Proteomic Identification of Protein Targets for 15-Deoxy-Δ12,14-Prostaglandin J2 in Neuronal Plasma Membrane
14-Prostaglandin J2 in
Neuronal Plasma Membrane. PLoS ONE 6(3): e17552. doi:10.1371/journal.pone.0017552
Proteomic Identification of Protein Targets for 15-Deoxy- 12,14 D -Prostaglandin J2 in Neuronal Plasma Membrane
Howard Gendelman, University of Nebraska, United States of America
15-deoxy-D12,14-prostaglandin J2 (15d-PGJ2) is one of factors contributed to the neurotoxicity of amyloid b (Ab), a causative protein of Alzheimer's disease. Type 2 receptor for prostaglandin D2 (DP2) and peroxysome-proliferator activated receptorc (PPARc) are identified as the membrane receptor and the nuclear receptor for 15d-PGJ2, respectively. Previously, we reported that the cytotoxicity of 15d-PGJ2 was independent of DP2 and PPARc, and suggested that 15d-PGJ2 induced apoptosis through the novel specific binding sites of 15d-PGJ2 different from DP2 and PPARc. To relate the cytotoxicity of 15d-PGJ2 to amyloidoses, we performed binding assay [3H]15d-PGJ2 and specified targets for 15d-PGJ2 associated with cytotoxicity. In the various cell lines, there was a close correlation between the susceptibilities to 15d-PGJ2 and fibrillar Ab. Specific binding sites of [3H]15d-PGJ2 were detected in rat cortical neurons and human bronchial smooth muscle cells. When the binding assay was performed in subcellular fractions of neurons, the specific binding sites of [3H]15d-PGJ2 were detected in plasma membrane, nuclear and cytosol, but not in microsome. A proteomic approach was used to identify protein targets for 15d-PGJ2 in the plasma membrane. By using biotinylated 15d-PGJ2, eleven proteins were identified as biotin-positive spots and classified into three different functional proteins: glycolytic enzymes (Enolase2, pyruvate kinase M1 (PKM1) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH)), molecular chaperones (heat shock protein 8 and Tcomplex protein 1 subunit a), cytoskeletal proteins (Actin b, F-actin-capping protein, Tubulin b and Internexin a). GAPDH, PKM1 and Tubulin b are Ab-interacting proteins. Thus, the present study suggested that 15d-PGJ2 plays an important role in amyloidoses not only in the central nervous system but also in the peripheral tissues.
Competing Interests: JK, NO, TS, MF and TY are employed by Shionogi and Co., Ltd. There are no patents, products in development or marketed products to
declare. This does not alter the authors adherence to all the PLoS ONE policies on sharing data and materials.
Eicosanoids are divided into two groups, according to their
mechanism of action: the conventional eicosanoids, e.g.,
prostaglandin D2 (PGD2) and the cyclopentenone-type PGs, e.g.,
15-deoxyD12,14-PGJ2 (15d-PGJ2). PGD2 has been considered to be a
proinflammatory mediator in inflammatory diseases such as
Alzheimers disease (AD) and Asthma. In AD, PGD2 formation increased
in the frontal cortex of the patients when compared with those of the
healthy subjects . AD is characterized pathologically by cortical
atrophy, neurodegeneration and deposits of amyloid protein in the
various regions of brain such as cerebral cortex . Amyloid b (Ab)
generated PGD2 from cortical neurons before inflammation .
However, the toxicity of PGD2 via its GTP-binding protein-coupled
PGD2 receptors does not occur. First, the PGD2 receptor blocker
did not inhibit PGD2-induced neuronal cell death . Second, little
mRNA of the PGD2 receptor is observed in the rat  and human
 cerebral cortex. Third, few binding sites of [3H]PGD2 were
detected in the plasma membranes from rat cortices . Fourth, the
extent of specific [3H]PGD2 in total biding is much lower (3040%)
than that of [3H]15d-PGJ2 (.80%), although binding sites of PGD2
have been reported in synaptosomes of rat  and human brains
. Fifth, the LD50 value (8.2 mM) of PGD2 is much higher than the
affinity for PGD2 receptor (dissociation constant = 14 nM) .
Finally, PGD2 required a latent time to exert toxicity. PGD2 was
non-enzymatically metabolized to prostaglandin J2 (PGJ2),
D12PGJ2 and 15d-PGJ2 . Among PGD2 metabolites, 15d-PGJ2
exhibited most potent inflammatory effects . Taken together,
PGD2 appeared to mediate inflammation via 15d-PGJ2 in the
The surface receptors specific for 15d-PGJ2 have not been
identified, and 15d-PGJ2 is believed to be actively transported into
cells. It possesses an a, b-unsaturated carbonyl group in the
cyclopentane ring that can form covalent adducts with free thiols
in proteins by Michael addition. 15d-PGJ2 covalently binds to
Cys285 of its nuclear receptor , peroxysome-proliferator
activated receptorc (PPARc) , . Recently, 15d-PGJ2 has
been implicated in the antiproliferation independently from
PPARc. Moreover, 15d-PGJ2 inhibits the NF-kBdependent
gene expression through the covalent modification at Cys179 in
IkB kinase . Previously, we have found the novel binding sites
of 15d-PGJ2 on the cell surface . [3H]15d-PGJ2 bound
specifically to plasma membranes of cortical neurons. Among
PGD2 metabolites, 15d-PGJ2 exhibited the highest affinity for the
specific binding sites. Other eicosanoids and PPAR agonists did
not affect the specific binding sites. 15d-PGJ2 regulated cell
numbers in primary cultures of rat cortical neurons. The
neurotoxicity of 15d-PGJ2 was the most potent among PGD2
and its metabolites, whereas little effect of other eicosanoids and
PPAR agonists was detected. In peripheral tissues, 15d-PGJ2 also
exhibited toxicity independently of PPARc. In response to basic
fibroblast growth factor, bronchial smooth muscle cells (BSMC)
proliferate and remodel airway in asthma . 15d-PGJ2 inhibits
proliferation in a PPARc-independent manner. Thus, the
identification of cell surface targets for 15dPGJ2 is required to clear
how 15d-PGJ2 induces cell toxicity and involves in amyloidoses.
In the present study, we identified cell surface targets for
15dPGJ2 in cortical neurons. In general, glycolytic enzymes, molecular
chaperones and cytoskeletone identified as membrane targets for
15d-PGJ2 are known to localize in the cytosol, but their roles on
the cell surface have not been elucidated sufficiently. Here, we
propose hypothetical role of membrane targets for 15d-PGJ2 on
the cell toxicity and amyloidoses.
Materials and Methods
Dulbeccos modified Eagles medium, Leibovitzs L-15 medium,
Roswell Park Memorial Institute 1640 medium, MCDB, CS-C,
trypsin, deoxyribonuclease I, fetal bovine serum (FBS), horse
serum (HS), penicillin, and streptomycin were obtained from
Invitrogen (Carlsbad, CA). Ab (2535) was purchased from
Bachem AG (Bubendorf, Switzerland). [3H]PGD2 (115 Ci/mmol)
and human hepatocytes was purchased from Perkin Elmer Life
Science Products (Boston, MA). Human BSMC and human
dermal fibroblasts were purchased from Lonza (Basel,
Switzerland). PGD2, PGJ2, D12-PGJ2, 15d-PGJ2 and biotinylated
15dPGJ2 were obtained from Cayman Chemicals (Ann Arbor, MI;
Cabru, Milan, Italy). ImmobilineTM DryStrip Gels (pH 310),
Amersham ECL PlusTM Western Blotting Detection Reagents,
were obtained from GE Healthcare Bio-Sciences Corp.
(Piscataway, NJ). Iodoacetamide, dithiothreitol (DTT), ethyleneglycol bis
tetraacetic acid (EGTA) and ATP (disodium salt) were from
Sigma-Aldrich (Milan, Italy). Sequence grade modified trypsin was
purchased from Promega (Madison, WI; Milan, Italy), and
N-(1pyrenyl) iodoacetamide was from Molecular Probes (Eugene, OR).
Horseradish peroxidase-linked antibody against biotine was
obtained from Cell Signaling Technology (Boston, MA). The
protein concentration was measured using the BCA protein assay
reagent obtained from Thermo Fisher Scientific. (Rockford, IL).
All other chemicals were of reagent grade.
All procedures were conducted in accordance with NIH
guidelines concerning the Care and Use of Laboratory Animals
and with the approval of the Animal Care Committee of the Himeji
Dokkyo University. Rat cortical neurons, human BSMC, human
hepatocytes and human dermal fibroblasts were cultured as
previously reported . Cerebral cortices from the cerebral cortex
of day-19 Sprague-Dawley rat embryos were dissociated in isotonic
buffer with 4 mg/ml trypsin and 0.4 mg/ml deoxyribonuclease I.
Cells were plated at a density of 2.56105 cells/cm2 on
poly-L-lysinecoated dishes in conditioning medium, Leibovitzs L-15 medium
supplemented with 5% FBS and 5% horse serum at 37uC in 5%
CO2 and 9% O2. On day 1 after plating, cultures were treated with
0.1 mM arabinosylcytosine C. On day 4, cortical cultures were
immunostained with anti-MAP2 specific for neurons, anti-GFAP
for astrocytes, and anti-microglial antigen (OX-42). Cultures
prepared by this method, consisted of approximately 95% neurons.
Human BSMC were cultured at a density of 3.56103 cells/cm2 on
48-well plates in Molecular, Developmental, and Cellular Biology
medium supplemented with 5% FBS, 50 mg/ml gentamicin, 50 ng/
ml amphotericin. Human hepatocytes were cultured at a density of
56104 cells/cm2 on 48-well plates in CS-C medium (Applied Cell
Biology Research Institute) supplemented with 10% FBS. Human
dermal fibroblasts were cultured at a density of 56104 cells/cm2 on
48-well plates in DMEM supplemented with 10% FBS, 50 units/ml
penicillin, and 50 mg/ml streptomycin.
Aggregation assessment of fAb
A stock solution of fibrillar Ab (2535) (fAb) was prepared by
dissolving Ab at 1 mM in deionized water and incubating Ab at
37uC for 25 days to aggregate the peptide and stored at 220uC
until use . The aggregation state of fAb was assessed in two
ways. First, light microscopy was used to identify the presence of
precipitated peptides both in stock solutions and after their
addition to tissue culture wells; the observations were confirmed by
three observers. Second, the aggregation state of fAb was assessed
by migration patterns after sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE). Samples of fAb stock solutions
were added to reducing buffer, heated at 100uC for 3 min, and
electrophoresed on 15% SDSPAGE at 70 V.
Two different methods were employed for assessment of cell
viability as previously reported . First, the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide dye (MTT) reduction
assay reflecting mitochondrial succinate dehydrogenase activity
was employed. Second, residual cells were counted according to
morphologic criteria; neurons with intact neurites and a smooth,
round soma were considered viable, whereas those with
degenerated neurites and an irregular soma were considered nonviable.
BSMC with extended cell bodies and their bright phase-contrast
appearance were considered viable, whereas those with shrank
and round cell bodies were considered nonviable.
Cell fractionation was performed as previously reported .
Cerebral cortices from rat brains were homogenized in 3 volumes of
ice-cold STEA solution (0.25 M sucrose, 5 mM Tris-HCl (pH 7.5),
1 mM EGTA and 50 karikllein units/ml aprotinin). The
homogenate was filtered through three meshes and centrifuged at 7006g
for 10 min. Fractionations of nuclear and plasma membrane; The
pellet was resuspended in 120 ml of STEA solution by gentle
homogenization, and the resuspension was dispersed in 1080 ml of
isosmotic Percoll solution (15.7% Percoll, 0.25 M sucrose, 1 mM
EGTA, 50 karikllein units/ml aprotinin and 10 mM Tris HCI
(pH 7.5)). The mixture was centrifuged at 35,0006g for 30 min.
The resulting pellet was suspended in HEA solution (50 mM
HepesNaOH (pH 7.4), 1 mM EGTA and 50 kIU/ml aprotinin) as the
nuclear fraction. On the other hand, the second band from the
surface in the supernatant was collected, washed by dilution with 2
3 volumes of HEA solution and centrifuged at 10,0006g for 30 min.
The pellet was suspended in HEA solution as the plasma membrane
fraction and stored in liquid nitrogen until used . Fractionations
of cytosol and microsome: The supernatant was centrifuged at
7,0006g for 10 min. The resulting supernatant was recentrifuged at
100,0006g for 1 h. The pellet was used as the microsomal fraction.
The supernatant was used as the cytosolic fraction.
Binding assay of [3H]15d-PGJ2
Binding assay of [3H]15d-PGJ2 were performed as previously
reported . The standard reaction mixture of 10 nM
[3H]15dPGJ2 contained 50 mM Tris-HCl buffer (pH 8.0), 100 mM NaCl
and plasma membranes (10 mg) in a total volume of 100 ml.
Incubation was initiated by addition of the reaction mixture to
plasma membranes, and was carried out at 4uC for 24 h. We
determined non-specific binding by performing incubations with
[3H]15d-PGJ2 in the presence of 100 mM unlabeled 15d-PGJ2.
The specific binding was calculated by subtraction of the
nonspecific binding from the total binding. Data are expressed as
means 6 standard error of the mean (S.E.M.) values (n = 4).
Protein separation by two-dimensional electrophoresis
Membrane preparation and binding assay of biotinylated
15dPGJ2 were conformed to Binding assay of [3H]15d-PGJ2. The
standard reaction mixture of 1 mM biotinylated 15d-PGJ2 contained
50 mM Tris-HCl buffer (pH 8.0), 100 mM NaCl and plasma
membranes (400 mg) in a total volume of 4 ml. Incubation was
initiated by addition of the reaction mixture to plasma membranes,
and was carried out at 4uC for 24 h in the presence or absence of
unlabeled 15d-PGJ2. We determined non-specific binding by
performing incubations with biotinylated 15d-PGJ2 in the presence
of 100 mM unlabeled 15d-PGJ2. According to the method of Toda
and Kimura , two-dimensional electrophoresis was performed
with the CoolPhoreStar Horizontal Gel Electrophoresis Unit
IPGIEF (Anatech: Tokyo, JP). The samples containing 400 mg of
membrane lysates were dissolved in a rehydration buffer (5 M urea,
2 M thiourea, 2%(w/v) CHAPS, 2%(w/v) SB3-10, 2% Pharmalytes
and 65 mM DTT) for the first dimensional isoelectric focusing (IEF).
The pH range of the IEF was 310. Before IEF was performed, the
gel strips were incubated with a swelling buffer (6 M urea, 2 M
thiourea, 2%(w/v) TritonX-100, 2%(w/v) SB3-10, 2% Pharmalytes,
2.5 mM acetic acid, 0.0025% BPB and 13 mM DTT). After IEF was
performed, the gel strips were incubated with an SDS buffer (6 M
urea, 32 mM DTT, 2%(w/v) SDS, 0.0025% BPB, 30%(v/v)
glycerol, and 25 mM Tris-HCl pH 6.8) for 10 min, and then with
an alkylation buffer (6 M urea, 243 mM iodoacetamide, 2%(w/v)
SDS, 0.0025% BPB, 30%(v/v) glycerol, and 25 mM Tris-HCl
pH 6.8) for 10 min. For the second dimensional electrophoresis,
polyacrylamide gel (12% acrylamide, 0.4% bis-acrylamide, 10.6%
glycerol, 0.1% SDS, 1.2% APS, 0.1% (v/v) TEMED and 369 mM
Tris-HCl pH 8.8) was used. All procedures followed the
manufacturers protocol. Separated proteins were then fixed in the gel using 1)
40% methanol and 10% acetic acid, 2) 10% methanol and 7% acetic
acid, and 3) 10% methanol and 8% acetic acid. Then, they were
stained with SYPRO Ruby protein gel stain, and scanned using the
FluoroPhoreStarH 3000 (Anatech: Tokyo, JP). The protein spots were
visualized by Progenesis Same Spots (Nonliner Dynamics Ltd:
Newcastle upon Tyne, UK). For immunoblotting, gels were
transferred to polyvinylidene fluoride membranes (Millipore Co.,
Bedford, USA). The membranes were incubated with
phosphatebuffered saline containing 0.1% Tween20 (PBS/Tween) and 5%
skim milk for blocking and washed with PBS/Tween. This procedure
was followed by the addition of horseradish peroxidase-conjugated
anti-biotin antibody and ECL reagents (GE Healthcare
BioSciences). The spots were visualized by LAS-3000 (Aisin Seiki Co.,
Ltd., Aichi, Japan).
Identification of 15d-PGJ2-targeted proteins
Gel pieces were washed in 50 mM ammonium bicarbonic acid
containing 50% acetonitrile for 10 min, twice. Then, they were
dried in block incubator Bl-516S (ASTEC Co., ltd.; Tokyo, JP) at
95uC for 10 min. Each sample was proteolyzed with 10 ml 1 mM
ammonium bicarbonic acid containing 200 ng trypsin overnight at
37uC. The peptide in each gel was extracted with 50% acetonitrile
containing 0.1% TFA followed by sonication for 15 min. The
supernatant was collected, and peptides were further extracted
with 75% acetonitrile containing 0.1% TFA followed by
sonication for 15 min. Peptide extracts were concentrated to
,10 ml using Speedvac concentrator. Then, they were desalted
with Ziptip (Millipore Co.) and mixed with an equal volume of
5 mg a-cyano-4hydroxycinnamic acid (Shimadzu GLC ltd.;
Tokyo, JP) dissolved in 0.5 ml 50% acetonitrile containing 0.1%
TFA. One micro liter samples were spotted onto a matrix assisted
laser desorption/ionization (MALDI) plate. After air drying, spots
were identified by MALDI time of flight mass spectrometry
(MALDI-TOF MS: Shimazu, AXIMA TOF2TM). MS spectra
were collected over m/z 5003500. The acquisition parameters
were Tunig mode: Reflectron, Mass range: 13500, Max Laser
Rep Rate: 10.0, CID: off, Power: 75, Profiles: 200, Shots: 5, Ion
gate: Blank 900, P. Ext: 2500, Scenario: Advanced, Profile
average: All profiles, Peak width: 2 chans, Smoothing method:
Gaussian, Smoothing filter width: 2 chans, Baseline filter width: 16
chans, Peak detection method: Thresh hold Apex, Thresh hold
offset 0.500 mV, Use monoisotopic peak picking, Minimum mass
500, Maximam mass: 3500, Resolution of the MS analyzer was
1,000 (01k Da), 5,000 (1 kDa2 kDa) and 10,000 (.2
kDa).Minimum isotope: 1, Maximum intensity variation: 90 and
Overlapping distributions Minimum peak percent: 10. Proteins
were identified with the MASCOT (Matrix Science, London)
searching algorithms using the Swiss-plot database.
Probabilitybased MOWSE scores were estimated by comparison of search
results against estimated random match population and were
reported as-10* log 10(p), where p is the absolute probability.
Scores greater than 50 were considered significant, meaning that
for scores higher than 50 the probability that the match is a
random event is lower than 0.05. The sequence version of the
Swiss-Prot were heat shock cognate 71 kDa protein (Hspa8): 1,
Internexin a: 2, Tubulin b2b: 1, glial fibrillary acidic protein
(GFAP): 2, keratin, type I cytoskeletal 20 (CK20): 2, T-complex
protein 1 subunit a (TCP1a): 1, pyruvate kinase M1 (PKM1): 3,
Enolase 1: 4, Enolase 2: 2, Actin b: 1, F-actin-capping protein
subunit a-2 (CapZa2): 1 and glyceraldehyde-3-phosphate
dehydrogenase (GAPDH): 3. The interrogation parameters were Type
of search: Peptide Mass Fingerprint, Enzyme: Trypsin, Fixed
modifications: Carbamidomethyl (C), Variable modifications:
Gln.pyro-Glu (N-term Q), Glu-.pyro-Glu (N-term E), Oxidation
(M), Mass values: Monoisotopic, Protein Mass: Unrestricted,
Peptide Mass Tolerance: 60.5 Da, Peptide Charge State: 1+ and
Max Missed Cleavages: 1. Angiotensin II and ACTH were used as
an internal standard. All protein identifications were in the
expected size and PI range based on position in the gel.
The standard reaction mixture of 1 mM biotinylated 15d-PGJ2
contained 50 mM Tris-HCl buffer (pH 8.0), 100 mM NaCl and
plasma membranes (400 mg) in a total volume of 4 ml. Incubation
was initiated by addition of the reaction mixture to plasma
membranes, and was carried out at 4uC for 24 h. Membrane
lysates were incubated with Streptavidin Agarose beads
(Invitrogen, Carlsbad, CA) at room temperature for 30 min. The beads
were rinsed three times with lysis buffer. The proteins were eluted
by boiling the beads in Laemmli sample buffer and analysed by
SDS-PAGE followed by immunodetection with antibodies to
GAPDH (rabbit polyclonal, abcam [ab9485], Cambridge, UK),
PKM1 (goat polyclonal, abcam [ab6191]), Enolase 2 (goat
polyclonal, Santa Cruz [sc-31859], Santa Cruz, CA), Tubulin b
(rabbit polyclonal, Santa Cruz [sc-9104]), TCP1a (mouse
monoclonal, Enzo Life Sciences [ADI-CTA-191-D], New York,
NY), Internexin a (mouse monoclonal, Millipore [MAB5224],
Billerica, MA) and Actin b (mouse monoclonal, abcam [ab8226]).
This procedure was followed by the addition of horseradish
peroxidase-conjugated secondary antibody and ECL reagents.
Data are given as means 6 S.E.M. (n = number of
observations). Data were analyzed statistically by use of Students
non-paired t test for comparison with the control group, and data
on various inhibitors and blocker groups were analyzed statistically
by use of two-way ANOVA followed by Dunnetts test for
comparison with the PG group (15d-PGJ2, D12-PGJ2, PGJ2, PGD2
and 15d-PGD2). The half maximal inhibitory concentration
(IC50), the half maximal lethal dose (LD50) and the half maximal
lethal time (LT50) were calculated by Microsoft Excel Fit.
Susceptibilities of various cell lines to amyloid protein
Sensitivities of various cell lines to amyloid protein were
examined in the central nervous system and peripheral tissues.
Cortical neurons, BSMC, hepatocytes and dermal fibroblasts were
exposed to fAb or vehicle (ionized water) for 48 h, and their
viability was quantified by the MTT-reducing activity. In
comparison with vehicle, fAb significantly reduced the viability
of cortical neurons and BSMC at 10 mM. On the other hand, fAb
did not significantly affect the viability of hepatocytes and dermal
fibroblasts (Figure 1A). In neuronal cells and BSMC among tested
cell lines, amyloid protein inhibited the cell viability in a
concentration-dependent manner (Figure 1B).
Apoptotic effects of 15d-PGJ2related compounds were correlated to their affinities for DP1, DP2 and the specific binding sites for [3H]15d-PGJ2 (SBJ). LD50: The
concentration of 15d-PGJ2related compounds required to induce apoptosis in the half of neurons which were cultured for 24h in the absence of serum. LT50: The time
of 10 mM 15d-PGJ2related compounds required to induce apoptosis in the half of neurons which were cultured in the absence of serum. IC50: The concentration of
15d-PGJ2-related compounds required to inhibit half of the specific binding of [3H]15d-PGJ2 to SBJ. LD50, LT50 and IC50 were calculated from Yagami et al.. These data
on Ki: The Ki values of 15d-PGJ2-related compounds to DP1 and DP2 were referred from Sawyer et al.
Sensitivities of various cell lines to 15d-PGJ2
We examined susceptibilities to 15d-PGJ2 in cortical neurons,
BSMC, hepatocytes and dermal fibroblasts. These cell lines were
exposed to 15d-PGJ2 or vehicle (0.1% ethanol), and their viability
was quantified by the MTT-reducing activity. In comparison with
vehicle, 15d-PGJ2 significantly reduced the viability of cortical
neurons and BSMC at 10 mM. On the other hand, 15d-PGJ2 did
not significantly affect cell viability of hepatocytes and dermal
fibroblasts (Figure 2A). As well as amyloid protein, 15d-PGJ2 also
reduced the cell viability of neuronal cells and BSMC, but neither
hepatocytes nor dermal fibroblasts in a concentration-dependent
manner (Figure 2B).
In control cultures, neurons had extended neurites and smooth,
round cell bodies (Figure 3A). On the other hand, some cell bodies
shrank and lost their bright phase-contrast appearance in
15d-PGJ2treated cultures. By 24 h, there were markedly fewer cells, and
extensive debris was seen attached to the substratum (Figure 3B). In
control cultures, BSMC extended cell bodies and exhibited their
bright phase-contrast appearance (Figure 3C). When BSMC were
cultured, we confirmed that the cell density was increased (data not
shown). This increment was significantly prevented by 10 mM
15dPGJ2 (Figure 3B and 3D). In 15d-PGJ2-treated cultures, some cell
bodies shrank and became round (Figure 3D). Thus, there was a
close correlation between susceptibilities to 15d-PGJ2 and amyloid
protein, suggesting an involvement of 15d-PGJ2 in the amyloid
Effects of PGD2 and Its metabolites on the viability of
cortical neurons and BSMC
MTT assay is a colorimetric assay for measuring the activity of
enzymes that reduce MTT or close dyes to formazan dyes. These
reductions take place only when reductase enzymes in
mitochondria are active, and therefore conversion is often used as a measure
of viable (living) cells. Previously, we have reported that there was
a linear relationship between cell density and MTT-reducing
activities in cortical neurons . As well as the MTT-reducing
activity, the cell density was reduced by 10 mM 15d-PGJ2 in
cortical neurons and BSMC (Figure 4A). MTT-reduction assay is
also established for various cell types other than neurons to enable
accurate, straightforward quantification of changes in their cell
In most experiments, the neurotoxicity of 15d-PGJ2 was
evaluated at 10 mM for 24 h in the presence of serum. Since
PGD2 can be non-enzymatically metabolized to PGJ2, D12-PGJ2
and 15d-PGJ2 in the present culture medium , it is very difficult
to compare their neurotoxic potencies. When serum was deprived
from culture medium to decelerate the metabolism of PGD2, we
have succeeded in detecting their neurotoxic hierarchy by the
treatment with each PG at 10 mM for 8 h. We observed that
serum-deprivation did not induced neuronal cell death within 8 h.
The growth-inhibitory effect of PGD2 and its metabolites at
10 mM was 15d-PGJ2 . D12-PGJ2 . PGJ2 & PGD2 in sequence
(Figure 4B). On the other hand, 15-deoxy-D12,14-PGD2
(15dPGD2) did not affect MTT-reducing activity of neuronal cells. In
BSMC, 15d-PGJ2 significantly decreased MTT-reducing
activities. Although D12-PGJ2 showed a tendency to decrease
MTTreducing activity, the inhibitory effect was significantly detected in
neither 15d-PGD2, D12-PGJ2, PGJ2 nor PGD2.
Specific binding sites of 15d-PGJ2 in the plasma
membranes of cortical neurons and BSMC
Cortical neurons were fractionated into nuclear, plasma
membrane, cytosol and microsome. Binding assay of
[3H]15dPGJ2 was performed at room temperature for 1 h. The ratio of
specific binding of [3H]15d-PGJ2 to total binding were 78%, 66%,
45% and 4% in the fraction of plasma membrane, nuclear, cytosol
and microsome, respectively (Figure 5A). Previously, we have
reported the binding assay of [3H]15d-PGJ2 in the plasma
membrane under optimal conditions at 4uC for 24 h . The ratio
of specific binding of [3H]15d-PGJ2 to total binding was more than
80% in the cortical neuron. The inhibitory effect of
15d-PGJ2related compounds at 100 mM was 15d-PGJ2 . D12-PGJ2 . PGJ2
& PGD2 in sequence (Figure 5B). 15d-PGJ2 displaced the specific
binding of [3H]15d-PGJ2 in a concentration-dependent manner
(Figure 5B). In BSMC, 15d-PGJ2 also inhibited the specific binding
of [3H]15d-PGJ2 in a concentration-dependent manner (Figure 5B).
Figure 8. MALDI-TOF mass spectrum of the tryptic digest of spot 8. Spot 8 from Figure 7D was digested in gel with trypsin, and the resulting
peptides were analyzed by MALDI-TOF MS as detailed in the experimental section. (A) Typical mass spectrum from a representative experiment. (B)
Probability based Mowse Score. (C) Positions of matched peptides in the sequence of GFAP.
The IC50 value of 15d-PGJ2 to the specific binding of [3H]15d-PGJ2
in BSMC was 31 mM, and 20-fold higher than that (1.6 mM) in
neuronal cells. The binding sites of 15d-PGJ2 in cortical neurons
could also be recognized by D12-PGJ2 and PGJ2, whereas those in
BSMC could be specifically done by 15d-PGJ2 (Figure 5B). In the
two cells, the MTT-reducing activities of 15d-PGJ2 and its
precursors were paralleled to the affinities of these ligands for the
membrane specific binding sites of 15d-PGJ2.
Comparison of the specific binding sites for [3H]15d-PGJ2
in plasma membranes to authentic receptors, DP1 and
In peripheral tissues including nerves, chemoattractant
receptorhomologous molecule expressed on Th2 cells has been identified as a
type 2 receptor for PGD2 (DP2), and reported to be also a membrane
receptor for 15d-PGJ2 . We compared characterization of specific
binding sites for [3H]15d-PGJ2 (SBJ) and DP2. According to LD50
and LT50, the apoptotic effect of 15d-PGJ2-related compounds was
15d-PGJ2 . D12-PGJ2 . PGJ2 . PGA2 & PGD2 . 15d-PGD2 in
sequence (Table 1). In the view of IC50, the affinity of 15d-PGJ2
related compounds for SBJ was 15d-PGJ2 . D12-PGJ2 . PGJ2 .
PGA2 & PGD2 . 15d-PGD2 in sequence (Table 1). On the other
hand, the affinity of 15d-PGJ2related compounds for DP2 was
PGD2 . 15d-PGD2 .15d-PGJ2 . PGJ2 . D12-PGJ2 & PGA2 in
sequence (Table 1). In addition, the affinity of 15d-PGJ2related
compounds for DP1 was PGJ2 .PGD2 & D12-PGJ2
.15dPGJ2.15d-PGD2 . PGA2 in sequence (Table 1). Thus, the
apoptotic effect of 15d-PGJ2-related compounds was correlated to
their affinities for SBJ, but not to those for DP1 or DP2.
Isolation of Targets for 15d-PGJ2
To identify target proteins for 15d-PGJ2, membrane proteins
were labeled with biotinylated 15d-PGJ2 under the serum-free
condition to reduce non-specific binding. Under this condition,
biotinylated 15d-PGJ2 induced neuronal cell death in a
concentration- dependent manner as well as 15d-PGJ2. Their LD50 values
were almost 1 mM (Figure 6A). Biotinylated 15d-PGJ2 suppressed
the extension of neurites and shrank cell bodies in a similar fashion
to 15d-PGJ2 (Figure 6B). Next, neuronal plasma membranes were
incubated with 1 mM biotinylated 15d-PGJ2 in the absence or
presence of 15d-PGJ2 at the indicated concentrations. Then,
membrane proteins modified with biotinylated 15d-PGJ2 were
separated by two-dimensional gel electrophoresis. The patterns that
were given by western blot analysis probed with anti-biotin
antibody-HRP and SYPRO Ruby fluorescence staining are shown
in Figure 7. Several biotinylated 15d-PGJ2-protein conjugates were
detected as biotin-positive spots (Figure 7A and 7B). 15d-PGJ2
inhibited the modification of proteins with the biotinylated
15dPGJ2 in a concentration-dependent manner (Figure 7C and 7D). At
100 mM, 15d-PGJ2 eliminated almost completely the biotin-positive
spots (Figure 7D). After superimposition of both patterns, the
SYPRO Ruby-stained proteins that coincided with the
biotinpositive spots were excised from two-dimensional gels (Figure 7E),
subjected to trypsin digestion, and then successfully analyzed by
MALDI-TOF MS fingerprint analysis (Figure 8A).
Identification of Targets for 15d-PGJ2
Spot 8 corresponding to a 50 kDa 15d-PGJ2-protein conjugate
was one of the targets of the modification by biotinylated
15dPGJ2, as seen in Figure 8. Using MASCOT, the probability based
MOWSE score was 267 for GFAP (p,0.05) (Figure 8B), with 28
Spots that were excised from the gel show in Figure 7E were identified by tryptic digestion and MALDI-TOF MS. Shown are the spot number, name of the identified
protein, the accession number in the SwissProt database, the theoretical molecular mass and isoelectric point, the probability based MOWSE score, the number of
peptides matched according to the Mascot database, the percentage of the protein sequence that is covered by the identified peptides.
peptide matches (error 60.02%) (Figure 9), which represents 56%
sequence coverage (Figure 8C). Table 2 lists the identity of 22
protein spots, which could be identified in three independent
experiments. The multiple gel spots for a single identification
could be ascribed to posttranslational modification, such as
phosphorylation. For example, spot 6 could contain 3
phosphorylation sites (T129, T130 and Y283), which represented the
probability based MOWSE score59, 16 peptide matches, 32%
sequence coverage. Spot 7 could contain 1 phosphorylation site
(Y283), which represented the probability based MOWSE score
188, 31 peptide matches, 51% sequence coverage. On the other
hand, the phosphorylation site of spot 8 was not detected. The
identified proteins fall into several different functional classes,
including glycolytic enzymes (Enolase 1, Enolase 2, GAPDH and
PKM1), molecular chaperones (Hsp8a and TCP1a) and
cytoskeltones (Tubulin b2b, Actin b, Internexin a, GFAP and CapZa2).
Next, we attempted to detect the 15d-PGJ2-target adducts in the
plasma membranes exposed to the biotinylated 15d-PGJ2. by
streptavidin agarose pull-down assays. Western blot revealed that
15d-PGJ2 interacted with Actin b, Enolase 2, GAPDH, Internexin
a, PKM1, TCP1a and Tubulin b2b (Figure 10). Since plasma
membranes were prepared from adult cerebral cortices including
neurons and astrocytes, non-neuronal enolase1 and GFAP
appeared to be derived from astrocytes.
Regions homologous to the binding site of 15d-PGJ2 in
Several lines of evidences indicate the covalent binding sites of
15d-PGJ2 in previous target proteins. To ascertain whether the
cysteine residue in the present target proteins responded to the
covalent binding sites of 15d-PGJ2 in previous target proteins or
not, homologous regions were searched (Table 3). As query
sequences, we used the amino acid sequences of the previous
target proteins, in which the covalent binding sites of 15d-PGJ2 are
identified: Cys374 of Actin b (P60711) , Cys269 of c-Jun
(NP_068607) , Cys184 of H-ras (NP_001091711) , Cys179
of IkB-kinase b (Q9QY78) 8, Cys285 of PPARc (NP_619725) ,
Cys35 and Cys69 of thioredoxin (NP_446252) . Hspa8
contained Cys603 responded to the Cys179 of IkB-kinase b. The
amino sequence of Hspa8 from Lys597 to Leu610 was homologous
to that of IkB-kinase b from Lys171 to Leu186. Based on the
comparison between the two sequences, the initial score, the
optimal score and the identity were 15, 29 and 31%, respectively.
In a similar fashion, Internexin a, Tubulin b2b, GFAP, CK20,
TCP1a, PKM1, Enolase 1, Enolase 2, Actin b, CapZa2 and
GAPDH contained the cysteine residue responded to Cys69 of
thioredoxin, Cys184 of H-ras, Cys269 of c-Jun, Cys69 of
thioredoxin, Cys35 of thioredoxin, Cys35 of thioredoxin, Cys184
of H-ras, Cys184 of H-ras, Cys374 of actin b, Cys179 of IkB-kinase
b8, Cys35 of thioredoxin, respectively. Thus, the present target
proteins also contained the cysteine residue responded to the
previous covalent binding site of 15d-PGJ2, and exhibited
homologous sequences around the specific binding site.
Cortical neurons and BSMC sensitive to amyloid protein were
susceptible to 15d-PGJ2. [3H]15d-PGJ2 bound specifically to the
two cells, suggesting that 15d-PGJ2 played an important role in
amyloidoses not only in the central nervous system but also in the
peripheral tissues. The specific binding sites of [3H]15d-PGJ2 were
detected in the neuronal subcellular fractions of nuclear, cytosol
and plasma membrane, but not in the microsomal fraction.
15dPGJ2 binds to the nuclear receptor, PPARc  and the cytosolic
protein, Ras . In peripheral tissues including nerves,
chemoattractant receptor-homologous molecule expressed on
Th2 cells has been identified as a type 2 receptor for PGD2
(DP2), and reported to be also a membrane receptor for 15d-PGJ2
. Contrary to its mRNA, little protein of DP2 has yet been
detected in the central nerve. Furthermore, we ruled out the
possibility that the specific binding site of 15d-PGJ2 in the plasma
membrane of cortical neurons was DP2. First, few binding sites of
[3H]PGD2 are detected in plasma membranes from rat cortices
. Although binding sites of [3H]?12-PGJ2 and [3H]PGJ2 are also
detected in plasma membranes, those are displaced most potently
by 15d-PGJ2 among PGD2 metabolites . Second, a DP2
selective agonist, 15d-PGD2 do not affect the cell number of
neuronal cells and BSMC (Figure 3B and Table 1). Third, the
LD50 value (.10 mM) of PGD2 is much higher than the affinity for
PGD2 receptor (dissociation constant = 8.8 nM) .
In the present study, we identified membrane proteins targeted
for 15d-PGJ2 including glycolytic enzymes, molecular chaperones
and cytoskeletons (Table 2 and Figure 10). GAPDH, Enolase 1,
Enolase 2 and PKM1 were previously believed to perform
exclusively house-keeping glycolysis. GAPDH is not only found
in the cytoplasm, but also closely associated with the plasma
membrane . GAPDH catalyses the conversion of
glyceraldehyde 3-phosphate to D-glycerate 1,3-bisphosphate. Reduction in
glycolysis precedes cognitive dysfunction and is therefore believed
to be an important early event in AD development . Apart
from its glycolytic role, overexpression of the particular
membrane-associated GAPDH has a direct role in neuronal apoptosis
 (Figure 11). GAPDH is located in amyloid plaques ,
Query 171 KELDQGSLCTSFVGTL 186
Sbjct 597 KELEK VCNPI I T KL 610
Query 60 DDCQDVAADCE 70
Sbjct 173 EEV QRLR ARCE 183
Query 183 SCKCV 187
Sbjct 126 SCDCL 130
Query 261 RNRI AAS KCRKRKL 274
Sbjct 284 RRQLQALTC DLE S L 297
Sbjct 136 I KDAQ I ENARCVLQ 149
Query 59 VDDCQDVAADCEVK 72
Query 34 PCKMI KPFFH 43
Sbjct 124 ACKEAVRYIN 133
Query 35 CKMI 38
Sbjct 165 CKVV 168
Query 178 GPGCMSCKCV L 188
Sbjct 331 AAGE KSCNCL L 341
Query 183 SCKCVL 188
Sbjct 336 ACNCLL 341
Query 356 WISKQEYDESGPSIVHRKCF 375
Sbjct 356 WISKQEYDESGPSIVHRKCF 375
Query 169 YAKELDQGSL CT SFVGTLQ 187
Sbjct 131 YVKEHYPNGVCTVYGKKVD 149
Query 35 CKMIKP 40
Sbjct 245 CRLEKP 250
Homologies were determined with Lipman-Pearson searching algorithms using
the Swiss-plot database. As query sequences, we used the amino acid
sequences of the previous target proteins, in which the covalent binding sites
of 15d-PGJ2 are identified: Cys374 of Actin b (P60711) , Cys269 of c-Jun
(NP_068607) , Cys184 of H-ras (NP_001091711) , Cys179 of IkB-kinase b
(Q9QY78) , Cys285 of PPARc (NP_619725) , Cys35 and Cys69 of thioredoxin
(NP_446252) . As subject sequences, we used the amino acid sequences of
our target proteins. The listed sequences exhibited the highest score in the
initial score, the optimal score and the identity.
interacts with the C-terminal region of amyloid precursor protein
(APP) , and co-precipitates with fAb. Furthermore,
GAPDH associates tightly with Enolase 2 and Hspa8, and makes
up trans-plasma-membrane oxidoreductases (PMOs), the
extracellular redox sensor for signaling external oxidative stress to the cell
Enolase 1 and Enolase 2 belong to a superfamily of abundantly
expressed carbon-oxygen lyases known for the catalysis of
2phosphoglycerate to phosphoenolpyruvate. Ubiquitous enolase1
and neuron specific enolase 2 exist as monomers and also as
dimmers on the neuronal membrane surface . Recent studies
have demonstrated that enolases possess different regulatory
properties from glycolysis in the brain . Enolase1 is one of
the most consistently up-regulated and oxidatively modified
proteins in brain of subjects of early-onset AD . Enolase1
and enolase 2 are autoantigen targets in post-streptococcal
autoimmune disease of central nervous system (Figure 11). The
anti-enolase antibodies induce neuronal apoptosis . Enolase 2
is part of neuronal PMOs, and the anti-enolase2 antibody can
inhibit PMO activity on the plasma membrane .
Pyruvate kinase transfers a phophate from
phosphoenolpyruvate to ADP. Pyruvate kinase is also defined as the autoantigen,
and its antibodies induce neuronal apoptosis  (Figure 10). The
significant increase in pyruvate kinase activity is found in frontal
and temporal cortex of AD brains . Pyruvate kinase is elevated
in the cortical neurons undergoing Ab-mediated apoptosis .
Pyruvate kinase is co-precipitated with fAb . Biotinylated
15dPGJ2 binds to PKM1 in mesangial cells , supporting our
Hsp8a is dnaK-type molecular chaperone heat shock protein
72-ps1 in the PMO complex . It is located in the cytoplasm
, but nuclear localization and accumulation near or at the
plasma membrane in stressed cells and in synaptosomal
membranes has been observed . Hsp8a binds to the cytoplasmic
domain near the post-transmembrane region of APP (Figure 11).
TCP1a is a selective molecular chaperone in tubulin biogenesis,
by that nascent tubulin subunits are bound to TCP1a and released
in assembly competent forms. Cytoskeletal proteins are deficient
and aggregated in AD. When TCP1a is related to its natural and
specific substrate tubulin b, the ratio is significantly decreased in
the temporal, frontal, parietal cortex and in thalamus of AD
patients . Relatively decreased molecular chaperoning of
tubulin b by TCP1a is suggested to lead to misfolded tubulin
aggregating and accumulating in plaques and tangles, a hallmark
of AD (Figure 11).
Tubulin has been identified as a membrane component of
synaptosomes and various plasma membranes. Both tubulin a and
b have been shown to associate with the amyloid deposits of
familial amyloidosis  and to bind to the Ab sequence of APP
. Moreover, tubulin b is retained by a monomeric Ab column
, and co-precipitated with fAb  (Figure 11). The tau
protein interacts with tubulin to stabilize microtubules and
promote tubulin assembly into microtubules. PGJ2 induces
caspase-mediated cleavage of tau, generating Dtau, an aggregation
prone form known to seed tau aggregation prior to neurofibrillary
tangle formation . Hyperphosphorylation of the tau protein
(tau inclusions) can result in the self-assembly of tangles of paired
helical filaments and straight filaments, which are involved in the
pathogenesis of AD . Biotinylated 15d-PGJ2 binds to tubulin b
in mesangial cells , supporting our results.
AD-linked human Ab synergistically enhances the ability of
wild-type tau to promote alterations in the actin cytoskeleton
(Figure 11) and neurodegeneration . The ability of globular
actin to rapidly assemble and disassemble into filaments is critical
to many cell behaviors. F-actin-capping protein subunit a-2
(CapZa2) regulates growth of the actin filament by capping the
barbed end of growing actin filaments (Figure 11). Members of the
actin-depolymerizing factor (ADF)/cofilin family are important
regulators of actin dynamics. ADF and cofilins ability to increase
actin filament dynamics is inhibited by their phosphorylation on
Ser3 by LIM kinase 1 and other kinases  Ab dystrophy
requires LIM kinase 1-mediated phosphorylation of ADF/cofilin
and the remodeling of the actin cytoskeleton . Biotinylated
15d-PGJ2 covalently binds to actin b in various cells  other
than neurons, supporting our results in neurons.
Internexin ais classified as a type IV neuronal intermediate
filament. Internexin a also co-assembles with the neurofilament
(NF) triplet proteins . The protein is expressed by most, if not
all, neurons as they commence differentiation and precedes the
expression of the NF triplet proteins . Although the interaction
of internexin a with amyloid proteins has not yet been reported,
Internexin a, and not NF triplet, ring-like reactive neurites are
present in end-stage AD cases, indicating the relatively late
involvement of neurons that selectively contain Internexin a
(Figure 11). Another intermediate filament protein, GFAP is
expressed exclusively in astrocytes. Ab increased the total number
of activated astrocytes, and elevated the expression of GFAP by
Ab-induced spontaneous calcium transients . 15d-PGJ2
suppresses inflammatory response by inhibiting NF-kB signaling
at multiple steps as well as by inhibiting the PI3K/Akt pathway
independent of PPARc in primary astrocytes .
In conclusion, membrane target proteins for 15d-PGJ2 were
factors associated with the two remarks of AD, the amyloid plaque
and the neurofibrillary tangle. Beyond classical roles as glycolytic
enzymes and molecular chaperones, GAPDH, enolase 2 and
Hsp8a can form the antioxidant complex of PMOs responded to
the extracellular oxidative stress. 15d-PGJ2 might regulate the
activity of PMOs during inflammation and degeneration. Apart
from glycolysis, pyruvate kinase and enolase might be involved in
the 15d-PGJ2induced apoptosis as autoantigens. Thus, the
present study sheds light on the ecto-enzymes targeted for
15dPGJ2 as a prelude to the death receptor stimulated by 15d-PGJ2 or
the antioxidant complex regulated by 15d-PGJ2.
Conceived and designed the experiments: TY. Performed the experiments:
YY KT JK M. Fujita NO TS. Analyzed the data: YY KT TY. Contributed
reagents/materials/analysis tools: M. Fujimoto. Wrote the paper: YY TY.
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