(Received for publication, April 19, 1995; and in revised form, June 7, 1995)
From the
The cellular form of the prion protein (PrP) is a
glycoprotein anchored to the cell membrane by a
glycosylphosphatidylinositol moiety. An aberrant form of PrP
that is partially resistant to proteases, PrP
, is a
hallmark of prion diseases, which in humans include Creutzfeldt-Jakob
disease (CJD), Gerstmann-Sträussler-Scheinker
syndrome, and fatal familial insomnia. We have characterized the major
forms of PrP in normal and pathological human brains. A COOH-terminal
fragment of PrP
, designated C1, is abundant in normal and
CJD brains as well as in human neuroblastoma cells. Sequence analysis
revealed that C1 contains alternative NH
termini starting
at His-111 or Met-112. Like PrP
, C1 is glycosylated,
anchored to the cell membrane, and is heat-stable. Consistent with the
lack of the NH
-terminal region of PrP
, C1 is
more acidic than PrP
and does not bind heparin. An
additional fragment longer than C1, designated C2, is present in
substantial amounts in CJD brains. Like PrP
, C2 is
resistant to proteases and is detergent-insoluble. Our data indicate
that C1 is a major product of normal PrP
metabolism,
generated by a cleavage that disrupts the neurotoxic and amyloidogenic
region of PrP comprising residues 106-126. This region remains
intact in C2, suggesting a role for C2 in prion diseases.
The cellular prion protein (PrP) (
)is a
glycoprotein anchored to the cell surface by a
glycosylphosphatidylinositol (GPI) moiety (Stahl et al.,
1987). It is encoded by a single gene on human chromosome 20 and
contains 253 amino acids with two potential N-linked
glycosylation sites (Kretzschmar et al., 1986; Liao et
al., 1986; Puckett et al., 1991). Mature human PrP
spans residues 23-231 of the polypeptide predicted from the
PrP cDNA sequence (Oesch et al., 1985; Kretzschmar et
al., 1986; Liao et al., 1986). It results from the
removal of the 22-amino acid NH
-terminal signal peptide
(Hope et al., 1986; Turk et al., 1988) and the
cleavage of the 23-amino acid COOH-terminal hydrophobic peptide with
the simultaneous addition of the GPI anchor (Stahl et al.,
1987, 1990). In mouse neuroblastoma cells, PrP
turns over
with a half-life of 3-6 h (Caughey et al., 1989;
Borchelt et al., 1990). A chicken homologue of mammalian
PrP
has been shown to recycle between the plasma membrane
and an endosomal compartment with concurrent generation of truncated
PrP
intermediates (Shyng et al., 1993). The role
of PrP
in normal cellular function remains elusive.
Expression of PrP
was found to correlate with neuronal
differentiation (Wion et al., 1988) and embryonic development
(Mobley et al., 1988; Lazarini et al., 1991; Manson et al., 1992). Transgenic mice devoid of PrP
lack
detectable defects in development and behavior
(Büler et al., 1992), but display an
impairment of synaptic function in the hippocampus (Collinge et
al., 1994).
An aberrant isoform of PrP, PrP,
characterized by relative resistance to proteolysis and by insolubility
in nondenaturing detergents, is a hallmark of the prion diseases, which
include familial, transmitted, and sporadic forms of neurodegenerative
disorders such as Creutzfeldt-Jakob disease (CJD),
Gerstmann-Sträussler-Scheinker syndrome, kuru, and
fatal familial insomnia in humans and scrapie and bovine spongiform
encephalopathy in animals (for reviews, see Prusiner(1991) and Prusiner
and DeArmond (1994)). Studies using scrapie-infected mouse
neuroblastoma cells suggest that PrP
is formed from
PrP
, either at the cell surface (Caughey and Raymond, 1991)
or along the endocytic pathway (Borchelt et al., 1992). Since
there are no detectable differences in primary structure between the
two PrP isoforms (Stahl et al., 1993), the formation of
PrP
is thought to result from the conversion of PrP
to PrP
through a conformational transition.
Consistent with this notion is the difference in secondary structure
between PrP
and PrP
: PrP
is rich
in
-helices, while PrP
has a higher content of
-pleated sheets (Caughey et al., 1991a; Safar et
al., 1993; Pan et al., 1993). Several predicted
-helical domains of PrP
form
-sheet structures
and aggregate into amyloid-like fibrils when synthesized as
polypeptides (Gasset et al., 1992; Tagliavini et al.,
1993). In a recent study, Kocisko et al.(1994) succeeded in
converting PrP
to a protease-resistant form in a cell-free
system, suggesting that the conversion process results from a direct
interaction between PrP
and PrP
.
The
molecular mechanism that leads to this conversion and to subsequent
prion replication remains unclear. An essential step toward the
complete clarification of the conversion of PrP to
PrP
is the detailed knowledge of PrP
metabolism. In this study, we have characterized the PrP forms
present in normal and pathological human brains and in neuroblastoma
cells. Using epitope mapping and radiosequencing, we have demonstrated
the presence of truncated forms of PrP
under normal
conditions, in addition to full-length PrP
. Moreover, an
additional NH
-terminally truncated fragment that is
insoluble in detergents and resistant to protease is present in the
brains of subjects with prion diseases.
To release GPI-anchored membrane proteins,
the P2 fraction was resuspended in a hypotonic buffer (20 mM Tris, 2 mM EDTA, pH 7.5). The sample was then adjusted to
20 mM Tris, 150 mM NaCl, 2 mM EDTA, pH 7.5
(TNE buffer), and incubated for 2 h at 37 °C with 1 unit/ml
recombinant Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PIPLC) purified
according to Deeg et al.(1992) or obtained from Oxford
Glycosystems. The mixture was then diluted with 10 volumes of TNE
buffer and centrifuged at 100,000 g for 1 h to
separate PIPLC-released proteins (supernatant) from the rest of the
membrane components (pellet).
To examine the detergent solubility
and protease resistance of PrP in normal and CJD brains, the membrane
fraction (P2) was resuspended in lysis buffer (without protease
inhibitors) and centrifuged at 100,000 g to obtain
detergent-soluble (supernatant) and detergent-insoluble (pellet)
fractions. Protease treatment was carried out by incubation of samples
in lysis buffer with 10 µg/ml proteinase K at 37 °C for 1 h.
Figure 1:
Characterization of truncated forms of
PrP in normal human brain. A, shown is a schematic
representation of human PrP, with the underlined regions
recognized by the three main antibodies used in this study. The
amyloidogenic and neurotoxic domain (residues 106-126)
(Tagliavini et al., 1993; Forloni et al., 1993) of
PrP is shaded. Post-translational modifications of PrP include
two N-linked glycosylation sites (indicated by Y-shaped
symbols) at residues 181 and 197, respectively, and a GPI linkage
at the COOH-terminal residue 231 (Prusiner, 1991). Numbering
corresponds to human PrP amino acid sequence (Kretzschmar et
al., 1986). B, shown is the presence of truncated forms
of PrP in the normal human brain. Brain homogenates were
either left untreated(-) or treated (+) with PNGase F to
remove N-linked oligosaccharides. Samples were analyzed on
immunoblots with the anti-N (a-N), 3F4, and anti-C (a-C) antibodies. A prominent PrP fragment, designated C1,
reacted with anti-C, but not with 3F4 and anti-N. C, PrP
immunoreactivity was eliminated following absorption of anti-N and
anti-C with their respective peptide
antigens.
Figure 2:
Subcellular localization of C1 in human
brain. A, a postnuclear brain fraction (S1) was used
to prepare cytosol (S2) and membrane (P2) fractions.
The fractions were deglycosylated prior to immunoblotting with anti-C
and 3F4. C1 was recovered mainly in the membrane fraction, while a
small amount was detectable in the cytosol. B, the membrane
fraction (P2) obtained in A was treated with PIPLC and then
centrifuged at 100,000 g for 1 h. The resulting
fractions were analyzed in immunoblots with anti-C to detect PrP forms
released by PIPLC (supernatant) and those remaining associated
with the membrane (pellet). Like PrP
, C1 was
released by PIPLC and recovered in the
supernatant.
Small amounts of C1 and PrP were also present in the
cytosolic fraction (Fig.2A, S2). Both of
these forms may represent PrP
molecules that have slowly
lost their GPI linkage to the membrane as observed previously in
primary cultures of neonatal hamster brain (Borchelt et al.,
1993).
Figure 3: Presence of C1 on the cell surface of human neuroblastoma cells and increased expression with differentiation. M17 BE(2)-C cells were cultured in the absence(-) or presence (+) of retinoic acid for 7-10 days. Cells were then incubated in fresh medium containing PIPLC for 2 h at 37 °C to release PrP molecules from the cell surface. The samples were analyzed on immunoblots with anti-C (lanes 1-4) or 3F4 (lanes 5-8) before (-PNGase) or after (+PNGase) deglycosylation.
Since human
PrP is encoded by a single exon of a single copy gene on
chromosome 20 (Liao et al., 1986; Puckett et al.,
1991), C1 is likely to be a product of PrP
processing. The
lack of C1 immunoreactivity with 3F4, whose epitope includes PrP
residues 109-112, suggests that C1 is generated by a cleavage
beyond residue 109. This cleavage may constitute an important event in
normal PrP
metabolism. To characterize the exact cleavage
site(s) in PrP
that generated C1, we partially purified C1
from the medium of PIPLC-treated cells by reverse-phase HPLC,
radioiodinated Tyr residues of C1 with
I and IODO-GEN,
and performed NH
-terminal radiosequencing of C1. After
radioiodination and SDS-PAGE, C1 appeared as a doublet of
22 kDa (Fig.4A, lane2). This shift in
mobility is not surprising since incorporation of
I into
Tyr residues results in an increased mass of C1 and could produce
additional mobility shifts due to perturbation in SDS binding. To
characterize the doublet bands of C1 (designated as C1-upper and
C1-lower, respectively), they were each subjected to
NH
-terminal radiosequencing. The results show that both
bands have the same NH
termini. Both C1-upper and C1-lower
produced major
I peaks at cycles 17 and 18, consistent
with C1 starting at either His-111 or Met-112 (Fig.4B). Therefore, C1 is generated by an alternative
cleavage around His-111 of PrP
. Since no apparent doublet
is seen in unlabeled C1 (Fig.4A, lane1), the observed
I-C1 doublet may represent
the same C1 molecules with anomalous behavior on SDS-PAGE due to
radioiodination.
Figure 4:
Radiosequencing of C1. C1 from
PIPLC-conditioned culture medium was separated from other proteins by
its resistance to heat and by chromatography on a reverse-phase HPLC
column. A, C1 was either left untreated (lane1) or radioiodinated with NaI by the
IODO-GEN method (lane2). Unlabeled C1 was detected
on immunoblots with anti-C (lane1), while labeled C1
was immunoprecipitated with anti-C and visualized by autoradiography (lane2). After iodination, C1 appeared as doublet
bands at
22 kDa designated as C1-upper and C1-lower, respectively. B, Problott membranes
containing the C1-upper and C1-lower bands shown in A were
excised and radiosequenced. The sequences of C1 molecules beginning at
His-111 and Met-112 are superimposed to illustrate the concordance of
radioactivity with positions at which tyrosine residues should
occur.
To compare the biochemical properties of C1 to
those of full-length PrP present in cultured cells, we
first examined the capacity of these two molecules to bind heparin.
PrP
has been shown to interact with heparin and other
glycosaminoglycans, which prevent the formation of PrP
(Gabizon et al., 1993; Caughey and Raymond, 1993;
Caughey et al., 1994). Fig.5shows that at variance
with PrP
(lane6), C1 did not bind
heparin (lane4). The lack of the PrP
NH
-terminal region in C1 suggests that this region is
important for heparin binding. Taking advantage of the different
heparin binding characteristics of C1 and PrP
, we have
attempted to clarify the changes in electrophoretic mobility of these
two proteins following deglycosylation. It is clear that the 18-kDa
deglycosylated C1 isoform stems from its 28-kDa glycoform (lanes3 and 4), while 27-kDa PrP
is
derived from a glycosylated 35-kDa form (lanes5 and 6). The 8-10-kDa reduction in molecular mass for both C1
and PrP
following deglycosylation is consistent with the
presence of two Asn-linked glycans at residues 181 and 197 (Endo et
al., 1989; Haraguchi et al., 1989).
Figure 5:
Binding of C1 and PrP to
heparin. Medium containing PIPLC-released proteins from cells was
incubated with heparin-agarose for 1 h at room temperature, followed by
centrifugation at 500
g. An aliquot of the untreated
sample served as control (ORIGINAL). Samples were analyzed on
immunoblots with anti-C before(-) and after (+)
deglycosylation with PNGase F. C1 did not bind heparin and was
recovered in the supernatant (UNBOUND), while full-length PrP
was bound to heparin immobilized on agarose beads (BOUND).
We also studied
the heat resistance of PrP isolated from cultured cells.
PIPLC-released proteins were boiled in the presence of protease
inhibitors, followed by cooling and centrifugation to pellet the
heat-sensitive protein aggregates. Both C1 and full-length PrP
were recovered in the supernatant as heat-stable proteins (Fig.6, lanes3 and 4). In contrast,
most of the other proteins were heat-sensitive and were recovered in
the pellet.
Figure 6:
Thermal stability of PrP and
C1. Cells were treated with fresh Opti-MEM containing PIPLC for 2 h at
37 °C. The medium was collected and adjusted to 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 2 mM EDTA, 1 mM dithiothreitol, 100 mM Tris, pH 7.5.
After boiling for 15 min followed by cooling for 1 h at 4 °C, the
sample was centrifuged at 16,000
g for 15 min at 4
°C. The resulting supernatant (heat-stable) and pellet (heat-sensitive) were analyzed on immunoblots with anti-C
before(-) and after (+) deglycosylation with PNGase
F.
Two-dimensional gel electrophoresis of the
PIPLC-released and deglycosylated proteins from neuroblastoma cells
revealed that C1 and full-length PrP migrated to regions of
the NEPHGE gel of pH 6.0 and 7.8, respectively (Fig.7). Thus,
C1 is more acidic than PrP
. Since the
NH
-terminal region of PrP
contains several
basic amino acid residues, an acidic shift in the pI of C1 is
consistent with the loss of this region. Some charge microheterogeneity
was evident for both C1 and full-length PrP
. This may be
due to the presence of sialic acid in some of the GPI anchors of
PrP
(Stahl et al., 1992).
Figure 7:
Two-dimensional gel electrophoresis of C1
and PrP. PIPLC-released proteins from M17 BE(2)-C cells
were concentrated and deglycosylated prior to NEPHGE for 4 h at 400 V,
followed by SDS-PAGE (14% separating gel). An immunoblot was prepared
using anti-C. C1 (arrowhead) and PrP
migrated to
NEPHGE regions of pH
6.0 and 7.8,
respectively.
Figure 8: Presence of the C2 fragment in CJD brains. Brain homogenates from one control (CO) and three CJD subjects were deglycosylated and analyzed on immunoblots with anti-C and 3F4. In the CJD brains, there were significant amounts of the C2 fragment, which reacted with both anti-C and 3F4.
PrP is known to be insoluble in
nondenaturing detergents, a property that allows it to be separated
from normal PrP
by differential centrifugation (Caughey et al., 1991b; McKinley et al., 1991). To investigate
the possibility that C2 shares these characteristics with
PrP
, detergent-soluble and detergent-insoluble membrane
fractions from a CJD brain as well as from a normal brain were obtained
by extraction with lysis buffer and centrifugation. As shown by
immunoblots (Fig.9) with anti-C (upperpanel)
and 3F4 (lowerpanel), C2 was found only in the
detergent-insoluble fraction of the CJD brain (lanes2), along with PrP
. Deglycosylated 27-kDa
PrP
was seen as a doublet (lanes2).
The two bands of the doublet reacted with both anti-C and 3F4 (Fig.9) as well as with anti-N (data not shown), suggesting
that they may be the same in length, but differ in an unidentified
post-translational modification. Immunoblots of the detergent-insoluble
fraction, following proteinase K digestion (lanes6),
revealed a single band that had the same electrophoretic mobility and
immunostaining characteristics as C2. Both C1 and full-length PrP
were recovered in the detergent-soluble fraction (lanes1 and 3) and were sensitive to the protease
treatment (lanes5 and 7). Therefore, C2 is
likely to correspond to the PrP
fragment generated in
vitro by proteinase K digestion, which includes residues
90-231 (Bolton et al., 1987). The amount of C2 appeared
to correlate with the amount of abnormal PrP
fragment
generated in vitro by proteinase K treatment (data not shown).
Our findings indicate that the NH
-terminally truncated
PrP
form, likely to be the same as that generated in
vitro from brains of patients with prion diseases following
proteinase K digestion, is already present in CJD brains in
vivo. This conclusion is consistent with the observation that a
PrP
isoform truncated at residue 90 is present in
scrapie-infected mouse neuroblastoma cells (Caughey et al.,
1991b).
Figure 9:
Insolubility in detergent and resistance
to protease of the C2 fragment. Membrane fractions of a CJD brain and
of a normal brain (Control) were prepared as described under
``Materials and Methods.'' The P2 fraction was resuspended in
detergent-containing lysis buffer (DET) and centrifuged at
100,000 g for 1 h at 4 °C. The resulting
supernatant and pellet were referred to as detergent-soluble (S) and detergent-insoluble (I) fractions,
respectively. They were treated in the absence(-) or presence
(+) of 10 µg/ml proteinase K (PK) in the above buffer
for 1 h at 37 °C. All samples were then deglycosylated prior to
immunoblotting with anti-C (upperpanel) and 3F4 (lowerpanel). C2 and PrP
were
insoluble in detergents, while C1 and PrP
were
soluble.
C2 was found to be more basic than C1 on two-dimensional gel electrophoresis (Fig.10). This finding is consistent with the known presence of additional basic amino acids between residues 90 and 110. C2 thus contains residues 106-126, the region that has been shown in vitro to be highly amyloidogenic and neurotoxic (Tagliavini et al., 1993; Forloni et al., 1993).
Figure 10: Two-dimensional gel electrophoresis of the C2 fragment. Brain homogenate from a CJD brain was deglycosylated prior to NEPHGE for 4 h at 400 V, followed by SDS-PAGE (14% separating gel). Immunoblots were prepared using anti-C and 3F4. C2 (open arrowheads) migrated to a more basic NEPHGE region than C1 (solidarrowhead), while full-length PrP migrated to an even more basic region at 27 kDa.
This study indicates that a COOH-terminal fragment of
PrP, which we have termed C1, is present in significant
amount in human brain (Fig.1). This fragment is unlikely to be
the product of post-mortem autolysis since it is also present in brain
tissue obtained by biopsy and in a human neuroblastoma cell line. C1 is
NH
-terminally truncated and is the main product of normal
PrP
processing. It shares many features with the
COOH-terminal half of PrP
, including glycosylation,
membrane attachment by a GPI anchor, and heat stability in aqueous
solutions. The thermal stability of C1 and PrP
in solution,
which to our knowledge has never been reported, provides a simple way
to purify PIPLC-released PrP
forms because of the
relatively small number of heat-stable proteins.
Biochemical
differences between C1 and full-length PrP are related to
the lack of the NH
-terminal region of PrP
in
C1. C1 polypeptide exhibited a relatively acidic pI when compared with
PrP
. This shift in electrophoretic mobility is to be
expected as a result of the loss of the NH
-terminal region
of PrP
, which contains 10 basic residues up to position
110. Basic residues have also been shown to be crucial for heparin
binding (Margalit et al., 1993), providing a possible
mechanism for the lack of heparin binding of C1. On the basis of
antibody competition, PrP
-heparin binding had been proposed
to occur between residues 142 and 174 (Gabizon et al., 1993).
How the polyclonal antibody recognizing PrP residues 142-174
affected heparin binding in that study is not clear. Our present
finding argues that the first 110 NH
-terminal residues of
PrP
are necessary for heparin binding since C1 lacks
affinity for heparin even though it contains residues 142-174.
The difference in heparin binding affinity allows for the separation of
C1 from full-length PrP
.
The cellular compartment in
which human PrP is cleaved to generate C1 in vivo is unknown. PrP fragments comparable to C1 have been noted
previously. In mouse neuroblastoma cells transfected with chicken
PrP
constructs, PrP
has been shown to recycle
between the cell surface and the endosomal compartment to produce a
COOH-terminal fragment (Shyng et al., 1993) by a cleavage
between chicken PrP
residues 116 and 137 (Harris et
al., 1993), corresponding to human PrP
residues
109-130 (Gabriel et al., 1992). A similar fragment in
hamster brain has been suggested to result from a cleavage between
hamster PrP
residues 111 and 138, corresponding to residues
112-134 of the human PrP sequence (Pan et al., 1992). We
have performed NH
-terminal radiosequencing and found that
human PrP
is cleaved to produce C1 that has the NH
terminus starting at either His-111 or Met-112. The sequence
information obtained from the present study will help to reconstruct
the individual steps of PrP
metabolism and C1 formation.
There is mounting evidence that the region between residues 100 and
130, containing the C1 cleavage site, plays a critical role in the
conformational changes underlying the conversion of PrP into PrP
, the abnormal isoform present in all prion
diseases. Gasset et al.(1992) have identified the PrP
sequence comprising residues 109-122 as one of the putative
-helical regions that, as synthetic peptide, acquires a
-sheet structure and forms amyloid-like fibrils, a property shared
by PrP
purified from scrapie-infected brains (Caughey et al. 1991a; Gasset et al., 1992; Safar et
al., 1993). Tagliavini et al.(1993) have also shown that
a synthetic peptide encompassing human PrP
residues
106-126 forms fibrils similar to those present in the PrP amyloid
plaques of prion diseases such as CJD and
Gerstmann-Sträussler-Scheinker syndrome. Moreover,
this peptide is toxic to neurons and causes astrocytic proliferation in
culture (Forloni et al., 1993).
Our data demonstrate that
in normal human brain and in neuroblastoma cells, PrP undergoes a proteolytic cleavage that breaks the potentially
pathogenic region. Thus, the cleavage that generates C1 from PrP
may be the crucial step in the constitutive pathway of PrP
metabolism that precludes the accumulation of a pathogenic
fragment (Hope and Chong, 1994). It is thus critical to know whether
perturbations in this metabolic pathway play a role in prion diseases.
In scrapie, PrP
and PrP
have been shown to
have identical amino acid sequence and are thought to differ only in
conformation. Hence, prion replication would occur by conversion of
PrP
into PrP
through dimerization (Cohen et al., 1994) or nucleation-dependent polymerization (Jarrett
and Lansbury, 1993). In brains from subjects with sporadic CJD, a PrP
COOH-terminal fragment that we named C2 was markedly increased compared
with control brains (Fig.8). C2 is insoluble in nondenaturing
detergents (Fig.9), a property shared by PrP
(Bolton et al., 1987). C2 is recognized by
90-104, 3F4, and anti-C and has the same electrophoretic
mobility as the protease-resistant core fragment of PrP
that spans residues 90-231 (Bolton et al., 1987;
Stahl et al., 1993). Therefore, C2 is the ``in vivo homologue'' of the PrP
fragment generated in vitro by proteinase K digestion and is likely to derive
from limited NH
-terminal trimming of PrP
in
CJD brains. The concurrent presence of both full-length PrP
and C2 is consistent with this notion. The finding that
NH
-terminal truncation of PrP
occurs in
vivo in scrapie-infected mouse brain (Hope et al., 1988)
and scrapie-infected neuroblastoma cells (Caughey et al.,
1991b) further supports this conclusion. Alternatively, C2 may derive
from PrP
through activation of an otherwise minor metabolic
pathway that differs from the constitutive pathway generating C1 and
that preserves the pathogenic PrP
region comprising
residues 106-126. In scrapie-infected cells, it has been shown
that a PrP
COOH-terminal fragment, comparable to C2, can be
directly converted into a protease-resistant form (Rogers et
al., 1993). Therefore, the possibility that in CJD and in other
prion diseases the production of significant amounts of C2 through an
abnormal metabolic pathway is an important pathogenetic event, and C2
is directly converted to the insoluble form, cannot be ruled out.
Regardless of which of these two possibilities is correct, the present
findings suggest that normal processing of PrP
in human
brain occurs at a cleavage site within the amyloidogenic region of
PrP
, generating C1. In prion disease, either PrP
or PrP
is also cleaved at a different site,
resulting in a longer fragment, C2, which contains the entire
amyloidogenic region.
The pathogenic region of PrP bears
resemblance to the amyloid
-protein (Come et al., 1993),
a pathogenic peptide of 39-42 amino acids derived from a large
precursor protein (amyloid
-precursor protein) (Selkoe, 1994).
Amyloid
-precursor protein is constitutively cleaved at residue 16
of amyloid
-protein, a cleavage that prevents the production of
intact amyloid
-protein (Selkoe, 1994).
Amyloid -protein,
upon conformational change, may become toxic and resistant to proteases
and form amyloid fibrils (Nordstedt et al., 1994; Maury,
1995). Although small amounts of amyloid
-protein are secreted by
cells in vitro, intact amyloid
-protein is not detectable
in normal human brains. However, it is abundantly present in the brains
of subjects with Alzheimer's disease (Tabaton et al.,
1994). The presence of significant amounts of a pathogenic peptide,
perhaps as a result of altered metabolic processing, might be a
critical event in both Alzheimer's and prion diseases.