(Received for publication, January 30, 1996; and in revised form, March 4, 1996)
From the
Prion diseases are thought to be caused by the conversion of the
normal, or cellular, prion protein (PrP) into an abnormal
protease-resistant conformation (PrP
). There are three
familial forms of human prion disease, Creutzfeldt-Jakob disease (CJD),
Gerstmann-Straussler-Scheinker syndrome, and fatal familial insomnia
(FFI) which are all expressed at advanced age despite the congenital
presence of the mutant prion protein (PrP
). The cellular
mechanisms that result in the age-dependent conversion of PrP
into PrP
and the unique phenotypes associated with
each PrP
are unknown. FFI and a familial type of
Creutzfeldt-Jakob disease (CJD
), share the D178N mutation
in the PrP gene but have distinct phenotypes linked to codon 129, the
site of a methionine/valine polymorphism (129M/V). We analyzed PrP
processing in cells transfected with constructs reproducing the FFI and
CJD
genotypes. The D178N mutation results in instability
of the mutant PrP which is partially corrected by N-glycosylation. Hence, only the glycosylated forms of
PrP
reach the cell surface whereas the unglycosylated form
is degraded. The unglycosylated PrP
is also
under-represented in the brain of FFI patients validating the cell
model. These results offer new insight into the effect of the D178N
mutation on the metabolism of the prion protein.
Mutations underlying an increasing number of inherited diseases are being discovered. The task now is to define the individual steps through which the mutated protein causes a specific disease, in some cases by becoming a pathogen, often after a symptom-free interval of several decades (Hamilton et al., 1992).
The prion protein
(designated PrP) has been implicated in a variety of human
and animal diseases referred to as prion diseases, spongiform
encephalopathies, or transmissible amyloidoses (Prusiner and DeArmond,
1994). Prion diseases can occur either sporadically or by infectious
transmission in both humans and animals. In addition, 18 mutations in
the PrP
gene, identified as PRNP, have been
reported to be associated with inherited forms of prion diseases in
humans (Parchi and Gambetti, 1995).
The inherited prion diseases
associated with PRNP mutations fall into three major groups:
Creutzfeldt-Jakob disease (CJD), ()Gerstmann-Sträussler-Scheinker
syndrome, and the recently discovered fatal familial insomnia (FFI)
(Parchi and Gambetti, 1995). Despite their phenotypic differences, FFI
and one familial type of CJD (CJD
) are both linked to a
single mutation of PRNP at codon 178 resulting in the
substitution of asparagine for aspartic acid (D178N) (Goldfarb et
al., 1992). They differ, however, at the PRNP codon 129,
a natural polymorphic site encoding either methionine or valine
(Goldfarb, Petersen et al., 1992). Codon 129 of the mutant
allele specifies the disease phenotype associated with the D178N
mutation: the 129M,D178N haplotype is linked to FFI, the 129V,D178N
haplotype to CJD
(Goldfarb, Petersen et al.,
1992).
A variety of data clearly demonstrate the importance of the
mutant PrP (PrP) in the pathogenesis of FFI and CJD
and other inherited prion diseases. They provide no information,
however, on the precise metabolic events that lead to the disease. In
this study we used transfected human neuroblastoma cells to examine the
effect of the PRNP D178N mutation associated with either the
129M or 129V codon on the synthesis and metabolism of the
PrP
.
Figure 1: Schematic representation of the prion protein and the cell lines used in this study. A, linear representation of the human prion protein indicating pertinent features: signal sequence, amino-terminal epitope, 3F4 epitope, the polymorphic codon 129 (methionine or valine), codon 178 site of the aspartic acid to asparagine mutation, glycosylation sites at residues 181 and 197, carboxyl-terminal cleavage site for GPI anchor addition, carboxyl-terminal epitope, and termination site. B, the cell types listed with the genotype of the transfected constructs.
Cells were tested for the presence
of proteinase K-resistant PrP in the following manner. Whole cell
homogenates were prepared from 10 cells, after washing the
plates with cold PBS, by lysing with 9 volumes of 100 mM NaCl,
10 mM Tris, 10 mM EDTA, 0.5% Nonidet P-40, and 0.5%
sodium deoxycholate, pH 7.4. The postnuclear supernatant was digested
with proteinase K in either of 2 ways: 2
10
cell
equivalents were digested in a 20-µl reaction for 1 h with 0.5, 1,
5, or 10 µg/ml proteinase K (Boehringer Mannheim) or 2
10
cell equivalents were digested with 5 µg/ml for 5,
10, 15, or 20 min. The proteinase K reaction was terminated by addition
of PMSF to 3 mM. The samples were precipitated with 4 volumes
of methanol, resuspended in sample buffer, resolved on 14% SDS-PAGE,
blotted onto Immobilon P, and detected with the 3F4 antibody.
Brain
tissue was homogenized in 9 volumes of 0.32 M sucrose, 20
mM Tris, pH 7.5, containing 2 µg/ml PMSF, 1 µg/ml
leupeptin, 1 µg/ml pepstatin. The supernatant was clarified by
centrifugation at 1000 g for 10 min, and recentrifuged
at 100,000
g for 1 h to obtain a membrane fraction.
The total homogenate and the membrane fraction, before and after
deglycosylation, were resolved on 12% SDS-polyacrylamide gels,
transferred to Immobilon P, and probed with an anti-amino-terminal
antibody (a polyclonal rabbit serum to a synthetic peptide, human PrP
23-40; Chen et al.(1995)) provided by B. Ghetti, Indiana
University, Indianapolis, IN. Quantitative analyses of the immunoblots
was performed with a computer-assisted laser scanner (LKB Ultrascan
XL).
We used human
neuroblastoma cells transfected with DNA constructs expressing the
human prion protein sequence to determine the effect of the D178N
mutation on the metabolism of PrP (Fig. 1). The constructs are
based on an episomal vector, so hygromycin-resistant bulk cultures,
rather than cloned cell lines, were used. The advantage of this system
is that the expression level is not influenced by the site of
integration facilitating direct comparison in metabolic studies of the
PrP expressed by different constructs. Initially, we compared the cell
surface forms of PrP and PrP
expressed in
transfected cells following cleavage of the GPI anchor with the enzyme
PI-PLC (Fig. 2A).
Figure 2:
Less modified forms of PrP,
the unglycosylated (U) and intermediate (I) forms are
under-represented on the surface of cells. A, transfected
cells were treated with PI-PLC and the released protein was
concentrated by precipitation. The protein was then resolved on
SDS-polyacrylamide gels, blotted onto Immobilon P, and reacted with the
monoclonal antibody 3F4. The amount of mutant protein loaded was three
times that of the normal. The antibody reaction was detected using
horseradish peroxidase-conjugated sheep anti-mouse IgG followed by a
chemiluminescent substrate. M, mature glycosylated PrP; I, an intermediate migrating form of PrP; U,
unglycosylated PrP. B, the samples in panel A were
treated with the enzyme peptide:N-glycosidase F to remove the
sugars and analyzed as above. A single band that comigrates with the
unglycosylated form and a small amount of a 20-kDa product are observed
in the normal cell lines.
The PrP released from
transfected control cells shows three distinct glycoforms which are
similar to those previously described in non-transfected mouse
neuroblastoma cells (Fig. 2A; Caughey et al.,
1988; Scott et al., 1988). Since our cells overexpress
PrP
we compared, in a separate experiment, the ratio of
PI-PLC released glycoforms with those of a non-transfected human cell,
M17 BE2C a clonal line from the same tumor, to ensure that we were not
observing a processing bias toward less glycosylated forms due to an
overload of the glycosylation system. The non-transfected control cells
express the three PrP glycoforms in a ratio similar to that of our
transfected cells expressing normal PrP. This indicates that the
processing observed in the transfected cells is an accurate reflection
of the normal pathway (data not shown). PrP
released from
the cells transfected with the D178N mutant coding sequence and either
the 129M codon (129M,D178N; FFI haplotype) or the 129V codon
(129V,D178N; CJD haplotype) show two differences when compared with the
control cells (Fig. 2A): 1) an overall decrease in the
PI-PLC-released PrP
(the loading of the samples from the
mutant cells was adjusted by a factor of 3 so that they would be
comparable to the control cells) and 2) a selective decrease in the
forms that migrate most rapidly in gels. Densitometric analyses
demonstrated that only one-third the amount of PrP was released from
the surface of the cells expressing PrP
compared to cells
expressing PrP
(1:0.34 ± 0.18; n =
5; p < 0.0002) and the most rapidly migrating form, which
accounts for 8.3 ± 3.6% (n = 5) of the PI-PLC
released PrP in the control cells, is virtually undetectable in the
mutant cells. The cells transfected with the vector alone show no
immunoreactivity, indicating that the PrP expressed in the transfected
cells is coded entirely by the inserted constructs.
After treatment
of the PI-PLC released PrP with PNGase F, which cleaves N-linked oligosaccharides, the PrP migrates as a single band (Fig. 2B) which has the same mobility as the most
rapidly migrating form in the untreated preparations. This finding
confirms that the three PrP forms differ in N-glycosylation
and that the most under-represented PrP glycoform at the
cell surface is the unglycosylated form.
To confirm that the
unglycosylated PrP was missing from the cell surface, and
not merely resistant to PI-PLC, we surface labeled cells with biotin
that were untreated or previously treated with PI-PLC. As shown in Fig. 3, cells expressing PrP
showed a marked
reduction in surface labeling after treatment with PI-PLC indicating
that mutant PrP
is not resistant to PI-PLC. Thus, the
under-representation of unglycosylated PrP
released from
the surface of the cells by PI-PLC (Fig. 2) is not a result of
an inability to cleave the GPI anchor of PrP
.
Figure 3: Mutant PrP is efficiently cleaved by PI-PLC. Cells that express 129M,D178N (Mutant) PrP, untreated or treated with PI-PLC, were biotinylated and the cell surface protein was recovered from the cell extract by immunoprecipitation with the monoclonal antibody 3F4 after which the biotin-labeled protein was detected on blots using horseradish peroxidase-coupled streptavidin. M, mature; I, intermediate; U, unglycosylated.
At the end of the
labeling period (time 0; Fig. 4A), all cell lysates
exhibit the same immunoprecipitable bands in comparable amounts
including three distinct bands and a slowly migrating smear. The three
discrete bands are a high mobility band corresponding to the
unglycosylated PrP and two bands with slightly lower gel mobility. The
latter two bands are sensitive to digestion with endoglycosidase H
(endo H), indicating that they correspond to the PrP forms glycosylated
with the high mannose core and that they are still located in the ER or
the Golgi complex (data not shown; Caughey et al.(1989)).
After a 30-min chase, as a result of modifications to the sugar chains,
these two forms become endo H resistant (data not shown). The slowly
migrating smear is the PrP on which the carbohydrates have been highly
modified (Caughey et al., 1989). At 2 h, only mature forms of
PrP are detected (Fig. 4A). At the 2-h time point
PrP is under-represented in the cells expressing either the
129M,D178N or the 129V,D178N. The average amount of unglycosylated
PrP
from both mutant cell types accounts for 1.9 ±
2.3% of the total PrP
while the unglycosylated PrP
accounts for 8.2 ± 2.6% in control cells (n = 6; p < 0.003; Fig. 4C).
Although there is also an apparent under-representation of the
intermediate form, as noted above, the species migrating at this
position change with time making quantitation difficult. Similar
results are obtained using antibodies directed against the amino
(anti-N) and carboxyl (anti-C) termini, except that anti-N recognizes
an additional band at 20 kDa which is an unglycosylated,
carboxyl-terminal truncated form of PrP and anti-C detects a truncated
form of the unglycosylated PrP, not recognized by 3F4, that migrates at
approximately 18 kDa (data not shown; Chen et al.(1995)).
Truncated forms lacking the amino terminus have been described (Pan et al., 1992; Harris et al., 1993; Chen et
al., 1995).
Figure 4:
Unglycosylated PrP is not
retained in cells (A) nor does it efficiently reach the cell
surface (B). A, cells were labeled for 30 min at 37
°C with Trans
S-label, the media was removed, and cells
were chased for the times indicated above the lane. PI-PLC was added 30
min before the end of the chase after the chase media was removed. The
cell homogenate was immunoprecipitated with monoclonal antibody 3F4.
The precipitated PrP was resolved on 16% SDS-polyacrylamide gels and
visualized by fluorography. M, mature; I,
intermediate; U, unglycosylated. B, the media from
the PI-PLC-treated cells was immunoprecipitated and analyzed in the
same manner as the cell homogenate. M, mature; I,
intermediate; U, unglycosylated; 20kd, minor
proteolytic product. C and D, graphical
representation of the glycoforms of PrP present in the cell or on the
cell surface as a percent of total PrP (panel C: *, p < 0.003; panel D: *, p < 0.008, p < 0.0008).
The unglycosylated PrP is a significant
fraction of the total PrP
at the zero time point indicating
that this form is normally produced by all cell types (18.0 ±
2.2% versus 23.3 ± 2.2% in control cells; n = 8). At the zero time point, the labeled PrP is still in
the ER-Golgi complex region as shown by the barely detectable quantity
of PrP that is PI-PLC-released from the cell surface (Fig. 4B). The decrease of the intracellular
unglycosylated PrP
occurs at the 30-min and 2-h time points
when the glycosylated PrP
forms are still intracellular or
are being inserted into the plasma membrane. When analyzed separately,
the unglycosylated cell surface PrP
is undetectable after 2
h in the 129M mutant cells (n = 4), but accounts for
0.6 ± 0.4% of total PrP in 129V mutant cells (n = 4; Fig. 4D). In contrast, the
unglycosylated form accounts for 3.2 ± 1% and 5.3 ± 0.6%
in 129M and 129V control cells (n = 4; p <
0.008 (129 M) and p < 0.0008 (129V) two tailed t test; Fig. 4D). These findings, and the
previous finding that PrP
is under-represented at the cell
surface in the steady state, indicates that PrP
is
inefficiently transported through the secretory pathway. This is
especially evident for the less modified glycoforms. The absence of the
truncated, unglycosylated 20-kDa form on the surface of the mutant
cells also supports this conclusion (Fig. 4B).
In
addition to the surface and intracellular forms of PrP detailed above,
we detected a small amount of PrP (<5% of the total) in the chase
medium without addition of PI-PLC (data not shown). Epitope mapping
indicates that the secreted PrP corresponds to the previously described
PrP form lacking the last four carboxyl-terminal residues and the GPI
anchor (Stahl et al., 1993). The secreted PrP is
under-represented in the media from the mutant cells relative to the
normal cells, indicating that the low amount of PrP at the
surface of the mutant cells is not the result of increased secretion.
Figure 5:
Tunicamycin impairs transport to the cell
surface and alters stability of PrP. A, cells were
preincubated in media lacking methionine and cysteine with or without 2
µg/ml tunicamycin. The media was removed and the cells were labeled
for 60 min in media containing Trans
S-label and 2
µg/ml tunicamycin. 30 min into the labeling period PI-PLC was added
to one plate. After the labeling period the media was removed, a cell
homogenate was immediately prepared from the plate treated with PI-PLC
which was immunoprecipitated with the monoclonal antibody 3F4. Media
lacking the
S label was added to the second plate, after
3.5 h at 37 °C, PI-PLC was added and following an additional 30-min
incubation at 37 °C the media was removed and a cell homogenate was
prepared and immunoprecipitated with 3F4. The immunoprecipitated
samples were analyzed on SDS-polyacrylamide gels and visualized by
fluorography. M, mature; I, intermediate; U,
unglycosylated. B, the media from the PI-PLC-treated cells was
immunoprecipitated with monoclonal antibody 3F4, analyzed on
SDS-polyacrylamide gels, and visualized by fluorography. M,
mature; I, intermediate; U, unglycosylated; 20kd, minor proteolytic product. C, graphical
representation of the amount of PrP present on the surface of mutant
and control cells treated with tunicamycin expressed as a percent of
the total labeled PrP at time 0 or after a 4-h chase. *, p < 0.0045; two-tailed t test).
The finding that when glycosylation is
prevented PrP barely reaches the cell surface and is
undetectable inside the cell shortly after its synthesis lends
additional support to the notion that the unglycosylated form of the
D178N PrP
is degraded in an intracellular compartment and
that PrP
, but not PrP
, requires glycosylation
to facilitate transport to the cell surface.
Figure 6:
Unglycosylated 129M PrP is
less stable than 129V PrP
in cells treated with brefeldin
A. A, cells were preincubated in media lacking methionine and
cysteine and labeled for 30 min in media containing
Trans
S-label and 1 µg/ml brefeldin A. Cells were
chased for the times indicated above the lanes, cell homogenates were
prepared and immunoprecipitated with monoclonal antibody 3F4. The
immunoprecipitated samples were resolved on SDS-polyacrylamide gels and
visualized by fluorography. The fastest migrating band is the
unglycosylated PrP; the two upper bands are glycosylated with the high
mannose core which is modified to endoglycosidase H resistance over the
course of the 2-h chase. G, glycosylated; U,
unglycosylated. B, graphical representation of the decrease in
the unglycosylated PrP as a percent of total PrP after a 2-h chase in
brefeldin A-treated cells (*, p <
0.003).
Figure 7:
Unglycosylated PrP is
under-represented in the brain of a subject affected by FFI. Membrane
fractions obtained from the occipital cortex of a control subjects (lane 1) and one FFI subject heterozygous for a deletion of
one of the octarepeats within PRNP codons 76 and 91 (lane
2). The samples were immunoblotted and stained with an antibody
that recognizes the amino-terminal region of PrP. As expected the
unglycosylated form migrates as two uneven bands in the FFI subjects
because of the deletion, whereas in the control the unglycosylated form
migrates as a single band. The samples were treated with the enzyme
PNGase F to remove the N-linked sugars and analyzed as above
(control, lane 3; FFI, lane 4). In the FFI subject
the two bands, which now contain all the original glycoforms, are
comparable indicating that the unglycosylated form is selectively
decreased. (U, unglycosylated; M,
mature).
The central event in the pathogenesis of the prion diseases
is thought to be a change in the conformation of PrP (Prusiner and DeArmond, 1994) that renders the PrP
protease resistant (PrP
) (Hope et al.,
1986; Caughey and Raymond, 1991; Pan et al., 1993; Safar et al., 1993). The abnormally conformed PrP
is
believed to act as a template for the conversion of newly synthesized
PrP
into PrP
. This mechanism is thought to be
shared by all forms of prion diseases: sporadic, transmitted, and
inherited. In the sporadic form, the change in conformation of
PrP
would be the consequence of either a somatic mutation
or of a stochastic event involving the direct conformational
modification of a PrP
molecule. In the transmitted forms,
the conversion would be triggered by the exogenous PrP
.
In the inherited prion diseases the pathogenic mutation presumably
predisposes the PrP
to spontaneous conversion into
PrP
, however, this conversion occurs as a function of age
even though mutant protein is produced throughout the life of the
individual.
The study of the metabolism of the PrP in
cells transfected with constructs homologous to the two PRNP haplotypes linked to FFI and CJD
was undertaken to
assess the metabolic differences, if any, between the two forms of
PrP
and PrP
. Studying cell models of FFI and
CJD
is of special interest since FFI and CJD
share the same D178N mutation in PRNP, but have two
different phenotypes providing a striking example of phenotypic
heterogeneity (Goldfarb, Petersen et al., 1992). Since the
only heterogeneity in the PRNP coding sequence between these
two diseases is at codon 129 of the mutant allele, the phenotypic
differences are likely to be due to the amino acid present at position
129 of PrP
which in all FFI subjects examined to date is
methionine and in all CJD
subjects is valine (Goldfarb,
Petersen et al., 1992; Gambetti et al., 1995). These
differences extend to the PrP
associated with the two
diseases (Monari et al., 1994). The ratio of the three
glycoforms and the size of the PrP
fragment generated by
proteinase K digestion are different. In FFI the unglycosylated form is
under-represented and the PrP
fragment generated by
proteinase K treatment is smaller than in CJD
(Monari et al., 1994). The difference in size of the PrP
fragments, which is due to different sites of cleavage by the
proteinase K in the two PrP
forms, is consistent with the
hypothesis that the PrP
present in FFI and CJD
have distinct conformations. The simplest explanation for these
findings, based on a large body of experimental data, is that the
presence of methionine or valine at position 129 of the PrP
results in PrP
that differ in the ratios of the
glycoforms and in conformation. In turn, these differences determine
two distinct disease phenotypes (Monari et al., 1994; Gambetti et al., 1995).
The metabolism of PrP we
observed in the human cell line expressing the normal PRNP is
in accord with the data previously reported for the mouse neuroblastoma
N2A cells (Caughey et al., 1989; Taraboulos et al.,
1992; Harris et al., 1993). In our transfected neuroblastoma
cells, the ratio of the three glycoforms is essentially the same as the
ratio found in non-transfected cells (data not shown). We also observed
an
20-kDa band in the cells transfected with the normal construct
which corresponds to one of the N-terminally truncated form described
previously (Harris et al., 1993; Chen et al., 1995).
This band was more pronounced following treatment of the cells with
tunicamycin or digestion of the samples with PNGase F, indicating that
most of this truncated form is glycosylated and consequently
co-migrates with the higher molecular weight forms as demonstrated by
Chen(1995). In addition to this fragment, we also observed an
additional
20-kDa form that was immunoprecipitated by the anti-N
serum to the amino-terminal region of PrP
.
This
fragment is currently being characterized.
The cell lines
transfected with the D178N mutant coding sequence and either the
methionine or valine codon at position 129 synthesize three high
mannose PrP glycoforms in amounts comparable to those of
control cells. The mutant transfectants also express three mature PrP
forms, one unglycosylated and the other two glycosylated. However, in
contrast to controls, these three forms are all decreased in amount at
or after they have passed the trans Golgi compartment and have
been transported to the cell surface. Moreover, the decrease
preferentially affects the unglycosylated form which is vastly
under-represented at the cell surface. This change is more severe in
the cells bearing the FFI 129M,D178N coding sequence than in the cells
with the CJD
129V,D178N sequence. The most reasonable
explanation is that the decreased amount of all forms of D178N
PrP
and the selective under-representation of the
unglycosylated form are due to the decreased stability and/or impaired
transport of the PrP
.
The experiments with tunicamycin
and BFA provide insight into the stability and transport
characteristics of the two D178N PrP that we examined.
Following treatment with tunicamycin, which abolishes N-glycosylation, cells expressing PrP
show a
single PrP
unglycosylated form (Elbien, 1987). Although, as
previously reported, total PrP
is decreased in the
tunicamycin-treated control cells (Caughey et al., 1989), the
time required for transport to the surface of the unglycosylated form
is comparable in treated and untreated cells, indicating that transport
of PrP
to the plasma membrane is relatively unaffected by
tunicamycin. In tunicamycin-treated cells expressing either the
129M,D178N or the 129V,D178N coding sequence, intracellular PrP
is readily detectable at the zero time point in both cell types,
but becomes essentially undetectable at 4 h. These findings demonstrate
that the lack of glycans destabilizes PrP
and are
consistent with the conclusion derived from the previous experiments
that transport of the unglycosylated PrP
form to the cell
surface is preferentially impaired in both D178N cells.
The BFA
experiment provides information concerning the cellular locale in which
the PrP is digested. In the presence of BFA, N-linked glycoproteins are retained in the BFA-generated
ER-Golgi compartment (Sampath et al., 1992). However, in this
hybrid compartment the oligosaccharide chains acquire endo H resistance
(Kornfield and Kornfield, 1985). As expected, all three PrP
glycoforms are present in significant amounts inside the cell,
presumably in the ER-Golgi compartment. They are relatively stable over
a 2-h period and are not transported to the cell surface. BFA has a
slight but significantly different effect on the unglycosylated form of
PrP
expressed in the cells transfected with the 129M,D178N
and 129V,D178 constructs. While the unglycosylated form of PrP
is degraded or glycosylated in the 129M,D178N cells at a slightly
greater rate than the unglycosylated form PrP
in control
cells, it is relatively constant in the 129V,D178N cells. Thus, some of
the unglycosylated ``FFI-like'' PrP
may be
digested in the ER-Golgi (Fra and Sitia, 1993). The finding that the
unglycosylated form of the PrP
is relatively stable in this
compartment suggests that during normal processing in untreated cells
the unglycosylated PrP
is digested in the
endosomal-lysosomal system. Our data also support the hypothesis that N-linked oligosaccharides aid in the folding process and
provide structural stability to proteins (Helenius, 1994; Jethmalani et al., 1994).
Taken together these data indicate that the
D178N PrP is unstable and this instability is partially
corrected by N-glycosylation. Thus, while the glycosylated
forms are synthesized and transported to the cell surface in a fairly
normal fashion, although in reduced amounts, the unglycosylated form
remains unstable and is broken down before it reaches the cell surface.
In contrast to the recent reports by Lehmann and Harris(1995, 1996) on
the metabolism of mouse homologues of several pathogenic human
mutations in Chinese hamster ovary cells, the PrP
expressed
in our cells was released from the cell surface by PI-PLC suggesting a
normal association with the cell membrane. In addition, the PrP
produced in our cells was fully digested by proteinase K. Whether
the discrepancies between these results are the consequence of
homologous versus heterologous systems or the sequence
differences between the human and mouse prion protein remains to be
determined.
We examined the PrP from the brain of a FFI
subject with the D178N mutation and one octapeptide repeat deletion in
the mutant allele in an area of the brain lacking PrP
(Bosque et al., 1992; Parchi et al., 1995).
Because of the 8-residue difference, the PrP
and PrP
can be separated by gel electrophoresis. The unglycosylated
PrP
was present at one-third of the amount of the
unglycosylated PrP
. These findings are consistent with the
conclusion that the unglycosylated form of the 129M,D178N PrP
is also unstable in the brain cells of FFI patients and that the
neuroblastoma cells transfected with the 129M,D178N construct
recapitulate the early metabolic events of the PrP
occurring in brain cells of the FFI patients.
The reduced
stability of the D178N PrP may be based on the spatial
proximity of residues 178 and 129. Secondary structure predictions
generated by the alignment of PrP
sequences place these two
residues in close proximity of each other in the hydrophobic core
(Huang et al., 1994; Nguyen et al., 1995). If this
model of PrP secondary structure is correct, methionine and valine at
position 129 would be strategically located to influence the
conformation of the D178N PrP
(Huang et al.,
1994). The brefeldin A experiment suggests that a subtle but
significant difference exists in the processing of PrP
in
129M and 129V cells. The minor differences do not immediately shed
light on the different phenotypes observed in FFI and
CJD
, however, this may reflect the complexity of the
disease process. Within the more complex cellular environment provided
by the intact brain, and the longer time frame needed to develop the
disease, the subtle differences we observe between the 129M and 129V
PrP
proteins may be sufficient to result in two distinct
diseases.
Finally, although the present study provides no direct
data as to the mechanism by which PrP is converted into
PrP
in FFI and CJD
, it suggests that in the
FFI brain the ratios of PrP
and PrP
glycoforms are comparable. As expected from the studies by
Caughey et al.(1990), the prion protein must reach the cell
surface for it to be converted to PrP
explaining the
observation that in FFI very little unglycosylated PrP
is
detected (Monari et al., 1994). Therefore, the
under-representation of the unglycosylated PrP
in FFI
appears to result solely from the degradation of the unglycosylated
form before it reaches the cell surface.