Nature Cell Biology
1, 55 - 59 (1999)
doi:10.1038/9030
Strain-specific prion-protein conformation determined by metal ionsJonathan D.F. Wadsworth1, Andrew F. Hill1, Susan Joiner1, Graham S. Jackson1, Anthony R. Clarke1, 2
& John Collinge11 MRC Prion Unit and Department of Neurogenetics, Imperial College School of Medicine at St Mary's, Norfolk Place, London W2 1PG, UK 2 Department of Biochemistry, School of Medical Sciences, University of Bristol, University Walk, Bristol BS8 1TD, UK
Correspondence should be addressed to John Collinge j.collinge@ic.ac.ukIn animals infected with a transmissible spongiform encephalopathy, or
prion disease, conformational isomers (known as PrPSc proteins)
of the wild-type, host-encoded cellular prion protein (PrPC)
accumulate. The infectious agents, prions, are composed mainly of these conformational
isomers, with distinct prion isolates or strains being associated with different
PrPSc conformations and patterns of glycosylation. Here we
show that two different human PrPSc types, seen in clinically
distinct subtypes of classical Creutzfeldt−Jakob disease, can be interconverted
in vitro by altering their metal-ion occupancy. The dependence of PrP
Sc conformation on the binding of copper and zinc represents a new
mechanism for post-translational modification of PrP and for the generation
of multiple prion strains, with widespread implications for both the molecular
classification and the pathogenesis of prion diseases in humans and animals.
The transmissible spongiform encephalopathies are a group of transmissible
neurodegenerative diseases that include Creutzfeldt−Jakob disease (CJD)
in humans and scrapie and bovine spongiform encephalopathy (BSE) in animals.
These diseases have attracted wide interest not only because of their unique
biology, but also because of the appearance of new variant CJD 1,
which appears to be caused by exposure to the causative agent of BSE 2,
3,
4. The diseases are associated with the accumulation in affected
brains of a conformational isomer (PrPSc) of host-derived prion
protein (PrPC). According to the protein-only hypothesis of
prion propagation, PrPSc is the principal or sole component
of transmissible prions 5. The formation of PrPSc
from PrPC is associated with an increase in the amount of  -sheet
secondary structure in the protein 6, and PrPSc
is recognized biochemically by its acquisition of partial resistance to digestion
by proteinase K. Although the structure of PrPC has been determined 7, the insolubility of PrPSc, which is often present
in brain as amyloid fibrils and which is isolated from tissue in a highly
aggregated state, has so far precluded high-resolution structural analysis
of PrPSc.
The existence of multiple strains or isolates of prions, encoding distinct
disease phenotypes, has been difficult to accommodate within a protein-only
model of prion propagation. However, considerable evidence now indicates that
the diversity of prion strains may be encoded within PrP itself. Distinct
PrPSc conformations 2,
8,
9,
10,
11 and patterns
of glycosylation 2 are associated with different prion strains
and these biochemical properties can be transmitted to experimental animals 2,
10. The elucidation of cellular mechanisms that may influence PrP
Sc conformation is thus of considerable interest. Here we show that
strain-specific human PrPSc molecules are isolated from diseased
brain in a metal-ion-occupied form and can undergo conformational change as
a result of binding metal ions. We propose that this simple post-translational
mechanism may be of widespread importance in conferring strain-specific properties
to distinct PrPSc conformers.
Results
Human prion strain types.
We previously identified
four human PrPSc types or strains that are associated with
distinct forms of sporadic or acquired CJD 2. Type-4 PrP
Sc characterizes new variant CJD, which is causally related to BSE 2,
3,
4; however, there is no evidence for an animal origin for the
prion strains causing classical, or sporadic, CJD (PrPSc types
1−3) 12. Following limited proteolysis with proteinase
K and western blotting, these distinct types of PrPSc can be
easily distinguished by their differing fragment sizes or by relative differences
in intensities of the three PrP glycoforms (corresponding to amino-terminally
truncated cleavage products generated from di-, mono-, or non-glycosylated
PrPSc) 2 (Fig. 1). A common
PrP polymorphism (the presence of either methionine (M) or valine (V) at residue
129) contributes to genetic susceptibility to both sporadic and acquired human
prion disease 13,
14. So far, PrPSc types 1 and
4 have been found only in individuals of the MM genotype; type 2 is seen individuals
of all genotypes (MM, MV and VV); and type-3 PrPSc accompanies
only the MV or VV genotypes (ref. 2, 15, 16 and A.F.H., S.J. and
J.C., unpublished observations).
 | | |
In an earlier study 17 of PrPSc types in classical
CJD, only two types of PrPSc were described and these authors
have argued that the types 1 and 2 that we describe correspond to their type
1, while our type-3 pattern corresponds to that of their type-2 protein 18. However, these authors concede a degree of heterogeneity in their
type-1 cases 18. We have performed a large-scale study of PrP
Sc types in CJD in conjunction with the UK National CJD Surveillance
Unit. Comprehensive phenotypic assessment of patients and PrPSc
typing were performed blind. A detailed study will be published elsewhere,
but we showed that patients classified as type 1 and type 2 using our criteria
have quite distinct phenotypes (Fig. 2), confirming
the validity of our molecular classification. Type-1 human CJD is a distinct
human prion disease with an aggressive clinical course and remarkably short
clinical duration. These observations are consistent with PrPSc
conformation being the foundation of prion strain diversity.
| | |
Conformations of PrPSc types 1 and 2 depend on metal
ions.
In an attempt to elucidate the molecular basis of strain
variation, we investigated the biochemical properties of type-1 and type-2
human PrPSc. In all patients studied, both alleles of the PrP
C-encoding gene, PRNP, coded for PrP with methionine at position
129 (genotype MM). When we treated type-1 and type-2 PrPSc
from these patients with 20 mM of the metal-ion chelator EDTA before treatment
with proteinase K, the pattern of cleavage was changed. Rather than producing
their distinct patterns, both types gave indistinguishable and common fragment
sizes (Fig. 3a). As these digestion products had lower
relative molecular masses than the cleavage products of either type-1 or type-2
PrPSc, we designated these products type 2-.
In marked contrast, treatment with EDTA did not alter the generation of characteristic
cleavage products from PrPSc types 3 or 4 (
Fig. 3b).
| | Figure 3. Digestion of human PrPSc by proteinase K in the presence
of metal chelators. | | |  | a, b, Effects of EDTA on digestion of PrPSc
types 1−4. 10% w/v brain homogenates prepared in cold
lysis buffer were treated with proteinase K directly (-) or after (+)
adjustment of EDTA in the buffer to a final concentration of 20 mM. a,
Lanes 1, 2, type-1 PrPSc; lanes 3, 4, type-2 PrPSc
MM. b, Lanes 1, 2, type-1 PrPSc genotype MM; lanes 3,
4, type-2 PrPSc genotype MM; lanes 5, 6, type-3 PrP
Sc genotype MV; lanes 7, 8, type-4 PrPSc genotype MM.
c, The effect of EDTA on type-1 PrPSc is consistent in
different buffers. 10% w/v brain homogenates from a patient
with type-1 PrPSc were prepared in cold lysis buffer (CLB;
lanes 1, 2), PBS (lanes 3, 4) or N-ethylmorpholine buffer (M; lanes
5, 6) and were digested with proteinase K before (-) or after (+) adjustment
with EDTA to a final concentration of 20 mM. d, EDTA exposes a new
site of cleavage by proteinase K (PK) on type-1 PrPSc. Aliquots
of a 10% w/v PBS brain homogenate from a patient with type-1
PrPSc were western blotted directly (no proteinase-K treatment)
in the absence (lane 1) or presence (lane 2) of 25 mM EDTA. In lanes 3−5,
aliquots of a 10% w/v PBS brain homogenate from a type-1 patient
were treated with proteinase K in the absence (lanes 3, 5) or presence (lane
4) of 25 mM EDTA. Following proteolysis, the sample in lane 5 was boiled in
SDS sample buffer and subsequently adjusted to 25 mM EDTA before electrophoresis.
e, f, Effects of different chelators on the digestion of type-1
PrPSc. Aliquots of a 10% w/v N-ethylmorpholine
buffer brain homogenate from a type-1 PrPSc patient were treated
with proteinase K in the absence (lane 1) or presence (lanes 2−7) of
different chelators. The chelators and their final concentrations were:
e, lane 2, 20 mM EDTA; lane 3, 20 mM EGTA; lane 4, 20 mM dipicolinic acid;
lane 5, 20 mM bathophenanthroline disulphonic acid; lane 6, 20 mM neocuproine;
lane 7, 20 mM 1,10 phenanthroline; f, lane 2, 20 mM EDTA; lane 3, 20
mM EGTA; lane 4, 20 mM triethylenetetramine; lane 5, 20 mM dipicolinic acid;
lane 6, 10 mM triethylenetetramine plus 10 mM dipicolinic acid; lane 7, 10
mM triethylenetetramine plus 10 mM bathophenanthroline disulphonic acid. The
'types' are the types of PrPSc cleavage products
produced, that is, types 1, 2, 3, 4 or 2-.
 Full Figure and legend (57K) |
| |
The generation of type-2- cleavage products from type-1
PrPSc typically required final EDTA concentrations in the range
of 15−20 mM; no further change was elicited by higher chelator
concentrations (data not shown). This effect of EDTA was fully reproducible
(>60 repetitions using samples from nine type-1 patients) and occurred irrespective
of the buffer in which brain homogenates were prepared (Fig.
3c). We analysed nine homogenates from type-1 and type-2 patients before
and after treatment with EDTA. In each case, we detected the expected shift
of type-1 or type-2 products to type-2- products. We estimated
the shift in apparent relative molecular mass from type-1 to type-2
- products and from type-2 MM to type-2− products
to be 1,100  300 (mean s.d.; n = 9) and 650
300 (mean s.d.; n = 9), respectively. There was no significant
alteration in the ratios of the three principal PrP glycoforms.
We excluded the possibility that EDTA itself directly influenced electrophoretic
mobility. Without protease digestion, type-1 PrPSc samples
migrated equivalently in the presence or absence of EDTA (
Fig. 3d). Similarly, application of EDTA to type-1 PrPSc
samples after proteolysis had no effect (Fig. 3d). These
findings indicate that the respective conformations of type-1 PrPSc
and type-2 PrPSc MM may depend upon the presence of
metal ions and that metal-ion chelation may induce a conformational change
in the protein, exposing a new proteolytic cleavage site that is apparently
common to both metal-ion-depleted conformers.
Effects of metal-ion-selective chelators.
As the N-terminal
octapeptide repeat region of PrP binds Cu2+ (19− 24), we thought that
Cu2+ might be involved in determining metal-ion-dependent PrP
Sc conformation. However, the use of various metal-selective chelating
agents revealed a more complex situation. EDTA is a broad-specificity chelator
with high affinity for many divalent metal ions; other, more selective chelators,
including those with high affinity for Cu2+ (EGTA and triethylenetetramine),
Cu+ (neocuproine), Zn2+ (dipicolinic acid and
1,10 phenanthroline) or Fe2+ (1,10 phenanthroline and bathophenanthroline
disulphonic acid) were unable to mirror the effects of EDTA precisely (Fig. 3e, f). However, the effectiveness of combined application
of triethylenetetramine and dipicolinic acid (Fig. 3f)
indicated that chelation of both Cu2+ and Zn2+
may be required for generation of type-2- cleavage products
from type-1 PrPSc.
Both Cu2+ and Zn2+ interact with PrP
Sc.
We developed an alternative method for probing the
metal-ion dependency of PrPSc conformation, by washing the
homogenates to strip bound metal from the protein. Washing type-1 PrP
Sc homogenates with N-ethylmorpholine buffer (equivalent to
a ~5,000-fold dilution) before digestion by proteinase K readily resulted
in the formation of type-2- digestion products (
Fig. 4a, b). Repetition of this procedure using buffers supplemented
with various metal ions at the total concentrations observed in serum 25 convincingly showed that the type-1 conformation is dependent on
metal ions. In the maintained presence of Cu2+ or Zn
2+, digestion products closely resembled those generated from untreated
type-1 PrPSc (Fig. 4a); that is, the
original PrPSc conformation was retained. Other metal ions,
when present at their respective total concentration found in serum, had no
effect, when applied either separately (data not shown) or together (Fig. 4a). We can exclude the occurrence of anomalous electrophoretic
mobility of cleavage products in the presence of Cu2+ or Zn
2+: exposure of metal-ion-depleted and proteinase-K-digested type-1
PrPSc samples to either Cu2+ or Zn2+
before and during electrophoresis had no effect (
Fig. 4b). Together, these findings (coupled with the results obtained
using metal-ion-selective chelators) implicate Cu2+ or Zn
2+ as the most relevant metal ions that interact with type-1 PrP
Sc in prion-diseased brain. Notably, the concentrations of Cu
2+ that we find to be effective in maintaining type-1 PrPSc
conformation correlate closely with the dissociation constant of
14 µM for binding of Cu2+ to recombinant full-length
hamster PrP 23.
| | Figure 4. Both Cu2+ and Zn2+ interact with PrP
Sc. | | |  | PrPSc types 1 and 2 (PRNP genotype MM) were washed
in the presence of various metal ions. a, Type-1 PrPSc.
A 10% w/v brain homogenate from a type-1 patient was prepared
in PBS and aliquots were digested with proteinase K before (lane 1) or after
washing with N-ethylmorpholine buffer alone (lane 2) or the same buffer
containing 20 µM FeCl3, 1 mM MgCl2, 1 µM
NiCl2, 2 mM CaCl2, 0.05 µM MnCl2 and
0.03 µM CoCl2 (lane 3); 10 µM ZnCl2 (lane
4); 20 µM ZnCl2 (lane 5); 10 µM CuSO4 (lane
6); 20 µM CuSO4 (lane 7); or 25 µM CuSO4
(lane 8). b, Type-1 PrPSc. A 10% w/v brain
homogenate from a type-1 patient was prepared in PBS and aliquots were digested
with proteinase K before (lane 1) or after (lanes 2−4) washing with
N-ethylmorpholine buffer. Following proteolysis and before electrophoresis,
samples in lanes 3 and 4 were washed with N-ethylmorpholine buffer
containing either 20 µM ZnCl2 (lane 3) or 25 µM CuSO
4 (lane 4). c, Type-1 PrPSc. A 10% w/
v brain homogenate from a type-1 patient was prepared in N-ethylmorpholine
buffer and aliquots were digested with proteinase K after washing with
N-ethylmorpholine buffer alone (lane 1) or the same buffer containing
20 µM ZnCl2 (lane 2), 30 µM NiCl2 (lane
3), 30 µM CoCl2 (lane 4), 30 µM MnCl2 (lane
5), or 30 µM FeCl3 (lane 6). d, Type-2 PrP
Sc. A 10% w/v brain homogenate from a type-2 patient
was prepared in cold lysis buffer and aliquots were digested with proteinase
K before (lane 1) or after washing with N-ethylmorpholine buffer alone
(lane 2) or the same buffer containing 20 µM ZnCl2 (lane
3) or 25 µM CuSO4 (lane 4). Lanes 5, 6 show digestion products
from a type-1 PrPSc PBS brain homogenate that was proteinase-K-treated
directly (lane 5) or after addition of 25 mM EDTA (lane 6).
Full Figure and legend (50K) |
| |
Although both Cu2+ and Zn2+ are present at
much higher total concentrations in normal brain compared with in serum (discussed
in ref. 23, 25),
these ions would exist predominantly in protein complexes rather than as free
ions. Transient total extracellular concentrations of Zn2+
can reach as high as 300 µM in brain during sustained neuronal activity 26, but the proportion that exists as free ions is uncertain. In the
case of Cu2+, it is unlikely that micromolar levels of free
ions will occur in any cell compartment in the physiological state, and it
thus remains to be shown how PrPC might acquire Cu2+
in vivo. From our findings, however, the pathological relevance
of metal-ion binding to PrPSc is clear: PrPSc
types 1 and 2 are isolated from diseased brain in metal-ion-occupied form.
These results could indicate that the concentrations of Cu2+
and Zn2+ in prion-diseased brain are grossly perturbed. This
has recently been shown to be the case in Alzheimer's disease, where
Cu2+, Zn2+ and Fe2+ are highly
concentrated within the periphery and core of senile plaque deposits 27. Moreover, micromolar concentrations of Cu2+ and
Zn2+ can induce marked aggregation of amyloid A protein 25. In the latter study, at the total concentrations of metal ions
found in serum, only Cu2+ and Zn2+ were able
to induce marked aggregation of A protein; however, at supraphysiological
concentrations (30 µM), Ni2+ and Co2+
were also effective. We have also tested the effectiveness of 30 µM
Ni2+, Co2+ or Mn2+ in maintaining
the conformation of type-1 PrPSc; at this concentration, only
Ni2+ can effectively substitute for Cu2+ or
Zn2+ (Fig. 4c). These results further
reinforce our deduction that Cu2+ and Zn2+ are
likely to be the most important metal ions that interact with PrPSc
in prion-diseased brain.
Interconversion of human PrPSc types 1 and 2.
Interestingly, whereas the conformation of type-1 PrPSc
could be easily maintained in the presence of 10 µM Zn2+,
higher concentrations of Cu2+ were required to have the same
effect. We studied a range of Cu2+ concentrations (10−25
µM); in the presence of 20 µM Cu2+, a pattern of
cleavage products was produced that migrated with a mobility similar to that
of the type-2 products, that is, intermediate between type 1 and type 2
- (Fig. 4a, compare lanes 6−8). This intermediate
pattern could also be discerned after digestion of type-1 PrPSc
in the presence of the copper-selective chelators EGTA or triethylenetetramine
(Fig. 3e, f), or after washing and digestion of type-1
PrPSc in the presence of 30 µM Co2+ (Fig. 4c). As the mobility of these intermediate fragments
resembled that of type-2 cleavage products, these results indicate that the
conformations of type-1 PrPSc and type-2 PrPSc
may differ principally with respect to the relative occupancy of their metal-ion-binding
sites.
To explore this possibility further, we studied the effects of applying
exogenous metals to type-2 PrPSc. Consistent with the results
obtained for type-1 PrPSc, washing insoluble aggregates of
type-2 PrPSc with buffer alone gave type-2-
cleavage products (Fig. 4d). However, in the maintained
presence of different concentrations of Cu2+ or Zn2+
, we observed either the original type-2 pattern of digestion products,
or a new pattern of higher-molecular-mass cleavage fragments similar to those
generated from untreated type-1 PrPSc (Fig. 4d). These
findings indicate that the conformations of type-1 PrPSc and
type-2 PrPSc may be interchangeable and depend on the level
of occupancy by these metal ions.
Discussion
The demonstration that phenotypically distinct types of CJD are associated
with the biochemically distinct PrPSc types 1 and 2 clarifies
earlier confusion on classification of CJD subtypes 18. However,
the precise aetiology of sporadic CJD remains obscure. The spontanous conversion
of PrPC to PrPSc in a rare stochastic event,
or somatic mutation of the PRNP gene, resulting in expression of a
pathogenic PrP mutant 12, are possible causes. However, epidemiological
studies have not ruled out the possibility that environmental exposure to
human or animal prions 28 causes at least some cases. Sporadic
CJD may have multiple aetiologies. Our results immediately allow a more precise
molecular classification of human prion disease, with important implications
for epidemiological studies into the aetiology of sporadic CJD. Re-analysis
of epidemiological data using these molecular subtypes may reveal important
risk factors that are obscured when sporadic CJD is analysed as a single entity.
Our results also define a potential molecular mechanism for strain variation.
The ability of metal ions to influence PrPSc conformation directly
has widespread implications for understanding strain diversity in human and
animal prion diseases. Our demonstration of an interaction between PrP
Sc and Cu2+ not only supports recent work that indicates
that Cu2+ may stabilize PrPSc conformation 29, but also provides further evidence that the neuropathology of
prion diseases may be related to abnormalities in copper metabolism 22,
30,
31,
32 and raises the possibility that drugs that influence
copper metabolism may have therapeutic potential in prion disease.
Methods
Western blot analysis.
All procedures were carried
out in a microbiological-containment level-3 facility with strict adherence
to safety protocols. 10% w/v brain homogenates from human brain
tissue obtained at autopsy from patients with CJD were prepared in the following
solutions: cold lysis buffer (10 mM Tris and 10 mM EDTA, pH 7.4, containing
100 mM NaCl, 0.5% w/v NP-40 and 0.5% w/v sodium
deoxycholate); phosphate-buffered saline (PBS) (Dulbecco's sterile PBS
lacking Ca2+ and Mg2+; Sigma); N-ethylmorpholine
buffer (25 mM N-ethylmorpholine, pH 7.4, containing 0.5% w/
v NP-40). Samples were adjusted to a final concentration of 50 µg
ml-1 proteinase K (Merck) and incubated at 37 °C for 1
h. Digestion was terminated by addition of an equal volume of 2  SDS
sample buffer (125 mM Tris-HCl and 20% v/v glycerol, pH 6.8,
containing 4% w/v SDS, 4% v/v 2-mercaptoethanol,
8 mM 4-(2-aminoethyl)-benzene sulphonyl fluoride and 0.02% w/v
bromophenol blue) and immediate transfer to a 99 °C heating block for
10 min. Samples were analysed by electrophoresis and western blotting using
anti-PrP monoclonal antibody 3F4 as described 2.
Chelation studies.
Chelators were added to brain homogenates
as aliquots from stock solutions. EDTA was prepared as either a 100-mM or
a 250-mM stock in water and titrated to pH 8.0 with NaOH. Other chelators
(Fig. 3) were prepared similarly as 100-mM stock solutions,
pH 8.0, with the exception of 1,10 phenanthroline and neocuproine which were
prepared as 100-mM stocks in 50% v/v ethanol in water. All chelators
were obtained from Sigma. Physical properties of the chelators used and the
stability constants of complexes formed with various metal ions have been
described 33.
Metal-ion-supplementation studies.
10-µl aliquots
of 10% w/v brain homogenates were centrifuged for 10 min at
14,000 r.p.m. in a microfuge (Eppendorf), after which supernatants were removed
and discarded. Pellets were thoroughly resuspended in 500 µl N-ethylmorpholine
(pH 7.4, 25 mM) containing 0.5% w/v NP-40; the N-ethylmorpholine
either lacked or contained various metal salts as described in
Fig. 4. Following incubation for ~10 min at room temperature, samples
were centrifuged (15 min at 14,000 r.p.m. in a microfuge), after which the
supernatant was discarded. Each aspirated pellet was resuspended appropriately
with the analogous solution to a final volume of 10 µl and treated with
proteinase K. In some experiments samples were washed with metal solutions
after digestion by proteinase K.
Received 11 January 1999; Accepted 15 March 1999
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Acknowledgements
This work was funded by the Medical Research Council and Wellcome Trust.
We thank R. Will, J. Ironside and colleagues at the National CJD Surveillance
Unit for help with this study.
Correspondence and requests for materials should be addressed to J. C.
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