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Regular Articles |
From the Department of Pathology,*
New York University
Medical Center, New York, New York; the Istituto Nazionale Neurologico
Carlo Besta,
Milano, Italy and the Department
of Pathology and Laboratory Medicine,
Indiana
University School of Medicine, Indianapolis, Indiana
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Prion diseases are neurodegenerative disorders of humans and animals pathologically characterized by spongiosis, gliosis, neuronal loss, and variable amyloid deposition.12 Human prion diseases encompass sporadic Creutzfeldt-Jakob disease (CJD); genetic forms linked to the PrP gene (PRNP), such as familial CJD, Gerstmann-Sträussler-Scheinker disease (GSS), and fatal familial insomnia (FFI); and infectious forms such as kuru, iatrogenic CJD, and new variant CJD.13 Most of these disorders may be experimentally transmitted through exposure to pathological tissues. Extensive purification of the infectious agent of scrapie, a prototypic prion disease of sheep, led to the isolation of a major and possibly sole protein component.14 Subsequent studies identified that protein as a relatively insoluble and protease-resistant abnormal PrP isoform, termed PrPSc or PrPres.15,16
PrPSc derives from the normal cellular form (PrPC) via a post-translational process17 that is thought to lead to a conformational transition within the central hydrophobic and carboxyl-terminal regions of PrP resulting in a striking increase in ß-sheet secondary structure.18 The efficiency of this reaction may be influenced by certain amino acid substitutions in the PrP sequence, such as the codon 129 polymorphism19,20,21 and the mutations linked to familial prion diseases.22 The overrepresentation of PrP codon 129 homozygotes in iatrogenic and sporadic CJD as well as the restriction of new variant CJD to Met/Met subjects show that the codon 129 polymorphism affects the individual susceptibility to prion diseases.19,20,23 Moreover the presence of Val or Met at codon 129 of PRNP in phase with the D178N mutation determines the phenotypic presentation as CJD or FFI, respectively.21 Recent data suggest that distinct host molecules (factor X) also are implicated in the conversion.24 The most likely candidates are PrP-binding proteins with chaperone activity,25 but other alternatives cannot be excluded.
Characteristics of the proteolytic cleavage of PrPC in humans have been only partially addressed.26 As particular domains appear to be relevant to the conformational change that distinguishes PrPSc from PrPc, distinct proteolytic pathways of PrPC might yield peptides with different susceptibility to conversion.27-30 Likewise, knowledge of proteolytic processing of PrPSc isoforms is limited to the identification and characterization of fragments generated by proteinase K (PK), a nonphysiological method of cleavage. This approach has allowed a differentiation of prion disorders based on the molecular weight and degree of glycosylation of their protease-resistant PrP cores, including two types of sporadic CJD, a third type characteristic of peripherally acquired CJD, and a fourth type found only in the new variant CJD.23,31 In this paper we show that the endogenous proteolytic cleavage of PrPC in normal and in Alzheimer disease (AD) control brains follows a stereotypic pattern that is similar in brain cortex and cerebellum and not modified by the codon 129 genotype. However, we found significant differences in PrPC proteolysis, and probably in glycosylation and GPI composition, between neural and non-neural (tonsils and platelets) tissues. Most remarkably, we observed characteristic proteolytic pathways in brains affected with type 1 and type 2 sporadic CJD and GSS cases carrying the F198S mutation (GSS F198S), which generated PrPSc fragments that appear to be specific for each particular disorder.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Neural tissues were obtained at autopsy from 10 AD patients (AD1-10; age, 74.3 ± 2.1 years; postmortem intervals of 4 to 16 hours), 9 sporadic CJD cases (CJD1-9;age, 63.5 ± 6.9 years; postmortem intervals of 18 to 24 hours), 2 GSS patients carrying the F198S mutation (GSS1 and GSS2; ages 58 and 77 years; postmortem intervals of 5 and 7 hours), and 2 neurologically normal subjects (N1 and N2; ages 74 and 78 years; postmortem intervals of 6 and 8 hours). Samples consisted of fragments of brain cortex (N1, AD1-3, AD7, AD8, CJD1-9, GSS1, and GSS2) and cerebellum (N2, AD4-6, AD9, AD10, CJD3, CDJ9, and GSS1). AD was diagnosed according to CERAD criteria.32 The diagnosis of sporadic CJD and GSS was based on clinical, neuropathological, biochemical, and genetic criteria. Tonsils were collected after surgical removal from three otherwise normal children. Fresh platelets from three healthy individuals were purified by gel filtration as described.33 All samples were frozen immediately after collection and stored at -80°C.
Molecular Genetic Analysis
DNA was extracted from brain by standard procedures and analyzed to determine the genotype at codon 129 of the PrP gene.34 Polymerase chain reaction (PCR) was performed using oligonucleotide forward (5'-AAG AAG CGC CCG AAG CCT GGA GGA TGG-3') and reverse (5'-ATC CTG CAG GGG CCT GTA CAC TTG GTT-3') for 30 cycles, each consisting of a denaturation step at 94°C for 30 seconds, an annealing step at 50°C for 30 seconds, and an elongation step at 72°C for 90 seconds. PCR products of 438 bp were separated on 5% polyacrylamide gels and visualized by ethidium bromide staining. After confirming successful amplification, PCR products were digested with MaeII (Boehringer-Mannheim, Indianapolis, IN) following the manufacturer's conditions. The resulting fragments were resolved on 5% polyacrylamide gels and visualized by ethidium bromide staining under ultraviolet light.
Homogenization and Sarkosyl Solubility
Tissues were homogenized using a mixer pellet pestle in 6 vol of lysis buffer (10 mmol/L Tris, 150 mmol/L NaCl, 1% Nonidet P-40, 0.5% deoxycholic acid, 0.1% SDS, 5 mmol/L EDTA, pH 8) in the presence of protease inhibitors (Complete, Boehringer-Mannheim). After centrifugation at 12,000 rpm for 15 minutes, pellets were discarded and supernatants (S) were stored in aliquots at -80°C.
To test Sarkosyl solubility tissues were homogenized in 9 vol of sucrose buffer (0.25 mmol/L sucrose, 10 mmol/L Tris, 5 mmol/L EDTA, pH 7.5). After centrifugation at 1000 x g for 10 minutes, pellets were discarded and supernatants (S1) were centrifuged again at 100,000 x g for 1 hour at 4°C. The resultant pellets (P2) were resuspended in 1 vol of TNE buffer (20 mol/L Tris, 150 mmol/L NaCl, 2 mmol/L EDTA, pH 7.5), diluted with 9 vol of the same buffer containing 2% Sarkosyl, and centrifuged at 100,000 x g for 1 hour to obtain soluble (S3) and insoluble (P3) fractions. S3 was concentrated with 4 vol of ice-cold methanol, and then both S3 precipitate and P3 were resuspended in 1 vol of TNE.
Enzymatic Deglycosylation
Sample aliquots were mixed with 1/10 volume of denaturing buffer (20 mmol/L Tris, 150 mmol/L NaCl, 2 mmol/L EDTA, 10% ß-mercaptoethanol, 5% SDS, pH 7.5) and boiled for 15 minutes. After addition of Nonidet P-40 to 1%, Asn-linked oligosaccharides were cleaved by incubation with recombinant PNGase-F for 4 hours at 37°C and 12 hours at room temperature, as specified by the supplier (Biolabs, Beverly, MA).
PK Treatment
Aliquots of S fractions were precipitated with 4 vol of ice-cold methanol. Pellets were resuspended in 1 vol of TNE and incubated with 50 µg/ml PK (Boehringer-Mannheim) at 37°C. The reaction was stopped after 1 hour by addition of 3 mmol/L phenylmethylsulfonyl fluoride.
Immunoblot Analysis
Sample aliquots were mixed with 1/2 volume of 3X loading buffer
(240 mmol/L Tris, 6% SDS, 6% ß-mercaptoethanol, 30% glycerol,
0.06% bromophenol blue, pH 6.8) and boiled for 5 minutes. After
centrifugation at 12,000 rpm for 1 minute, samples were electrophoresed
on 16.5% polyacrylamide Tris-tricine gels and electroblotted onto
nitrocellulose membranes for 1 hour at 400 mA using 10 mmol/L
3-cyclohexylamino-1-propanesulfonic acid buffer, pH 11, containing 10%
methanol. The membranes were blocked with 5% nonfat dried milk in TBST
(10 mmol/L Tris, 150 mmol/L NaCl, 0.1% Tween 20, pH 7.5) for 2 hours
at room temperature and then incubated under the same conditions with
antibodies 3F4 or 
-SP214 diluted 1:50,000 and 1:100, respectively.
3F4 is a monoclonal antibody directed to human PrP residues 109 to
112.35
-SP214 is a monoclonal antibody raised against a
synthetic peptide corresponding to human PrP residues 214 to 231 that
was synthesized and characterized by analytical reverse-phase
high-pressure liquid chromatography, amino acid analysis, and
desorption spectrometry by Chiron Corp. (Emeryville, CA). The
monoclonal antibody to this peptide was custom produced by the Tissue
Culture/Hybridoma Facility at State University of New York (Stony
Brook, NY). The membranes were washed with TBST and then incubated for
1 hour with a horseradish-peroxidase-conjugated rabbit anti-mouse
antibody (Amersham, Arlington Heights, IL) at a dilution of 1:5000. The
membranes were washed again, developed using an enhanced
chemiluminescent substrate (SuperSignal, Pierce, Rockford, IL), and
detected with autoradiography film (Reflection, Dupont NEN, Boston,
MA). For quantitative analyses, blots were scanned using the UMAX 2.4.1
Magic Scan, and the density of the bands was measured using NIH Image
software.
Protease Inhibition
Aliquots of normal brain cortex (N1) sucrose homogenate were
diluted with 5 vol of phosphate-buffered saline, pH 7.2, incubated at
37°C for 24 hours with protease inhibitors (Table 1)
, metal ions (Ca2+,
Cu2+, Fe2+, Mg2+, or
Zn2+), or both at a 20:1/inhibitor:metal ion molar ratio
and then deglycosylated and analyzed by immunoblot with antibody
-SP214. The bands corresponding to full-length PrP were quantified
by densitometry, and the results were expressed as a percentage related
to the control, a nonincubated aliquot.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To study the proteolytic processing of PrPC in brain we analyzed 10 AD and 2 normal autopsy brains. The basis for the selection of AD brains was, namely, 1) the absence of detectable levels of PrPSc in AD brains36 and 2) the existence of specific and widely accepted diagnostic criteria for AD.32 As the polymorphism at codon 129 of the PrP gene affects the phenotypic expression of prion disorders and the susceptibility to particular strains,19,20,21,23,31 we compared the different genotypes: 1) N2 and AD cases 4, 5, 7, and 10 were heterozygous Met/Val; 2) N1 and AD cases 2, 3, 8, and 9 were homozygous for Met; and 3) AD cases 1 and 6 were homozygous for Val.
The AD and normal brains were tested by immunoblot analysis with 3F4
and -SP214, two monoclonal antibodies directed to the central
(residues 109 to 112) and carboxyl-terminal (residues 214 to 231)
regions of human PrP, respectively. In contrast to these domains, the
amino-terminal region of PrP is labile and not needed for
infectivity.37
Sample aliquots were standardized to yield
signals of approximately equal intensity in immunoblots probed with
3F4; these quantities were employed in all assays.
Untreated AD and normal brain homogenates tested with 3F4 contained two
broad immunoreactive bands of 35 and 33 kd and a faint band of 27 kd
(Figure 1A)
. All samples exhibited a
similar 3F4 pattern characterized by a decreasing intensity from high
to lower molecular weight bands. However, the immunoblots developed
with -SP214 showed a marked heterogeneity (Figure 1A)
. Eight samples
(N1, N2, and AD cases 1, 4, and 7 to 10) had a pattern comparable to
that with 3F4 (pattern 1). The remaining cases (AD cases 2, 3, 5 and 6)
contained major bands of 35 and 27 kd (pattern 2) and two fainter
signals at 22 and 18 kd (Figure 1A)
. The postmortem intervals of all
pattern 2 samples were longer than 8 hours (range, 8 to 16 hours),
whereas pattern 1 cases had intervals of 4 to 10 hours.
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-SP214 (Figure 1B)
. The 27-kd band is
consistent with the molecular weight of deglycosylated full-length
PrP,38
whereas the remaining signals correspond to
amino-truncated fragments.26
The 35- and 33-kd bands were
not detected after PNGase-F treatment, which indicates that they are
glycosylated species. There were no significant molecular weight
differences between the deglycosylated PrPC fragments of
normal and AD brains or of different PrP codon 129 genotypes or
of neocortical and cerebellar tissues (Figure 1C)
. However, there was
an obvious heterogeneity in the intensity of the 18-kd band, with
pattern 2 samples presenting a higher signal than pattern 1 cases
(Figure 1C
, AD6). The intensity of the 21 to 22-kd bands appeared to
vary randomly with the immunoblot profile of untreated homogenates.
As pattern 2 samples had longer postmortem intervals and a
proportionally higher content of 18-kd fragment than pattern 1 cases,
we suspected that the observed differences were related to postmortem
lysis. To address the endogenous proteolysis of the PrPC
isoform, intact normal and AD brain tissues were incubated for 24 to 48
hours at room temperature before homogenization. Brain samples thus
treated showed a progressive increase in the deglycosylated 18-kd
fragment and a concomitant decrease in the 27- and 21- to 22-kd species
(see the example of N1, a pattern 1 case, in Figure 1D
). These
quantitative changes reflected a conversion of pattern 1 to pattern 2
(Figure 1D)
.
Metalloproteases Are Involved in the Proteolytic Processing of PrPC in Brain
Taking advantage of the effects of postmortem lysis, we then tried
to identify the enzymatic activities involved in PrPC
degradation. Aliquots of normal brain (N1) homogenate were incubated
for 24 hours at 37°C in the presence of different protease inhibitors
(Table 1)
, deglycosylated, and analyzed by immunoblot with -SP214.
The 27-kd bands corresponding to full-length PrP were quantified by
densitometry, and results were expressed as percentage of intensity
related to control, not incubated samples. As shown in Figure 2, A and B
, the metal-chelating agents EDTA and
EGTA were the most effective in blocking PrPC degradation.
The effect of EDTA was reversed by adding divalent metal ions
(Ca2+, Mg2+, Fe2+,
Cu2+, and Zn2+) in molar excess (not shown),
but only Fe2+, Cu2+, and to a far lesser degree
Zn2+ were active when added at a 20:1/EDTA:metal ion molar
ratio (Figure 2C)
.
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Prion disease brains contain both PrPC and
PrPSc isoforms, which can be differentiated by the relative
PK resistance and insolubility in nondenaturing detergents of the
latter.14,16
Our sporadic CJD cases could be divided into
two groups by the size of their PK-resistant cores.23,31
Four cases (CJD1 to -3 and CJD7), all of them homozygous for Met at
codon 129, contained three bands of 31, 28, and 22.5 kd in blots probed
with 3F4 and -SP214, corresponding to di-, mono-, and unglycosylated
species, respectively (type 1 sporadic CJD; Figure 3, A and B
). A second group included cases with
Met/Val (CJD4 and CJD9), Met/Met (CJD6), and Val/Val (CJD5 and CJD8)
genotypes, and in each case the PK-treated samples had similar bands of
30, 26, and 21 kd (type 2 sporadic CJD; Figure 3, A and B
). The
patterns of our type 1 and type 2 sporadic CJD groups were in agreement
with type 1 and type 2 PrPres 31
although with
minor differences in electrophoretic mobility, probably due to
experimental variability. PK-treated GSS F198S brain tissue (GSS1 and
GSS2, both with Met/Val codon 129 genotype) contained bands of 27 to
29, 18 to 20, and 8 kd detectable only by 3F4 and a faint 33-kd signal
revealed by both antibodies (Figure 3A)
.39
As expected,
protease-resistant species in CJD and GSS brains were similar in
cortical and cerebellar tissues and insoluble in 2% Sarkosyl (not
shown), whereas PK treatment of normal and AD brains led to a lack of
immunoreactive bands in blots probed with both 3F4 and -SP214
(Figure 3A)
.
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After deglycosylation, type 1 sporadic CJD brain cortex and cerebellum
had species of 27 and 22.5 kd detectable by 3F4 and -SP214 and an
18-kd fragment detected only with -SP214 (Figure 4, A and B)
.26
The pattern of
deglycosylated type 2 CJD samples was indistinguishable from that of
normal and AD brains (Figure 4, A and B)
, except for the presence of
PrP aggregates in tissues with high amyloid content (Figure 5B)
. GSS F198S brains had a distinctive
3F4 profile, composed of peptides measuring 27 to 30, 21 to 22, 19 to
20, and 9 kd, and a diffuse high molecular weight signal (Figure 4A)
.39
GSS samples tested with -SP214 contained species
of 27 to 30, 21 to 22, and 18 kd.
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. The results of type 2 CJD brains were more
heterogeneous. Cerebral cortex from Val/Val patients mirrored the
profile of normal brain, with a decrease in full-length PrP and 21- to
22-kd species and an increase in the 18-kd peptide (Figure 4C)
. In
contrast, tissues from a Met/Val case with high amyloid burden
presented higher amounts of the 21- to 22-kd fragments (Figure 4D)
. GSS
F198S samples had a decrease in full-length PrP and 21- to 22-kd
species and an increase in the 20- and 9-kd peptides; in addition, a
carboxyl-terminal fragment of 10 kd was detected (Figure 4C)
.
To further discriminate between PrPC and PrPSc
in CJD and GSS brains, tissues were homogenized in 2% Sarkosyl,
centrifuged to obtain soluble and insoluble fractions, and
deglycosylated. The soluble fractions of type 1 sporadic CJD brains
contained a small quantity of full-length PrP and all of the detectable
18-kd fragment. The insoluble fractions, enriched in PrPSc,
included the majority of full-length PrP and the 22.5-kd fragment
(Figure 5A)
. The soluble fractions of type 2 CJD brains contained
intact PrP and fragments of 21 to 22 and 18 kd, whereas the insoluble
fractions included PrP aggregates, full-length PrP, and 21- to 22-kd
products (Figure 5, A and B)
. Of note, the relative amounts of PrP in
each fraction were again more heterogeneous in type 2 than in type 1
CJD. Type 2 CJD samples with high amyloid content, such as cerebellum
from Met/Val heterozygotes, had the greatest quantities of insoluble
PrP (Figure 5B)
, whereas brain cortex from Val/Val patients contained
minimal amounts of insoluble PrP, mainly in the form of full-length
species (Figure 5a
, CJD8). These results correlate with those of PrP
degradation by endogenous proteases and may be explained by the
reported variation of PrPres content in type 2 CJD
tissues.31
Comparable assays using GSS F198S brains showed
the presence of full-length PrP and the 21- to 22- and 18-kd fragments
in the supernatants; the pellets contained the bulk of the
immunoreactive material, composed of PrP aggregates, full-length PrP,
and truncated products of 19 to 20 and 9 kg (Figure 5A)
.
Tissue-Specific Properties of the PrPC Isoform in Non-Neural Tissues
The mechanisms controlling the neurotropism of prions are still
unclear. As prion replication requires a
PrPC-PrPSc interaction that is affected by
homology,41
tissue-specific attributes of PrPC
may modulate, and eventually preclude, the pathological conversion.
Lymphoid tissue (tonsils) and platelets have significant amounts of
PrP.8,42
Immunoblot analysis of untreated homogenates of
platelets and tonsils from neurologically normal subjects revealed
three 3F4 and -SP214 immunoreactive bands of 38, 33, and 26 kd
(Figure 6A)
. Fully glycosylated platelet
and tonsil PrPC had a higher molecular weight than the
brain homologue. Conversely, deglycosylated, full-length
PrPC in non-neural tissues had a lower molecular weight
(approximately 26 kd) than in brain (27 kd; Figure 6B
). Tonsil samples
contained additional deglycosylated fragments of 20 to 21 kd detected
with 3F4 and minimally with -SP214 and an 18-kd fragment detectable
only by -SP214 (Figure 6B)
. Deglycosylated platelet PrPC
fragments were identified at 16 kd with -SP214 and at 19 kd with
both 3F4 and -SP214 (Figure 6B)
.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Different PrPC proteolytic pathways may originate fragments with distinct susceptibility to conversion, as prediction models,28 comparative structural and immunological studies of PrPC and PrPSc,18,29 and nuclear magnetic resonance experiments with recombinant PrP30,47,48 suggest that only certain PrP domains, especially residues 90 to 125, are essential for the conformational changes that differentiate PrPSc from PrPC. Cleavages within these critical regions could preclude the conversion of PrPC into PrPSc. Therefore, studies of PrPC proteolytic processing may help elucidate the phenomena of disease susceptibility and tissue specificity.
The present study shows the existence of two major PrPC
proteolytic cleavages in normal and AD brain cortex and cerebellum
(Figure 7)
. The cleavage sites were
identified by immunoblot analysis of deglycosylated samples using
monoclonal antibodies 3F4 and -SP214, which revealed a spectrum of
PrP fragments besides the full-length GPI-containing species of 27
kd.38
We inferred that these assays reflect proteolysis
because the entire open reading frame of the human PrP gene is
contained within a single exon, which excludes mRNA
splicing.49
The dominant cleavage yields a
carboxyl-terminal fragment of 18 kd that does not include the 3F4
epitope; thus, it is amino truncated and starts after Met109. This
fragment has been previously denominated C1 and its amino terminus
identified as His111-Met112 in neuroblastoma cell lines.26
The second pathway yields heterogeneous peptides of 21 to 22 kd that
contain both 3F4 and -SP214 epitopes and therefore are also amino
truncated but start before or at Met109. Molecular weight estimates by
immunoblot analysis suggest that the amino termini of these fragments
are within residues 80 to 100. It remains to be established whether
both pathways are independent or whether the 21- to 22-kd species are
obligatory intermediaries for the generation of C1 (Figure 7)
. In
addition to the major internal cleavages, the bands adjacent to
full-length PrPC in blots probed with 3F4 and -SP214
indicate the existence of products with limited amino- and
carboxyl-terminal truncation.
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As a first attempt to characterize the enzymatic activities involved in the proteolysis of PrPC in brain, we asked which protease inhibitors are most effective in blocking PrPC degradation. The metal-chelating agents EDTA and EGTA showed the highest activity against PrPC protease(s), followed by the inhibitors of thiol proteases. The addition of Fe2+ or Cu2+ at a 20:1/EDTA:metal ion molar ratio reversed the effect of EDTA, indicating the selectivity of the inhibition. Although our results were reproducible, they should be interpreted cautiously because we used whole-brain homogenates, which contain a multiplicity of enzymes not necessarily relevant under physiological conditions. However, the presence of metal-binding properties of PrP, an intriguing and recurrent observation in the literature, may influence PrP conformation.52 Our data also suggest a role for metal ions in the proteolytic processing of PrP. The high ferritin accumulation in microglial cells surrounding PrP amyloid plaques53 could reflect among other functions the overregulation of metalloproteases involved in amyloid formation or degradation.
In contrast to the stereotypic characteristics of PrPC in normal and AD brains, we found that the PrPSc isoforms in different prion disorders are heterogeneous. Initially, as previously reported,23,31 we observed that the different molecular weights of the PK-resistant PrPSc cores (PRP27–30) in sporadic CJD brains correlated with their codon 129 genotypes. A group of CJD cases, homozygous for Met at codon 129, had three PK-resistant bands of 31, 28, and 22.5 kd, corresponding to di-, mono-, and unglycosylated PrP species, respectively. A second group of CJD samples, composed of Met/Val, Met/Met, and Val/Val codon 129 genotypes, contained proteins of 30, 26, and 21 kd. Thus, our first and second groups correspond to the type 1 and type 2 PrPres, respectively, described by Parchi et al in sporadic CJD.31 Of note, the deglycosylated 21-kd fragment described above in normal and AD brains has the same mobility and immunoreactivity as the unglycosylated PK-resistant core of type 2 CJD brain tissue; therefore, it is a physiological PrP27-30-like species. GSS F198S brains, characterized by PrP amyloid deposition associated with neurofibrillary tangles, had four PK-resistant bands of 33, 27 to 29, 18 to 20, and 8 kd.39
However, a significant limitation of PK digestion is that it is an unphysiological hydrolytic process that yields enzymatic end-products and no elucidative intermediary species. Thus, it cannot be used to characterize naturally occurring PrP species, nor does it allow study of effects of disease on PrPC. To investigate the full spectrum of PrP isoforms in prion-affected brains we used an alternative approach based on deglycosylation, degradation by endogenous proteases, and Sarkosyl solubility. Deglycosylation alone allows the identification of the multiplicity of PrP peptides present in tissues, and PrPSc isoforms can be further distinguished by their relative protease resistance and insolubility in nondenaturing detergents.15,16
According to our data, type 1 sporadic CJD PrPSc species
are in the form of full-length PrP and a prominent amino-truncated
fragment of 22.5 kd (molecular weights refer to deglycosylated species)
(Figure 7)
. This 22.5-kd product has the same mobility and
immunoreactivity as the C2 fragment identified by Chen et al in
sporadic CJD brains26
and represents a natural proteolytic
product that coincides with the protease-resistant core obtained after
PK digestion. In contrast, we describe for the first time type 2
sporadic CJD PrPSc in the form of full-length PrP and
variable amounts of amino-terminal-truncated fragments of 21 to 22 kd
and PrP aggregates (Figure 7)
. These 21- to 22-kd products cannot be
distinguished by size from homologue species of normal and AD brains,
which suggests that PrPSc proteolytic pathways in type 2
CJD are similar, if not identical, to those of PrPC in
normal brain. However, whereas normal PrPC is readily
cleaved further at the amino-terminus to yield the C1 fragment,
PrPSc isoform in type 2 CJD is not degraded as efficiently
and eventually may accumulate as a 21-kd protease-resistant core.
Interestingly, type 2 CJD cases with Met/Val codon 129 genotype and
marked amyloid deposition contained higher amounts of PrP aggregates
and 21- to 22-kd peptides than Val/Val cases with no or minimal amyloid
accumulation. These data show that the exact profile of
PrPSc peptides in prion-affected tissues, including the
relative amounts of the different species and not merely the
characteristics of the protease-resistant cores, correlates with the
neuropathological phenotype.
The most distinctive profile was found in GSS F198S brain tissue, which
included PrPSc aggregates, full-length PrPSc,
and amino- and carboxyl-terminal truncated fragments of 19 to 20 and 9
kd (Figure 7)
. Although the 22.5-kd fragment in type 1 CJD and the 19-
to 20- and 9-kd products in GSS F198S brains were not found in
significant amounts in normal tissue, that does not necessarily imply
that they proceed from novel pathogenic cleavages. They may represent
normally transient proteolytic intermediaries that are stabilized under
pathological conditions. In addition to PrPSc,
prion-affected brains also had variable quantities of PrPC
species, which were identified by their solubility in Sarkosyl and a
normal proteolytic pattern characterized by an increase in C1 after
incubation without protease inhibitors.
Taken together, our data demonstrate that the PrPSc isoforms of different prion disorders have both common and distinct properties. First, the protease-resistant cores in sporadic CJD and GSS F918S brains were all detectable by 3F4, indicative of the intactness of the C1 cleavage site (His111-Met112). This holds even for the smallest amino- and carboxyl-truncated fragments of 7 and 11 kd present in GSS F198S amyloid.54,55 Therefore, a pivotal difference between PrPC and PrPSc isoforms appears to be the increased resistance to cleavage of the C1 site of PrPSc species. The preservation of the C1 site, presumably reflective of the pathological conformation of the abnormal proteins, precludes the disruption of a conserved hydrophobic region (residues 106 to 126) involved in toxicity and fibrillogenesis56,57 and may lead to reduced clearance and subsequent accumulation of deleterious PrP species. This hypothesis is supported by recent structural data that indicate that the 90 to 112 PrPC region has no defined structure30 and thus may be a critical domain for the conformational change that differentiates PrPSc from PrPC.
In contrast to this common feature of the PrPSc isoforms, we observed characteristic proteolytic cleavages that originated fragments specific for each disorder. The possible implications of this finding are exemplified by studies of Alzheimer amyloid ß peptide (Aß). Amyloid Aß deposits are composed of a spectrum of peptides with heterogeneous amino and carboxy termini, derived by proteolysis from a larger precursor. Current evidence indicates that Aß variants exhibit a partially different distribution in tissues.58 Previous studies of prion diseases have shown that there are differences in the molecular weight and degree of glycosylation of the PK-resistant PrPSc cores in the distinct clinicopathological syndromes. Herein we show that there are fragments comparable to those identified after PK digestion also in intact tissues, in agreement with the results of Piccardo et al39 for GSS F198S and of Chen et al26 for type-1 CJD. These data indicate that the different PrPSc isoforms, when exposed to the endogenous proteases, display patterns of cleavage that are characteristic of each disorder and therefore are associated with distinct sets of fragments. By analogy with the Aß variants, the different PrPSc fragments may exhibit particular biological and physicochemical properties. As a consequence, phenotypic expression in prion diseases may be defined in part by the molecular variability of proteolytic processing, which in turn probably is determined by the particular conformation of the different PrPSc isoforms. This does not exclude the role of additional factors in phenotypic expression, in particular, differences in glycosylation that may be especially relevant in the new variant CJD
The pathology of prion diseases is limited mainly to neural tissues
despite the ubiquitous expression of PrPC.3,12
As the factors underlying this restricted distribution are unknown, we
also investigated the existence of tissue-specific attributes of
PrPC and its fragments. Lymphoid tissue (tonsils) and
peripheral blood platelets from neurologically normal patients were
selected, based on their possible role in the pathogenesis of prion
disorders, especially in the peripherally acquired
forms.42,59
Immunoblot analyses showed a proteolysis of
PrPC analogous but not identical to that in brain.
Deglycosylated platelet proteins had a prominent amino-truncated
PrPC product of 19 kd and minimal amounts of a 16-kd
carboxyl-terminal fragment. The 19-kd fragment probably corresponds to
the deglycosylated form of a PrP27-30-like species released from normal
platelets that starts at Gly90.42
After deglycosylation,
tonsil samples contained PrPC peptides of 20 to 21 kd
detected with antibody 3F4, and an 18-kd peptide signaled only by
-P214. There also were slight disparities in the molecular weight of
the bands corresponding to deglycosylated full-length PrPC,
possibly due to heterogeneity in the composition of the GPI anchor,
differences in PNGase-F accessibility to Asn-linked carbohydrates,
limited terminal truncation, or other undefined post-translational
modifications. These modifications may affect the position of the lower
bands, and thus pattern variability may be due to factors other than
the site of cleavage. In addition, the distinct molecular weight of the
glycosylated PrPC species in platelets and tonsils relative
to brain PrPC suggests heterogeneity in the composition of
the Asn-linked carbohydrates.
Thus, we found several variances in PrPC proteolytic cleavage, and probably in the glycosylation and GPI composition of PrPC, between neural and non-neural tissues. As PrPC-PrPSc interaction appears to be a crucial pathogenic step promoted by homology,41 the observed differences may modify the rate of prion replication. In agreement with this idea, recent studies have shown that the glycosylation of the PrPC isoform in transgenic mice60 and cell culture systems61 affects the efficiency of its conversion to the abnormal isoform. Whether this is a mechanism underlying tissue specificity remains an open question. The presence of these differences also is compatible with the hypothesis that replication of peripherally acquired prions first occurs in non-neural tissues, most likely lymphoid organs, where they acquire distinctive molecular features.23
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Acknowledgements |
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Footnotes |
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Supported by the National Institutes of Health grants AR 02594 and AG 08721 to B. Frangione, the Italian Ministry of Health, Department of Social Services and Telethon-Italy (grant E.250) to F. Tagliavini and NS29822 to B. Ghetti.
Accepted for publication July 29, 1998.
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