Download Identification of proteins co-purifying with scrapie infectivity

J O U RN A L OF P R O TE O MI CS 7 2 (2 0 0 9 ) 6 9 0–6 9 4
a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / j p r o t
Identification of proteins co-purifying with scrapie infectivity
S. Petrakis a,1 , A. Malinowska b , M. Dadlez b , T. Sklaviadis a,⁎
a
Prion Disease Research Group, Laboratory of Pharmacology, Department of Pharmaceutical Sciences, Aristotle University of Thessaloniki,
54124, Thessaloniki, Greece
b
Mass Spectrometry Laboratory, Institute of Biochemistry and Biophysics, Polish Academy of Science, Pawinskiego 5a, 02 106 Warsaw, Poland
AR TIC LE D ATA
ABSTR ACT
Article history:
PrPC, the cellular isoform of prion protein, is widely expressed in most tissues. Despite its
Received 18 September 2008
involvement in several bioprocesses it still has no apparent physiological role. During
Accepted 19 January 2009
propagation of Transmissible Spongiform Encephalopathies, PrPC is converted to the
pathological isoform, PrPSc, in a process believed to be mediated by unknown host factors.
Keywords:
PrPSc has altered biochemical properties and forms amyloid aggregates that display
Prion protein
infectious characteristics. PrPSc is also the major component in biochemically enriched
2-DE
infectious samples. Other molecules co-purify with it, but the protein content of these
LC-MS/MS
aggregates remains unknown. The goal of this project was to identify other host molecules
with high affinity for PrPSc. Here, we present the identification of protein molecules that copurify with PrPSc isolated from naturally scrapie-infected ovine brain tissue. The infectious
preparations were analyzed by two-dimensional gel electrophoresis and unknown proteins
were identified by LC-MS/MS. These proteins may prove to be strategic targets for
prevention and therapy of prion diseases.
© 2009 Elsevier B.V. All rights reserved.
1.
Introduction
Transmissible Spongiform Encephalopathies (TSE) are a group
of fatal neurodegenerative diseases characterized by the
formation of amyloid aggregates and vacuolation of brain
tissue. They are caused by the conversion of a physiological
cellular protein, PrPC, to an insoluble and infectious isoform,
termed PrPSc [1]. The conversion mechanism remains undefined, but it is proposed to be mediated by as yet unknown
host-encoded factors [2]. PrPSc represents the main component of the infectious agent and has the ability to accumulate
in extracellular fibrillar aggregates.
PrPC, the physiological prion protein is attached to the
outer cell membrane via a glycosylphosphatidylinositol (GPI)
anchor. Even though it is expressed in most tissues [3,4], still it
has no definite biological function. Several attempts have
been made to identify molecules that interact with PrPC in
order to understand its function and the mechanism of its
conversion to PrPSc [5–8]. However, little is known concerning
proteins that co-purify with TSE aggregates.
In this study we tried to identify proteins in highly-enriched
infectious preparations, using a proteomic approach. Proteins
associated with the pathological isoform of PrP, originating
from scrapie-infected brain tissue, were identified by the
combination of two-dimensional gel electrophoresis (2-DE)
Abbreviations: TSE, transmissible spongiform encephalopathies; GPI, glycosylphosphatidylinositol; ECM, extracellular matrix; IEF,
isoelectric focusing; PK, Proteinase K; BRAL1, brain link protein-1; DSG1, desmoglein1; JUP, plakoglobin; CAMK2A, alpha subunit of the
calcium/calmodulin-dependent protein kinase 2; FTL and FTH, ferritin light and heavy chain; PG, proteoglycan.
⁎ Corresponding author. Laboratory of Pharmacology, School of Pharmaceutical Sciences, Aristotle University of Thessaloniki, 54124
Thessaloniki, Greece. Tel.: +302310997615; fax: +302310997720.
E-mail address: [email protected] (T. Sklaviadis).
1
Current address: Max-Delbrück-Center for Molecular Medicine (MDC), Department of Neuroproteomics, Robert-Rössle-Str. 10, 13125,
Berlin-Buch, Germany.
1874-3919/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.jprot.2009.01.025
J O U RN A L OF P R O TE O MI CS 72 ( 20 0 9 ) 6 9 0–6 9 4
and mass spectrometry. The results showed that amyloid
aggregates contain components of the extracellular matrix
(ECM) and proteins related to it. The results presented here
suggest that these molecules correlate with prion infectivity
and may participate in the pathogenesis of TSE.
2.
Materials and methods
2.1.
PrPSc enrichment
Ovine cortex from normal or scrapie infected sheep were
kindly provided by Dr. P. Toumazos (Veterinary Services
Laboratory, Cyprus). The tissues were used for PrPSc enrichment, as previously described [9]. In brief, 500 mg brain tissue
was homogenized in nine volumes of 10% w/v Sarkosyl, 25 mM
Tris–Cl pH 7.6 with the addition of 0.5 mM PMSF. Homogenization was performed using a polytron homogenizer
(Kinematica, Switzerland) at setting 3, for 3 × 15 s at room
temperature. The homogenates were centrifuged at 25 × 103 g
for 20 min at 4 °C. The supernatants were transferred to
polyallomer tubes and ultracentrifuged at 215 × 103 g for 2 h
30 min at 20 °C. The ultracentrifugation pellet was resuspended in Buffer A (10% w/v NaCl, 0.05% w/v Sarkosyl, 25 mM
Tris–Cl pH 7.6) overnight at room temperature. The resuspension was centrifuged at 20 × 103 g for 30 min and was washed
twice in Buffer B (0.05% Sarkosyl, 25 mM Tris–Cl pH 7.6). The
final pellet was treated with or without Proteinase K (PK) and
analyzed by electrophoresis.
2.2.
Proteinase K treatment
The final pellet was resuspended in 100 µl Buffer B and treated
with 30 µg/ml PK for 1 h at 37 °C with constant shaking
(800 rpm/min). PMSF was added (0.5 mM), followed by
centrifugation at 20 × 103 g for 30 min. The pellet was
resuspended in 500 µl Buffer A for 2 h at room temperature
and then centrifuged at 20 × 103 g for 30 min. The final pellet
was washed twice in Buffer B before analyzing in twodimensional electrophoresis.
2.3.
Two-dimensional gel electrophoresis
The final pellet was resuspended in 6 M Urea, 2 M Thiurea, 4%
w/v CHAPS, 60 mM DTT, 2% ampholytes pH 3–10, 0.1% w/v
Bromophenol Blue and sonicated in a bath sonicator for 5 min.
For isoelectric focusing (IEF) using immobilized pH gradient
(IPG) strips in the first dimension, precast gels with a nonlinear pH range of 3–10 (Zoom, Invitrogen) were reswollen for
16 h in 180 µl of the resuspended sample. IEF was carried out at
room temperature for 20 min at 200 V, 15 min at 450 V, 15 min
at 750 V and 105 min at 2000 V for, using a cooled
electrophoresis unit (Zoom IPG runner, Invitrogen). For the
second dimension, IPG strips were equilibrated for 15 min in
6 M Urea, 2 M Thiurea, 60 mM DTT Invitrogen LDS sample
buffer, followed by equilibration for 15 min in 6 M Urea, 2 M
Thiurea, 2.5% w/v Iodoacetamide Invitrogen LDS sample
buffer. The strips were placed on 4–12% gradient NuPAGE
691
Novex Bis–Tris gels and proteins were analyzed at 200 V for 1 h
in MES buffer.
2.4.
Silver protein staining
Silver protein staining was performed as previously described
[10]. In brief, proteins were fixed in the gel with 50% v/v
methanol, 5% v/v acetic acid for 1 h, followed by incubation
with 50% v/v methanol for 15 min and dH2O for another
15 min. After fixation, the gels were immersed in 0.02% w/v
sodium thiosulfate for 3 min and washed with dH2O for
2 × 1 min. The gels were then incubated with 0.15% w/v silver
nitrate for 45 min. After 2 × 1 min washes in dH2O, proteins
were visualized with 0.04% v/v formaldehyde, 2% w/v sodium
bicarbonate. The reaction was quenched with 5% v/v acetic
acid and the gels were stored in 10% v/v methanol, 1% v/v
acetic acid.
2.5.
Western blotting
Proteins were electrotransferred onto nitrocellulose (NC)
membranes (Pall Life Sciences) at 100 V for 2 h. The
membranes were blocked with 5% w/v BSA (Sigma-Aldrich)
in PBST for 1 h at room temperature. Prion protein was
detected with anti-PrP mAb 6H4 (Prionics, 1:3000 in PBST)
overnight at 4 °C. The membrane was washed in PBST and
incubated with rabbit anti-mouse alkaline-phosphatase conjugated secondary antibody (Pierce, 1:5000 in PBST), for 1 h at
room temperature. After washes with PBST, blots were
developed with Nitro Blue Tetrazolium/5-Bromo-4-Chloro-3Indolyl Phosphate disodium salt (NBT/BCIP) (Sigma-Aldrich).
For non-specific staining NC membranes were stained with
Aurodye (Amersham Pharmacia) according to manufacturer's
instructions.
2.6.
LC-MS/MS analysis and protein identification
Proteins co-purifying with PrPSc were analyzed in 2-DE and
stained with silver solution. Prior to analysis the gel slices
containing the desired spots, were subjected to a standard
procedure during which proteins were reduced with 100 mM
DTT for 30 min at 56 °C, alkylated with iodoacetamide in
darkness for 45 min at room temperature and digested
overnight with trypsin. The resulting peptides were eluted
from the gel with 0.1% TFA and 2% ACN. Peptide mixtures
were applied to an RP-18 pre-column (Waters) using 0.1% TFA
in dH2O as mobile phase and then transferred to a nano-HPLC
(NanoACQUITY UPLC, Waters) RP-18 column (75 µM i.d,
Waters.) with an acetonitrile gradient (0%–30% AcN in
45 min) in the presence of 0.1% formic acid (flow rate 250 nl/
min). The column outlet was directly coupled to the ESI
(Finningan Nanospray, Thermo) ion source of the LTQ-FT
(Thermo) mass spectrometer working in the regime of data
dependent MS to MS/MS switch. A blank run ensuring lack of
cross contamination from previous samples preceded each
analysis. Source voltage and current were set to 1.6 kV and
100 µA, respectively. The capillary voltage was 42 V and tube
lens 120 V. FTMS resolution was set to 12,500 and m/z values
from range 300–2000 Th were acquired. Minimal signal
required to trigger MS to MS/MS switch was set to 500,
692
J O U RN A L OF P R O TE O MI CS 7 2 (2 0 0 9 ) 6 9 0–6 9 4
activation Q was 0.250 and normalized collision energy was 35.
The spectrometer was working in positive polarity mode and
charge states accepted for sequencing were 2+, 3+ and 4+.
Dynamic ion exclusion was enabled and exclusion time was
set to 300 s.
After preprocessing of the raw data with Mascot Distiller
software (version 2.1.1, Matrix Science), output lists of
precursor and product ions were compared to National Center
for Biotechnology non-redundant (NCBInr) database (containing 3695564 sequences; 1269795892 residues) using Mascot
database search engine (version 2.1, Matrix Science). Search
parameters included semi-trypsin enzyme specificity, one
missed cleavage site, cysteine carbamidomethyl fixed modification and variable modifications including methionine
oxidation and lysine carbamidomethylation. Protein mass
and taxonomy were unrestricted, peptide mass tolerance was
200 ppm and the MS/MS tolerance was 0.8 Da. Proteins
containing peptides with Mascot cut-off scores > 50, indicating
identity or extensive homology (p < 0.05) of peptide, were
considered positive identifications.
3.
Results and discussion
In order to identify novel molecules that may participate in TSE
pathogenesis, scrapie-infected ovine brain tissue was homogenized and enriched in the pathological isoform of prion
protein, PrPSc. The final preparation, a pellet obtained after
ultracentrifugation at 215 × 103 g for 2 h 30 min, contains almost
95% of the infectivity and displays unique infectious characteristic [9]. The presence of PrPSc was verified by western
blotting (Fig. 1A). Additionally, the same preparation was
stained with non-specific protein staining solution (Fig. 1B)
which showed that these samples are highly enriched in the
pathological isoform.
In order to identify the unknown proteins that, together
with PrPSc, precipitated at 215 × 103 g, samples were digested
with PK and analyzed in a 2-DE. The protein content of the
sample (5 times higher than the amount used for 1-DE gel
electrophoresis) was focused in non-linear pH 3–10 IPG strips
Fig. 1 – PrPSc enrichment in scrapie-infected brain preparations.
Ultracentrifugation pellets were prepared as described in
Materials and methods and enriched in the infectious isoform,
PrPSc. Samples (100 mg b.e.) were treated with or without
Proteinase K and then probed for either A. PrPSc with anti-PrP
mAb 6H4 or B. other proteins present in the preparation, using
non-specific protein staining solution.
Fig. 2 – 2D gel electrophoresis and non-specific protein
staining of ultracentrifugation pellets. PrPSc enriched brain
preparations (500 mg b.e.) were digested with PK, analyzed in
a two-dimensional gel electrophoresis and stained with
mass-spectrometry compatible silver staining solution. The
proteins indicated with numbers were excised form the gel
and identified by LC-MS/MS.
before separation in a pre-cast gradient 4–12% NuPAGE gel.
After 2-DE analysis, proteins were stained with mass-spectrometry compatible silver nitrate solution. Control experiments
were performed in brain preparations originating from
healthy individuals, in which a significantly lower amount
and pattern of protein was detected (Supplementary Fig. 1).
However, the pellets resulting from healthy and infected
individuals have totally different biochemical features and are
not comparable. As shown in Fig. 2, several proteins (including
PrPSc) were detected in the infectious preparation after PK
treatment. Experiments were performed three times and in
each case the observed protein pattern was reproducible. As
this pellet contains the major infectivity components, we were
interested in identifying most of the molecules that participate in its formation and are good candidates to be part of the
infectious agent. The molecules indicated by numbers were
manually excised from the gel and analyzed by LC-MS/MS
(Table 1). Proteins were mainly identified by similarities with
homologous proteins from other species, as the ovine genome
is not completely sequenced and many ovine proteins are not
available in the database. The mass spectrometry score for
each molecule was calculated based on the ion score of the
peptides identified and attributed to the respective protein. It
is important to note that the electrophoretic mobility of most
spots was not consistent with the expected molecular weight
of the identified proteins, as the sample was treated with PK
before analysis, possibly resulting in partial fragmentation of
the molecules. In three of the spots we identified various types
of collagen (spots 1, 3 and 5). Additionally, we identified
components of the desmosomes, such as desmoglein 1 (DSG1,
spots 2 and 3) and plakoglobin (JUP, spot 2). The infectious
pellet also contained ubiquitin (spot 9), versican V3, a splicing
variant of a large chondroitin sulfate proteoglycan (spot 4) and
brain link protein-1 (BRAL1), a protein which is directly related
to versican (spot 9). In spots 6–8 we identified the alpha
subunit of the calcium/calmodulin-dependent protein kinase
2 (CAMK2A, spot 8), the light and heavy chains of ferritin (FTL
and FTH, spots 6 and 8, respectively) and prion protein, as
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Table 1 – Proteins identified in PrPSc enriched pellets.
Spot
Identified
proteins
NCBI accession
code
Protein
score
Matching
peptides
Peptide sequence
Mass
m/z
(charge)
1
2
COL6A1
DSG1
gi|6753484
gi|4503401
58
126
1
3
2
3
JUP
COL1A2
gi|1122889
gi|27806257
91
147
1
2
3
DSG1
gi|55647309
105
2
4
Versican V3
gi|3253306
268
4
5
6
COL1A2
FTL
gi|27806257
gi|42564199
76
173
1
2
6
7
8
PrP
PrP
CAMK2A
gi|2330622
gi|2330622
gi|4836795
86
82
242
1
1
3
8
8
9
PrP
FTH
BRAL1
gi|2330622
gi|1305505
gi|11141887
92
50
158
1
1
2
9
Ubiquitin
gi|161281
91
1
IALVITDGR
EMQDLGGGER + Ox(M)
YQGTILSIDDNLQR
YVMGNNPADLLAVDSR + Ox(M)
ALMGSPQLVAAVVR + Ox(M)
GPSGPQGIR
GEAGPAGPAGPAGPR
QEPSDSPMFIINR + Ox(M)
VGDFVATDLDTGRPSTTVR
LLASDAGR
YTLNFEMAQK
LATVGELQAAWR
VSVPTHPEDVGDASLTMVK
VSVPTHPEDVGDASLTMVK + Ox(M)
GEAGPAGPAGPAGPR
TQDAMEAALLVEK + Ox(M)
QNYSTEVEAAVNR
YPNQVYYR
GENFTETDIK
NTTIEDEDTK
TIEDEDTKVR
ITQYLDAGGIPR
GENFTETDIK
QNYHQDSEAAINR
LDASLVIAGVR
LEGVVFPYQPSR
TITLEVEPSDTIENVK
956.5655
1106.4662
1634.8264
1749.8356
1426.7966
867.4563
1260.6211
1548.7242
2006.0069
801.4345
1243.5907
1313.7092
1980.9826
1996.9776
1260.6211
1433.7072
1479.6954
1101.5243
1152.5299
1164.5146
1204.5936
1302.6932
1152.5299
1544.6968
1112.6554
1390.7245
1786.92
479.294 (+ 2)
554.224 (+ 2)
818.434 (+ 2)
875.954 (+ 2)
714.413 (+ 2)
434.738 (+ 2)
631.326 (+ 2)
775.383 (+ 2)
669.683 (+ 3)
401.727 (+ 2)
622.809 (+ 2)
657.868 (+ 2)
661.335 (+ 3)
666.674 (+ 3)
631.323 (+ 2)
717.87 (+ 2)
740.862 (+ 2)
551.773 (+ 2)
577.276 (+ 2)
583.268 (+ 2)
603.309 (+ 2)
652.36 (+ 2)
577.274 (+ 2)
515.91 (+ 3)
557.336 (+ 2)
696.382 (+ 2)
894.485 (+ 2)
Sc
Table shows PrP associated proteins which were identified in each spot after LC-MS/MS analysis. It also indicates the protein score, the number
of matching peptides used for protein identification, the sequence, mass, m/z values and charge of each peptide.
expected. The identification of more than one protein in some
spots is possibly due to sample overlapping, especially in the
lower molecular weight area of the gel.
The goal of this project was to identify proteins that copurify with the infectious isoform of prion protein in PrPSc
enriched brain preparations. Despite the fact that several
ligands of the physiological prion protein, PrPC, have already
been identified, the protein composition of TSE aggregates
remains unknown. These aggregates display unique infectious characteristics, as they can effectively transmit the
disease to healthy individuals. The results showed that the
infectious preparation contains mostly proteins of the ECM,
such as various types of collagen, proteoglycans (versican V3)
and molecules related to them (BRAL1). Additionally, we
identified components of the desmosomes (DSG1, JUP),
ubiquitin, ferritin and CAMK2A.
To minimize non-specific protein isolation, we used very
stringent conditions, washing the pellet extensively with
buffers containing high concentration of salts (10 w/v NaCl).
However, there is always a possibility that some of these
molecules may not specifically associate with PrPSc, but
coincidentally co-purify with it because of the experimental
procedure we followed. The identification of glycoprotein
binding molecules (versican V3 and BRAL1) or CAMK2A, which
is involved in ER stress under neurodegenerative conditions
[11] indicates the specificity of PrPSc co-purifying proteins. The
detection of ubiquitin in prion aggregates provides another
piece of evidence, as protein aggregates involved in most
neurodegenerative diseases are highly ubiquitinated [12]. It
also has to be mentioned that this fraction is one of the most
extensively purified infectivity preparations that is reported in
TSE field. Thus, the presence of the identified proteins in this
preparation makes them candidate molecules directly associated with prion infectivity.
ECM consists mainly of glycosylated proteins, such as
collagen, proteoglycans (PGs) and hyaluronic acid. It is located
in the space between cells and has multiple roles. It provides
the cells with the essential mechanical support, as it is
connected through the integrins to the cytoskeleton. The
whole system requires the presence of the desmosomes and
hemidesmosomes which allow intercellular connections and
stabilize the cells to the ECM, respectively. ECM also regulates
cellular communication and activity of the ion channels
located in the cell membrane. The regulation mechanism
requires the interaction of PGs with membrane glycoproteins,
which activate intracellular phosphorylation systems, such as
protein kinase II [13].
The PGs located in the ECM seem to be involved in
Alzheimer's disease, as they can act as seed cores for the
formation of amyloids [14]. Additionally, heparan sulfate, the
most abundant proteoglycan of ECM, accumulates in the
amyloid plaques of Gerstmann–Straussler syndrome disease
patients [15] and is directly related to prion infectivity [16]. It is
therefore, possible that proteins such as versican V3 and
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J O U RN A L OF P R O TE O MI CS 7 2 (2 0 0 9 ) 6 9 0–6 9 4
BRAL1, which were identified in the infectious preparations,
participate in the conversion of PrPC to PrPSc and the
propagation of prions to the central nervous system.
Identification of ferritin in the infectious preparation is also
quite interesting. Experimental data show that ferritin and
PrPSc form complexes and are co-transported through receptors to epithelial cells of the gastrointestinal tract [17].
Hypothetically, a similar mechanism could also exist for the
transportation of the infectious isoform to the CNS and the
crossing of the blood brain barrier.
In summary, the experimental data presented in this paper
show that the infectious isoform of prion protein is directly
related to ECM and proteins associated with it. These proteins
may prove to be therapeutical targets for the prevention of
prion diseases. Further experiments are needed in order to
study in detail their potential participation in the conversion
mechanism of PrPC to PrPSc and pathogenesis of TSE.
Acknowledgments
The authors wish to thank Dr. C. H. Panagiotidis (CERTH-INA)
for the revision of the manuscript. This work was supported by
the European Union (FOOD-CT-2004-506579 NEUROPRION),
the Greek Ministry of Development (IRAKLEITOS) and the
Bodossaki Foundation and is a part of S. P.'s thesis.
Appendix A. Supplementary data
Supplementary data associated with this article can be found,
in the online version, at doi:10.1016/j.jprot.2009.01.025.
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