(Received for publication, September 30, 1994; and in revised form, November 23, 1994)
From the
Scrapie is a transmissible spongiform encephalopathy of sheep and other mammals in which disease appears to be caused by the accumulation of an abnormal form of a host protein, prion protein (PrP), in the brain and other tissues. The process by which the normal protease-sensitive form of PrP is converted into the abnormal protease-resistant form is unknown. Several hypotheses predict that oligomeric forms of either the normal or abnormal PrP may act as intermediates in the conversion process. We have now identified a 60-kDa PrP derived from hamster PrP expressed in murine neuroblastoma cells. Peptide mapping studies provided evidence that the 60-kDa PrP was composed solely of PrP and, based on its molecular mass, appeared to be a PrP dimer. The 60-kDa PrP was not dissociated under several harsh denaturing conditions, which indicated that it was covalently linked. It was similar to the disease-associated form of PrP in that it formed large aggregates. However, it resembled the normal form of PrP in that it was sensitive to proteinase K and had a short metabolic half-life. The 60-kDa PrP, therefore, had characteristics of both the normal and disease-associated forms of PrP. Formation and aggregation of the 60-kDa hamster PrP occurs in uninfected mouse neuroblastoma cells, which suggests that hamster PrP has a predisposition to aggregate even in the absence of scrapie infectivity. Similar 60-kDa PrP bands were identified in scrapie-infected hamster brain but not in uninfected brain. Therefore, a 60-kDa molecule might participate in the scrapie-associated conversion of protease-sensitive PrP to protease-resistant PrP.
Creutzfeldt-Jakob disease,
Gerstmann-Sträussler-Scheinker disease, and kuru in
humans, bovine spongiform encephalopathy in cattle, and scrapie in
sheep are members of a family of infectious neurodegenerative mammalian
diseases known as the transmissible spongiform encephalopathies. During
disease pathogenesis, a protease-resistant form of prion protein (PrP) ()accumulates in the brain and other tissues of infected
animals and appears to be responsible for the pathogenic
effects(1) . Although the exact nature of the etiologic agent
is unknown, it is resistant to inactivation by various harsh
treatments(2, 3) . These studies led Griffith to
propose that the scrapie agent was a protein and contained no nucleic
acid(4, 5, 6) . Subsequently, the
protease-resistant form of PrP, PrP-res, was found to be closely
associated with infectivity(7, 8, 9) . This
led to the hypothesis that PrP-res itself might be the infectious
agent(8, 10, 11) . However, this hypothesis
is still controversial(12, 13, 14) .
An
endogenous protease-sensitive form of PrP, PrP-sen, is the precursor to
PrP-res(15, 16, 17) . PrP-res differs from
PrP-sen in that PrP-res aggregates and is resistant to digestion with
proteinase
K(8, 11, 18, 19, 20) .
These aggregates accumulate and appear eventually to lead to cell death
and the spongiform changes observed in scrapie-infected brains. There
are no known post-translational modifications that can account for
these different properties of PrP-sen and
PrP-res(21, 22) . Recent spectroscopic analysis of
PrP-res suggests that PrP-res contains a higher -sheet content
than that predicted for PrP-sen(23, 24, 25) .
This secondary structure change could be important in the aggregation
and accumulation of PrP-res into amyloid-like aggregates of stacked
-sheet structures, which are partially resistant to protease
degradation.
Conversion of PrP-sen to PrP-res in a cell-free system using substantially purified components has provided evidence that PrP-res derives from direct PrP-sen/PrP-res interactions(17) . However, the exact mechanism by which the disease-associated conversion of PrP-sen into PrP-res occurs is unknown. Genetic studies of scrapie pathogenesis in mice led Dickinson and Outram (26) to propose, as early as 1979, that a dimeric protein might be important in scrapie replication in vivo. Current models of the role of PrP in scrapie pathogenesis also predict that oligomeric forms of PrP, such as dimers, could facilitate a more rapid conversion of PrP-sen into PrP-res(21, 27, 28, 29) . Consistent with these predictions, apparent dimers of PrP-res have been observed in scrapie-infected hamster brains(30, 31) , and there is one report of a PrP-sen molecule similar in size to that predicted for a PrP-sen dimer(32) . However, no attempt has been made to further study any of these molecules.
In the present studies, we identified a unique 60-kDa hamster PrP-sen molecule, which appeared to be a covalently linked dimer of two 30-kDa PrP monomers. The 60-kDa PrP was found in heterogeneous high molecular mass aggregates similar to proteinase K-resistant PrP-res from scrapie-infected hamsters. However, the 60-kDa PrP showed no resistance to proteinase K. Therefore, the 60-kDa PrP appeared to have properties of both PrP-res and PrP-sen. Additionally, our recent data showed the 60-kDa PrP could be converted to PrP-res in a cell-free system(17) . Thus, the 60-kDa PrP may prove to be an important participant in the scrapie-associated generation of PrP-res in vivo. The unique properties of the 60-kDa PrP dimer suggest that it may also be a useful molecular model to study the intermediate steps, such as dimerization and aggregation, which are believed to be involved in the conversion of PrP-sen to PrP-res.
Figure 1:
A 60-kDa HaPrP molecule is synthesized
in MNB cells expressing the HaPrP gene. PanelA, PrP
was immunoprecipitated with the monoclonal antibody 3F4 from S-methionine/cysteine cell lysates of normal MNB cells,
MNB cells expressing an exogenous HaPrP gene (HaPrP-46, HaPrP-D4), or
MNB cells expressing a mutated MoPrP gene containing the 3F4 epitope
(MoPrP-A5). The lane labeled MoPrP-A5 is derived from a
different experiment than the other lanes. The results were
reproducible over several experiments. The faint 25-, 30-, and
32-40-kDa HaPrP bands are due to the lower level of expression of
these forms of HaPrP in the HaPrP-46 cells when compared with the
60-kDa HaPrP protein. Molecular mass markers, in kilodaltons, are shown
on the left, and the immunoprecipitated forms of PrP and their
sizes are indicated on the right. PanelB,
PrP was immunoprecipitated from
S-labeled HaPrP-46 cells
using four different anti-PrP peptide rabbit polyclonal antibodies
(
)(34) . The amino acid residues for the synthetic PrP
peptides used to make each antisera are indicated above each
pair of lanes, and their location within the PrP protein are indicated
on the map of PrP in the bottom half of the panel. -,
antibody alone; +, antibody preabsorbed with the synthetic peptide
to which it was made. The PrP-specific bands are indicated on the left.
Figure 2: The 60-kDa protein is composed of PrP. The 60-, 30-, and 25-kDa forms of HaPrP were immunoprecipitated, using the monoclonal antibody 3F4 from MNB cells expressing the HaPrP gene, and partially digested with the endoprotease Lys-C (Lys-C) or endoprotease Glu C (Glu-C) according to the method of Cleveland(39) . For each panel, molecular mass markers are shown on the left. Squares designate partial digestion products. PanelA, 60- and 30-kDa HaPrP molecules from HaPrP-D4 cells. Lanes1 and 2 contain undigested 60- and 30-kDa HaPrP. Lanes3 and 4 are the 60- and 30-kDa HaPrP molecules digested with 0.1 mg/ml Lys-C. PanelB, 60- and 30-kDa HaPrP molecules from HaPrP-46 cells. Lanes1 and 2 contain undigested 60- and 30-kDa HaPrP molecules. Lanes3 and 4 are the 60- and 30-kDa HaPrP molecules digested with 0.025 mg/ml Glu-C. Lane4 was exposed 26 times longer than the other lanes. PanelC, 60- and 25-kDa HaPrP molecules from HaPrP-46 cells. Lanes1 and 2 contain undigested 60- and 25-kDa HaPrP molecules. Lanes3 and 4 are 60- and 25-kDa HaPrP molecules digested with 0.025 mg/ml Glu-C. This gel was not electrophoresed as long as the gel in PanelB, and the resolution of the peptide bands is not as high. The 25-kDa lanes were exposed 4 times longer than the 60-kDa lanes. The results were reproducible over several experiments.
Figure 3:
Treatment of immunoprecipitated HaPrP with
DTT, formic acid, and 8 M urea. HaPrP-D4 cells were labeled
with S methionine/cysteine as described under
``Experimental Procedures.'' After radioimmunoprecipitation
of HaPrP from the cell lysate, samples were either left untreated (none) or treated with 175 mM DTT or 96% formic acid (FA). These samples were then separated on a 12.5% SDS-PAGE
gel. Although there was a slight decrease in the amount of 60-kDa PrP
protein in the presence of DTT, a similar decrease is apparent with the
other forms of PrP. The observed decrease was therefore probably due to
sampling error. These results were reproducible over several
experiments. A final sample was treated with 8 M urea and
electrophoresed on a separate 8 M urea SDS-PAGE gel. The sizes
of the HaPrP molecules are indicated.
Figure 4:
The 60-kDa HaPrP molecule contains only
high mannose glycans. HaPrP-46 cells were labeled with S
methionine/cysteine and chased for the indicated periods of time as
detailed under ``Experimental Procedures.'' HaPrP was
immunoprecipitated using the monoclonal antibody 3F4 and incubated at
37 °C with (+) or without(-) endoglycosidase H (endoH). The HaPrP-specific bands and their molecular masses
are indicated on the left, and molecular mass markers are
shown on the right. The faint 25-, 30-, and 32-40-kDa
HaPrP bands are due to the lower level of expression of these forms of
HaPrP in the HaPrP-46 cells when compared with the 60-kDa HaPrP
protein. The results were reproducible over several
experiments.
Figure 5:
Kinetics of 60-kDa HaPrP and 25-40-kDa
HaPrP biosynthesis in HaPrP-46 cells. Confluent 25-cm flasks of HaPrP-46 cells were labeled with
S
methionine/cysteine for 10 min and chased in complete medium for the
indicated periods of time as described under ``Experimental
Procedures.'' HaPrP was immunoprecipitated from cell lysates using
the monoclonal antibody 3F4. PanelA, kinetics of
biosynthesis of the 60-kDa HaPrP. The gel was exposed for 6 h. PanelB, kinetics of biosynthesis of 25-40-kDa
HaPrP. The 25-40-kDa forms of HaPrP and their sizes are indicated
on the left. The data are from the same gel and experiment as
in PanelA, but the exposure time was 11 days, 44
times longer than in PanelA.
Figure 6:
The 60-kDa HaPrP protein is not expressed
on the cell surface. HaPrP-46 cells were radiolabeled with S methionine/cysteine and treated with PIPLC (A)
or PK (B) as described under ``Experimental
Procedures.'' After PIPLC or PK treatment, HaPrP was
immunoprecipitated from either the cell lysate (cells) or the
cell culture medium (medium).
Figure 7: The 60-kDa HaPrP is proteinase K-sensitive. Individual aliquots of a HaPrP-46 cell lysate were treated with increasing concentrations of proteinase K. Proteinase K was inactivated with protease inhibitors, and HaPrP was precipitated in methanol. The resultant pellet was sonicated into sample buffer, an aliquot was electrophoresed on a 20% SDS-PAGE PHAST gel, and HaPrP was detected by immunoblot using the hamster-specific monoclonal antibody 3F4. The 60-kDa HaPrP and the 25-40-kDa HaPrP are indicated on the left. The data are from the same gel, but the exposure time for the 25-40-kDa HaPrP was 20 times longer than for the 60-kDa HaPrP.
Figure 8: The 60-kDa HaPrP is part of a heterogeneous cellular aggregate. HaPrP-46 cells were lysed, and an aliquot of the lysate was spun through a 15-40% sucrose gradient. Fractions were collected from the gradient, the amount of HaPrP in each fraction was assayed by immunoblot using the monoclonal antibody 3F4, and the relative integrated intensities of the 60-kDa HaPrP and the 25-40-kDa HaPrP were determined using densitometry. The amount of the 60-kDa HaPrP or the 25-40-kDa HaPrP (HaPrP-sen) present in each fraction are plotted as a percentage of the total amount of each type of HaPrP present in the whole gradient. The data shown are derived from a single gradient but were reproducible over several experiments. The migration of standard molecular mass markers (Pharmacia) through a parallel gradient are indicated.
Figure 9:
The 60-kDa HaPrP pellets as a particle
ranging in size from 400 to 7 S. HaPrP-46 cells and hamster
scrapie-infected hamster brains were lysed. The infected hamster brain
lysate was treated with 25 µg/ml PK for 1 h at 37 °C to remove
the HaPrP-sen. Aliquots of the lysates were centrifuged through a 5%
sucrose cushion at the indicated Xg for 45 min, and the amount of 60kDa
HaPrP or HaPrP-res present in the pellet was determined by immunoblot
as detailed under ``Experimental Procedures.'' The range of S
values for particles that would pellet under the conditions used are
indicated. S values were estimated according to the
manufacturer's instructions (Beckman) using the equation t = k/S where t equals the time for each
centrifugation, k is a measure of the rotor's relative
pelleting efficiency in water at 20 °C, and S is the sedimentation
coefficient. The percentage of the total 60-kDa HaPrP present in the
HaPrP-46 lysate that pelleted at the bottom of the centrifuge tube is
shown (shadedbars), and the percentage of the total
HaPrP-res in the infected hamster brain lysate that pelleted is
indicated (openbars). Under the conditions used,
monomeric HaPrP-sen (25-40 kDa) did not pellet. The results are
the average of three independent experiments (HaPrP-46) or two
independent samples (infected hamster brain), and S.D. are
indicated by bars. The 7 S data are from centrifugation at
353,000 g for 2 h instead of 1
h.
Figure 10:
A 60-kDa
HaPrP molecule is expressed in scrapie-infected hamster brain. HaPrP
was extracted from uninfected (Sc) or
scrapie-infected (Sc
) hamster brain (Ha
Brain) from age-matched animals as described
previously(36) . The samples were not treated with proteinase
K. One brain was used per sample. Equivalent amounts of protein were
loaded in each lane and separated on a 20% SDS-PAGE PHAST gel, and the
HaPrP was detected by immunoblot using the monoclonal antibody 3F4. For
comparison, HaPrP was extracted from HaPrP-46 cells and assayed on the
same gel (HaPrP-46). The HaPrP species synthesized in HaPrP-46
cells are indicated on the left.
We have described a 60-kDa form of HaPrP that is expressed at high levels in MNB cells which synthesize HaPrP. Based on peptide mapping and reactivity to a series of anti-PrP peptide antibodies, we conclude that the 60-kDa protein band is composed of HaPrP. The size of the band is consistent with the size expected if two molecules of HaPrP are linked together to form a dimer. Based on the observations reported in this article, we suggest that the 60-kDa HaPrP is a dimeric form of normal PrP.
One of the most striking properties of the 60-kDa HaPrP molecule is its ability to form large aggregates. These aggregates are not scrapie-specific, because they are present in uninfected MNB cells. Aggregation of PrP-sen in scrapie-infected animals, however, may contribute to disease pathogenesis. For example, as predicted in the nucleation-dependent polymerization model of PrP-res accumulation, the presence of dimers of PrP-sen or PrP-res may greatly accelerate the formation of an ordered nucleus of PrP molecules(27) . This ordered nucleus of aggregated PrP molecules could act as a seed for the formation of large amounts of PrP-res(27, 28) . Alternatively, if large aggregates of PrP-sen were present in a cell infected with scrapie, a small amount of PrP-res could interact with the aggregate to induce the rapid conversion of a large amount of PrP-sen into PrP-res. This is similar to the scrapie replication site hypothesis proposed by Dickinson and Outram (26) in that dimers or larger multimers could provide a greater number of available ``replication sites'' for the scrapie agent.
The aggregation and proteinase K resistance of PrP-res appear to be closely linked. When the PrP-res aggregate is exposed to increasing concentrations of harsh denaturants, PrP-res becomes more sensitive to proteinase K(7, 17, 46) . If the PrP-res aggregate is allowed to renature, the resistance of PrP-res to proteinase K is restored(17) . The 60-kDa HaPrP molecule forms aggregates of heterogeneous size, the largest of which are similar in size to PrP-res aggregates from scrapie-infected brains. Unlike PrP-res, however, the 60-kDa HaPrP is not proteinase K-resistant. Therefore, the data presented here demonstrate that aggregation and proteinase K resistance are not necessarily linked. This separation of aggregation and proteinase K resistance implies that the PK resistance of PrP-res may be a scrapie-specific phenomenon, whereas aggregation may be a property of certain forms of PrP-sen, which is independent of scrapie infection.
An alternative explanation for the observed differences between the dimer and PrP-res may involve the association of the dimer with other molecules, such as glycosaminoglycans, which alter the structure of the aggregates formed. In this instance, it is the interaction with different types of secondary molecules that dictate aggregate size and protease sensitivity. A similar mechanism could explain the different properties of some scrapie strains. In fact, it has been shown that different strains of hamster scrapie derived from the transmissable mink encephalopathy scrapie agent can have different PK sensitivities and aggregate sizes(48) . This also suggests that molecules other than PrP may contribute to the aggregation and protease sensitivity of all forms of PrP, including the dimer.
Further evidence for the relevance of the 60-kDa PrP as a potential intermediate form of PrP in scrapie pathogenesis can be found in the fact that a 60-kDa form of PrP is also present in scrapie-infected hamster brains. This molecule is PK-resistant when isolated from scrapie-infected brains (30, 31) (data not shown), indicating that a 60-kDa PrP-sen molecule can be converted to PrP-res and may contribute to disease pathogenesis. We have recently reported that the 60-kDa HaPrP detected in HaPrP-D4 cells acts in a manner similar to the 60-kDa HaPrP detected in vivo in that it can be converted in a cell-free system into PrP-res(17) . Thus, the 60-kDa HaPrP fulfills many of the characteristics that might be expected of a dimeric intermediate in PrP-res formation as follows: 1) it forms large aggregates, 2) it is present in scrapie-infected hamster brains, and 3) it can be converted into PrP-res.
It is not known why a HaPrP dimer is expressed in uninfected mouse neuroblastoma cells. One explanation may be that overexpression of PrP is necessary for the formation of a PrP dimer. For example, the formation of PrP dimers could be due to the association of a high concentration of HaPrP monomers in cellular membranes. Alternatively, overexpression could lead to improper processing of the PrP monomer during biosynthesis or incorrect folding of HaPrP by chaperonins or other accessory proteins. However, we have assayed several clonal mouse neuroblastoma cell lines that express HaPrP at low levels, and all synthesize the dimer (data not shown). It is therefore unlikely that overexpression is the sole explanation for the formation of a HaPrP dimer in mouse neuroblastoma cells.
It is unclear where or how two HaPrP monomers could be linked to form a 60-kDa dimer. It does not appear that intermolecular disulfide bonds are necessary. Nevertheless, the two molecules of PrP appear to be covalently linked. Some evidence suggests that the linkage may be near the amino terminus of the molecule. The 60-kDa HaPrP expressed in HaPrP-D4 cells can be converted to a 23-kDa proteinase K-resistant form similar in size to PrP-res(17) , not the 46-kDa expected if two molecules of PrP-res were still linked. Thus, digestion of the PK-resistant 60-kDa PrP by PK may have removed the portion of the molecule responsible for linking two HaPrP proteins together. Proteinase K removes the amino-terminal 67 amino acids of PrP-res(21, 49) . Linkages involving lysines are among the most common types of protein cross-links(50) , and three lysine residues are among the 67 amino acids removed by PK digestion at the amino terminus of hamster PrP. Thus, it is possible that one or more of these residues could be involved in the covalent linkage of two 30-kDa monomer HaPrP molecules.
The identification of a 60-kDa dimer of PrP with the unusual features described here may offer valuable insights into scrapie pathogenesis. For example, its ability to form large aggregates of HaPrP might shorten the course of clinical disease by increasing the rate at which PrP-res accumulates. If, as its properties suggest, the 60-kDa HaPrP molecule is a form of PrP intermediate between those of PrP-sen and PrP-res, the metabolic pathways involved in the conversion of PrP-sen to PrP-res might be elucidated by characterizing the cell biology of the 60-kDa HaPrP dimer. The interactions of the 60-kDa PrP with cellular accessory proteins, its precise location within the cell, the part of the cell in which dimerization occurs, and the nature of the covalent link binding two 30-kDa PrP molecules together might clarify the manner in which differential processing of PrP can lead to the formation of the abnormal forms of PrP associated with scrapie pathogenesis.