(Received for publication, November 21, 1995; and in revised form, December 4, 1995)
From the
Prion diseases are unusual neurodegenerative disorders that can
be both infectious and inherited. Both forms are hypothesized to result
from a posttranslational structural alteration in the cell surface
glycoprotein PrP (cellular isoform of the prion protein)
that converts it into the protease-resistant isoform PrP
(scrapie isoform of the prion protein). However, a direct
comparison of molecular events underlying these two manifestations of
prion diseases has not been possible, because there has been no cell
culture model for the familial forms. We report here that when mutant
prion proteins associated with three different inherited prion
disorders of humans are expressed as their murine homologues in
cultured Chinese hamster ovary cells, the proteins are
protease-resistant and detergent-insoluble, two biochemical properties
characteristic of infectious PrP
. In addition, each mutant
protein remains tightly associated with the plasma membrane after
enzymatic cleavage of its glycosylphosphatidylinositol anchor, a
property that we now show is also typical of infectious
PrP
. The cell culture system described here is the first in vitro model for familial prion diseases and provides
compelling evidence that infectious and genetic cases share common
molecular features.
Prion diseases are fatal neurodegenerative disorders of human
beings and animals characterized by dementia, ataxia, myoclonus, and
spongiform deterioration of the brain and spinal
cord(1, 2) . A unique feature of these disorders is
their manifestation as infectious, genetic, and sporadic forms.
Infectious cases, including kuru, experimental scrapie, and iatrogenic
Creutzfeldt-Jakob disease (CJD), ()are thought to be caused
by prions (3) . These particles contain little or no nucleic
acid and are composed primarily of PrP
(4) , a
posttranslationally modified isoform of a glycolipid-anchored membrane
protein of the host called PrP
(5) . Familial prion
diseases, including Gerstmann-Sträussler syndrome
(GSS), fatal familial insomnia (FFI), and about 10% of the cases of
CJD, display an autosomal dominant pattern of inheritance with nearly
complete penetrance and are linked to insertional and point mutations
in the chromosomal gene that encodes PrP
(6) .
Both infectious and inherited forms of prion disease are
characterized by accumulation in the central nervous system of
protease-resistant PrP that is capable of transmitting the
disease to laboratory animals(2) . It is uncertain, however,
whether the cellular mechanisms underlying production of PrP
are the same in the two disease states. This uncertainty is due,
in part, to the absence of a cell culture model of familial prion
diseases, analogous to the ones that have been developed for analysis
of scrapie infection (7, 8, 9, 10) .
It is hypothesized that all prion diseases result from a
posttranslational change in the conformation of PrP
(11, 12, 13, 14) , but it is
possible that distinct cellular compartments and molecular components
are involved in inherited and infectious forms. To resolve this issue,
it will be necessary to employ cultured cell models in addition to
transgenic mice (15) and cell-free systems(16) .
To
develop such a model, we have constructed stably transfected Chinese
hamster ovary (CHO) cells that express murine homologues of mutant PrPs
associated with human prion diseases. We previously reported that one
of these mutants, a Creutzfeldt-Jakob homologue containing an insertion
of six additional octapeptide repeats, displays an abnormal mode of
attachment to the plasma membrane(17) . Unlike wild-type PrP,
this protein remains tightly bound to the cell surface, even after
enzymatic cleavage of its glycosylphosphatidylinositol (GPI) anchor. In
the present work, we have analyzed CHO cells expressing mutant PrPs
related to all three inherited prion diseases of humans, in order to
determine whether these proteins acquire biochemical properties of
infectious PrP. In addition, we have investigated whether
PrP
synthesized in scrapie-infected neuroblastoma cells
has a membrane attachment similar to that of the mutant PrPs.
Scrapie-infected N2a mouse neuroblastoma cells were kindly provided by Dr. Byron Caughey (Rocky Mountain Laboratories, Hamilton, MT) and were grown as described(19, 20) .
To assess the decrease in electrophoretic mobility of moPrPs after
cleavage of the GPI anchor, CHO cells were labeled for 20 min with
TranS-label and chased for 3 h in Opti-MEM. Cell lysates
were incubated first with N-glycosidase F as described above
and then with 1 unit/ml of PIPLC for 1 h at 37 °C, following which
moPrP was immunoprecipitated using antibody P45-66 and analyzed by
SDS-PAGE.
The mutant moPrP constructs employed here are listed in Table 1, along with their human homologues and the phenotypes
with which they are associated. M128V moPrP was analyzed as a negative
control, because a Met Val substitution at codon 129 is a
nonpathogenic polymorphism in the human population(26) .
To
test the detergent solubility of the moPrPs, lysates of metabolically
labeled CHO cells expressing each protein were centrifuged at 265,000
g for 40 min, a protocol that sediments PrP
but not PrP
(19) . We found that significantly
more PG11, P101L, D177N, and E199K moPrPs than wild-type moPrP
sedimented (Fig. 1, A and B). We noted that
E199K moPrP was more soluble than the other mutants (60% versus 80-90% in the pellet). Importantly, very little M128V moPrP
sedimented, indicating that the effect on detergent solubility was
specific for disease-related mutations.
Figure 1:
MoPrPs carrying disease-related
mutations are detergent-insoluble and protease-resistant. A,
detergent lysates of metabolically labeled CHO cells expressing each
moPrP were centrifuged first at 16,000 g for 5 min and
then at 265,000
g for 40 min. MoPrP in the
supernatants (S) and pellets (P) from the second
centrifugation were immunoprecipitated and analyzed by SDS-PAGE. The arrowheads indicate the positions of PrP-specific bands,
immunoprecipitation of which is blocked when the antibody is
preincubated with the peptide immunogen (not shown). Molecular mass
markers are in kilodaltons. B, PrP bands from the experiment
shown in A, and from two additional experiments, were
quantitated using a PhosphorImager, and the percentage of each protein
in the pellet was calculated. Each bar represents the mean
± S.D. Values that are significantly different from wild-type
moPrP by t test (p < 0.001) are indicated by an asterisk. C, CHO cells expressing each protein were
labeled for 3 h with Tran
S-label and chased for 4 h in
Opti-MEM. Proteins in cell lysates were either digested at 37 °C
for 10 min with 3.3 µg/ml of proteinase K (+ lanes)
or were untreated (- lanes), prior to recovery of moPrP
by immunoprecipitation. Five times as many cell equivalents were loaded
in the + lanes as in the - lanes. D, PrP bands
shown in C, and from two additional experiments using each
protein, were quantitated using a PhosphorImager. The amount of PrP
remaining after digestion was expressed as a percentage of the amount
observed in the absence of protease. For lanes 2, 4, 6, 8, 10,
and 12, only the region of the gel between 27 and 30 kDa,
where the bulk of the protease-resistant PrP migrated, was included in
the quantitation. Each bar represents the mean ± S.D.
Values that are significantly different from wild-type moPrP by t test (p < 0.001) are indicated by an asterisk. These differences are not attributable to variations
in the amount of protein substrate, since similar amounts of both total
protein and radiolabeled moPrP were present in all samples before
digestion. WT = wild-type
moPrP.
To assess the protease
resistance of the moPrPs, lysates of metabolically labeled cells were
treated for 10 min with 3.3 µg/ml of proteinase K, conditions
similar to those that have been used for digestion of PrP derived from some scrapie strains(32, 33) . We
observed that PG11, P101L, D177N, and E199K moPrPs were cleaved by the
protease to yield fragments that migrated between 27 and 30 kDa, the
same size as the protease-resistant core of PrP
(4, 5) (Fig. 1C). In contrast,
wild-type moPrP, as well as M128V moPrP, were completely degraded under
these conditions (Fig. 1, C and D). In a
separate study, we have shown that proteinase K truncates the PG11
protein within a segment of
20 amino acids following the
octapeptide repeats, the same region within which cleavage of
infectious PrP
occurs. (
)
More than half of
each of the mutant PrPs is present on the cell surface at steady state,
as determined by susceptibility to externally applied trypsin (data not
shown). To test whether the proteins can be released from the surface
by cleavage of their GPI anchors, cells were biotinylated with a
membrane-impermeant reagent and incubated with the bacterial enzyme
PIPLC, which cleaves the diacylglycerol portion of the anchor. We found
that 5% of PG11, P101L, and D177N moPrPs were released by PIPLC,
compared with
90% for wild-type and M128V moPrPs (Fig. 2, A and B). An intermediate amount (
50%) of E199K
moPrP was released, which correlates with the higher solubility of this
protein compared to the other disease-related mutants (Fig. 1B). Confirming that the GPI anchor on each
protein had been cleaved, we observed a characteristic decrease in the
electrophoretic mobility of the protein after PIPLC
treatment(17, 34) (Fig. 2C). The
structural features that account for lack of PIPLC release are also
likely to confer protease resistance on mutant PrP molecules, since we
found that the pool of E199K protein that was retained on the cell
surface after PIPLC treatment was protease-resistant (Fig. 2D, lanes 4 and 8), while the released
pool was completely digested (Fig. 2D, lanes 3 and 7). Several possible explanations may account for retention of
mutant PrPs on the cell surface following GPI anchor cleavage,
including integration of the PrP polypeptide chain into the lipid
bilayer, tight association with other membrane components, or
aggregation(17) .
Figure 2: MoPrPs carrying disease-related mutations are retained on the cell surface after cleavage of their GPI anchors. A, surface-biotinylated CHO cells expressing each protein were treated with PIPLC (1 unit/ml) for 2 h at 4 °C prior to lysis. MoPrP in the PIPLC incubation media (M lanes) and cell lysates (C lanes) was immunoprecipitated, separated by SDS-PAGE, and visualized by developing blots of the gel with horseradish peroxidase-streptavidin and ECL. B, PrP bands from the ECL film shown in A, and from two additional experiments, were quantitated by densitometry, and the amount of PrP released by PIPLC was plotted as a percentage of the total amount of PrP (medium + cell lysate). Each bar represents the mean ± S.D. Values that are significantly different from wild-type moPrP by t test are indicated by single (p < 0.01) and double (p < 0.001) asterisks. C, lysates of metabolically labeled cells expressing each protein were incubated with (+ lanes) or without (- lanes) PIPLC. MoPrP was then immunoprecipitated and analyzed by SDS-PAGE. PrP-specific bands (arrowheads) are reduced in electrophoretic mobility after PIPLC treatment. D, CHO cells expressing wild-type and E199K moPrPs were surface-biotinylated and treated with PIPLC as described in the legend of A. Proteins in the PIPLC incubation media (M lanes) and cell lysates (C lanes) were then either digested at 37 °C for 10 min with 3.3 µg/ml of proteinase K (PK) (+ lanes) or were left untreated (- lanes), prior to recovery of moPrP by immunoprecipitation. Biotinylated moPrP was then visualized as described in the legend of A. Five times as many cell equivalents were loaded in the + lanes as in the - lanes. Only the fraction of E199K moPrP not released by PIPLC (lane 4) generates a protease-resistant fragment (bracket, lane 8). WT, wild-type moPrP.
Although all of the moPrPs carrying disease-related mutations were more detergent-insoluble and protease-resistant, and less PIPLC-releasable than wild-type moPrP, we noted several biochemical features that distinguished the mutant PrPs from each other. First, the protease-resistant fragments of the D177N and E199K proteins consistently migrated 1-2 kDa more slowly than the fragments of the other mutant proteins (Fig. 1C), suggesting that the cleavage site may not be identical for all of the mutants. Second, the glycosylation patterns of the mutants were different from each other, although all of the disease-related mutants displayed a higher proportion of lower molecular mass glycoforms than wild-type PrP (Fig. 2A). Third, E199K moPrP was more detergent-soluble and PIPLC-releasable than the other mutants (Fig. 1B and 2B). These observations raise the possibility that mutant PrPs possess ``strain-specific'' molecular properties that account for the different clinical and neuropathological phenotypes with which they are associated(32, 33, 35, 36, 37, 38, 39) .
Although numerous reports have emphasized the hydrophobicity of
infectious PrP(40, 41, 42) ,
there has been uncertainty about how this isoform is associated with
cell membranes. Scrapie-infected mouse neuroblastoma cells have been
used extensively to analyze the biochemical mechanisms underlying prion
generation, since these cells produce PrP
that is both
protease-resistant and infectious (Fig. 3A; Refs. 7, 8,
19, and 23). To determine whether infectious PrP
, like
mutant PrP
, remains membrane-bound after PIPLC treatment,
we surface-biotinylated scrapie-infected and uninfected neuroblastoma
cells and incubated them with the phospholipase. In agreement with
previous studies(43, 44) , the majority of the
protease-sensitive moPrP in both types of cells was released by PIPLC (Fig. 3B, lanes 1-4). In contrast,
protease-resistant PrP
was retained on the surface of
infected cells after PIPLC treatment (Fig. 3B, lanes 7 and 8). This behavior is similar to that of the mutant
PrPs and suggests an important similarity between the membrane
topologies of mutant and infectious molecules. The fact that PrP
can be biotinylated in these cells indicates that at least some
must be present on the plasma membrane and must therefore be accessible
to externally applied PIPLC, a result consistent with previous
reports(20, 45, 46) .
Figure 3:
PrP synthesized in
scrapie-infected neuroblastoma cells is retained on the cell surface
after digestion with PIPLC. A, uninfected (Sc
) and scrapie-infected (Sc
) N2a cells were metabolically labeled for
5 h with Tran
S-label and chased for 16 h in Opti-MEM. Cell
lysates were treated with proteinase K (PK) as described in Fig. 1C, except that digestion was carried out for 30
min with 10 µg/ml of the protease. The bracket indicates a
triplet of bands in lane 4 that is characteristic of PrP
27-30 from these cells(19, 23) . B, the
same biotinylation experiment described in Fig. 2D was
performed on uninfected (Sc
) and
scrapie-infected (Sc
) cells, except that 10
µg/ml proteinase K (PK) was used for 30 min. The bracket indicates the position of the PrP 27-30 triplet,
which is present in lane 8 but absent in lane
7.
Several results
argue strongly that the cell culture system employed here is a faithful in vitro model of familial prion diseases. First, the mutant
PrPs synthesized in this system display all of the major biochemical
properties of authentic PrP, including detergent
insolubility, protease resistance, abnormal attachment to the plasma
membrane, and, as shown in a separate study,
metabolic
stability. Second, these PrP
-like biochemical properties
are detected in four different moPrP mutants whose human homologues are
associated with each of the known forms of inherited prion disorder
(familial CJD, GSS, and FFI). Third, a mutation in moPrP (M128V) whose
human homologue represents a nonpathogenic polymorphism fails to induce
PrP
properties. Fourth, there are variations among the
disease-related mutants in their biochemical properties, consistent
with the concept that structurally distinct PrP ``strains''
are associated with different disease
phenotypes(32, 33, 35, 36, 37, 38, 39) .
It might be argued that there is a difference between our cell
culture system and the brains of patients afflicted with inherited
prion diseases in the time course and extent of PrP production. The human disorders evolve slowly, not manifesting
themselves clinically until adulthood, and even in the terminal stages
only a small proportion of the available PrP
is converted
to PrP
(6) . In contrast, we find that CHO cells
convert a substantial fraction of mutant PrP they express into
PrP
within a matter of hours.
Possible
explanations for this discrepancy are that cell- and tissue-specific
factors influence the efficiency of the conversion reaction and that
PrP
accumulates in the brain for a period of time before
pathological damage and clinical symptoms ensue.
We now plan to test
whether mutant moPrPs expressed in CHO cells are infectious, as are PrP
molecules from the brains of some patients with familial prion
diseases. It has been observed, however, that inherited prion diseases
as a group display lower rates of transmission to rodents and primates
than do cases having an infectious or sporadic etiology(47) .
This result may simply reflect variations in titer among brain regions
from which inocula are derived, or it may imply that some forms of
PrP can produce neurological damage without being
infectious. If it should turn out that the mutant PrPs produced in our
system are not infectious, we would hypothesize that CHO cells are
capable of carrying out the initial steps in biochemical conversion of
PrP
to PrP
, but that additional processes are
necessary for the generation of finished prion particles.
Although
infectious prion diseases arise by a species-specific interaction
between exogenous PrP and endogenous
PrP
(48, 49) , while inherited cases are
presumed to involve a spontaneous transformation of mutant PrP into
PrP
(2) , the results obtained with our in
vitro system provide compelling evidence that the two kinds of
diseases share key molecular features. We have shown here that mutant
and infectious PrP
molecules have remarkably similar
biochemical properties, including an unusually tight attachment to the
plasma membrane that had not been previously appreciated. In addition,
acquisition of at least some of these properties occurs
posttranslationally, as demonstrated by pulse-chase labeling studies of
mutant
and infectious PrPs (20, 23) in
cultured cells. Taken together, these results strongly support the
hypothesis(11, 12, 13, 14) that all
prion diseases are initiated by a conformational transition of the PrP
polypeptide chain that profoundly alters its biochemical properties
following synthesis, and at least in most cases, endows it with
infectivity. It remains to be determined whether this transition occurs
along the same cellular pathways in all forms of the disease and
whether the same set of molecular intermediates and accessory proteins
is involved. Further analysis of PrP
production in
infected cell lines and in cultured cells expressing mutant PrPs will
make it possible to resolve these issues and for the first time to
directly compare the cellular and biochemical mechanisms underlying two
major etiologies of prion disease.