From the National Institute of Animal Health,
Tsukuba, Ibaraki 305-0856, Japan, § Nippi Research Institute
of Biomatrix, Adachi, Tokyo 120-8601, Japan, ¶ Brain Science
Institute, RIKEN, Wako, Saitama 351-0198, Japan, and
Institute
for Virus Research, Kyoto University, Sakyo-ku,
Kyoto 606-8507, Japan
Received for publication, September 25, 2000, and in revised form, January 8, 2001
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ABSTRACT |
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The central event in prion disease is thought to
be conformational conversion of the cellular isoform of prion protein
(PrPC) to the insoluble isoform PrPSc. We
generated polyclonal and monoclonal antibodies by immunizing PrPC-null mice with native PrPC. All seven
monoclonal antibodies (mAbs) immunoprecipitated PrPC, but
they immunoprecipitated PrPSc weakly or not at all, thereby
indicating preferential reactivities to PrPC in solution.
Immunoprecipitation using these mAbs revealed a marked loss of
PrPC in brains at the terminal stage of illness. Histoblot
analyses using these polyclonal antibodies in combination of
pretreatment of blots dissociated PrPC and
PrPSc in situ and consistently demonstrated the
decrease of PrPC at regions where PrPSc
accumulated. Interestingly, same mAbs showed immunohistochemical reactivities to abnormal isoforms. One group of mAbs showed reactivity to materials that accumulated in astrocytes, while the other group did
so to amorphous plaques in neuropil. Epitope mapping indicated that
single mAbs have reactivities to multiple epitopes, thus implying dual
specificities. This suggests the importance of octarepeats as a part of
PrPC-specific conformation. Our observations support the
notion that loss of function of PrPC may partly underlie
the pathogenesis of prion diseases. The conversion of PrPC
to PrPSc may involve multiple steps at different sites.
Prion diseases, such as scrapie in sheep and goats and
Creutzfeldt-Jakob disease in humans, are transmissible
neurodegenerative disorders (spongiform encephalopathy). A major
component of the infectious agent responsible for these diseases is
thought to be a post-translationally modified form of a host-encoded
membrane glycoprotein
PrPC,1 termed
PrPSc (1). Conformational differences are observed between
PrPC and PrPSc. PrPSc has a large
number of It is also ideal to use antibodies distinguishing conformations of
PrPC and PrPSc. However, since PrPC
is phylogenetically highly conserved and is expressed in various tissues, including immune systems (6, 7), immune tolerance means
restriction in generating antibodies. To overcome this problem, other
workers have used mice devoid of PrPC to generate
monoclonal antibodies (mAbs) to prion protein (PrP) (8-13). Almost all
mAbs so far obtained recognize both PrPSc and
PrPC. Exceptionally, one of the mAbs (clone 15B3) was
reported to be specific for PrPSc (11). To gain insight
into conversion processes, it is important to characterize epitopes
reflecting conformational differences between PrPC and
PrPSc.
PrPC, expressed on the cell surface with a C-terminal
glycosylphosphatidylinositol anchor, is expressed in most tissues of uninfected animals. The function of PrPC has remained
obscure. However, it has been suggested that PrPC has a
role for normal synaptic function (14, 15). Furthermore, ablation of
the prion protein gene (Prnp) caused cell death in some
circumstances in vivo and in vitro (16, 17).
Although recent studies suggested that the ectopic expression of the
Prnd gene, encoding a homolog of PrPC,
was involved in cell death (32), expression of PrPC
antagonized cell death signaling in both cases (17, 18). These results
raised the possibility that functional loss of PrPC might
partly underlie the pathogenesis of prion diseases. However, the
expression level of the mRNA of the Prnp gene is
unaltered in cases of scrapie infection (19). Thus, the equilibrium of PrPC and PrPSc in the pathogenesis remained to
be examined.
We immunized Prnp Generation of Prnp Antibodies--
Fluorescein isothiocyanate-conjugated anti-mouse
IgG and IgM, horseradish peroxidase (HRP)-conjugated anti-mouse IgM,
fluorescein isothiocyanate-conjugated anti-rabbit IgG, and
HRP-conjugated anti-rabbit IgG antibodies were purchased from Jackson
Immunoresearch Laboratories. Biotinylated anti-mouse IgM antibody was
purchased from Vector Laboratories. Anti-PrP peptide (position of the
peptide on mouse PrP was amino acid residues 213-226) rabbit serum
(Ab.Mo-VI) (21) was used as a reference antibody.
Western Blot Analysis (WB)--
WB was carried out, as described
(21). In brief, mouse brain extracts were fractionated by
SDS-polyacrylamide gel electrophoresis and blotted onto polyvinylidene
difluoride membrane (Immobilon-P; Millipore Corp.). After blocking the
membrane with Block Ace (Dainippon Seiyaku), the blots were immersed in
primary antibodies at 37 °C for 1 h. Then after washing, the
blots were incubated with either HRP-conjugated anti-mouse IgG
(1:5,000), anti-mouse IgM (1:5,000), or anti-rabbit IgG (1:5,000) and
developed in an ECL Western blotting detection reagent (Amersham
Pharmacia Biotech).
Immunization and Fusion Protocols--
For immunization, 10%
brain homogenates or 2 × 107 thymocytes of
Prnp+/+ mice were given intraperitoneally to
Prnp PrP recombinant Baculovirus--
Autographa
californica nuclear polyhedrosis virus (AcNPV) and
recombinant virus stocks were grown and assayed in monolayers of
Spodoptera frugiperda ovary cells IPLB-SF-21AE
(SF21AE) (23) in TC100 medium containing 10% (v/v) fetal bovine serum.
The Prnp open reading frame fragment was amplified by
polymerase chain reaction and subcloned into the BamHI site
of the transfer vector pAcYM1S. Primers used were
5'-GGGATCCAGTCATCATGGCGAACCT-3' and 5'-GGGATCCACGAGAAATGCGAAGGAA-3'.
SF21AE cells were cotransfected with AcNPV DNA and transfer vector DNA
by Lipofectin (Life Technologies, Inc.), and then the recombinant
baculovirus PrP-AcNPV was selected, as described (24). The expression
of recombinant PrP was confirmed by indirect fluorescent assay (IFA)
and WB by using Ab.Mo-VI (21). PrP-AcNPV-infected cells were washed
with phosphate-buffered saline and then fixed with acetone for 5 min at
room temperature. These fixed cells were used for screening of
hybridomas, using an IFA test (see details below). The antigenicity of
the recombinant PrP was evaluated by immunizing 7-8-week-old
Prnp Scrapie Prions and Animals--
The Obihiro strain of scrapie
prion (PrPSc), which had been passaged in ICR/Slc mice more
than 10 times, was prepared from infected brains homogenized in
phosphate-buffered saline and intracerebrally inoculated into
3-week-old C57BL/6 (Prnp+/+) and
Prnp Immunohistochemical Analysis--
Mouse brains were frozen in
liquid N2 and then cut into 5-µm-thick cryosections. The
sections were fixed with acetone for 10 min and then reacted with
primary antibodies for 30 min. After washing three times in
phosphate-buffered saline, the sections were incubated with fluorescein
isothiocyanate-conjugated anti-mouse IgG or IgM. Recombinant
baculovirus-infected cells were also examined using the same procedure.
Immunoprecipitation (IP)--
The brains were homogenized
(10%, w/v) in 0.5% Nonidet P-40, 0.5% sodium deoxycholate, 100 mM NaCl, 10 mM EDTA, in 10 mM
Tris-HCl (pH 7.5) and then precleared by centrifugation at 11,000 × g for 30 min at 4 °C. The samples were incubated with
antibodies at 4 °C for 1 h. The antigen-antibody complexes were
collected on beads. Immuno Assist MG-PP (Kanto Chemical) and protein G
beads (Amersham Pharmacia Biotech) were used for mAbs and Ab.Tg,
respectively. After washing three times, the beads were mixed with
SDS-sample buffer and boiled for 5 min. The precipitated PrP was
detected by WB, using biotinylated Ab.Tg and streptavidin-HRP (Life
Technologies, Inc.).
Histoblot Analysis--
Histoblot analysis was done as described
(25). Briefly, mouse and hamster brains were frozen in liquid
N2, and 8-µm-thick cryosections were prepared and placed
on glass slides. The glass slides carrying the sections were
immediately pressed onto Immobilon-P membranes for 1 min. The membranes
were used with one of the following pretreatments: 100 µg/ml
proteinase K for 1 h at 37 °C or hydrated autoclaving
pretreatment for 10 min at 115 °C. To detect PrPSc,
hydrated autoclaving pretreatment was done following proteinase digestion. The pretreated membranes were incubated with Ab.Tg. Bound
antibodies were detected using HRP-conjugated anti-mouse IgG and
chemiluminescence, as described for WB.
Epitope Analysis--
To determine the epitopes, antibodies were
incubated with a gridded array of peptides comprising 122 polypeptides
of 13 amino acids, shifted by 2 amino acids and covering the entire
mouse sequence. The peptides were covalently attached at COOH termini to a cellulose support, as individual spots (Jerini Biotools, Berlin).
Bound antibodies were detected using HRP-conjugated anti-mouse IgG or
IgM and chemiluminescence, as described for WB.
Generation of Mice Devoid of PrPC--
We generated
mice devoid of PrPC by gene targeting. As shown in Fig.
1, the entire open reading frame and
3'-end of the second intron were replaced with the
pgk-neo gene cassette. The genotype of mice was
determined by either Southern blotting or polymerase chain reaction
analysis of DNA prepared from tails of the mice. An example of Southern
blot analysis of tail DNAs from crosses between heterozygotes is shown
in Fig. 1B. Immunoblot analysis of lysates from cerebra
showed the absence of PrPC in
Prnp Generation of Polyclonal and Monoclonal Antibodies to
PrP--
Prnp Characterization of mAbs--
All of these mAbs reacted to neither
PrPC nor PrPSc in WB and showed only a weak
reaction to PrPSc fractions, using ELISA. These mAbs were
IgMs. Despite evidence that the mAbs did not react with PrP in WB, the
immunoreactivity of mAbs was confirmed by immunoprecipitation.
Immunoprecipitates from brain lysates with mAbs were size-fractionated,
blotted onto a membrane, and probed with Ab.Tg. All mAbs efficiently
immunoprecipitated PrP from brain samples of wild-type healthy mice.
Two mAbs, 4A3 and 11H1, representing two distinct groups that showed
different properties in IFA, as described later, were extensively
characterized. Representative data from mAbs 4A3 and 11H1 are shown in
Fig. 3A (lane
2). These mAbs reacted weakly to brain homogenates from scrapie-affected mice (Fig. 3A, lanes
3). No signals were detected with
Prnp Dynamics of PrPC to PrPSc Conversion in the
Brain--
The findings that the mAbs immunoprecipitated small amounts
of PrP from infected brains (Fig. 3A) suggested the
exhaustion of PrPC caused by the exponential conversion of
PrPC to PrPSc. Thus, we measured the
equilibrium of PrPC and PrPSc during the
pathogenesis. Homogenates from mouse brains at 0, 9, 12, 15, and 20 weeks postinfection, immunoprecipitated with mAbs and Ab.Tg, were
probed using the biotinylated Ab.Tg (Fig. 3, C and
D). The immunoprecipitates with Ab.Tg might represent the
total amount of PrP (Fig. 3C). The total amount of PrP was also assessed by WB without proteinase K pretreatment (Fig.
3B). Proteinase K resistant products (PrPSc)
accumulated in the brains at terminal stages of illness (Fig. 3B). The PrPSc was first detected at 15 weeks
postinfection and dramatically increased by 20 weeks postinfection
(Fig. 3B). Consistent with the WB analysis, the total PrP
immunoprecipitated with Ab.Tg increased within 20 weeks postinfection
(Fig. 3C). In contrast, the mAbs-related immunoprecipitates
were reduced dramatically by 20 weeks postinfection (Fig.
3D). The results were obtained with mAb 11H1 and similar results were seen with mAb 4A3 (data not shown). These results suggested that PrPC is exhausted by conversion to
PrPSc, at the terminal stage of illness.
Spatial Distribution of PrPC and
PrPSc--
To gain insight into the regional distribution
of PrPC and PrPSc, we attempted to define
conditions to discriminate PrPC and PrPSc,
using histoblot analysis. The pan-PrP-specific Ab.Tg was used as a
probe. Without any pretreatment of the membrane, diffuse signals were
observed in both scrapie-affected and mock-affected wild-type mice
(Fig. 5A, first
column from the left). These signals disappeared
by proteinase K treatment (Fig. 5A, second
column). With autoclave pretreatment, the signal from
scrapie-affected mice was enhanced (Fig. 5A,
third column), while the signal from mock
affected mice was slightly reduced. When the autoclave pretreatment was
followed by proteinase K digestion (Fig. 5A,
fourth column), the signal from scrapie affected
mice was further enhanced. The same treatment completely eliminated the
signal from the mock affected mice. No signals were detected in case of
Prnp Immunohistochemical Dissociation of Multiple Isoforms--
Ab.Tg
recognized PrPC in Prnp+/+ mice
(Fig. 6, panel 2). Homogeneous
signals representing PrPC were distributed throughout the
entire brain areas except for nuclei. In the scrapie-affected mouse
brain, granulous and/or amorphous signals appeared (Fig. 6,
panel 1). Seven mAbs were classified into two
groups in terms of reactivities to distinct materials in IFA. All mAbs
showed homogeneous and weak signals in the normal unaffected mouse
brain (Fig. 6, panels 5 and 8, and
Table II) in agreement with their
reactivities to PrPC by IP. However, for the
scrapie-affected samples, four (clones 1H8, 4A3, 6B5, and 9A8) showed
stellar structures (Fig. 6, panel 4, and Table
II), while the others (7H8, 8G6, and 11H1) showed plaque structures in
neuropil (Fig. 6, panel 7, and Table II). Double
staining of the former group together with the anti-GFAP antibody
indicated that stellar materials were PrPs that had accumulated in
astrocytes (data not shown). There were no signals in
Prnp Epitope Mapping--
A single mAb recognizes PrPC,
under a certain condition (in solution) but recognizes abnormal
isoforms under another condition (acetone-fixed tissue section). To
elucidate the molecular basis for these observations, we determined the
epitopes recognized by mAbs and Ab.Tg, using a gridded array of
synthetic peptides consisting of 122 13-residue peptides,
sequentially shifted in steps of 2 amino acids and covering the whole
mouse PrP sequence (Fig. 7A).
Ab.Tg recognized distinct clusters of polypeptides, probably reflecting
the conformation of native PrPC used as an immunogen. Four
discontinuous regions appeared to constitute dominant epitopes.
Interestingly, all mAbs examined recognized multiple discontinuous
peptides, as summarized in Fig. 7B. Two mAbs (4A3 and 11H1)
as well as Ab.Tg recognized sequences around octarepeat sequences. In
the 4A3-1 segment, a pair of tripeptides ((T/G/S/)WG; positions
55-57, 63-65, 71-73, 79-81, and 87-89) seemed to constitute an
epitope. Two perfect repeats (GQPHGGGWG; positions 57-65 and 81-89),
but not repeats containing a substituted residue
(GQPHGGSWG; positions 65-73 and 73-81), were preferential epitopes recognized by Ab.Tg. mAb 4A3 also recognized a different region (4A3-2, GNDWEDR; positions 141-147), the sequence of which completely overlapped with the epitope of mAb 15B3-1, which is specific
for PrPSc (11). mAbs 11H1 and 8G6 also recognized segments
(11H1-2: MIHFGND (positions 137-143); 8G6-1: MSRPMIHFGNDWE (positions
133-145)) that partially overlapped with the 15B3-1. Furthermore, mAb
11H1 recognized the third segment (11H1-3: YYRPVDQYS (positions
161-169)), which is a complete overlap with the second epitope of mAb
15B3 (15B3-2). mAb 8G6 recognized two other segments (8G6-2: QVYYRPVDQ (positions 159-167); 8G6-3: SNQNNFVHDCV (positions 169-179)). In
addition to the octarepeat region, Ab.Tg also recognized Ab.Tg-2 (QWNKP; positions 97-101) and Ab.Tg-3 (DWEDRYYRE; 143-151). The position of Ab.Tg-3 was close to that of 15B3-1. To examine the reproducibility of the experimental system, we analyzed sera from four
independent immune mice. Ab.Tg-1 and -2 were detected with all sera,
while Ab.Tg-3 was detected with three of four immune sera samples.
These highly reproducible results indicate the reliability of our
experimental system.
To gain insight into epitopes representing PrPC
conformation and in vivo conversion processes of
PrPC to PrPSc, we immunized
Prnp Although all of the mAbs we used showed preferential affinities for
PrPC in solution, they were divided into two groups in
terms of reactivities to tissue sections. In immunohistochemistry for
acetone-fixed scrapie brain, some mAbs (1H8, 4A3, 6B5, and 9A8)
recognized a stellar structure (Fig. 6, panel 4),
while other mAbs (7H8, 8G6, and 11H1) reacted with plaque structures in
neuropil (Fig. 6, panel 7). In mock samples, a
weak and diffuse signal was evident. Stellar and plaque structures were
never observed (Fig. 6, panels 5 and
6). Thus, these stellar and plaque structures might
represent disease-specific isoforms of PrP. mAb 4A3 recognized an
isoform that accumulates in astrocytes (Fig. 6, panel
4). The PrP may be an intermediate form in converting to the
fully pathogenic PrPSc. It has been reported that
PrPSc first accumulates in astrocytes prior to development
of neuropathological changes (26) and that the astrocyte-specific
induction of PrP in Prnp The mAbs were preferentially reactive to PrPC in IP but
were immunohistochemically reactive to abnormal isoforms of PrP. One of
the intriguing observations is that all mAbs recognized multiple discontinuous linear peptides that show no apparent similarities (Fig.
7). A similar unusual nature was noted for a PrPSc-specific
clone 15B3 (11). Clone 15B3 recognized three distinct linear
polypeptides. At least one of each of the epitopes recognized by three
mAbs examined in this study overlapped with one of the 15B3 epitopes.
Especially, the segments of 4A3-2 and 11H1-3 completely overlapped with
epitopes 15B3-1 and 15B3-2, respectively. Epitope 8G6-2 is closely
related to the 15B3-2 epitope. We suggest that these epitopes largely
contributed to reactivities of these mAbs to abnormal isoforms in
acetone-fixed tissue sections. Epitopes 15B3-1/4A3-2 and
15B3-2/11H1-3/8G6-2 may represent isoforms accumulating in astrocytes
and in the neuropil, respectively.
Regarding epitopes specific to PrPC, the octarepeat region
is notable. Two of three mAbs, 4A3 and 11H1, reacted to the region of
octarepeats. Furthermore, results from polyclonal antibody Ab.Tg
suggested that this region is likely to be one of the major epitopes on
PrPC. Because native PrPC was used as an
immunogen, the epitopes recognized by Ab.Tg may reflect conformation of
PrPC. A pair of tripeptides ((T/G/S/)WG; positions
55-57, 63-65, 71-73, 79-81, and 87-89) seemed to constitute an
epitope of mAb 4A3. Ab.Tg recognized two perfect repeats (GQPHGGGWG;
positions 57-65 and 81-89) but not other repeats that carried a
substitution (GQPHGGSWG; positions 65-73 and 73-81). The
substitutions at 71 and 79 residues (Gly to Ser; underlined) affect the
immunoreactivity of Ab.Tg. A model suggested that the octarepeat region
is constrained by four Cu(II)-coordinating histidines into a compact
structure (29). The tripeptides involved in 4A3 reactivity are located
at centers of loops generated by copper binding (29). The epitopes for some mAbs generated by DNA-mediated immunization of
Prnp It is noteworthy that the polyclonal antibodies from immunized mutant
mice revealed limited regions with regard to being effective epitopes,
although the Prnp Data from experiments using these mAbs and polyclonal antibodies we
developed revealed a marked decrease of PrPC in brains of
animals at the terminal stage of illness, thereby providing a molecular
basis for the hypothesis that loss of function of PrPC may
have some role in the pathogenesis of prion diseases. Results from
epitope mapping and our immunohistochemical studies suggest that
conversion of PrPC to PrPSc may involve
multiple steps at different sites.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-sheets and a diminished
-helical content compared with
PrPC (2-4); hence, it is relatively resistant to
protease digestion. The protease resistance of PrPSc has
been widely accepted as the physico-chemical basis to distinguish between PrPC and PrPSc. A recent study
demonstrated that plasminogen has selective affinity to
PrPSc but not to PrPC (5).
/
mice with
native PrPC from wild type mice
(Prnp+/+) and established mAbs to PrP. These
mAbs specifically recognized PrPC in solution with a marked
decrease of PrPC at the terminal stage of illness. Epitope
mapping of these mAbs and polyclonal antibodies from mice immunized
with native PrPC suggested the importance of octarepeats as
part of PrPC specific conformation.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
Mice--
Prnp
/
mice were
generated by homologous recombination, as described (20). The entire
Prnp gene open reading frame as well as 3'-end of the intron
2 was replaced with a pgk-neo gene cassette. The resulting
allele is almost identical to that generated by another group (16). The
homologous recombinant ES clones were injected into blastocysts of
C57BL/6 mice to give rise to chimeras. Mice heterozygous for the
mutation (Prnp+/
) were obtained by crossing
the chimeras with C57/BL6J mice. The heterozygotes
(Prnp+/
) were further intercrossed to obtain
mutation homozygotes (Prnp
/
).
The mouse line has been maintained by backcrossing to C57BL/6. Genotypes of the mice were determined by Southern blot analysis or by
polymerase chain reaction analysis of DNAs prepared from tails of the
mice. The primers used were 5'-GTACAGTAGACCAGTTGCTC-3' and
5'-CAGAGTCAAGATCTCCTAGT-3' for the wild-type allele and
5'-CTCGTGCTTTACGGTATCGC-3' and 5'-CAGAGTCAAGATCTCCTAGT-3' for the
mutated allele.
/
mice four times at
2-3-week intervals. The brains were homogenized with RPMI medium and
then centrifuged at 3,000 rpm for 30 min. The supernatant fraction was
emulsified with Freund's complete and incomplete adjuvants for first
and subsequent immunizations, respectively. Thymocytes were given
intraperitoneally without adjuvants. The spleen cells of the immunized
mice were fused to P3U1 mouse myeloma cells (P3 × 63Ag8U.1) and
cultured, as described (22). Subclasses of the mAbs were determined
using a mouse mAb isotyping kit (Amersham Pharmacia Biotech).
/
, and
Prnp+/+ mice with PrP-AcNPV-infected cells.
/
mice, as described (21).
At appropriate time points, the brains were collected and used for
PrPSc extraction and immunoprecipitation. Noninfected
brains of Prnp
/
and
Prnp+/+ mice served as controls. For histoblot
analysis, we used the Sc237 strain of scrapie prion, which had been
passaged through Syrian Golden hamsters more than 10 times.
Three-week-old hamsters were intracerebrally inoculated with Sc237
prion, as described (21). PrPSc extraction from infected
animal brains and an enzyme-linked immunosorbent assay (ELISA) were
done, as described (21).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
mice and a reduced level
in Prnp+/
mice compared with findings in
Prnp+/+ mice (Fig. 1C). Homozygous
mutant mice showed no obvious behavioral changes and at a young age
appeared to have normal motor activity. However, the aged mice showed
tremor and ataxia, as reported (16).
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Fig. 1.
Mutation in the Prnp
locus. A, schematic representations of the
wild-type and mutated alleles. The entire coding sequence in exon 3 of
Prnp, including the splicing acceptor sequence, was replaced
with the pgk-neo gene cassette by gene targeting. Genotypes
of the resulting mice were determined either by Southern blots or
polymerase chain reaction. The 3' probe used is indicated by
thick bars. The primer sets for wild-type and
mutant alleles are indicated by short arrows.
B, Southern blot analysis for tail DNAs of littermates
obtained by crossing between heterozygotes. C, Western blot
analysis of brain lysates with anti-pan-PrP antibody, Ab.Tg.
B, BamHI; E, EcoRI +/+,
wild type; +/ , heterozygote;
/
, homozygote.
/
mice were
immunized with brain homogenates and thymocytes of
Prnp+/+ mice. The sera from immunized mice
recognized PrP in ELISA, WB, and IFA (Table
I). In contrast, the sera from
Prnp+/+ mice inoculated with the same antigens
showed no reactivities in these assays. Antisera (Ab.Tg) served as
anti-pan-PrP polyclonal antibodies throughout this study. We then
attempted to generate mAbs, using a conventional B cell hybridoma
technique. In initial studies, we wanted to screen the hybridomas by
IFA against thymocytes from Prnp+/+ mice and by
ELISA, using brain homogenates of Prnp+/+ mice,
but we did not detect candidate clones secreting mAbs reactive to PrP.
This failure was attributed to limited concentrations of antigens.
Thus, we decided to use recombinant proteins expressed by
baculoviruses. The recombinant baculovirus (PrP-AcNPV) carrying murine
Prnp open reading frame, under the promoter of the
polyhedrin gene, was plaque-purified. SF21AE cells infected with
PrP-AcNPV showed expression of recombinant PrP revealed by IFA, using
Ab.Tg (Fig. 2A). WB analysis
using Ab.Tg detected an immunoreactive band of the expected size in
homogenates of infected SF-21 cells (Fig. 2B). Furthermore,
Prnp
/
mice immunized with
lysates of PrP-AcNPV-infected SF21 cells generated antibodies reactive
to PrP, in various forms (Table I). Thus, PrP-AcNPV infected SF-21
cells could serve as an antigen for screening mAbs. Splenocytes from
mice immunized with brain homogenates were fused with P3U1 mouse
myeloma cells, and the resulting hybridomas were screened by IFA
against PrP-AcNPV-infected SF21 cells. Noninfected SF21 cells were
served as controls. Seven clones of hybridomas were established.
Immunoreactivities of sera from Prnp/
mice immunized with
various antigens
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Fig. 2.
Expression of recombinant PrP with a
baculovirus vector. SF21AE cells infected with recombinant
baculovirus PrP-AcNPV were fixed with acetone, and the expression of
recombinant PrP was examined using Ab.Tg and fluorescein
isothiocyanate-conjugated anti-mouse IgG and IgM (A).
Expressed proteins were examined by WB with Ab.Tg (B).
SF21AE cells (lane 1), AcNPV-infected SF21AE
cells (lane 2), and PrP-AcNPV-infected SF21AE
cells (lane 3).
/
brain homogenates (Fig.
3A, lanes 1), thus indicating the PrP specificity of the immunoreactive bands. On the other hand, Ab.Tg detected larger amounts of PrP from the scrapie-infected brains (Fig.
3A, lane 3). These results suggested
that PrPC was selectively identified by IP, using these
mAbs. However, none of the mAbs showed cell surface staining of
wild-type thymocytes (data not shown), but Ab.Tg did show staining.
Sequential IP with mAbs and Ab.Tg indicated that the PrPC
immunoprecipitated with the mAbs is a subfraction of PrPC
immunoprecipitated with Ab.Tg (Fig. 4).
On the other hand, sequential immunoprecipitation of 4A3 and 11H1
suggested that the fraction precipitated by these mAbs is probably
identical (Fig. 4). We observed similar results with all the mAbs used.
These results suggest that these mAbs are specific to a subfraction of
PrPC.
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Fig. 3.
A, immunoprecipitation of
scrapie-affected and mock-affected mouse brains with two mAbs and
Ab.Tg. Brain homogenates of
Prnp /
mice (lane
1), mock-infected Prnp+/+ mice
(lane 2), and scrapie-affected
Prnp+/+ mice (lane 3) at
20 weeks postinfection were immunoprecipitated and analyzed by WB,
using biotinylated Ab.Tg. B, WB with Ab.Tg of PrP from the
scrapie-affected mouse brain. Scrapie-affected mice were killed at 0, 9, 12, 15, and 20 weeks postinfection. Brain homogenates were examined
with (+) or without (
) proteinase K (PK) treatment. PK(
)
samples represent the total amounts of PrPC and
PrPSc, while PK(+) samples represent the amounts of
PrPSc. Note the increase of proteinase K-resistant PrP at
20 weeks postinoculation. C, immunoprecipitates from
samples same as in B. D, brain homogenates used
for B and C were immunoprecipitated with
11H1 mAb. In C, same homogenates were
immunoprecipitated with anti-pan-PrP (Ab.Tg) antibody. 11H1-reactive
PrP was markedly decreased at 20 weeks postinoculation, while Ab.Tg
detected a larger amount of PrP (PrPC and
PrPSc) even in the same sample.
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Fig. 4.
Sequential immunoprecipitation of
PrPC. Brain homogenates were sequentially
immunoprecipitated with 11H1 and 4A3 mAbs and polyclonal Ab.Tg in the
order indicated. Ab.Tg precipitated a large amount of PrPC
from the samples that had been immunoprecipitated with mAbs. On the
other hand, mAbs precipitated a limited amount of PrPC from those
samples.
/
mice, regardless of
pretreatment conditions. These results indicate that signals on the
membrane (without pretreatment) preferentially represent
PrPC, while PrPSc was specifically detected on
the membrane exposed to a combination of hydrated autoclaving and
proteinase K digestion (Fig. 5A, fourth column). It should be noted that the PrPC signal
was reduced in the scrapie-affected sample, particularly in the
cerebral cortex (Fig. 5A, first
column). To better understand temporal and spatial
relationships between amounts of PrPC and
PrPSc, we inoculated scrapie prion Sc237 into the brains of
hamsters, which were killed at 38 and 80 days after inoculation,
respectively. Mock-infected hamsters were also examined, as controls.
As shown in Fig. 5B, PrPC but not
PrPSc was present in normal hamster brains.
PrPSc deposition was first detected in the thalamus at 38 days postinoculation, and the PrPC signal decreased
specifically in that region. At the terminal stage of illness,
PrPSc had spread to all brain regions, and the
PrPC signal was decreased in those areas (Fig.
5B). The distribution of PrPSc revealed by the
histoblot was consistent with the distribution of PrPSc
plaque deposits detected by immunohistochemistry, using the reference antibody (Ab.Mo-VI) (data not shown). The results from histoblot and IP
analyses consistently indicated the loss of PrPC at the
terminal stage of illness.
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Fig. 5.
Regional distribution of PrPC and
PrPSc. A, discrimination of
PrPC and PrPSc by histoblot analysis, using
Ab.Tg. The blots of scrapie-affected Prnp+/+,
mock-affected Prnp+/+, and
Prnp /
mouse brain samples were
reacted with Ab.Tg., with or without pretreatment of hydrated
autoclaving and proteinase K. PrPC was detected on
nonpretreated membranes (far left).
PrPSc was specifically detected by the combination of
autoclaving and proteinase K treatments (right-hand column).
B, temporal examination of PrPC and
PrPSc in scrapie-affected hamsters, using histoblots.
Hamsters were inoculated intracerebrally with scrapie prion Sc237. At
postinfection day 38 and 80, brain samples were taken.
Mock-infected brain was also included (day 0). Note the decrease of
PrPC at sites where PrPSc was
accumulated.
/
brain samples (Fig. 6,
panels 3, 6, and 9). These
results suggested that the mAbs recognized not only PrPC
but also abnormal isoforms in the acetone-fixed sections.
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Fig. 6.
Indirect fluorescent assay with Ab.Tg and
mAbs 4A3 and 11H1 to scrapie-affected
Prnp+/+ mice. Scrapie-affected and
mock-affected Prnp+/+ mouse brains were rapidly
frozen and then cryosectioned.
Prnp /
mice served as
controls. Brain samples were examined using Ab.Tg (panels
1-3), mAb 4A3 (panels 4-6), and 11H1 (panels
7-9) as primary antibodies. Ab.Tg detected the PrPC
throughout the entire brain areas as indicated by smear signals
(panel 2). In addition to similar signals,
granulous and amorphous signals representing PrPSc were
observed in the scrapie-affected brain (panel 1).
mAbs 4A3 and 11H1 also weakly revealed PrPC in the
mock-affected brain (panels 5 and 8).
These mAbs detected stellar structures and plaques, respectively
(panels 4 and 7). No signal was
observed in Prnp
/
mice, using
these antibodies (panels 3, 6, and
9).
Characteristics of monoclonal and polyclonal antibodies
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Fig. 7.
Epitopes associated with
PrPC. A, the mAbs and Ab.Tg were reacted
with a gridded array of peptides comprising 122 polypeptides of 13 amino acids, shifted by 2 amino acids and covering the entire mouse
sequence. Epitope numbers for each antibody are indicated by
lines. Polypeptide numbers are indicated on the
left. B, epitope positions are indicated with
boxes under the sequences of mature
murine PrPC (positions 23-231) (31). Sequence numbers are
indicated on the right. End sequence numbers are also
indicated. Octarepeats (positions 51-90) are lined
above the sequences.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
mice with native
PrPC, prepared as brain homogenates or intact thymocytes
from wild-type mice. With this attempt, we obtained polyclonal
antibody, named Ab.Tg, and seven mAbs. The Ab.Tg was highly reactive to
both PrPC and PrPSc (Table II); therefore, it
was used as a pan-PrP-specific antibody. Unlike the Ab.Tg, none of the
mAbs detected any isoforms of PrP by WB analysis; they showed only weak
reactivities to partially purified PrPSc samples in ELISA
(Table II). However, all of these mAbs efficiently immunoprecipitated
PrP in brain lysates from noninfected mice but not from
scrapie-infected mice, indicating preferential affinity to
PrPC in solution. We observed that the immunoprecipitable
PrPC was markedly decreased in the infected brain by
one-fifth to one-sixth of the level in noninfected brain,
depending on PrPSc accumulation. At this time point, we did
not observe cell loss to this magnitude. Thus, the loss of
PrPC probably reflects a reduction of PrPC per
cell rather than a loss of cells in diseased brains. Consistent results
were also obtained by histoblot analyses. The Ab.Tg detected diffusely distributed PrPC on the histoblot, without any
pretreatment (Fig. 5A). In contrast, the same antibody
specifically detected PrPSc on the blot exposed to hydrated
autoclaving and partial digestion with proteinase K (Fig.
5A). The time course study revealed that the site of the
PrPC-decrease correlated with the site at PrPSc
depositing (Fig. 5B). Although the expression level of
mRNA of the Prnp gene is unchanged with scrapie
infection (19), our results suggest that the amount of PrPC
in infected brains is exhausted by conversion of PrPC to
PrPSc, thus providing a molecular basis for the notion that
the loss of function of PrPC may partly account for
pathogenesis of the central nervous system (14-18).
/
mice
restores susceptibility to scrapie (27). These data suggest that
astrocytes play an important role in scrapie pathogenesis. The mAb 4A3
may be useful to study the roles of astrocytes in scrapie disease. The
mAb 11H1 may recognize the isoform closely related to the fully
pathogenic PrPSc. The immunohistochemical staining pattern
of the mAb 11H1 resembled results obtained with rabbit polyclonal
antibodies to synthetic peptide or scrapie-associated fibrils in
formalin-fixed samples treated with either hydrated autoclaving (21) or
guanidine (28). Either guanidine or hydrated autoclaving pretreatment
has been found essential for immunofluorescent detection of
PrPSc plaques. The mAbs and Ab.Tg facilitated detecting
abnormal isoforms of PrP, with solely acetone fixation. The distinct
immunohistochemical staining patterns of two groups of mAbs suggests
that conversion from PrPC to PrPSc involves
multiple steps at different sites in vivo.
/
mice were also located at
an octarepeat region (8). These results suggest that the octarepeats
are important for antigenicity of PrPC, and copper may
contribute to related characteristics. Consistent with this notion, it
has been reported that copper can enhance restoration of proteinase K
resistance and infectivity after the denaturation of PrPSc
with guanidine hydrochloride (30).
/
mice were
immunologically intolerant of PrP. We examined sera from four
independent immune mice and observed reproducible results among the
sera against the PrPC immunogen. Epitopes 1 and 2 of Ab.Tg
(Ab.Tg-1 and Ab.Tg-2) were always detected, and the third one (Ab.Tg-3)
was detected in three of four immune sera, indicating their dominance
as epitopes. The NH2-terminal sequences between positions
23 and 40 seldom functioned as an epitope, and therefore were
unstable. These results suggested that the amino-terminal half of
PrPC has a characteristic conformation, in a good agreement
with findings with the mAbs we characterized. We also observed distinct
epitope patterns on PrPSc
fractions.2 This experimental
strategy may aid in understanding species barrier mechanisms and
differences in infectious prion strains replicated in a given animal species.
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ACKNOWLEDGEMENTS |
---|
We thank M. Matsuura for technical assistance; H. Oohashi, M. Nagatsuka, S. Mizukoshi, H. Nakamura, and other staff in the animal section of the National Institute of Animal Health for maintaining the mouse colony; H. Sentsui, M. Narita, and N. Yuasa for encouragement; and M. Ohara and M. J. Schmerr for reading the manuscript.
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FOOTNOTES |
---|
* This work was supported by grants from the Science and Technology Agency of Japan, and the Ministry of Agriculture, Forestry, and Fisheries, Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
** To whom correspondence should be addressed. Tel.: 81-48-467-9724; Fax: 81-48-467-9725; E-mail: sitohara@brain.riken.go.jp.
Published, JBC Papers in Press, January 10, 2001, DOI 10.1074/jbc.M008734200
2 T. Yokoyama and S. Itohara, unpublished observation.
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ABBREVIATIONS |
---|
The abbreviations used are: PrP, prion protein; mAb, monoclonal antibody; WB, Western blot; AcNPV, A. californica nuclear polyhedrosis virus; IFA, indirect fluorescent assay; ELISA, enzyme-linked immunosorbent assay; IP, immunoprecipitation.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Prusiner, S. B. (1991) Science 252, 1515-1522[Medline] [Order article via Infotrieve] |
2. | Caughey, B. W., Dibg, A., Bhat, K. S., Ernst, D., Hayes, S. F., and Caughey, W. S. (1991) Biochemistry 30, 7672-7680[Medline] [Order article via Infotrieve] |
3. | Pan, K.-M., Baldwin, M., Nguyen, J., Gasset, M., Serban, A., Groth, D., Mehlhorn, I., Huang, Z., Fletterick, R. J., Cohen, F. E., and Prusiner, S. B. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 10962-10966[Abstract] |
4. |
Safar, J.,
Roller, P. P.,
Gajdusek, D. C.,
and Gibbs, C. J., Jr.
(1993)
J. Biol. Chem.
268,
20276-20284 |
5. | Fischer, M. B., Roeckl, C., Parizek, P., Schwarz, H. P., and Aguzzi, A. (2000) Nature 408, 479-483[CrossRef][Medline] [Order article via Infotrieve] |
6. | Cashman, N. R., Loertscher, R., Nalbantoglu, J., Shaw, I., Kascsak, R. J., Bolton, D. C., and Bendheim, P. E. (1990) Cell 61, 185-192[Medline] [Order article via Infotrieve] |
7. | Horiuchi, M., Yamazaki, N., Ikeda, T., Ishiguro, N., and Shinagawa, M. (1995) J. Gen. Virol. 76, 2583-2587[Abstract] |
8. | Krasemann, S., Groschup, M. H., Harmeyer, S., Hunsmann, G., and Bodemer, W. (1996) Mol. Med. 2, 725-734[Medline] [Order article via Infotrieve] |
9. |
Williamson, R. A.,
Peretz, D.,
Pinilla, C.,
Ball, H.,
Bastidas, R. B.,
Rozenshteyn, R.,
Houghten, R. A.,
Prusiner, S. B.,
and Burton, D. R.
(1998)
J. Virol.
72,
9413-9418 |
10. |
Williamson, R. A.,
Peretz, D.,
Smorodinsky, N.,
Bastidas, R.,
Serban, H.,
Mehlhorn, I.,
DeArmond, S. J.,
Prusiner, S. B.,
and Burton, D. R.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
7279-7282 |
11. | Korth, C., Stierli, B., Streit, P., Moser, M., Schaller, O., Fischer, R., Schulz-Schaeffer, W., Kretzschmar, H., Raeber, A., Braun, U., Ehrensperger, F., Hornemann, S., Glockshuber, R., Riek, R., Billeter, M., Wüthrich, K., and Oesch, B. (1997) Nature 390, 74-77[CrossRef][Medline] [Order article via Infotrieve] |
12. |
Zanusso, G.,
Liu, D.,
Ferrari, S.,
Hegyi, I.,
Yin, X.,
Aguzzi, A.,
Hornemann, S.,
Liemann, S.,
Glockshuber, R.,
Manson, J. C.,
Brown, P.,
Petersen, R. B.,
Gambetti, P.,
and Sy, M.-S.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
8812-8816 |
13. | Demart, S., Fournier, J.-G., Creminon, C., Frobert, Y., Lamoury, F., Marce, D., Lasmézas, C., Dormont, D., Grassi, J., and Deslys, J.-P. (1999) Biochem. Biophys. Res. Commun. 265, 652-657[CrossRef][Medline] [Order article via Infotrieve] |
14. | Collinge, J., Whittington, M. A., Sidle, K. C., Smith, C. J., Palmer, M. S., Clarke, A. R., and Jefferys, J. G. (1994) Nature 370, 295-297[CrossRef][Medline] [Order article via Infotrieve] |
15. | Whittington, M. A., Sidle, K. C., Gowland, I., Meads, J., Hill, A. F., Palmer, M. S., Jefferys, J. G., and Collinge, J. (1995) Nat. Genet. 9, 197-201[Medline] [Order article via Infotrieve] |
16. | Sakaguchi, S., Katamine, S., Nishida, N., Moriuchi, R., Shigematsu, K., Sugimoto, T., Nakatani, A., Kataoka, Y., Houtani, T., Shirabe, S., Okada, H., Hasegawa, S., Miyamoto, T., and Noda, T. (1996) Nature 380, 528-531[CrossRef][Medline] [Order article via Infotrieve] |
17. | Kuwahara, C., Takeuchi, A. M., Nishimura, T., Haraguchi, K., Kubosaki, A., Matsumoto, Y., Saeki, K., Matsumoto, Y., Yokoyama, T., Itohara, S., and Onodera, T. (1999) Nature 400, 225-226[CrossRef][Medline] [Order article via Infotrieve] |
18. | Nishida, N., Tremblay, P., Sugimoto, T., Shigematsu, K., Shirabe, S., Petromilli, C., Erpel, S. P., Nakaoke, R., Atarashi, R., Houtani, T., Torchia, M., Sakaguchi, S., DeArmond, S. J., Prusiner, S. B., and Katamine, S. (1999) Lab. Invest. 79, 689-697[Medline] [Order article via Infotrieve] |
19. | Oesch, B., Westaway, D., Wälchli, M., McKinley, M. P., Kent, S. B. H., Aebersold, R., Barry, R. A., Tempst, P., Teplow, D. B., Hood, LE., Prusiner, S. B., and Weissmann, C. (1985) Cell 40, 735-746[Medline] [Order article via Infotrieve] |
20. | Gomi, H., Yokoyama, T., Fujimoto, K., Ikeda, T., Katoh, T., Itoh, T., and Itohara, S. (1995) Neuron 14, 29-41[Medline] [Order article via Infotrieve] |
21. | Yokoyama, T., Kimura, K., Tagawa, Y., and Yuasa, N. (1995) Clin. Diagn. Lab. Immunol. 2, 172-176[Abstract] |
22. | Yokoyama, W. M. (1991) in Current Protocols in Immunology (Coligan, J. E. , Kruisbeek, A. M. , Margulies, D. H. , Shevach, E. M. , and Strober, W., eds), Vol. 1 , John Wiley & Sons, New York |
23. | Vaughn, J. L., Goodwin, R. H., Tompkins, G. J., and McCawley, P. (1977) In Vitro 13, 213-217[Medline] [Order article via Infotrieve] |
24. | Inumaru, S., and Yamada, S. (1991) Virus Res. 21, 123-139[Medline] [Order article via Infotrieve] |
25. | Taraboulos, A., Jendroska, K., Serban, D., Yang, S.-L., DeArmond, S. J., and Prusiner, S. B. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 7620-7624[Abstract] |
26. | Diedrich, J. F., Bendheim, P. E., Kim, Y. S., Carp, R. I., and Haase, A. T. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 375-379[Abstract] |
27. |
Raeber, A. J.,
Race, R. E.,
Brandner, S.,
Priola, S. A.,
Sailer, A.,
Bessen, R. A.,
Mucke, L.,
Manson, J.,
Aguzzi, A.,
Oldstone, M. B. A.,
Weissmann, C.,
and Chesebro, B.
(1997)
EMBO J.
16,
6057-6065 |
28. | Taraboulos, A., Serban, D., and Prusiner, S. B. (1990) J. Cell Biol. 110, 2117-2132[Abstract] |
29. |
Viles, J. H.,
Cohen, F. E.,
Prusiner, S. B.,
Goodin, D. B.,
Wright, P. E.,
and Dyson, H. J.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
2042-2047 |
30. |
McKenzie, D.,
Bartz, J.,
Mirwald, J.,
Olander, D.,
Marsh, R.,
and Aiken, J.
(1998)
J. Biol. Chem.
273,
25545-25547 |
31. | Westaway, D., Goodman, P. A., Mirenda, C. A., McKinley, M. P., Carlson, G. A., and Prusiner, S. B. (1987) Cell 51, 651-662[Medline] [Order article via Infotrieve] |
32. |
Weissmann, C.,
and Aguzzi, A.
(1999)
Science
286,
914-915 |