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Journal of General Virology (1995), 76, 2583 2587. Printedin Great Brita#l
2583
A cellular form of prion protein (PrP c) exists in many non-neuronal
tissues of sheep
Motohiro Horiuchi,* Noriko Yamazaki, Tetsuya Ikeda, N a o t a k a Ishiguro and
Morikazu Shinagawa
Department of Veterinary Public Health, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho,
Obihiro, Hokkaido 080, Japan
A cellular form of the prion protein (PrP c) is thought to
be a substrate for an abnormal isoform of the prion
protein (PrP s~') in scrapie. PrP c is abundant in tissues of
the central nervous system, but little is known about the
distribution of PrP c in non-neuronal tissues of sheep, the
natural host of scrapie. This study investigated the tissue
distribution of PrP c in sheep. Although PrP c was
abundant in neuronal tissues, it was also detected in
non-neuronal tissues such as spleen, lymph node, lung,
heart, kidney, skeletal muscle, uterus, adrenal gland,
parotid gland, intestine, proventriculus, abomasum and
mammary gland. Neither PrP c nor PrP mRNA was
detected in the liver. The tissue distribution of PrP c
appears to be inconsistent with the tissues which possess
scrapie infectivity, suggesting that factor(s) specific to
certain cell types may be required to support multiplication of the scrapie agent.
Scrapie is a transmissible, neuro-degenerative disease
which occurs naturally in sheep and goats. One of the
characteristics of scrapie is that a proteinase-resistant
abnormal isoform of a host-encoded cellular membrane
protein, referred to as prion protein (prpSc), accumulates
in the central nervous system (CNS) and lymphoid
tissues of sheep during the disease (Rubenstein et al.,
1987; Ikegami et al., 1991). PrP s° is thought to be derived
from the normal cellular membrane protein (PrP c) by an
unknown post-translational modification that probably
occurs at the plasma membrane or along an endocytic
pathway to the lysosomes (Caughey & Raymond, 1991).
The exact nature of the scrapie agent is still controversial;
the association of PrP s~ with highly purified infectious
preparations suggests that the scrapie agent is composed
largely, if not entirely, of PrP s~ (Bolton et al., 1982).
Oral/alimentary transmission is proposed as a mode
of infection of some of the transmissible spongiform
encephalopathies (TSEs) such as bovine spongiform
encephalopathy (Wilesmith et al., 1988), transmissible
mink encephalopathy (Hartsough & Burger, 1965) and
kuru (Alpers, 1987). Oral/alimentary transmission has
also been suggested as a mode for the natural spread of
scrapie in sheep (Hadlow et al., 1982), but the source of
the scrapie infectivity is unclear. In addition, the sequence
of events in the infectious process such as the site of entry
and primary multiplication, and the mechanism by
which the agent is transported to the CNS remain
obscure. Despite relatively low titres, scrapie infectivity
can be detected in non-neuronal tissues (Hadlow et al.,
1982), and PrP sc is also found in non-neuronal tissues
of scrapie-infected sheep (Ikegami et al., 1991). PrP c is
considered to be a substrate for the production of scrapie
infectivity (Btieler et al., 1993), and therefore the tissue
distribution of PrP c in sheep may help to understand
how the infectivity spreads in the course of natural
infection.
In the hamster, PrP c is reported to be found in almost
all tissues (Bendheim et al., 1992); however, the tissue
distribution of PrP c in sheep, the natural host of scrapie,
is unknown. Here we report the tissue distribution of
PrP c in sheep. PrP c was present in many extracerebral
tissues of sheep including the proventriculus and the
abomasmn.
Four synthetic peptides used as immunogens were as
follows: B-103, corresponding to bovine PrP codons
103-121,
NH2-QGGTHGQWNKPSKPKTNMKCOOH; B-146, corresponding to bovine PrP codons
146-159, NH~-SRPLIHFGSDYEDR-COOH; B-225,
corresponding to bovine PrP codons 225-241, NH 2CITQYQRESQAYYQRGA-COOH;
M-90,
corresponding to mouse PrP codons 90-104, NH 2GQGGGTHNQWNKPSK-COOH.
An
additional
cysteine residue was added at the carboxy-termini of
B-103, B-146 and M-90 to conjugate the peptides to a
*Author for correspondence.Fax +81 155 49 5402.
0001-3279 © 1995SGM
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kDa
44
28
B-103
(Rabbit)
B-225
(Rabbit)
M-90
(Sheep)
BSPX-54
(MAb)
carrier protein, egg albumin. Sheep were inoculated with
M-90, and the other synthetic peptides were administered
to rabbits or mice to obtain monoclonal antibodies
(MAbs). The splenocytes from immunized mice were
fused with myeloma cells P3-X63.Ag8.653 or P3X63 .Ag8.U 1 by using polyethylene glycol 1500 according
to the supplier's instruction (Boehringer Mannheim).
Hybridomas were screened by an ELISA using the
immunizing peptide as an antigen. Hybridomas that
were positive for ELISA were further screened by
immunoblot analysis, and cloned by limiting dilution.
A crude membrane fraction (CMF) was prepared as
described below. Samples of sheep tissue (4 g) were
homogenized to 10% (w/w) in CMF buffer [0'25Msucrose, 10 mM-Tris-HC1, pH 7"5, 2.5 mM-EDTA, 1 mMdithiothreitol (DTT), and a cocktail of protease
inhibitors (1 mM-phenylmethylsulphonyl fluoride, 2 gg/
ml leupeptin, 1 lag/ml pepstatin, 1 ~tg/ml aprotinin,
2 laM-E-64 and 2 gM-bestatin)]. The homogenates were
centrifuged at 2000 g for 15 rain, and the supernatants
were centrifuged at 100000g for 1 h. The resulting
pellets were solubilized with 2 ml of lysis buffer (1%
Triton X-100, 50 mM-Tris-HC1, pH 7"5, 2.5 mM-EDTA,
150 mM-NaC1, 1 mM-DTT and the cocktail of protease
inhibitors), and the solutions were centrifuged at
100000 g for 1 h. The resulting supernatants were used as
the Triton X-100 solubilized fractions of the CMF (TXCMF). All procedures were done at 4 °C. To obtain the
prpC-enriched fractions, TX-CMF from brain was
partially purified by heparin affinity chromatography on
an Econo-Pac heparin cartridge (Bio-Rad).
Affinity purified B-103 rabbit antibodies (5 lag) were
added to 1 g tissue equivalent of TX-CMF and incubated
for 4 h at 4 °C. The immune-complexes adsorbed by
protein A-Sepharose (Pharmacia) were solubilized in
100 ~tl of sample buffer (2% SDS, 0'5 M-fl-mercaptoethanol, 10% glycerol, 0.01% bromophenol blue,
50 mM-Tris-HC1, pH 6.8). The proteins were subjected
to SDS-12.0% polyacrylamide gel electrophoresis
(PAGE), and transferred electrophoretically to nitro-
B-225-101
(MAb)
Fig. 1. Reactivities of MAbs and antisera. PrP cenriched fractions (bovine, sheep and mouse),
prepared by heparin affinity chromatography,
were subjected to SDS-PAGE and transferred
onto nitrocellulose membranes. The MAbs or
antisera used for immunostaining are indicated at
the bottom of each panel.
cellulose membranes. The blots were visualized with
ECL Western blotting detection reagents (Amersham).
Total RNA was extracted from sheep tissues by using
an acid guanidinium thyocyanate-phenol-chloroform
method (Chomczynski & Sacchi, 1987). Poly(A)+ RNA
Was purified with Oligotex-dT 30 (Takara). Electrophoresis and transfer of RNA to nylon membranes were
performed as described by Sambrook et al. (1989).
Amino acid polymorphisms were determined by DNA
sequencing of PCR-amplified sheep PrP DNA as
described elsewhere (Ikeda et al., 1995).
Hybridomas BSPX-54 and B-225-101, which secreted
MAbs against synthetic peptides B-146 and B-225,
respectively, were established. No MAb against synthetic
peptide B-103 was obtained. Fig. 1 shows the reactivities
of antisera and MAbs in Western blot analysis. MAb
B-225-101 (isotype IgM) reacted with bovine and sheep
PrP e, but not with mouse PrP c. MAb BSPX-54 (isotype
IgG2b) showed similar reactivity; however, it did react
slightly with mouse PrP c when the MAb was used in high
concentration (data not shown). MAb BSPX-54 appears
to react more effectively with bovine PrP c than sheep
PrP c when the reactivity is compared with that of MAb
B-225-101. This difference may be caused by the amino
acid sequences of the synthetic peptides; B-225 was
based on the deduced amino acid sequence of bovine
PrP c but it is identical to the corresponding sequence of
sheep PrP c, while synthetic peptide B-146 differed by one
amino acid from the corresponding sequence of sheep
PrP c (aa 153: Ser ~Asn/bovine--*sheep). Thus the
difference may affect the affinity of BSPX-54 for bovine
and sheep PrP e. Rabbit antisera against synthetic
peptides B-103 and B-225 reacted with PrP c from three
hosts. As expected, M-90 sheep serum reacted with
mouse PrP c but not with sheep and bovine PrP c. B-103
rabbit serum reacted with cell surface PrP c of N2a mouse
neuroblastoma cells and PC-12 rat pheochromocytoma
cells as demonstrated by flow cytometric analysis (data
not shown). The bovine PrW had a slightly greater
molecular mass than sheep and mouse PrP c.
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C
K
L
S
Lu
A
Ln
Pg
P
U
H
M
2585
kDa
~iiiiii
i -- 44
Sheep
I
.~i~
C
K
L
S
Lu
A
Ln
Pg
P
H
M
!~
I
~-
Shi]eP
44
28
N
C
Sheep
III
K
L
S
A
Pg
Ln
P
Ru
Re Om
i
First, we tried to detect PrP c directly in the TX-CMF
of various sheep tissues by Western blot analysis;
however, PrP c was detected only in the CNS (data not
shown). N e x t we tried to detect PrP c by immunoprecipitation followed by Western blot analysis. A
combination of B-103 rabbit antibodies for immunoprecipitation and MAb B-225-101 for immuno-staining
was the best of several combinations of antibodies tested.
Thus, this combination was used in the following
analysis. The use of two antibodies which recognize
different epitopes in PrP c made it possible to produce
reliable results. Fig. 2 shows the distribution of PrP c in
various sheep tissues. When 200 mg tissue equivalent of
immunoprecipitate was used for the analysis, PrP c was
detected in the cerebrum, spleen, lung, adrenal gland,
lymph node, uterus, heart, skeletal muscle and parotid
gland (sheep I, Blue-de-Dorset; PrP genotype, Prp~A~Q/
Prp~v~q). No PrP c was detected in the pancreas, liver
and kidney. No band was detected when immunoprecipitation was performed with normal rabbit IgG
instead of B-103 rabbit IgG (data not shown). To
examine the distribution of PrP c further, we analysed
another sheep (sheep II, Suffolk; PrP genotype, PrWARQ/
PrpMARR). Similar to sheep I, PrP c was detected in many
non-neuronal tissues. PrP c was detected in the kidney
but only when 400 mg of kidney equivalent was used
Ab
PBS
Fig. 2. Detection of PrP c in various sheep tissues
by immunoprecipitation-Western blot (IP-WB)
analysis. A 200 mg tissue equivalent sample of
immunoprecipitate was loaded in each lane. PBS
indicates that immunoprecipitation was done with
PBS instead of TX-CMF. Molecular mass markers are indicated. Abbreviations: C, cerebrum;
K, kidney; L, liver: S, spleen; Lu, lung; A, adrenal
gland; Ln, lymph node; Pg, parotid gland; P~
pancreas; U, uterus; H, heart; M, skeletal muscle;
I, intestine; Ru, rumen; Re, reticulum; Om,
omasum; Ab, abomasum.
(data not shown). PrP c was not detected in the liver and
pancreas even when 400 mg tissue equivalent was used.
PrP c was also detected in the rumen, reticulum, omasum
and abomasum (sheep III, Suffolk; PrP genotype,
PrpMARQ/PrPMA~). However, some differences in the
distribution ofPrP c were observed in sheep III: PrP c was
not detected in the adrenal gland and lymph node, and
the molecular mass of PrP c in the spleen of sheep III
(20-26 kDa) was less than in other sheep. This band was
still detected by immunostaining with MAb BSPX-54
(data not shown). In addition, heterogeneity of PrP c was
observed among tissues and among sheep. We do not
have any evidence to explain the heterogeneity; it is
conceivable that the composition of oligosaccharide
chains differs among the cell types or that the PrP
genotype also affects the biochemical properties of PrP c;
however, further analysis is required to assess these
possibilities. PrP c was also detected in mammary gland
(data not shown).
As described above, PrP c was not detected in liver and
pancreas. To examine whether PrP m R N A was expressed
in these tissues, Northern blot analysis was performed.
As shown in Fig. 3, an m R N A of about 4-1 kb was
detected in all tissues except for liver and pancreas. In
addition to a 4.1kb mRNA, a 2.1kb m R N A was
detected in kidney, spleen, lung, adrenal gland, lymph
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2586
Short communication
C
K
L
S
Lu
A
Ln
Pg
P
H
M
I
kb
-4.7
e~
o
e~
-l.9
1 0.20 ND 0"13 O12 0"34 0.08 0.45 ND
ND
ND
..=
'6
9
e~
node and parotid gland. The 2.1 kb m R N A appeared to
be due to the differential usage of a poly(A) addition
signal as demonstrated by using a probe which corresponds to a region 3' downstream of the sheep PrP O R F
(data not shown). Expression of PrP m R N A in the
pancreas could not be assessed here, because no
significant poly(A) + R N A was loaded. Comparison of
PrP m R N A and PrP c from the same tissues of sheep II
revealed that the proportional relationship between PrP
m R N A and PrP c varied between the brain and the other
tissues. For instance, the brain expressed PrP m R N A at
a level about fivefold higher than in the kidney (Fig. 3).
On the other hand, PrP c was detected in 0.5 mg brain
equivalent of T X - C M F , but not at all in the kidney even
when 20 mg tissue equivalent of T X - C M F was used,
indicating that the brain contains at least fortyfold more
PrP c than the kidney (data not shown). Thus, the
translational efficiency or the course of PrP c synthesis
including degradation may differ between the brain and
the other cell types that express PrP c.
In this paper, we have shown that PrP c is present in
m a n y non-neuronal tissues of sheep. Widespread distribution of PrP c in hamsters has also been reported
(Bendheim et al., 1992), indicating that the pattern of
distribution seems to be basically comparable a m o n g
mammals. Quantitative comparison of this distribution
could not be done because of the differences in the
antibodies used. An apparent difference was observed in
PrP m R N A expression: the PrP m R N A in mice and
hamster spleens was less than 1% of that in the brain
(Caughey et al., 1988), while PrP m R N A in sheep spleen
was estimated to about 10 % of that in the brain (Fig. 3).
Divergence between the sheep and rodent PrP gene
promoter regions (Westaway et al., 1994) m a y explain
ND
Fig. 3. Detection of PrP mRNA in sheep tissues.
Poly(A)+ RNA prepared from 4(~120 lag of total
RNA from tissues of sheep I1 was loaded and the
membrane was hybridized with a ~zP-labelled
BamHI Hincll fragment (nt 771-1292) from the
sheep PrP open reading frame (DDBJ accession
no. D38179). DNA fragments of 4.7 and 1.9 kb to
which the probe can hybridize were used as
markers. The same filter was reprobed with a
fragment of the mouse fl-actingene corresponding
to nt703 1131 (Tokunaga et al., 1986). The
radioactivities were analysed on a Bio-imaging
Analyzer BAS 2000 (Fuji) and the amounts of
RNA were normalized to the radioactivity of the
fl-actin gene. The relative levelsof PrP mRNA are
indicated at the base of the upper panel. Those of
pancreas, heart, skeletalmuscleand intestine were
not calculated (ND)because of their relatively low
radioactivities. Abbreviations are as in the legend
to Fig. 2.
this difference. Expression of PrP m R N A , therefore, is
not likely to be comparable between sheep and rodents.
PrP c is reported to be involved in long term potentiation
in the hippocampus (Collinge et al., 1994); however, the
occurrence of PrP c in nearly all tissues suggests that the
normal function of PrP c is not unique to neuronal
tissues.
Although the conditions under which the conversion
of PrP c to PrP sc occurs remain to be elucidated, the
existence of a substrate for PrP s° in a variety of tissues
indicates that PrP s° may be produced in m a n y tissues;
the scrapie agent might potentially be able to replicate in
a variety of tissues. The high infectivity in the CNS and
low infectivity in the liver of scrapie-affected sheep or
goats, and BSE-affected cattle (Hadlow et af., 1979;
Danner, 1993), agree well with the distribution of prpc;
however, the distribution of PrP c in non-neuronal tissues
appeared to be inconsistent with the tissue distribution of
infectivity reported so far. For instance, infectivities in
the heart and skeletal muscles have been reported to be
undetectable (Hadlow et al., 1979; Danner, 1993),
whereas PrP c was apparently present in these tissues.
This m a y indicate that factor(s) specific to certain cell
types are required for the multiplication of scrapie
infectivity; or possibly, the scrapie agent m a y not reach
the heart and skeletal muscle during the course of
infection. Comparison of the cells expressing PrP c with
those in which PrP s~ accumulates should help to resolve
this question. Furthermore, such information and the
sensitive PrP detection method described here will greatly
contribute to the improvement of pre-clinical diagnosis
(Ikegami et al., 1991).
This work was supported by a Grant-in Aid for Scientific Research
from the Ministry of Education, Science and Culture of Japan
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(04454113), and Special Coordination Funds of the Science and
Technology Agency of the Japanese Government.
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