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From: TSS (216-119-139-126.ipset19.wt.net)
Subject: Re: Molecular Basis of Barriers for Interspecies Transmissibility of Mammalian Prions [FULL TEXT]
Date: April 8, 2004 at 7:31 pm PST
In Reply to: Molecular Basis of Barriers for Interspecies Transmissibility of Mammalian Prions posted by TSS on April 8, 2004 at 11:18 am:
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Subject: Molecular Basis of Barriers for Interspecies Transmissibility of Mammalian Prions [FULL TEXT]
Date: Thu, 08 Apr 2004 21:28:11 -0500
From: "Terry S. Singeltary Sr."
To: bse-l
Molecular Cell, Vol. 14, 139145, April 9, 2004, Copyright ?2004 by Cell
Press
Short Article Molecular Basis of Barriers
for Interspecies Transmissibility
of Mammalian Prions
barriers in prion propagation has been brought to the
public eye by recent epidemics of mad cow disease
and concerns regarding potential transmission of animal
prion diseases to humans (Bruce et al., 1997; Hill et
al., 1997). However, the molecular basis of the species
David L. Vanik,1 Krystyna A. Surewicz,1
and Witold K. Surewicz*
Department of Physiology and Biophysics and
Department of Chemistry
Case Western Reserve University
Cleveland, Ohio 44106 barrier and its relationship to prion strain
specificity remain
poorly understood.
Recent experiments have provided a wealth of information
about prion-like phenomena in yeast and fungi Summary
(Chien and Weissman, 2001; Chien et al., 2003; Derkatch
et al., 1996; Uptain and Lindquist, 2002; Wickner et al., Spongiform
encephalopathies are believed to be
transmitted by a unique mechanism involving self- 2000). However,
studies aimed at understanding the
mechanism of mammalian prion propagation, in general, propagating
conformational conversion of prion protein
into a misfolded form. Here we demonstrate that and molecular aspects of
species barrier and strain diversity,
in particular, have been hampered by difficulties fundamental aspects of
mammalian prion propagation,
including the species barrier and strain diversity, in propagating the
PrPC?PrPSc conversion in vitro. The
cell-free conversion assay developed by Caughey and can be reproduced in
vitro in a seeded fibrillization of
the recombinant prion protein variant Y145Stop. Our coworkers has
provided fundamentally important insight
into molecular barriers in the transmissibility of data show that
species-specific substitution of a single
amino acid in a critical region completely changes TSE agents (Bessen et
al., 1995; Caughey, 2001; Kocisko
et al., 1995; Raymond et al., 1997). However, an intrinsic the seeding
specificity of prion protein fibrils. Furthermore,
we demonstrate that sequence-based barriers limitation of this assay is
that the conversion yields are
perplexingly low, below the level required to sustain that prevent
cross-seeding between proteins from different
species can be bypassed, and new barriers es- continuous
self-propagation of the prion state. These
yields could be improved in the presence of additional tablished, by a
template-induced adaptation process
that leads to the emergence of new strains of prion cellular cofactors
(Lucassen et al., 2003). Recently, we
have shown that, in contrast to the recombinant full- fibrils. Although
the seeding barriers observed in this
study do not fully match those seen in animals, the length human prion
protein, the Y145Stop mutant
(huPrP23144), which is associated with a familial prion present
findings provide fundamental insight into
mechanistic principles of these barriers at a molecu- disease, undergoes
an efficient nucleation-dependent
conversion to the amyloid state in vitro (Kundu et al., lar level.
2003). Limited attempts to transmit this variant of the
disease to mice have not yet been successful (Tateishi Introduction
et al., 1996). Nevertheless, the huPrP23144 model
opens up new possibilities for studying the propagation Transmissible
spongiform encephalopathies (TSEs) are
a group of fatal neurodegenerative diseases that affect of
conformationally altered PrP states in vitro. Here, we
have used this system to explore the molecular basis humans and animals.
These disorders are associated
with conformational conversion of the cellular prion pro- of the species
barrier and its relationship to prion strains.
tein, PrPC, into an abnormal form, PrPSc (Caughey and
Chesebro, 2001; Collinge, 2001; Prusiner, 1998). The Results
latter protein is characterized by a high content of
sheet structure, partial resistance to proteolytic diges- Fibrillization
and Seeding Specificity of PrP23144
tion, and a propensity to aggregate into amyloid-like Is Strongly
Species Dependent
fibrils and plaques. The protein-only hypothesis asserts We first asked
how species-dependent differences in
that the transmission of TSEs does not require nucleic the primary
structure affect the propagation and seeding
acids and that PrPSc itself is the infectious agent (Prusi- specificity
of the amyloid state of PrP23144. To address
ner, 1982, 1998). This highly unusual pathogen is be- this issue, we
expressed and purified recombinant prolieved
to self-propagate by binding to cellular prion pro- teins corresponding
to residues 23144 of human (hu),
tein and catalyzing its conversion to PrPSc. Within the mouse (mo), and
Syrian hamster (Sha) prion proteins.
context of this model, two features of TSEs are particu- The conversion
of these proteins to amyloid fibrils was
larly perplexing. The first one is the existence of multiple followed by
the thioflavine T (ThT) fluorescence assay
prion strains that produce distinct disease phenotypes (Naiki et al.,
1989). Consistent with our recent study
within the same animal species (Bruce and Fraser, 1991). (Kundu et al.,
2003), huPrP23144 formed self-seeded
The second is the species barrier that restricts transmis- fibrils
through a process characterized by distinct lag
sion of prion diseases between different mammalian and growth phases.
Under the present experimental
species (for a review see Collinge, 2001). The issue of conditions, this
lag phase was approximately 3.6 hr (Figure
1). A similar conversion was observed for the mouse
and hamster proteins. However, the lag phases were *Correspondence:
witold.surewicz@case.edu
1These authors contributed equally to this work. dramatically different,
increasing to approximately 16 hr
Molecular Cell
140
We next examined the efficiency of seeding between
proteins belonging to different species. Human and
mouse proteins were found to be fully compatible, i.e.,
huPrP23144 fibrils readily seeded mouse protein and
moPrP23144 fibrils acted as an efficient seed for fibrillization
of human PrP23144 (Figures 2A and 2B). In contrast,
a strong barrier was found between human and
hamster proteins. The lag phases for huPrP23144 fibrillization
in the presence of ShaPrP23144 seeds, or
ShaPrP23144 polymerization in the presence of
huPrP23144 seeds were very similar to those for nonseeded
reactions (Figures 2A and 2C), indicating that
no cross-seeding occurs between human and hamster
PrP23144. Similar cross-seeding experiments between
mouse and Syrian hamster PrP23144 revealed an intriguing
asymmetry in seeding specificity for this pair of
proteins. While preformed ShaPrP23144 fibrils readily
seed polymerization of mouse protein, fibrilar moPrP23
144 shows no detectable capacity to nucleate the conversion
of ShaPrP23144 (Figures 2B and 2C).
Amino Acids at Position 138 and 139 Are
Critical Determinants of Species-Dependent
Seeding Specificity
The data shown above demonstrate substantial differences
in amyloidogenic propensities of PrP23144 corresponding
to human, mouse, and Syrian hamster sequences
and, most importantly, indicate remarkable
specificity in cross-seeding between proteins that belong
to different species. As shown in Figure 3, there
are a number of amino acid differences between human,
mouse, and hamster PrP23144. However, our recent
study points to a critical role in huPrP23144 conversion
of a very short segment encompassing amino acid resi-
Figure 1. Spontaneous Fibrillization of PrP23144 Representing Dif-
dues 138141 (Kundu et al., 2003). Two positions
ferent Species within this segment138 and 139show species-
(A) Time course of the increase in thioflavine T fluorescence in the
dependent variability (Figure 3). In human protein both
presence of huPrP23144 (), moPrP23144 (), ShaPrP23144 (),
positions are occupied by isoleucine, while in mouse I138M huPrP23144
(), and I138M/I139M huPrP23144 (). The
PrP Ile138 is replaced by methionine. Syrian hamster kinetic curves were
obtained in 50 mM phosphate buffer (pH 6.4)
protein shows further divergence from human PrP, with at a protein
concentration of 400 M.
(B) Mean values of lag phases for nonseeded fibrillization based on Met
present both at position 138 and 139. This suggests
four to eleven experiments. a potentially crucial role for residues 138
and/or 139 in
(C) Electron micrograph showing fibrils of huPrP23144. The bar
determining the species-dependent specificity of
corresponds to 200 nm. The electron micrographs for mouse and
PrP23144 conformational conversion. Syrian hamster proteins showed
similar morphology of amyloid fi-
To test this hypothesis, we have prepared two brils.
huPrP23144 variants: one in which Ile138 was replaced
by Met and one in which both Ile138 and Ile139 were for moPrP23144 and
38 hr for ShaPrP23144 (Figures
substituted with Met. As shown in Figure 1, the single 1A and 1B). In
each case, the formation of fibrils was
I138M mutation had a profound effect on the rate of confirmed by
electron microscopy (Figure 1C).
spontaneous huPrP23144 fibrillization extending the Addition of a small
amount (2% w/w) of preformed
lag phase from 3.6 to 18 hr, very similar to that observed huPrP23144
fibrils to the solution of the monomeric
for moPrP23144. In the case of the double mutant huPrP23144 resulted
in elimination of the lag phase
I138M/I139M, the lag phase was further increased to (Figure 2A). Similar
behavior was observed for both
39 hr, becoming very similar to that found for sponta- moPrP23144 and
ShaPrP23144. Even though the lag
neous fibrillization of hamster prion protein. Guided by phases for
spontaneous fibrillization of these proteins
these findings, we next tested the seeding specificity were much longer,
these lag phases were completely
of the single and double mutants of huPrP23144. Also abolished when 2%
w/w preformed moPrP23144 or
in this regard, the behavior of the I138M and I138M/ ShaPrp23144
fibrils were added to solutions containing
I139M variants paralleled the properties of moPrP23 monomers of the
respective proteins (Figures 2B and
144 and ShaPrP23144, respectively. Akin to moPrP23 2C). These results
indicate that in all three cases fibril
144, the fibrillar form of the I138M variant efficiently formation
requires oligomeric seeds and has characterseeded
fibrillization of moPrP23144 and huPrP23144, istics of
nucleation-dependent polymerization (Come et
al., 1993). but could not seed the conversion of Syrian hamster
Molecular Basis of Barriers in Prion Propagation
141
Figure 2. Species- and Mutation-Dependent Seeding Specificities of
PrP23144 Amyloid Fibrils
(AC) Human (hu), mouse (mo), and Syrian hamster (Sha) PrP23144 seeded
with preformed fibrils of proteins representing different species.
Seeds of huPrP23144, moPrP23144, and ShaPrP23144 are denoted as [hu],
[mo], and [Sha], respectively.
(DF) Human, mouse, and Syrian hamster proteins seeded with preformed
fibrils of I138M and I138M/I139M variants of huPrP23144. I138M
huPrP23144 and I138M/I139M huPrP23144 seeds are denoted as [138] and
[138/139], respectively.
(G and H) I138M huPrP23144 and I138M/I139M huPrP23144 seeded with
preformed fibrils of I138M and I138M/I139M variants of huPrP23144.
Each experiment was repeated at least four times.
protein. Furthermore, akin to ShaPrP23144, preformed served when
I138M/I139M variant was incubated in the
I138M/I139M huPrP23144 fibrils seeded fibrillization of presence of
I138M huPrP23144 fibrils (Figures 2G and
ShaPrP23144 and moPrP23144, but failed to seed 2H). Thus, these data
demonstrate that mouse or hamhuman
PrP23144 (Figures 2D2F). Finally, analogous ster PrP-specific amino
acid substitutions at position
to the behavior of the moPrP23144-ShaPrP23144 pair, 138 and 139 are
sufficient to change essential amyloidothe
double mutant fibrils readily seeded polymerization genic properties of
huPrP23144, with the mutant proof
the single mutant protein, but no seeding was ob- teins adopting seeding
specificities of PrP corresponding
to different species.
To further test the role of residues 138 and 139 in
PrP23144 fibrillization, we have performed a series of
experiments with Syrian hamster protein in which both
Met138 and Met139 were substituted with human PrPspecific
Ile residues (Figure 3). The lag phase for nonseeded
conversion of this humanized ShaPrP23144 Figure 3. Sequence
Comparison for Human (hu), Mouse (mo), and
dropped from 38 hr to approximately 3.5 hr, a value very Syrian Hamster
(Sha) PrP Highlighting Amino Acid Differences in
the 23144 Region similar to that found for huPrP23144. Furthermore, the
Molecular Cell
142
[mo]Sha to seed fibrillization of hamster PrP is not due
to residual ShaPrP23144 seeds which are present at a
concentration of 0.04%, an amount far too small to affect
the conversion reaction. Furthermore, residual ShaPrP23
144 seeds obviously could not account for the lost ability
of [mo]Sha to nucleate conversion of human prion protein.
As expected from the data of Figure 2B, the second
generation [mo]Sha fibrils could also nucleate conformational
conversion of moPrP23144. Importantly, the thirdgeneration
product of this reaction retained the seeding
specificity of [mo]Sha, i.e., it could nucleate the conversion
of ShaPrP23144, but not that of huPrP23144 (data
not shown for brevity). The only logical explanation of the
present data is that the properties of second-generation
moPrP23144 fibrils prenucleated with ShaPrP23144
seeds are different from those of moPrP23144 fibrils
formed spontaneously or those nucleated with moPrP23
144 seeds. Clearly, seeding with hamster PrP led to the
formation of a new strain of moPrP23144 fibrils. This
newstrain acquired the templating properties of the parent
seed and stably propagated in subsequent rounds of
seeded fibrillization. Importantly, the altered templating
properties allowed the new strain to overcome the origi-
Figure 4. The Emergence of New Strains of PrP Amyloid Fibrils with nal
barrier in cross-seeding the conversion between pro-
Different Seeding Specificities
teins belonging to different species.
(A and B) Second generation fibrils of moPrP23144 were obtained The
remarkable ability of PrP23144 fibrils to propa- by seeding moPrP23144
with preformed fibrils of ShaPrP23144.
gate in vitro as different strains could also be demon- These second
generation fibrils, denoted [mo]Sha, could not seed
strated using the I138M and I138M/I139M variants of huPrP23144 (A) but
seeded ShaPrP23144 (B).
(C and D) Second generation fibrils of I138M huPrP23144 were human
prion protein. Thus, second-generation fibrils of
obtained by seeding I138M huPrP23144 with preformed fibrils of I138M
huPrP23144 preseeded with the I138M/I139M
I138M/I139M huPrP23144. These second generation fibrils, de- variant
acquired the properties of the seed: unlike originoted
[138]138/139, were used to seed either huPrP23144 (C) or I138M/ nal
I138M huPrP23144 fibrils, they could nucleate poly- I139M huPrP23144
(D). First generation seeds of moPrP23144,
merization of the I138M/I139M variant (or ShaPrP23 and I138M
huPrP23144 are denoted as [mo] and [138], respectively.
144), but not that of the wild-type huPrP23144 (see Both [mo]Sha and
[138]138/139 could seed moPrP23144 and I138M
huPrP23144 (data not shown for brevity). Each experiment was Figures 4C
and 4D).
repeated at least four times.
Discussion
mutation completely altered the seeding specificity of Studies with
transgenic animals have demonstrated that
the protein: preformed M138I/M139I ShaPrP23144 fi- species barriers for
TSE transmissibility are closely rebrils
could no longer seed Syrian hamster protein but lated to differences in
prion protein amino acid seacquired
the ability to seed huPrP23144 (data not quences between the donors and
recipients of infection
shown for brevity). Thus, M138I/M139I ShaPrP23144 (Scott et al., 1993).
The importance of sequence homoladopted
the properties of human PrP23144. ogy in prion propagation has also
been underscored in
experiments in vitro. Following the initial observation
with short synthetic PrP fragments (Come et al., 1993), Cross-Seeding
Leads to the Emergence of New
Strains of PrP23144 Fibrils sequence specificity of PrPC-PrPSc
interactions has been
extensively documented in a series of studies employing The asymmetry in
seeding specificity observed for the
moPrP23144-ShaPrP23144 pair (Figures 2B and 2C) noncontinuous
cell-free conversion assay (Caughey,
2001; Kocisko et al., 1995; Raymond et al., 1997), as well provided us
with the unique opportunity to study the
relationship between the species barrier and strain di- as in
experiments with scrapie-infected neuroblastoma
cells (Priola and Chesebro, 1995; Priola et al., 2001). The versity. To
this end, we have seeded moPrP23144 with
ShaPrP23144 fibrils, and used the product of this reac- present data
provide a new insight into the relationship
between PrP sequence and species barriers, pointing tiondenoted
[mo]Shaas second-generation seeds to
nucleate polymerization of human, mouse, and hamster to the extremely
tight control of prion protein conversion
by individual amino acid residues. Remarkably, species- proteins. If
these second-generation moPrP23144 fibrils
retain the original properties of mouse protein, they specific
substitution of a single amino acid in the critical
region of human or hamster PrP23144 is sufficient to should seed
huPrP23144, but not ShaPrP23144. Data
of Figure 4 clearly show that this is not the case. The completely
change the behavior of these polypeptides,
with mutant polypeptides acquiring fibrillization kinetics
second-generation moPrP23144 fibrils, [mo]Sha, have
lost the ability to seed fibrillization of human prion pro- and seeding
specificities of proteins corresponding to
different species. Apart from implications for a molecu- tein and have
gained the ability to nucleate fibrillization
of hamster PrP23144. This newly acquired capacity of lar basis of the
species barrier, the finding that point
Molecular Basis of Barriers in Prion Propagation
143
mutations can drastically alter seeding specificity of mately related
phenomena is in line with the observations
regarding transmission of BSE and CJD prions to prion protein may also
help to explain puzzling differences
in infectivity of inherited prion diseases. While experimental animals
(Collinge, 2001).
The present study was performed using the C-trun- sporadic
Creutzfeldt-Jakob disease (CJD) and many familial
CJD cases can be readily transmitted to primates cated PrP23144 variant
that corresponds to a rare form
of familial prion disease. Similar studies with the recom- and
transgenic mice, attempts to transmit other forms of
hereditary prion diseases have not yet been successful binant
full-length PrP have not yet been feasible due
to practical difficulties in propagating efficient seeded (Kong et al.,
2004). The latter group includes the Y145Stop
variant, as well as most familial GSS cases with conversion of the
latter protein, although certain aspects
of seeded conversion of PrP23231 or PrP90231 could point mutations. It
is possible that some of these mutations
could affect the seeding specificity of the prion be reproduced using a
reduction-oxidation process (Lee
and Eisenberg, 2003), or under conditions requiring high protein,
creating new transmissibility barriers that prevent
disease transmission to wild-type animals. Con- concentrations of
denaturants and very vigorous shaking
(Baskakov, 2003). The apparently low conversion ceivably, transmission
of such cases could only be possible
in animals carrying prion protein with a mutation propensity of the
full-length PrP may reflect evolutionary
selection toward preventing conversion of this ubiqui- matching that of
the donor.
It is well recognized that passage of prions through tous protein into
the extremely toxic PrPSc state, likely
by shielding of critical amyloidogenic determinant(s) by animals may
lead to the establishment of new prion
strains (for a review see Bruce and Fraser, 1991; Col- intramolecular
contacts with the folded C-terminal domain
(Kundu et al., 2003). Although the crucial role of linge, 2001). The
existence of multiple prion strains has
for many years presented a major challenge to the pro- amino acid
residue 139 (equivalent to 138 in mouse or
142 in goat PrP) in prion protein conversion is in line tein-only
hypothesis, providing arguments for models
advocating scrapie-specific nucleic acids. It was only with previous
observations in scrapie-infected neuroblastoma
cells (Priola and Chesebro, 1995) and animal recently that experimental
data started to emerge suggesting
that multiple prion strains could be rationalized studies (Goldmann et
al., 1996), it should be noted that
the seeding barriers in the present study with PrP23144 within the
framework of the protein-only model, with
individual strains likely representing different PrPSc con- do not fully
match the pattern of transmission barriers
observed in vivo. In particular, mice appear to be resis- formations
(Bessen and Marsh, 1994; Parchi et al., 2000;
Peretz et al., 2002; Telling et al., 1996; Safar et al., 1998). tant to
hamster prions (Kimberlin and Walker, 1978),
whereas no barrier was observed for seeding of Here we have shown that
the phenomenon reminiscent
of prion strain diversity, as observed in animal studies, moPrP23144
with ShaPrP23144 fibrils. The lack of a
perfect match is not at all surprising since, in addition can be
reproduced in a simple system consisting solely
of highly purified mammalian prion protein variant. This to residues
138/139, amino acids beyond the 23144
region of PrP are also known to play a role in the species in vitro
system provided us with the unique opportunity
to explore the molecular basis of strain diversification, barrier for
prion transmission (Caughey, 2001; Kocisko
et al., 1995; Priola et al., 2001; Scott et al., 1993). Further- as well
as the relationship between strains and barriers
for interspecies transmissibility of mammalian prions. more, chaperone
proteins or other cellular cofactors
may be involved in the species specificity in vivo. How- While
reaffirming the importance of individual amino
acids in PrP as determinants of the species barriers, ever, the purpose
of the present study was not to quantitatively
reproduce the pattern of naturally occurring spe- the present study
clearly demonstrates that sequence
compatibility between host prion protein and the donor cies barriers
but, rather, to develop a model for
understanding basic mechanistic principles of these of infection is only
part of the equation. As shown in
our experiments, original sequence-based barriers that barriers at a
molecular level. This model allowed us to
demonstrate a very close relationship between species prevent
cross-seeding of conformational conversion between
proteins representing species A and B may be barriers and strains,
revealing the mechanism of an adaptation
process that leads to the emergence of new readily bypassed and new
barriers may be established
by a simple adaptation process. This process, involving prion strains
which can bypass original sequence-based
barriers for interspecies transmissibility of mammalian preseeding of
protein A with PrP23144 fibrils corresponding
to the third species C, leads to the emergence prions. The findings for
the C-truncated PrP variant are
conceptually similar to those recently reported for a of a new strain of
fibrils A. Remarkably, even though
this new strain has the amino acid sequence of PrP structurally
unrelated yeast protein Sup35, the determinant
of the yeast prion state [PSI] (Chien and Weiss- corresponding to
species A, it adopts seeding specificity
of protein C. Given the identity of protein primary man, 2001; Chien et
al., 2003). This striking similarity
strongly suggests that the mechanistic principles estab- structure in
both strains, the altered seeding specificity
of the new strain must be rooted in different fibril confor- lished in
our model are of general validity, also applying
to the full-length mammalian prion protein. mation. The precise nature
of conformational differences
that determine seeding specificity of individual The principle of strain
adaptability could explain some
of the most puzzling observations regarding mammalian strains is under
current investigation. Regardless of the
nature of these differences, the present data strongly prion propagation
in vivo, including that of species-specific
differences in transmissibility of classical human suggests that
barriers in TSE transmissibility are not so
much a simple function of species-dependent differ- CJD and vCJD prions
(Bruce et al., 1997; Collinge, 2001;
Hill et al., 1997). While classical CJD prions encounter ences in PrP
primary sequence but, rather, they are a
conformational property of individual prion strains. The a significant
barrier for transmission to wild-type mice,
they transmit very efficiently to transgenic mice express- notion that
species barriers and prion strains are inti-
Molecular Cell
144
Received: January 5, 2004 ing only human PrP. In contrast, vCJD prions
transmit
Revised: February 5, 2004 readily to wild-type mice, whereas their
transmission to
Accepted: February 13, 2004 transgenic mice is relatively inefficient.
The transmission
Published: April 8, 2004
properties of vCJD are very similar to those of BSE
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