Protease-resistant and Detergent-insoluble Prion Protein Is Not
Necessarily Associated with Prion Infectivity*
Gideon M.
Shaked
,
Gilgi
Fridlander§,
Zeev
Meiner
,
Albert
Taraboulos§, and
Ruth
Gabizon
¶
From the
Department of Neurology, Hadassah University
Hospital, Jerusalem 91120, Israel and the § Department of
Molecular Biology, Hebrew University Medical School, Jerusalem
91120, Israel
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ABSTRACT |
PrPSc, an abnormal isoform of
PrPC, is the only known component of the prion, an agent
causing fatal neurodegenerative disorders such as bovine spongiform
encephalopathy (BSE) and Creutzfeldt-Jakob disease (CJD). It has been
postulated that prion diseases propagate by the conversion of
detergent-soluble and protease-sensitive PrPC molecules
into protease-resistant and insoluble PrPSc molecules by a
mechanism in which PrPSc serves as a template. We show here
that the chemical chaperone dimethyl sulfoxide (Me2SO) can
partially inhibit the aggregation of either PrPSc or that
of its protease-resistant core PrP27-30. Following Me2SO removal by methanol precipitation, solubilized PrP27-30 molecules aggregated into small and amorphous structures that did not resemble the rod configuration observed when scrapie brain membranes were extracted with Sarkosyl and digested with proteinase K. Interestingly, aggregates derived from Me2SO-solubilized PrP27-30
presented less than 1% of the prion infectivity obtained when the same
amount of PrP27-30 in rods was inoculated into hamsters. These results suggest that the conversion of PrPC into protease-resistant
and detergent-insoluble PrP molecules is not the only crucial step in
prion replication. Whether an additional requirement is the aggregation
of newly formed proteinase K-resistant PrP molecules into uniquely
structured aggregates remains to be established.
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INTRODUCTION |
PrPSc, an abnormal isoform of PrPC (1), is
the only known component of the prion, an agent causing fatal
neurodegenerative disorders such as BSE1 and CJD (2). It
has been postulated that prion diseases propagate by the conversion of
PrPC molecules into protease-resistant and -insoluble
PrPSc molecules by a mechanism in which PrPSc
serves as a template (3). The pathway for PrPSc synthesis
may feature the formation of PrPC-PrPSc
heterodimers (4). Alternatively, the nucleation-dependent protein polymerization model argues that the formation of new PrPSc molecules depends on the presence of a seed composed
of aggregated PrPSc molecules and that new
PrPSc molecules join previously assembled prion polymers
(5). Although many lines of evidence suggest that PrPSc is
the crucial and even the only prion component, until today infectivity
could not be associated with PrPSc like PrP molecules
produced by an array of in vitro conversion protocols
(6-8).
The organic solvent dimethyl sulfoxide
(Me2SO) was shown to block
the formation of amyloid fibrils by A
peptide in vitro (9). After a single dose of Me2SO, the urine of human
amyloidotic patients contained fibrils with the tinctorial properties
of amyloids, suggesting that Me2SO can either break large
amyloid fibrils or inhibit their formation, resulting in smaller
structures that can be mobilized from the connective tissue and
eliminated by the kidneys (10). Me2SO was also shown to
inhibit the accumulation of PrPSc in scrapie-infected
neuroblastoma cells (11), suggesting that Me2SO, in its
function as a "chemical chaperone," stabilized the conformation of
PrPC molecules, thereby preventing them from undergoing the
conformational changes required for the conversion of PrPC
to PrPSc.
In this work, we investigated whether the hallmark properties of
PrPSc, i.e. resistance to proteases and
insolubility in detergents, are affected by in vitro
treatment with Me2SO. These biochemical properties of PrP
have been traditionally linked to the presence of prion infectivity
(12), although in some experimental setups, protease-resistant PrP
could not be found in samples that contain prion infectivity (13-15).
Interestingly, it was shown lately that in prion strains with long
incubation times, PrPSc is considerably less resistant to
proteases than in short incubation time strains (16).
Our results show that when membranes prepared from brains of hamsters
terminally ill with scrapie were incubated in the presence of
Me2SO and detergents, as opposed to detergent only, part of the PK-resistant PrP molecules could neither be precipitated by high
speed centrifugation nor did they aggregate into very large structures.
Me2SO, although it can inhibit the aggregation of protease-resistant PrP molecules, could not solubilize previously aggregated PrPSc. These soluble PrP27-30 molecules will
aggregate upon the removal of Me2SO, albeit not to the
characteristic rod structure obtained when scrapie brain membranes are
extracted with Sarkosyl and digested with proteinase K (PK) (17). When
inoculated into hamster brains, only traces of scrapie infectivity were
associated with this prion-specific isoform.
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EXPERIMENTAL PROCEDURES |
Sucrose Gradients--
Three hundred microliters of 10, 15, 20, 25, 30, and 60% sucrose in phosphate-buffered saline were loaded into
TLS-55 ultracentrifuge tubes (Beckman Instruments) to form a zonal
gradient. Microsomes from brains of scrapie-infected hamsters (60-80
µl containing about 15 µg/ml protein) were diluted with STE buffer
(100 mM NaCl, 10 mM Tris, pH 7.4, 1 mM EDTA) to 240 µl. Sarkosyl (2%) and when appropriate
Me2SO (10%) were added to the mixture and incubated for
16 h at 4 °C before loading on top of the gradient and
centrifuged at 100,000 × g for 1 h at 20 °C.
After the centrifugation, gradient fractions of equal volume were
collected and immunoblotted with anti-PrP mAb 3F4.
In Vivo Infectivity Experiments--
Top and bottom fractions
from sucrose gradients (after PK digestion) with and without
Me2SO were precipitated by 4 volumes of methanol (to
discard the Me2SO) and resuspended in 100 µl of saline,
10% bovine serum albumin. 10 µl of each sample was serially diluted
in STE (3 × 10), and all samples were immunoblotted with mAb 3F4.
After comparing the protease-resistant PrP signal in control
microsomes, bottom fractions from sucrose gradients with and without
Me2SO, as well as top fractions of the Me2SO
gradient, the top samples were diluted 10 times and the rest 100 times
to produce solutions with similar concentrations of PrP27-30.
Four-week-old male Syrian hamsters were inoculated intracerebrally with
samples to be tested for prion infectivity (50 µl). Each sample was
inoculated into five hamsters, and all experiments were performed in
duplicates. The hamsters were tested daily. Prion titers were measured
by monitoring the incubation period until the appearance of symptoms (18).
Cross-linking by DSS--
Disuccinimidyl suberate, an
N-hydroxy succinimide ester homobifunctional cross-linker
(reacting with NH2 groups), was dissolved in
Me2SO (to 1 M) and subsequently diluted into
double-distilled water to 500 µM. Samples of sucrose
gradient fractions were incubated with DSS at a final concentration of
125 µM DSS (30 min, room temperature). Following the
incubation, the reaction was terminated by the addition of 1 M Tris. The samples were precipitated by methanol and
immunoblotted with mAb 3F4.
Histoblots--
Histoblots were carried out as described by
Taraboulos et al. (19). Shortly, glass slides carrying
8-µm thick cryostat sections were quickly thawed and immediately
pressed onto nitrocellulose membrane saturated with lysis buffer. The
membranes were thoroughly air-dried, rehydrated for 1 h in TBST
(10 mM Tris, pH 8, 100 mM NaCl, 1.5% Tween
20), and then subjected to limited proteolysis in digestion buffer
containing 40 µg/ml PK for 1 h at 37 °C, followed by
incubation of the blots in 3 M guanidine thiocyanate/10
mM Tris-HCl, pH 7.8. Subsequently, the blots were processed
as for immunoblotting.
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RESULTS |
10- µl microsomes (20) (15 µg/ml protein) prepared from the
brains of Syrian hamsters infected with experimental scrapie 263K (21)
were diluted to 100 µl in STE buffer (100 mM NaCl, 10 mM Tris, pH 7.4, 1 mM EDTA) and incubated with
2% sodium sarcosinate (Sarkosyl) in the presence or absence of 10%
Me2SO at 4 °C for 16 h before centrifugation for
1 h at 100,000 × g. Me2SO was removed from the supernatants by methanol precipitation, and pellets were rinsed once with 70% methanol. Ethanol or methanol precipitation are
established methods to precipitate PrPSc and prions for
infectivity assays as well as for biochemical manipulations (22-25).
All samples were resuspended in STE with 2% Sarkosyl, incubated with
proteinase K (40 µg/ml, 1 h, 37 °C), and then analyzed by
SDS-PAGE followed by immunoblotting with
PrP mAb 3F4 (26).
Proteinase K completely digests PrPC and concomitantly
converts PrPSc into PrP27-30(1). In the presence of
Me2SO (Fig. 1, lane
1), a considerable part of the protease-resistant PrP remained in the supernatant; otherwise almost all of it pelleted in the high speed
spin (lane 3). Me2SO was almost ineffective in
solubilizing PrPSc if added 2 h after the detergent,
when most PrPSc molecules were already aggregated (compare
lanes 2 and 4). This suggests that although
Me2SO can inhibit PrPSc aggregation, it does
not solubilize previously aggregated PrPSc.
Me2SO was also unable to solubilize purified PrP27-30 or
PrPSc 33-35, which are already aggregated (not shown).

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Fig. 1.
Me2SO inhibits the aggregation of
PrPSc molecules. Membranes from scrapie-infected
brains were incubated with 2% Sarkosyl in the presence or absence of
10% Me2SO at 4 °C. Following the incubation, all
samples were digested with 40 µg/ml PK for 1 h at 37 °C and
centrifuged at 100,000 x g. Pellets and supernatants
(sup) were tested for the presence of PK-resistant PrP by
immunoblotting with PrP mAb 3F4. Lane 1, incubation with
Sarkosyl and Me2SO for 16 h. Lane
2, Me2SO was added 2 h after Sarkosyl, and
the sample was incubated for 14 additional hours. Lane 3, incubation with Sarkosyl alone for 16 h. Lane 4, Sarkosyl was added to this sample 2 h before the end of the
incubation.
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To test whether the results obtained in Fig. 1 resulted from the
in vitro conversion of PrPC to a
protease-resistant species by the Me2SO treatment or
whether traces of Me2SO inhibit the activity of PK,
microsomes from scrapie-infected hamster brains were pretreated with PK
before the incubation in the presence or absence of Me2SO.
The effect of Me2SO on PrPSc aggregation was
identical regardless of whether Me2SO was applied before or
after digestion with PK (Fig. 2). We
conclude therefore that Me2SO does not confer protease
resistance to otherwise PK-sensitive PrP molecules but rather inhibits
the aggregation of PrPSc molecules. Whether
Me2SO just reveals and amplifies a pre-existing difference
between two populations of PrPSc molecules (such as
preaggregation) or actively partitions PrPSc molecules into
two distinct populations remains to be established.

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Fig. 2.
Me2SO (DMSO)
inhibits the aggregation of PrP27-30 molecules. Membranes from
scrapie-infected hamsters were incubated with Sarkosyl and in the
presence (lanes b and d) and absence (lanes
a and c) of Me2SO (as in Fig. 1). The
samples presented in lanes a and b were digested
for 1 h with 40 µg/ml PK before the addition of
Me2SO and Sarkosyl. In lanes c and d,
samples were digested with PK after Me2SO treatment (as in
Fig. 1). After Me2SO incubation and PK treatment (or vice
versa), all samples were centrifuged and immunoblotted as in Fig. 1.
Lanes a-d represent the supernatants of all samples after
the centrifugation.
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To estimate the degree of aggregation of Me2SO-solubilized
PrPSc, normal and scrapie-infected hamster brain microsomes
were incubated for 16 h in Sarkosyl in the presence or absence of
Me2SO, and the resulting lysates were sedimented through
10-60% sucrose gradients containing 2% Sarkosyl. Molecules migrating
to the bottom of such gradients are either in very large aggregates or
of very large molecular weight, and detergent-soluble proteins of small
size are expected to remain in the upper gradient fractions. As can be
seen in Fig. 3, PrPC from
normal microsomes, which solubilizes readily in the presence of
detergents (20), remained in the upper fractions of the gradient in the
presence or absence of Me2SO. In addition, the incubation of PrPC with Me2SO treatment did not convert
the normal prion isoform into protease-resistant PrP (Fig. 3,
a-d).

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Fig. 3.
Me2SO-solubilized
PrPSc presents as a low density oligomer. Membranes
from normal or from scrapie-infected brains were incubated with 2%
Sarkosyl in the presence and absence of 10% Me2SO and were
subjected to a size-separating sucrose gradient as described under
"Experimental Procedures." Fractions collected from the gradients
were digested in the presence and absence of PK and immunoblotted with
mAb 3F4. a, normal hamster brain membranes. b, normal
hamster brain membranes digested for 1 h with 40 µg/ml PK.
c, Me2SO-treated normal hamster brain.
d, Me2SO-treated normal hamster brain membranes
digested for 1 h with 40 µg/ml PK. e, scrapie hamster
brain membranes. f, scrapie hamster brain membranes digested
for 1 h with 40 µg/ml PK. g,
Me2SO-treated scrapie hamster brain membranes.
h, Me2SO-treated scrapie hamster brain membranes
digested for 1 h with 40 µg/ml PK.
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Microsomes from scrapie-infected brains, which are believed to contain
both PrPC and PrPSc (20), yielded a bimodal PrP
distribution in the absence of Me2SO; about half of the PrP
(presumably PrPC) was found in the top of the gradient,
whereas the rest migrated to the bottom (Fig. 3e). However,
only the PrP at the bottom of the gradient resisted the action of
proteinase K, as expected from PrPSc (Fig.
3f).
When Me2SO was present during the lysis of scrapie
microsomes, a profound change in the distribution of protease-resistant PrP was observed (Fig. 3h). A considerable portion of the
PrP molecules at the top of the gradient resisted proteinase
K-catalyzed proteolysis, which instead produced PrP27-30, the
protease-resistant core of PrPSc. Thus, the action of
Me2SO on scrapie microsomes yielded protease-resistant, prion-specific PrP structures that are contained in low degree oligomers.
To reinforce the conclusion that the Me2SO-soluble
PrP27-30 is the protease-resistant core of PrPSc and not a
partially resistant PrPC promoted either by the
Me2SO incubation or by methanol precipitation, the three
lightest fractions from sucrose gradients c and
g, respectively, in Fig. 3 were combined, precipitated with
methanol, resuspended in Sarkosyl, and subsequently digested with low
PK concentrations for short periods. It has been shown lately that
under such mild conditions of PK digestion, PrPC reveals a
PK-resistant core of a lower Mr than PrP27-30,
since the 3F4 epitope (residues 108-111) is absent from this peptide (27). These PK-digested samples were immunoblotted either with mAb 3F4
or with mAb 13A5, which reacts with residue 138 of hamster PrP. As can
be seen in Fig. 4, while in the
protease-resistant core of Me2SO-treated PrP in the scrapie
fractions, both epitopes were present after 1 h PK digestion, and
this was not the case for PrP from normal brain. These results show
that Me2SO-solubilized PrP from scrapie brains was indeed
PrPSc which, unlike PrPC, produced upon mild or
harsh PK digestion a protease-resistant core of the same molecular
weight as aggregated PrPSc (see also Figs. 3 and 8). In
addition, these results also show that methanol precipitation does not
confer or reduce protease resistance.

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Fig. 4.
Me2SO-solubilized PK-resistant
PrP is not derived from PrPC. Membranes from normal
and scrapie-infected hamsters were incubated in the presence of
Me2SO and subjected to sucrose gradients as described in
Fig. 3 and under "Experimental Procedures." The three lightest
fractions from the normal and the scrapie gradient were combined,
precipitated by methanol (4 volumes), and subsequently resuspended in
STE + 2% Sarkosyl before PK digestion at 20 µg/ml for 0, 5, 10, or
60 min and immunoblotting with either mAb 3F4 (a) or mAb
13A5 (b).
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To investigate the degree of oligomerization of soluble PrP27-30 in
Fig. 3, PK-digested Me2SO-treated scrapie microsomes were resolved on a sucrose gradient as described above, and the diverse gradient fractions were subsequently cross-linked by DSS. The rationale
of this experiment is that although monomeric PrPSc will
not cross-link, aggregated PrPSc will cross-link heavily
and thereby not enter the polyacrylamide gel. Indeed, as can be seen in
Fig. 5, while treatment of the heavy
fractions from the Me2SO gradient with DSS resulted in a reduction in the PrP27-30 band seen by 3F4 immunoblotting, PrP27-30 in the light fractions was resistant to cross-linking.

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Fig. 5.
Me2SO-solubilized
PrPSc does not cross-link. Me2SO-treated
scrapie-infected membranes were subjected to a sucrose gradient as in
Fig. 3. Fractions from this gradient were incubated in the presence or
absence of 125 µM DSS for 30 min at room temperature. The
reaction was stopped by the addition of 1 M glycine.
Samples were subjected to immunoblotting with mAb 3F4.
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To test whether Me2SO-solubilized PrPSc
molecules will either aggregate or remain soluble following the removal
of Me2SO, fractions from the top and from the bottom of
sucrose gradients were incubated with PK and then precipitated with
methanol. Me2SO is very soluble in this alcohol. Methanol
pellets of top and of bottom fractions were resuspended in 2%
Sarkosyl, and suspensions were either incubated overnight at 4 °C or
mixed prior to incubation. Suspensions were then resolved again in the
same kind of sucrose gradient. Most of the soluble protease-resistant
PrP27-30 (from the top fractions) sedimented to the bottom of the
gradient under these conditions (Fig. 6).
These results show that the Me2SO-solubilized PrP retained its propensity to aggregate upon Me2SO removal. When,
instead of removal by methanol precipitation, Me2SO was
dialyzed from the light fractions at 4 °C against a Sarkosyl
containing buffer, only part of the PrP27-30 could be recovered from
the dialysis tube. Aggregated PrPSc or PrP27-30 molecules
are known for their nonspecific adhesion properties (28). Even under
these conditions, only a fraction of the recoverable protease-resistant
PrP was present in the light fractions of the new sucrose gradient
(Fig. 6d), suggesting the reaggregation of PrP27-30 upon
Me2SO removal precipitation is an intrinsic property of
Me2SO-solubilized PrPSc.

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Fig. 6.
Me2SO-solubilized PrP becomes
insoluble after Me2SO removal. Fractions
from a sucrose gradient of Me2SO-treated scrapie brain
hamsters (a) were either precipitated with methanol (4 volumes) and resuspended in 2% Sarkosyl for 2 h or dialyzed
against STE + 2% Sarkosyl overnight. Light and heavy fractions by
themselves or combined with each other were subjected to an additional
sucrose gradient. b, heavy fraction after methanol
precipitation. c, light fraction after Me2SO
removal by precipitation. d, light fractions after
Me2SO removal by dialysis. e, combined heavy and
light fractions after Me2SO removal by precipitation.
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When the aggregates produced by Me2SO-solubilized PrP27-30
were looked upon by electron microscopy and compared with those in the
original heavy fractions, a profound difference in structure was
observed. Although PrP27-30 from the heavy fractions aggregated into
the familiar rod-like structure (29), the new aggregates were amorphous
(Fig. 7).

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Fig. 7.
Me2SO-solubilized
PrPSc aggregates into non-rod structures. Fractions
from a sucrose gradient loaded with Me2SO-treated scrapie
brain membranes were immunostained with mAb 3F4 and protein A gold (10 nM) and negatively stained with uranyl acetate and examined
under an electron microscope. All samples were digested with 40 µg/ml
PK. a, gradient heavy (bottom) fractions
(bar, 100 nm). b, light fractions after
Me2SO removal (bar, 100 nm). c,
sample b at a higher magnification (bar, 10 nm).
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PK-digested heavy and light fractions of sucrose/Sarkosyl gradients
with and without Me2SO were tested for prion infectivity. The three lightest and heaviest fractions (out of 12) of each gradient
were combined, concentrated by methanol, and then resuspended in 10%
bovine serum albumin in saline. The same protocol was applied on a
control sample consisting of scrapie brain microsomes. Before inoculation, the control, both heavy fractions, as well as the light
fraction of the Me2SO gradient were adjusted by dilution to
contain comparable concentrations of PrP27-30 (as judged by immunoblotting with the mAb 3F4). The light fraction prepared in the
absence of Me2SO, which did not contain visible
PrPSc, was diluted for inoculation identically to the
Me2SO-treated light fraction. Parallel samples were
re-digested with PK and were shown to be identically resistant to
digestion as the ones inoculated into hamsters. All samples were
inoculated intracerebrally, and the hamsters were monitored for
clinical signs of scrapie. The results are summarized in Fig.
8. Regardless of the Me2SO treatment, most of the infectivity (<99%) was found in the heavy fractions (that contained rods), whereas only about 1% of the infectivity was associated with light fractions. Thus,
Me2SO-soluble PrP27-30 was associated with very low, if
any, prion infectivity. Interestingly, although the amount of
protease-resistant PrP present in the light fractions prepared in the
presence of Me2SO was at least 100 times higher than in the
light fractions without Me2SO (as judged by
immunoblotting), no significant difference in the infectivity
associated with these fractions could be observed.

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Fig. 8.
Prion infectivity is mostly associated with
rods like PrPSc. PK-resistant control scrapie
microsomes and fractions from sucrose gradients of scrapie-infected
membranes with and without Me2SO as in Fig. 3 were
precipitated with 4 volumes of methanol and resuspended so that they
contain identical concentrations of PrP27-30. Light fractions prepared
without Me2SO, which do not contain PrP27-30, were diluted
similarly as the Me2SO light fractions. 50 µl of sample
C and bars 1-4 in the immunoblot, shown in the
figure, were inoculated into Syrian hamster brains and monitored for
signs of prion infection. Infectivity titers were calculated from
incubation time (days) according to Prusiner et al. (29).
C, control scrapie microsomes. Bar 1, light
fractions of gradient without Me2SO. Bar
2, light fraction of gradient with Me2SO.
Bar 3, heavy fraction of bar 1.
Bar 4, heavy fraction of bar 2.
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A possible explanation for the infectivity results presented above is
that Me2SO produced a profound change in the conformation of PrPSc, therefore generating a new prion strain (30, 31).
To test this possibility, we looked for evidence of strain-specific
parameters in the hamsters that were inoculated with samples from the
top and the bottom fractions of Me2SO-treated and untreated
microsomes. Clinical signs observed in all groups were similar and were
characteristic of the parent Sc263K strain (32). PrP27-30 banding
(Fig. 9a) and
PrPSc histoblot patterns (Fig. 9b) (33-35) were
also similar in all the four groups. However, since there is a residual
infectivity in the light fractions of untreated microsomes, these
results do not rule out the presence, in the Me2SO-treated
samples, of a new prion strain with a much longer incubation time than
that of the parent Sc263 strain.

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Fig. 9.
a, PrPSc banding pattern in
brains of hamsters inoculated with Me2SO-treated or
untreated samples. Ten percent brain homogenate of hamsters dying of
scrapie due to infection with the samples described in Fig. 7 were
digested with 40 µg/ml PK and immunoblotted with mAb 3F4. Lane
C, brain inoculated with control scrapie microsomes. Lane
1, brain inoculated with light fraction of gradient without
Me2SO. Lane 2, brain inoculated with
light fraction of gradient with Me2SO. Lane 3, brain inoculated with heavy fraction of lane 1.
Lane 4, brain inoculated with heavy fraction of
lane 2. b, brain histoblots of
hamsters inoculated with Me2SO-treated or untreated light
and heavy gradient fractions. Hippocampal histoblots of Syrian hamster
brains inoculated with light fraction of gradient without
Me2SO (blot 1). Light fraction of gradient with
Me2SO (blot 2). Heavy fraction of blot
1 (blot 3). Heavy fraction of blot 2 (blot 4).
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DISCUSSION |
Although there is little doubt that PrPSc plays a
crucial role in prion diseases (2), the mechanism by which
PrPC converts into the scrapie isoform of the prion
protein, as well as the specific role of the PrP isoforms before and
during the disease, is still unknown. A puzzling point to the prion
hypothesis is the lack of correlation between the number of
PrPSc molecules in a sample and its infectivity as revealed
by animal bioassays. In most cases, the infectious unit is associated
with as many as 105 PrPSc molecules (36).
However, in some cases, prion infectivity was transmitted even in the
apparent absence of protease-resistant PrPSc molecules (13,
37). One explanation for such an effect depends on the different
biochemical characteristics of distinct prion strains. It has been
shown that each prion strain can be associated with a characteristic
protease-resistant core which probably results from a defined
PrPSc conformation for each strain (33, 38-40). It was
also shown that the prion incubation time depends on the degree of
protease resistance of PrPSc in each such strain (16).
Indeed, PrP and infectivity in some CJD stains shows very little
protease resistance (15), whereas the hamster 263K produces the most
PK-resistant PrP identified among prion strains (16).
It is more difficult to speculate on how samples that contain the same
prion strain can present different incubation times for the same
concentration of PrPSc. One explanation for this effect is
that disaggregated PrPSc could display a higher specific
infectivity, perhaps because more infectious centers would exist in
such a sample or perhaps due to the increased surface area of such a
disaggregated agent. For instance, dispersion of prion rods into DLPC
resulted in increased prion titer, suggesting that smaller and more
open aggregates may have increased access to the molecules in the brain
which participate in the transmission of infectivity (41). As opposed to dispersion into DLPC, treatment of PrPSc with the
amyloid-binding dye Congo Red reduces prion titer by stabilizing the
aggregated state of PrPSc (42, 43). Based on these facts,
we assumed that samples containing Me2SO-solubilized
PrPSc would display very high specific prion titers.
Surprisingly, this was not the case. The light samples in the gradients
shown in Fig. 3, with and without Me2SO, presented the same
low prion titer, even though the control sample had no apparent
PrPSc in contrast to the Me2SO-treated sample,
which contains large quantities of the protease-resistant PrP isoform.
It should be noted that the DLPC dispersion experiments were performed
mostly on preaggregated rods, which as described above contain a
priori all the infectivity. In addition, it is possible that the
inoculation of PrPSc in DLPC, although dispersed into
monomers or small oligomers, represents a favorable pharmacological
pathway to infect cells with prions. Me2SO-solubilized
PrPSc may not present such a biological advantage.
Our results show that prion-specific PrP molecules can be
differentiated into two distinct species of disparate physicochemical properties: "classical" PrPSc and
Me2SO-soluble PrPSc. This demonstrates that
prion-specific PrP molecules can exist as a soluble species and yet
possess the protease-resistant core PrP27-30. Apart from being
resistant to proteases, these soluble molecules differ from
PrPC in that they have not lost their propensity to
aggregate (albeit to amorphous, non-rod structures) when
Me2SO is removed. However, this species is not associated
with prion infectivity.
We have also shown here that soluble PrPSc can be
dissociated from prion infectivity only when aggregation is inhibited
during membrane extraction and not by dissociation from previously
aggregated PrPSc. This is the fundamental difference
between the experiments presented here and other approaches which
failed to show non-infectious protease-resistant PrPSc
(44). As shown above, non-infectious and infectious PrPSc
have similar aggregation properties, and therefore, once aggregated in
the presence of detergent they are biochemically indistinguishable.
Me2SO-solubilized PrPSc may be a metabolic
intermediate in the formation of infectivity-associated
PrPSc. If so, then the process of prion replication may be
composed of more than one irreversible step, the conversion of
PrPC to a protease-resistant species being only one of
them. In vitro converted PrPSc may also be such
an intermediate, since although presenting the biochemical properties
of PrPSc, it has not been shown to be infectious (7, 8).
Prion infectivity may only be associated with the final step, which
would involve the specific aggregation of PrPSc into a
structure with the pharmacological properties required for the biology
of the infectious process.
Although it is not impossible that Me2SO caused a profound
change in the structure of part of the PrPSc molecules, the
fact that Me2SO-soluble PrPSc could only be
generated from microsomes and not from preformed rods is more
consistent with the possibility of more than one prion-specific
PrPSc existing before the addition of Me2SO and
the extraction of scrapie brain membranes with detergents. Regardless
of the mechanism, our results show that not all PK-resistant PrP
molecules are associated with prion infectivity.
Me2SO-soluble PrPSc species, although not
infectious, could still play an important role in the neuropathology of
prion diseases. At the last stages of the disease, when the load of
total PrPSc molecules in the brain is large, non-infectious
PrPSc molecules may replace most of the PrPC
molecules or otherwise inhibit their normal function. A large load of
non-infectious PrPSc molecules may also be neurotoxic or
contribute to brain degeneration in as yet unknown mechanisms.
 |
FOOTNOTES |
*
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.
¶
To whom correspondence should be addressed: Dept. of
Neurology, Hadassah University Hospital, Jerusalem 91120, Israel. Fax: 972-2-6429441; E-mail: gabizonr{at}hadassah.org.il.
 |
ABBREVIATIONS |
The abbreviations used are:
BSE, bovine
spongiform encephalopathy;
CJD, Creutzfeldt-Jakob disease;
Me2SO, dimethyl sulfoxide;
PK, proteinase K;
mAb, monoclonal antibody;
DSS, disuccinimidyl suberate;
DLPC, detergent
lipid protein complex.
 |
REFERENCES |
-
Oesch, B.,
Westaway, D.,
Walchli, M.,
McKinley, M. P.,
Kent, S. B.,
Aebersold, R.,
Barry, R. A.,
Tempst, P.,
Teplow, D. B.,
Hood, L. E.,
Prusiner, S. B.,
and Weismann, C.
(1985)
Cell
40,
735-746[Medline]
[Order article via Infotrieve]
-
Prusiner, S. B.
(1998)
Brain Pathol.
8,
499-513[Medline]
[Order article via Infotrieve]
-
Pan, K. M.,
Baldwin, M.,
Nguyen, J.,
Gasset, M.,
Serban, A.,
Groth, D.,
Mehlhorn, I.,
Huang, Z.,
Fletterick, R. J.,
Cohen, F. E.,
and Prusiner, S. B.
(1993)
Proc. Natl. Acad. Sci. U. S. A.
90,
10962-10966[Abstract]
-
Prusiner, S. B.,
Scott, M.,
Foster, D.,
Pan, K. M.,
Groth, D.,
Mirenda, C.,
Torchia, M.,
Yang, S. L.,
Serban, D.,
Carlson, G. A.,
Hoppe, P. C.,
Westaway, D.,
and De Armond, S. J.
(1990)
Cell
63,
673-686[Medline]
[Order article via Infotrieve]
-
Come, J. H.,
Fraser, P. E.,
and Lansbury, P. T., Jr.
(1993)
Proc. Natl. Acad. Sci. U. S. A.
90,
5959-5963[Abstract]
-
Kaneko, K.,
Peretz, D.,
Pan, K. M.,
Blochberger, T. C.,
Wille, H.,
Gabizon, R.,
Griffith, O. H.,
Cohen, F. E.,
Baldwin, M. A.,
and Prusiner, S. B.
(1995)
Proc. Natl. Acad. Sci. U. S. A.
92,
11160-11164[Abstract]
-
Kocisko, D. A.,
Come, J. H.,
Priola, S. A.,
Chesebro, B.,
Raymond, G. J.,
Lansbury, P. T.,
and Caughey, B.
(1994)
Nature
370,
471-474[CrossRef][Medline]
[Order article via Infotrieve]
-
Hill, A. F.,
Antoniou, M.,
and Collinge, J.
(1999)
J. Gen. Virol.
80,
4-11
-
Shen, C. L.,
and Murphy, R. M.
(1995)
Biophys. J.
69,
640-651[Abstract]
-
Ravid, M.,
Kedar, I.,
and Sohar, E.
(1977)
Lancet
1,
730-731[Medline]
[Order article via Infotrieve]
-
Tatzelt, J.,
Prusiner, S. B.,
and Welch, W. J.
(1996)
EMBO J.
15,
6363-6373[Abstract]
-
Bolton, D. C.,
McKinley, M. P.,
and Prusiner, S. B.
(1984)
Biochemistry
23,
5898-5906[Medline]
[Order article via Infotrieve]
-
Lasmezas, C. I.,
Deslys, J. P.,
Robain, O.,
Jaegly, A.,
Beringue, V.,
Peyrin, J. M.,
Fournier, J. G.,
Hauw, J. J.,
Rossier, J.,
and Dormont, D.
(1997)
Science
275,
402-405[Abstract/Free Full Text]
-
Xi, Y. G.,
Ingrosso, L.,
Ladogana, A.,
Masullo, C.,
and Pocchiari, M.
(1992)
Nature
356,
598-601[CrossRef][Medline]
[Order article via Infotrieve]
-
Manuelidis, L.,
Sklaviadis, T.,
and Manuelidis, E. E.
(1987)
EMBO J.
6,
341-347[Abstract]
-
Safar, J.,
Wille, H.,
Itri, V.,
Groth, D.,
Serban, H.,
Torchia, M.,
Cohen, F. E.,
and Prusiner, S. B.
(1998)
Nat. Med.
4,
1157-1165[CrossRef][Medline]
[Order article via Infotrieve]
-
McKinley, M. P.,
Meyer, R. K.,
Kenaga, L.,
Rahbar, F.,
Cotter, R.,
Serban, A.,
and Prusiner, S. B.
(1991)
J. Virol.
65,
1340-1351[Medline]
[Order article via Infotrieve]
-
Prusiner, S. B.,
Cochran, S. P.,
Groth, D. F.,
Downey, D. E.,
Bowman, K. A.,
and Martinez, H. M.
(1982)
Ann. Neurol.
11,
353-358[Medline]
[Order article via Infotrieve]
-
Taraboulos, A.,
Jendroska, K.,
Serban, D.,
Yang, S. L.,
DeArmond, S. J.,
and Prusiner, S. B.
(1992)
Proc. Natl. Acad. Sci. U. S. A.
89,
7620-7624[Abstract]
-
Meyer, R. K.,
McKinley, M. P.,
Bowman, K. A.,
Braunfeld, M. B.,
Barry, R. A.,
and Prusiner, S. B.
(1986)
Proc. Natl. Acad. Sci. U. S. A.
83,
2310-2314[Abstract]
-
Kimberlin, R. H.,
and Walker, C. A.
(1986)
J. Gen. Virol.
67,
255-263[Abstract]
-
Prusiner, S. B.,
Groth, D.,
Serban, A.,
Stahl, N.,
and Gabizon, R.
(1993)
Proc. Natl. Acad. Sci. U. S. A.
90,
2793-2797[Abstract]
-
Gabizon, R.,
McKinley, M. P.,
Groth, D. F.,
Kenaga, L.,
and Prusiner, S. B.
(1988)
J. Biol. Chem.
263,
4950-4955[Abstract/Free Full Text]
-
Prusiner, S. B.
(1982)
Science
216,
136-144[Medline]
[Order article via Infotrieve]
-
Safar, J.,
Ceroni, M.,
Piccardo, P.,
Liberski, P. P.,
Miyazaki, M.,
Gajdusek, D. C.,
and Gibbs, C. J., Jr.
(1990)
Neurology
40,
503-508[Abstract]
-
Kascsak, R. J.,
Rubenstein, R.,
Merz, P. A.,
Tonna DeMasi, M.,
Fersko, R.,
Carp, R. I.,
Wisniewski, H. M.,
and Diringer, H.
(1987)
J. Virol.
61,
3688-3693[Medline]
[Order article via Infotrieve]
-
Buschmann, A.,
Kuczius, T.,
Bodemer, W.,
and Groschup, M. H.
(1998)
Biochem. Biophys. Res. Commun.
25,
693-702
-
Prusiner, S. B.,
McKinley, M. P.,
Groth, D. F.,
Bowman, K. A.,
Mock, N. I.,
Cochran, S. P.,
and Masiarz, F. R.
(1981)
Proc. Natl. Acad. Sci. U. S. A.
78,
6675-6679[Abstract]
-
Prusiner, S. B.,
McKinley, M. P.,
Bowman, K. A.,
Bolton, D. C.,
Bendheim, P. E.,
Groth, D. F.,
and Glenner, G. G.
(1983)
Cell
35,
349-358[Medline]
[Order article via Infotrieve]
-
Bruce, M. E.,
and Dickinson, A. G.
(1987)
J. Gen. Virol.
68,
79-89[Abstract]
-
Pattison, I. H.,
and Millson, G. C.
(1961)
J. Comp. Pathol.
71,
101-108
-
Lowenstein, D. H.,
Butler, D. A.,
Westaway, D.,
McKinley, M. P.,
DeArmond, S. J.,
and Prusiner, S. B.
(1990)
Mol. Cell. Biol.
10,
1153-1163[Medline]
[Order article via Infotrieve]
-
Telling, G. C.,
Parchi, P.,
DeArmond, S. J.,
Cortelli, P.,
Montaga, P.,
Gabizon, R.,
Mastrianni, J.,
Lugaresi, E.,
Gambetti, P.,
and Prusiner, S. B.
(1996)
Science
274,
2079-2082[Abstract/Free Full Text]
-
Hill, A. F.,
Desbruslais, M.,
Joiner, S.,
Sidle, K. C.,
Gowland, I.,
Collinge, J.,
Doey, L. J.,
and Lantos, P.
(1997)
Nature
389,
448-450[CrossRef][Medline]
[Order article via Infotrieve]
-
Hecker, R.,
Taraboulos, A.,
Scott, M.,
Pan, K. M.,
Yang, S. L.,
Torchia, M.,
Jendroska, K.,
DeArmond, S. J.,
and Prusiner, S. B.
(1992)
Genes Dev.
6,
1213-1228[Abstract]
-
Beekes, M.,
Baldauf, E.,
and Diringer, H.
(1996)
J. Gen. Virol.
77,
1925-1934[Abstract]
-
Hsiao, K. K.,
Groth, D.,
Scott, M.,
Yang, S. L.,
Serban, H.,
Rapp, D.,
Foster, D.,
Torchia, M.,
Dearmond, S. J.,
and Prusiner, S. B.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
9126-9130[Abstract]
-
Kascsak, R. J.,
Rubenstein, R.,
Merz, P. A.,
Carp, R. I.,
Robakis, N. K.,
Wisniewski, H. M.,
and Diringer, H.
(1986)
J. Virol.
59,
676-683[Medline]
[Order article via Infotrieve]
-
Bessen, R. A.,
and Marsh, R. F.
(1992)
J. Virol.
66,
2096-2101[Abstract]
-
Collinge, J.,
Beck, J.,
Campbell, T.,
Estibeiro, K.,
and Will, R. G.
(1996)
Lancet
348,
56
-
Gabizon, R.,
McKinley, M. P.,
and Prusiner, S. B.
(1987)
Proc. Natl. Acad. Sci. U. S. A.
84,
4017-4021[Abstract]
-
Caspi, S.,
Halimi, M.,
Yanai, A.,
Ben Sasson, S.,
Taraboulos, A.,
and Gabizon, R.
(1998)
J. Biol. Chem.
273,
3484-3489[Abstract/Free Full Text]
-
Ingrosso, L.,
Ladogana, A.,
and Pocchiari, M.
(1995)
J. Virol.
69,
506-508[Abstract]
-
Sklaviadis, T. K.,
Manuelidis, L.,
and Manuelidis, E. E.
(1989)
J. Virol.
63,
1212-1222[Medline]
[Order article via Infotrieve]
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