Received for publication, August 4, 2000, and in revised form, November 7, 2000
Gerstmann-Sträussler-Scheinker
disease (GSS) is a cerebral amyloidosis associated with mutations in
the prion protein (PrP) gene (PRNP). The aim of this study
was to characterize amyloid peptides purified from brain tissue of a
patient with the A117V mutation who was Met/Val heterozygous at codon
129, Val129 being in coupling phase with mutant
Val117. The major peptide extracted from amyloid fibrils
was a ~7-kDa PrP fragment. Sequence analysis and mass spectrometry
showed that this fragment had ragged N and C termini, starting mainly
at Gly88 and Gly90 and ending with
Arg148, Glu152, or Asn153. Only Val
was present at positions 117 and 129, indicating that the amyloid
protein originated from mutant PrP molecules. In addition to the
~7-kDa peptides, the amyloid fraction contained N- and C-terminal PrP
fragments corresponding to residues 23-41, 191-205, and 217-228.
Fibrillogenesis in vitro with synthetic peptides corresponding to PrP fragments extracted from brain tissue showed that
peptide PrP-(85-148) readily assembled into amyloid fibrils. Peptide
PrP-(191-205) also formed fibrillary structures although with
different morphology, whereas peptides PrP-(23-41) and
PrP-(217-228) did not. These findings suggest that
the processing of mutant PrP isoforms associated with
Gerstmann-Sträussler-Scheinker disease may occur
extracellularly. It is conceivable that full-length PrP and/or large
PrP peptides are deposited in the extracellular compartment, partially
degraded by proteases and further digested by tissue endopeptidases,
originating a ~7-kDa protease-resistant core that is similar in
patients with different mutations. Furthermore, the present data
suggest that C-terminal fragments of PrP may participate in amyloid formation.
 |
INTRODUCTION |
Gerstmann-Sträussler-Scheinker disease
(GSS)1 is an adult-onset
neurodegenerative disorder (1, 2) that is inherited as an autosomal
dominant trait and segregates with variant genotypes resulting from the
combination of a pathogenic mutation (P102L, P105L, A117V, F198S,
D202N, Q212P, and Q217R) and a common polymorphism at codon 129 (Met/Val) in the prion protein (PrP) gene (PRNP) (3-9). The
pathological hallmarks of the disease are the accumulation of altered
forms of PrP, termed PrPSc, and the deposition of PrP
amyloid in the central nervous system (10-15). The main
physicochemical properties that distinguish PrPSc from the
normal cellular isoform (PrPC) are a high content of
-sheet secondary structure, insolubility in nondenaturing
detergents, and partial resistance to protease digestion (16). In the
presence of detergents, the protease-resistant core of
PrPSc (termed PrPres) assembles into
amyloid-like fibrils (16).
In previous studies we have determined the biochemical composition of
amyloid fibrils extracted from brain tissue of patients with mutations
F198S and Q217R in PRNP. The smallest amyloid subunit was
found to correspond to a ~7-kDa PrP fragment spanning residues ~81
to ~150 (17, 18). Although this fragment did not include the region
with the amino acid substitution, it originated from mutant molecules,
suggesting that the codon 198 and 217 mutations are a dominant factor
for amyloidogenesis (18).
The allelic origin of the altered forms of PrP involved in the
pathologic process has been investigated in other genetic prion diseases including GSS P102L, fatal familial insomnia, and
Creutzfeldt-Jakob disease (CJD) associated with point or insertional
mutations (19-23). These studies showed that only mutant PrP is
detergent-insoluble and protease-resistant in GSS P102L (19, 20), fatal
familial insomnia, and CJD D178N (21), whereas both the mutant and
wild-type proteins have these properties in CJD V210I (22) and CJD with five or six extra copies of the octapeptide repeat (21). CJD E200K
illustrates an intermediate state, as the wild-type PrP is insoluble
but protease-sensitive (23). Thus, the recruitment of the wild-type
protein into the disease process may reflect the effects of
conformational changes due to specific mutations, which allow
interaction between mutant and normal PrP.
The present study was undertaken to characterize the amyloid protein
and establish its allelic origin in a patient with GSS A117V from a
previously reported Alsatian family (24, 25).
 |
EXPERIMENTAL PROCEDURES |
Immunohistochemistry--
Specimens of cerebral cortex were
fixed in 4% formaldehyde and embedded in paraplast. Seven µm thick
serial sections were stained with hematoxylin-eosin, Congo Red, and
thioflavine S or were immunostained with antibodies to the N terminus,
mid-region, and C terminus of human PrP. These included a rabbit
antiserum to a synthetic peptide homologous to residues 23-40
(PrP-(23-40)) (18), the monoclonal antibody 3F4 which recognizes the
epitope 109-112 (26, 27), and a monoclonal antibody to a synthetic peptide spanning residues 214-231 (SP214) (28). The antibodies were
used at a dilution of 1:200. Before immunostaining, the sections were
treated with 98% formic acid for 60 min. The immunoreactions were
revealed using the EnVision Plus-horseradish peroxidase system for
rabbit or mouse immunoglobulins (Dako, Carpenteria, CA) and diaminobenzidine as chromogen (29).
Immunoblot Analysis of Crude Brain Extracts--
Specimens of
cerebral cortex and basal ganglia were homogenized in 9 volumes of
lysis buffer (10 mM Tris-HCl, pH 7.5, 100 mM
NaCl, 10 mM EDTA, 0.5% Nonidet P-40, 0.5% sodium
deoxycholate), sonicated for 1 min, and centrifuged at 16,000 × g at 4 °C for 15 min. To test the protease resistance of
PrP, aliquots of supernatant corresponding to 100 or 250 µg of
protein were digested with proteinase K (PK) at concentrations ranging
from 5 to 50 µg/ml at 37 °C for 1 h. The digestion was
carried out either in lysis buffer or in Laemmli's sample buffer
containing 2% SDS. After PK digestion, aliquots were deglycosylated
using recombinant peptide N-glycosidase F (New
England Biolabs Inc., Beverly, MA) at 37 °C for 12 h, following the manufacturer's instructions. The samples were fractionated by 12.5 or 16.5% Tricine/SDS-polyacrylamide gel electrophoresis (Tricine/SDS-PAGE) under reducing conditions, transferred to
polyvinylidene difluoride membranes (Millipore Corp., Bedford, MA), and
probed with antibodies PrP23-40 (1:1000), 3F4 (1:50,000), and SP214
(1:200). To test the solubility of PrP in nondenaturing detergents,
samples of cerebral cortex were homogenized in 9 volumes of 20 mM Tris, pH 7.5, 0.32 mM sucrose, 5 mM EDTA, and centrifuged at 1000 × g for
10 min. The resulting supernatant was further centrifuged at
100,000 × g at 4 °C for 1 h. The pellet
containing cell membranes was resuspended in lysis buffer, sonicated
for 1 min, and centrifuged at 100,000 × g at 4 °C
for 1 h to obtain detergent-soluble and detergent-insoluble
fractions. Soluble and insoluble fractions were analyzed by Western
blot using the antibody 3F4.
Isolation of Amyloid Cores--
Amyloid plaque cores were
isolated from 30 g of cerebral cortex. After removal of
leptomeninges and large vessels, the tissue was homogenized with a
Brinkmann homogenizer in buffer A (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 2 mM EGTA,
0.4 mM phenylmethylsulfonyl fluoride, 1% Triton X-100) at
a sample to buffer ratio of 1:5 (w/v). The homogenate was sieved
through 1-mm nylon mesh, and the filtrate was centrifuged at
10,000 × g for 20 min. The pellet was rehomogenized in
buffer B (as for buffer A, with NaCl replaced by 0.6 M KI
and the concentration of Triton X-100 reduced to 0.5%) and centrifuged
at 10,000 × g for 20 min. The pellet was rehomogenized in buffer C (as for buffer B, with KI replaced by 1.5 M
KCl) and centrifuged at 10,000 × g for 20 min. The
pellet was washed three times in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, and centrifuged at 70,000 × g
for 30 min. The pellet was resuspended in 50 mM Tris-HCl,
pH 7.5, 10 mM CaCl2, 3 mM
NaN3 at a sample to buffer ratio of 1:25 (w/v), and
subjected to collagenase digestion (Collagenase 3.4.24.3 type 1, Sigma)
at 37 °C for 18 h, using a 1:100 (w/w) ratio of enzyme to
pellet. After centrifugation at 70,000 × g for 60 min,
the pellet was resuspended and washed three times in 50 mM
Tris-HCl, pH 7.5, loaded on a discontinuous sucrose gradient (1.0, 1.2, 1.4, 1.7, and 2.0 M sucrose in 10 mM Tris-HCl,
pH 7.5), and centrifuged at 100,000 × g for 180 min.
Each interface was collected, washed, and pelleted three times in 50 mM Tris-HCl, pH 7.5. Aliquots of each pellet were assessed
for presence of amyloid fibrils by polarized light microscopy after
Congo Red staining, fluorescence microscopy after thioflavine S
treatment, and electron microscopy after negative staining with 2%
aqueous phosphotungstic acid. Amyloid plaque cores were recovered in
the 1.7 M sucrose fraction.
Purification and Characterization of Amyloid Proteins--
The
amyloid-enriched pellet was suspended in 99% formic acid and sonicated
four times for 20 s. After addition of 2 volumes of distilled
water, the sample was centrifuged at 10,000 × g for 10 min, and the supernatant was applied to a calibrated Sephadex G-100
column (1.2 × 120 cm) equilibrated with 3 M formic
acid. Protein peaks were pooled and concentrated with a speed vacuum concentrator. The purity and molecular weight of the fractions were
determined by 12.5% Tricine/SDS-PAGE under reducing conditions and by
immunoblot analysis with the 3F4 antibody. The amyloid fraction was
further analyzed with rabbit antisera to synthetic peptides homologous
to residues 58-71, 90-102, 127-147, and 151-165 of human PrP
(1:1000 dilution) as described previously (18). Gel filtration fraction
5 that contained the major amyloid subunit was further purified by high
performance liquid chromatography (HPLC) on a reverse-phase C4 column
(214TP104, Vydac) with a 0-80% linear gradient of acetonitrile
containing 0.1% (v/v) trifluoroacetic acid, pH 2.5. The column eluents
were monitored at 214 nm, and protein peaks were lyophilized. Following
Tricine/SDS-PAGE and immunoblot analysis, aliquots of the
HPLC-purified, PrP-immunoreactive peptides were dissolved in 25 mM Tris-HCl, pH 8.5, 1 mM EDTA, and incubated
at 37 °C for 24 h with endoproteinase Lys-C (Roche Molecular
Biochemicals) at an enzyme to substrate ratio of 1:30 (w/w). The
peptides generated from enzymatic digests were separated by HPLC on a
reverse-phase Delta-Pak C18 column (0.39 × 30 cm, Waters,
Milford, MA) with a 0-70% linear gradient of acetonitrile containing
0.1% (v/v) trifluoroacetic acid, pH 2.5.
Amino Acid Sequencing--
Sequence analyses of the intact
amyloid protein and peptides generated by enzymatic digestion were
carried out on a 477A microsequencer, and the resulting
phenylthiohydantoin amino acid derivatives were identified using the
on-line 120A PTH analyzer and the standard program (PE Biosystems,
Foster City, CA).
Matrix-assisted Laser Desorption Mass Spectrometry--
N- and
C-terminal fragments of the amyloid protein generated by endoproteinase
Lys-C digestion were reduced, repurified by HPLC on a reverse-phase C4
column, and prepared for matrix-assisted laser desorption/ionization
mass spectrometry using the dried droplet method (30). The matrix used
was
-cyano-4-hydroxycinnamic acid (Sigma), which was purified
by recrystallization. To produce the dried droplets, a saturated
solution of matrix was prepared in 2:1 aqueous 0.1% trifluoroacetic
acid:acetonitrile at room temperature. The sample was added to this
solution so that the final sample concentration was 1-10
mM. One-half microliter of the solution was placed on the
probe of the mass spectrometer and allowed to dry. The sample was then
ready for analysis. The mass spectrometer was a custom-built linear
time-of-flight mass spectrometer with a 1-m flight tube with a wire ion
guide operated at
50 V DC. The ion source operated at +40 kV ion
acceleration potential. The laser used was an LSI-337ND (Laser Science,
Boston, MA) nitrogen laser (2 ns pulse width), which was focused onto a
200-µm diameter fiber optic, conducted down 1 m of fiber optic, and then imaged onto the matrix deposit with a doublet of positive lens
(magnification = 1). The detector of the mass spectrometer was a
combination of a microchannel plate (the input stage) and a discrete
dynode electron multiplier (amplification stage). The custom data
system uses a Lecroy 9350M oscilloscope configured for dynamic range
enhancement to improve the fidelity of small signals.
Fibrillogenesis in Vitro--
Peptides homologous to residues
23-41, 85-148, 191-205, and 217-228 of human PrP were synthesized
by solid-phase chemistry and purified by reverse-phase HPLC as
described previously (31). Purity was greater than 95%. The peptides
were suspended in deionized water or in 20 mM Tris, pH 7.0, at a concentration of 1, 5, and 10 mg/ml. Following incubation at
37 °C for 1 h or at 20 °C for 1, 3, and 7 days, 50 µl of
each sample were air-dried on gelatin-coated slides, stained with 1%
aqueous thioflavine S, and analyzed by fluorescence microscopy. For
electron microscopy, 5 µl of suspension were applied to
Formvar-coated nickel grids, negatively stained with 5% (w/v) uranyl
acetate, and observed in an electron microscope model 410 (Philips
Electronic Instruments, Mahwah, NJ) at 80 kV.
 |
RESULTS |
The patient selected for this study was a 24-year-old man, whose
clinico-pathologic and genetic profile has been reported in detail
previously (24, 25). He started at age 20 with a pseudobulbar syndrome,
followed by cerebellar, extrapyramidal, and pyramidal signs, and
cognitive deterioration. Neuropathologic examination revealed massive
deposition of PrP-immunoreactive deposits in the cerebral cortex,
thalamus, basal ganglia, and cerebellum. Most PrP deposits exhibited
the tinctorial and optical properties of amyloid, i.e.
birefringence after Congo Red staining and fluorescence following
thioflavine S treatment. Genetically, the patient carried the silent A
to G transition at the third position of PRNP codon 117 and
a C to T transition at the second position of that codon, resulting in
Ala
Val substitution. Furthermore, he was heterozygous Met/Val at
codon 129, Val129 being in coupling phase with mutant
Val117 (24).
To define the PrP species involved in the pathologic process,
immunohistochemical and biochemical studies were undertaken. The
immunohistochemical analysis of formalin-fixed, paraplast-embedded brain sections showed that the amyloid cores were immunoreactive with
antibodies to the central region of the molecule, whereas antibodies to
the N and C termini immunostained only the periphery of the cores (Fig.
1). Western blot analysis of homogenates
of cerebral cortex and basal ganglia revealed the presence of a
PK-resistant PrP fragment of ~7 kDa (Fig.
2A). This fragment was
immunoreactive with antibodies to the central region while unreactive
with antibodies to N- and C-terminal domains, and its electrophoretic
mobility was unmodified by N-deglycosylation. The protease
resistance of the 7-kDa peptide was observed under standard conditions
(50 µg/ml PK in lysis buffer, 37 °C, 1 h) and was abolished
by the addition of 2% SDS to the reaction buffer (data not shown). A
similar 7-kDa fragment was present in brain homogenates before PK
digestion, as a prominent component of a complex PrP pattern composed
of ~35-, ~33-, and ~28-kDa full-length PrP (i.e. di-,
mono-, and nonglycosylated species), 21-22-kDa N-terminal-truncated
peptides, and 16-17-kDa N- and C-terminal truncated fragments (Fig.
2A). As previously observed in the Indiana kindred of GSS
with F198S mutation (32), the relative abundance of the low molecular
weight fragment was not dependent upon the extent of amyloid burden. The analysis of detergent solubility showed that ~40% of full-length PrP partitioned in the insoluble fraction, whereas the 21-22-kDa N-terminal truncated peptides were mostly soluble, and the N- and
C-terminal truncated fragments of 16-17 and 7 kDa were entirely insoluble (Fig. 2B). Digestion of the insoluble fraction
with PK verified that only the 7-kDa peptide was protease-resistant (data not shown).

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Fig. 1.
PrP immunohistochemistry of cerebral amyloid
deposits. A and B are adjacent sections of
cerebral cortex from the A117V patient, immunolabeled with the
antibodies 3F4 (A) and SP214 (B) to PrP residues
109-112 and 214-231, respectively. Magnification bar, 100 µm.
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Fig. 2.
Western blot analysis of PrP peptides in
brain tissue homogenates and amyloid fraction, and HPLC purification of
amyloid peptides. A, extract of cerebral cortex of the
A117V patient before (lane 1) and after (lane 2)
PK digestion or PK digestion followed by deglycosylation (lane
3). The sample has been fractionated on 16.5%
Tricine-polyacrylamide minigel and probed with the antibody 3F4.
B, total homogenate (lane 1), detergent-soluble
(lane 2), and detergent-insoluble (lane 3)
fractions, separated on 12.5% Tricine-polyacrylamide minigel and
probed with the antibody 3F4. C, Western blot analysis of
the amyloid protein fraction obtained from gel filtration
chromatography, using antisera to synthetic peptides PrP-(58-71)
(lane 1), -90-102 (lane 2), -109-112
(lane 3), -127-147 (lane 4), and -151-165
(lane 5). The major amyloid protein component migrated as a
broad band centered at ~7 kDa. This band was immunoreactive with
antibodies spanning PrP residues 90-147 (lanes 2-4) and
unreactive with antisera to residues 58-71 and 151-165 (lanes
1 and 5). Each lane contained 10 µg of proteins.
Molecular mass markers expressed in kilodaltons are shown to the
left. D, HPLC purification of fraction 5 obtained
from gel filtration chromatography. One major (peak 1) and
several minor protein peaks were present in the chromatogram.
Immunoblot analysis showed that peak 1 was immunoreactive
with antibodies to PrP. Aliquots of this peak were used for sequence
analysis and enzymatic digestion with endoproteinase Lys-C.
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Amyloid cores were isolated from cerebral cortex by a procedure
combining buffer extraction, sieving, collagenase digestion, and
sucrose gradient fractionation. As shown by light and electron microscopic analysis, amyloid cores were the main component of the
final preparations; minor contaminants included lipofuscin granules and
small microvessel fragments. Proteins were extracted from amyloid
plaque cores with formic acid and fractionated on a Sephadex G-100
column. Gel filtration yielded two major peaks, the void volume
(fraction 1) and a low molecular weight peak (fraction 5) that was
present as a broad band centered at ~7 kDa on Tricine/SDS-PAGE minigels. In addition, minor intermediary peaks (fractions 2-4) were
observed in the chromatograms (data not shown). Immunoblot analysis of
the fractions obtained by gel filtration showed that the 7-kDa band was
immunoreactive with antibodies to PrP residues 90-102, 109-112, and
127-147 and was unreactive with antibodies to residues 58-71 and
151-165, suggesting that it corresponded to an internal fragment of
the molecule (Fig. 2C).
Further purification of fraction 5 by HPLC on a reverse-phase C4 column
yielded one major and several minor peaks (Fig. 2D). Immunoblot analysis showed that the major peak (Fig. 2D, peak 1) was immunoreactive with antibodies to PrP. Amino acid
sequencing revealed that peak 1 was composed of a PrP fragment with a
ragged N terminus, the major signals corresponding to residues 88 and 90. Peak 1 was digested with endoproteinase Lys-C; the enzymatic digest
was fractionated by HPLC on a reverse-phase C18 column and the peptides
subjected to microsequencing and laser desorption mass spectrometry.
Only two cleavage sites (i.e. positions 106/107 and 110/111)
resulting in three peptides were predicted on the basis of molecular
weight, antigenic profile, N-terminal sequence analysis, and the known
resistance to cleavage of Lys-Pro bonds at positions 101/102 and
104/105 of PrP. Microsequencing and mass spectrometry of the
HPLC-purified peptides revealed a spectrum of N termini spanning
residues 85-95, although Gly88, Gly90, and
Gly92 were the predominant components (Fig.
3, A and B).
Moreover, the C terminus was heterogeneous corresponding to
Arg148, Glu152, or Asn153 (Fig.
3C). Although the patient was heterozygous for the Ala
Val mutation at codon 117 and the Met/Val polymorphism at codon 129, only Val was found at these positions (Fig.
4).
In addition to the ~7-kDa amyloid peptide, fraction 5 obtained by gel
filtration chromatography also contained N- and C-terminal PrP
fragments corresponding to residues 23-41, 191-205, and 217-228. To
investigate whether these fragments could contribute to amyloid fibril
formation, we compared the fibrillogenic properties of homologous
synthetic peptides with those of the synthetic amyloid peptide spanning
residues 85-148. The peptides were incubated in deionized water or in
20 mM Tris, pH 7.0, for 1, 24, 72, and 168 h at a
concentration of 1, 5, and 10 mg/ml and analyzed by fluorescence
microscopy following thioflavine S staining and by electron microscopy
after negative staining. The peptide PrP-(85-148) formed dense
networks of straight, unbranched, ~13 nm diameter fibrils after
1 h incubation at 37 °C at a concentration of 1 mg/ml (Fig.
5A). Under the same
conditions, peptide PrP-(191-205) also formed fibrillary structures,
although morphologically different; they were more narrow and less
regular in size, both in diameter (range ~4.5-10 nm) and length (100 nm to 1-2 µm) (Fig. 5B). The PrP-(85-148) and
PrP-(191-205) aggregates exhibited fluorescence after thioflavine S
staining. Conversely, peptides PrP-(23-41) and PrP-(217-228) did not
generate amyloid-like fibrils in vitro even at the highest
concentration after 1 week.
The analysis of PrP extracted from amyloid plaque cores showed that the
amyloid protein corresponded to an ~7-kDa fragment spanning residues
85-153. Similar to previous studies on GSS F198S and Q217R, this
fragment was derived from the mutant allele since only Val was found at
position 117 and 129 (18). This finding is consistent with the
observation that the detergent-soluble fraction from total brain
homogenates contained ~60% of full-length PrP which was
protease-sensitive, whereas the remainder of full-length molecules as
well as the N- and C-terminal truncated fragments of 16-17 and 7 kDa
partitioned in the insoluble fraction. Mass spectrometric analysis
revealed that the amyloid peptide had ragged N and C termini; in
particular, the N terminus was remarkably heterogeneous, starting at
each position between residue 85 and 95. The amyloid fraction also
contained peptides corresponding to the 19 N-terminal amino acids of
full-length PrP and two C-terminal fragments comprising residues
191-205 and 217-228. This finding was consistent with the observation
that the periphery of the amyloid plaque cores was strongly
immunoreactive with antibodies to N- and C-terminal domains. The
copurification of these small peptides with the larger amyloid protein
was likely due to insufficient resolution of the gel filtration column
within this molecular weight range; alternatively, these peptides could
have been bound to the amyloid protein. Collectively, these data
suggest that in GSS A117V, mutant PrP is at least partially degraded by
proteases in the extracellular compartment, with formation of a major
amyloid peptide whose size and sequence is similar to that found in
patients with different mutations. Other fragments generated by partial degradation of the amyloid precursor protein contribute to plaque formation and may also participate in amyloidogenesis, since we found
that the peptide comprising residues 191-205 was able to assemble into
amyloid-like fibrils in vitro.
Studies with cell-free translation systems containing endoplasmic
reticulum-derived microsomal membranes have revealed that PrP may exist
in multiple topological forms, including a secretory form that is fully
translocated into the endoplasmic reticulum lumen and two transmembrane
forms with opposite membrane orientation (35-37). A specific
transmembrane form was found to be increased in transgenic mice
expressing the A117V mutation and was detected in brain tissue of
patients with GSS A117V (34). This form could be recognized by limited
proteolysis of brain homogenates using PK at low temperature in the
absence of ionic detergents; conversely, no protease-resistant PrP
peptides were found under conditions used to detect PrPres
in other prion diseases. Based on these observations it has been proposed that the transmembrane form of PrP rather than
PrPres plays a central role in neuropathological changes
observed in GSS A117V (34) as well as in other prion diseases (38). In the present study we found a remarkable accumulation of PrP amyloid in
brain tissue by immunohistochemistry and substantial amounts of a
PK-resistant, low molecular weight PrP peptide both in brain tissue
homogenates and in purified amyloid fractions by Western blot analysis.
Whether this protease-resistant PrP peptide originates from
transmembrane, glycosylphosphatidylinositol-anchored or secreted forms
of PrP has to be investigated.
In conclusion, our data from the present and previous studies on GSS
patients with different PRNP mutations indicate that the
minimal PrP segment essential for amyloidogenesis is an N- and
C-terminal-truncated fragment spanning residues ~88 to ~146 (Fig.
6). The major part of this fragment
corresponds to the end of the flexible N-terminal domain of
PrPC (39) which is thought to undergo a profound
conformational change in the conversion of the normal protein into
disease-specific species. The C terminus of the amyloid peptides is
similar to the truncated PrP found in a patient carrying an amber
mutation at PRNP codon 145 (40, 41). The conformational
plasticity of this region and its tendency to adopt
-sheet secondary
structure is supported by studies with synthetic peptides (42-44).
Interestingly, GSS amyloid peptides are derived from a PrP region that
is essential for disease transmissibility (Fig. 6). Early studies
determined that the protease-resistant core of PrPSc from
scrapie-infected brains, which is N-terminally truncated near residue
90, can transmit disease (45). Propagation and transmissibility of the
pathologic process is also sustained by a redacted version of PrP
lacking residues 23-88 and 141-176 (46, 47). Recently, Kaneko and
co-workers (48) addressed the question as to whether a PrP fragment
similar to the GSS amyloid peptide may initiate prion disease. They
used a synthetic peptide homologous to mouse PrP residues 89-143
(i.e. human residues 90-144) with the P101L substitution
corresponding to GSS-linked P102L mutation. Intracerebral inoculation
of a
-sheet-rich form of this peptide into transgenic mice
expressing low levels of the P101L mutation resulted in neurologic
dysfunction and neuropathological changes consistent with GSS.
Conversely, larger doses of a non-
-form of the same peptide failed
to induce these changes, emphasizing the importance of protein
conformation in disease initiation and propagation. Indeed, although
the 7-kDa PrP amyloid fragment is a component of the minimal PrP
sequence required for disease transmissibility, it is unlikely that
this peptide per se might support an efficient interaction
with PrPC and PrPC > PrPSc
conversion due to its insoluble
-sheet secondary structure and aggregation state.
We are grateful to Dr. R. J. Kascsak
(Institute for Basic Research in Developmental Disabilities, Staten
Island, NY) for donating the monoclonal antibody 3F4.
Published, JBC Papers in Press, November 21, 2000, DOI 10.1074/jbc.M007062200
The abbreviations used are:
GSS, Gerstmann-Sträussler-Scheinker disease;
PrP, prion protein;
PRNP, prion protein gene;
PrPC, normal cellular
form of the prion protein;
PrPSc, disease-specific isoform
of the prion protein;
PrPres, protease-resistant core of
PrPSc;
CJD, Creutzfeldt-Jakob disease;
PK, proteinase K;
PAGE, polyacrylamide gel electrophoresis;
HPLC, high
performance liquid chromatography;
Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine;
PTH, phenylthiohydantoin.
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