Cleavage of the Amino Terminus of the Prion Protein by Reactive Oxygen Species*

Hilary E. M. McMahonDagger , Alain MangéDagger §, Noriyuki NishidaDagger , Christophe Créminon||, Danielle CasanovaDagger , and Sylvain LehmannDagger **

From the Dagger  Institut de Génétique Humaine, CNRS U.P.R. 1142, 141 Rue de la Cardonille, 34396 Montpellier Cedex 5, France and the ||  Commissariat à l'Energie Atomique (Saclay, France), Service de Pharmacologie et d'Immunologie, Saclay, 91191 Gif Sur Yvette Cedex, France

Received for publication, August 10, 2000, and in revised form, October 26, 2000



    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Relatively limited information is available on the processing and function of the normal cellular prion protein, PrPC. Here it is reported for the first time that PrPC undergoes a site-specific cleavage of the octapeptide repeat region of the amino terminus on exposure to reactive oxygen species. This cleavage was both copper- and pH-dependent and was retarded by the presence of other divalent metal ions. The oxidative state of the cell also decreased detection of full-length PrPC and increased detection of amino-terminally fragmented PrPC within cells. Such a post-translational modification has vast implications for PrPC, in its processing, because such cleavage could alter further proteolysis, and in the formation of the scrapie isoform of the prion protein, PrPSc, because abnormal cleavage of PrPSc occurs into the octapeptide repeat region.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Prion diseases or transmissible spongiform encephalopathies (TSEs)1 are a group of neurodegenerative diseases affecting both animals and humans (1). Such disorders include scrapie in sheep, bovine spongiform encephalopathy in cattle, and Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker syndrome, and fatal familial insomnia in humans (2, 3). TSEs may be of sporadic, genetic, or infectious origin, and the agent believed to be responsible for these disorders is a protein molecule termed PrPSc, which is a conformational variant of the normal cellular prion protein (PrPC) (4). PrPC is a 33-35-kDa glycosyl-phosphatidylinositol-anchored protein that is expressed by most tissues and in particular at high levels by neuronal cells. Unlike PrPC, PrPSc possesses a high beta -sheet content and is partially protease-resistant (5). It still remains unclear how PrPC is converted to PrPSc and whether the infectious agent consists solely of PrPSc, because the infectivity of in vitro converted PrPSc remains to be established (6). However, it is becoming increasingly evident that PrPC is essential for disease development on infection (7), although the function of the protein still remains obscure.

The amino terminus of PrPC contains a series of octapeptide repeats possessing the following consensus sequence: PHGGGWGQ. This region, which is among the most conserved regions of mammalian PrP (8), has been implicated in the binding of divalent metal ions, and in particular copper (9). Whether this binding is of structural or functional significance is not known. However, NMR studies have shown that the recombinant protein possesses a flexible amino terminus lacking any given structure (10), and it is reported that copper binding not only adds structure to this region (11) but may also lend stability to the carboxyl terminus (12). Because of the capacity of PrPC to bind copper, this protein has been implicated in copper transport and metabolism (13) and in the defense mechanism of the cell against oxidative insult, possibly through a regulation of the Cu,Zn superoxide dismutase activity (14). However, a recent study may lead to a re-evaluation of the link between PrP and copper metabolism (15). In addition, although it would appear that the octapeptide repeat region may not be essential for disease development (16), the insertion of additional octapeptide repeats, which could increase metal binding, results in a pathogenic mutation (17). Thus the role of copper binding by this highly conserved region of PrPC still remains unclear.

Copper is an essential redox transition element and a crucial component of various enzyme systems (18), and yet under conditions of oxidative stress copper ion-mediated damage to proteins through reactive oxygen species (ROS) is very significant. Links between oxidative stress, ROS, and neurological disorders have been reported in Parkinson's and Alzheimer's diseases, amylotrophic lateral sclerosis (19), and aging itself. ROS, such as the superoxide radical (O&cjs1138;2) and hydrogen peroxide (H2O2), which although itself not an ROS is an important mediator of oxidative stress in neurons (20), can oxidatively modify biomolecules (21). The generation of such ROS is promoted by the presence of copper and iron through the Fenton reaction (22). In addition, when the metal ion involved is protein bound, the oxidative-reductive reaction can locally generate oxygen species that may react at specific sites in the protein itself, impairing activity or resulting in cleavage (23). Because oxidative stress may play a role in TSEs (24), it is possible that metal-catalyzed oxidation of PrPC, at its octapeptide repeat region, may be an important factor in TSEs. In fact in Alzheimer's disease, the beta -amyloid precursor protein, which is another copper-binding protein, reduces Cu2+ on binding, and it is believed that its processing occurs under conditions of oxidative stress (25). Considering this, and that PrPC may also possess the capacity to reduce Cu2+ (26), we examined the effect of ROS on the processing of PrPC.

The cleavage of PrPC in the conserved middle region of the molecule, as well as the cleavage of PrPSc close to the octapeptide region, has been well documented both in cultured cells and in the brain (27-29). However, the regulation and the physiological and pathological significance of these cleavages remain largely unknown. Here we demonstrate, in the presence of ROS, that the octapeptide repeat region of PrPC undergoes a copper-dependent cleavage. This cleavage can be inhibited by the presence of additional alternative divalent metal ions. It is possible that such ROS cleavage may be involved in PrPC processing, a post-translational modification that could also result in the activation of the protein. Additionally, because abnormal cleavage of PrPSc occurs into the repeats, such cleavage of PrPC may be important in TSE disease development.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Reagents and Antibodies-- Opti-MEM and trypsin were from Life Technologies, Inc.; MEM-alpha was from ICN; and fetal calf serum was from BioWhittaker. Secondary antibodies were from Jackson ImmunoResearch (West Grove, PA). All other reagents were from Sigma. The antibody against actin (1-19) was from Santa Cruz Biotechnology. Rabbit polyclonal antibody P45-66 raised against a synthetic peptide encompassing mouse PrP (MoPrP) residues 45-66 has been described earlier (30). Scrapie-associated fibril (SAF) 32, 60, 69, and 70 are four monoclonal antibodies produced by the group of J. Grassi (Commissariat à l'Energie Atomique (Saclay, France)). They were obtained using as immunogens SAFs that were prepared from infected hamster brains. In enzyme immunometric assays, SAF 60, 69, and 70 were characterized as recognizing the peptide epitope 142-160 of hamster PrP, and SAF 32 was characterized as recognizing the epitope 78-91. A mixture consisting of an equal volume of ascitis of SAF 60, 69, and 70 antibodies, namely SAF mix, was used to improve PrPC detection. MAB 8G8 recognizes epitope 94-109 of MoPrP (31).

Chinese Hamster Ovarian (CHO) Cell Cultures-- Construction of cDNA encoding the wild-type PrP that is derived from the Prn-pa allele and the generation and cultures of stably transfected lines of CHO cells expressing wild-type MoPrP have been described previously (30).

Preparation of Conditioned Medium-- Subconfluent MoPrP CHO cells were rinsed twice with phosphate-buffered saline and were then overlaid with opti-MEM and incubated for 24 h at 37 °C in a 5% CO2 humidified atmosphere. Medium was then removed and centrifuged for 5 min at 10,000 rpm and 4 °C; the supernatant was then recovered (conditioned medium) and used immediately, after the addition of the protease inhibitors (0.5 mM phenylmethylsulfonyl fluoride, 1 µg/ml pepstatin, and 1 µg/ml leupeptin), for assay purposes.

Effect of Reagents on PrPC Detection-- Conditioned medium was incubated with the reagents tested under the conditions indicated under "Results". Proteins were then methanol-precipitated, resuspended in sodium dodecyl sulfate (SDS)-sample buffer and boiled for 5 min before 12% SDS-polyacrylamide gel electrophoresis (PAGE). The latter was then followed by electroblotting onto Immobilon membranes, and PrPC was detected by using the antibodies (see above) indicated under "Results" and a peroxidase-conjugated goat anti-mouse, or as appropriate anti-rabbit, secondary antibody. The blots were developed using enhanced chemiluminescence. Films were analyzed using Images Analysis software. The metal ions when used were CuSO4, MnSO4, CoCl2, ZnSO4, and CaCl2.

To analyze the direct effect of H2O2 on cells, confluent MoPrP CHO cells were rinsed twice with phosphate-buffered saline and overlaid with opti-MEM containing H2O2 at the concentrations indicated under "Results". After 24 h of incubation at 37 °C, the medium was removed, and the cells were lysed in lysis buffer (150 mM NaCl, 0.5% Triton X-100, 0.5% sodium deoxycholate, 50 mM Tris-HCl, 1 µg/ml pepstatin, 1 µg/ml leupeptin, 0.5 mM phenylmethylsulfonyl fluoride, and 2 mM EDTA) for 20 min at 4 °C. The lysates were then spun at 14,000 rpm for 4 min, and the protein level in the supernatant was determined with the BCA protein assay kit (Pierce). After the addition of 2× SDS-sample buffer, samples were analyzed by SDS-PAGE followed by immunoblotting.


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

PrPC Undergoes a Site-specific Cleavage in the Presence of H2O2-- PrPC and the epitopes that are recognized by the antibodies used in this study are diagrammatically presented in Fig. 1A. Subconfluent stably transfected MoPrP CHO cells, which were incubated for 24 h in Opti-MEM, released into the medium (conditioned medium) a large quantity of soluble full-length glycosylated PrPC lacking its glycosyl-phosphatidylinositol anchor (data not shown). Western blots of conditioned medium showed a major band at 33 kDa that reacted with P45-66, SAF 32, 8G8, and SAF mix antibodies (Fig. 1, B, C, D, and E, lane 1, respectively). In addition, a set of bands corresponding to amino-terminal fragments of PrPC around 6.5 kDa was detected using the antibodies P45-66, SAF 32, and 8G8 (Fig. 1, B, C, and D, respectively). The size of the amino-terminal fragments and the fact that they were recognized by both P45-66 and 8G8 could indicate that a mixture of amino-terminal fragments are released into the medium (this was confirmed using 15% Tricine gel; data not shown). Such fragments could arise from multiple cleavage of the amino terminus of PrPC, from cleavage into the octapeptide repeat region and the conserved region. A band around 14 kDa, corresponding most likely to carboxyl-terminally truncated PrP, was also detected by using SAF mix (Fig. 1E).



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Fig. 1.   PrPC undergoes a site-specific cleavage of the amino terminus by H2O2 A, presented is a diagrammatic representation of MoPrP with the regions recognized by the antibodies employed in this study. Conditioned medium (the preparation of which is described under "Experimental Procedures") was incubated with 5 mM H2O2 at pH 7.0 (0.05 M HEPES) for 0-2 h in the presence of 10 µM Cu2+ at 37 °C. After methanol precipitation PrP was detected by 12% SDS-PAGE, followed by immunoblotting with the antibodies P45-66 (B), SAF 32 (C), 8G8 (D), and SAF mix (E). Results are representative of three independent experiments. PrP signals (, P45-66; black-square, SAF 32; black-triangle, 8G8; , SAF mix) from B, C, D, and E were quantified by densitometry and were plotted as a percentage of the control (time 0) (F). Molecular mass markers in kDa are indicated on the left of the panels.

To examine the effect of H2O2 on PrPC, conditioned medium (in the presence of protease inhibitors) was incubated with 5 mM H2O2 at pH 7.0 and 37 °C in the presence of 10 µM Cu2+ over varying time periods (0-2 h). Exposure to H2O2 induced a slight increase in the molecular mass of PrPC (Fig. 1B, compare lanes 1 and 2) (this observation will be discussed later). Within 5 min a dramatic reduction in the full-length PrPC P45-66 signal was detected (Fig. 1B) concomitant with the appearance of a P45-66-negative but SAF 32-, 8G8-, and SAF mix-positive truncated PrPC at 28.5 kDa (Fig. 1, B, C, D, and E, respectively). This latter fragment remained relatively unaffected by further exposure, indicating a H2O2 cleavage that was not random, but one that occurred in the repeat region. In addition, within 5 min of exposure the intensity of the signal for the amino-terminal band (as detected by P45-66) increased by 18% (Fig. 1B, compare lanes 1 and 2). This latter increase occurred in conjunction with the loss of full-length P45-66-positive PrPC and could consequently represent an increase in detection of a cleavage product. After a further 25 min this amino-terminal band was no longer detected (Fig. 1B, lane 4); this may be a consequence of additional cleavage of the fragment by H2O2.

Effect of H2O2 Concentration, Chelators, Me2SO, and Metal Ions on Cleavage-- To further examine PrPC cleavage by H2O2, the effect of varying concentrations of this oxidant (0-5 mM H2O2) on the loss of full-length PrPC (P45-66 signal) was determined (Fig. 2A). To observe an effect with lower concentrations of H2O2, an incubation period of 24 h at 37 °C was chosen. H2O2 induced a concentration-dependent loss of the P45-66 signal, with a complete loss at a concentration of 2.5 mM (Fig. 2B).



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Fig. 2.   Effect of H2O2 concentration, chelators, dimethyl sulfoxide, and metal ions on PrPC cleavage. Conditioned medium was incubated at pH 7.0 and 37 °C with varying concentrations of H2O2 (0, 5.0, 4.0, 2.5, 1.0, 0.5, and 0.1 mM H2O2, lanes 1-7, respectively). Samples were then methanol-precipitated, and PrP was separated by 12% SDS-PAGE and detected by immunoblotting with the antibody P45-66 (A). The H2O2-dependent loss of full-length PrPC from A was quantified by densitometry and plotted as a percentage of the control (no H2O2) (B). C, conditioned medium was also incubated at pH 7.0 and 37 °C with 1 mM EDTA (lanes 4, 8, and 13), 1 mM bathocuproine (BC) (lanes 5, 9, and 14), 1 mM DTPA (lanes 6, 10, and 15), and Me2SO (DMSO; 10% v/v) (lanes 7, 11, and 16) in the presence (+ lanes) or absence (- lanes) of Cu2+ (10 µM) and/or H2O2 (2.5 mM). D, conditioned medium was also incubated at pH 7.0 and 37 °C with 10 µM Co2+ (lanes 3 and 7), 10 µM Zn2+ (lanes 4 and 8), 10 µM Mn2+ (lanes 5 and 9), and 10 µM Ca2+ (lanes 6 and 10) in the presence (+ lanes) or absence (- lanes) of 10 µM Cu2+ and/or H2O2 (2.5 mM). PrPC was then detected after methanol precipitation of samples by 12% SDS-PAGE, followed by immunoblotting with P45-66. Results are representative of three individual experiments. Molecular mass markers in kDa are indicated on the left of the panels.

To facilitate an elucidation of the mechanism and dependence of the reaction, the effect of chelators, Me2SO, and metal ions on the cleavage was investigated (Fig. 2, C and D). In the presence of 10 µM Cu2+ and 2.5 mM H2O2 (the concentration of H2O2 allowing for a complete disappearance of the P45-66 signal in 24 h at 37 °C (Fig. 2A)), the P45-66 signal was lost (Fig. 2C, lane 12). Incubation of PrPC with 10 µM Cu2+ alone at 37 °C did not result in cleavage (Fig. 2C, lane 2), indicating that the cleavage with H2O2 was most probably a non-enzymatic event. In addition, in the absence of added copper, H2O2 induced a slight increase in PrPC molecular mass without inducing cleavage (Fig. 2C, lane 3). The chelator (at a 1 mM concentration) EDTA; a broad range metal chelator, bathocuproine disulfonate; a Cu1+ chelator, diethylenetriaminepentaacetic acid (DTPA); a Cu2+ chelator; and the reagent Me2SO (10% v/v), a hydroxyl radical (·OH) scavenger, had no effect on their own on the migration pattern (Fig. 2C, lanes 4-7) or on the shift-up observed with H2O2 in the absence of added copper (Fig. 2C, lanes 8-11). This would indicate that the increase in molecular mass induced by H2O2 was metal ion-independent. It is possible that the increase may have been due to amino acid oxidation, a phenomenon observed with H2O2 (21); this is currently under investigation. Recently it was reported that storage of PrPC under oxidative conditions induced amino acid modifications that resulted in a similar increase in the Mr of PrPC (32); this is consistent with the idea that the increased Mr observed on H2O2 exposure may also be due to amino acid modification. Importantly, both EDTA and DTPA protected PrPC from cleavage induced by H2O2 (Fig. 2C, lanes 13 and 15); in contrast bathocuproine disulfonate and Me2SO were without effect (Fig. 2B, lanes 14 and 16).

In addition, because it has been reported that PrPC may interact with metal ions other than copper (33), Cu2+ was replaced by 10 µM Co2+, Zn2+, Mn2+, or Ca2+ (Fig. 2D, lanes 3-6). Cleavage by H2O2 was only detectable with Cu2+ (Fig. 2D, lane 2); interestingly, when the other ions were added in combination with Cu2+, all but Co2+ had a significant protective effect on H2O2 cleavage (Fig. 2D, lanes 7-10).

Effect of pH on the Cleavage of PrPC by H2O2-- It has been shown that binding of copper by PrPC is highly pH-dependent (34). Therefore, because cleavage of the amino terminus by H2O2 appeared to be dependent on Cu2+, the effect of pH on PrPC cleavage by H2O2, in the presence of 10 µM Cu2+, was determined by following the P45-66 signal of full-length PrPC (Fig. 3A). Although H2O2 can induce significant cleavage of PrPC at low concentrations (Fig. 2A), a concentration of 5 mM H2O2 was selected for this experiment to increase the rate of cleavage. Loss of signal by H2O2 was highly pH-dependent, with the rate of cleavage decreasing with decreasing pH (Fig. 3B).



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Fig. 3.   Cleavage of PrPC by H2O2 is pH-dependent. Conditioned medium was incubated at pH 7.0 (0.05 M HEPES), pH 6.0 (0.05 M MES), or pH 5.0 (0.05 M acetate buffer) at 37 °C for varying time periods (0-24 h) in the presence of 10 µM Cu2+ and 5 mM H2O2. PrPC was detected after methanol precipitation, followed by 12% SDS-PAGE and by immunoblotting with the P45-66 antibody (full-length PrPC is presented) (A). B, the MoPrP-specific bands in A were quantified by densitometry and plotted as a percentage of the control (A, lane 1); , pH 7.0; black-triangle, pH 6.0; black-square, pH 5.0. Results are representative of three independent experiments.

Cleavage of the Amino Terminus of PrPC in the Presence of Superoxide Ions (O&cjs1138;2)-- It has been reported (35) that recombinant PrPC has a copper-dependent superoxide dismutase activity. Superoxide dismutases are involved in the dismutation of O&cjs1138;2 to H2O2; therefore, because H2O2 effects a cleavage of the amino terminus of the prion protein, the effect of O&cjs1138;2 on the processing of PrPC was investigated. Conditioned medium was incubated in the presence of xanthine (1 × 10-4 M) and xanthine oxidase (0.02 units/ml) (O&cjs1138;2 producers) for 1 and 24 h in the presence and absence of 10 µM Cu2+ (Fig. 4). After 1 and 24 h of incubation at 37 °C, in the presence and absence of Cu2+, the P45-66 signal was lost by 90, 40, 42, and 26%, respectively (Fig. 4, A and B), whereas the SAF 32 signal was lost by 99, 65, 90, and 40%, respectively (Fig. 4, C and D). Similar to H2O2, O&cjs1138;2 induced a loss of the P45-66 signal, and this loss of signal was augmented by the presence of Cu2+. In addition, the loss of the P45-66 signal was more rapid than that of SAF 32, which is similar to the cleavage of PrPC by H2O2 in Fig. 1.



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Fig. 4.   O&cjs1138;2 also induces a site-specific cleavage of the PrPC amino terminus. Conditioned medium was incubated for 0, 1, and 24 h at 37 °C in the presence (+ lanes) or absence (- lanes) of Cu2+ and/or the O&cjs1138;2-producing system (xanthine (1 × 10-4 M) and xanthine oxidase (0.02 units/ml)). The samples were then methanol-precipitated, and PrPC was separated by 12% SDS-PAGE and detected by immunoblotting with P45-66 (A) or SAF 32 (C). PrPC-specific antibody signals from A and C (lanes 1-5) were quantified by densitometry and plotted, ± standard deviations, as a percentage of the control (B and D, respectively). Results are representative of three independent experiments. Molecular mass markers in kDa are indicated on the left of the panels.

Effect of H2O2 on PrPC in Cells-- To confirm the physiological relevance of the finding that PrPC can be cleaved by H2O2 into the repeat region, confluent MoPrP CHO cells were exposed for 24 h to a range of H2O2 concentrations (0, 10, and 100 µM, which is within the range reported to be reached in rat brain under oxidative stress (25-160 µM) (36)) (Fig. 5). Because the concentration of H2O2 used on the cells was lower than that used in previous experiments, it was possible to observe a concentration-dependent increase in the detection of an amino-terminal fragment within cells (Fig. 5, A and B). For the latter observation a long film exposure was employed, but with a reduced exposure a decrease in full-length PrPC was also observed (Fig. 5C, compare lane 1 with lanes 2 and 3). This would indicate an increased cleavage of full-length PrPC under conditions of oxidative stress within the cell. In addition, because the protein level of another protein, actin, was unaffected by this treatment (Fig. 5D), it can be inferred that exposure to H2O2 did not result in an increase of all protein cleavage.



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Fig. 5.   H2O2 induces a cleavage of PrPC in cultured cells. Confluent MoPrP CHO cells were incubated with an overlay of Opti-MEM containing H2O2 (0, 10, or 100 µM) for 24 h at 37 °C. The medium was then removed, and the cells were lysed as described under "Experimental Procedures." PrPC in lysates was separated by 12% SDS-PAGE and detected by immunoblotting with the antibody P45-66 or actin (1-19). To visualize varying levels of the P45-66-positive amino-terminally cleaved PrPC fragment, a high film exposure is presented (A), and this fragment is indicated by an arrow. A lower exposure showing full-length PrPC is also shown (C). Bars in B represent a densitometric analysis of the amino-terminal fragment in A and are expressed as a percentage of the control, ± standard deviations (**, p < 0.01; Student's t test). The effect of H2O2 treatment on the levels of actin within the cell is also shown (D). Results are representative of three independent experiments. Molecular mass markers in kDa are indicated on the left of the panels.



    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The cell surface glycosylated protein, PrPC, has been recognized as a metal-binding protein since 1992 (5). Although the role of copper binding by PrPC remains obscure, it may be structural, because copper binding appears to induce structure to the otherwise flexible amino terminus of the protein (11). The present study suggests that copper binding by PrPC allows for the processing of the protein to occur under conditions of oxidative stress. In the presence of H2O2 and copper, PrPC was observed to be cleaved into the octapeptide repeat region, yielding a protein of 28.5 kDa. H2O2 cleavage was most efficient in inducing a loss of the full-length P45-66 signal; exposure also affected the SAF 32 signal (albeit at a lower rate) and resulted in further cleavage of the amino-terminal P45-66-positive fragment that was formed during the reaction. This would indicate that oxidative processing of PrPC most likely occurred within each of the metal-binding repeats, but that highest affinity was toward the repeat region after amino acid residue 66.

This H2O2 cleavage of PrPC was Cu2+-dependent, because EDTA (a broad range metal ion chelator) and DTPA (a Cu2+ chelator) inhibited cleavage, whereas bathocuproine disulfonate (a Cu1+ chelator) was without effect. Under oxidative stress ·OH is one of the most destructive biological molecules, and it is primarily generated through the breakdown of H2O2 in the presence of copper or iron in the Fenton reaction (22). Although copper ions are invariably protein bound in vivo, binding does not prevent copper from participating in oxygen radical reactions but limits radical formation to the site of copper binding (23). Importantly, Me2SO, a known trapping agent for ·OH, did not inhibit cleavage. Therefore, it can be assumed that in the case of PrPC ·OH radical formation does not occur in solution; rather oxidation of PrP-copper by H2O2 occurs at the site of copper binding. Additionally, because binding of Cu2+ by PrPC is highly pH-dependent (34, 37), the pH dependence of the H2O2 cleavage of PrPC (Fig. 3) would further emphasize the participation of protein-bound copper in the reaction. Thus, similar to Cu,Zn superoxide dismutase in amylotrophic lateral sclerosis (23) and beta -amyloid precursor protein in Alzheimer's disease (25), the results presented here would suggest that PrPC is also subject to a copper-dependent cleavage in the presence of H2O2 with the following proposed reactions.
<UP>PrP-Cu<SUP>2+</SUP></UP>+<UP>H<SUB>2</SUB>O<SUB>2</SUB> ↔ PrP-Cu<SUP>+</SUP></UP>+<UP>O&cjs1138;<SUB>2</SUB></UP>+<UP>2H<SUP>+</SUP></UP>

<UP><SC>Reaction 1</SC></UP>

<UP>PrP-Cu<SUP>+</SUP></UP>+<UP>H<SUB>2</SUB>O<SUB>2</SUB> → PrP-OH<SUP>⋅</SUP></UP>+<UP>OH<SUP>−</SUP> → PrP cleavage</UP>

<UP><SC>Reaction</SC> 2</UP>
Because cleavage of PrPC, in the presence of Cu2+, was antagonized by additional divalent metals (Zn2+, Ca2+, and Mn2+, but not Co2+), it is possible that PrPC can interact with more than one metal ion at a time or that the presence of additional metal ions could interfere with Cu2+ binding. Either way the other metal ions are not known to participate in the Fenton reaction. In fact Mn2+ has the capacity to inhibit it (38); therefore, the binding of these other metal ions could inhibit ROS cleavage by preventing the site-specific generation of ·OH radicals.

Like H2O2, O&cjs1138;2 also has the capacity to interact with bound metal ions. Recently, it was reported that PrPC may have a copper-dependent superoxide dismutase-like activity (35). Cu,Zn superoxide dismutase has the capacity to dismutate O&cjs1138;2 to H2O2, and under normal circumstances the enzyme is believed to possess protective amino acids at its active site that protect the protein from cleavage by its dismutation product, H2O2. The results presented here would indicate that PrPC does interact with O&cjs1138;2 in a copper-dependent manner, but unlike for Cu/Zn superoxide dismutase, this interaction leads to cleavage of the protein.

Both O&cjs1138;2 and H2O2 are available within the cell, and their levels are regulated by interdependent oxidative enzymes (39). With age ROS levels are known to increase (40), and under conditions of oxidative stress it is reported that H2O2 can reach 26-160 µM in the brain (36). Substantial cleavage of PrPC was observed at 100 µM H2O2 (Fig. 2A), and exposure of cells to H2O2 at a concentration as low as 10 µM induced a significant increase in PrPC cleavage. Consequently, it is most likely that PrPC could be susceptible in vivo to ROS-mediated cleavage of its amino-terminal octapeptide repeat region.

In the brain normal PrPC cleavage is believed to occur within the conserved region, yielding a fragment called C1, whereas PrPSc is thought to be cleaved outside this region, yielding a 27-30-kDa species (27). Jimenez-Huete et al. (28), on studying PrPC proteolytic cleavage using deglycosylated PrPC, were able to detect a 21-22-kDa fragment. Although amino-terminal sequencing is not available for this fragment, its size would indicate that cleavage of PrPC can occur physiologically into the repeats without cleavage of the conserved region (such cleaved PrP has also been observed in our cell models (data not shown)). However, it was not known if the 21-22-kDa species could be an obligatory intermediate for C1 generation. One possibility is that the 21-22 kDa fragment may have been generated through oxidative cleavage. Such cleavage could then initiate or facilitate further proteolysis of the molecule. In fact, it has been reported that such modified proteins are more susceptible to proteolysis than their native counterparts (21). Jimenez-Huete et al. (28) also demonstrated that PrP cleavage producing amino-truncated peptides could be stopped by EDTA and EGTA. Interestingly, a return of cleavage could only be achieved by adding the metal ions Cu2+ and Fe3+ but not by adding Mg2+, Zn2+, or Ca2+. Hence because both Cu1+ and Fe2+ can participate in the Fenton reaction, it is possible that the chelators may not have inhibited an enzymatic event but rather a reactive oxygen species event.

Under normal conditions, amino-terminally truncated PrPC, resulting from ROS cleavage, could be rapidly degraded within the cell or on the cell surface. However, because PrPSc proteinase K digestion results in cleavage into the octapeptide repeats and because additional repeats in PrPC lead to abnormal properties in cells (41) and CJD in familial TSEs, it is possible that oxidative cleavage of the octapeptide repeat region may be a key factor in TSEs. This theory is further emphasized because ROS, oxidatively modified proteins, and CJD phenotypic expression increase with age. Additionally, amino-terminal sequence data available for PrPSc, without proteinase K digestion, would indicate that a quantitative cleavage in the repeat region of PrPSc does occur in vivo (29, 42). The cleavage patterns that have been reported for size variants of mouse scrapie-associated fibrils are far from random, occurring three to four amino acid residues away from the copper-binding histidine in each of the octapeptide repeat regions. Because increasing evidence indicates that oxidative stress is involved in TSEs (43, 44) and because H2O2 at a concentration as low as 10 µM altered PrPC processing, it is likely that PrPSc is susceptible to ROS cleavage, and it is possible that such cleavage may account for in vivo amino-terminal truncation. In addition, because a number of reports have now separated PrPSc proteinase K resistance from infectivity and neurodegeneration (45, 46), it is possible that ROS modification of the molecule could modulate its toxicity and/or its effect on PrP-mediated signal transduction, for example (47, 48).

Shmerling et al. (48) observed that expression of amino-terminally truncated PrP in Zurich Prnpo/o mice led to abnormalities that were reversed by the introduction of a single PrP allele. They proposed that the carboxyl terminus of PrPC may interact with a ligand and that the full-length amino terminus, through an attachment to a secondary site on the same ligand, may induce a cell survival signal. It was also proposed that in Zurich Prnpo/o mice an additional PrP-like molecule named pi  could carry out the same function but that the presence of truncated PrP could also interact with the ligand, at a higher affinity than pi , preventing cell survival signaling. Additionally, it was recently suggested (49) that expression of Doppel, a PrP-like molecule, except that it resembles truncated PrPC lacking the repeat region (50), could also result in disease through a mechanism similar to that proposed for truncated PrPC. From the data presented here, we proposed that an increase in ROS-truncated PrPC may be pathogenic and lead to an alteration in cellular signaling. Additional repeats within PrPC, as is associated with CJD in familial TSEs, could increase copper binding and as a result susceptibility to ROS. Consequently, in CJD cases disease may present with age through a mechanism similar to that proposed by Shmerling et al. (48).

Whether ROS cleavage is fundamental to the function of PrPC or to the production of PrPSc remains to be elucidated. However, it may be proposed that such ROS processing, at a rate allowing for the formation of a low level of amino-terminally cleaved PrPC, may be important for the function of the protein, because a number of proteins require ROS post-translational modification for activation. It has been suggested that PrPC may be involved in synaptic transmission, and it was reported recently that synaptic activity can be stimulated by H2O2 in a PrPC-dependent manner (51). The H2O2 effect was believed to be due to the higher synaptosomal copper content of PrPC mice over PrP-/- mice. In light of the results reported here, it is possible that the activation may have been due to a direct interaction between PrPC and H2O2, leading to PrPC cleavage and possibly increased copper release. The results presented here show for the first time that PrPC processing occurs under conditions of oxidative stress. Because cleavage of PrPC by ROS occurs into a region similar to that in PrPSc, it is possible that such cleavage may be relevant in TSEs. Consequently, the findings presented here may lead to further approaches in the prevention and treatment of such diseases.


    ACKNOWLEDGEMENT

We are grateful to David Harris for providing us with the antibody P45-66.


    FOOTNOTES

* This work was supported by grants from the Cellule de Coordination Interorganismes sur les Prions and the CNRS.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.

§ Supported by Grant BIO4CT98-6055 from the European Community Biotech.

Supported by Grant BIO4CT98-6064 from the European Community Biotech.

** To whom correspondence should be addressed: IGH du CNRS, 141 Rue de la Cardonille, 34396 Montpellier Cedex 5, France. Tel.: 33 4 99 61 99 31; Fax: 33 4 99 61 99 01; E-mail: Sylvain.Lehmann@igh.cnrs.fr.

Published, JBC Papers in Press, November 1, 2000, DOI 10.1074/jbc.M007243200


    ABBREVIATIONS

The abbreviations used are: TSE, transmissible spongiform encephalopathy; CJD, Creutzfeldt-Jakob disease; PrP, prion protein; PrPSc, scrapie isoform of PrP; PrPC, cellular isoform of PrP; ROS, reactive oxygen species; MoPrP, mouse PrP; SAF, scrapie-associated fibril; CHO, Chinese hamster ovarian; SDS-PAGE, SDS-polyacrylamide gel electrophoresis; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; DTPA, diethylenetriaminepentaacetic acid; MES, 4-morpholineethanesulfonic acid.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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