1 Laboratorium für Molekulare Biologie-Genzentrum-Institut für
Biochemie der LMU München, Feodor-Lynen Str. 25, D-81377 Munich,
Germany
2 Veterinary Medicine and Primate Husbandry, German Primate Center, Kellnerweg
4, D-37077 Goettingen, Germany
* Author for correspondence (e-mail: Weiss{at}lmb.uni-muenchen.de)
Accepted 1 August 2002
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Summary |
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Key words: Prion protein, PrP mutants, Proteinase K resistance, Semliki-Forest virus (SFV) system, Processing, Glycosylation
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Introduction |
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The physiological function of PrP is unclear. It has been proposed that PrP
plays a role in synaptic processes
(Collinge et al., 1994), in the
regulation of circadian activity rhythms and sleep
(Tobler et al., 1996
) and in
copper transport (Hornshaw et al.,
1995
). Later, a function in the survival of purkinje cells
(Sakaguchi et al., 1996
) was
suggested, but very recently it has been reported that upregulation of the
prion-like protein doppel causes Purkinje cell degeneration in
Prnp0/0 mice instead of PrP depletion
(Moore et al., 1999
). More
recently, a superoxide dismutase activity
(Brown et al., 1999
) and a role
in signal transduction (Mouillet-Richard, 2000) for PrP have been suggested.
Owing to the absence of any phenotype for PrP described in various reports
(Bueler et al., 1992
;
Lledo et al., 1996
;
Manson et al., 1994
), the only
confirmed role of PrPc is in development of TSEs, which it appears
to be essential for (Bueler et al.,
1993
). Cellular PrP is synthesized in the rough endoplasmatic
reticulum (rER) and is transported via the Golgi and secretory granules to the
surface of neuronal cells where it is anchored to the plasma membrane by its
glycosyl phosphatidylinositol (GPI) moiety
(Rogers et al., 1991
). Three
different PrP glycoforms differing in their glycosylation degree have been
observed on the cell surface: diglycosylated (70%), monoglycosylated (25%) and
unglycosylated PrP (5%) (Caughey et al.,
1989
; Monari et al.,
1994
; Petersen et al.,
1996
). Recently, we identified the 37 kDa laminin receptor
precursor (LRP) as an interactor for the prion protein
(Rieger et al., 1997
) (for
reviews, see Gauczynski et al.,
2001a
; Rieger et al.,
1999
). Cell-binding and internalization studies on neuronal and
non-neuronal cells have demonstrated that the 37 kDa/67 kDa laminin receptor
acts as the cell-surface receptor for the cellular prion protein
(Gauczynski et al., 2001b
).
Direct and heparan sulfate proteoglycan (HSPG)-dependent interaction sites
mediating the binding of cellular PrP to its receptor have been identified
(Hundt et al., 2001
).
Additional heparan sulfate (HS)-binding domains in PrPc have been
described (Warner et al.,
2002
). Cell culture experiments demonstrated the 37 kDa/67 kDa
laminin-receptor-dependent binding and internalization of recombinant
GST::human PrP generated in insect cells and glycosylated human PrP
synthesized in BHK cells transfected with recombinant SFVRNA
(Gauczynski et al.,
2001b
).
High-level expression and purification of recombinant, glycosylated prion
proteins in mammalian cells are essential for a better understanding of the
physiological function of PrPc and biochemical processes
responsible for familial prion diseases. The synthesis and study of wild-type
as well as mutant PrP in cell culture systems allows a better insight into the
biology of these proteins, owing to the presence of important organelles,
membranes and other cellular co-factors that are necessary for the correct
processing, trafficking and localization of the protein. Therefore, we used
the Semliki-Forest virus (SFV) system to express high amounts of glycosylated
wild-type and mutant disease-associated prion protein in cultured mammalian
cells. The SFV system supplies a multitude of advantages for the expression of
recombinant proteins in mammalian cells: (i) large-scale production for up to
72 hours post-transfection; (ii) a broad host range; (iii) modifications such
as glycosylation in a correct and sufficient way; and (iv) an easy and fast
transfection procedure with in-vitro-transcribed RNA. SFV is an insect-borne
alphavirus and belongs to the family of Togaviridae (Schlesinger, 1986). Its
viral genome consists of capped and polyadenylated single-stranded RNA of
positive polarity and encodes its own RNA polymerase. SFV expression vectors
are based on a cDNA copy of the viral genome. Here, viral structural genes are
deleted and replaced by the gene of interest. Owing to the remaining viral
replicase, which leads to an efficient production of recombinant RNA within
the cell, a high-level synthesis of the foreign protein occurs
(Liljestrom and Garoff,
1991).
In the work described here, we have generated recombinant disease-related mutant isoforms of human PrP in BHK cells transfected with recombinant SFV RNAs. The FFI-associated mutant PrP D178N/M129 and the CJD-related PrP+9OR were efficiently and highly glycosylated when expressed in cultured cells. We further examined biochemical features, such as the glycosylation status and proteinase K resistance of recombinant mutated PrP in comparison to recombinant wild-type PrP, as well as subcellular localization in transfected BHK cells. We observed that both mutants were proteinase K resistant at 8 µg/ml and share this biochemical hallmark with infectious PrPSc. Therefore, the SFV system acting as a powerful expression system for high-level production of glycosylated prion proteins also functions as an appropriate cell culture model for inherited human prion diseases.
To facilitate purification and to introduce an additional epitope for immunodetection, we introduced a FLAG-tag at different positions in the cellular human and bovine PrP. FLAG-tag insertions at the C-terminus of PrP were located two amino acids nearer to the N-terminus than the GPI-anchorage site and allowed the purification of predominantly diglycosylated human and bovine PrP from cell lysates by anti-FLAG antibody affinity chromatography.
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Materials and Methods |
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Recombinant pSFV plasmid constructions
pSFV-1 (Liljestrom and Garoff,
1991), pSFV3-lacZ (Life Technologies) and the ORFs from human PrP
and bovine PrP were used. The plasmid DNAs pSFV1-boPrP1-264, pSFV1-huPrP1-253
(Krasemann et al., 1996
),
pSFV1-huPrP-FFI (encoding aa 1-253 of the human PrP containing a
FFI-associated mutation at codon 178 Asp
Asn plus Met at position 129)
and pSFV1-huPrP+9OR (encoding aa 1-253 of the human PrP including nine
additional octapeptide repeats) (Krasemann
et al., 1995
) were used. pSFV1-huPrP22FLAG was generated by the
QuikChangeTM site-directed mutagenesis method (Stratagene) employing
pSFV1-huPrP1-253 DNA as a template. Construction of pSFV1-huPrP227FLAG
(insertion of the FLAG-tag encoding sequence between codons 227 and 228 of the
human PrP sequence) was described elsewhere
(Hundt et al., 2002
).
Construction of pSFV1-boPrP239FLAG
The insertion of a FLAG-tag-encoding sequence between codon 239 and 240 of
the bovine PrP sequence was performed by PCR using the pSFV1-boPrP1-264
plasmid DNA as a template. A 125 bp fragment (with the FLAG-encoding sequence
inserted) that encodes the C-terminus of boPrP was amplified, introducing a
HinfI restriction site (at codons 232-233 of the endogenous sequence)
at the 5' end, the tag-encoding sequence between codon 239 and 240 as
well as a SmaI site at the 3' end. This PCR fragment encoding
the C-terminal part of boPrP was digested with HinfI and
SmaI and ligated via the HinfI restriction site to a 707 bp
fragment encoding the N-terminal part of boPrP from pSFV1-boPrP1-264 digested
with HinfI and SmaI. The ligated DNA fragments were cloned
into the expression plasmid pSFV1 via the SmaI restriction sites
resulting in pSFV1-boPrP1-239FLAG240-264. All constructs were confirmed by
dideoxy sequencing.
SFV-mRNA generation by in vitro transcription
DNAs pSFV3-lacZ (Life Technologies), pSFV1-huPrP1-253, pSFV1-huPrP-FFI,
pSFV1-huPrP+9OR, pSFV1-huPrP22FLAG, pSFV1-huPrP227FLAG, pSFV1-boPrP1-264 and
pSFV1-boPrP239FLAG were linearized with SpeI following purification
by phenol-chloroform extraction. Transcriptions were carried out in a total
volume of 50 µl containing 1.5 µg linearized plasmid DNA, 10xSP6
transcription buffer (0.4 M Tris-HCl, pH 8.0 at 20°C; 60 mM
MgCl2; 100 mM dithiothreitol; 20 mM spermidine), 1 mM of each ATP,
CTP and UTP, 500 µM of GTP, 1 mM of m7G(5')ppp(5')G,
50 units of RNasin and 50 units of SP6 RNA polymerase and incubated for 2
hours at 37°C. The correct length of the transcripts was verified by
agarose gel electrophoresis. RNA was stored at -20°C.
Cell transfection
For transfection, BHK cells were trypsinized, washed once in PBS (w/o
MgCl2 and CaCl2) and resuspended in PBS, resulting in
1x107 cells per ml. 800 µl of the cell suspension was
mixed with the individual SFV-RNA and then transferred to a 0.4 cm cuvette.
Transfections were carried out by electroporation at room temperature by two
consecutive pulses at 0.8 kV/25 µF using a Gene Pulser (BioRad). Cells were
diluted in complete growth medium and plated on 10 cm cell culture dishes. For
immunofluorescence microscopy, a maximum one-tenth of the volume of the
electroporated cells (8x105) was diluted in complete growth
medium and transferred into 35 mm wells containing a sterile glass coverslip.
Cells were incubated for at least 24 hours. Transfection efficiencies as
determined by transfecting SFV3-lacZ control RNA followed by X-gal staining
were almost 100% for BHK cells.
Deglycosylation assays
8x106 BHK cells were transfected with recombinant SFV RNA
as described above and plated on 10 cm culture dishes. 24 hours
post-transfection, cells were washed once with PBS, scraped off in PBS,
harvested by centrifugation and finally lysed in 250 µl N-glycosidase F
buffer by repeated freezing and thawing. The crude lysates were obtained by
centrifugation at 20,200 g 4°C for 15 minutes. A 20 µl
aliquot of the supernatants was then treated with 2 units of N-glycosidase F
(Roche Diagnostics) at 37°C over night. In addition, aliquots of 50 µl
of the supernatants were incubated with or without 0.5 units/ml
endoglycosidase H (Roche Diagnostics) at 37°C for 3 hours. The reactions
were stopped by addition of SDS-loading buffer and heating at 95°C for 5
minutes. Deglycosylation was monitored by SDS-PAGE followed by western
blotting and detection with the monoclonal anti-PrP antibody 3B5.
Proteinase K digestion
8x106 BHK cells were transfected with recombinant SFV RNA
as described above and plated on 10 cm culture dishes. 24 hours post
transfection cells were washed once with PBS, scraped off in PBS and harvested
by centrifugation. Cells were resuspended in lysis buffer (10 mM Tris pH 7.5,
100 mM NaCl, 10 mM EDTA, 0.5% Triton-X100, 0.5% DOC) and finally lysed on ice
for 15 minutes. The crude lysates were obtained by centrifugation at 20,200
g 4°C for 15 minutes. Proteinase K (Roche Diagnostics) was
added to a 20 µl aliquot to get the final concentrations of 2, 4 and 8
µg/ml. Reactions were carried out at 37°C for 30 minutes, stopped with
0.5 mM pefabloc and analysed by western blotting using the anti-PrP mAB 3F4.
Protein concentrations were determined using an ELISA reader.
Western blot analysis
After addition of Laemmli buffer, protein samples were separated on 12%
SDS-polyacrylamide gels by SDS-PAGE and transferred to PVDF membranes
Immobilon P (Millipore Corp.) for 1.5 hours at 55 V. The membranes were
blocked with I-Block (Tropix) in Tris-buffered saline pH 7.5 supplemented with
0.05% Tween, probed with one of the monoclonal anti-PrP antibodies (3B5; 3F4)
or the polyclonal anti-LRP antibody W3 and thereafter with an appropriate
peroxidase-coupled secondary antibody. The immunoreactivity was visualized by
enhanced chemiluminescence (NENTM Life Science Products) on Kodak BioMax
MR-1 films and by staining with diaminobenzidine-tetrahydrochloride
(Sigma).
Immunofluorescence analysis
To investigate the cellular localization of recombinant prion proteins in
BHK cells, we used immunofluorescence microscopy. 24 hours post-transfection,
cells were washed three times with PBS and fixed with 4% paraformaldehyde.
Non-permeabilized cells were fixed with 2% paraformaldehyde. After rinsing
three times with PBS, cells were permeabilized for cytoplasmic staining with
0.2% Triton X-100 (10 minutes for 4°C). The preparation was saturated with
a 10% FCS solution (in PBS) for 1 hour at room temperature, washed and
incubated with the primary antibody mAB 3B5 diluted in PBS with 10% FCS for 1
hour at room temperature. After washing three times with PBS, the preparations
were diluted in saturation buffer and incubated in the dark for 45-60 minutes
with the secondary antibody (goat anti mouse) conjugated with indocarbocyanine
(Cy3) (red). For nuclear staining 1 µg/ml
4'-6-diamidine-2-phenylindole (DAPI) for 10 minutes at room temperature
was used. Please note that both the primary and secondary antibodies were
added after fixing the cells. The coverslip was mounted with aqueous mounting
medium (Fluoromount®), and the slides were examined using an axioviert
fluorescence microscope (Zeiss) with appropriate filters. Immuno fluorescence
images were processed using Metamorph software®.
Purification of FLAG-tagged prion proteins by anti-FLAG antibody
chromatography
Transfection of BHK cells with the recombinant SFV-huPrP227FLAG or the
recombinant SFV-boPrP239FLAG RNA was performed as described above. The total
volume of the electroporated cells was plated on 10 cm dishes containing 15 ml
of complete growth medium followed by incubation for at least 48 hours at
37°C. 48 hours post-transfection, cells were harvested, washed once with
PBS and then lysed in PBS supplemented with 0.1% Triton-X100 by repeated
freezing and thawing. The crude lysates were obtained by centrifugation at
20,200 g 4°C for 15 minutes. The FLAG-tagged proteins from
the supernatants were bound over night to anti-FLAG M2 affinity gel (Sigma) by
rotating at 4°C. Beads with immobilized protein were then washed four
times with TBS, eluted overnight by competition with 1 ml TBS containing 100
µg/ml FLAG peptides (Sigma) and dialyzed against 20 mM HEPES, pH 7.4. The
purity and the concentration of the proteins were checked by SDS
polyacrylamide gel electrophoresis followed by silver staining of the gel.
Antibodies
For western blot analyses, monoclonal anti-PrP antibodies 3F4 (Chemicon) or
3B5 (G. Hunsmann) and the polyclonal anti-LRP antibody W3
(Gauczynski et al., 2001b)
were used. For immunofluorescence analyses, mAb 3B5 and a secondary Cy3
(indocarbocyanine)-conjugated antibody (used at 1:200 dilutions) (Jackson
Laboratories and Southern Biotechnology, respectively) were used.
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Results |
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Proteinase K status of recombinant cellular PrP and
disease-associated PrP variants
The partial resistance to proteinase K is a major hallmark of
PrPSc. By contrast, cellular PrP is completely proteinase K
sensitive. To investigate whether recombinant FFI- and CJD-associated PrP
isoforms synthesized in SFV-transfected BHK cells share PrPSc-like
properties, proteinase K was added to total cell extracts at increasing
concentrations of 2, 4 and 8 µg/ml followed by incubation at 37°C for
30 minutes (Fig. 2, upper
panel). Both mutant PrPs, the FFI-associated PrP as well as the CJD-related
insertion mutant, were resistant towards proteinase K amounts up to 8
µg/ml, yielding proteinase-K-resistant fragments with molecular weights
ranging from 23 to 25 kDa (Fig.
2, lanes 8 and 12). By contrast, wild-type PrP was completely
digested after treatment with 8 µg/ml proteinase K
(Fig. 2, lane 4). Comparable
signals for the 37 kDa-LRP (in the absence of proteinase K) revealed that
equal amounts of protein have been loaded
(Fig. 2, bottom panel). Protein
concentrations in the samples digested with proteinase K were adjusted.
|
Cellular localization of recombinant prion proteins in transfected
BHK cells
In order to prove whether or not recombinant prion proteins, either
wild-type or mutant, are transported to the surface of transfected BHK cells,
non-permeabilized cells (Fig.
3, left panels) were analyzed by immunofluorescence (IF)
microscopy. In cells, wild-type human or bovine PrP traffics to the cell
surface where it appears partially in a punctuated manner
(Fig. 3B,E). Virtually all
cells synthesizing PrP+9OR (Fig.
3C) or PrP-FFI (Fig.
3D) showed similar staining patterns and comparable expression
levels on the cell surface, suggesting that mutated PrP variants are correctly
processed to the cell surface. To visualize PrP in the interior of transfected
cells, cells were permeabilized prior to immunostaining. Expression of
wild-type human and bovine PrP (Fig.
3B,E, right panels) as well of both mutated PrPs
(Fig. 3C,D, right panels)
revealed a wide-spread staining pattern including areas surrounding the
nucleus and regions of the cytoplasm. Thus, regarding the subcellular
localization, no differences between PrP mutants and the cellular forms were
detectable. Cells transfected with SFV RNA encoding the viral replicase showed
no detectable PrP staining on either the cell surface or in the interior of
permeabilized cells (Fig. 3A),
suggesting that BHK cells express no or only marginal amounts of endogenous
PrPc.
|
Expression of glycosylated FLAG-tagged prion protein
To facilitate purification of human and bovine prion proteins and to
introduce an additional epitope for immunodetection, we inserted a FLAG-tag at
the C-terminus near the GPI-anchor adhesion site resulting in the constructs
huPrP227FLAG and boPrP239FLAG and at the N-terminus behind the signal peptide
resulting in huPrP22FLAG (Fig.
4A). FLAG-tagged PrP isoforms expressed were highly glycosylated
in transfected BHK cells (Fig. 4B, lanes
1-3 and 4C, lanes 1 and 2). To confirm the glycosylation state,
samples were deglycosylated by treatment with N-glycosidase F, resulting in
augmentation of the non-glycosylated PrP forms
(Fig. 4B, lanes 4-6 and 4C, lanes 3 and 4). The correct processing of FLAG-tagged prion proteins to the
cell surface was further analyzed by treatment with endoglycosidase H (Endo H)
(Fig. 4D). A portion of the
non-mutated FLAG tagged prion protein huPrP22FLAG is resistant to Endo H and
therefore reaches the Golgi in which high mannose sugars are modified
(Fig. 4D, lane 2). By contrast,
both PrP forms carrying the tag close to the GPI attachment site, the
huPrP227FLAG and primarily the boPrP239FLAG, feature Endo H sensitivity,
suggesting that only low amounts of the highly glycosylated proteins are
properly transported via ER and Golgi to the cell surface
(Fig. 4D, lanes 4 and 6).
|
Cellular localization of FLAG-tagged prion proteins in transfected
BHK cells
To investigate whether FLAG-tagged prion proteins are localized at the
surface of transfected BHK cells, non-permeabilized cells were stained with
anti-PrP antibody 3B5 and analyzed by IF microscopy. As already shown in
Fig. 3, human and bovine
wild-type PrP were efficiently transported to the cell surface of transfected
BHK cells (Fig. 3B,E;
Fig. 5B,E). Owing to the
overexpression of recombinant protein in recombinant SFV RNA-transfected
cells, wild-type human and bovine PrP were also detectable inside
permeabilized cells (Fig. 5B,E,
right panels). Permeabilization of transfected cells prior to immunostaining
reveals intracellular deposits of both wild-type and FLAG-tagged prion
proteins (Fig. 5, right
panels). Whereas huPrP22FLAG was detectable on the cell surface at comparable
levels (Fig. 5C, left panel),
both prion proteins carrying the FLAG-tag insertion close to the
GPI-attachment site were transported efficiently only in a minority of cells
to the surface of transfected BHK cells
(Fig. 5D,F), which is in
agreement with the negligible Endo H resistance of these proteins
(Fig. 4D), suggesting that the
FLAG-tag at this position hampers trafficking of PrP to the cell surface.
Indeed, most of the protein remains within the interior of the cell as shown
by IF analysis on permeabilized cells (Fig.
5D,F, right panels). BHK cells transfected with SFV RNA encoding
for the viral replicase showed neither surface nor intracellular staining
(Fig. 5A), confirming that no
or only a negligible amount of cellular PrP is synthesized by BHK cells.
|
Purification of glycosylated FLAG-tagged PrP
Purification of FLAG-tagged prion proteins was performed by anti-FLAG
antibody chromatography. FLAG-tagged proteins from cell lysates were
immobilized to anti-FLAG M2-conjugated agarose beads
(Fig. 6A,B, lanes 2).
Silver-stained SDS gels revealed that mainly the diglycosylated PrP form was
purified by this one-step purification process
(Fig. 6C). Both kinds of
recombinant proteins were recognized by the PrP-specific antibody 3B5
(Fig. 6A,B, lanes 3). The
huPrP22FLAG could hardly be purified by either anti-FLAG M1 or M2 antibody
chromatography probably due to insufficient binding of the FLAG epitope to the
anti-FLAG antibody immobilized on sepharose under native conditions.
|
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Discussion |
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Processing and trafficking of wild-type and mutated human prion
proteins
Correctly processed PrP traffics to the cell surface where it is anchored
to the plasma membrane by its GPI moiety
(Rogers et al., 1991). We
investigated the subcellular location of recombinant wild-type and
disease-associated mutant PrP by IF microscopy. Very recently, it has been
observed that mutant PrP molecules linked to familial prion diseases partially
are retained in the ER (Ivanova et al.,
2001
). This led to the conclusion that several pathogenic
mutations, that is, point and insertion mutations within the Prn-p
gene, exert influence on the trafficking of PrP and that protein quality
control might play an important role in TSEs
(Ivanova et al., 2001
) (for
summary see Table 1). For
several mutations an additional mode of membrane association resulting in
resistance to enzymatic cleavage of the GPI-anchor was reported
(Lehmann and Harris, 1995
;
Lehmann and Harris, 1996a
), a
phenomenon that is also typical for infectious PrPSc. Previous
studies have already shown that mutant prion proteins are inefficiently
processed to the cell surface and accumulate in intracellular compartments
such as the ER, Golgi and endosomes/lysosomes
(Capellari et al., 2000
;
Jin et al., 2000
;
Petersen et al., 1996
;
Singh et al., 1997
;
Zanusso et al., 1999
) (for
summary see Table 1). By
contrast, IF analysis of non-permeabilized, transfected BHK cells expressing
huPrP-FFI or huPrP+9OR revealed similar surface staining in comparison to
cells expressing wild-type human PrP at their cell surface. In addition, the
intracellular staining of wild-type as well as both mutant PrP isoforms
revealed the same wide-spread pattern encompassing nearly the entire interior
of the cell. This effect might be caused by the SFV system, owing to the
hyperexpression of recombinant protein while competing out the host
translation machinery. Therefore, it might be difficult to detect subcellular,
trapped PrP in cells overexpressing this protein. However, we cannot exclude
the possibility that mutant PrP molecules are partially retained in cellular
compartments such as the ER. Endoglycosidase H (Endo H) digestions, however,
revealed that the FFI- and CJD-associated PrP mutants are partially Endo H
resistant, suggesting that high mannose sugars have been added to most of the
recombinant prion proteins followed by modifications on their way through the
ER and the Golgi apparatus to the cell surface. This finding is in contrast
with the hypothesis that the mutant prion proteins might be trapped in
compartments of the secretory pathway and instead confirms the finding
obtained by IF analyses (Fig.
3) that mutant prion proteins are located at the cell surface.
|
Proteinase K resistance of mutated human prion proteins
One major biochemical characteristic of PrPSc is its resistance
to proteinase K. Some mutant isoforms of the mouse prion protein carrying
mutations homologous to familial human prion diseases were produced in stably
transfected CHO cells and displayed scrapie-like biochemical properties such
as proteinase K resistance (for summary see
Table 1) and detergent
insolubility (Daude et al.,
1997; Lehmann and Harris,
1996a
; Lehmann and Harris,
1996b
). Furthermore, hamster insertion mutants encompassing
additional octarepeats showed protease resistance when synthesized in cultured
cells (Table 1)
(Priola and Chesebro, 1998
).
To investigate whether genetically linked human PrP mutants expressed in
transiently transfected BHK cells behave in a similar manner to
PrPSc, total cell extracts were treated with proteinase K at final
concentrations of 2, 4 and 8 µg/ml for 30 minutes at 37°C. Under these
conditions, we detected a protease-resistant fragment of FFI-associated and
CJD-related human PrP with a molecular weight ranging from 23 to 25 kDa.
Previous reports describing partial PK resistance for disease-associated PrP
used lower proteinase K concentrations and/or shorter incubation times (for a
summary, see Table 1). The
mouse PG14 PrP mutant encompassing nine additional octarepeats was proteinase
K sensitive at a concentration of 2 µg/ml when synthesized in stably
transfected BHK cells using the Sindbis replicon
(Ivanova et al., 2001
). In
addition, the human PrP mutant D178N/M129 was totally degraded when digested
with low amounts of PK for 1 hour or with 5 µg/ml PK for at least 5 minutes
(Table 1). It is difficult to
compare the characteristics of mutant PrPs of different species, expression
systems and cells and the diverse conditions of proteinase K digestion.
Employing the SFV system, we were able to synthesize glycosylated,
cell-surface orientated and proteinase-K-resistant (up to 8 µg/ml) human
PrP mutants, recommending its capability as a cellular model system for
familial human prion diseases. Bioassays will prove whether these PK-resistant
PrP molecules will harbor endogeneous infectivity.
Processing, trafficking and purification of FLAG-tagged human and
bovine prion proteins
To purify prion proteins rapidly and simply from total cell extracts and to
introduce an epitope suitable for specific immunogenic detection, a FLAG-tag
was inserted within the human PrP-encoding sequence close to the GPI anchorage
site at residue 227 or at the N-terminus following the signal peptide sequence
at residue 22. A homologous FLAG-tag insertion within the bovine PrP sequence
termed boPrP239FLAG was also constructed. The locations of the FLAG-tag
insertion consisting of the eight amino-acid sequence
Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys, influenced the processing of bovine and human
PrPc in transfected BHK cells. HuPrP22FLAG was processed normally
and expressed at high levels similar to wild-type PrP on the cell surface of
transfected cells. By contrast, introduction of the FLAG-tag close to the
GPI-attachment site hampered the transport of huPrP227FLAG and boPrP239FLAG to
the cell surface. Only low amounts of both proteins reached the cell surface.
One possible explanation for this phenomenon might be the hampering effect of
the FLAG-tag, which was inserted close to the GPI attachment site, leading to
a disruption of the transit of huPrP227FLAG and boPrP239FLAG to the cell
surface and a retention of these proteins within cellular compartments of the
secretory pathway such as the ER. This explanation is supported by the finding
that huPrP227FLAG and boPrP239FLAG are both sensitive towards Endo H
digestion, whereas huPrP22FLAG is more Endo H resistant. Recombinant
glycosylated huPrP227FLAG and boPrP239FLAG have been purified from crude
lysates by anti-FLAG-antibody chromatography. Such an epitope-tagged prion
protein, which can be purified simply and rapidly from total cell extracts
might represent a useful tool to elucidate the physiological function of
PrPc and its role in the pathological mechanisms of TSEs.
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Acknowledgments |
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References |
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