality of copper delivery into ceruloplasmin. The LEC rat might lack a putative factor essential to the transportation of the copper atom to the L P, I cellular sites where copper is incorporated into ceruloplasmin. In the brindled mouse, which is an inborn error of copper metabolism and an animal model of Menkes disease (33), a 60 48-kDa cytosolic copper-binding protein has been suggested as a protein responsible for the primary defect in abnormal copper accumulation in kidney (34). Hepatocytes may possess C 0 401 U F344 a similar protein involved in intracellular copper delivery. + L E C Metallothionein levels are elevated as thecopper-bound form E in LEC rat liver (35 and Fig. 5). It should be noted here that the elevated metallothionein is supposed to be a secondary 0 200 400 600 800 1000 effect accompanied by the elevated copper levels rather than the primary defect. Time (min) Our data in the 64Cu efflux experiment indicate that the FIG. 7. Efflux of ‘“Cu from hepatocytes. Hepatocytes were abnormality of the copper delivery mechanism in LEC rat preloaded by incubating with 5 pCi of %u in Earle’s balanced salt solution plus 10%fetal calf serum for6 h. After the preloading period, hepatocytes also causes a reduced copper efflux from hepatothe media were discarded,and the cells were washed three times with cytes, leading to theexcess hepatic copper accumulation. The Hanks’ balanced salt solution. %u-free medium (Earle’s balanced major route of copper efflux from hepatocytes is thought to salt solution plus 10% fetal calf serum) was added and cultured for be copper excretion into bile. Based on our previous data that the indicated times. After culturing, the cells were washed and proc- the two anomalies, the deficiency in serum ceruloplasmin essed as described under “Materials and Methods” to determine the activity and the hepatic copper accumulation, are correlated M C efflux ~ fromnormal (open circles) and LEC (closed circles) rat hepatocytes. Each point and bar represents the mean +- S.E. of completely (29), copper delivery into thebiliary copper excretriplicate observations. The experiments were repeated for three tion pathway appears to be impaired in the LEC rat through separate hepatocytes prepared from different animals in both normal the primary defect associated with the abnormality of copper and LEC rats, and this is representative of the three similar results. delivery into ceruloplasmin. Furthermore, from the data of 64Cuaccumulation by hepatocytes, this primary defect in the 960 min after reculturing in 64Cu-freemedia. In contrast,LEC LEC rat also seems to cause an inhibition of the copper rat hepatocytes exhibited a reduced efflux of 64Cufrom he- transportation from the cytosol to noncytosolic compartpatocytes. Student’s t test confirmed that the value of the ments. We assume that themajor noncytosolic compartments fraction ofY!u retained was significantly different between should be the dense polysome fraction on the rough endonormal and LEC rats at 960 min after reculturing (36.7 f 9.1 plasmic reticulum and lysosomes. The possibility of the rough and 85.3 & 10.2% in three separate hepatocyte preparations endoplasmic reticulum is partly supported by evidence that of normal and LEC rats, respectively). This result, together the copper atoms areincorporated only into newly synthesized with the datashowing the reduction in the efficiency of copper ceruloplasmin and thatde nouo synthesis of the ceruloplasmin transport from the cytosolic to noncytosolic fractions, indi- appears to occur in a dense polysome fraction on the rough cated an abnormality of the copper delivery mechanism in endoplasmic reticulum (18). The possibility of lysosomes is LEC rat hepatocytes. We therefore postulate the hypothesis partly supported by evidence that the pathway for biliary that theabnormality in copper delivery into theceruloplasmin copper efflux involves the lysosomes (36). molecule is a primary cause for the deficiency in the serum The kinetic parameter Vmaxfor copper uptake in LEC rat ceruloplasmin activity. In addition, the abnormality seems to hepatocytes was significantly lower than that in normal rat, cause an inhibition of biliary copper excretion and,asa whereas the kinetic parameter K, was the same in LEC and consequence, abnormal copper accumulation in LEC rat he- normal rats (datanot shown). This indicates that in the LEC patocytes (see “Discussion”). rat an intracellular putative copper carrier protein, to which copper binds initially after the passive copper entrance, has DISCUSSION an identical affinity for copper but decreases copper binding Our present study demonstrates thatthe deficiency in sites as compared with the normal rat. The apparent lower serum ceruloplasmin activity in the LEC mutant rat iscaused Vmaxof copper uptake for hepatocytes in the LEC rat would by the inhibition of the incorporation of copper atoms into be explained by the occupation of copper binding sites on a ceruloplasmin, which is essential to its catalytic activity. The putative cytosolic factor, since the rate in copper transportaquestion arises: what is the primary defect for the inhibition tion from cytosolic to noncytosolic fractions was reduced in of copper incorporation into ceruloplasmin which leads to the LEC rat liver. However, a reduction of Vmaxmight be exdeficiency in serum ceruloplasmin activity? Indeed, the ceru- plained by the fact that thecapacity of the copper binding in loplasmin gene existed normally on the LEC rat genome, and the noncytosolic fraction has been filled in LEC rat hepatothe expression process to protein occurred normally in LEC cytes. Therefore, itseems important that we present evidence rat liver. We considered the possibility that theceruloplasmin thatthe reduction in the copper transportrate is nota molecule might possess a mutation by which the ceruloplas- secondary effect of the accumulated copper in noncytosolic min molecule cannot bind copper. However, we have obtained fraction. The total copper content in LEC rat fetal liver was the result that theceruloplasmin gene is not the gene causing shown to be lower (1,000 pmol/mg of protein) than that of the deficiency in serum ceruloplasmin activity by genetic normal rat fetus (7,700pmol/mg of protein) (data notshown), analysis using restriction fragment length polymorphism of probably reflecting the lower serum copper content in the the ceruloplasmin gene detected with KpnI (29). From our pregnant LEC rat than in a normal pregnant rat. The ratio (D

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Inhibition of Incorporation Copper Ceruloplasmin into of the cytosolic to noncytosolic copper content in the LEC rat fetus is similar to that of the adult, in spite of the total hepatic copper content in the fetus being smaller than thatin the normal rat fetus. These data suggest that the reduction in the copper transport rate is a primary inherent defect in the LEC rat irrespective of the copper accumulation. Based on the results in our present study together with others (24), we consider that the pathogenesis of the hepatic disorder in the LEC rat is very similar to that of human Wilson's disease (37-39). First, the deficiency in serum ceruloplasmin activity in the LEC rat is caused by the inhibition of copper incorporation into ceruloplasmin, and the biliary copper excretion is also inhibited in the LEC rat. These data are consistent with the data reported for Wilson's disease (40). Second, dissociation of the ceruloplasmin gene from a putative gene causing the deficiency in serum ceruloplasmin activity in the LEC rat (29) is consistent with thedata reported for Wilson's disease, namely, the ceruloplasmin gene is located on chromosome 3, whereas aputative Wilson's disease gene is located on chromosome 13 (41-43). Further detailed genetic and molecular analyses in the LEC rat may elucidate the pathogenesis of human Wilson's disease as well as a specific biochemical process responsible for the intracellular copper delivery. REFERENCES 1. Ryden, L., and Bjork, I. (1976) Biochemistry 16,3411-3417 2. Orena, S. J., Goode, C. A., and Linder,M. C. (1986) Biochem. Biophys. Res. Commun. 139,822-829 3. Takahashi, N., Ortel, T. L., and Putnam, F. W. (1984) Proc. Natl. Acad. Sei. U. S. A. 81,390-394 4. Goldstein, I. M., Kaplan, H. B., Edelson, H. S., and Weissmann, G. (1979) J. Biol. Chem. 264,4040-4045 5. Win e D R (1984) Semin. Liuer Dis. 4,239-251 6. Frieteh, E. (1986) Clin. Physiol. Biochem. 4, 11-19 7. Samokyszyn, V. M., Miller, D. M., Reif, D. W., and Aust, S. D. (1989) J. Bwl. Chem. 264,Zl-26 8. Folkman, J., and Klagsburn, M. (1987) Science 236,442-447 9. Walker, F. J., and Fay, P. J. (1990) J. Biol. Chem. 266, 1834-1836 10. Gutteridge, J. M.C., and Stocks, J. (1981) CRC Crit. Reu. Clin. Lab. Sci. 14,257-329 11. Cousins, R.J. (1985) Physiol. Reu. 66,238-309

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