From the Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110
Received for publication, July 27, 2000, and in revised form, September 28, 2000
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ABSTRACT |
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The prion protein (PrP), a glycolipid-anchored
membrane glycoprotein, contains a conserved hydrophobic sequence that
can span the lipid bilayer in either direction, resulting in two
transmembrane forms designated NtmPrP and
CtmPrP. Previous studies have shown that the proportion of
CtmPrP is increased by mutations in the membrane-spanning
segment, and it has been hypothesized that CtmPrP
represents a key intermediate in the pathway of prion-induced neurodegeneration. To further test this idea, we have surveyed a number
of mutations associated with familial prion diseases to determine
whether they alter the proportions of NtmPrP and
CtmPrP produced in vitro, in transfected cells,
and in transgenic mice. For the in vitro experiments, PrP
mRNA was translated in the presence of murine thymoma microsomes
which, in contrast to the canine pancreatic microsomes used in previous
studies, are capable of efficient glycolipidation. We confirmed that
mutations within or near the transmembrane domain enhance the formation of CtmPrP, and we demonstrate for the first time that this
species contains a C-terminal glycolipid anchor, thus exhibiting an
unusual, dual mode of membrane attachment. However, we find that
pathogenic mutations in other regions of the molecule have no effect on
the amounts of CtmPrP and NtmPrP, arguing
against the proposition that transmembrane PrP plays an obligate role
in the pathogenesis of prion diseases.
Prion diseases are neurodegenerative disorders characterized by
spongiform destruction of brain tissue and the presence of cerebral
amyloid plaques (1, 2). These disorders include kuru and
Creutzfeldt-Jakob disease in humans, "mad cow disease" in cattle,
and scrapie in sheep. Prion diseases can have an infectious or genetic
origin, or can arise spontaneously. The infectious agent is
hypothesized to be
PrPSc,1 a
conformationally altered isoform of a normal cell-surface glycoprotein of unknown function called PrPC (3). PrPSc from
dietary or other infectious sources is thought to act as a catalyst or
template to convert endogenous PrPC into more
PrPSc, which then accumulates, eventually causing disease.
Familial forms of prion disease result from germline mutations in the
PrP gene on chromosome 20, which are believed to favor conversion of
the protein to the PrPSc form (4). Sporadic cases may be
due to rare, spontaneous conversion of wild-type PrPC to
PrPSc. No covalent modifications that distinguish
PrPSc from PrPC have been detected (5), but the
conformations of the two isoforms are dramatically different, with
PrPSc having a much higher content of PrPSc is found in the brain in most cases of infectious,
familial, and sporadic prion disease (3). However, in some inherited prion diseases, for example cases of Gerstmann-Sträussler
syndrome due to an A117V mutation in PrP, PrPSc has not
been detected and brain material does not appear to be infectious when
injected into rodents (9-11). These exceptions raise the interesting
possibility that PrPSc, although it is the infectious form
of the protein, may not be the proximate cause of neurodegeneration in
at least some forms of prion disease.
Recently, it has been proposed that an alternate form of PrP that is
distinct from PrPSc may play an important role in prion
pathogenesis. This form, designated CtmPrP, has an unusual
transmembrane topology (12). Most molecules of PrPC do not
span the lipid bilayer and are attached to the cell surface exclusively
by a glycosyl phosphatidylinositol (GPI) anchor appended to the C
terminus of the polypeptide chain (13, 14). In contrast, CtmPrP is thought to span the membrane once, with its C
terminus on the exofacial surface and a highly conserved, hydrophobic
region in the center of the molecule (amino acids 111-134) serving as a transmembrane anchor. Another form of PrP, NtmPrP, has
also been described with the same transmembrane segment, but the
reverse orientation (N terminus on the exofacial surface) (12). It has
been proposed that the relative proportions of these three topological
variants is influenced by as yet unidentified accessory proteins
that interact with the translocation apparatus in the endoplasmic
reticulum (ER) (15, 16).
Recent studies have brought the potential biological relevance of
transmembrane forms of PrP into sharper focus. These species were
originally observed only after translation of PrP mRNA in vitro on rabbit reticulocyte or wheat germ ribosomes in the
presence of canine pancreatic microsomes (17-21). In more recent
investigations, however, CtmPrP has been identified in
brain membranes from transgenic mice that express PrP molecules
carrying mutations within or near the transmembrane domain; in
vitro translation experiments had indicated that these mutations
increased the relative proportion of CtmPrP (12, 22).
Transgenic mice expressing such CtmPrP-favoring mutations
at high levels develop a spontaneous neurodegenerative illness that
bears some similarities to scrapie, but without the presence of
PrPSc (12, 22). There is also indirect evidence that
CtmPrP accumulates in mice expressing wild-type PrP during
the course of scrapie infection (22). Based on these results, it has
been hypothesized that CtmPrP represents a common
intermediate in the pathogenesis of both infectious and genetic prion
diseases (22). In this view, CtmPrP is the ultimate cause
of neurodegeneration, and PrPSc acts indirectly by
increasing the amount of CtmPrP.
We have previously carried out extensive studies of the properties of
mutant PrP molecules expressed in cultured cells and transgenic mice
(23-27). The mutations analyzed in those investigations lie outside of
the conserved hydrophobic region that serves as a transmembrane anchor
in CtmPrP and NtmPrP. To test the hypothesis
that transmembrane PrP is part of a general pathway for prion-related
neurodegeneration, we undertook here to determine whether these
mutations induce the formation of NtmPrP and
CtmPrP in vitro, in cultured cells, and in
transgenic mice. We confirm that mutations in the central, hydrophobic
region enhance formation of transmembrane PrP, and we demonstrate for
the first time that CtmPrP contains a C-terminal GPI anchor
as well as a transmembrane segment, thus exhibiting two modes of
membrane attachment. However, we find that pathogenic mutations in
other regions of the molecule have no effect on the amounts of
CtmPrP and NtmPrP, arguing against the
possibility that transmembrane PrP plays an obligate role in the
pathogenesis of prion diseases.
Antisera--
P45-66 antibody, raised against a synthetic
peptide encompassing amino acids 45-66 of mouse PrP, has been
described previously (14). Monoclonal antibody 3F4, which recognizes an
epitope from hamster and human PrP encompassing residues 109-112 (28),
was a gift of Richard Kascsak (Institute for Basic Research, New York). R20 antibody, raised against a synthetic peptide comprising residues 218-232 of mouse PrP (29), was a gift of Byron Caughey (Rocky Mountain
Laboratories, Hamilton, MT). Anti-calnexin antibody was from
Stressgen (Vancouver, British Columbia, Canada).
PrP Plasmids and mRNA Synthesis--
All mouse PrP cDNAs
were cloned into the vector pcDNA3 (Invitrogen), and carried an
epitope tag for monoclonal antibody 3F4 created by changing residues
108 and 111 to methionine. The following mutations were introduced into
the wild-type PrP cDNA using polymerase chain reaction as described
previously (14): PG11 (six-octapeptide insertion), PG14
(eight-octapeptide insertion), K109I/H110I, 3AV (Ala In Vitro Translation--
Messenger RNAs were translated with
[35S]methionine in a final volume of 25 µl containing
50% nuclease-treated, rabbit reticulocyte lysate (Promgea, Madison,
WI) according to the manufacturer's instructions. Canine pancreatic
microsomal membranes were purchased from Promega, or were prepared by
the method of Walter and Blobel (30). Microsomes from BW5147.3 mouse
thymoma cells were prepared as described (31), except that cells were
lysed by Dounce homogenization and passage through a 27-gauge needle.
Pancreatic and thymoma microsomes were used at 1.5 and 5 µl per
translation reaction, respectively, to equalize the amounts of ER
marker proteins added. In some reactions, an oligosaccharide acceptor
peptide (Bz-N-G-T-Ac; Bachem, Torrance, CA) was used at a final
concentration of 0.5 mM to inhibit N-linked
glycosylation. Translation reactions were incubated at 30 °C for 60 min.
To detect protease-protected products, 5 µl aliquots of translation
reactions were incubated in a final volume of 50 µl with 100 µg/ml
PK (Roche Molecular Biochemicals) in 50 mM Tris-HCl (pH
7.5) and 1 mM CaCl2 for 60 min at 4 °C,
followed by addition of 5 mM phenylmethylsulfonyl fluoride
to terminate digestion. Some digestion reactions also contained 0.5%
Triton X-100 to solubilize membranes.
Samples were either analyzed directly by SDS-PAGE and autoradiography,
or they were first subjected to immunoprecipitation or treatment with
phosphatidylinositol-specific phospholipase C (PIPLC) as described
below. Radioactive bands on gels were quantitated using a
PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
Immunoprecipitation--
Aliquots of the translation reactions
were boiled in the presence of 1% SDS for 5 min to denature the
proteins, and were then diluted with 10 volumes of radioimmune
precipitation assay buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1% Triton X-100, 0.5% sodium deoxycholate,
0.1% SDS) plus protease inhibitors (pepstatin A and leupeptin, 1 µg/ml; phenylmethylsulfonyl fluoride, 5 mM). One µl of
the appropriate anti-PrP antibody was added, and samples incubated on
ice for 60 min. Protein A-Sepharose beads were added, and samples were
rotated at 4 °C for 30 min. Beads were collected by low speed
centrifugation and washed three times with radioimmune precipitation
assay buffer, after which proteins were eluted with 1% SDS in 50 mM Tris-HCl (pH 7.5) and subjected to deglycosylation with
peptide:N-glycosidase F (New England Biolabs,
Beverly, MA) according to the manufacturer's directions. Following
methanol precipitation, proteins were analyzed by SDS-PAGE and autoradiography.
PIPLC Treatment--
Five µl aliquots of translation reactions
were diluted to 200 µl with 1% Triton X-114 (precondensed as
described (Ref. 32)) in phosphate-buffered saline (PBS). After
incubation at 4 °C for 20 min, samples were subjected to phase
partitioning by incubation at 37 °C for 10 min, followed by
centrifugation at 16,000 × g for 10 min to separate
the phases. The aqueous phase was removed, and the detergent phase
diluted to 200 µl with PBS containing protease inhibitors and 100 µg/ml bovine serum albumin. One unit of PIPLC from Bacillus
thuringiensis (prepared as described in Ref. 33) was added to one
half of the diluted detergent phase, and both halves were incubated at
4 °C for 2 h. The phase separation was repeated, and proteins
in the second set of aqueous and detergent phases either were
precipitated with methanol and analyzed by SDS-PAGE or were subjected
to immunoprecipitation with 3F4 antibody.
Transfected Cells--
BHK cells were maintained in Brain Membranes--
Fresh brain tissue (~0.5 g wet weight)
was homogenized using a Dounce apparatus in 5 ml of Buffer B (0.25 M sucrose, 10 mM HEPES (pH 7.5), 100 mM KOAc, 1 mM Mg(OAc) 2, 1 µg/ml
pepstatin, 1 µg/ml leupeptin), followed by passage (five times each)
through 16-, 18-, 21-, 23-, and 27-gauge needles. The homogenate was
centrifuged first at 12,000 × g for 10 min in a
microcentrifuge, and then at 541,000 × g for 20 min in
a Beckman TLA100.3 rotor, and the microsomal pellet was resuspended in
250 µl of Buffer B. PK protection assays were carried out as
described above for transfected cells, except that PK was used at 50 µg/ml.
Microsomes from Canine Pancreas and Murine Thymoma Cells Differ in
Their Efficiency of GPI Anchoring--
PrP is attached to the cell
surface via a C-terminal GPI anchor (13, 14). In carrying out studies
of the membrane topology of PrP after in vitro translation,
it would thus be desirable to utilize microsomal membranes that are
capable of attaching the GPI anchor to newly synthesized polypeptides.
Previous investigations have employed microsomes derived from canine
pancreas, which do not effectively carry out GPI anchor addition (35).
We therefore tested microsomes derived from BW5147.3 murine thymoma
cells, which have been reported to be much more efficient in GPI
anchoring (31, 36).
We translated mouse PrP mRNA in vitro using rabbit
reticulocyte lysate in the presence of pancreatic or thymoma
microsomes, and then incubated Triton X-114 lysates of the membranes
with or without PIPLC, a bacterial phospholipase that cleaves the GPI anchor. By partitioning the lysate into detergent and aqueous phases,
we could then score the amount of PrP that had been rendered hydrophilic by removal of the anchor. As shown in Fig.
1, all translation reactions produced two
groups of products of 32 and 25 kDa, representing, respectively,
core-glycosylated and unglycosylated forms of PrP. The latter
correspond to molecules that had not been translocated into the lumen
of the microsomes, since they are susceptible to digestion with PK (see
below). Less than 10% of the PrP chains translated in the presence of
canine pancreatic microsomes shifted into the aqueous phase after PIPLC
treatment, indicating that very few molecules carried a GPI anchor
(lanes 1-4). In contrast, about 50-60% of the
glycosylated protein produced in the presence of thymoma microsomes
shifted into the aqueous phase, indicating relatively efficient GPI
anchoring (lanes 5-8). Untranslocated PrP
chains, although they lack a GPI anchor, are retained in the detergent
phase, presumably because they contain both N- and C-terminal signal
peptides that are hydrophobic. Of note, PrP molecules shifted into the
aqueous phase migrated with a slightly lower mobility than those that
remained in the detergent phase (compare lanes 7 and 8), a phenomenon that is typical for polypeptides that
have lost their GPI anchor (37). We believe that the actual efficiency
of anchor addition by thymoma microsomes may be even higher than 60%,
and that the persistence of some glycosylated PrP in the detergent
phase after PIPLC treatment is likely to reflect inefficient PIPLC
digestion and phase partitioning. In support of this idea, when samples
were denatured in SDS prior to PIPLC treatment, to improve
accessibility of proteins to the phospholipase, essentially all of the
glycosylated PrP was converted to the more slowly migrating band (not
shown). In contrast, PIPLC treatment after SDS denaturation did not
alter the gel mobility of PrP synthesized with pancreatic
microsomes.
Transmembrane Forms of PrP Are Produced in Both Microsome
Preparations, and Their Proportion Is Increased by Mutations in the
Hydrophobic Region--
To detect transmembrane forms of PrP,
translation products were subjected to digestion by PK, which cleaves
off portions of the polypeptide chain residing on the external side of
the microsomal membrane and leaves transmembrane and lumenal domains
intact. Samples were then immunoprecipitated with anti-PrP monoclonal antibody 3F4 to eliminate globin contamination contributed by the
reticulocyte lysate, which obscures visualization of low molecular weight PrP fragments. Proteins were also subjected to enzymatic deglycosylation to reveal differences in the sizes of PrP species independent of glycosylation state. After PK digestion of translation reactions containing either canine pancreatic or thymoma microsomes, wild-type PrP was resolved into three forms of 25, 19, and 15 kDa (Fig.
2, A and B,
lane 2). The 25-kDa form is the same size as PrP
before protease treatment (lane 1) and therefore
represents molecules that are completely protected from digestion, and
which therefore have been fully translocated into the microsome lumen; for consistency with an earlier report (12), we refer to this form as
SecPrP. The two smaller fragments are derived from
transmembrane species whose cytoplasmic domains have been digested by
the protease, and whose lumenal and transmembrane domains have been
protected by the microsome. No PrP is detected after PK treatment in
the presence of a detergent that disrupts the microsomal membrane (lane 3), confirming that the 19- and 15-kDa
fragments do not represent intrinsically protease-resistant portions of
the molecule. As will be shown below, the 19-kDa fragment derives from
CtmPrP, a species whose C terminus resides in the ER lumen,
and the 15-kDa fragment from NtmPrP, a species whose N
terminus is lumenal.
Consistent with previous studies (12, 22), we find that two mutations
in the central hydrophobic region of the PrP molecule increase the
relative amount of CtmPrP with little change in the amount
of NtmPrP (Fig. 2, A and B,
lanes 4-9; Table
I). A116V is the mouse homologue of a
mutation in human PrP (A117V) that is associated with
Gerstmann-Sträussler syndrome (38, 39); 3AV is a triple alanine
We found that, for wild-type PrP, as well as for the two mutant PrPs,
both NtmPrP and CtmPrP were produced in
considerably smaller amounts in the presence of thymoma microsomes than
in the presence of pancreatic microsomes (Fig. 2, compare A
and B; Table I). This difference is not attributable to an
inhibitory effect of GPI addition on the generation of transmembrane PrP in thymoma microsomes, since we have found that elimination of the
C-terminal GPI addition signal does not alter the amounts of
NtmPrP and CtmPrP produced in this system (data
not shown). Presumably, there are other cell type- or species-related
differences between the two microsome preparations that affect PrP topology.
Determination of the Topology of NtmPrP and
CtmPrP--
Two kinds of experiments were carried out.
First, we investigated whether or not NtmPrP and
CtmPrP were glycosylated by carrying out translations of
3AV PrP in the presence and absence of a peptide inhibitor of
N-linked glycosylation (Bz-N-G-T-Ac). We observed that the
protease-protected fragment of CtmPrP shifted from 24 to 19 kDa upon inclusion of the inhibitor, while the migration of the
NtmPrP fragment was unchanged (data not shown). This result
indicates that CtmPrP but not NtmPrP is
glycosylated. Since the two consensus sites for
N-glycosylation lie at positions 180 and 196, and the
presumed transmembrane segment at positions 111-134, the data indicate
that the C terminus of CtmPrP and the N terminus of
NtmPrP lie in the lumen of the microsome.
In a second experiment, we carried out epitope mapping to define the
topology of NtmPrP and CtmPrP (Fig.
3). Translations of both wild-type and
3AV PrP were carried out in the presence of thymoma microsomes, and
protease-protected products were immunoprecipitated with three
different antibodies: P45-66, which recognizes the octapeptide repeats
in the N terminus of PrP (14); 3F4, which reacts with an epitope
encompassing residues 108-111 (28) (this epitope, which is not
normally present in mouse PrP, was introduced into all of our PrP
constructs by changing residues 108 and 111 to methionine); and R20,
which recognizes residues 218-231 (29). We found that the
NtmPrP fragment was recognized by P45-66 and 3F4 but not
by R20, indicating that the N terminus of NtmPrP through
residue 111 is protected by the microsome. In contrast, the
CtmPrP fragment was recognized by 3F4 and R20 but not by
P45-66, indicating that the C terminus of CtmPrP starting
from residue 108 is protected. As expected, SecPrP, which
represents the full-length form of the protein, immunoprecipitates with
all three antibodies. These mapping results, in conjunction with the
observed sizes of the protected fragments, indicate that NtmPrP and CtmPrP span the membrane once, but
in opposite directions, via the central hydrophobic segment (Fig.
3B). Similar results were obtained when pancreatic rather
than thymoma microsomes were used.
CtmPrP Has a GPI Anchor--
Since the C terminus of
CtmPrP is lumenal, this form of the protein has the
potential to be GPI-anchored. To test this possibility, we carried out
translations of wild-type and 3AV PrP in the presence of thymoma cell
microsomes, which attach GPI anchors efficiently. As in Fig. 1, we
utilized Triton X-114 phase partitioning after PIPLC treatment to
assess the presence of the anchor. Translation reactions were treated
with protease prior to detergent lysis to reveal the protected fragment
derived from CtmPrP, which migrates at 24 kDa because it
has not been deglycosylated in this experiment. We found that this
fragment, as well as fully protected SecPrP (32 kDa), were
shifted into the aqueous phase after PIPLC treatment, consistent with
the presence of a GPI anchor on both species (Fig.
4, lanes 4 and
8). The change in partitioning of the 24-kDa fragment was
especially easy to appreciate for 3AV PrP (lane
8), which produces larger amounts of CtmPrP than
wild-type PrP. Consistent with loss of the GPI anchor after
phospholipase treatment, both SecPrP and CtmPrP
in the aqueous phase displayed a slightly reduced gel mobility compared
with the same species remaining in the detergent phase (compare
lanes 7 and 8). About 40-50% of the
CtmPrP fragment was shifted into the aqueous phase by
PIPLC, about the same proportion seen for SecPrP. As
discussed above, we believe that the presence of residual PrP in the
detergent phase after phospholipase treatment reflects inefficient
PIPLC digestion and phase partitioning. These results indicate that
CtmPrP has a dual mechanism of membrane attachment,
including both a transmembrane segment and a GPI anchor. From Fig. 4 it
would appear that the transmembrane segment is not sufficiently
hydrophobic to prevent segregation of the CtmPrP fragment
into the aqueous phase once the GPI anchor is removed by PIPLC.
Mutations Outside of the Central, Hydrophobic Domain Do Not Alter
the Amount of Transmembrane PrP--
It might be predicted that
mutations such as 3AV and A116V would affect the proportions of
CtmPrP or NtmPrP by altering the capacity of
the central, hydrophobic region to serve as a transmembrane anchor. We
wondered whether the same is true of mutations that lie outside of the
hydrophobic region. We therefore carried out in vitro
translations in the presence of thymoma microsomes of a number of PrP
mutants whose human homologues are associated with familial prion
diseases. The mutations analyzed are located both N- and C-terminal to
the central, hydrophobic segment. As positive controls, we analyzed PrP
containing the 3AV and A116V mutations, as well as another artificial
mutation in the central region (K109I/H110I, referred to as KH-II) that has been reported to increase the amount of transmembrane PrP (12, 22).
We found that the proportion of CtmPrP was not increased
over wild-type levels by any of the mutations outside of the central,
hydrophobic domain (Fig. 5, A
and B). As expected, the 3AV, A116V, and KH-II mutations
resulted in significantly increased levels of CtmPrP.
Since the amount of CtmPrP associated with wild-type PrP in
thymoma microsomes is small (~10%) and close to the limits of
detectability, we carried out similar experiments on mutant PrPs
translated with canine microsomes, which generate larger amounts of
CtmPrP. In this system as well, the amount of
CtmPrP was not significantly altered by pathogenic
mutations outside of the central region (Fig. 5B).
When samples were subjected to immunoprecipitation so that
NtmPrP could be visualized, the amount of this species
produced in the presence of either thymoma or canine microsomes was not
changed by any of the mutations (data not shown).
Little CtmPrP Can Be Detected in Transfected
Cells--
To test whether transmembrane PrP can be detected in
cultured cells, we carried out protease protection experiments on
membranes in post-nuclear supernatants derived from transiently
transfected BHK cells. As shown in Fig.
6, CtmPrP was not detectable
in cells expressing wild-type PrP, although small amounts of a 19-kDa
band that is likely to represent the deglycosylated, protease-protected
fragment of CtmPrP were present in cells expressing 3AV or
A116V PrPs. The identity of this band was confirmed by its reactivity
with 3F4 and R20, but not with P45-66 (data not shown). Cells
expressing PrP molecules with mutations outside of the hydrophobic
region did not produce detectable amounts of CtmPrP,
indicating that either these mutations do not increase the amount of
transmembrane PrP, or if they do, their effect is less pronounced than
that of 3AV and A116V. No NtmPrP was detected in any of the
transfected cells. The integrity of the membrane vesicles in these
experiments was confirmed by the production of a 70-kDa,
protease-protected fragment of calnexin, which represents the
transmembrane and lumenal domains of the protein (40). Results similar
to those shown in Fig. 6 were also obtained in transiently transfected
CHO cells (data not shown). We conclude from these experiments that
amount of transmembrane PrP produced in cultured cells is considerably
lower than after in vitro translation, with
CtmPrP representing <2% of the total PrP even for
transmembrane-favoring mutations.
CtmPrP Is Not Detectable in the Brains of Transgenic
Mice Expressing a Mutant PrP--
To determine whether transmembrane
forms of PrP could be detected in brain tissue, we also carried out
protease protection experiments on microsomal membranes prepared from
Tg(WT) transgenic mice that express wild-type PrP, or from Tg(PG14)
mice that express a mutant PrP carrying a nine-octapeptide insertion.
Tg(PG14) mice spontaneously develop a neurological illness
characterized by ataxia, granule cell apoptosis, synaptic PrP
deposition, and accumulation of a weakly protease-resistant form of PrP
(26, 27). As was the case in cultured cells, neither CtmPrP
nor NtmPrP were observable in the brains of mice expressing
either wild-type PrP or PrP carrying the nine-octapeptide insertion
(Fig. 7). Again, the integrity of the
microsomes was confirmed by the presence of the 70-kDa
protease-protected fragment of calnexin. Thus, in contrast to mutations
in the hydrophobic region, which have been shown to produce detectable
amounts of CtmPrP in the brains of transgenic mice (12,
22), a pathogenic mutation in the octapeptide repeat region does not
increase the amount of transmembrane PrP above the level of
detectability.
3AV PrP, but Not PG14 PrP, Can Be Released from the Cell Surface by
PIPLC--
Since CtmPrP is attached to the lipid bilayer
by both a transmembrane and a GPI anchor, it would not be released from
the membrane after PIPLC cleavage of the anchor. To see if the
increased amount of CtmPrP associated with the 3AV mutation
caused a reduction in the proportion of the mutant protein that could
be released from the cell surface by PIPLC, we labeled transfected CHO
cells with a membrane-impermeant biotinylation reagent (Fig.
8). We found that >90% of the surface
biotinylated 3AV PrP was released by the phospholipase (lanes 3 and 4), a proportion similar
to that for wild-type PrP (lanes 1 and
2). Thus, the 3AV mutation has no observable effect on the
PIPLC releasability of surface PrP. In contrast, virtually no
biotinylated PrP carrying the PG14 mutation could be released by PIPLC
(lanes 5 and 6), even though this
mutation does not affect the amount of CtmPrP (Fig. 5).
Similar results were obtained with transfected BHK cells (data not
shown).
We show that PrP chains translated in vitro or
expressed in cultured cells can adopt one of three possible topologies
(Fig. 9). Molecules with the
SecPrP orientation have been fully translocated into the ER
lumen and conjugated to a GPI anchor that serves as their sole means of
attachment to the lipid bilayer. This is the topology that is
presumably displayed by the majority of molecules present on the
surface of cells. In contrast, NtmPrP and
CtmPrP span the lipid bilayer once via the central
hydrophobic region (residues 111-134), with either the N terminus or C
terminus, respectively, in the ER lumen. CtmPrP also
acquires a C-terminal GPI anchor, and thus displays an unusual, dual
mode of membrane attachment. We find that several mutations within or
near the central, hydrophobic region increase the amount of
CtmPrP, but pathogenic mutations outside of this region
have no effect. The results presented here confirm and significantly
extend previous reports on the membrane topology of PrP (12, 22).
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-sheet (6, 7).
There are several biochemical properties that distinguish
PrPSc from PrPC, the most prominent being
protease resistance. After treatment with proteinase K (PK),
PrPSc is cleaved near amino acid 90 to yield a
protease-resistant core fragment known as PrP 27-30, while under the
same conditions PrPC is completely degraded (8).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Val at 112, 114, and 117), A116V, D177N, V179I, F197S, E199K, and V209I. Plasmids
were linearized with XbaI and gel-purified. In
vitro transcriptions were performed with the mMessage mMachine T7
kit (Ambion, Austin, TX).
-minimal
essential medium supplemented with 10% fetal calf serum, nonessential
amino acids, and penicillin/streptomycin. CHO cells were grown in
-minimal essential medium supplemented with 7.5% fetal calf serum
and penicillin/streptomycin. Transfections were performed with
LipofectAMINE (Life Technologies, Inc.) according to the
manufacturer's instructions. Cells were harvested 24 h after
transfection by brief trypsinization or by mechanical detachment,
rinsed twice with PBS, and resuspended in 250 µl of 0.25 M sucrose, 10 mM HEPES (pH 7.4), 1 µg/ml
pepstatin A, and 1 µg/ml leupeptin. After 5 min on ice, cells were
lysed by 10 passages through silastic tubing (0.3 mm, inner diameter) connecting two syringes with 27-gauge needles (34). A post-nuclear supernatant was prepared by centrifugation at 5,000 × g for 2 min. PK protection assays were performed by
incubating post-nuclear supernatants in 50 mM Tris-HCl (pH
7.5), 250 µg/ml PK, and in some cases 0.5% Triton X-100. After 60 min at 22 °C, digestion was terminated by addition of 5 mM phenylmethylsulfonyl fluoride. Samples were
deglycosylated with peptide:N-glycosidase F prior to
analysis by Western blotting.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Microsomes from canine pancreas and murine
thymoma cells differ in their efficiency of GPI anchoring.
Messenger RNA encoding wild-type PrP was translated in vitro
using rabbit reticulocyte lysate supplemented with the indicated
microsome preparation. Translation reactions were then lysed in 1%
Triton X-114 at 4 °C and were phase-separated by warming to
37 °C. One half of the detergent phase was incubated with PIPLC at
4 °C for 2 h (lanes 3, 4,
7, and 8), and the other half was incubated in
the absence of PIPLC (lanes 1, 2,
5, 6). The phase separation was repeated, and
proteins in the detergent phase (D lanes) and
aqueous phase (A lanes) were analyzed by SDS-PAGE
and autoradiography. The open and filled
arrowheads to the right of the gel indicate the
positions of glycosylated and unglycosylated PrP, respectively.
Molecular size markers are given in kilodaltons.
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Fig. 2.
Mutations in the hydrophobic region increase
the proportion of two transmembrane forms of PrP. Messenger RNA
encoding WT, 3AV, or A116V PrP was translated in rabbit reticulocyte
lysate supplemented with microsomes from canine pancreas (A)
or murine thymoma cells (B). Aliquots were then incubated
with (lanes 2, 3, 5,
6, 8, and 9) or without
(lanes 1, 4, and 7) PK in
the presence (lanes 3, 6, and
9) or absence (lanes 1, 2,
4, 5, 7, and 8) of Triton
X-100 (Det). PrP was then immunoprecipitated with 3F4
antibody, enzymatically deglycosylated, and analyzed by SDS-PAGE and
autoradiography. The positions of full-length SecPrP and
the protease-protected fragments of CtmPrP and
NtmPrP (seen in lanes 2,
5, and 8) are indicated by arrowheads
to the right of the gels.
valine mutation at residues 112, 114, and 117 that is not found in
human PrP but that produces neurological disease in transgenic mice
(12). The effect of the A116V mutation was weaker than that of the 3AV
mutation.
Proportions of three topological forms of PrP produced by in vitro
translation
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Fig. 3.
Epitope mapping of NtmPrP and
CtmPrP. A, messenger RNA encoding WT or 3AV
PrP was translated in rabbit reticulocyte lysate supplemented with
microsomes from murine thymoma cells. Aliquots were then incubated with
(lanes 1, 3-5, and 7-10)
or without (lanes 2 and 6) PK in the
presence (lanes 1 and 10) or absence
(lanes 2-9) of Triton X-100 (Det).
PrP was then immunoprecipitated with the indicated antibody,
enzymatically deglycosylated, and analyzed by SDS-PAGE and
autoradiography. The positions of protease-protected products derived
from SecPrP, CtmPrP, and NtmPrP are
indicated by arrowheads to the right of the gel.
B, schematic of the postulated structures of full-length
SecPrP, and the protease-protected fragments of
NtmPrP and CtmPrP. The molecular sizes given in
parentheses correspond to the polypeptides without
oligosaccharides. The epitopes recognized by each antibody are shown.
The black segment represents the transmembrane
domain. The positions of the C terminus of NtmPrP and the N
terminus of CtmPrP are approximate.
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Fig. 4.
CtmPrP has a GPI anchor.
Messenger RNA encoding WT or 3AV PrP was translated in reticulocyte
lysate supplemented with murine thymoma microsomes. After treatment
with PK, translation reactions were solubilized in 1% Triton X-114 at
4 °C and were then phase-separated by warming to 37 °C. One half
of the detergent phase was incubated with PIPLC at 4 °C for 2 h
(lanes 3, 4, 7, and
8), and the other half was incubated in the absence of PIPLC
(lanes 1, 2, 5, and
6). The phase separation was repeated, and PrP in the
detergent phase (D lanes) and aqueous phase
(A lanes) was immunoprecipitated with 3F4
antibody and analyzed by SDS-PAGE and autoradiography. The positions of
protected products derived from SecPrP and
CtmPrP are indicated by the arrowheads to the
right of the gel. The NtmPrP product is present
in only small amounts and is not seen on this gel.
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Fig. 5.
Mutations outside of the hydrophobic domain
do not alter the amount of transmembrane PrP. A,
messenger RNA encoding WT or mutant PrP was translated in reticulocyte
lysate supplemented with murine thymoma microsomes. Samples were then
incubated with (+ PK lanes) or without ( PK lanes) proteinase K in the presence (+ Det lanes) or absence (
Det
lanes) of Triton X-100. Samples were then analyzed by
SDS-PAGE and autoradiography without immunoprecipitation of PrP. The
SecPrP band is indicated by open
arrowhead, and the CtmPrP band by the
filled arrowhead. On the film exposures shown
here, the CtmPrP fragment is visible only for KH-II, 3AV,
and A116V PrP; however, small amounts of this fragment can be detected
above background levels using the PhosphorImager (see panel B). The NtmPrP product is not detectable
because its migration is obscured by globin. The human homologues of
the mutations are associated with the following familial prion
diseases: Creutzfeldt-Jakob disease (PG11, PG14, V179I, E199K, V209I),
Gerstmann-Sträussler syndrome (A116V, F197S), and fatal familial
insomnia (D177N). B, translations were carried out with
either thymoma or pancreatic microsomes as in panel
A, and the CtmPrP and SecPrP
fragments produced after PK digestion were quantitated by
PhosphorImager analysis of SDS-PAGE gels. The percentage of
CtmPrP was expressed as
CtmPrP/(CtmPrP + SecPrP) × 100. NtmPrP was not included in this calculation because it
cannot be visualized without first immunoprecipitating the samples to
eliminate contaminating globin, which interferes with the migration of
low Mr species. The horizontal
dashed lines indicate the amount of
CtmPrP observed for wild-type PrP. Each bar
represents the mean ± S.E. of 2-15 replicates. Values that are
significantly different (p < 0.05) from wild-type PrP
are indicated by an asterisk.
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Fig. 6.
Only small amounts of CtmPrP are
produced in transfected cells. BHK cells were transiently
transfected with plasmids encoding WT or mutant PrPs. Post-nuclear
supernatants prepared from cells 24 h after transfection were
incubated with (+ PK lanes) or without ( PK lanes) proteinase K in the presence (+ Det lanes) or absence (
Det
lanes) of Triton X-100. Proteins were then solubilized in
SDS, enzymatically deglycosylated, and subjected to Western blotting
with 3F4 antibody. The SecPrP band is indicated by an
open arrowhead, and the CtmPrP band
(seen for 3AV and A116V) by the filled arrowhead.
The bands indicated by the bracket for PG14 PrP represent
nonspecific cleavage products produced by cellular proteases. In each
experiment, samples were also Western-blotted with an antibody to
calnexin to confirm the integrity of the ER membranes. A representative
calnexin blot from an experiment on cells expressing wild-type PrP is
shown in the lower right panel.
Full-length calnexin migrates at 90 kDa (
PK/
Det lane). The 70-kDa band (+ PK/
Det lane) represents the transmembrane and
lumenal domains of calnexin, which are protected from protease
digestion. The 65-kDa band (+ PK/+ Det
lane) is an intrinsically protease-resistant fragment of
calnexin (40).
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Fig. 7.
CtmPrP is not detectable in the
brains of Tg(WT) or Tg(PG14) mice. Brain microsomal membranes were
incubated with (lanes 2, 3,
5, and 6) or without (lanes
1 and 4) PK in the presence (lanes
3 and 6) or absence (lanes
1, 2, 4, and 5) of Triton
X-100 (Det). Proteins were then solubilized in SDS,
enzymatically deglycosylated, and subjected to Western blotting with
3F4 antibody. The SecPrP band is indicated by an
open arrowhead; no CtmPrP fragment
(19 kDa) is visible in lanes 2 and 5.
The bands marked by a single asterisk represent
PrP that has not been completely deglycosylated. Samples were also
Western-blotted with an antibody to calnexin to confirm the integrity
of the ER membranes. Microsomes from the Tg(PG14) mouse contain
variable amounts of the 70-kDa protected fragment of calnexin even in
the absence of PK treatment (lane 4,
double asterisk), presumably due to endogenous
protease activity. The Tg(PG14) mouse used for this experiment was
terminally ill.
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Fig. 8.
3AV PrP, but not PG14 PrP, can be released
from the cell surface by PIPLC. CHO cells were transiently
transfected with plasmids encoding WT, 3AV, or PG14 PrP. Twenty-four
hrs after transfection, cells were surface-biotinylated and then
treated with PIPLC for 2 h at 4 °C. PrP was immunoprecipitated
from PIPLC incubation media (M lanes) and cell
lysates (C lanes) after enzymatic deglycosylation
and was subjected to Western blotting with 3F4 antibody. Blots were
developed with horseradish peroxidase-streptavidin to identify
biotinylated PrP molecules.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 9.
Models of the three topological forms of
PrP. NtmPrP is presumed to lack a GPI anchor, although
we do not have direct experimental evidence for this.
All previous studies of PrP membrane topology by in vitro translation utilized canine pancreatic microsomes (12, 17-22), which we show here do not efficiently attach GPI anchors to newly synthesized polypeptide chains. GPI anchor addition occurs in the ER soon after completion of the polypeptide chain, and involves a transamidation reaction in which a hydrophobic, C-terminal sequence is cleaved and replaced by the pre-formed anchor structure (41). Presumably, the transamidase or other enzymes involved in GPI synthesis or addition are relatively inactive in microsomes derived from canine pancreas, or else endogenous phospholipases are more active. In our experiments, we have employed microsomes prepared from BW5147.3 mouse thymoma cells, which attach GPI anchors to the majority of PrP chains. Using this system, we find that both SecPrP as well as CtmPrP become GPI-anchored. The demonstration of a GPI anchor on CtmPrP is a novel finding and indicates that this species is attached to the membrane by both a bilayer-spanning segment as well as a C-terminal GPI structure that is covalently linked to the outer leaflet of the bilayer. The existence of such a dual mechanism of membrane attachment is unusual, although several other proteins have been proposed to adopt such a topology (42-44).
We and others have previously observed that PrP molecules carrying pathogenic mutations are resistant to release from the cell surface by PIPLC (14, 23, 45-47). The presence of a dual membrane anchor on the mutant proteins does not explain this phenomenon, since most mutations do not increase the amount of CtmPrP. In fact, PrP molecules carrying the 3AV mutation, which enhances formation of CtmPrP, are fully releasable by PIPLC, suggesting that SecPrP is the major species on the cell surface. Presumably, this is because the amount of CtmPrP that is present in cells is small in comparison to the amount of SecPrP, or because CtmPrP does not reach the cell surface. Our previous studies indicate that the most likely explanation for the PIPLC resistance of mutant PrPs is that the GPI anchors of these molecules are physically inaccessible to the phospholipase, possibly as a result of the conformational change that accompanies conversion to the PrPSc state (45).
It had been previously reported that several mutations in or around the
central, hydrophobic region increase the amount of CtmPrP
produced in translation reactions in vitro or in brain
tissue (12, 22), but the effect of mutations outside of this area had
not been examined. The previously studied mutations included one
(A116V) whose human homologue (A117V) is associated with
Gerstmann-Sträussler syndrome (38, 39), and three (N107I,
K109I/H110I, and 3AV) that are artificial and have not been described
in humans. The data reported here confirm that these mutations increase
the amount of CtmPrP. A plausible explanation for this
effect is that the amino acid changes alter the hydrophobicity,
-helical structure, or some other physical property of the central
region in such a way as to make it a more suitable transmembrane
segment. Two other artificial mutations in the central region of the
molecule have been found to have the opposite effect, eliminating
formation of NtmPrP and CtmPrP and causing
exclusive production of SecPrP (12, 22). One of these
(G122P) is likely to disrupt the formation of a transmembrane
-helix, and the other (
103-111) eliminates a segment of
hydrophilic amino acids (designated "stop transfer effector") that
strongly influences the topology of the adjacent transmembrane segment
(19, 20). Taken together, the available data indicate that the central
region of the PrP molecule acts as a crucial determinant of membrane
topology, presumably in conjunction with the N-terminal signal
sequence, but that other sections of the molecule, including those
where many pathogenic mutations lie, are topologically inert. Exactly
how the topology-determining domains of PrP function at the level of
the ER translocon, and how the final topology of the protein is
regulated will require further investigation (15, 16).
Our observation that most pathogenic mutations do not affect CtmPrP production has important implications for the role of this transmembrane species in the pathogenesis of prion diseases. Hegde et al. (22) have hypothesized that the generation of CtmPrP represents a final common pathway for neurodegeneration in both transmissible and familial forms of prion disease. This idea is based upon their observations that transgenic mice expressing CtmPrP-favoring mutations at high levels develop a spontaneous neurodegenerative illness in the absence of PrPSc, and that the amount of CtmPrP increases during the course of scrapie infection of mice expressing wild-type PrP. However, our observation that seven different pathogenic mutations fail to alter the amount of CtmPrP makes it unlikely that this species is an obligatory intermediate in all cases of prion-associated neurodegeneration. It has been speculated that some mutations like E200K indirectly enhance formation of CtmPrP by first causing accumulation of PrPSc (22). If this were the case, it might be argued that these mutations would not have an observable effect on CtmPrP levels in our experiments if PrPSc were not being produced. However, five of the pathogenic PrPs (PG11, PG14, D177N, F197S, and E199K) do in fact assume a protease-resistant form that resembles PrPSc when expressed in transfected CHO and BHK cells (2, 23),2 while three of mutants (A116V, V179I, and V209I) are protease-sensitive and display other properties characteristic of PrPC.3 This comparison makes it clear that there is no correlation between the efficiency with which a mutation promotes conversion to a PrPSc-like state in cell culture and its potency in inducing CtmPrP. A similar pattern is seen in vivo, since the human homologues of some of the mutations we have examined are associated with infectious PrPSc in the brains of patients while others are not (4). These results argue against the proposition that PrPSc causes disease by enhancing formation of CtmPrP. In cases where neither PrPSc nor CtmPrP are present, it seems likely that another form of PrP is the primary pathogenic entity.
Whether CtmPrP is an irrelevant byproduct of PrP
biogenesis, or whether it plays some more restricted role in the
disease process remains to be determined. One possibility is that
CtmPrP is responsible for a subset of inherited prion
diseases due to mutations within or near the transmembrane segment.
Alternatively, when CtmPrP is present, it may accelerate or
enhance neurodegeneration triggered by PrPSc. To evaluate
these and other hypotheses, it will be crucial to understand more about
the cell biology of transmembrane forms of PrP. We have observed that
the amount of CtmPrP produced in transfected cells is
considerably less than after in vitro translation. This may
reflect differences in the efficiency of transmembrane PrP synthesis
between the in vitro and in vivo situations, or,
alternatively, it may be due to more rapid degradation of transmembrane
PrP in cells. It is also possible that neuronal cells are more
efficient at synthesizing CtmPrP than the BHK and CHO cells
we have used here. It will be important now to analyze how
transmembrane forms of PrP are generated during translation, where they
are localized at the subcellular level, and how they are metabolized,
to shed further light on the role of these unique species in the
biology of prion diseases.
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ACKNOWLEDGEMENTS |
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We thank Richard Kascsak and Byron Caughey for their generous gifts of antibodies and Mike Mueckler for samples of canine pancreatic microsomes.
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Note Added in Proof |
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We find that the P101L mutation, whose human homologue is associated with Gerstmann-Sträussler syndrome, does not alter the amount of CtmPrP produced by in vitro translation performed as in Fig. 5A.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant R01 NS35496.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by National Institutes of Health Training Grant T32 NS07129.
§ 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: 314-747-0940; E-mail: dharris@cellbio.wustl.edu.
Published, JBC Papers in Press, October 25, 2000, DOI 10.1074/jbc.M006763200
2 R. S. Stewart and D. A. Harris, unpublished results.
3 R. S. Stewart and D. A. Harris, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: PrPSc, scrapie isoform of prion protein; ER, endoplasmic reticulum; GPI, glycosyl phosphatidylinositol; PIPLC, phosphatidylinositol-specific phospholipase C; PK, proteinase K; PrP, prion protein; PrPC, cellular isoform of prion protein; CtmPrP, C-terminal transmembrane form of prion protein; NtmPrP, N-terminal transmembrane form of prion protein; SecPrP, secretory form of prion protein; PBS, phosphate-buffered saline; BHK, baby hamster kidney; CHO, Chinese hamster ovary; WT, wild-type; PAGE, polyacrylamide gel electrophoresis.
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