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
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ABSTRACT |
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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.
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 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.
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 PrP 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 (
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).
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.
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.
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).
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).
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.
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
To explore the effect of octapeptide deletions, we first analyzed an
N-terminal truncated form of PrP (
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.
PrP
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.
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 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 Several other experimental manipulations besides copper addition have
been shown to increase the 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.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helical content whereas
PrPSc is rich in
-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.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
32-80 were provided by C. Weissmann (28).
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 (
51-82) was constructed by re-inserting the sequence for the octapeptide PHGGGWGQ into the
51-90 template using overlapping polymerase chain reaction. All cDNAs were cloned into the expression vector pcDNA3 (Invitrogen).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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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.
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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.
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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.
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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.
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[in a new window]
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.
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).
View larger version (25K):
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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 PrP 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.
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 PrP
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.
51-90, which contains an exact deletion of all 5 octapeptide
repeats, remained detergent soluble in the presence of copper. In
contrast, PrP
51-82, which contains a single repeat (PHGGGWGQ) was
rendered insoluble by copper. Taken together with the results for
PrP
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).
View larger version (73K):
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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),
PrP 51-82 containing 1 octapeptide repeat (PHGGGWGQ), or
PrP
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.
View larger version (34K):
[in a new window]
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
-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.
-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.
-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
-rich, protease-resistant forms that are distinct from
PrPSc.
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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 51-82 and
51-90 plasmids.
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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.
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.
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