From the Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, NIAID, National Institutes of Health, Hamilton, Montana 59840
Received for publication, October 22, 2002, and in revised form, January 8, 2003
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ABSTRACT |
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Prion protein (PrP) is usually bound to membranes
by a glycosylphosphatidylinositol (GPI) anchor that associates with
detergent-resistant membranes, or rafts. To examine the effect of
membrane association on the interaction between the normal
protease-sensitive PrP isoform (PrP-sen) and the protease-resistant
isoform (PrP-res), a model system was employed using PrP-sen
reconstituted into sphingolipid-cholesterol-rich raft-like liposomes
(SCRLs). Both full-length (GPI+) and GPI
anchor-deficient (GPI Prion protein (PrP)1 is
a glycoprotein usually bound to membranes by a
glycosylphosphatidylinositol (GPI) anchor (1). Like other
GPI-anchored proteins, PrP is enriched in sphingolipid- and
cholesterol-rich membrane microdomains known as detergent-resistant membranes (DRMs), or rafts (2). Several lines of evidence from biochemical and molecular biological approaches suggest raft
association is required for conversion of the normal protease-sensitive
isoform (PrP-sen) to the transmissible spongiform
encephalopathy-associated protease-resistant isoform (PrP-res) in a
cell culture model of infection (2-7). Although cell-free studies
using purified PrP molecules have provided new insights into binding
and conversion of PrP-sen by PrP-res (reviewed in Ref. 8), few studies
have considered the membrane-associated nature of PrP (9-13) and the influence of this association on PrP-sen/PrP-res interactions (14).
Given the complex composition of cellular raft membranes, which contain
molecules other than PrP-sen that might influence interactions with
PrP-res, investigations into the effect of PrP-sen association with
rafts on these interactions would benefit from the use of a defined
system that replicated raft membranes in the absence of other
raft-associated molecules. One candidate system involves the use of
sphingolipid-cholesterol-rich raft-like liposomes (SCRLs) containing
phosphatidylcholine, sphingomyelin, brain cerebrosides, and
cholesterol. SCRLs have been shown to resemble rafts in several
respects, including major lipid composition and low buoyant density,
which permits their isolation by floatation through density gradients
(15). GPI-anchored proteins reconstituted into SCRLs or related
sphingolipid-rich liposomes acquire properties of their cell-associated
counterparts, most notably insolubility in cold Triton X-100 (15, 16).
Hence, SCRLs serve as a reasonable approximation of the natural
membrane environment of PrP in the absence of other raft-associated molecules.
Our previous work examined the effect of PrP-sen association with rafts
on interactions with PrP-res using raft membranes prepared from
neuroblastoma cells (14). These experiments showed that raft-bound
PrP-sen resisted conversion to PrP-res until PrP-sen was released from
rafts by phospholipase digestion or the PrP-res was inserted into
contiguous membranes. To examine the effect of PrP-sen membrane
association on its interactions with PrP-res under more defined
conditions and to determine if membrane association itself inhibits
conversion of PrP-sen by exogenous PrP-res, we have employed a model
system using PrP-sen reconstituted into SCRLs. While developing this
system, two groups recently reported a novel property of recombinant
PrP-sen expressed in Escherichia coli: binding to model
membranes of various compositions (10, 13). We had also observed this
phenomenon in our system using GPI anchor-deficient PrP-sen expressed
in mammalian cell lines and have further characterized the nature of
this binding activity using various forms of PrP-sen that may more
closely represent the native state of the molecule, particularly with
respect to the addition of N-linked glycans. Furthermore, we
have directly tested the effect of the two methods of PrP-sen
association with membranes (i.e. GPI
anchor-dependent and -independent) on its interactions with
exogenous PrP-res molecules as assayed by its ability to serve as a
substrate for conversion to the protease-resistant state under
cell-free conditions. Our results indicate that the method of PrP-sen
association with model membranes has strikingly different effects on
its ability to interact with PrP-res.
Cells and Purification of 35S-Labeled
PrP-sen--
Hamster PrP-sen was derived from mouse fibroblast cell
lines expressing either full-length (GPI+ PrP-sen) or GPI
anchor-deficient (GPI Preparation of Liposomes--
Sphingolipid-cholesterol-rich
liposomes (SCRLs) were prepared essentially as described previously
(15) with exceptions as noted below. All lipids were obtained from
Avanti Polar Lipids. SCRLs contained bovine liver phosphatidylcholine
(PC)/brain sphingomyelin (SM)/brain cerebrosides (CB)/cholesterol
(Chol) in a 2:1:1:2 molar ratio. For some experiments, modified SCRLs
were prepared consisting of PC/CB/Chol (1:1:1), PC/SM/Chol (1:1:1),
PC/SM/CB (2:1:1), SM/CB/Chol (1:1:2), or PC/Chol (1:1). For each type
of modified SCRL, the total moles of lipid used in the preparation as
well as the molar ratio of phospholipid:sphingolipid:cholesterol was
matched to that contained in SCRLs where possible. SCRLs were usually
prepared in citrate-buffered saline (CBS, 10 mM citrate,
137 mM NaCl, pH 6.0) except for the experiments with
modified SCRLs (Tricine-buffered saline, pH 7.8) and the reconstitution
experiments to evaluate the effect of pH where saline buffered with
acetate (pH 5.0), phosphate (pH 7.0), or Tricine (pH 7.8) was also
used. After dialysis, SCRLs were either used for reconstitution or
binding studies as outlined below.
Reconstitution of [35S]PrP-sen into
SCRLs--
PrP-sen was reconstituted into SCRLs using a protocol
adapted from Schroeder et al. (15).
[35S]PrP-sen was adjusted to contain 137 mM
NaCl and 0.8% Sarkosyl just before addition to SCRLs that had
been freeze-thawed three times. The samples were rapidly mixed and
dialyzed immediately in Slide-a-lyzer cassettes (Pierce) against the
buffer in which the SCRLs were prepared (usually CBS). The cassettes
were manually agitated approximately hourly for the first few hours of
dialysis to keep the liposomes well dispersed. The final concentration of Sarkosyl in the initial PrP-sen/SCRL mixture was
Small scale preparations (scaled down 1/10 for amount of PrP-sen and
lipids) were used to evaluate the effect of lipid composition and pH on
incorporation efficiency. Small scale gradients for analytical purposes
contained 30 µl (for five steps) or 50 µl (for three steps) per
step for the overlaid fraction and were centrifuged in a Beckman TLS-55
rotor at 25,000 rpm for 90 min at 4 °C in polycarbonate tubes. Six
fractions were collected from these gradients starting from the top:
five gradient fractions of 30 µl each and one fraction corresponding
to the bottom (i.e. the volume of sample loaded in 10%
Optiprep). For five-step gradients, the lipid band formed at the
1%/2.5% Optiprep interface and the majority of the lipid was
collected in fractions 1 and 2. For three-step gradients, the lipid
band formed at the 1%/4% interface and the majority of the lipid was
collected in fraction 2. The fractions were mixed with 20 µg of
thyroglobulin, and proteins were precipitated with 4 volumes of cold
methanol. After centrifugation at 21,000 × g for 20 min, methanol pellets were resuspended in SDS-PAGE sample buffer,
boiled for 5 min, and analyzed on Novex pre-cast acrylamide gels.
Radioactive proteins were visualized and quantitated using a Storm
PhosphorImager instrument (Amersham Biosciences).
Binding of [35S]PrP-sen and PI-PLC-released
Proteins to SCRLs in the Absence of Detergent--
SCRLs were first
washed in CBS and concentrated by centrifugation as described above.
For experiments for testing the pH dependence of the binding, SCRLs
were washed and resuspended in buffered saline of the appropriate pH
(i.e. acetate (pH 5.0), citrate (pH 6.0), HEPES (pH 7.0), or
Tricine (pH 7.8) each at 10 mM). To bind PrP-sen to SCRLs
in the absence of detergent, [35S]PrP-sen (10,000-15,000
cpm or ~1-2 ng) was added to SCRLs (~67 µg of lipid) in binding
buffer consisting of a final concentration of 50 mM buffer
of appropriate pH (usually citrate), 137 mM NaCl. To bind
35S-labeled PI-PLC-released/secreted proteins to SCRLs in
the absence of detergent, PI-PLC culture supernatant (1/25 to 1/50
equivalents released from a T-25 flask of cells) was mixed with SCRLs
(~67 µg of lipid) in 50 mM CBS (final concentration).
Binding reactions were incubated at 37 °C for 1-2 h unless
indicated otherwise and were mixed at ~15-min intervals to keep the
liposomes well dispersed. The reactions were then chilled on ice,
adjusted to 10% Optiprep, and processed on small scale analytical
gradients as described above. In some cases, binding was quantitated by
liquid scintillation counting of the gradient fractions and included
boiling the empty centrifuge tube in a volume of SDS-PAGE sample buffer
(30 µl) equal to the volume of the "bottom" fraction to ensure
recovery of any residual protein. These results are expressed as the
mean percentage of counts recovered in the low density fractions (1-4) versus total counts recovered (sum of the six gradient
fractions and protein recovered after boiling the empty tube in
SDS-PAGE sample buffer). Where indicated, gradient fractions were
deglycosylated with PNGase F prior to methanol precipitation or
immunoprecipitation with 3F4 antibody and SDS-PAGE as above. Binding
reactions were scaled up to contain ~200 ng of
[35S]GPI Treatments to Extract SCRL-bound Proteins from
SCRLs--
Protein-containing SCRLs were harvested from Optiprep
gradients, washed in buffered saline, and centrifuged in an
ultracentrifuge as described above to recover the SCRLs. Liposome
pellets were usually resuspended directly in extraction buffer
consisting of either 0.1 M sodium carbonate (pH 11.5), 1 or
3 M NaCl in 10 mM citrate buffer (pH 6.0), or
1% Triton X-100 in CBS. In some cases, an SCRL pellet was resuspended
in CBS and mixed with one volume of 2% Triton X-100 in CBS or 2 M NaCl in citrate buffer. Extractions with NaCl or sodium
carbonate were incubated on ice for 30-60 min. Extractions with Triton
X-100 were either incubated on ice or at 37 °C for 20 min. Samples
were then adjusted to contain 10% Optiprep and processed on small
scale analytical gradients as above.
Cell-free Conversion Reactions--
Cell-free conversion
reactions were performed essentially as described previously in 50 mM citrate (pH 6.0), 137 mM NaCl, and 5 mM MgCl2 (conversion buffer) supplemented with
100 µg/ml heparan sulfate and protease inhibitors (0.1 mM
phenylmethylsulfonyl fluoride, 0.7 µg/ml pepstatin A, 1 µg/ml
aprotinin) with exceptions noted below (14). Crude brain microsomes
were prepared either from mice infected with 87V mouse-adapted scrapie
or hamsters infected with 263K hamster-adapted scrapie and used as a
source of PrP-res (14). Microsomes from normal uninfected animals were used as a control. [35S]PrP-sen-containing SCRLs were
washed and resuspended in CBS prior to addition to conversion reactions
(~10,000-15,000 cpm/reaction). Reactions containing the different
forms of GPI Immunoblotting--
Immunoblot detection of PrP was performed as
described by Horiuchi et al. (24) using a mouse/human
recombinant monoclonal antibody Fab (R1, 0.36 µg/ml) that binds
hamster PrP between residues 220 and 231 (25) and was a generous gift
from Drs. Anthony Williamson and Dennis Burton (The Scripps Research
Institute). Bound R1 antibody was detected using alkaline
phosphatase-conjugated goat anti-human IgG secondary antibody (Sigma
(A8542)) at a 1:10000 dilution.
PrP-sen Reconstitution into SCRLs--
We adapted a previously
described technique (15) to allow reconstitution of PrP-sen into SCRLs.
Hamster PrP-sen was expressed in fibroblasts as either full-length
GPI-anchored protein (GPI+) or a GPI anchor-deficient
(GPI PrP-sen Associates with SCRLs via Hydrophobic
Interactions--
The nature of the binding interactions of the
PrP-sen molecules with SCRLs was characterized by subjecting the
PrP-bound SCRLs to various extraction conditions. Treatments with
either high salt (1 M NaCl (Fig.
3A) or 3 M NaCl
(data not shown)) or 0.1 M sodium carbonate (pH 11.5) (Fig.
3B), which extract peripheral membrane proteins, failed to
remove either type of PrP-sen molecule from SCRLs. A significant
fraction (~40-50%) of GPI+ PrP-sen resisted extraction
with Triton X-100 at 4 °C (Fig. 3C, lanes 1 and 2 versus lane 6) but not 37 °C
(Fig. 3D, lanes 1 and 2 versus lane 6), resembling the behavior of
GPI-anchored proteins in rafts (14, 26). Treatment of the cold-Triton
X-100-extracted SCRLs with PI-PLC to cleave the GPI anchors caused the
release of a majority (~60%) of the formerly GPI-anchored PrP-sen
molecules from the SCRLs upon re-extraction with cold Triton (data not
shown). However, GPI
Because others have reported various factors affecting the interaction
of PrP-sen with membranes using model systems different from ours, we
examined the effect of some of these factors on the binding observed in
our system. Given evidence PrP may exhibit preferential binding to
specific lipids (12, 27), we tested the incorporation efficiency of
PrP-sen into modified SCRLs. No specific interactions with any SCRL
lipids were detected as both types of PrP-sen incorporated similarly
into modified SCRLs lacking any of the individual lipid components or
sphingolipids (i.e. PC/Chol liposomes) (data not shown).
Other investigators have shown a pH-dependent binding of
recombinant PrP-sen to liposomes (10, 13). We determined the
incorporation efficiency over a range of pH in our system. Association
of both GPI+ and GPI PrP-sen Binds SCRLs in the Absence of Detergent--
Given the
reconstitution protocol involved the incubation of PrP-sen with SCRLs
in the presence of a low concentration of detergent, it was possible
that the GPI anchor-independent binding was mediated by a minor,
transient perturbation of the liposomes by the detergent. To address
this issue, binding studies were conducted by directly mixing
GPI
On the contrary, the transient Sarkosyl treatment had a clear effect on
the nature of GPI+ PrP-sen association with SCRLs. When
incubated with SCRLs in the absence of Sarkosyl, GPI+
PrP-sen efficiently bound to the SCRLs but, similar to the behavior of
GPI Glycosylated, GPI Anchor-deficient PrP-sen Binds to SCRLs--
The
GPI Residues from the N-terminal Half of PrP Contribute to
GPI SCRL Binding Is Specific to PrP-sen and a Subset of
PI-PLC-released/secreted Proteins--
To determine if GPI
anchor-independent membrane binding might be an activity common among
GPI-anchored proteins, we incubated SCRLs with culture supernatants
from PI-PLC-treated fibroblast cells expressing GPI+
PrP-sen to simultaneously assay the complement of (formerly) GPI-anchored proteins for SCRL-binding activity. Only a small subset of
PI-PLC-released proteins and proteins secreted during the PI-PLC
treatment period were found to bind SCRLs (Fig.
7A, fractions
1-3), an observation more easily visualized for gradient fractions deglycosylated with PNGase F prior to SDS-PAGE (Fig. 7B, fractions 1-3). Interestingly, one of these
proteins (indicated by a bracket in Fig. 7A and
by an arrow in Fig. 7B) had an apparent molecular
mass and glycoform profile (as revealed by PNGase F digestion) similar
to PrP-sen. By immunoprecipitation from the gradient fractions with
anti-PrP monoclonal antibody (3F4), one of the proteins was identified
as PrP-sen (Fig. 7C). We also confirmed that the
PI-PLC-released proteins were binding to the SCRLs in a fashion similar
to purified PrP-sen by concentrating the combined protein-containing
SCRL fractions by centrifugation, extracting the samples with cold
Triton X-100 or 1 M NaCl, and re-floating the samples on a
new density gradient. As with purified PrP-sen, the bound proteins were
largely removed by treatment with cold Triton X-100 (Fig.
7D) but not high salt (data not shown). No evidence of SCRL
binding of proteins corresponding to PrP-sen was observed when PI-PLC
culture supernatants from cells expressing the Cell-free Conversion of SCRL-associated GPI+ PrP-sen by
Exogenous PrP-res--
To measure the effect of the different types of
membrane association on the interaction of PrP-sen with exogenous
PrP-res, we used a previously described near-physiological cell-free
conversion assay (14) containing a crude brain microsome fraction from 263K scrapie-infected hamsters as a source of PrP-res. This assay is
capable of generating new protease-resistant PrP exhibiting the ~6-
to 8-kDa decrease in apparent molecular mass after PK digestion that is
characteristic of PrP-res synthesized in vivo. The fate of
the input 35S-labeled PrP-sen in the reactions is
specifically monitored by autoradiography. For reactions using
GPI+ PrP-sen, the PrP-sen was reconstituted into the SCRLs
in the presence of Sarkosyl followed by extraction with cold Triton
X-100 to ensure that the PrP-sen was associated with the SCRLs in a GPI
anchor-dependent manner. As observed with DRMs prepared
from a mouse PrP-overexpressing neuroblastoma cell line (14),
SCRL-bound GPI+ PrP-sen was not converted to PrP-res until
PI-PLC, which cleaves the GPI anchor of PrP-sen but not PrP-res
molecules (1, 29-30), was added to the reaction (Fig.
8A, lane 10), or
the combined membrane fractions were treated with a high concentration
(30%) of the membrane-fusing agent polyethylene glycol (PEG) (Fig.
8A, lane 9). Reactions assisted by PI-PLC often
exhibited a higher conversion efficiency than reactions with 30% PEG
(27% versus 9.1% conversion for the experiment shown in
Fig. 8A), possibly due to a higher efficiency of PI-PLC
cleavage than PEG-assisted membrane fusion. When compared with
PEG-assisted reactions, the PI-PLC-assisted conversion products
exhibited a slightly reduced electrophoretic mobility (Fig.
8A, lane 10 versus lane 9),
suggesting the PI-PLC-assisted converting species lacked a GPI anchor
(14, 29, 31). A similar requirement for 30% PEG or PI-PLC was observed in reactions using SCRL-reconstituted mouse GPI+ PrP-sen
and brain microsomes from a mouse infected with the murine-adapted scrapie strain 87V (Fig. 8B, lanes 7 and
8). These data show that SCRL-incorporated GPI+
PrP-sen behaves similarly to GPI+ PrP-sen in cellular raft
membranes in being resistant to conversion by exogenous PrP-res
molecules without the assistance of PI-PLC or a membrane-fusogenic
concentration of PEG (14, 32, 33). Our data also show that the
requirements for conversion of membrane-associated GPI+
PrP-sen are conserved between PrP-res molecules associated with two
different strains of scrapie agent, adapted to either hamsters (263K)
or mice (87V).
Cell-free Conversion of SCRL-associated GPI We have examined the interaction of PrP-sen with model raft
liposomes and described two modes by which this association can occur.
The first is mediated by insertion of the GPI anchor of PrP-sen into
the liposome membranes after reconstitution in the presence of
detergent (Fig. 10B). The
second is a newly described method that does not require a GPI anchor
(Fig. 10A) but can also occur when GPI+
PrP-sen is incubated with SCRLs in the absence of detergent (Fig. 10C) and may provide a dual mode of membrane attachment for
GPI+ PrP-sen (not depicted).
) PrP-sen produced in fibroblasts
stably associated with SCRLs. The latter, alternative mode of membrane
association was not detectably altered by glycosylation and was
markedly reduced by deletion of residues 34-94. The SCRL-associated
PrP molecules were not removed by treatments with either high salt or
carbonate buffer. However, only GPI+ PrP-sen resisted
extraction with cold Triton X-100. PrP-sen association with SCRLs was
pH-independent. PrP-sen was also one of a small subset of
phosphatidylinositol-specific phospholipase C (PI-PLC)-released proteins from fibroblast cells found to bind SCRLs. A cell-free conversion assay was used to measure the interaction of SCRL-bound PrP-sen with exogenous PrP-res as contained in microsomes. SCRL-bound GPI+ PrP-sen was not converted to PrP-res until PI-PLC was
added to the reaction or the combined membrane fractions were treated
with the membrane-fusing agent polyethylene glycol (PEG). In contrast, SCRL-bound GPI
PrP-sen was converted to PrP-res without
PI-PLC or PEG treatment. Thus, of the two forms of raft membrane
association by PrP-sen, only the GPI anchor-directed form resists
conversion induced by exogenous PrP-res.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
PrP-sen) wild-type (wt) PrP-sen
(17, 18). The corresponding N-terminal hamster PrP-sen deletion mutants
were derived from cell lines created by Lawson and co-workers (19).
Wild-type GPI+ mouse PrP-sen was isolated from a mouse
neuroblastoma cell line described elsewhere (20). PrP-sen molecules
were immunoprecipitated from cells metabolically labeled with
[35S]methionine as described previously (21). Mouse
PrP-sen was immunoprecipitated using rabbit antiserum (R30) against a
PrP synthetic peptide (amino acids 89-103) (22). Hamster
PrP-sen was immunoprecipitated with 3F4 monoclonal antibody (23) or rabbit polyclonal antiserum (R20) raised against a C-terminal (amino
acids 218-232) PrP synthetic peptide (22). The R20 antiserum was used specifically to isolate PrP-sen molecules (both wt and mutants) for experiments involving the N-terminal deletion mutants, because one mutant (
124) lacks the 3F4 epitope. Cell culture supernatants containing phosphatidylinositol-specific phospholipase C
(PI-PLC)-released proteins from metabolically labeled cells expressing
GPI+ PrP-sen were prepared as described previously (14).
Where indicated, PI-PLC-released hamster PrP-sen (referred to as
GPIPI-PLC PrP-sen) was immunoprecipitated from the
supernatants as described elsewhere with 3F4 or R20 (14). For some
experiments, PI-PLC culture supernatant proteins were deglycosylated
with PNGase F (New England BioLabs) as per the manufacturer's
instructions prior to PrP-sen immunoprecipitation.
0.08%. Large scale preparations usually contained about 200 ng of
[35S]PrP-sen (~1-2 × 106 cpm) and 2 mg (3.2 × 10
6 mol) of lipids. After overnight
dialysis, the PrP/liposome mixture was adjusted to 10% Optiprep
(Invitrogen) in the appropriate buffered saline. The sample was then
overlaid with a linear step gradient of Optiprep in buffered saline
consisting of either five steps (1%, 2.5%, 4%, 5.5%, and 7%) or
three steps (1%, 4%, and 7%). Large scale gradients contained 300 µl (for five steps) or 500 µl (for three steps) per step for the
overlaid fraction and were centrifuged in a Beckman SW50.1 rotor at
21,000 rpm for 90 min at 4 °C in polycarbonate tubes. To prepare
Triton X-100-treated SCRLs containing GPI+ PrP-sen, the
dialyzed PrP-sen/SCRL mixture was first diluted with 1 ml of CBS and
centrifuged in a Beckman SW50.1 rotor at 40,000 rpm for 60 min at
4 °C to pellet the liposomes. The pellet was then resuspended in 300 µl of 1% Triton X-100 in CBS and incubated for 20 min on ice prior
to adjustment to 10% Optiprep and centrifugation as above. After
centrifugation, the SCRL lipid band was collected and stored on ice
until use. Relative to the input cpm, typically ~40-60% of the
GPI
or GPI+ PrP-sen was associated with the
SCRLs (without Triton X-100 treatment) and ~20-30% (of
GPI+ PrP-sen) was recovered in the SCRL fractions after
Triton X-100 extraction.
PrP-sen and 2 mg of lipid and
fractionated on a 600-µl gradient (200 µl each of 1%, 4%, and 7%
Optiprep) in a TLS-55 rotor as above to prepare sufficient
PrP-containing SCRLs for use in cell-free conversion reactions.
PrP-sen were adjusted to contain equivalent
amounts of input radioactivity and simply involved mixing the
PrP-sen-containing SCRLs or purified PrP-sen with microsomes in
conversion buffer with heparan sulfate and protease inhibitors. Also,
the [35S]GPI
PrP-sen used in all these
reactions was derived from a single preparation of protein divided into
three parts: one part was added directly to the reactions (free
PrP-sen), a second part was used to prepare PrP-sen bound to SCRLs in
the presence of Sarkosyl, and the remaining portion was used to prepare
PrP-sen bound to SCRLs in the absence of Sarkosyl. Reactions using
GPI+ PrP-sen-containing SCRLs were set up as described for
[35S]DRMs in our previous study, although we modified our
protocol to include a dilution step (addition of 50 µl of conversion
buffer) after the 5-min incubation at room temperature with
polyethylene glycol (PEG) or conversion buffer to allow efficient
recovery of membranes from PEG-treated reactions in the subsequent
pelleting step (14). Conversion reactions were incubated at 37 °C
for either 2 days or 1 day (in the case of some of the
GPI
PrP-sen reactions) and processed as described
previously with incubation at 37 °C for 20 min in 50 mM
Tris-HCl (pH 8.0), 0.5% Triton X-100, 0.5% sodium deoxycholate, and
137 mM NaCl (1× extraction buffer) followed by
proteinase K (PK) digestion for 1 h with 33 µg/ml PK (14). In
some cases, the samples were deglycosylated after PK digestion but
before methanol precipitation by treatment with PNGase F (New
England BioLabs) as per the manufacturer's instructions.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
) form lacking the GPI anchor addition sequence.
Purified PrP-sen was mixed with SCRLs in the presence of a low
concentration (
0.08%) of Sarkosyl and immediately dialyzed against
buffered saline. SCRLs and SCRL-bound proteins were then isolated by
floatation through a density gradient. GPI+ PrP-sen stably
associated with SCRLs in the low density fractions (1-4) of the
gradient (Fig. 1A).
Surprisingly, control experiments with GPI
PrP-sen
revealed that this derivative also avidly associated with SCRLs (Fig.
1B). When fractionated on gradients in the absence of SCRLs,
all of the PrP-sen (both GPI+ and GPI
) was
found in the bottom fraction (fraction 6) indicating that floatation of
PrP-sen in the gradient is dependent upon SCRLs (data not shown). To
verify the SCRL association was not due to trapping of PrP-sen inside
the liposomes, we incubated the PrP-containing SCRLs with proteinase K
(PK). As shown in Fig. 2, the vast
majority (~90%) of SCRL-bound PrP-sen was susceptible to PK
digestion without detergent-mediated disruption of the liposomes,
indicating that the bulk of the protein was surface-accessible. These
data show that PrP-sen exhibits an alternative, GPI anchor-independent
method of associating with model membranes, a conclusion consistent
with other recent studies (10, 13).
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Fig. 1.
Reconstitution of PrP-sen into SCRLs.
GPI anchor-containing (A) or -deficient (B)
[35S]PrP-sen was mixed with SCRLs in the presence of
0.08% Sarkosyl followed by immediate dialysis. The dialyzed sample was
fractionated by floatation on five-step Optiprep gradients as described
under "Experimental Procedures." Fraction numbers for fractions
collected from the gradients are indicated above each lane.
Molecular mass markers are indicated in kDa on the left. The
data are representative of several experiments.
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Fig. 2.
PrP-sen reconstituted into SCRLs is
surface-localized. SCRLs containing reconstituted
[35S]PrP-sen as described in Fig. 1 were digested with PK
(10 µg/ml) in the presence or absence of detergent (1% Triton X-100)
at 37 °C for 50 min. Results are representative of two independent
experiments, each performed in duplicate.
PrP-sen was readily extracted with
cold Triton X-100 without PI-PLC treatment (Fig. 3C,
lanes 7 and 8 versus lane
12), suggesting that this GPI anchor-independent method of
attachment was mediated by hydrophobic protein-lipid interactions. A
very small proportion of GPI
PrP-sen apparently resisted
extraction by cold Triton X-100, possibly due to use of an insufficient
amount of detergent or inversion of the liposomes during extraction
leading to trapping/reorienting of PrP-sen inside the liposomes. In
support of this proposal, we have observed a small decrease (~5%) in
the amount of surface-localized PrP-sen in GPI+
PrP-sen-containing SCRLs after extraction with cold Triton X-100 (data
not shown).
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Fig. 3.
PrP-sen associates with SCRLs via hydrophobic
interactions. SCRLs containing reconstituted
[35S]PrP-sen as described in Fig. 1 were treated with 1 M NaCl (A), 0.1 M sodium carbonate
(Na2CO3, pH 11.5) (B), 1% Triton
X-100 at 4 °C (C), or 1% Triton X-100 at 37 °C
(D) and fractionated by floatation on five-step Optiprep
gradients. The results are representative of an experiment performed in
duplicate.
PrP-sen with SCRLs was
pH-independent over the range of pH 5-7.8 (data not shown). Therefore,
the factors influencing PrP-sen binding to membranes in our SCRL system
differ from those reported previously.
PrP-sen with SCRLs in the absence of detergent
followed by SCRL isolation by floatation. Under these conditions,
GPI
PrP-sen readily associated with SCRLs (Fig.
4A). Kinetic analysis of the
association within the time constraints of the floatation assay
indicated this binding occurred rapidly, achieving near maximal levels
with a 5-min incubation (41% of input PrP-sen) (Fig. 4A),
although there was an additional ~20-min delay before initiating the
centrifugation due to the time required to prepare the density
gradients. Extending the incubation time for the reaction to 60 min
increased the recovery of GPI
PrP-sen in the SCRL
fractions to 64% of the PrP-sen input into the reaction (Fig.
4A). Also, as described above, the binding was
pH-independent (Fig. 5), and bound
protein was only removed by extraction with cold Triton X-100 (data not
shown), suggesting the binding is mediated by hydrophobic protein-lipid
interactions. Therefore, the GPI anchor-independent SCRL binding by
GPI
PrP-sen was not dependent on incubation in the
presence of detergent. Nevertheless, unless otherwise indicated, all
remaining studies with GPI anchor-deficient PrP-sen were performed
using these binding conditions in the absence of detergent.
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Fig. 4.
GPI anchor-deficient PrP-sen binds to SCRLs
in the absence of detergent. [35S]PrP-sen lacking a
GPI anchor either due to expression in a form deleted for the GPI
anchor addition sequence (GPI PrP-sen) (A) or
to PI-PLC digestion (GPIPI-PLC PrP-sen) (B) was
mixed with SCRLs in the absence of detergent, incubated for the time
indicated, and fractionated by floatation on three-step density
gradients. No increase in binding was observed after overnight
incubation (not shown). The data are representative of two independent
experiments, each performed in duplicate.
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Fig. 5.
GPI PrP-sen binding to SCRLs is
pH-independent. [35S]GPI
PrP-sen was
mixed with SCRLs as in Fig. 4 in buffers at a range of pH. Binding
reactions were fractionated on three-step density gradients, and the
amount of PrP-sen in each fraction was quantitated by liquid
scintillation counting. Results are expressed as mean percentage of
counts recovered in the low density fractions (1-4)
versus total counts recovered. The error bars
indicate range (n = 2). The data are representative of
two independent experiments each performed in duplicate.
PrP-sen, was also readily extracted by treatment with
cold Triton X-100 (data not shown). This shows that GPI+
PrP-sen can exhibit GPI anchor-independent-like SCRL binding, however,
this can be clearly distinguished from GPI anchor-dependent binding by extracting the PrP-bound SCRLs with cold Triton X-100.
PrP-sen used above was generated by deletion of the
GPI anchor addition sequence and is synthesized in a predominantly unglycosylated form. GPI anchor-deficient PrP-sen can also be generated
by treatment of cells with phosphatidylinositol-specific phospholipase
C (PI-PLC) to remove the diacylglycerol moiety of the GPI anchor, which
releases some PrP-sen from the cell surface where it is found in a
predominantly fully glycosylated form. This allowed us to test the
effect of glycosylation on GPI anchor-independent SCRL binding.
Purified PI-PLC-released PrP-sen (GPIPI-PLC PrP-sen) bound
to SCRLs with similar kinetics and efficiency as GPI
PrP-sen with 48% or 62% of the input GPIPI-PLC PrP-sen
being recovered in the SCRL fractions after a 5- or 60-min incubation,
respectively (Fig. 4B). This indicates that glycosylation does not detectably influence SCRL binding in our assay.
PrP-sen Binding to SCRLs--
PrP-sen contains a
hydrophobic stretch of amino acids from residues 112 to 135 that
comprises a transmembrane domain in certain rare forms of PrP (28) and
was a candidate for the region mediating the hydrophobic, GPI
anchor-independent SCRL association. To evaluate the role of N-terminal
residues of GPI
PrP-sen in SCRL binding and to determine
the contribution of this hydrophobic region, we tested the binding of
two readily available mutants containing deletions from amino acid
residues 34-94 (
94) and 34-124 (
124). Both mutants bound SCRLs
less efficiently than the wild-type GPI
PrP-sen control,
the
124 mutant showing a slightly reduced binding compared with the
94 mutant (Fig. 6A).
Western blot analysis of the PrP-sen samples verified equivalent
amounts of each type of PrP-sen were used in the binding reactions
(Fig. 6B). Because these deletions failed to completely
inhibit binding, our data suggest that residues outside of this region
may also contribute to SCRL binding.
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Fig. 6.
N-terminal deletions decrease
GPI PrP-sen binding to SCRLs.
[35S]GPI
PrP-sen either without N-terminal
deletions (wt) or deleted for residues 34-94 (
34) or
34-124 (
124) were assayed for binding to SCRLs as described in Fig.
4. A, binding was quantitated as in Fig. 5. Error
bars indicate standard deviation (n = 3).
B, immunoblot analysis of total PrP-sen in preparations used
for binding assays in A. All samples have equivalent amounts
of unglycosylated PrP-sen (indicated by brackets). In
addition, cell lines expressing the deletion mutants produce higher
levels of partially and fully glycosylated forms (indicated by
upper two bands in lanes 2 and 3)
compared with wt.
94 (Fig.
7E) or
124 (Fig. 7F) mutant PrP-sen molecules
were used, consistent with the poor binding observed using the purified proteins (Fig. 6A). To control for the possibility that
SCRL-binding activity was simply due to an excess of PrP-sen relative
to other proteins in the supernatant, we tested a generic protein
(purified recombinant murine/human Fab fragment D13) for SCRL binding.
Even when incubated with SCRLs at a protein:lipid molar ratio >20-fold higher than the PrP-sen:lipid ratio present in PrP-containing reactions, the recombinant Fab fragment failed to bind to SCRLs (data
not shown). Altogether, these data demonstrate GPI anchor-independent SCRL binding is not a ubiquitous property of GPI-anchored proteins and
that SCRL binding is not an artifact of using purified PrP-sen.
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Fig. 7.
Binding of PI-PLC-released/secreted
proteins to SCRLs. Culture supernatants from metabolically
labeled, PI-PLC-treated fibroblast cells expressing either full-length
(A-D) or N-terminally-deleted (E and
F) GPI+ PrP-sen were incubated with SCRLs in the
absence of detergent and fractionated on three-step density gradients.
Fractions were treated with PNGase F where indicated. A and
B, the bracket and arrow indicate
bands (most abundant in fraction 2) with apparent molecular
mass and glycoform pattern similar to PrP-sen. C, gradient
fractions were immunoprecipitated with anti-PrP monoclonal antibody
prior to SDS-PAGE. The arrow indicates the band
corresponding to PrP-sen. D, SCRL fractions
(1-3) as in A were combined, pelleted, extracted
with cold 1% Triton X-100, and fractionated by floatation on a second
density gradient. Fractions from this second gradient are shown.
E and F, lanes labeled
"PrP-sen" correspond to PrP-sen immunoprecipitated from
a PNGase F-treated aliquot of PI-PLC culture supernatant equivalent to
one-tenth the amount added to the SCRL binding reactions.
Arrows indicate bands corresponding to PrP-sen.
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Fig. 8.
PEG and PI-PLC assist cell-free conversion of
SCRL-associated GPI+ PrP-sen. Cell-free conversion
reactions were performed as described under "Experimental
Procedures" using either hamster (A) or mouse
(B) [35S]GPI+ PrP-sen
reconstituted into SCRLs as in Fig. 1 and isolated by floatation on
density gradients after extraction with cold 1% Triton X-100. Samples
were treated with various concentrations of PEG or PI-PLC as indicated.
PK lanes contain a one-tenth
aliquot of each reaction mixture before PK digestion.
PK+ lanes contain a nine-tenths
aliquot of each reaction mixture after PK digestion. A,
samples were deglycosylated with PNGase F after conversion to aid
visualization of PrP bands. Arrows indicate newly generated
PK-resistant PrP-res bands (lanes 9 and 10). The
upper two arrows correspond to incompletely deglycosylated
conversion products. Results are representative of two independent
experiments each performed in duplicate. B, all reactions
contained PrP-res. Brackets indicate PrP-sen
(PK
panel) and newly generated
PrP-res (PK+ panel). Results are
representative of a single experiment performed in duplicate.
PrP-sen
by Exogenous PrP-res--
Next, we tested the effect of the GPI
anchor-independent form of PrP-sen association with SCRLs on
conversion. In contrast to GPI+ PrP-sen, GPI
PrP-sen pre-bound to SCRLs in the presence or absence of Sarkosyl was
converted to new PrP-res without any additional treatments to the
reactions (Fig. 9A,
lanes 8 and 12). Overall, the conversion efficiencies were comparable to reactions containing free
GPI
PrP-sen without SCRLs (Fig. 9B). One
potential exception involved GPI
PrP-sen bound to SCRLs
in the absence of Sarkosyl, which converted with a similar efficiency
to free GPI
PrP-sen after a 1-day reaction but was
marginally more efficient after a 2-day reaction (Fig. 9B).
In addition, GPI
PrP-sen bound to SCRLs in the presence
of Sarkosyl was converted with a slightly reduced efficiency compared
with free GPI
PrP-sen after a 1-day reaction but not a
2-day reaction. Hence, we cannot exclude the possibility that Sarkosyl
modifies the binding of GPI
PrP-sen to SCRLs in a subtle
way not detected by our salt/detergent treatments to extract PrP-sen
above. Nevertheless, it is notable that conversion products were
generated in all of these reactions. Thus, the mode of membrane
association (GPI anchor-dependent versus -independent) had dramatically different effects on the ability of
PrP-sen to serve as a substrate in the conversion reaction.
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Fig. 9.
Cell-free conversion of SCRL-associated
GPI PrP-sen. Cell-free conversion reactions were
incubated for 1 or 2 days using [35S]GPI
PrP-sen either in the absence of SCRLs (free) or pre-bound to SCRLs in
the presence (as in Fig. 1) or absence (as in Fig. 4) of Sarkosyl.
A, representative images of conversion reactions using each
type of GPI
PrP-sen after conversion for 1 day.
Brackets indicate newly generated PrP-res (lanes
4, 8, and 12). B, quantitation of
newly generated PrP-res. Results are expressed as mean percent
conversion ± S.D. (n = 3).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 10.
Summary of modes of PrP-sen association with
SCRLs. A, GPI anchor-deficient PrP-sen likely
associates with SCRLs by partial insertion into the lipid bilayer.
B, GPI-anchored PrP-sen can associate with SCRLs by
insertion of the GPI anchor into the lipid bilayer when reconstituted
in the presence of detergent. C, when bound to SCRLs in the
absence of detergent, GPI-anchored PrP-sen binds SCRLs in a manner not
directed by the GPI anchor similar to GPI anchor-deficient PrP-sen. The
GPI anchors may self-associate as pseudomicelles and be unavailable for
insertion into SCRLs.
This alternative, GPI anchor-independent mode of membrane association
was detected in control experiments with GPI PrP-sen
during our attempts to reconstitute GPI+ PrP-sen in SCRLs.
These reconstitution experiments included the mixing of PrP-sen with
SCRLs in the presence of a low concentration of Sarkosyl as transient
exposure to detergent is commonly required to permit insertion of
GPI-anchored proteins into liposomes (15, 16, 34, 35). Presumably, the
detergent is required to disrupt the pseudomicelles formed by
GPI-anchored proteins in the absence of detergent (36) and/or to
transiently perturb the liposome membranes to facilitate insertion of
the GPI anchor. The acquisition of resistance to solubilization in cold
Triton X-100 by a significant fraction of GPI+ PrP-sen
indicates that we were successful in reconstituting this type of
PrP-sen in a GPI anchor-directed fashion. This was confirmed by the
fact that PI-PLC pretreatment allowed the solubilization in cold Triton
of a majority of these formerly Triton X-100-resistant molecules (data
not shown). Because treatment of GPI+ PrP-sen-containing,
cold Triton-extracted SCRLs with PI-PLC alone released only a small
percentage of the PrP-sen (data not shown), simultaneous association
via GPI anchor-dependent and -independent methods might be
possible. Alternatively, there may be re-binding of PrP-sen by the
GPI-independent mechanism after release by PI-PLC cleavage. The
extraction of some of the GPI+ PrP-sen in cold Triton X-100
(Fig. 3C, lane 6) may indicate that our protocol
used suboptimal levels of Sarkosyl to promote GPI anchor-directed
insertion into the SCRLs and that this extractable population was
associated solely via the GPI anchor-independent mode. Arguably, the
presence of the detergent could have artifactually assisted the
association of GPI
PrP-sen with the SCRLs. However, the
occurrence of GPI
PrP-sen binding to SCRLs in the absence
of detergent with a comparable efficiency with respect to the input
PrP-sen showed that detergent was not required for the effect.
Furthermore, we have demonstrated the specificity of the SCRL binding
activity of GPI anchor-deficient PrP-sen by showing only very few
PI-PLC-released or secreted proteins share this activity (Fig. 7,
A, B, E, and F). Consistent
with our specificity studies, detergent-assisted reconstitution of another GPI-anchored protein (bovine intestine alkaline phosphatase) into raft-like liposomes was shown to be GPI anchor-dependent (34).
Admittedly, we cannot entirely rule out the possibility of another
PI-PLC-released protein indirectly facilitating the SCRL binding of
PI-PLC-released PrP-sen, although our experiments with purified PrP-sen
would indicate that other accessory proteins are not required for this
binding activity.
Two other groups have recently published studies of binding of GPI anchor-deficient PrP-sen to liposomes, but with significant differences in experimental design that complicate comparisons to the present study (10, 13). The previous studies relied predominantly on spectroscopic techniques to assay binding activity, whereas we have used a simple direct binding assay by floatation on density gradients. Perhaps most importantly, both of the previous studies made use of recombinant PrP-sen derived from E. coli and refolded in the absence of copper. We have used forms of PrP-sen expressed in mammalian cells (mouse fibroblasts). This allowed us the advantage of using a source of PrP-sen that is synthesized and folded under native conditions and, when expressed with a GPI-anchor, also contains the appropriate N-glycosylation of the native protein. Furthermore, this expression method also allowed the use of cell surface PrP-sen as released into culture medium (phosphate-buffered balanced salts solution) without purification. The binding of this PI-PLC-released PrP-sen to SCRLs (Fig. 7, A-C) shows the binding is not an artifact of using purified PrP-sen that may contain conformational alterations not detected by low resolution structural analyses (e.g. circular dichroism).
Morillas and co-workers (10) showed full-length (amino acids 23-231) human PrP-sen binds to acidic lipid-containing liposomes (phosphatidylcholine/phosphatidylserine (PC/PS)) with the strongest binding occurring at acidic pH. Using two truncated forms of human PrP-sen, a pH-dependent component of the binding was localized to residues 90-231, whereas pH-independent binding was observed for a fragment consisting of residues 23-145 (10). These data are consistent with our results in detecting pH-independent liposome binding (Fig. 5 and data not shown) and localizing a portion of PrP-sen membrane binding to the N terminus (Fig. 6A), although we did not observe a requirement for acidic lipids.
The second study made use of liposomes with sphingomyelin and
cholesterol that are related to SCRLs but with minor differences (13).
In comparison to SCRLs, these liposomes lacked cerebrosides and
contained a phospholipid:sphingolipid:cholesterol ratio differing from
that found in rafts isolated from cells (26). These differences or
perhaps one of those described above, might account for their observation of binding of PrP-sen to raft-like liposomes only at
neutral pH (13). However, Sanghera and Pinheiro only studied the
binding of the 90-231 fragment of hamster PrP-sen at pH 5.0 and 7.0. The most closely related hamster PrP-sen species in our study, lacking
residues 34-94 (94), was only tested for binding at pH 6.0 and
showed residual binding activity (Fig. 6A). It is possible
then that binding of PrP-sen to raft-like liposomes may consist of a
pH-dependent component via the C terminus and a
pH-independent component directed by the N terminus. Despite the
differences, both this and a previous study have provided independent
evidence for the hydrophobic nature of the binding of GPI
anchor-deficient PrP-sen to raft-like liposomes (13).
Membrane insertion activity is usually restricted to proteins with specialized functions like pore-forming toxins or viral fusion proteins, for example. Hence, the hydrophobic nature of the GPI anchor-independent membrane association is particularly interesting, suggesting that the binding is mediated by at least partial insertion of PrP-sen into the membrane and raising the possibility of a mechanism for direct cytotoxicity of PrP in some conformation, as proposed recently (13). Indeed, Sanghera and Pinheiro (13) obtained spectroscopic evidence for such activity. The involvement of residues 34-94, which contain the glycosaminoglycan-binding (37-40) and copper-binding octapeptide repeats (41-48), suggest that the SCRL binding affected by this region of PrP-sen may be regulated by interactions with glycosaminoglycans and/or copper, although this has yet to be tested. Obviously, this is a model system, and the extent to which this form of GPI anchor-independent binding occurs in cells remains to be determined. However, these observations may provide an explanation for the observation of incomplete release of cell-surface PrP-sen from cultured cells treated with PI-PLC. Armed with a better understanding of how this binding is regulated using our model system, we will be better equipped to investigate these processes directly in cells.
The observation of conformational changes in PrP-sen occurring on binding to liposomes (10, 13) only emphasizes the importance of understanding the effects of PrP-sen membrane association in its various forms on interactions with PrP-res. We have directly evaluated the effect of both GPI anchor-dependent and -independent methods of PrP-sen SCRL binding on the ability of PrP-sen to serve as a substrate for conversion to PrP-res and shown that the method of membrane association strongly influences the conversion reaction. Our previous work suggested that association of murine PrP-sen with DRMs prepared from a mouse PrP-overexpressing neuroblastoma cell line inhibited conversion by exogenous murine PrP-res as contained in a crude brain microsome fraction from scrapie-infected mice (14). Generation of new PrP-res in these DRM-based reactions required the addition of PI-PLC or pulse treatment with 30% PEG to induce membrane fusion, suggesting either removal of PrP-sen from membranes or insertion of PrP-res into contiguous membranes was a prerequisite for conversion. We have recapitulated these results using GPI+ PrP-sen reconstituted into SCRLs with two different scrapie strains, showing that the lack of conversion of DRM-bound PrP-sen by exogenous PrP-res was not due to inhibition by other DRM-associated molecules and that these observations are common to multiple scrapie strains.
Binding of recombinant PrP-sen to raft-like liposomes of PC/SM/Chol
leads to a higher content of -helical structure and has been
proposed to stabilize PrP-sen in an
-helical conformation (13).
Although we lack sufficient protein to perform a structural determination of SCRL-bound PrP-sen in this study, our data show no
evidence for a dramatic effect of pre-association of PrP-sen with SCRLs
on conversion (Fig. 9B). Thus, if any conformational changes
in PrP-sen occurred on binding SCRLs, they did not detectably influence
the conversion reaction under the conditions used here.
The clearly contrasting effects of GPI anchor-dependent versus -independent membrane association of PrP-sen on conversion demonstrate that it is not membrane binding per se but the mode of membrane binding that controls the interaction with exogenous PrP-res. Our observations support the use of SCRLs as model raft membranes and suggest that membrane association specifically directed by the GPI anchor inhibits conversion of membrane-bound PrP-sen by exogenous PrP-res. It is possible that when tethered to the membrane via the GPI anchor, the PrP-res binding site on PrP-sen is occluded and incapable of binding to PrP-res in a manner leading to conversion until the PrP-res is inserted into a membrane contiguous with PrP-sen. Consistent with this proposal, previous studies have localized residues clustered near the C terminus in the NMR structure of PrP-sen as potentially contributing to the PrP-res binding site (8, 24, 49-51). When PrP-sen is modeled in a GPI anchor-directed membrane-bound state (11), it is conceivable that the PrP-res binding region is not readily accessible for interaction with large aggregates of exogenous PrP-res. Future studies will address whether conversion of raft-bound GPI+ PrP-sen by exogenous PrP-res is blocked at the level of binding or acquisition of protease resistance (24).
Our study of GPI anchor-deficient forms of PrP-sen led to the detection of an alternative mode by which PrP-sen associates with membranes. Although we have found that GPI+ PrP-sen can also exhibit this alternative membrane-binding mode under certain circumstances, the characterization of this binding activity is obviously simplified by the use of GPI anchor-deficient forms of PrP-sen where we can definitively rule out any contribution from the GPI anchor to any effects we observe. However, given that the majority of PrP-res generated in infected animals contains a GPI anchor (1), it is not immediately clear whether GPI anchor-deficient forms of PrP-sen themselves play any role in PrP biology.
On the other hand, several studies support the possibility that
GPI-anchorless forms of PrP are physiologically important. The release
of apparently soluble forms of PrP from cells was first demonstrated
many years ago (29, 53, 55). Borchelt and co-workers (56) later showed
that low levels of GPI forms of PrP-sen (comprising
~10% of total PrP-sen) are generated in various cultured cells and
hamster brain homogenates. More recently, similar results have been
obtained using splenocytes or cerebellar granule neurons from
PrP-overexpressing transgenic mice (57). Furthermore, Stahl et
al. (54) reported that ~10-20% of PrP-res molecules purified
from infected hamsters are truncated at glycine-228, similar to the
GPI
PrP-sen used in the present study, which is truncated
at residue 231 (18). Although it is unclear whether this small fraction of PrP-res molecules was generated from a GPI
PrP-sen
precursor, GPI
PrP-sen has been shown to be capable of
acting as a precursor for the synthesis of new PrP-res in
scrapie-infected mouse neuroblastoma cells, albeit at a significantly
reduced apparent efficiency compared with the wild-type protein
(52).
When considered together with our data, these observations suggest
possible scenarios whereby GPI forms of PrP-sen might
participate in the initiation of infection of cells. Because insertion
of exogenous PrP-res into contiguous membranes appears to be a
prerequisite for conversion of membrane-associated forms of
GPI+ PrP-sen (14 and this study) but not
membrane-associated forms of GPI
PrP-sen, exogenous
PrP-res molecules might initially interact with and induce the
conversion of membrane-bound GPI
PrP-sen. This process
could promote the association of the PrP-res aggregates with host cell
membranes and might facilitate the insertion of the PrP-res molecules,
the majority of which are GPI-anchored, into the membrane where they
would then become competent to convert the GPI+ PrP-sen
molecules to new PrP-res. Alternatively, the newly converted GPI
PrP-res might directly interact with and seed the
conversion of GPI+ PrP-sen molecules thereby acting as a
"bridge" between exogenous PrP-res and cell-associated
GPI+ PrP-sen. Whether any of these scenarios are important
in transmissible spongiform encephalopathy infections remains to
be determined.
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ACKNOWLEDGEMENTS |
---|
We thank Gregory and Lynne Raymond for technical assistance and David Kocisko, Ravindra Kodali, Srisailam Sampath, and Jay Silveira for critical reading of the manuscript. We thank Bruce Chesebro, Victoria Lawson, and Sue Priola for providing cell lines expressing hamster PrP N-terminal deletion mutants.
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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.
Supported by post-doctoral fellowships from the Natural Sciences
and Engineering Research Council of Canada and the Canadian Institutes
of Health Research.
§ To whom correspondence should be addressed. Tel.: 406-363-9264; Fax: 406-363-9286; E-mail: bcaughey@nih.gov.
Published, JBC Papers in Press, February 19, 2003, DOI 10.1074/jbc.M210840200
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ABBREVIATIONS |
---|
The abbreviations used are:
PrP, prion protein;
GPI, glycosylphosphatidylinositol;
DRM, detergent-resistant membrane;
PrP-sen, protease-sensitive PrP;
PrP-res, protease-resistant PrP;
SCRL, sphingolipid-cholesterol-rich liposome;
GPI+, GPI anchor
containing;
GPI, GPI anchor-deficient by deletion of GPI
signal sequence;
PI-PLC, phosphatidylinositol-specific phospholipase C;
GPIPI-PLC, GPI anchor-deficient by PI-PLC cleavage;
CBS, citrate-buffered saline;
PEG, polyethylene glycol;
PK, proteinase K;
wt, wild-type;
PC, phosphatidylcholine;
SM, brain sphingomyelin;
CB, brain cerebrosides;
Chol, cholesterol;
Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine;
PS, phosphatidylserine;
PNGase F, peptide N-glycosidase
F.
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REFERENCES |
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