Copper Converts the Cellular Prion Protein into a Protease-resistant Species That Is Distinct from the Scrapie Isoform*

Elena QuaglioDagger, Roberto Chiesa§, and David A. Harris

From the Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110

Received for publication, October 23, 2000, and in revised form, November 29, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Several lines of evidence have suggested that copper ions play a role in the biology of both PrPC and PrPSc, the normal and pathologic forms of the prion protein. To further investigate this intriguing connection, we have analyzed how copper ions affect the biochemical properties of PrPC extracted from the brains of transgenic mice and from transfected cells. We report that the metal rapidly and reversibly induces PrPC to become protease-resistant and detergent-insoluble. Although these two properties are commonly associated with PrPSc, we demonstrate using a conformation-dependent immunoassay that copper-treated PrP is structurally distinct from PrPSc. The effect of copper requires the presence of at least one of the five octapeptide repeats normally present in the N-terminal half of the protein, consistent with the idea that the metal alters the biochemical properties of PrP by directly binding to this region. These results suggest potential roles for copper in prion diseases, as well as in the physiological function of PrPC.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Prions, the causative agents of neurodegenerative diseases in humans and other mammals, are composed of PrPSc,1 a post-translationally modified form of a normal cellular protein designated PrPC (1, 2). Spectroscopic studies reveal that PrPC has a high alpha -helical content whereas PrPSc is rich in beta -sheets (3-6). These two isoforms also differ biochemically, with PrPSc displaying reduced solubility in nondenaturing detergents and partial resistance to protease digestion (7, 8). Although a great deal is known about PrPSc and its role in the disease process, the physiological function of PrPC has remained enigmatic.

Recently, however, several lines of evidence have suggested that the essential trace metal, copper, may play a key role in the biology of PrPC. Most importantly, several laboratories have shown that copper ions bind with low micromolar affinity to the octapeptide repeat region in the N-terminal half of mammalian PrPC which in mouse contains four copies of the sequence PHGG(G/S)WGQ and one copy of the sequence PQG GTWGQ (9-16). Binding of copper is pH-dependent, and induces a conformational change in this normally unstructured region of the molecule. In addition, we have shown that copper rapidly and reversibly stimulates endocytosis of PrPC from the cell surface, raising the possibility that PrPC normally serves as a receptor for cellular uptake or efflux of copper (17). An enzymatic function for PrPC has also been claimed based on the observation that copper binding confers superoxide dismutase activity on the protein (18).

Other connections between PrPC and copper have been proposed but have proven to be controversial. PrPC was postulated to be a major copper-binding protein in brain based on the observation that the content of copper is 5-50% of normal in membrane fractions derived from the brains of mice which carry a disrupted PrP gene (16, 19). The activity of SOD1 was also reported to be 50% of normal in the brains of these mice, and neurons cultured from the animals were found to be more susceptible to oxidative stress, suggesting a role for PrPC in protection from oxidative damage (20, 21). However, we have failed to observe any differences in brain copper content or in the activities of SOD1 and a second cuproenzyme, cytochrome oxidase, among mice that express 0, 1, and 10 times the normal levels of PrP (22).

There are also several results which suggest interactions between PrPSc and copper, and a possible role for the metal in prion diseases. First, copper facilitates restoration of protease resistance and infectivity during refolding of guanidine-denatured PrPSc (23). Second, the protease cleavage pattern of PrPSc derived from the brains of patients with Creutzfeldt-Jakob disease is altered by addition or chelation of copper and zinc, suggesting a role for metal occupancy in determining prion strain properties (24). Finally, it was reported almost 25 years ago that administration of the copper chelating agent cuprizone to mice caused a spongiform degeneration of the brain similar to scrapie (25).

To further investigate the interaction of PrP with copper, we have analyzed how copper affects the biochemical properties of PrPC extracted from the brains of transgenic mice and from transfected cells. We report that the metal causes PrPC to assume a protease-resistant and detergent-insoluble form that is similar to, but conformationally distinct from PrPSc. Surprisingly, this effect requires only a single octapeptide repeat in the N-terminal half of the protein.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Transgenic Mice-- Tg(WT) and Tg(PG14) mice that express 3F4-tagged, wild-type, or PG14 mouse PrP, respectively, under the control of the Prn-p promoter have been described previously (26, 27). The experiments reported here were performed on Tg(PG14+/+) mice of the A2 and A3 lines, as well as on Tg(WT+/+) mice of the E1 line, all generated by breeding onto the C57BL/6J × 129/Prn-p0/0 background. Tgd11 mice expressing mouse PrPDelta 32-80 were provided by C. Weissmann (28).

Antibodies-- Monoclonal antibody 3F4 recognizes an epitope consisting of residues 109-112 of hamster and human PrP (29); this epitope was introduced into mouse PrP by mutation of homologous residues 108 and 111 to methionines. Rabbit polyclonal antibody P45-66 was raised against a synthetic peptide encompassing mouse PrP residues 45-66 (30). Rabbit polyclonal antibodies R20 and R30 are directed against mouse PrP residues 218-232 and 89-103, respectively (31). To detect doppel (32), a rabbit antibody was raised against the peptide GIKHRFKWNRKVLPSSGGQCG (corresponding to residues 28 to 46 with CG appended at the C terminus) that had been conjugated to keyhole limpet hemocyanin.

Plasmids-- Construction of a cDNA encoding wild-type mouse PrP that contains a 3F4 epitope tag has been described previously (30). A cDNA encoding mouse PrP that contains an exact deletion of all 5 octapeptide repeats (Delta 51-90) was constructed by overlapping polymerase chain reaction using the wild-type plasmid as a template. A cDNA encoding mouse PrP that contains a deletion of the first 4 octapeptide repeats (Delta 51-82) was constructed by re-inserting the sequence for the octapeptide PHGGGWGQ into the Delta 51-90 template using overlapping polymerase chain reaction. All cDNAs were cloned into the expression vector pcDNA3 (Invitrogen).

The open reading frame of mouse doppel was amplified by polymerase chain reaction using DNA extracted from CD1 mouse tail as a template. The sense primer contained a HindIII restriction site and had the sequence: 5'- GACCAGAAGCTTATGAAGAACCGGCTGGGTACATGG-3'. The antisense primer contained a BamHI restriction site and had the sequence: 5'-GACCAGGGATCCTTACTTCACAATGAACCAAACG-3'. The polymerase chain reaction product was digested with HindIII and BamHI and ligated into HindIII-BamHI-digested pcDNA3.

Transfected Cells-- CHO cells were grown in Dulbecco's modified Eagle's medium containing 7.5% fetal bovine serum and penicillin/streptomycin in an atmosphere of 5% CO2, 95% air. For the experiment shown in Fig. 7, CHO cells were transfected with the appropriate plasmids using LipofectAMINE (Life Technologies) according to the manufacturer's instructions, and analyzed 2 days later. For preparation of stable lines expressing doppel (Fig. 8), CHO cells were transfected with the doppel plasmid and were then selected in medium containing G418 (300 µg/ml). CHO cells stably transfected to express wild-type mouse PrP with a 3F4 tag were previously described (30).

Detergent Insolubility Assay-- Ten percent (w/v) homogenates of Tg mouse tissues were prepared with a Teflon-glass apparatus (10 strokes at 1,000 rpm) in ice-cold phosphate-buffered saline (pH 7.2) containing 0.5% Triton X-100, 0.5% Nonidet P-40, 0.5% sodium deoxycholate, and 0.2% N-lauroylsarcosine (homogenization buffer). Homogenates were centrifuged for 5 min at 900 × g, and the protein concentration of the supernatant was measured with the BCA Protein Assay Kit (Pierce). Supernatants were diluted to a protein concentration of 0.5 mg/ml in homogenization buffer containing protease inhibitors (pepstatin and leupeptin, 1 µg/ml; PMSF, 2 mM), and after incubation for 20 min at 4 °C were centrifuged at 16,000 × g for 5 min. Supernatants were incubated with CuSO4, ZnSO4, or MnCl2 at 20 °C and then centrifuged at 186,000 × g for 40 min. PrP in the pellet and supernatant fractions was analyzed by Western blotting using antibody 3F4.

Transfected CHO cells were lysed in homogenization buffer at 4 °C for 20 min, centrifuged at 16,000 × g for 5 min, and the protein concentration of the cleared supernatants was determined. Metal ion treatment and ultracentrifugation were carried out as described above.

Protease Resistance Assay-- Brain homogenates were diluted to 4 mg/ml in homogenization buffer without protease inhibitors and incubated for 20 min at 4 °C. Aliquots corresponding to 80 µg of protein were treated with metal ions at 20 °C and then digested with proteinase K for 30 min at 37 °C. Digestion was terminated by addition of PMSF to a final concentration of 5 mM and boiling in SDS-PAGE sample buffer.

Conformation-dependent Immunoprecipitation of PrP-- Brain homogenates were diluted to 4 mg/ml in 300 µl of phosphate-buffered saline containing 0.5% Nonidet P-40, 0.5% sodium deoxycholate, and incubated for 20 min at 4 °C. After clearing by centrifugation at 16,000 × g for 2 min, the lysates were divided into two aliquots. One aliquot was denatured in 1% SDS by incubation at 95 °C for 10 min, then Nonidet P-40 was added to a final concentration of 1% to bind the SDS. The other aliquot was processed in parallel, but the denaturation step was omitted. Samples were precleared by incubation with protein A-Sepharose and incubated with or without 400 µM CuSO4 for 30 min at 20 °C. Fifty-µl aliquots were collected to test detergent insolubility and proteinase K resistance as described above. To immunoprecipitate PrP, samples were incubated for 1 h at 4 °C with 3 µl of antibody 3F4, and the immune complexes were collected with protein A-Sepharose. Protein A-Sepharose pellets were washed 4 times in RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS). For copper-treated samples, 400 µM CuSO4 was added to the RIPA buffer to ensure that the copper-induced conformation was maintained during washing of the beads. Proteins were eluted in sample buffer at 95 °C for 10 min, separated by SDS-PAGE, and blotted onto polyvinylidene fluoride membranes. Blots were incubated with biotinylated 3F4 prepared as described (33), and visualization of the bound antibody was achieved using horseradish peroxidase-coupled streptavidin and enhanced chemiluminescence (Amersham Pharmacia Biotech).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Copper Causes PrPC to Become Detergent Insoluble-- Detergent lysates were prepared from the brains of Tg(WT) mice that express wild-type mouse PrP carrying an epitope tag for the monoclonal antibody 3F4 (26, 27). Lysates were incubated with increasing concentrations of CuSO4 for 30 min at 20 °C and then PrP was tested for detergent insolubility by ultracentrifugation. While PrP was completely soluble in the absence of metal, incubation with 200 µM copper shifted ~50% of the protein into the pellet, and treatment with 300 or 400 µM copper rendered 80-90% of the PrP insoluble (Fig. 1A). Within this concentration range, copper did not cause nonspecific precipitation of protein, since actin (Fig. 1A) and tubulin (not shown) remained entirely in the supernatant.



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Fig. 1.   Cu2+ renders PrP detergent insoluble. A, detergent extracts of Tg(WT) mouse brain were incubated with the indicated concentration of CuSO4 for 30 min at 20 °C and subsequently centrifuged at 186,000 × g for 40 min. Proteins in supernatants (S lanes) and pellets (P lanes) were separated by SDS-PAGE and immunoblotted with either anti-PrP antibody 3F4 or anti-actin antibody. B, detergent lysates of Tg(WT) mouse brain extracts were incubated with (lanes 5-8) or without (lanes 1-4) CuSO4 for 30 min. Each sample was then divided in two aliquots and incubated with (lanes 3, 4, 7, and 8) or without (lanes 1, 2, 5, and 6) 1 mM BCS for 5 min prior to ultracentrifugation. Proteins in supernatants (S) and pellets (P) were separated by SDS-PAGE and immunoblotted with 3F4 antibody. C, detergent extracts of heart and skeletal muscle from a Tg(WT) mouse were incubated with the indicated concentrations of CuSO4 for 30 min and ultracentrifuged. PrP in the supernatants (S lanes) and pellets (P lanes) was detected by immunoblotting with 3F4. Size markers are given in kDa.

The effect of copper on the solubility of PrP was rapid and reversible. Between 80 and 90% of the protein was recovered in the pellet after treatment with 300 µM copper regardless of whether the lysate was subjected to ultracentrifugation immediately after addition of metal, or was incubated with metal for 30 min prior to ultracentrifugation (data not shown). To test the reversibility of the effect, brain lysates were incubated with 300 µM CuSO4 for 30 min, and then the copper chelator bathocuproinedisulfonate (BCS) was added for 5 min prior to ultracentrifugation. We found that BCS completely shifted PrP back into the supernatant fraction (Fig. 1B). Copper ions also induced insolubility of PrP in tissue homogenates prepared from the heart and skeletal muscle of Tg(WT) mice, indicating that the effect was not restricted to PrP expressed in brain (Fig. 1C).

Since there is evidence for interaction between PrP and both Zn2+ and Mn2+ (34), we tested the ability of these ions to confer detergent insolubility on PrP. We found that Zn2+ (200-500 µM) caused PrP to become detergent insoluble, while Mn2+ did not (Fig. 2). Neither metal altered the solubility of actin.



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Fig. 2.   Zn2+ but not Mn2+ renders PrP detergent insoluble. Detergent extracts of Tg(WT) mouse brain were incubated with the indicated concentrations of ZnSO4 (upper panels) or MnCl2 (lower panels) for 30 min and subsequently centrifuged at 186,000 × g for 40 min. Proteins in supernatants (S lanes) and pellets (P lanes) were separated by SDS-PAGE and immunoblotted with either anti-PrP antibody 3F4 or with anti-actin antibody.

Copper Converts PrPC into a Protease-resistant Form-- Since detergent insolubility is a characteristic property of PrPSc, we tested whether copper caused PrP to acquire a second PrPSc-like property, protease resistance. Proteinase K (PK) cleaves PrPSc near residue 90 to produce a core fragment of 27-30 kDa, while under the same conditions PrPC is completely digested. We found that, in the absence of copper, PrP in brain lysates from Tg(WT) mice was completely digested with a PK concentration as low as 3 µg/ml. In contrast, after incubation with 200 or 300 µM CuSO4, PrP became resistant to high concentrations of PK (500 or 800 µg/ml), producing a fragment that migrated at 27-30 kDa (Fig. 3A). The effect of copper was specific, since actin (Fig. 3A), as well as tubulin and most other proteins observable on Coomassie-stained gels (not shown), were completely digested by PK concentrations of 500 or 800 µg/ml. Under our experimental conditions neither Zn2+ nor Mn2+ induced conversion of PrP to a PK-resistant form (data not shown).



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Fig. 3.   Cu2+ causes PrP to become PK resistant. A, detergent extracts of Tg(WT) mouse brain were incubated with the indicated concentrations of CuSO4 for 30 min and then digested with different amounts of PK for 30 min at 37 °C. After termination of the digestion with PMSF, proteins were separated by SDS-PAGE and immunoblotted with either anti-PrP antibody 3F4 or anti-actin antibody. The lanes containing undigested samples (0 µg/ml PK) represent 8 µg of protein, and the other lanes 40 µg of protein. B, detergent lysates of Tg(WT) mouse brain were incubated with 200 µM CuSO4 for 30 min at 20 °C, and were then either digested with 160 µg/ml PK (lanes 2 and 3), or were left undigested (lane 1). Prior to digestion, one sample was denatured by boiling in 0.5% SDS (lane 3). PrP was detected by immunoblotting using 3F4 antibody. Lane 1 represents 8 µg of protein, and lanes 2 and 3 represent 40 µg of protein. C, detergent lysates of Tg(WT) mouse brain were treated for the indicated times with 300 µM CuSO4. Aliquots were then incubated with (lanes 3 and 5) or without (lanes 1, 2, and 4) 1 mM BCS for 5 min. Finally, samples were digested for 30 min with 200 µg/ml PK for 30 min (lanes 2-5), or left undigested (lane 1). After addition of PMSF, proteins were separated by SDS-PAGE and immunoblotted with anti-PrP antibody 3F4. The undigested sample (lane 1) represents 8 µg of protein, and the other lanes 40 µg of protein.

To rule out the possibility that appearance of the 27-30-kDa band was due to inhibition of PK activity by copper, brain lysates that had been incubated with the metal were denatured by boiling in 0.5% SDS before adding PK. After denaturation, PrP was completely digested by the protease (Fig. 3B), indicating that copper does not affect PK activity. This experiment also suggests that production of the PrP 27-30 fragment depends on a native conformation of the protein.

To test the time course and reversibility of the copper effect on protease resistance, samples treated with copper for 15 or 30 min were incubated with or without BCS for 5 min, and then digested with PK. As shown in Fig. 3C, the 27-30-kDa fragment was detected after either 15 or 30 min of copper treatment, and was not present in samples incubated with BCS. PK treatment immediately after addition of copper also led to the appearance of the 27-30-kDa band (not shown). Thus, the effect of copper on PK resistance, as well as on detergent insolubility, occurs within minutes and is readily reversible by removal of the metal.

To characterize the protease-resistant fragment induced by copper, we analyzed its immunoreactivity to antibodies directed against different regions of the protein. As shown in Fig. 4, the 27-30-kDa fragment was detected by antibodies 3F4 and R20 directed against residues 108-111 and 218-232, respectively, but not by antibody P45-66 which reacts with residues 45-66 within the octapeptide repeats. These results indicate that the PK cleaves copper-treated PrP at a location between the end of the octapeptide repeats and residue 108, the same region where authentic PrPSc is cleaved. This cleavage site is distinct from one within the central hydrophobic region (residues 110-120) that is utilized by cellular proteases and that produces a C-terminal fragment that does not react with 3F4 antibody (27, 35, 36).



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Fig. 4.   Epitope mapping of the PK-resistant fragment produced by copper treatment. Detergent extracts from Tg(WT) mouse brain were incubated for 30 min with (lanes 3 and 4) or without (lanes 1 and 2) CuSO4 (300 µM). Aliquots corresponding to 120 µg of total protein were digested with the indicated concentrations of PK for 30 min. Proteins were then separated by SDS-PAGE, and immunoblotted with antibodies specific for three different regions of PrP. The amino acid residues comprising the antibody epitopes are indicated in parentheses below the antibody designation. The lanes containing undigested samples (0 µg/ml PK) represent 8 µg of protein.

The Protease-resistant Form of PrP Induced by Copper Is Structurally Distinct from PrPSc-- There is evidence that the epitope recognized by antibody 3F4 becomes buried upon conversion of PrPC to PrPSc (37, 38). Thus, PrPSc in the native state reacts with 3F4 much more poorly than does PrPC, whereas both forms react equally well after denaturation. To test whether copper converts PrPC to a form that is structurally similar to PrPSc, we compared the 3F4 reactivity of the protein by immunoprecipitation before and after copper treatment. We found that treatment with 400 µM CuSO4 had no effect on the 3F4 reactivity of PrP in brain lysates from Tg(WT) mice (Fig. 5A, lanes 5 and 7) and that the copper-treated protein reacted equally well in the native and denatured states (Fig. 5A, lanes 7 and 8). We confirmed that, under the conditions of the experiment, copper had converted PrP to a form that was both protease resistant (Fig. 5B) and detergent insoluble (Fig. 5C). In contrast to copper-treated PrP, authentic PrPSc from scrapie-infected hamster brain reacted with 3F4 much more weakly than PrPC from uninfected brain (Fig. 5A, lanes 1 and 3), and the reactivity of the PrPSc form was enhanced by denaturation (Fig. 5A, lanes 3 and 4). As expected, untreated PrPC from both hamster and mouse brain reacted equally well with 3F4 in both the native and denatured states (Fig. 5A, lanes 1 and 2, 5 and 6). These results indicate that although copper-treated PrP is protease-resistant and detergent-insoluble, its conformation is likely to be distinct from that of PrPSc.



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Fig. 5.   Conformation-dependent immunoprecipitation of PrP in the presence of Cu2+A, detergent extracts were prepared from the brains of uninfected hamsters (lanes 1 and 2), hamsters infected with the 263K strain of scrapie (lanes 3 and 4), and Tg(WT) mice (lanes 5-8). The mouse brain extracts were incubated with (lanes 7 and 8) or without (lanes 5 and 6) CuSO4 (400 µM) for 30 min. Samples in lanes 2, 4, 6, and 8 were denatured in SDS prior to immunoprecipitation. PrP was immunoprecipitated from all samples using 3F4 antibody, and after separation by SDS-PAGE, blots of the gel were reacted with biotinylated 3F4 antibody followed by visualization using horseradish peroxidase-streptavidin and ECL. B and C, prior to immunoprecipitation, aliquots of mouse brain lysates that had been incubated with (lanes 3 and 4) or without (lanes 1 and 2) CuSO4 were tested for PK resistance (B) and detergent insolubility (C), as described in the legends to Figs. 3 and 1, respectively. The concentration of PK used in panel B was 100 µg/ml.

A Single Octapeptide Repeat Is Sufficient for Mediating the Effects of Copper-- There is considerable evidence that copper binds to the octapeptide repeat region of PrP (9-16). We therefore investigated whether the effects of copper on the detergent insolubility and protease resistance of PrP were dependent on the number of octapeptide repeats. For these experiments, we used PrP molecules carrying octapeptide expansions or deletions that were expressed in either transgenic mice or transfected cells.

To test the effect of extra octapeptide repeats, we analyzed lysates from the brains of Tg(PG14) mice which express PrP molecules carrying a nine-repeat insertion (a total of 14 repeats). This mutation is associated with a form of Creutzfeldt-Jakob disease in humans and with a neurological illness in the transgenic mice (26, 27). As reported previously, PG14 PrP is weakly protease resistant in the absence of copper, producing a 27-30-kDa fragment when digested with 3 µg/ml PK (Fig. 6A). However, addition of 100-300 µM CuSO4 greatly enhanced the protease resistance of the mutant protein, so that a resistant fragment was detectable at 500 or 800 µg/ml PK. We noted that PG14 PrP appeared to be slightly more sensitive to copper than wild-type PrP, since a highly PK-resistant fragment was detected at copper concentrations of >= 100 µM for the mutant protein (Fig. 6A), compared with >= 200 µM for the wild-type protein (Fig. 3A). Investigation of the effect of copper on the detergent insolubility of PG14 PrP is not feasible, since more than half of the protein is detergent insoluble even in the absence of copper (27).



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Fig. 6.   Effect of octapeptide expansions and deletions on the copper-induced PK resistance of PrP from transgenic mouse brains. A, detergent lysates of Tg(PG14) mouse brain were incubated with the indicated concentrations of CuSO4 for 30 min and then digested with different amounts of PK for 30 min at 37 °C. After termination of the digestion with PMSF, proteins were separated by SDS-PAGE and immunoblotted with either anti-PrP antibody 3F4 or anti-actin antibody. The lanes containing undigested samples (0 µg/ml PK) represent 8 µg of protein, and the other lanes 40 µg of protein. B, detergent lysates prepared from the brains of Tgd11 mice expressing PrPDelta 32-80 were incubated with 300 µM CuSO4 for 30 min at 20 °C, and were then either digested with 200 µg/ml PK (lanes 2 and 3) or were left undigested (lane 1). Prior to digestion, one sample was denatured by boiling in 0.5% SDS (lane 3). PrP was detected by immunoblotting using antibody R30. Lane 1 represents 8 µg of protein, and lanes 2 and 3 represent 40 µg of protein.

To explore the effect of octapeptide deletions, we first analyzed an N-terminal truncated form of PrP (Delta 32-80) containing only a single octapeptide repeat (PHGGGWGQ) that is expressed in the brains of the Tgd11 line of transgenic mice (28). This protein is missing 19 amino acids N-terminal to the octapeptide repeats as well as the first 4 of the repeats. Surprisingly, we observed that PrPDelta 32-80 became protease resistant (Fig. 6B) and detergent-insoluble (not shown) in the presence of copper, effects that were abolished when protein was first denatured in SDS.

To further investigate the effect of reduced numbers of octapeptide repeats, we examined the detergent insolubility of PrP expressed in transiently transfected CHO cells (Fig. 7). We confirmed that copper caused wild-type PrP synthesized in CHO cells to become detergent insoluble, although the concentrations of metal required to observe this effect (400-600 µM) were somewhat higher than for PrP expressed in transgenic mouse brains. Since the CHO cell and brain lysates are utilized at equivalent total protein concentrations, the difference in copper sensitivity is presumably attributable to differences in the composition of the two kinds of lysate, including possibly the higher expression of PrP in brain compared with transfected cells. PrPDelta 51-90, which contains an exact deletion of all 5 octapeptide repeats, remained detergent soluble in the presence of copper. In contrast, PrPDelta 51-82, which contains a single repeat (PHGGGWGQ) was rendered insoluble by copper. Taken together with the results for PrPDelta 32-80 in Tgd11 mice, this observation indicates that a single histidine-containing repeat is sufficient to confer copper-dependent insolubility on PrP. Similar results were obtained when the same constructs were transiently expressed in N2a neuroblastoma cells (data not shown).



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Fig. 7.   A single octapeptide repeat is sufficient to render PrP detergent insoluble in the presence of Cu2+. Detergent lysates of transiently transfected CHO cells expressing wild-type mouse PrP (5 octapeptide repeats), PrPDelta 51-82 containing 1 octapeptide repeat (PHGGGWGQ), or PrPDelta 51-90 containing 0 octapeptide repeats were incubated with the indicated concentrations of CuSO4 for 30 min and subsequently centrifuged at 186,000 × g for 40 min. Proteins in supernatants (S lanes) and pellets (P lanes) were separated by SDS-PAGE and PrP was visualized by immunoblotting with 3F4 antibody.

Doppel is a recently identified PrP paralogue that lacks the N-terminal half of the PrP sequence, including the octapeptide repeat region and the central hydrophobic domain (32, 39). To investigate the effect of copper on the biochemical properties of doppel, we generated stably transfected CHO cell lines expressing mouse doppel and tested the solubility of the protein in detergent cell lysates incubated with or without copper. As shown in Fig. 8, incubation with CuSO4 did not alter the solubility of doppel, which was recovered entirely in the supernatant after ultracentrifugation, while PrP was rendered partially insoluble.



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Fig. 8.   Cu2+ induces detergent insolubility of PrP but not of doppel. Detergent lysates of stably transfected CHO cells expressing wild-type mouse PrP (A) or mouse doppel (B) were incubated with (lanes 3 and 4) or without (lanes 1 and 2) 400 µM CuSO4 for 30 min and centrifuged for 40 min at 186,000 × g. Proteins in supernatants (S lanes) and pellets (P lanes) were separated by SDS-PAGE and immunoblotted with 3F4 antibody to detect PrP and with anti-doppel (28-46) to detect doppel.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We demonstrate here that copper ions induce PrPC derived from cells and tissues to adopt a protease-resistant and detergent-insoluble form that is distinct from PrPSc, and that this transformation requires only a single octapeptide repeat in the N-terminal half of the protein. This work significantly extends previous studies on the interaction of copper and PrP, most of which have utilized synthetic peptides or bacterially produced, recombinant PrP which lack modifications such as N-linked oligosaccharides or a glycolipid anchor that are normally present on cellular PrP (9-16, 40). Our results suggest possible mechanisms by which copper may play a role in prion diseases, and in the normal function of the protein.

What is the nature of the copper-induced change in the physical state of PrPC? We have found that addition of copper ions to detergent lysates prepared from brain and peripheral tissues or from transfected cells causes PrPC to become insoluble during ultracentrifugation and resistant to digestion with high concentrations of PK (up to 800 µg/ml). This effect occurs within minutes, is completely reversible, and requires that PrPC be in a native state. Although copper at millimolar concentrations is known to cause the aggregation and precipitation of proteins (41), the effects on detergent insolubility and protease resistance observed here occur at 100-300 µM copper, a concentration range that is likely to exist in tissues (41), and they are not seen for tubulin, actin, or other unidentified Coomassie-stainable proteins in the lysates. It thus seems likely that copper is causing a relatively specific effect on either the conformation and/or aggregation state of PrP. Consistent with this idea, copper binding has been shown to favor formation of beta -sheet structures in recombinant PrP, and to induce fibrillation and aggregation of the protein (11, 42). In agreement with our results, copper has also been reported to enhance the protease resistance of recombinant PrP (42, 43) as well as PrP in brain microsomes (42), although protease cleavage of the recombinant protein occurred between residues 111 and 121, a site distinct from the one cleaved in PrP 27-30. Qin et al. (42) correlate the copper-induced change in protease resistance of PrP with spontaneous deamidation of the asparagine residue at position 107, an effect that occurs with aging of the protein. In contrast, deamidation is unlikely to be a prerequisite for the biochemical changes that we observe here, since they occur independently of the age or storage conditions of the lysates.

Do the copper-induced biochemical alterations we have observed in PrP indicate that the protein has been converted to the PrPSc state? Detergent insolubility and protease resistance are operational properties commonly used to recognize PrPSc, the infectious form of PrP that is found in most cases of infectious, inherited, and sporadic prion disease (2). But because these properties are merely biochemical markers, their presence does not necessarily indicate that the protein has acquired the PrPSc conformation, which is known to be rich in beta -sheets. A more direct structural indicator of the PrPSc state is protection of the epitope for the 3F4 monoclonal antibody which lies near the central, hydrophobic region of the molecule (37, 38). This epitope is accessible in PrPC, but is inaccessible in PrPSc unless the protein is denatured. In contrast, we find that the 3F4 epitope is fully reactive in native, copper-treated PrP, indicating that although the protein has been rendered detergent-insoluble and protease-resistant by the metal, its conformation is distinct from that of authentic PrPSc. The reversibility of the copper effect also argues against the possibility that we have generated PrPSc, since the only agents known to render PrPSc protease sensitive are chemicals like guanidine and SDS which irreversibly denature the protein (44, 45). The most definitive test of whether copper treatment converts PrPC to the PrPSc state would be to assay the infectivity of the protein using animal bioassays, but this would be difficult to do using our current experimental procedure because of the presence of detergents in the tissue and cell lysates.

Several other experimental manipulations besides copper addition have been shown to increase the beta -sheet content of PrP or induce protease resistance and aggregation. These include exposing recombinant PrP to denaturants, alterations in solvent conditions, pH or temperature, and reduction of the disulfide bond (46-51), as well as expression of PrP in the yeast cytoplasm (52), and treatment of PrP-expressing cells with dithiothreitol and/or glycosylation inhibitors (52, 53). In none of these cases has it been shown that the protein produced is infectious. It therefore seems likely that a variety of conditions can induce PrP to adopt beta -rich, protease-resistant forms that are distinct from PrPSc.

Do the changes in detergent insolubility and protease resistance that we have observed result from binding of copper to PrP itself? Since all of our experiments were carried out in detergent lysates of tissues or cultured cells, we cannot rule out the possibility that the metal affects PrP indirectly by binding to other molecules in the lysates. However, we think a direct interaction between PrP and copper is more likely. The most compelling piece of evidence in support of this contention is our observation that copper-induced alterations in the properties of PrP require the presence of at least one histidine-containing octapeptide repeat in the N terminus of the molecule. PrP molecules in which all five of the peptide repeats have been deleted are immune to the effects of copper, as is the doppel protein, a PrP paralogue that lacks the octapeptide repeat region as well as the central hydrophobic domain. In contrast, PrP molecules containing a single copy of the PHGGGWGQ repeat, expressed in either cultured cells or in transgenic mouse brain, are rendered detergent insoluble or protease resistant by copper. Copper is known to bind to the octapeptide repeat region of PrP, and as mentioned above, this binding induces conformational and biochemical changes in the purified protein. There is still uncertainty about which atoms within the octapeptide repeat region serve to coordinate copper, but some studies favor a stoichiometry in which each PHGGGWGQ unit binds a single copper ion, with one of imidazole nitrogens of the histidine residue and two amide nitrogens contributed by the glycine residues serving as coordination ligands (13). In this model, PrP containing one copy of the repeat would still be capable of binding copper, consistent with the results reported here.

We found that zinc rendered PrPC detergent insoluble but not protease resistant, while manganese had no effect on the protein. These results indicate that zinc affects the properties of PrPC in a way that is different from copper. Although some studies find that binding of transition metals to PrPC is highly selective for copper (11), other studies report that the protein also binds zinc, nickel, and manganese (34). Both copper and zinc stimulate endocytosis of PrPC from the cell surface (17), and both metals have been found to induce changes in the biochemical properties of PrPSc strains (24). Thus, several different metals may interact with PrP, although their affinities, binding sites, and biochemical effects may differ. In contrast to our study, a report by Brown et al. (34) states that manganese but not copper renders PrP synthesized by cultured astrocytes protease resistant. However, these authors treated intact cells with the metals prior to testing the protease resistance of PrP that had been immunoprecipitated from cell lysates, a procedure which is likely to result in significant loss of metal from the protein.

Our results have significance for understanding the pathological as well as the physiological properties of PrP. The fact that copper-treated PrP is protease resistant but 3F4-reactive raises the possibility that this form of the protein represents a physical state that is intermediate between that of PrPC and PrPSc. Thus, some additional chemical treatment might be capable of converting copper-bound PrP fully and irreversibly to the scrapie form. Intermediate states of PrP have been postulated on the basis of thermodynamic considerations (54), and have been detected experimentally in cultured cells expressing mutant PrP molecules (55). In addition, alternate forms of PrP that are distinct from both PrPC and PrPSc have been postulated to be the primary neurotoxic species in some prion diseases (27, 56). It is thus possible to envisage that copper either initiates or modulates the production of pathogenic PrP molecules in prion diseases, and that manipulation of copper levels may represent a strategy for treating these disorders. The fact that expression of PrP lacking the octapeptide repeat region fails to restore scrapie susceptibility to Prn-p0/0 mice (57), or else produces an atypical disease phenotype (58) is consistent with an important role for copper binding in the pathogenic process.

An equally intriguing possibility is that copper-induced changes in the biochemical properties of PrPC are not related to the pathway of PrPSc formation, but instead to a normal function of PrPC in copper metabolism. For example, copper-induced oligomerization of PrPC could be a mechanism by which the metal stimulates endocytic trafficking of the protein, a process that we have postulated to be important if PrPC serves as a receptor for cellular uptake of copper ions (17). Copper-induced conformational changes could also play a role in enzymatic or other functions of PrPC.


    ACKNOWLEDGEMENTS

We thank Charles Weissmann for providing Tgd11 mice, as well as Richard Kascsak and Byron Caughey for supplying antibodies. We also acknowledge Bettina Drisaldi for assistance with one of experiments, Cheryl Adles for maintaining the mouse colony, and Sylvain Lehmann for construction of the PrP triangle 51-82 and triangle 51-90 plasmids.


    FOOTNOTES

* This work was supported by National Institutes Health Grant R01 NS40061 (to D. A. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Current address: Istituto di Ricerche Farmacologiche "Mario Negri," Via Eritrea 62, 20157 Milano, Italy.

§ Recipient of fellowships from the Comitato Telethon Fondazione Onlus and the McDonnell Center for Cellular and Molecular Neurobiology at Washington University. Current address: Istituto di Ricerche Farmacologiche "Mario Negri," Via Eritrea 62, 20157 Milano, Italy.

To whom correspondence should be addressed: Dept. of Cell Biology and Physiology, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, MO 63110. Tel.: 314-362-4690; Fax: 314-362-7463; E-mail: dharris@cellbio.wustl.edu.

Published, JBC Papers in Press, January 18, 2001, DOI 10.1074/jbc.M009666200


    ABBREVIATIONS

The abbreviations used are: PrPSc, scrapie isoform of PrP; BCS, bathocuproinedisulfonate; PK, proteinase K; PrP, prion protein; PrPC, cellular isoform of PrP; CHO, Chinese hamster ovary; PMSF, phenylmethylsulfonyl fluoride; PAGE, polyacrylamide gel electrophoresis.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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