Previous Article | Next Article 
Journal of Virology, December 2000, p. 11928-11934, Vol. 74, No. 24
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Affinity-Tagged Miniprion Derivatives Spontaneously
Adopt Protease-Resistant Conformations
Surachai
Supattapone,1,2
Hoang-Oanh B.
Nguyen,1,2
Tamaki
Muramoto,1,
Fred E.
Cohen,1,2,3,4
Stephen J.
DeArmond,1,5
Stanley B.
Prusiner,1,2,4,* and
Michael
Scott1,2
Institute for Neurodegenerative
Diseases1 and Departments of
Neurology,2 Cellular and Molecular
Pharmacology,6
Medicine,3
Pathology,5 and Biochemistry and
Biophysics,4 University of California at San
Francisco, San Francisco, California 94143
Received 14 July 2000/Accepted 22 September 2000
 |
ABSTRACT |
An abridged PrP molecule of 106 amino acids designated PrP106 can
form infectious miniprions in transgenic (Tg) mice (29). Addition of six-histidine (His6) affinity tags to selective
sites within PrP106 resulted unexpectedly in new PrP proteins that
spontaneously adopted protease-resistant conformations when expressed
in neuroblastoma cells and Tg mice. Acquisition of protease resistance
depended on the length, charge, and placement of the affinity tag.
Introduction of the disease-linked mutation E200K into the sequence of
PrP106(140/6His) increased the recovery of protease-resistant PrP
fivefold, whereas introduction of the mutations C213A and 
214-220
did not affect the recovery of protease-resistant PrP. Treatment of
cultured cells expressing affinity-tagged PrP106 mutants with
polypropyleneimine dendrimer rendered these proteins sensitive to
protease digestion in a manner similar to wild-type PrPSc.
We conclude that certain affinity-tagged PrP106 proteins spontaneously fold into conformations partially resembling, yet distinct from, wild-type PrPSc. These proteins might be useful tools in
the identification of new disease-causing mutations as well as for
screening compounds for therapeutic efficacy.
| |
INTRODUCTION |
Prion diseases are disorders of
protein conformation in which the cellular form of the prion protein,
PrPC, undergoes a pathogenic conformational change into an
infectious isoform, PrPSc (20-22). During this
conformational change, full-length PrPC containing ~40%
-helix and little 
-sheet folds into PrPSc that is
composed of ~30% -helix and ~40% -sheet (17, 18, 24). Attempts to identify a chemical modification that drives this profound conformational change that is responsible for
PrPSc formation have been unrewarding (28).
Earlier studies showed that the protease-resistant core of
PrPSc designated PrP 27-30 polymerizes into amyloid fibrils
(23). PrP amyloid, like all other known amyloids, possesses
a high -sheet content (3, 7).
Determining the high-resolution three-dimensional molecular structures
of PrPC and PrPSc is an important step toward
deciphering the mechanism underlying prion diseases. Recombinant PrP
molecules derived from Escherichia coli refolded into
conformations with a high -helical content that appear to
approximate the structure of PrPC (8, 9, 14). On
the other hand, investigation of PrPSc structure has been
hindered by the extreme insolubility and molecular heterogeneity of
this isoform (23, 32).
To elucidate the tertiary structure of PrPSc, we attempted
to design smaller infectious PrPSc molecules that might be
more amenable to structural investigation. We recently reported that a
PrP deletion mutant MHM2(23-88,141-176), designated PrP106,
successfully forms infectious miniprions in Prnp0/0 mice
(29). PrP106 contains only 106 amino acids, compared to the
208 residues in full-length PrP. In order to facilitate purification of
PrPSc106, we incorporated an affinity tag
consisting of six histidine residues (His6) at
various sites within the PrP106 backbone. Unexpectedly, we found that
some of these affinity-tagged derivatives spontaneously adopted
conformations with the same level of protease resistance as
PrPSc106.
| |
MATERIALS AND METHODS |
Explanation of nomenclature.
MHM2 is a full-length chimeric
construct that differs from wild-type MoPrP at positions 108 and 111 (27). Substitution at these positions with the corresponding
residues (109 and 112, respectively) from the Syrian hamster (SHa) PrP
sequence creates an epitope for the anti-PrP 3F4 monoclonal antibody
(MAb) (12), which does not recognize wild-type MoPrP, and
hence facilitates specific detection of the transgene by Western blot.
Mature MHM2 and MoPrP comprise residues 23 to 230 after processing
because residues 1 to 22 are removed by a signal peptidase and residues
231 to 254 are removed during the addition of a GPI anchor. PrP106
refers to a truncated MHM2 molecule, in which residues 23 to 88 and
residues 141 to 176 have been removed, and can also be designated as
MHM2(23-88,141-176). Additional sequences inserted into MHM2 or
PrP106 are denoted by the following convention: N-terminal residue of
attachment and/or additional sequence in parenthesis. For example,
PrP106(140/GASGAS) includes the following residues: 89-140-Gly-Ala-Ser-Gly-Ala-Ser-177-230.
Construction of DNA plasmids and transgenic (Tg) mice.
Most
of the new constructs described here were created by standard cassette
mutagenesis of pCOMBO3 (Mike Scott) or psp72PrP106 (Tamaki Muramoto),
using oligonucleotides obtained from Gibco-BRL. XbaI and
AspI were used to insert new sequences between residues 140 and 177. DNA sequencing (Perkin-Elmer) with T7 and SP6 primers was used
to verify the sequence of every new insert. The mutagenized PrP inserts
were removed from psp72 or pCOMBO plasmids by digestion with
BglII/XhoI and subcloned into
BamHI/XhoI-digested pSPOX.neo vector
(27) to create pSPOX N2a cell expression plasmids.
pSPOXPrP106(225/6His) was created by substitution of a
BstEII/XhoI-digested
pSPOX72MHM2(225/6His) (Kiyatoshi Kaneko) insert into
BstEII/XhoI-digested pSPOXPrP106 vector.
pSPOXPrP106(140/6His,E200K) was created by substitution of
BstEII/XhoI-digested pCOMBO2MHM2 E200K
insert into BstEII/XhoI-digested pSPOXPrP106(140/6His) vector. Qiagen Maxiprep columns were used to
purify pSPOX expression plasmids for transfection experiments.
CosTetPrP106(140/6His) and CosTetPrP106(225/6His) cosmids
were generated in a two-step process from their respective pSPOX
plasmids. First, a 400-bp
KpnI/
XhoI insert from
the appropriate
pSPOXPrP plasmid was ligated into
KpnI/
XhoI-digested psp72(
SalI)MHM2
vector (
27). Second, the
SalI/
XhoI PrP
insert from the modified
psp72(
SalI)PrP construct was cloned
into
XhoI-digested CosTet.neo.
Microinjection, breeding, and
screening of Tg animals was performed
as previously described
(
26).
Expression in neuroblastoma cells.
Stock cultures of N2a and
ScN2a cells were maintained in minimal essential medium with Earle's
salts plus 10% fetal bovine serum (FBS), 10% Glutamax (Gibco-BRL),
100 U of penicillin per ml, and 100 µg of streptomycin per ml. Cells
from a single confluent 100-mm dish were trypsinized and split into 10 separate 60-mm dishes containing Dulbecco's modified Eagle medium plus
10% FBS, 10% Glutamax, 100 U of penicillin per ml, and 100 µg of
streptomycin per ml (supplemented DME) 1 day prior to transfection. For
each construct, 15 µg of DNA was resuspended in 150 µl of sterile
HEPES-buffered saline (HBS) on the day of transfection. The DNA
solution was then mixed with an equal volume of 333 µg of DOTAP
(Boehringer Mannheim, Indianapolis, Ill.) per ml in HBS in Falcon 2059 tubes and incubated at room temperature for 10 min to allow formation of DNA-lipid complexes. Supplemented DME (2.5 ml) was added to the
mixture, and this was then pipetted onto drained cell monolayers. The
following day, the medium containing the DNA-lipid complexes was
removed and replaced with fresh supplemented DME.
Three days after transfection, cells were harvested by lysis in 1.2 ml
of 20 mM Tris (pH 8.0) containing 100 mM NaCl, 0.5%
NP-40, and 0.5%
sodium deoxycholate (DOC). Nuclei were removed
from the lysate by
centrifugation at 2,000 rpm for 5 min. This
lysate typically had a
total protein concentration of 0.5 mg/ml,
as measured by the
bicinchoninic acid protein assay (Pierce, Rockford,
Ill.).
Western blotting.
For samples not treated with proteinase K,
40 µl of whole lysate (representing 20 µg of total protein) was
mixed with 40 µl of 2× sodium dodecyl sulfate (SDS) sample buffer.
For proteinase K digestion, 1 ml of lysate was incubated with 20 µg
of proteinase K per ml (total protein/enzyme ratio = 25:1) for
1 h at 37°C. Proteolytic digestion was terminated by the
addition of 8 µl of 0.5 M phenylmethylsulfonyl fluoride in absolute
ethanol. Samples were then centrifuged for 75 min in a Beckman TLA-45
rotor (Fullerton, Calif.) at 100,000 × g at 4°C. The
pellet was resuspended by repeated pipetting in 80 µl of 1× SDS
sample buffer. The entire sample (representing 0.5 mg of total protein
before digestion) was boiled for 5 min and cleared by centrifugation
for 1 min at 14,000 rpm in a Beckman ultrafuge. For solubility studies,
the supernatant fractions of proteinase K-digested samples were
precipitated in 10 volumes of MeOH and resuspended in 80 µl of 1×
SDS sample buffer. SDS-polyacrylamide gel electrophoresis (PAGE) was
carried out in 1.5-mm, 15% polyacrylamide gels (13) or in
16% Tricine gels (Novex) as indicated.
Following electrophoresis, Western blotting was performed as previously
described (
25). Membranes were blocked with 5% nonfat
milk
protein in PBST (calcium- and magnesium-free PBS plus 0.1%
Tween 20)
for 1 h at room temperature. Blocked membranes were
incubated with
primary 3F4 MAb at a 1:5,000 dilution in PBST overnight
at 4°C.
Following incubation with primary antibody, membranes
were subjected to
three 10-min washes in PBST, incubated with
horseradish
peroxidase-labeled anti-mouse immunoglobulin G secondary
antibody
(Amersham Life Sciences, Arlington Heights, Ill.) diluted
1:5,000 in
PBST for 25 min at room temperature, and subjected
to three additional
10-min washes in PBST. After chemiluminescent
development with enhanced
chemiluminescence (ECL) reagent (Amersham)
for 1 to 15 min, blots were
sealed in plastic covers and exposed
to ECL Hypermax film (Amersham).
Films were processed automatically
in a Konica film
processor.
| |
RESULTS |
Protease resistance of affinity-tagged PrP106 derivatives in
neuroblastoma cells.
We incorporated His6 affinity
tags at three different sites within the PrP106 backbone. These sites
were: (i) the extreme N terminus at residue 89, (ii) the C terminus
between residues 225 and 226, and (iii) the junction of the
internal deletion bounded by residues 140 and 177. The new constructs
were expressed in both scrapie-infected (ScN2a) and uninfected (N2a)
neuroblastoma cells to assess protease resistance as previously
described (27). PrP106 expressed in either ScN2a or N2a
cells was resistant to proteinase K digestion for 30 min at 37°C
using a total protein/enzyme ratio of 71:1 (29), but it was
largely proteolyzed by more-stringent digestion for 1 h at a
protein/enzyme ratio of 25:1 (Fig. 1A, lane 2). Similarly, PrP106(89/6His) was fully proteolyzed under these stringent conditions (data not shown). In contrast, MHM2, which
forms full-length PrPSc, was resistant to proteinase K
digestion for 1 h at a protein/enzyme ratio of 25:1 in ScN2a
cells, as expected (Fig. 1A, lane 1). Surprisingly, PrP106(140/6His), PrP106(225/6His), and
PrP106(140/6His,225/6His) were all resistant to
proteinase K digestion under stringent conditions in both ScN2a (Fig.
1A, lanes 3 to 5) and N2a cells (data not shown).
View larger version (62K):
[in this window]
[in a new window]
|
FIG. 1.
Expression of His6-labeled PrP106
derivatives in neuroblastoma cells. ScN2a cells were transfected with
the following expression constructs: lane 1, MHM2; lane 2, PrP106; lane
3, PrP106(140/6His); lane 4, PrP106(140/6His,225/6His);
lane 5, PrP106 (225/6His); lane 6, mock transfection. (A) The minus
symbol indicates the whole lysate of an untreated sample; the plus
symbol indicates the pellet fraction of a sample digested with 20 µg
of proteinase K per ml for 1 h at 37°C. (B) Supernatant
fractions of proteinase K-digested samples precipitated in 10 volumes
methanol and resuspended in SDS sample buffer. Western immunoblotting
was performed with 3F4 MAb as described in Materials and Methods. The
apparent molecular masses based on the migration of protein standards
are given in kilodaltons.
|
|
The protease-resistant fragment of PrP106(140/6His) migrated on
SDS-PAGE with an apparent molecular mass of 19 kDa (Fig.
1A,
lane 3),
whereas the protease-resistant core of PrP106(225/6His)
had an
apparent molecular mass of 23 kDa (Fig.
1A, lane 5).
PrP106(140/6His)
could be distinguished from
PrP106(225/6His) after proteinase
K digestion not only by its
mobility on SDS-PAGE but also by its
solubility. In 0.5% NP-40-0.5%
DOC, approximately 25% of protease-resistant
PrP106(140/6His) and
10% of protease-resistant PrP106(140/6His,225/6His)
was
soluble (Fig.
1B, lanes 3 and 4), whereas PrP106(225/6His)
appeared to be largely insoluble (Fig.
1B, lane 5). Taken together,
these results suggest that PrP106(140/6His) might adopt a more
open conformation than PrP106(225/6His), thereby increasing its
solubility and exposing additional regions of the polypeptide
chain to
proteinase K digestion. Interestingly, protease digestion
of
PrP106(140/6His,225/6His) yielded both 19- and 23-kDa fragments
in a roughly 1:1 ratio (Fig.
1A, lane 4). Prolonged digestion
of
PrP106(140/6His,225/6His) did not alter the ratio of 19-kDa
to
23-kDa bands (data not shown), indicating that the 19-kDa band
does not
derive from proteolysis of the 23-kDa band. An alternative
explanation
is that PrP106(140/6His,225/6His) simultaneously adopts
two
distinct conformations, which are differentially proteolyzed.
The
ability of PrP molecules to adopt different protease-resistant
conformations has been well documented in the case of PrP
Sc
molecules derived from different prion strains (
2,
4,
31).
Mutagenesis of affinity-tagged PrP106 molecules.
Because the
presence of a His6 affinity tag at the N terminus did not
protect PrP106(89/6His) from stringent proteinase K digestion, we
concluded that appropriate placement of the affinity tag is one
requirement for formation of protease-resistant PrP106 derivatives. To
identify other requirements, we systematically altered the charge and
length of the tag in PrP106(140/6His) and measured the protease
resistance of the resulting molecules expressed in ScN2a cells.
Changing the composition of the tag from six histidines to six lysines
did not diminish protease resistance (Fig.
2B, compare lanes 1 and 5),
whereas substituting six glutamates, six alanines, or the mixed
amino acid spacer GASGAS completely abolished protease resistance (Fig.
2B, lanes 4, 6, and 7). The protease resistance of PrP106(140/6Lys)
suggests that positive charges in the affinity tag may facilitate the
formation of PrP106 protease-resistant fragments. Increasing the length
of the polyhistidine tag to seven residues resulted in less-intense
protease-resistant bands (Fig. 2B, lane 2), as did substituting
alternating alanine residues within the His tag (Fig. 2B, lane 3).
Taken together, these results indicate that appropriate insert
placement, spacing, and charge are all necessary for the production of
protease resistance in affinity-tagged PrP106 derivatives.
View larger version (98K):
[in this window]
[in a new window]
|
FIG. 2.
Expression of PrP106(140/6His) affinity tag mutants
in neuroblastoma cells. ScN2a cells were transfected with PrP106
expression constructs in which the following sequences replaced the
internal deletion 141-176; lane 1, His6; lane 2, His7; lane 3, HAHAHA; lane 4, GASGAS; lane 5, Lys6; lane 6, Glu6; lane 7, Ala6;
lane 8, mock transfection. (A) Whole lysates of undigested samples. (B)
Pellet fractions of samples digested with 20 µg of proteinase K per
ml for 1 h at 37°C. Western immunoblotting was performed with
3F4 MAb as described in Materials and Methods. Apparent molecular
masses based on the migration of protein standards are given in
kilodaltons.
|
|
Mutagenesis studies of full-length MHM2 expressed in ScN2a cells have
identified specific amino acids that appear to be important
for the
formation of full-length PrP
Sc. These include residue Q218
(
11), as well as cysteine residues
178 and 213, which form a
disulfide bridge (
16). Substitution
mutations of these amino
acids prevent the conversion of MHM2
into its scrapie isoform in ScN2a
cells. We sought to determine
the effect on protease resistance of
similar mutations introduced
into PrP106(140/6His). The mutations
C213A and
214-220 (removal
of Q218 along with two adjacent turns of
-helix C) each prevented
the formation of PrP
Sc when
introduced into MHM2 (Fig.
3B, lanes 5 and 6). However,
when these mutations were introduced into
PrP106(140/6His), the
resulting derivatives remained
protease-resistant in both N2a
and ScN2a cells (Fig.
3B, lanes 2 and
3).
View larger version (72K):
[in this window]
[in a new window]
|
FIG. 3.
Expression of PrP molecules with disruptive mutations in
neuroblastoma cells. ScN2a cells were transfected with the
following expression constructs. Lane 1, PrP106(140/6His);
lane 2, PrP106(140/6His,C213A); lane 3, PrP106(140/6His,214-220); lane 4, MHM2; lane 5, MHM2(C213A);
lane 6, MHM2(214-220); lane 7, mock transfection. (A) Whole lysates
of undigested samples. (B) Pellet fractions of samples digested with 20 µg of proteinase K per ml for 1 h at 37°C. Western
immunoblotting was performed with 3F4 MAb as described in Materials and
Methods. Apparent molecular masses based on the migration of protein
standards are given in kilodaltons.
|
|
Genetic studies have linked a number of PrP mutations to hereditary
forms of prion disease (for reviews, see references
6 and
19). We introduced one
such mutation, E200K, into PrP106(140/6His)
to evaluate the effect
on protease resistance. This mutation has
been linked to familial
Creutzfeldt-Jakob disease among a population
of Libyan Jews (
5,
10,
15). Whereas introduction of the
E200K mutation did not
affect the protease resistance of full-length
PrP or PrP106 (Fig.
4A, lane 3), it did increase the recovery
of protease-resistant PrP106(140/6His) by approximately fivefold
(Fig.
4A, lane 5).
View larger version (80K):
[in this window]
[in a new window]
|
FIG. 4.
Expression and clearance of mutant affinity-tagged
PrP106 proteins in neuroblastoma cells. (A) ScN2a cells were
transfected with the following constructs: lane 1, MHM2; lane 2, PrP106; lane 3, PrP106(E200K); lane 4, PrP106(140/6His);
lane 5, PrP106(140/6His,E200K). (B) ScN2a cells were transfected
with MHM2 (lanes 1 and 2) PrP106(225/6His) (lanes 3 and 4), and
PrP106(140/6His,E200K) (lanes 5 and 6). Cells from even-numbered
lanes were treated with 150 µg of PPI generation 4.0 (Aldrich) per ml
for 4 h prior to harvest. In both panels, the minus symbol
indicates the whole lysate of an untreated sample and the plus symbol
indicates the pellet fraction of a sample digested with 20 µg of
proteinase K per ml for 1 h at 37°C. Western immunoblotting was
performed with 3F4 MAb as described in Materials and Methods. Apparent
molecular masses based on the migration of protein standards are given
in kilodaltons.
|
|
Dendrimer-mediated clearance of protease-resistant
affinity-tagged PrP106 proteins.
Branched polyamines such as
polypropyleneimine (PPI) dendrimers render wild-type
PrPSc sensitive to protease digestion in ScN2a cells
(30). To investigate whether affinity-tagged PrP106
derivatives might share with PrPSc the property of being
susceptible to dendrimers, we treated ScN2a cells expressing
either full-length MHM2, PrP106(225/6His), or PrP106(140/6His,E200K) with PPI. The results indicate that
both PrP106(225/6His) and PrP106(140/6His,E200K)
were rendered protease sensitive by PPI (Fig. 4B).
Tg mice expressing affinity-tagged PrP106 derivatives.
We
expressed PrP106(225/6His) in Tg mice deficient for wild-type
PrP (Prnp0/0). Three separate lines of
Tg[PrP106(225/6His)]Prnp0/0 mice were
generated: two lines expressed the transgene at 32 times (32×) the
expression level of normal Syrian hamster (SHa) PrP, and one line
expressed the transgene at 8 times (8×) the level of SHaPrP.
Uninoculated mice from both lines of 32× expressor lines spontaneously
developed a fatal neurological disease at ~233 ± 8 days of age
(Table 1). The clinical signs of disease resembled scrapie and included ataxia, kyphosis, dull coat, masked facies, loss of deep pain sensation, proprioceptive defects, and weight
loss. Neuropathological examination revealed no vacuolation or nerve
cell loss and only mild gliosis (data not shown). However, after
hydrolytic autoclaving, numerous PrP deposits could be seen in nerve
cell bodies, as well as throughout the gray matter neuropil and white
matter (Fig. 5A and B).
Intracellular deposits measured ~5 µm in diameter at most, while
those in the gray and white matter neuropil were as large as 20 µm in
diameter. PrP106(225/6His) deposits were more abundant and
larger than the 1- to 10-µm PrP106 deposits seen in uninoculated
Tg(PrP106) mice (29). Congo red staining of
PrP106(225/6His) deposits was negative for amyloid (data not
shown). Western blot analysis of the brain homogenates from
Tg[PrP106(225/6His)]15961/Prnp0/0 mice
demonstrated the presence of a proteinase K-resistant, insoluble band
(Fig. 5C). A premorbid 40-day-old
Tg[PrP106(225/6His)]15961/Prnp0/0 mouse displayed
similar levels of proteinase K-resistant PrP as
~230-day-old, symptomatic counterparts (data not shown). We attempted
to transmit the spontaneous neurological disease of Tg[PrP106(225/6His)]Prnp0/0 mice to both
Tg(MoPrP)Prnp0/0 and Tg(PrP106)Prnp0/0 mice by
intracerebral inoculation of 1% brain homogenate, but all of the
animals remained alive and well >300 days after inoculation (Table 1).
In contrast to the 32× overexpressors, uninoculated Tg[PrP106(225/6His)]15947/Prnp0/0 mice expressing
the transgene at 8× the level found for SHaPrP in Syrian
hamsters survived >215 days without signs of disease (Table 1).
View larger version (182K):
[in this window]
[in a new window]
|
FIG. 5.
Accumulation of PrP in
Tg[PrP106(225/6His)]Prnp0/0 mice. (A)
High-magnification view of the neocortex of a spontaneously ill
Tg[PrP106(225/6His])15961/Prnp0/0 mouse at 225 days of age. A section stained by the periodic acid-Schiff (PAS)
histochemical method shows multiple PAS-positive deposits in a nerve
cell body (smaller arrow) and larger PAS-positive deposits in the
neuropil (larger arrow). (B) Lower-magnification view of the same
region of the neocortex immunostained for PrP106 by the hydrolytic
autoclaving method using 3F4 MAb and showing large numbers of deposits
scattered diffusely throughout neuropil. The bar in panel A is 25 µm,
and the bar in panel B is 50 µm. (C) Immunoblot of mouse brain
homogenates. Lanes: 1, normal Syrian hamster; 2, 60-day-old,
uninoculated Tg(PrP106)4290/Prnp0/0 mouse; 3, 120-day-old,
scrapie-affected Tg(PrP106)4290/Prnp0/0 mouse; 4, 225-day-old, uninoculated, ataxic
Tg[PrP106(225/6His)]15961/Prnp0/0 mouse. Brain
homogenates were prepared as previously described (29). The
minus symbol indicates the whole lysate of untreated sample; the plus
symbol indicates the pellet fraction of sample digested with 20 µg of
proteinase K per ml for 1 h at 37°C (total protein/enzyme
ratio = 50:1). Western immunoblotting was performed with MAb 3F4
as described in Materials and Methods. Apparent molecular masses based
on the migration of protein standards are given in kilodaltons.
|
|
We also generated one line of
Tg[PrP106(140/6His)]Prnp
0/0 mice with an expression
level that is 8× that of SHaPrP. These mice
survived >250 days
without any signs of spontaneous neurological
disease (Table
1). The
neuropathological examination of one healthy
60-day-old
Tg[PrP106(140/6His)]Prnp
0/0 mouse was unremarkable,
and hydrolytic autoclaving revealed only
moderate levels of 1- to
10-µm PrP deposits comparable to those
previously seen in
Tg(PrP106)Prnp
0/0 mice (
29) (data not
shown).
| |
DISCUSSION |
We previously observed that PrP106 expressed either in uninfected
N2a cells or in transgenic mice formed a partially protease-resistant fragment and that recombinant PrP106 refolded into a structure that was predominantly a -sheet (1, 29). These
observations raised the possibility that PrP106 might
spontaneously adopt a conformation resembling an intermediate on the
pathway to PrPSc formation (29). As reported
above, we unexpectedly discovered that incorporation of
His6 affinity tags into specific locations within the
PrP106 backbone produces molecules that spontaneously adopt even more
highly protease-resistant conformations resembling PrPSc.
How do affinity tags increase the protease resistance of PrP106?
These tags may promote the intrinsic folding of PrP106
derivatives into a more compact tertiary structure. Alternatively, they
may facilitate intermolecular interactions with other PrP molecules or
with cellular factors that promote compact folding of PrP. It is likely
that structural studies will be required to distinguish between these
possibilities. Location, charge, and spacing all influence the ability
of sequence tags to increase the protease resistance of PrP106.
Placement of the affinity tag either at the C terminus or in the gap
left by the internal deletion 141-176 increased the protease
resistance of PrP106. However, PrP106(140/6His) was more soluble
and displayed a shorter protease-resistant core than
PrPSc106 or PrP106(225/6His). Placement of the tag
at the N terminus did not increase the protease resistance of PrP106 at
all. Therefore, inserting the affinity tag at the C terminus
generates the derivative most similar to PrPSc106. Our
mutagenesis studies also demonstrated that building a tag with
six positively charged residues is optimal for acquisition of protease
resistance. Furthermore, the protease resistance of PrP106(140/6His) was increased by introduction of the
disease-associated mutation E200K located outside the tag. In contrast,
the protease resistance of PrP106(140/6His) was not affected by
mutations in -helix C that disrupt the conversion of full-length
PrPC to PrPSc. Taken together, our mutagenesis
studies suggest that PrP deletion mutants become increasingly protease
resistant as their structures become progressively destabilized. If
this explanation is correct, we predict that the internal and
C-terminal His6 tags are destabilizing elements for PrP106
and that the E200K mutation is a destabilizing element for
PrP(140/6His). Elucidating the precise mechanism by which
affinity-tagged PrP106 molecules become protease resistant will
ultimately require direct determination of their molecular structures.
Although affinity-tagged PrP106 proteins share some properties with
PrPSc, such as protease resistance, insolubility, and
susceptibility to dendrimers, there are several differences that
distinguish these PrP molecules. First, affinity-tagged PrP106
molecules can adopt their protease-resistant conformations
spontaneously in N2a cells and uninfected transgenic mice. In contrast,
the formation of either PrPSc106 or full-length
PrPSc always requires the presence of preexisting
infectious prions. Second, mutations that disrupt the formation of
PrPSc, such as 214-220 and C213A, do not alter the
protease-resistance of PrP106(140/6His) derivatives. Third,
the protease-resistant fragments of affinity-tagged PrP106
molecules are smaller than the protease-resistant core
PrPSc106. Finally, brains expressing protease-resistant
PrP106(225/6His) do not appear to be infectious.
Although affinity-tagged PrP106 molecules are not perfect models of
PrPSc, they may be potentially useful in several ways.
First, using affinity-tagged PrP106 proteins as a starting point, it
might be possible to generate spontaneous infectivity by
introducing disease-associated mutations such as E200K. Spontaneously
infectious PrP106 derivatives might be well suited for structural
studies because their structures could be compared with those of
noninfectious PrP106 proteins to identify structural elements that
generate infectivity. Second, it may be possible to use
PrP106(140/6His) to identify pathogenic PrP mutations. This
molecule may be partially destabilized in such a way that it is
capable of becoming more protease resistant when pathogenic PrP
mutations, such as E200K, are introduced into its sequence. Thus,
PrP106(140/6His) may be a sentinel molecule that can be used
in a simple assay to identify potentially pathogenic mutations. Third,
protease-resistant affinity-tagged PrP106 molecules might be convenient
substitutes for PrPSc in assays identifying novel
therapeutic compounds such as dendrimers.
In summary, affinity-tagged PrP106 molecules spontaneously adopt
conformations partially resembling PrPSc. These molecules
may prove to be useful research tools in areas of prion research such
as structural analysis, identification of pathogenic mutations, and
drug screening.
| |
ACKNOWLEDGMENTS |
We thank Chris Petromilli, Conny Heinrich, Darlene Groth, and
Patrick Tremblay for their expert contributions.
This work was supported by grants from the National Institutes of
Health (NS14069, AG02132, and AG10770), the American Health Assistance
Foundation, and a gift from the Leila and Harold Mathers Foundation.
Surachai Supattapone was supported by the Burroughs Wellcome Fund
Career Development Award and an NIH Clinical Investigator Development
Award (K08 NS02048-02).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Institute for
Neurodegenerative Diseases, Box 0518, University of California, San
Francisco, CA 94143-0518. Phone: (415) 476-4482. Fax: (415) 476-8386. E-mail: ind{at}itsa.ucsf.edu.
Present address: Tohoku University School of Medicine, 2-1 Seiryou-Machi, Aoba-ku, Sendai, 980 Japan.
| |
REFERENCES |
| 1.
|
Baskakov, I. V.,
C. Aagaard,
I. Mehlhorn,
H. Wille,
D. Groth,
M. A. Baldwin,
S. B. Prusiner, and F. E. Cohen.
2000.
Self-assembly of recombinant prion protein of 106 residues
Biochemistry.
39:2792-2804[CrossRef][Medline].
|
| 2.
|
Bessen, R. A., and R. F. Marsh.
1992.
Biochemical and physical properties of the prion protein from two strains of the transmissible mink encephalopathy agent.
J. Virol.
66:2096-2101[Abstract/Free Full Text].
|
| 3.
|
Caughey, B. W.,
A. Dong,
K. S. Bhat,
D. Ernst,
S. F. Hayes, and W. S. Caughey.
1991.
Secondary structure analysis of the scrapie-associated protein PrP 27-30 in water by infrared spectroscopy.
Biochemistry
30:7672-7680[CrossRef][Medline].
|
| 4.
|
Collinge, J.,
K. C. L. Sidle,
J. Meads,
J. Ironside, and A. F. Hill.
1996.
Molecular analysis of prion strain variation and the aetiology of "new variant" CJD.
Nature
383:685-690[CrossRef][Medline].
|
| 5.
|
Gabizon, R.,
Z. Meiner,
C. Cass,
E. Kahana,
I. Kahana,
D. Avrahami,
O. Abramsky,
G. Scarlato,
S. B. Prusiner, and K. K. Hsiao.
1991.
Prion protein gene mutation in Libyan Jews with Creutzfeldt-Jakob disease.
Neurology
41:160.
|
| 6.
|
Gambetti, P.,
R. B. Peterson,
P. Parchi,
S. G. Chen,
S. Capellari,
L. Goldfarb,
R. Gabizon,
P. Montagna,
E. Lugaresi,
P. Piccardo, and B. Ghetti.
1999.
Inherited prion diseases, p. 509-583.
In
S. B. Prusiner (ed.), Prion biology and diseases. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
|
| 7.
|
Gasset, M.,
M. A. Baldwin,
D. Lloyd,
J.-M. Gabriel,
D. M. Holtzman,
F. Cohen,
R. Fletterick, and S. B. Prusiner.
1992.
Predicted -helical regions of the prion protein when synthesized as peptides form amyloid.
Proc. Natl. Acad. Sci. USA
89:10940-10944[Abstract/Free Full Text].
|
| 8.
|
Hornemann, S., and R. Glockshuber.
1996.
Autonomous and reversible folding of a soluble amino-terminally truncated segment of the mouse prion protein.
J. Mol. Biol.
262:614-619.
|
| 9.
|
Hornemann, S.,
C. Korth,
B. Oesch,
R. Riek,
G. Wide,
K. Wüthrich, and R. Glockshuber.
1997.
Recombinant full-length murine prion protein, mPrP(23-231): purification and spectroscopic characterization.
FEBS Lett.
413:277-281[CrossRef][Medline].
|
| 10.
|
Hsiao, K.,
Z. Meiner,
E. Kahana,
C. Cass,
I. Kahana,
D. Avrahami,
G. Scarlato,
O. Abramsky,
S. B. Prusiner, and R. Gabizon.
1991.
Mutation of the prion protein in Libyan Jews with Creutzfeldt-Jakob disease.
N. Engl. J. Med.
324:1091-1097[Abstract].
|
| 11.
|
Kaneko, K.,
L. Zulianello,
M. Scott,
C. M. Cooper,
A. C. Wallace,
T. L. James,
F. E. Cohen, and S. B. Prusiner.
1997.
Evidence for protein X binding to a discontinuous epitope on the cellular prion protein during scrapie prion propagation Proc.
Natl. Acad. Sci. USA
94:10069-10074[Abstract/Free Full Text].
|
| 12.
|
Kascsak, R. J.,
R. Rubenstein,
P. A. Merz,
M. Tonna-DeMasi,
R. Fersko,
R. I. Carp,
H. M. Wisniewski, and H. Diringer.
1987.
Mouse polyclonal and monoclonal antibody to scrapie-associated fibril proteins.
J. Virol.
61:3688-3693[Abstract/Free Full Text].
|
| 13.
|
Laemmli, U. K.
1970.
Cleavage of structural proteins during the assembly of the head of bacteriophage T-4.
Nature.
227:680-685[CrossRef][Medline].
|
| 14.
|
Mehlhorn, I.,
D. Groth,
J. Stöckel,
B. Moffat,
D. Reilly,
D. Yansura,
W. S. Willett,
M. Baldwin,
R. Fletterick,
F. E. Cohen,
R. Vandlen,
D. Henner, and S. B. Prusiner.
1996.
High-level expression and characterization of a purified 142-residue polypeptide of the prion protein.
Biochemistry
35:5528-5537[CrossRef][Medline].
|
| 15.
|
Meiner, Z.,
R. Gabizon, and S. B. Prusiner.
1997.
Familial Creutzfeldt-Jakob disease Codon 200 prion disease in Libyan Jews.
Medicine
76:227-237[CrossRef][Medline].
|
| 16.
|
Muramoto, T.,
M. Scott,
F. E. Cohen, and S. B. Prusiner.
1996.
Recombinant scrapie-like prion protein of 106 amino acids is soluble.
Proc. Natl. Acad. Sci. USA
93:15457-15462[Abstract/Free Full Text].
|
| 17.
|
Pan, K.-M.,
N. Stahl, and S. B. Prusiner.
1992.
Purification and properties of the cellular prion protein from Syrian hamster brain.
Protein Sci.
1:1343-1352[Medline].
|
| 18.
|
Pergami, P.,
H. Jaffe, and J. Safar.
1996.
Semipreparative chromatographic method to purify the normal cellular isoform of the prion protein in nondenatured form.
Anal. Biochem.
236:63-73[CrossRef][Medline].
|
| 19.
|
Prusiner, S. B.
1994.
Inherited prion diseases.
Proc. Natl. Acad. Sci. USA
91:4611-4614[Free Full Text].
|
| 20.
|
Prusiner, S. B.
1982.
Novel proteinaceous infectious particles cause scrapie.
Science
216:136-144[Abstract/Free Full Text].
|
| 21.
|
Prusiner, S. B.
1996.
Prion diseases, p. 855-911.
In
N. Nathanson, R. Ahmed, F. Gonzalez-Scarano, D. Griffin, K. Holmes, F. A. Murphy, and H. L. Robinson (ed.), Viral pathogenesis. Raven Press, New York, N.Y.
|
| 22.
|
Prusiner, S. B.
1997.
Prion diseases and the BSE crisis.
Science
278:245-251[Abstract/Free Full Text].
|
| 23.
|
Prusiner, S. B.,
M. P. McKinley,
K. A. Bowman,
D. C. Bolton,
P. E. Bendheim,
D. F. Groth, and G. G. Glenner.
1983.
Scrapie prions aggregate to form amyloid-like birefringent rods.
Cell
35:349-358[CrossRef][Medline].
|
| 24.
|
Safar, J.,
P. P. Roller,
D. C. Gajdusek, and C. J. Gibbs, Jr.
1993.
Conformational transitions, dissociation, and unfolding of scrapie amyloid (prion) protein.
J. Biol. Chem.
268:20276-20284[Abstract/Free Full Text].
|
| 25.
|
Scott, M.,
D. Foster,
C. Mirenda,
D. Serban,
F. Coufal,
M. Wälchli,
M. Torchia,
D. Groth,
G. Carlson,
S. J. DeArmond,
D. Westaway, and S. B. Prusiner.
1989.
Transgenic mice expressing hamster prion protein produce species-specific scrapie infectivity and amyloid plaques.
Cell
59:847-857[CrossRef][Medline].
|
| 26.
|
Scott, M.,
D. Groth,
D. Foster,
M. Torchia,
S.-L. Yang,
S. J. DeArmond, and S. B. Prusiner.
1993.
Propagation of prions with artificial properties in transgenic mice expressing chimeric PrP genes.
Cell
73:979-988[CrossRef][Medline].
|
| 27.
|
Scott, M. R.,
R. Köhler,
D. Foster, and S. B. Prusiner.
1992.
Chimeric prion protein expression in cultured cells and transgenic mice.
Protein Sci.
1:986-997[Medline].
|
| 28.
|
Stahl, N.,
M. A. Baldwin,
D. B. Teplow,
L. Hood,
B. W. Gibson,
A. L. Burlingame, and S. B. Prusiner.
1993.
Structural analysis of the scrapie prion protein using mass spectrometry and amino acid sequencing.
Biochemistry
32:1991-2002[CrossRef][Medline].
|
| 29.
|
Supattapone, S.,
P. Bosque,
T. Muramoto,
H. Wille,
C. Aagaard,
D. Peretz,
H.-O. B. Nguyen,
C. Heinrich,
M. Torchia,
J. Safar,
F. E. Cohen,
S. J. DeArmond,
S. B. Prusiner, and M. Scott.
1999.
Prion protein of 106 residues creates an artificial transmission barrier for prion replication in transgenic mice.
Cell
96:869-878[CrossRef][Medline].
|
| 30.
|
Supattapone, S.,
H.-O. B. Nguyen,
F. E. Cohen,
S. B. Prusiner, and M. R. Scott.
1999.
Elimination of prions by branched polyamines and implications for therapeutics.
Proc. Natl. Acad. Sci. USA
96:14529-14534[Abstract/Free Full Text].
|
| 31.
|
Telling, G. C.,
P. Parchi,
S. J. DeArmond,
P. Cortelli,
P. Montagna,
R. Gabizon,
J. Mastrianni,
E. Lugaresi,
P. Gambetti, and S. B. Prusiner.
1996.
Evidence for the conformation of the pathologic isoform of the prion protein enciphering and propagating prion diversity.
Science
274:2079-2082[Abstract/Free Full Text].
|
| 32.
|
Wille, H.,
G.-F. Zhang,
M. A. Baldwin,
F. E. Cohen, and S. B. Prusiner.
1996.
Separation of scrapie prion infectivity from PrP amyloid polymers.
J. Mol. Biol.
259:608-621[CrossRef][Medline].
|
Journal of Virology, December 2000, p. 11928-11934, Vol. 74, No. 24
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Chiesa, R., Piccardo, P., Quaglio, E., Drisaldi, B., Si-Hoe, S. L., Takao, M., Ghetti, B., Harris, D. A.
(2003). Molecular Distinction between Pathogenic and Infectious Properties of the Prion Protein. J. Virol.
77: 7611-7622
[Abstract]
[Full Text]
-
Montalvetti, A., Bailey, B. N., Martin, M. B., Severin, G. W., Oldfield, E., Docampo, R.
(2001). Bisphosphonates Are Potent Inhibitors of Trypanosoma cruzi Farnesyl Pyrophosphate Synthase. J. Biol. Chem.
276: 33930-33937
[Abstract]
[Full Text]
Top
Abstract
Introduction
