Báo cáo Y học: Toxicity of novel C-terminal prion protein fragments and peptides harbouring disease-related C-terminal mutations pdf

Toxicity of novel C-terminal prion protein fragments and peptides
harbouring disease-related C-terminal mutations
Maki Daniels
1
, Grazia Maria Cereghetti
2
and David R. Brown
1
1
Department of Biochemistry, Cambridge University, UK;
2
Institute of Molecular Biology and Biophysics, ETH-Hoeneggerberg, Zu
¨
rich,
Switzerland
Mice expressing a C-terminal fragment of the prion protein
instead of wild-type prion protein die from massive neuronal
degeneration within weeks of birth. The C-terminal region
of PrP
c
(PrP121–231) expressed in these mice has an
intrinsic neurotoxicity to cultured neurones. Unlike PrP
Sc
,
which is not neurotoxic to neurones lacking PrP
c
expression,
PrP121–231 was more neurotoxic to PrP
c
-deficient cells.
Human mutations E200K and F198S were found to enhance

toxicity of PrP121–231 to PrP-knockout neurones and
E200K enhanced toxicity to wild-type neurones. The normal
metabolic cleavage point of PrP
c
is approximately amino-
acid residue 113. A fragment of PrP
c
corresponding to the
whole C-terminus of PrP
c
(PrP113–231), which is eight
amino acids longer than PrP121–231, lacked any toxicity.
This suggests the first eight amino residues of PrP113–121
suppress toxicity of the toxic domain in PrP121– 231.
Addition to cultures of a peptide (PrP112–125) correspond-
ing to this region, in parallel with PrP121–231, suppressed
the toxicity of PrP121–231. These results suggest that the
prion protein contains two domains that are toxic on their
own but which neutralize each other’s toxicity in the intact
protein. Point mutations in the inherited forms of disease
might have their effects by diminishing this inhibition.
Keywords: prion; neurotoxicity; circular dichroism; neuro-
degeneration.
PrP
c
is a normal cell surface glycoprotein expressed by many
cells including neurones and astrocytes [1–3], microglia [4],
oligodendroglia [2], leukocytes [5] and muscle cells [6].
PrP
c

is attached to the cell membrane via a GPI (glyco-
phosphoinositol) anchor [7]. Predominantly expressed at
synapses [8], it has been suggested that PrP
c
is important for
neuronal activity [9]. More recently it has been shown that
PrP
c
binds copper via an octameric repeat region [10]. PrP
c
has been shown to bind significant amounts of copper in
vivo and this copper binding may be necessary for its
normal form [11]. Recent work has suggested two functions
for the protein. PrP
c
influences uptake of copper into
neurones [12] where it can be utilized for synaptic release
[12] or incorporation into enzymes such as Cu/Zn super-
oxide dismutase [13]. Other data suggest that once PrP
c
has
bound copper, the protein can act as a superoxide dismutase
or superoxide scavenger [14].
During normal metabolism of PrP
c
, cleavage occurs in a
region around amino-acid residues 112– 114. The metabolic
C-terminal fragment of this protein can be detected nor-
mally in brain [15] and the N-terminal fragment, retaining
the copper binding region can be purified by metal affinity

chromatography from brain [16]. The rate of cleavage is
regulated by protein kinase C [17] and cleavage has been
suggested to be brought about by the metalloprotease
disintegrins ADAM10 and ADAM17 [18]. However, the
cellular fate or function of these fragments after cleavage
remains unknown. Conformational change in PrP
c
structure
results in a higher percentage of b sheet structure and
increased protease resistance, suggesting that the protein can
no longer be cleaved at this point.
Prion diseases are fatal neurodegenerative diseases. In
these diseases, PrP
c
is converted to a protease resistant form
(PrP
Sc)
that cannot be cleaved at the normal cleavage site.
PrP
Sc
represents an altered isoform that differs markedly in
conformation and accumulates to high levels in nervous
tissue [19]. PrP
Sc
is either a major part or the sole constituent
of the infectious agent of prion disease [20] and is also
neurotoxic when applied to cultured cells [21]. PrP
Sc
is
evidently the cause of neurodegeneration in vivo. However,

induction of neuronal loss both in vivo and in vitro requires
the expression of PrP
c
[21,22]. Mice lacking expression of
PrP
c
are resistant to both the toxicity of PrP
Sc
and its
neurodegenerative effects [22,23].
Attempts to understand the mechanism of PrP
Sc
neuro-
toxicity have focussed on a single peptide known as
PrP106–126 [24,25]. This peptide corresponds to the region
of the human protein that is normally cleaved during cellular
processing of PrP
c
but which becomes protease resistant
when PrP
c
is converted to PrP
Sc
. This peptide has many
features of PrP
Sc
including protease resistance, ability to
form fibrils and high b sheet content. The mechanism of the
action of this peptide has been studied in detail in culture by
many groups [24,26,27,28,29,30,31] including our own

[25,32,33,34,35,36,37] and has also been shown to be toxic
in vivo [38]. The basic mechanism by which PrP106–126
kills neurones in cerebellar cell cultures has been shown
to be the same as that by which PrP
Sc
acts. Both require
neuronal expression of PrP
c
[21,25,32] and the involvement
of a stress event such as superoxide production by activated
microglia [21,25]. PrP106–126 kills the neurones, probably
Correspondence to D. R. Brown, Department of Biology and
Biochemistry, University of Bath, Bath BA2 7AY, UK.
Fax: 1 44 1225 826 779, Tel.: 1 44 1225 323 133,
E-mail:
(Received 14 September 2001, accepted 1 October 2001)
Abbreviations: MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
tetrazolium bromide.
Eur. J. Biochem. 268, 6155–6164 (2001) q FEBS 2001
as a result of reduction in neuronal resistance to oxidative
stress [33]. Apoptosis induced by the peptide follows as a
result of increased calcium influx through
L-type channels
and NMDA receptors [34]. Attempts to define the toxic
region of the protein further have identified the region
AGAAAAGA as being necessary for both fibril formation
and PrP
Sc
-like toxicity [36]. The maximum toxicity was
found for a peptide corresponding to amino-acid residues

113–126 of the human sequence containing this palindrome
[36].
Mice have been generated that express truncated versions
of PrP
c
. In particular, a mouse expressing a prion protein of
106 amino acids [39] has been used to identify those parts of
the protein necessary for both infection and neurodegenera-
tion in prion disease. The truncated version of PrP
c
can be
converted into a truncated PrP
Sc
capable of infecting the
same transgenic mice and inducing neurodegeneration. The
106 amino acids comprise the amino-acid residues 89 –140
and from 177 to the C-terminus.
Although some mice expressing truncated forms of PrP
c
do not develop spontaneous disease [39,40] others do [41,42].
Of particular interest are mice with N-terminal deletions
lacking amino-acid residues up to 121 or 134 of the mouse
sequence. These mice express truncated protein at the cell
surface and develop degeneration in the cerebellum shortly
after birth. This disease is not prion disease but represents a
novel form of cerebellum-specific degeneration. Some mice
that have genetic ablations to prevent expression of all PrP
c
overexpress the prion protein homologue, Doppel [43]. Such
mice show Purkinje cell degeneration. Doppel is homo-

logous to the N-terminally truncated PrP that induces
cerebellar cell loss. Co-expression of full length PrP
c
with
either the N-terminally truncated PrP
c
in mice [42] or re-
introduced into the Doppel expressing PrP
c
-knockout mice
[44] prevents neurodegeneration. Therefore expression of
Doppel or a C-terminal region of PrP
c
(PrP121–231) in the
absence of the full length prion protein leads to
neurodegeneration. The mechanism of this is unknown.
The present investigation was carried out to determine if
the C-terminal region of PrP
c
(PrP121–231) has intrinsic
neurotoxicity and whether PrP
c
has an intrinsic mechanism
to inhibit this. Unlike PrP
Sc
PrP121–231 is more neurotoxic
to PrP
c
-deficient cells. Mutants of PrP121–231 carrying
known human mutations in the C-terminal region (E200K,

D178N and F198S) were also investigated. In particular,
E200K and F198S were also found to enhance toxicity.
However, PrP113–231 lacked any toxicity suggesting the
normal N-terminus of the C-terminal metabolic cleavage
product of the prion protein suppressed toxicity.
MATERIALS AND METHODS
Unless stated, pharmacological agents were purchased from
Sigma.
Animals
Prion protein knockout mice (Npu-Prnp8
/
8) used in this study
were those described by Manson et al. [45] or for some
experiments a different strain was used (Zrk-Prnp8
/
8) and
these were as described by Bu
¨
eler et al. [46]. The wild-type
mice used were either 129Ola mice (as control for Npu-
Prnp8
/
8) or descendants of an F1 generation mouse produced
by interbreeding the original parental strains (C57BL/6 J
and 129/Sv(ev) mice) used to generate Zrk-Prnp8
/
8 mice
originally.
Neuronal cell culture
Preparation of cerebellar cells from 6-day-old mice (P6) or

cortical cells from newborn mice (P0) was as previously
described [25,36]. Briefly, the cerebella were dissociated
in Hank’s Solution (Gibco) containing 0.5% trypsin (Sigma)
and plated at 1–2 Â 10
6
cells
:
cm
22
in 24-well trays (Falcon)
coated with poly
D-lysine (50 mg
:
mL
21
, Sigma). Cultures
were maintained in Dulbecco’s minimal essential medium
(Gibco) supplemented with 10% fetal bovine serum, 2 m
M
glutamine and 1% antibiotics (penicillin, streptomycin,
fungizone; Gibco). Cultures were maintained at 37 8C with
5% CO
2
for 10 days. The neuronal nature of these cultures
was confirmed by immunostaining of parallell wells with
neurofilament as previously described [3].
Peptides or proteins were added to cultures initially
and on the third day. Cell survival was determined on day
5 or 7. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazo-
lium bromide (MTT; Sigma) was diluted to 200 m

M in
Hanks’ solution (Gibco) and added to cultures for 1 h at
37 8C. The MTT formazan product was released from cells
by addition of dimethylsulfoxide (Sigma) and measured at
570 nm in a Unicam Helios spectrophotometer (ATI
Unicam). Relative survival in comparison to untreated
control cultures could then be determined.
For coculture experiments, cerebellar cells were plated as
normal in 24-well trays. Microglia were plated at 10 000
cells per well in Falcon insets with 0.4-mm pore diameter.
Peptides could then be applied directly to the lower wells
and to the insert. After peptide treatments, the inserts were
removed and MTT assays carried out on the cerebellar cells
as before.
Glia cell culture
Microglia were isolated as described previously [25]. Briefly,
cortices from newborn mice were dissociated with trypsin
and seeded into tissue culture flasks (Falcon). When cultures
were confluent, microglia were isolated by rapid shaking
for 2 hours. Microglia were isolated by shaking mixed
glial cultures for 2 h. The floating cells were collected and
plated for 30 min. The nonadhering cells were discarded.
The adhering cells were identified as pure cultures of micro-
glia by immunostaining for ferritin as previously described
[25].
PrP peptides and protein
Mouse prion protein peptides (PrP121 – 231), both wild-type
and those carrying human mutations, were generated as
previously described [47]. The wild-type peptide (PrP121–
231) was mutated to carry amino-acid substitutions

equivalent to the human mutations E200K, D178N and
F198S as previously described [47]. The numbering refers to
the human sequence but the equivalent amino-acid residue
of the mouse sequence was altered (one codon proximal in
each case).
Purification of these mutants from inclusion bodies was as
previously described [47] except that the bacteria were
6156 M. Daniels et al. (Eur. J. Biochem. 268) q FEBS 2001
grown at 26 8C to avoid major degradation of the protein and
no gradient was applied to the DE52/CM52 column during
purification. Full length protein (PrP23–231) and other
recombinant proteins equivalent to the complete metabolic
C-terminal cleavage product of PrP (PrP113–231) or dele-
tion mutants, PrP23–112, PrP79 – 231 and PrP105– 231,
were also purified and refolded as previously described [14].
PrP23–112, PrP113–231, PrP105–231 and PrP79–231
were generated by PCR-based mutagenesis using splint
oligonucleotides to introduce a restriction site before codon
113, 105 or 79 or after codon 112. Following digestion of the
pET-PrP construct with Nde I the N-terminus encoding
region was removed and religation of the vector lead to a
pET-PrP construct expressing the complete metabolic
C-terminal fragment of PrP as determined by sequencing.
The N-terminal fragment was prepared by digestion of the
mutated plasmid with Xho I, removing the C-terminal
encoding fragment and religating the plasmid. Fidelity of
the N-terminus of these deletion mutants was determined by
mass spectroscopy analysis.
Shorter peptides were synthesized in house by the PNAC
facility. These peptides based on the mouse prion protein

sequence with corresponding amino-acid residues were
PrP112–125(AGAAAAGAVVGGLG), 121–146(AVVGG
LGYMLGSAMSRPIIHFGSDYED), PrP147– 171(RYYRE
NMYRYPNQVYYRPVDQYSNQ), PrP163–184(RPVDQ
YSNQNNFVHDCVNITIK), PrP180– 198(NITIKQHTVTT
KGENFT) and PrP196–220(NFTETDVKMMERVVRQM
CVTQYQKE).
CD spectroscopy and analysis
CD spectra were recorded for prion proteins and peptides
using a CD6 spectropolarimeter (Jobin Yvon, Division
d’Instrumente S.A.) or a Jasco J-810 spectropolarimeter,
calibrated with ammonium (1)-camphor-10-sulfonate by a
method similar to that described previously [36]. Peptide
samples were diluted from high concentration dimethylsulf-
oxide stocks to a concentration of 2 mg
:
mL
21
in 10 mM
sodium phosphate (pH 7.4). These samples were measured
in cuvettes of 1 mm or 0.5 mm pathlength (Hellma). The
spectrum from 190 nm to 250 nm was analysed with step
resolution of 0.5 nm at a temperature of 23 8C. Five scans
were averaged and the background from buffer was
subtracted. Spectra are presented as molar ellipticity (u).
RESULTS
PrP121–231 is toxic to neurones
Prion proteins of different lengths were tested for toxicity by
application to cultures of cerebellar neurones. Cerebellar
cultures were prepared from 6-day-old mice. The proteins

were applied to the cultures and the cultures were
maintained in serum-free medium for 7-days. The medium
was changed every 3 days and replenished with prion
proteins. At the end of that time, survival was determined
using an MTT assay. Full length recombinant PrP
c
, PrP79–
231, PrP105– 231 and PrP113 –231 showed no toxicity to
wild-type cultures of cerebellar cells (Fig. 1A). However,
PrP121–231 was toxic to these cells in a dose-dependent
manner. The proteins were also applied to cultures of Npu-
Prnp8
/
8 cerebellar cells and the same results were obtained
(Fig. 1B). However, PrP121–231 was more toxic to Prnp8
/
8
cerebellar cells than to wild-type cells (Student’s t-test,
P , 0.05). Additionally, PrP121–231 was more toxic to
Zrk-Prnp8
/
8 cerebellar cells than wild-type cerebellar cells
Fig. 1. Toxicity of prion proteins. Cerebellar cell cultures from wild-
type (A) and Npu-Prnp8
/
8 (B) cerebellar cells were grown in serum-free
medium and treated with prion proteins at different concentrations for
7 days. After that time the survival of the cultures was measured with an
MTT assay. The values were compared to those for untreated cultures as
a percentage. Shown are PrP23–231 (W) PrP79–231 (O), PrP105–231

(A), PrP113–231 (K) and PrP121–231 (X). Bovine serum albumin (B)
was used as a negative control. (C) Wild-type and Zrk Prnp8
/
8 cerebellar
cells were either treated with
L-leucine methyl ester (grey bars) or
cocultured with additional microglia (black bars). Cerebellar cells were
treated with 1 mg
:
mL
21
PrP23–231 or PrP121–231 for 7 days. After
this time, the survival was measured using an MTTassay and comparing
values to those of untreated cultures (open bars). Shown are the
mean ^ SEM of four experiments (different cultures) with three
determinations (separate wells) each.
q FEBS 2001 Novel PrP peptide toxicity (Eur. J. Biochem. 268) 6157
(Fig. 1C). This confirms that the increased toxicity to
Prnp8
/
8 cerebellar cells is a result of the lack of PrP
c
expression and not the genetic background of the mice.
These results suggest that the toxicity of PrP121–231 does
not require the expression of native PrP
c
.
Previously, it has been shown that the synthetic peptide
fragment PrP106–126 is toxic and that this toxicity requires
microglia.

L-Leucine methyl ester selectively destroys
microglia [25]. Cultures of wild-type and Zrk- Prnp8
/
8
cerebellar cells were treated with 50 m
ML-leucine methyl
ester for 2 h before application of PrP121 – 231 or were
cocultured continuously with wild-type microglia during
application of PrP121– 231 for 7 days. At the end of this
time an MTT assay was carried out. Treatment with
L-leucine methyl ester had only a minor effect on the
toxicity of the protein. Addition of microglia had no signifi-
cant effect on the toxicity of PrP121– 231. These results
suggest that the toxicity of PrP121– 231 does not require
involvement of microglia.
In order to verify that PrP121–231 is toxic to neurones,
staining with Hoechst 33342 reagent was carried out during
treatment of cerebellar cell cultures. Dying neurones could
be determined by the presence of condensed and/or frag-
mented nuclei (Fig. 2). Treatment with PrP121–231 caused
a great increase in the number of fragmented nuclei present
after 4 days of treatment as compared to treatment with full
length protein (PrP23–231). The quantitation of this is
shown in Fig. 2C.
Peptide dissection of PrP121–231 toxicity
Five synthetic peptides corresponding to the majority of
PrP121–231 were synthesized. These peptides were
PrP121–146, PrP147–171, PrP163–184, PrP180–198 and
PrP196–220. Initially we attempted to synthesize a set of
four nonoverlapping peptides. However, one of these

peptides self terminated during synthesis. Therefore two
overlapping peptides were produced to cover the region
172–180. As for the recombinant proteins, the toxicity of
these peptides was tested on cultures of wild-type and
Npu-Prnp8
/
8 cerebellar cells (Fig. 3). Peptides PrP121–146,
PrP147–171 and PrP180–198 showed little or no toxicity to
wild-type cells whereas PrP196–220 and PrP163–184 were
both toxic to wild-type cerebellar cells (Fig. 3A). However,
when applied to Npu-Prnp8
/
8 cerebellar cells PrP147–171
showed toxicity. This suggests that this peptide is toxic in
the absence of PrP
c
expression. PrP163–184 showed reduced
toxicity. However, PrP196–220 also remained similarly
toxic (Fig. 3B). These results suggest that the toxicity of the
C-terminal domain of PrP
c
in particular is associated with
amino-acid residues 163–184 but that amino-acid residues
further N-terminal to this may also participate in the toxicity
to PrP
c
deficient cells. Additionally residues of PrP196–220
might also participate in this toxicity.
The peptides used in this analysis were studied using
the CD spectroscopic technique. The analysis of the struc-

tural content was determined by the curve fitting program
CNNR. The CD spectra of the five peptides appears in Fig. 4.
The analysis of the structural content of the peptides appears
in Table 1. As can be seen in Fig. 4, PrP180–198 has a
noticeably different spectrum with an unusual minimum at
230 nm. It is currently unclear what this minimum
represents. PrP196–200 has a high percentage of b sheet
structure. This peptide readily forms fibrils (data not
shown).
Mutants of PrP121 –231
Further recombinant mouse proteins based on PrP121–231
were analysed for toxicity. These new proteins contained the
equivalent of one of three human point mutations associated
with human prion disease. The mutations analysed were
E200K, D178N and F198S. For simplicity the mutants of
PrP121–231 shall be referred to by these mutation assign-
ments even though in the mouse sequence the location of the
residue is numerically one place closer to the N-terminus in
the sequence (e.g. E200K in human but in mouse the E is at
199). PrP121–231 will be referred to as wild-type protein.
Wild-type cerebellar cells were treated with the four
Fig. 2. PrP121–231 causes apoptotic cell death. Cerebellar cells
from wild-type mice were exposed to PrP23–231 (A) or PrP121–231
(B) for 2 days. The cells were then stained with the Hoechst reagent to
detect fragment or condensed nuclei. An increase in aberrant nuclei
(bright condensed, fragmented) was only detected in cultures treated
with PrP121–231. Scale bar, 50 m
M (C) Quantitation of aberrant nuclei
in culture treated with either PrP23 –231, PrP121–231 or the control.
Ten fields were counted on three coverslips for four separate

experiments. Shown and mean and SEM.
6158 M. Daniels et al. (Eur. J. Biochem. 268) q FEBS 2001
different proteins for 7 days, after which time an MTT assay
was used to quantitate survival. The E200K mutation was
the only mutation to increase the toxicity of PrP121–231
significantly above that of the wild-type protein (P , 0.05)
(Fig. 5A). Zrk-Prnp8
/
8 cerebellar cells were also treated with
the proteins and assayed for survival in the same manner
(Fig. 5B). Both E200k and F198S showed significantly
(P , 0.05) more toxicity to Zrk-Prnp8
/
8 cerebellar cells than
the wild-type PrP121 –231. The implication of these results
is that the mutations in the region of amino-acid residues
198–200 enhance the toxicity of the peptide PrP121–231.
Peptide inhibition of PrP121 –231 toxicity
In the paper by Shmerling et al. [42] it was suggested that
the in vivo toxicity of PrP121–231 was inhibited by the
coexpression of full length PrP
c
(PrP23–231). Therefore we
tested whether PrP23–231, PrP23–112, PrP105 – 231 or
PrP113–231 would inhibit the toxicity of PrP121 –231 to
Npu-Prnp8
/
8 cerebellar cells. Of these proteins only PrP23 –
231 inhibited toxicity completely as determined by MTT
assay (Fig. 5C). However, PrP105– 231 showed a low level

of inhibition. This result suggests that PrP23–231 contains a
domain inhibitory to the toxicity of PrP121–231, but this
domain does not lie in the unstructured N-terminus; possibly
it lies in the hydrophobic domain of PrP
c
.
A recent study on optimization of the neurotoxicity of the
well characterized prion protein peptide PrP106–126 [36]
indicated that optimal toxicity of this region is located in
amino-acid residues 113–126 of the human sequence. This
region is identical to amino-acid residues 112–125 of the
Fig. 4. Circular dichroism study of peptides. Peptides were freshly
prepared in 10 m
M phosphate buffer pH 7.4 at 2 mg
:
mL
21
. The
peptides shown are PrP112–125, PrP121–146, PrP147– 171, PrP163–
184, PrP180–198 and PrP196–220. Five sweeps were collected at
0.5-nm intervals. Spectra are presented as molar ellipticity (u).
Fig. 3. Toxicity of peptides to cerebellar neurones. Cerebellar cell
cultures from wild-type (A) and Npu-Prnp8
/
8 (B) cerebellar cells were
grown in serum free medium and treated with prion protein peptides at
different concentrations for 5 days. After that time the survival of the
cultures was measured with an MTT assay. The values were compared
to those for untreated cultures as a percentage. Shown are PrP121–146
(W) PrP147–171 (X), PrP163–184 (A), PrP180–198 (K) and PrP196–

220 (O). Shown are the mean ^ SEM of four experiments with three
determinations each.
Table 1. Analysis of CD of peptides. Values determined using the
CNNR program. Analysis based on the spectra between 190 and 250 nm.
Helix b Sheet b Turn Random coil
PrP121–146 12 14 8 56
PrP147–171 21 34 20 21
PrP163–184 33 24 17 26
PrP180–198 20 32 19 40
PrP196–220 20 41 20 20
q FEBS 2001 Novel PrP peptide toxicity (Eur. J. Biochem. 268) 6159
mouse sequence. This peptide is highly toxic to wild-type
cerebellar cells but is not at all toxic to Zrk-Prnp8
/
8
cerebellar cells [37]. The ability of this peptide to inhibit
the toxicity of PrP121–231 was analysed. Increasing
amounts of PrP112– 125 were mixed with fixed amounts
of E200K, D178N, F198S and wild-type PrP121–231 and
applied to Npu-Prnp8
/
8 cerebellar cells for 5 days. After
this time, an MTT assay was used to measure survival
(Fig. 5D). PrP112–125 suppressed the toxicity of wild-
type PrP in a dose-dependent manner reaching saturation
above a 1 : 1 molar ratio. PrP112–125 was not able to
completely suppress the toxicity of the mutant proteins.
There was no further effect of toxicity above a 1: 1 molar
ratio. The relative order of effective suppression was
wild-type . F198S E200K . D178N. Indeed, for

D178N there was no significant inhibition of toxicity
(P . 0.05).
CD analysis of peptide–protein interaction
The CD spectra of PrP121–231 and its mutants (in the
presence or absence of an equimolar amount of PrP112–
125) were measured (Fig. 6). In general, and as previously
described [47], the three point mutations did not alter the CD
spectra markedly (Fig. 6A). PrP112–125 has previously
been shown to have a predominantly random coiled
structure with some amount of b sheet (Fig. 4). The
mixture of wild-type PrP121–231 and PrP112–125 resulted
in a unique CD spectrum that did not resemble that of
PrP121–231, PrP112 –125 or an arithmetic sum of the two.
Fig. 5. Toxicity of PrP121–231 mutants. Cerebellar cell cultures
from wild-type (A) and Zrk-Prnp8
/
8 (B) cerebellar cells were grown in
serum free medium and treated with mutants of PrP121–231 at
different concentrations for 7 days. After that time the survival of the
cultures was measured with an MTT assay. The values were compared
to those for untreated cultures as a percentage. Shown are wild-type (W)
D178N (K), F198S (O) and E200K (X). (C) Npu-Prnp8
/
8 cerebellar
cells were either treated with 50 m
M of PrP121–231 and with
increasing concentrations of PrP23– 231 (X), PrP23–112 (W), PrP105–
231 (K) or PrP112–231 (O). After 5 days treatment the survival was
measured using an MTT assay and comparing values to those of
untreated cultures. Shown are the mean ^ SEM of four experiments

with three determinations each. (D) Npu-Prnp8
/
8 cerebellar cells were
either treated with PrP112–125 at varying concentrations alone (B)or
with the addition of 50 m
M of PrP121–231 (W) or PrP121–231
mutants, D178N (K), F198S (O) or E200K (X). After 5 days treatment
the survival was measured using an MTT assay and comparing values to
those of untreated cultures. Shown are the mean ^ SEM of four
experiments with three determinations each.
Fig. 6. Circular dichroism study of prion proteins. Proteins were
freshly prepared in 10 m
M phosphate buffer pH 7.4 at 0.1 mg
:
mL
21
.
Wild-type PrP121–231 (WT) and three mutants (E200K, F198S,
D178N) were measured either on their own (compared in A) or mixed
with an equimolar amount of PrP112–125 (B– E). The spectra of the
wild-type form of PrP121–231 and the mutants on their own (thin line)
are compared to spectra of the same proteins mixed with PrP112–125
(thick line). In addition the spectra for PrP105– 231 (F) and PrP113–
231 (G) were also determine on their own (thin line) or mixed with
PrP112–125 (thick line). The increased levels of structural elements
were determined by a CD spectra analysis program. Five sweeps were
collected at 0.5-nm intervals. Spectra are presented as molar ellipticity
[u]. The increased helical content in all the proteins induced by
PrP112–125 was determined and plotted in (H).
6160 M. Daniels et al. (Eur. J. Biochem. 268) q FEBS 2001

PrP112–125 had similar but lesser effect on the mutant
proteins E200K and F198S. There was little effect on the
spectrum of D178N. In particular, the mixtures showed
relative increases in helical content (Fig. 6H). This change
in spectrum possibly represents a change in the structure of
the protein as a result of an interaction between the protein
and the peptide.
In order to determine if the structure produced by the
interaction of PrP112–125 and PrP121–231 represents a
‘true’ PrP structure, the CD spectra of PrP105–231 and
PrP113–231 was determined (Fig. 6). The CD spectra of
both proteins appear similar but dissimilar to the mixtures
between PrP121–231 and PrP112–125. The spectra of
PrP113–231 and PrP105–231 were similar to that of
PrP121–231. However, both spectra show a loss of a strong
minima at 222 nm and an increase at 230 nm. However,
mixing these proteins with PrP112–125 did not cause any
change in the spectrum except a slight increase at 230 nm.
There was no increase in helical content (Fig. 6H).
DISCUSSION
The precise mechanism of neurodegeneration in prion
disease is unknown. Much research has concentrated on a
peptide PrP106–126 to understand neuronal death in
models of these diseases. Until this report there has been
no complete investigation of the prion protein for toxic
domains. It has been known for some time that the
N-terminus (amino-acid residues 23 –90) is not associated
with disease. We aimed to investigate whether the
C-terminal domain has inherent toxicity to neurones
because a recent publication by Shmerling et al. [42] has

shown that mice expressing a C-terminal fragment of PrP
c
(either deletion of amino-acid residues 32–121 or 32– 134)
die within several weeks of birth due to massive neuro-
degeneration in the granule cell layer of the cerebellum. A
different C-terminal fragment of PrP
c
(PrP112–231) is
generated by normal cleavage of the protein [15]. It has been
suggested that this fragment can be cycled back to the
cell surface where it may have a function independent of the
full length molecule [48]. We found that the fragment
PrP121–231 was highly neurotoxic. This seemed to us to be
paradoxical as one would not expect a normal cleavage
product to be potently neurotoxic. However, slightly longer
peptides (PrP105–231 and PrP113–231) showed no
toxicity. The region of the mouse protein containing
amino-acid residues 105 –125 is almost identical to the
sequence of human PrP, which is known as PrP106–126, the
neurotoxic peptide used by many laboratories as a model of
the neurotoxicity of PrP
Sc
. However, this region of the
protein, as part of a C-terminal fragment of PrP
c
, has no
demonstrable toxicity. Furthermore, addition of a peptide
with amino-acid residues 112–125 of the mouse PrP
c
sequence to cerebellar cultures in parallel with PrP121–231

neutralized the toxicity of PrP121 –231. The same result
was obtained with mouse PrP121–231 and the human
PrP106–126 (data not shown). Under these conditions, the
PrP106–126 toxicity was fully inhibited. Additionally we
also showed that PrP23–231 inhibits toxicity but that
neither the N-terminal fragment (PrP23–112) nor the full
C-terminal fragment (PrP113–231) inhibit the toxicity.
Studies of the N-terminus have shown that it is unstructured
and therefore it is unlikely to undergo a conformational
change when PrP23–231 is cleaved. However, PrP113–231
contains a very hydrophobic N-terminus (amino-acid
residues 113–135) and it is quite possible that this region
would be masked if it interacted with other hydrophobic
residues in the rest of the protein.
The normal cleavage product of PrP
c
metabolism is a
protein cleaved at around amino-residue 112–114 and not
121–122 [15,17,18]. Therefore separation of the hydro-
phobic, palindromic region of 112 –120 from the rest of the
C-terminus is artefactual. Such a fragment of the protein has
never been shown to exist in vivo. Furthermore, the paper by
Shmerling et al. [42] demonstrates that expression of a fuller
C-terminal cleavage product with a deletion only of residues
32–106 does not develop pathology. The toxicity of the
C-terminal part of PrP
c
has never been apparent before the
publication by Shmerling et al. [42] and most interest has
focussed on the known neurotoxic region PrP106–126

whose toxicity came to attention because of study of the
toxicity of PrP
Sc
[24]. If deletion of PrP
c
up to residue 121
creates a toxic protein then one would expect that deletion of
the C-terminus of the protein should similarly cause prob-
lems. Muramato et al. [41] showed that deletion of amino-
acid residues 177–200 or 201–217 results in neurodegen-
erative disease in mice expressing such modified PrPs.
However, the disease was not as severe as that described by
Shmerling et al. [42] and the cell death does not appear to be
due to the toxicity of the hydrophobic domain (amino-acid
residues 112–135). Our findings regarding PrP121–231
mediated neurotoxicity might explain the cerebellar
degeneration described by Shmerling et al. [42]. However,
although our results suggest that the neurodegeneration in
this model could be the result of direct neurotoxic effects of
PrP121–231, the full mechanism of this effect needs to be
elucidated.
The neurodegeneration seen in the 32–121 deleted mice
[42] was prevented by the presence of coexpressed full-
length PrP
c
. The previous interpretation of this result was
that PrP
c
has a ligand that interacts with it at two domains.
When the protein only interacts with PrP

c
at the C-terminal
domain, the resulting signal transduction leads to death.
However, the problem with this model is that the metabolic
C-terminal domain of PrP
c
(PrP112–231) exists in neurones
all the time. Even if it has a much lower affinity for the
‘ligand’ than full length PrP
c
then one would nevertheless be
causing the death of cerebellar granular cells. A better
hypothesis according to our data is that the C-terminal
domain (PrP121–231) as expressed by Shmerling et al. [42]
can interact with full length PrP
c
. This interaction via
binding to the hydrophobic domain of PrP
c
then inhibits the
toxicity of the C-terminus.
The normal N-terminal domain of the C-terminal
cleavage product of PrP
c
is conserved so highly as to be
unchanged from reptile to man [49,50]. This is very unusual,
especially for a region of the protein associated with
neurotoxicity. Therefore it is clearly essential to the normal
metabolism of PrP
c

. We have shown previously [11,37] that
this region of the protein is the site of interaction between
PrP
c
with PrP
Sc
. Therefore it is possible, under normal
metabolic conditions, that the hydrophobic-palindromic
region of PrP
c
interacts directly with the globular domain of
the C-terminus of another molecule of PrP
c
. However, we
consider it more likely that each molecule folds in such a
way that the very N-terminus of PrP’s C-terminal cleavage
q FEBS 2001 Novel PrP peptide toxicity (Eur. J. Biochem. 268) 6161
product folds into the globular domain. Due to the very
hydrophobic nature of this region, this may occurs directly
after cleavage of the protein.
Our studies of toxicity used wild-type neurones and
neurones deficient in PrP
c
expression in parallel. This
allowed us to distinguish fragments or peptides that were
toxic in absence of PrP
c
. This also allowed us to demonstrate
that the toxicity of PrP121 – 231 is different to that of PrP
Sc

,
as PrP
Sc
is nontoxic to cells lacking PrP
c
expression [21] but
the toxicity of PrP121–231 is increased to Prnp8
/
8 cerebellar
cells. In particular, the region of amino-acids 147–171
shows toxicity only to Prnp8
/
8 cerebellar cells and might
explain the increased toxicity of PrP121–231 to Prnp8
/
8
cerebellar cells. PrP peptides based on the hydrophobic
domain of PrP
c
have also been suggest to have effects not
mediated directly through PrP
c
[28,30] but this is the first
time the peptides based other regions of PrP have been
shown to be toxic to Prnp8
/
8 neurones.
Of importance are the studies with peptides that highlight
the region of the C-terminus involved in the toxicity of
PrP121–231 to cerebellar neurones. Two peptides, PrP163–

184 and PrP196–220, appeared to be the most toxic. This is
of considerable interest as the majority of point mutations
associated within inherited human prion disease are found
within these regions (e.g. D178N, V180I, T183A, F198S,
E200K, R208H, V201I, Q217R). Indeed, the two point
mutations studied in this paper, E200K and F198S, both map
to PrP196 –220 and both enhanced the toxicity of PrP121–
231. It is quite possible that these point mutations alter the
ability of the globular domain to interact with the conserved
hydrophobic region. This was demonstrated here by a
decreased ability of PrP112–125 to inhibit the toxicity of
the PrP121–231 mutants.
Many of these point mutations lie along a hydrophobic
cleft in the protein [47,51]. It is quite possible that the
N-terminal domain of the C-terminal cleavage product will
fold into this cleft. However, this report does not give any
information on this aspect of the structure of the protein.
Previously, NMR studies of PrP
c
have used either PrP121–
231 or PrP23–231 as the protein for analysis [47,52–55].
The general finding of these reports was that the C-terminal
of the protein contains three helices that fold together to
make a globular domain while the N-terminus has little
structure. For both the full length protein and the C-terminal
fragment the globular domain was very similar. However,
the protein PrP121–231 does not represent the true
C-terminal fragment of PrP as it does not include the
perfectly conserved amino-acid residues 112– 120 which
constitute the true N-terminus. Therefore it is probably of

vital importance that the C-terminal fragment containing
these additional amino acids be studied further, as the true
C-terminal fragment of PrP
c
may form a novel conformation
as yet unknown. Support for this comes from the analysis by
CD of two proteins PrP105–231 and PrP113–231 that have
moderately different spectra to that of PrP121–231. It has
previously been suggested that such proteins prepared
from bacteria are sensitive to protease digestion and the
amino-acid residues up to 121 are cleaved off [53]. We took
particular care to ensure that our proteins were not degraded
in this way.
Analysis of the effect of point mutations on the structure
of PrP
c
have focussed on their effect on the protein PrP121–
231. We have observed that two mutations alter the toxicity
of this protein but this is not likely to have direct relevance
to prion disease as the protein PrP121–231 does not
normally exist. However, if the amino-acid residues where
these mutations occur are central to refolding of the protein
after cleavage then it is possible that C-terminus could be
toxic in the inherited prion diseases. Alternatively, or in
addition, a misfolded C-terminal protein could somehow
influence formation of PrP
Sc
. Another alternative is that
interaction between the globular domain and the hydro-
phobic-palindromic region influences cleavage of the

protein. Then, a large amount of uncleaved mutant PrP
c
might remain and be sufficient to eventually aggregate and
begin conversion to PrP
Sc
.
In summary, we have shown that a C-terminal fragment of
PrP
c
commonly used in the investigation of PrP
c
structure
and known to induce neurodegeneration in mice lacking
expression of the full length PrP
c
is neurotoxic. This neuro-
toxicity does not require expression of PrP
c
. The neuro-
toxicity can be inhibited by the presence of the hydrophobic-
palindromic region (amino-acid residues 112–125) of the
protein either applied as a separate peptide or attached to the
protein. Known human point mutations enhance the toxicity
of this PrP
c
fragment. However, PrP121–231 might not
represent the true C-terminal cleavage produce of PrP
c
metabolism and re-investigation of the biology of the
C-terminus of PrP

c
is necessary.
ACKNOWLEDGEMENTS
The authors than Charles Weissmann for the Zrk1 prion protein
knockout mice and Jean Manson for the Npu prion protein knockout
mice. Thanks also to Rudi Glockshuber for PrP121 –231 mutant
proteins. In addition we thank Bill Broadhurst and Tim Daffron for help
with the CD analysis.
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