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INTRODUCTION |
The molecular basis of protein conformational diseases like prion
diseases, Alzheimer's disease, and polyglutamine diseases has not been
clarified so far, but protein misfolding, misprocessing, and
aggregation are common features of these diseases. Neuropathologic, genetic, and transgenic studies argue strongly in favor of a causal role of protein misfolding in the pathogenic process. Beyond this conceptual framework, prion diseases are exceptional in that they exist
as sporadic, genetic, and infectious forms. Prion diseases are
characterized by the formation of
PrPSc,1 an
abnormally folded isoform of the cellular prion protein
PrPC, a highly conserved protein mainly present at the
plasma membrane of neuronal and lymphatic cells. During transit through
the secretory pathway, the C-terminal domain of PrPC is
modified by the attachment of two N-linked complex
carbohydrate moieties (Asn180 and
Asn196) (1-4) and a glycosylphosphatidylinositol (GPI)
anchor at serine 230 (5).
Structural studies revealed that the mammalian prion protein is
composed of a flexibly disordered N terminus and a structured C-terminal domain. This autonomously folding domain contains three
-helical regions and a short, two-stranded
-sheet (6-8). Indeed, the C-terminal region seems to confer pathogenicity; after limited proteolysis of PrPSc, a C-terminal fragment, denoted PrP
27-30 (aa ~90-230), is formed which transmits the disease.
Furthermore, studies in transgenic mice indicated that N-terminally
truncated PrPC molecules (
23-89) support
PrPSc propagation (9, 10).
Molecular dynamics simulations of human PrP suggest that the structured
part of the protein is stabilized by the N-linked glycans.
Moreover, the two partially sialylated oligosaccharide moieties
generate a negative electrostatic field that covers the whole surface
of helix 2 and helix 3 (11). Regarding the possible physiological or
pathological relevance, PrP glycosylation has been shown to have an
impact on the conformational transition of PrPC
into PrPSc, to influence the selective neuronal targeting
of PrPSc, and to contribute to the phenomenon of strain
diversity (12-20).
The co- and posttranslational modifications of PrPC are
initiated with the cleavage of the N-terminal signal peptide (aa 1-22) and the transfer of core glycans while the nascent chain is still associated with the translocon. Shortly after the protein is fully translocated the GPI anchor is attached to the acceptor amino acid
close to the C terminus. The final maturation of PrPC
includes the processing of the core glycans into complex type glycans,
a series of enzymatic reactions in the endoplasmic reticulum (ER) and
Golgi compartment. Every asparagine present in the consensus sequence
Asn-X-Ser and Asn-X-Thr can serve as an acceptor
site for N-linked glycosylation; however, not all such sites
present in a polypeptide are actually modified. Similarly, it is
unknown why some of the core glycans, like those present in
PrPC, are terminally processed, whereas others remain as
high mannose structures. So far, it is assumed that the structure of
the polypeptide is an important determinant for both modifications.
In this study, we analyzed the in vivo folding and
maturation of PrPC and showed that helix 1 as well as
membrane attachment play a crucial role in the postranslational
modifications of PrPC.
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EXPERIMENTAL PROCEDURES |
Generation of PrP Mutants--
For ectopic expression of PrP,
pcDNA3.1-3F4 was used, which contains the mouse PRNP modified to
express PrP-L108M/V111M for immunostaining with the monoclonal antibody
3F4 (21). Amino acid deletions and substitutions were introduced by PCR
cloning techniques. The deletions include the following amino acids:
GPI, aa 230-254;
TM, aa 113-133;
H1, aa 141-171;
H1*, aa
144-156;
N, aa 27-89. In PrPmtGPI, the serines at positions
230-232 have been replaced by threonines. For the generation of
PrP-CD4, the CD4 transmembrane sequence and cytoplasmic tail (aa
395-457) were fused to the C terminus of PrP
GPI. CD4 sequences were
amplified by PCR from pSPOX-CD4PrP (22). All amino acid numbers refer to the mouse PrP sequence (GenBankTM accession number
M18070).
Antibodies--
The mouse monoclonal antibody 3F4 was described
earlier (23).
Cell Lines, Transfections, Endo H Digestion,
-Glucosidase, and
-Mannosidase Inhibitor Treatment--
N2a and ScN2a cells were
cultivated as described (24). SH-SY5Y is a human neuroblastoma cell
line (DSMZ number ACC 209). SH-SY5Y cells were grown in Dulbecco's
modified Eagle's medium containing 10% fetal calf serum. Cells were
transfected by a liposome-mediated method using LipofectAMINE Plus
reagent according to the manufacturer's instructions
(Invitrogen). 1-Deoxymannojirimycin (DMJ) or castanospermine (Calbiochem) was dissolved in phosphate-buffered saline (PBS) and added
to the cell culture medium (10 µg/ml). For endo H digestion, protein
lysates were adjusted to 0.5% SDS, boiled for 10 min, and then
digested with endoglycosidase H (New England Biolabs) for 1 h
at 37 °C as specified by the manufacturer.
Detergent Solubility Assay and Proteolysis Experiments--
As
described previously (25), cells were washed twice with cold PBS,
scraped off of the plate, pelleted by centrifugation, and lysed in cold
buffer A (0.5% Triton X-100 and 0.5% sodium deoxycholate in PBS). The
lysate was centrifuged at 15,000 × g for 20 min at
4 °C. After boiling in Laemmli sample buffer, supernatants and
pellets were examined by immunoblotting. For proteolysis experiments, cells were lysed as described above and incubated with proteinase K
(PK) (Roche Molecular Biochemicals) at the concentrations indicated in
Fig. 6 at 4 °C for 60 min. The reaction was terminated by the addition of Pefabloc SC (Roche Molecular Biochemicals) and boiling in
Laemmli sample buffer. Residual PrP was detected by Western blotting.
Northern Blot Analysis--
RNA was extracted and analyzed by
Northern blotting as described earlier (26). RNA bound to the membrane
was hybridized with a 32P-labeled DNA probe specific for
mouse BiP. As a positive control, mock-transfected cells were incubated
with tunicamycin (10 µg/ml) (Sigma) for 1 h prior to RNA extraction.
Western Blot Analysis and Indirect
Immunofluorescence--
Immunoblot analysis and immunofluorescence
were performed as described previously (26). A Leica TCSNT/DMIRB
confocal system (Heerbrugg, Switzerland) was used for confocal
laser-scanning microscopy. Quantification was performed using AIDA
1.0 image analysis software (Raytest).
Metabolic Labeling and Immunoprecipitation--
Cells were
starved for 30 min in methionine-free modified Eagle's medium
(Invitrogen) and subsequently labeled for 30 or 60 min with 300 µCi/ml Pro-mix L-[35S] in vitro cell
label mix (Amersham Biosciences; >37 TBq/mmol) in methionine-free
modified Eagle's medium medium. For the chase, the labeling medium was
removed, and the cells were washed twice and then incubated in complete
medium for the time indicated. Immunoprecipitation of PrP was carried
out as described previously (24). Immunoprecipitation products were
analyzed by SDS-PAGE. Gels were impregnated with Amplify (PerkinElmer
Life Sciences) and exposed to film.
Phospholipase C and Trypsin Treatment--
Cells were washed
twice with ice-cold PBS and then maintained on ice.
Phosphatidylinositol phospholipase C (PIPLC) (Roche Molecular
Biosciences) in PBS was added to the cells for 2 h at 4 °C (0.5 units/ml). The cells were washed extensively with PBS and then lysed
with cold buffer A. The distribution of PrP was analyzed by the
detergent solubility assay (see above). For PIPLC treatment of
extracts, cells were lysed in Triton buffer (0.1% Triton X-100 in
PBS), and PIPLC was added to the lysate. After incubation on ice for
1 h, extracts were fractionated by centrifugation and analyzed by
Western blotting. Trypsin treatment (0.25%, w/v) of the intact cells
was carried out in the cell culture dish on ice. The digest was
terminated by the addition of soy bean trypsin inhibitor (Invitrogen).
The cells were collected by a brief centrifugation, washed with trypsin
inhibitor, and then lysed in cold buffer A. PrP present in the cell
culture supernatant was precipitated by trichloroacetic acid and then
boiled in Laemmli sample buffer.
Expression and Characterization of Recombinant PrP--
Wild
type PrP and mutant PrP sequences were amplified by PCR and cloned into
the vector pPROEX-HTa (Amersham Biosciences) via the EheI
and HindIII restriction sites to add a N-terminal extension
of His6. Transformed Escherichia coli DH5
cells were grown at 37 °C to an OD = 0.6, and protein
expression was induced for 3 h by
isopropyl-1-thio-
-D-galactopyranoside (0.5 mM). PrP was purified under denaturing conditions (8 M urea, 50 mM Tris, pH 8) by
Ni2+-nitrilotriacetic acid chromatography. Proteins were
eluted (8 M urea, 100 mM sodium acetate, pH
4.6), and fractions containing PrP were pooled. For the formation of
the disulfide bonds, proteins were diluted (0.1 mg/ml) and air-oxidized
overnight using CuSO4 (1 µM) as a
catalyst (27). To concentrate PrP, proteins were adjusted to pH
8 with NaOH, rebound to an Ni2+-nitrilotriacetic acid
column, and eluted (8 M urea, 200 mM imidazole, 100 mM sodium phosphate, pH 7). Refolding was induced by
50-fold dilution into 100 mM sodium phosphate, pH 7. For
the limited proteolytic digestion, proteinase K (1 µg/ml; Roche
Molecular Biochemicals) was added, and the samples were incubated at
room temperature for the times indicated in Fig. 5. The reactions were
stopped by adding 5 mM phenylmethylsulfonyl fluoride (Roche
Molecular Biochemicals) and boiling in Laemmli sample buffer. Samples
were analyzed using SDS-PAGE and Coomassie Blue staining.
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RESULTS |
Helix 1 Directs Folding and Maturation of PrPC in the
Secretory Pathway--
To study PrPC folding in the
secretory pathway of mammalian cells, we expressed different PrP
mutants with deletions proximal to helices 2 and 3 in mouse
neuroblastoma (N2a) cells (Fig.
1A). The highly structured
helix 2-loop-helix 3 motif was left untouched, because it contains the
two complex N-linked glycans and the disulfide bridge,
modifications known to be important for proper folding (1, 2, 7).
Initially, we used the glycosylation status of the PrP mutants to
monitor the efficiency of folding. After transient expression in N2a
cells, the PrP mutants were analyzed by endo H digestion and
Western blotting (Fig. 1B). Endo H only cleaves high mannose
and hybrid structures; consequently, complex type glycans of wild type
PrP (wtPrP) could not be liberated by this enzyme (Fig. 1B,
wt). Similarly, PrP mutants devoid of the complete N
terminus (
N, aa 27-89 deleted) or the putative transmembrane domain
(
TM, aa 113-133 deleted) resisted endo H cleavage, revealing the
presence of complex N-linked glycans (Fig. 1B,
N and
TM). However, after endo H digestion,
the electrophoretic mobility of PrP
H1 (aa 141-171 deleted) and of
the double mutant PrP
N
H1 (aa 27-89 and 141-171 deleted)
increased, indicating that mutants lacking helix 1 were not
complex-glycosylated (Fig. 1B,
H1 and
N
H1). Since the
H1 deletion includes
helix 1 (aa 144-156) together with the second
-strand (aa 160-162)
of the prion protein, we also tested a construct in which only helix 1 has been deleted (PrP
H1*). PrP
H1* expressed in N2a cells showed
exactly the same phenotype as PrP
H1, indicating that the absence of
helix 1 was responsible for the observed effect. Of note, a faster
migrating PrP species present in cell extracts from PrP
TM-expressing
cells was also sensitive to endo H digestion (Fig. 1B,
TM).

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Fig. 1.
Helix 1 directs complex glycosylation of the
prion protein. N2a cells were transiently transfected with
different PrP deletion mutants depicted in A. Cell lysates
were prepared and either incubated with endo H or mock-treated
(EndoH +/ ) prior to Western blotting analysis using the
monoclonal anti-PrP antibody 3F4 (B). Molecular size markers
are indicated as bars on the left side
of the panels and represent 36, 30, and 16 kDa.
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To investigate the apparent lack of complex glycosylation of PrP
H1
in more detail, we performed metabolic labeling experiments and
analyzed PrP by immunoprecipitation. In addition, transfected cells
were treated with DMJ, which inhibits
-mannosidase I, thereby interfering with terminal processing of N-linked glycans
(28). After the pulse, two major PrP species were detected in wtPrP- and PrP
TM-expressing cells (Fig.
2A, lanes
1 and 3). The faster migrating bands represented
PrP containing high mannose N-linked glycans, which could be
released by endo H (data not shown). The upper diffuse band was
composed of PrP glycoforms that had undergone terminal processing
(i.e. contained complex type glycans). Notably, these
complex glycosylated PrP species of wtPrP and PrP
TM were missing in
cells that had been treated with DMJ (Fig. 2A,
lanes 2 and 4). In contrast, PrP
H1
was exclusively present as a faster migrating PrP species, identical to
PrP
H1 generated in the presence of DMJ (Fig. 2A,
lanes 5 and 6). Pulse/chase
experiments revealed that the majority of high mannose glycoforms of
wtPrP and PrP
TM were converted to complex structures within 30 min
(Fig. 2B, lanes 2 and 4).
In contrast, the N-linked glycans of PrP
H1 were not processed into complex structures (Fig. 2B, lane
6).

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Fig. 2.
Deletion of helix 1 interferes with the
conversion of high mannose N-linked glycans into
complex structures. N2a cells were transiently transfected with
wtPrP and two mutants lacking either helix 1 ( H1) or the
putative transmembrane domain ( TM). PrP was analyzed by
immunoprecipitation using the monoclonal antibody 3F4.
A, cells were cultivated in the presence (DMJ +)
or absence (DMJ ) of the -mannosidase inhibitor DMJ (10 µg/ml) and metabolically labeled with [35S]methionine
for 30 min. B, cells were metabolically labeled and either
analyzed directly (chase ) or incubated in fresh medium
for an additional 30 min (chase +) prior to the
immunoprecipitation of PrP.
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These initial experiments revealed that deletion of the complete N
terminus or of the internal putative transmembrane domain does not
interfere with the complex glycosylation of PrP. Deletion of helix 1, however, generates a high mannose glycoform of PrP that is not
terminally processed.
PrP
H1 Is Not Membrane-anchored and Is Secreted into the Cell
Culture Medium--
To analyze the cellular trafficking of PrP
H1,
intact cells were incubated with trypsin to remove all cell surface
proteins (Fig. 3A) or with
PIPLC to liberate GPI-anchored proteins specifically (Fig.
3B). After trypsin treatment, lysates prepared from wtPrP- or PrP
TM-expressing cells had lost most of their PrP, indicating that it was localized at the cell surface (Fig. 3A,
lanes 2 and 4). Similarly, after PIPLC
treatment, wtPrP and PrP
TM were found almost quantitatively in the
cell culture supernatant (Fig. 3B, wt,
TM, lane 4). In contrast, the
relative amount of PrP
H1 present in extracts prepared from trypsin-
or PIPLC-treated cells was not reduced when compared with the
mock-treated control, nor was the cell culture supernatant of
PIPLC-treated PrP
H1-expressing cells enriched in PrP (Fig. 3,
A, lane 6, and B,
H1, lane 4). Because the trypsin
and PIPLC treatments were performed at 4 °C, possible secretion of
PrP
H1 would not have been detected in these assays. Therefore,
transfected cells were cultivated in serum-free medium for 3 h at
37 °C, and then the cell culture supernatant was analyzed for the
presence of PrP. In contrast to wtPrP, PrP
H1 was efficiently
secreted under these conditions. Interestingly, the faster migrating,
endo H-sensitive glycoform of PrP
TM was also secreted into the cell
culture supernatant at 37 °C, indicating once more that the deletion
of the putative transmembrane domain generated a heterogeneous PrP
population, with the major fraction being folded correctly (Fig.
3B, lanes 5 and 6).

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Fig. 3.
Deletion of helix 1 interferes with the GPI
anchor attachment. A-C, N2a cells were transiently
transfected to express either wtPrP (wt) or PrP lacking the
putative transmembrane domain ( TM) or helix 1 ( H1) A, PrP H1 is not significantly
expressed on the cell surface. Cells were incubated on ice with trypsin
(Trypsin +) to remove cell surface proteins. After cell
lysis, residual PrP was detected by Western blotting. As a control,
mock-treated cells were analyzed in parallel (Trypsin ).
B and C, PrP H1 is not GPI-anchored.
B, live cells were incubated at 4 °C with PIPLC to
release GPI-anchored proteins from the cell surface or mock-treated
(PIPLC ) (lanes 1-4). In parallel
plates (lanes 5 and 6), cells were
incubated in fresh medium for 3 h at 37 °C. PrP present in the
cell lysate (Lysate) or cell culture supernatant
(Medium) was detected by Western blotting. C,
cell lysates were prepared in Triton buffer (0.1% Triton X-100 in PBS)
and incubated on ice with PIPLC or mock-treated (PIPLC
+/ ). After centrifugation, PrP present in the detergent-soluble
(Sup) or -insoluble (Pellet) fraction was
detected by Western blotting.
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The results of these studies indicated that PrP
H1 was not
GPI-anchored; instead, it was secreted into the cell culture medium. To
demonstrate further that PrP
H1 failed to receive a GPI anchor, we
performed an in vitro PIPLC digestion. After cell lysis in cold Triton X-100 buffer (0.1% in PBS) and subsequent centrifugation, wtPrP, like other GPI-anchored proteins, fractionated into the detergent-insoluble phase (Fig. 3C, Pellet,
lane 1). Incubation of the cell lysates with
PIPLC prior to centrifugation rendered the majority of wtPrP and
PrP
TM Triton X-100 soluble, indicative of their release from the GPI
anchor (Fig. 3C, Sup, lanes
2 and 4). PrP
H1, however, displayed a
different solubility profile. Interestingly, a substantial fraction of
PrP
H1 was readily solubilized in Triton X-100 buffer at 4 °C
(Fig. 3C, Sup, lane 5).
Moreover, after PIPLC digestion of the extracts, the relative amount of PrP
H1 found in the supernatant fraction was not increased (Fig. 3C, Sup, lane 6).
Consistent with the results described above (Fig. 3, A and
B), the endo H-sensitive glycoform of PrP
TM was also
partially soluble in 0.1% Triton X-100 (Fig. 3C,
Sup, lane 3).
In the next step, confocal laser-scanning microscopy was performed to
corroborate the findings from the biochemical analysis presented above.
As expected, wtPrP, PrP
TM, and PrP
N were located predominantly on
the surface of N2a cells. In cells expressing PrP
H1 or PrP
N
H1,
however, PrP immunoreactivity was found exclusively intracellularly,
following a distinct pattern indicative of the Golgi compartment (Fig.
4, N2a). Since PrP
H1 is
efficiently secreted, the intense staining of the post-ER compartment
is probably not due to an intracellular retention but rather
illustrates the highest local concentration of PrP
H1 and
PrP
N
H1 on their way to the cell surface. To exclude the
possibility that either the described effects were specific for N2a
cells or endogenous wtPrP might influence the processing of transfected
PrP mutants, we included SH-SY5Y cells in our analysis, an established
neuroblastoma cell line of human origin that does not express
detectable amounts of endogenous PrP (data not shown). Indeed, the
analysis of SH-SY5Y cells shown in Fig. 4 corroborated our findings in
N2a cells; in SH-SY5Y cells staining of PrP
H1 and PrP
N
H1 was
also most intense in the Golgi compartment, whereas wtPrP, PrP
TM,
and PrP
N were again targeted to the plasma membrane (Fig. 4,
SH-SY5Y).

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Fig. 4.
Localization of PrP in intact cells. N2a
and SH-SY5Y cells grown on glass coverslips were transfected with
plasmids encoding the indicated PrP mutants. Expression of PrP was
analyzed by indirect immunofluorescence of permeabilized cells using
confocal laser-scanning microscopy.
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Taken together, our data indicated that deletion of helix 1 has a
profound effect on posttranslational modifications and cellular trafficking of PrP; GPI anchor attachment is prevented, and a high
mannose glycoform of PrP
H1 is secreted into the cell culture medium.
A Highly Conserved Hydrophobic Residue in Helix 3 Is Required for
Correct Folding of PrPC--
Our data presented above
revealed that the presence of helix 1 is a prerequisite for correct
folding and trafficking of PrPC. We then thought of a way
to interfere with the formation of helix 1 without having to delete
this domain. Based on the NMR structure of mouse PrPC, a
hydrophobic side chain at residue 204 of helix 3 interacts with
phenylalanine 140, glutamic acid 145, tyrosine 148, and tyrosine 149 and thereby provides an essential stabilization of helix 1 structure
(Fig. 5A). Of note, this
residue, either methionine (human, mouse) or isoleucine (hamster), is
extremely well conserved between species (29). To disrupt helix 1 packing, we substituted serine or arginine for methionine 204 to create
PrP-M204S or PrP-M204R (Fig. 5B). The Western blot analysis
presented in Fig. 5C indicated that both PrP-M204S and
PrP-M204R did not receive complex glycans and were secreted into the
cell culture supernatant, similarly to PrP
H1. Support for the
assumption that wtPrP and PrP-M204S had different conformations was
provided by a biochemical characterization of the recombinant proteins.
Wild type PrP and PrP-M204S were expressed in E. coli,
purified, and oxidized as described under "Experimental Procedures"
(Fig. 5D). After dilution into an aqueous buffer, the
proteins were subjected to a limited proteolysis (Fig. 5E).
In the case of r-wtPrP, a fragment of about 15 kDa was generated, most
likely comprising the C-terminal globular domain. Western blot analysis
with an antibody specific for the C terminus of PrP supported this idea
(data not shown). In contrast, r-PrP-M204S was digested without a
detectable intermediate (Fig. 5E).

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Fig. 5.
An invariant hydrophobic amino acid in helix
3 is required for folding and maturation of PrPC.
A, methionine at residue 204 stabilizes helix 1 packing. The
overall structural organization of the prion protein is depicted
schematically by a worm representation of the protein backbone (in
gray). On the right, a close-up of the nonpolar
packing interactions of Met204 with neighboring amino acids
is shown. The side chain of Met204 is drawn in a
stick representation, whereas the side chains of
the interacting amino acids Phe140, Glu145,
Tyr148, and Tyr149 are shown additionally with
their accessible molecular surface superimposed. The surfaces were
calculated with a probe radius of 1.4 Å as implemented in the program
WebLab viewer (MSI). B and C, hydrophilic
replacements of Met204 prevent complex glycosylation and
membrane attachment. Two point mutants replacing Met204
either by Ser or Arg (B) were transiently expressed in N2a
cells (C). The cells were cultivated in fresh medium for
3 h at 37 °C. PrP present in the cell culture medium
(M) and the cell lysate (L) was analyzed by
Western blotting. To detect N-linked high mannose glycans,
the cell lysates were incubated with endo H prior to the Western blot
analysis (EndoH +). D and E, wtPrP and
PrP-M204S were expressed in and purified from bacteria. D,
after purification and oxidation, recombinant (r) PrP
was analyzed by SDS-PAGE and Coomassie Blue staining. E, PK
(1µ/ml) was added to r-PrP in aqueous buffer, and the samples were
incubated for the times indicated. The proteins were then analyzed by
SDS-PAGE and Coomassie Blue staining. A proteolytic intermediate that
appeared only after a limited digestion of r-wtPrP is indicated by an
arrow. Marker proteins are shown on the right
(M) and represent 50, 36, 22, and 16 kDa.
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Thus, PrPC maturation seems to be dependent on an invariant
hydrophobic amino acid located in helix 3, most likely due to a stabilization of helix 1 packing. A limited proteolytic digestion of
recombinant wtPrP and PrP-M204S indicated conformational differences between these proteins.
Disruption of Helix 1 Restores Core Glycosylation of
PrP
GPI--
Based on previous studies, deletion of the GPI anchor
results in the formation of detergent-insoluble PrP molecules that are not glycosylated (30-33). Notably, this misfolded PrP
GPI molecule is a preferred substrate for the formation of a PK-resistant PrP isoform in an in vitro-based conversion model. The phenotype
of full-length PrP lacking the C-terminal GPI anchor signal sequence is
illustrated in Fig. 6A;
PrP
GPI expressed in N2a cells was mainly unglycosylated and secreted
into the cell culture medium. Surprisingly, when we deleted helix 1 from PrP
GPI, we found that the double mutant was core-glycosylated
(Fig. 6A,
H1
GPI). Encouraged by
our previous results with the amino acid substitution at position 204, we introduced the same mutation into the GPI anchor-deficient PrP
construct. Similarly to the deletion of helix 1, the M204S substitution
prevented the formation of unglycosylated PrP
GPI; PrP-M204S
GPI was present as a high mannose glycoform sensitive to
endo H digestion (Fig. 6A, M204S
GPI). Support for the
assumption that PrP
GPI and PrP-M204S
GPI had different
conformations was provided by a limited proteolytic digestion. Whereas
PrP-M204S
GPI was PK-sensitive, a significant resistance against PK
digestion was observed for PrP
GPI (Fig. 6B).

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Fig. 6.
Glycosylation of PrP is mediated by the
C-terminal signal sequence, PrP folding, and membrane anchoring.
A, deletion or destabilization of helix 1 promotes core
glycosylation of PrP GPI. N2a cells were transiently transfected with
PrP mutants lacking the C-terminal GPI anchor attachment signal in the
wild type ( GPI) or disrupted helix 1 ( H1 GPI and M204S GPI)
background. Secretion of PrP at 37 °C into the cell culture medium
(M) and the glycosylation status of PrP present in the cell
lysate (L) were analyzed as described in the legend to Fig.
5. B, helix 1 directs the formation of PK-resistant
aggregates. N2a cells expressing PrP GPI or PrP-M204 GPI were lysed
in detergent buffer and subjected to a limited digestion with
proteinase K (1:100, 4 °C for 60 min). Remaining PrP was analyzed by
Western blotting. C, a nonfunctional GPI anchor sequence
leads to core glycosylated PrP. A PrP mutant with a mutated -site of
the C-terminal signal sequence (mtGPI) was expressed in N2a
cells and analyzed as described for A. D, an
artificial transmembrane anchor restores maturation of
PrPC. N2a cells were transiently transfected with PrP-CD4.
The supernatant cell culture medium (M) and the cell lysate
(L) were analyzed as described for A. To monitor
the glycosylation status of PrP-CD4, cell lysates were digested with
endo H (EndoH +). Localization of PrP-CD4 was visualized by
indirect immunofluorescence of permeabilized cells using confocal
laser-scanning microscopy as described in the legend to Fig. 4.
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These results revealed that deletion or destabilization of helix 1 restored core glycosylation of PrP
GPI in vivo. In
contrast to PrP
GPI, PrP-M204S
GPI and PrP
H1
GPI were
glycosylated and sensitive to a limited proteolytic digestion.
The C-terminal GPI Anchor Signal Sequence Mediates Core
Glycosylation of wtPrP--
From previous studies, it is known that
the location of the glycosylation consensus site with respect to its
distance from the C terminus influences the efficiency of
N-linked glycosylation (34). In case of the prion protein,
both acceptor sites for the core glycans are very close to the C
terminus (Asn180 and Asn196). To analyze the
role of the C-terminal GPI anchor signal sequence in the core
glycosylation of PrPC, we mutated the cleavage/attachment
site (
site) for the GPI anchor. In PrPmtGPI, the serines at
positions 230-232 have been replaced by threonines, which was expected
to impair the efficiency of GPI anchoring (35). In this way, the
original length of the nascent PrP polypeptide chain (aa 1-254) was
preserved without allowing the GPI anchor attachment to occur.
Interestingly, PrPmtGPI was glycosylated; however, the
N-linked glycans of PrPmtGPI were sensitive to endo H
(i.e. were not converted into complex structures (Fig.
6C, mtGPI, Endo H +)). To
further investigate the role of a membrane anchor in the formation of
complex glycosylated PrPC, we replaced the C-terminal GPI
anchor sequence with the heterologous CD4 transmembrane domain. As
expected from previous studies (22, 36), PrP-CD4 was complex
glycosylated and present at the plasma membrane (Fig.
6D).
By comparing the absence of the GPI anchor signal sequence with a
nonfunctional GPI anchor signal sequence, we found that the presence of
the GPI anchor signal sequence, whether functional or not, is necessary
to allow the attachment of the core glycans. However, interfering with
the native folding of PrPC by disrupting helix 1 relieves
the need for the extra stretch of C-terminal amino acids. For the
conversion of high mannose glycans to complex type glycans, membrane
anchoring of PrPC is required, be it via the authentic GPI
anchor or a heterologous transmembrane anchor.
Misfolded PrP Does Not Induce the Unfolded Protein Response and Can
Be Reinternalized by Heterologous Cells--
Removal of the three
terminal glucose residues from the core glycan is generally regarded as
an indication of a successful folding of the nascent chain (37, 38). To
monitor this modification, we inhibited the ER glucosidases by
castanospermine (39) and analyzed different PrP constructs by
immunoprecipitation. As expected, formation of complex glycosylated
wtPrP or PrP
TM was inhibited by castanospermine. Instead, immature
PrP species appeared, which were clearly distinguishable from high
mannose PrP generated in the presence of DMJ (Fig.
7A, wtPrP,
TM). The difference in electrophoretic mobility can be
attributed to the three additional glucose residues present in each of
the two core glycans. Similarly, PrP
H1 present in
castanospermine-treated cells was clearly larger than in the untreated
control, indicating that the initial processing of the core glycan
(i.e. the removal of the terminal glucose residues) was not
impaired in PrP
H1 (Fig. 7A,
H1). Similar
results were obtained for PrP-M204S and PrPmtGPI (data not shown).

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Fig. 7.
The immature PrP molecules undergo initial
trimming of the glucose residues and do not induce an unfolded protein
response. A, cells transfected with wtPrP, PrP TM, or
PrP H1 were cultivated in the presence of castanospermine
(CSP) or DMJ (10 µg/ml) and metabolically labeled with
[35S]methionine for 60 min. PrP was analyzed by
immunoprecipitation and compared with untreated controls ( ).
Different glycoforms are indicated. complex:
N-linked glycans with complex structure;
G3Man9GNA2,
core glycans composed of 2 N-acetylglucosamine
(GNA), 9 mannose (Man), and 3 terminal glucose
(G) residues;
Man9GNA2, core glycans
without the terminal glucose residues. B, Northern blot
analysis of RNA prepared from untransfected (mock) or
PrP-transfected cells using a BiP-specific probe. As a positive
control, mock-transfected cells were incubated with tunicamycin (10 µg/ml) for 1 h prior to RNA extraction. C, secreted
PrP can interact with heterologous cells. Cell culture medium collected
from N2a cells transiently transfected with wtPrP, PrP-M204S, PrPmtGPI,
or PrP GPI was filtered and then added to untransfected N2a cells.
These cells were lysed 24 h later, and PrP present in the
detergent insoluble (P) and soluble fraction (S)
was analyzed by immunoblotting with the anti-PrP antibody 3F4.
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To detect possible adverse effects of the expression of mutant PrP, we
analyzed the unfolded protein response pathway. Induction of the
unfolded protein response is indicative of the accumulation of immature
or misfolded protein in the ER and leads to the up-regulation of a
variety of proteins, such as the ER chaperone BiP (40, 41). However,
none of the PrP mutants induced increased transcription of BiP in a
detectable manner (Fig. 7B). As a positive control, strong
induction was found after tunicamycin treatment of N2a cells. To
analyze the fate of secreted PrP molecules further, we collected the
supernatant cell culture medium, removed any detached cells by
filtration, and applied the medium to untransfected N2a cells. 24 h later, the N2a cells were analyzed for the uptake of heterologous
PrP, which could be distinguished from endogenous PrP by the presence
of the 3F4 epitope. Interestingly, a significant amount of heterologous
PrP was present in the lysates of N2a cells which had been incubated
with the cell culture medium containing secreted PrP-M204S, PrPmtGPI,
or PrP
GPI, whereas no 3F4-positive PrP was present in cells
incubated with medium derived from cells transfected with wtPrP. It
should be noted that heterologous PrP was exclusively present in the
detergent-insoluble fraction of the N2a cells (Fig. 7C,
P).
In summary, these experiments indicated that the high mannose glycans
of the analyzed PrP mutants are initially processed in the ER by
glucosidases. Expression of these PrP mutants does not induce the
unfolded protein response; the immature PrP glycoforms lacking a GPI
anchor are rather secreted and can be reinternalized by heterologous cells.
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DISCUSSION |
A conformational transition of PrPC into
PrPSc is implicated in the pathogenesis of prion diseases.
Our study provides experimental evidence that deletion of the
N-terminal domain of PrPC including the putative
transmembrane region and the first
strand have no impact on the
formation of complex glycosylated and GPI-anchored PrPC.
Helix 1, however, plays a crucial role in the folding pathway of
PrPC; it seems to be essential for GPI anchoring and
complex glycosylation. Whereas the GPI anchor signal sequence allows
core glycosylation to occur, whether or not attachment of the GPI
anchor takes place, membrane anchorage of PrPC is necessary
for the terminal processing of glycans (Fig. 8).
Core Glycosylation of PrPC Is Dependent on the
C-terminal Signal Sequence--
The addition of the core glycans
occurs while the emerging polypeptide chain is still associated with
the translocon. Constraints are imposed by the location of the
glycosylation consensus site with respect to its distance from the C
terminus (34), and in PrP both glycosylation sites are close to the C
terminus (Asn180 and Asn196). Consequently,
PrP
GPI lacking the C-terminal amino acids of the GPI anchor signal
sequence was glycosylated very inefficiently, indicating that the
C-terminal signal sequence in addition to mediating the GPI anchor
attachment is also required for efficient core glycosylation. Indeed,
the expression of PrPmtGPI with a mutated, nonfunctional GPI anchor
signal sequence supported this hypothesis; in this case, core
glycosylation was restored, most likely by providing an extra stretch
of amino acids distal to the glycosylation sites. Interestingly, we
found that the C-terminal amino acids of the GPI anchor signal sequence
were only required to ensure glycosylation of wtPrP. Interfering with
the formation of helix 1, exemplified by PrP
H1 or PrP-M204S,
relieved the need for a C-terminal extension to allow core
glycosylation to occur; in contrast to PrP
GPI, PrP
H1
GPI and
PrP-M204S
GPI were efficiently core-glycosylated similarly to
PrPmtGPI. Unglycosylated PrP
GPI has also been observed in earlier
studies (30, 31, 33). In one study, the authors discussed the
possibility that PrP glycosylation and membrane anchorage are
cooperative processes (30). However, the fact that PrP
H1
GPI,
PrP-M204S
GPI, and PrPmtGPI can be core-glycosylated in the absence
of a membrane anchor points to a different scenario. In this context,
it is important to note that folding of PrP is one of the most rapid
folding reactions measured to date (42). It therefore is conceivable
that PrP
GPI, due to the C-terminal truncation, is released from the
translocon more rapidly than wtPrP or PrPmtGPI. Deletion or
destabilization of helix 1 in PrP
H1
GPI or PrP-M204S
GPI, on the
other hand, might delay cotranslational protein folding and thereby
prolong the association of these mutants with the translocon. Support
for the assumption that destabilization of helix 1 might be the cause
for the observed differences in folding was obtained by the biochemical
characterization of recombinant PrP. The appearance of a proteolytic
fragment after limited PK digestion of recombinant wtPrP but not
PrP-M204S indicated variant protein conformations.
GPI Anchor Attachment Is Dependent on PrP Folding and Is Required
for Complex Glycosylation--
A variety of proteins with diverse
physiological functions, such as signal transduction, neuronal
guidance, or cell-cell interaction, are linked to the plasma membrane
through a GPI anchor (43-45). GPI modification occurs in the ER
rapidly after synthesis, with the cleavage of the C-terminal signal
peptide followed by the transfer of the GPI anchor from a dolichol
carrier to the acceptor amino acid, called the
site (46). Like the
N-terminal signal peptides, the C-terminal GPI anchor signal sequences
do not have conserved sequences but rather characteristic features;
only a few amino acids with small side chains are allowed at the
,
the
+ 1, and the
+ 2 positions, and a hydrophobic domain is
required about 10-12 amino acids distal to the
site (35, 45,
47-49). It is known that a single amino acid substitution in the GPI
anchor signal sequence can significantly interfere with GPI anchor
attachment (50); however, much less is known about the role of the
protein conformation in this process.
Our study presents evidence that in addition to the amino acid sequence
of the GPI anchor signal sequence, protein folding has a major
impact on the efficiency of GPI anchor attachment. Despite the presence
of a correct signal sequence, PrP
H1 and PrP-M204S did not receive a
GPI anchor. To our knowledge, this is the first example to show that
protein folding can modulate the transfer
of a GPI anchor. The analysis of other PrP mutants supported this
hypothesis. Deletion of the complete N terminus, which lacks any
known structure, did not interfere with the GPI anchoring of PrP
N.
Interestingly, the majority of PrP
TM, with a deletion of the
putative transmembrane domain, was GPI-anchored, suggesting that this
domain has only a minor impact on the native folding of PrP. Of note,
an absence of the GPI anchor was not only seen for the mutant that
lacks helix 1 (PrP
H1) but also for PrP-M204S and PrP-M204R,
containing only a single amino acid substitution in helix 3. Based on
the NMR structure of wtPrP, the most likely explanation for this effect
is that helix 1 is destabilized by preventing hydrophobic interactions
with helix 3. Although the analysis of recombinant PrP implies
conformational differences between wtPrP and PrP-M204S, other
mechanisms, such as a direct effect on the GPI anchor transfer
reaction, can not be excluded at the moment. A detailed analysis of
recombinant PrP mutants with refined biophysical methods,
preferentially NMR, will help to answer this question.

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Fig. 8.
Determinants of PrP folding in
vivo. The scheme displays a summary of the PrP mutants
analyzed in this study and their degree of post-translational
modifications. Similarly to wtPrP, mutants lacking the unstructured N
terminus (PrP N) or the putative transmembrane
domain (PrP TM) were complex-glycosylated and
GPI-anchored. The GPI anchor could be replaced by a heterologous
transmembrane domain (PrP-CD4) without interfering with
postranslational modifications. A second group of mutants was
characterized by a disrupted helix 1. In this case, core glycans were
attached, and terminal glucose residues were trimmed; however, GPI
anchor attachment and conversion of the high mannose glycans into
complex structures failed. Interestingly, a nonfunctional C-terminal
GPI anchor signal sequence generated a similar PrP glycoform
(PrPmtGPI). A third phenotype was observed by deleting the
GPI anchor signal sequence from wtPrP. PrP GPI was mainly
unglycosylated and partially resistant to proteolytic digestion. Of
note, core glycosylation of PrP GPI could be restored by disrupting
helix 1 (PrP H1 GPI or
PrP-M204S GPI).
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Impairment of the GPI anchor attachment also had a major impact on
further posttranslational modifications, the conversion of the core
glycans into complex structures. None of the analyzed PrP mutants
without a GPI anchor was complex-glycosylated. However, complex
glycosylation was not specific for GPI-anchored PrP; PrP-CD4 fixed to
the membrane via the heterologous CD4 transmembrane domain was
complex-glycosylated as well. The absence of terminal glycosylation could be due either to an altered conformation of the PrP mutants, such
as PrP
H1 or PrP-M204S, or to the lack of a membrane anchor. In the
later scenario, membrane anchoring could provide a specialized environment (i.e. the membrane surface, necessary for native
folding of PrPC). The membrane components might directly
promote PrPC folding or indirectly prevent intermolecular
PrP interactions of hydrophobic domains. Support for the notion that
the membrane anchor might also influence the maturation of wtPrP was
provided by PrPmtGPI, which is characterized by a mutated GPI anchor
cleavage/attachment site. It can be assumed that this mutant has a
structure similar to wtPrP; nevertheless, it was not
complex-glycosylated. Interestingly, an influence of the membrane
environment on the structure of recombinant PrP was recently shown by
two in vitro studies (51, 52).
Implications for the Physiological Role of PrPC and the
formation of PrPSc--
Incomplete postranslational
modifications are often used as a diagnostic marker for proteins that
have failed to acquire their native conformation. Such immature
proteins are mainly degraded through a mechanism known as ER-associated
protein degradation (53-56). Previous studies showed that the
degradation of wild type and mutant PrP via the ER-associated protein
degradation pathway is possible (57-60). However, in our hands,
neither unglycosylated PrP
GPI nor the high mannose PrP mutants were
efficiently degraded via this pathway (data not shown). Instead, the
majority of these mutants were transported through the secretory
pathway and finally secreted, consistent with earlier reports (30, 31,
33). This might suggest that the PrP mutants were not sensed as
misfolded by the cellular quality control systems. Two observations
support such a hypothesis. First, the terminal glucose residues were
removed from the core glycans, and second, even the overexpression of PrP mutants did not result in the activation of the unfolded protein response. Interestingly, the secreted PrP mutants had a long lifetime and could interact with heterologous cells. Recently, several studies
shed light on possible physiological functions of PrPC,
including a role in signal transduction pathways, in protein-protein interactions or in copper homeostasis (61-66). Therefore, it will be
important to determine whether immature PrP species are physiologically active or might have a trans-dominant activity. Indeed, an earlier study with transgenic mice indicates a such a dominant activity. Expression of a mutant PrP lacking a part or the complete putative transmembrane domain caused ataxia and neuronal cell death (67). Similar to our studies in cell culture, this mutation did not abolish
the formation of complex glycosylated PrPC, but it might
well be that the secreted high mannose glycoform of PrP
TM we
observed was also present in these transgenic mice. It would be
interesting to analyze whether expression of PrP
H1 or PrPM204S,
generating exclusively the secreted high mannose glycoform, had a
similar or even aggravated phenotype in transgenic mice.