Lipocalin-type prostaglandin D synthase protects against ... · dysautonomia, Alzheimer’s disease and Parkinson’s disease [3,4]. A number of studies have suggested that the excessive

Biochem. J. (2012) 443, 75–84 (Printed in Great Britain) doi:10.1042/BJ20111889 75

Lipocalin-type prostaglandin D synthase protects against oxidativestress-induced neuronal cell deathAyano FUKUHARA*1, Mao YAMADA*1, Ko FUJIMORI†, Yuya MIYAMOTO*, Toshihide KUSUMOTO*, Hidemitsu NAKAJIMA‡ andTakashi INUI*2

*Laboratory of Biological Macromolecules, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan, †Laboratory of Biodefenseand Regulation, Osaka University of Pharmaceutical Sciences, Takatsuki, Osaka 569-1094, Japan, and ‡Laboratory of Veterinary Pharmacology, Graduate School of Life andEnvironmental Sciences, Osaka Prefecture University, Izumisano, Osaka 598-8531, Japan

L-PGDS [lipocalin-type PGD (prostaglandin D) synthase] is adual-functional protein, acting as a PGD2-producing enzymeand a lipid transporter. L-PGDS is a member of the lipocalinsuperfamily and can bind a wide variety of lipophilic molecules.In the present study we demonstrate the protective effect of L-PGDS on H2O2-induced apoptosis in neuroblastoma cell lineSH-SY5Y. L-PGDS expression was increased in H2O2-treatedneuronal cells, and the L-PGDS level was highly associated withH2O2-induced apoptosis, indicating that L-PGDS protected theneuronal cells against H2O2-mediated cell death. A cell viabilityassay revealed that L-PGDS protected against H2O2-inducedcell death in a concentration-dependent manner. Furthermore,the titration of free thiols in H2O2-treated L-PGDS revealedthat H2O2 reacted with the thiol of Cys65 of L-PGDS. The

MALDI–TOF (matrix-assisted laser-desorption ionization–time-of-flight)-MS spectrum of H2O2-treated L-PGDS showed a 32Da increase in the mass relative to that of the untreated protein,showing that the thiol was oxidized to sulfinic acid. The bindingaffinities of oxidized L-PGDS for lipophilic molecules werecomparable with those of untreated L-PGDS. Taken together,these results demonstrate that L-PGDS protected against neuronalcell death by scavenging reactive oxygen species without losingits ligand-binding function. The novel function of L-PGDScould be useful for the suppression of oxidative stress-mediatedneurodegenerative diseases.

Key words: apoptosis, lipocalin, neuroprotection, prostaglandinD synthase, reactive oxygen species (ROS), SH-SY5Y.

INTRODUCTION

Oxidative stress induced by the excessive production ofROS (reactive oxygen species) has been associated withischaemia/reperfusion injury [1,2]. ROS are produced duringand after cerebral ischaemia and cerebral infarction, havebeen shown to have a neurotoxic impact on the brain, andhave also been implicated in neurological disorders includingdysautonomia, Alzheimer’s disease and Parkinson’s disease[3,4]. A number of studies have suggested that the excessivegeneration of ROS underlies neuronal degeneration under diverseneurological conditions [5–8]. Among ROS, H2O2 is well knownto be involved in neuronal cell death [9]. Oxidative stress alsomodifies, reversibly or irreversibly, cellular components suchas DNA, lipids and proteins. Thus, to understand the protectivemechanism against oxidative stress in the central nervous system,it is necessary to elucidate the relationship between oxidativestress and the expression level of key proteins.

In the brain, L-PGDS [lipocalin-type PGD (prostaglandinD) synthase] is synthesized mainly at the rough endoplasmicreticulum membrane of arachnoid cells, chorioid plexus cells andoligodendrocytes, and then is secreted into the CSF (cerebrospinalfluid) as a second major protein in human CSF [10]. L-PGDS isa dual-functional protein, acting as a PGD2-synthesizing enzymeand as an extracellular transporter for lipophilic ligands [11].

The tertiary structure of L-PGDS exhibits a single eight-strandedantiparallel β-sheet closed back on itself to form a continuouslyhydrogen-bonded β-barrel and an α-helix [12,13]. Human L-PGDS possesses two free thiol groups, those of Cys65 and Cys167,and Cys65 is the active centre for the catalytic function of L-PGDS [14]. Protein thiol has been indicated to react with variouskinds of active oxygen, indicating that protein thiol modificationby ROS is important in biological defence mechanisms [15,16].These reports prompted us to investigate possible oxidative stress-mediated thiol modification of free cysteine residues in humanL-PGDS.

The L-PGDS concentration increases in the CSF of patientsseveral weeks after a stroke as well as in some tumourpatients [17]. The CSF level of L-PGDS is significantly lowerin patients with schizophrenia [18], a brain tumour [19], bacterialmeningitis [20] and normal pressure hydrocephalus [21]. L-PGDSis up-regulated in acute and massive brain injury resulting fromneonatal hypoxic ischaemic encephalopathy [22]. The L-PGDSlevels in the CSF of patients with aneurysmal subarachnoidhaemorrhage transiently increase to be 2-fold over normal within5 days [23]. All of these reports suggest that the L-PGDSconcentration is strongly related to various pathophysiologicalconditions. In the present study we demonstrate the protectiveeffect of human L-PGDS against ROS-induced neuronal cell deathby scavenging the active oxygen via the thiol of Cys65.

Abbreviations used: BV, biliverdin; CSF, cerebrospinal fluid; DAPI, 4′,6-diamidino-2-phenylindole; 15d-PGJ2,15-deoxy-�12,14-prostaglandin J2; DTNB,5,5′-dithiobis-(2-nitrobenzoic acid); HRP, horseradish peroxidase; LDH, lactate dehydrogenase; L-PGDS, lipocalin-type prostaglandin D synthase; MALDI–TOF, matrix-assisted laser-desorption ionization–time-of-flight; NC, negative control; PGD, prostaglandin D; RA, retinoic acid; ROS, reactive oxygen species;siRNA, small interfering RNA; TNB, 5-thiobenzoic acid; WT, wild-type.

1 These authors contributed equally to this work.2 To whom correspondence should be addressed (email [email protected]).

c© The Authors Journal compilation c© 2012 Biochemical Society

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76 A. Fukuhara and others

EXPERIMENTAL

Materials

DTNB [5,5′-dithiobis-(2-nitrobenzoic acid)], trichloroacetic acid,and guanidinium chloride were purchased from Wako PureChemical. Thrombin and DAPI (4′,6-diamidino-2-phenylindole)were purchased from Sigma. Sinapic acid was obtained fromAldrich. All other chemicals were of analytical grade.

Expression of recombinant L-PGDS in Escherichia coli andpurification

The open reading frame for human L-PGDS, which iscomposed of 190 amino acid residues (GenBank® accessionnumber M61900) [14], or that for mouse L-PGDS, composedof 189 amino acid residues (GenBank® accession numberX89222); [24], was ligated into the BamHI/EcoRI sites ofthe expression vector pGEX-2T (GE Healthcare Bio-Sciences)[30]. The N-terminal 22 amino acid residues correspondingwith the putative secretion signal peptide of both L-PGDSswere truncated. WT (wild-type) human L-PGDS possessesfour cysteine residues (Cys65, Cys89, Cys167 and Cys186), andtwo of them, Cys89 and Cys186, form a disulfide bridge thatis highly conserved among most lipocalins. C65A/C167A(ε280 = 25900 M− 1 · cm− 1), C89A/C167A/C186A (ε280 = 25700M− 1 · cm− 1) and C89A/C186A (ε280 = 25500 M− 1 · cm− 1)human L-PGDS, and C89A/C186A (ε280 = 22461 M− 1 · cm− 1)and Y63S/T67S/C89A/C186A (ε280 = 20892 M− 1 · cm− 1) mouseL-PGDS were expressed in E. coli BL21(DE3) (Toyobo). Site-directed mutagenesis was performed using a QuikChangeTM

site-directed mutagenesis kit (Stratagene). The DNA sequenceswere confirmed with a CEQ

TM

8000 Genetic Analysis System(Beckman Coulter) after cycle sequencing with a SequiThermCycle Sequencing kit (Epicentre Technologies). Each mutated L-PGDS was expressed as a glutathione transferase fusion protein.The fusion protein was bound to glutathione–Sepharose 4B(GE Healthcare Bio-Sciences) and incubated with thrombin(1 unit/1 μl) to release the L-PGDS. These recombinant proteinswere further purified to apparent homogeneity by HiLoad 16/60Superdex 75 (GE Healthcare Bio-Sciences) chromatography anddialysed against PBS.

Cell culture

SH-SY5Y cells, which are human neuroblastoma cells, werecultured in Dulbecco’s modified Eagle’s medium/Ham’s F12nutrient mixture (Gibco) containing 10% FBS (fetal bovineserum; Gibco), 100 units/ml penicillin, 100 μg/ml streptomycinand 0.25 μg/ml amphotericin B (Gibco) in a humidifiedatmosphere of 5 % CO2 and 95% air at 37 ◦C.

Cytotoxicity assay

SH-SY5Y cells were cultured in 96-well tissue culture platesat a density of 3 × 104 cells/well. After 48 h of cultivation, theappropriate amounts of H2O2 and/or L-PGDS were added tothe culture medium. The cells were further cultured for 24 h,and then WST-8 solution (Dojindo Laboratories) was added tothe culture medium; thereafter, the cells were incubated for 4 hat 37 ◦C. The absorbance of the formazan produced from WST-8was measured at 450 nm by using a microplate reader, Model 680(Bio-Rad Laboratories).

LDH (lactate dehydrogenase) activity was measured by use ofa Cytotoxicity Detection KitPLUS (Roche Diagnostics) according

to the manufacturer’s instructions. Absorbance was measured at492 nm by using a Microplate reader Lucy2 (Anthos).

Detection of DNA fragmentation by agarose gel electrophoresis

SH-SY5Y cells were cultured for 24 h in the presence or absenceof H2O2 (100 μM). Genomic DNA was isolated from the cellsby using Isogen (Nippon Gene) following the manufacturer’sinstructions, and resuspended in TE buffer [10 mM Tris/HCland 1 mM EDTA (pH 8.0)]. The purified DNA was loaded onto a 2% agarose gel in TAE [40 mM Tris/acetate, 1 mM EDTA(pH 8.0)] buffer and, following electrophoresis, it was stainedwith 200 μg/ml ethidium bromide to visualize the bands with a254-nm UV transilluminator.

DAPI staining

Cells were seeded on plates coated with 100 μg/ml poly-D-lysine, and then treated with H2O2 (100 μM) for 24 h. The cellswere washed with PBS and fixed for 10 min with 4 % (w/v)paraformaldehyde in phosphate buffer (pH 7.4). Then, cells werewashed three times with PBS and stained with 1 μg/ml DAPIin PBS including 0.05 % Tween 20. Fluorescence was detectedusing a fluorescence microscope (Nikon).

Preparation of RNA and quantification of mRNA levels

Total RNA was extracted by use of TriPure IsolationReagent (Roche Diagnostics) according to the manufacturer’sdirections. First-strand cDNAs were synthesized by usingReverTra Ace Reverse Transcriptase (Toyobo) primed byrandom-hexamer as described previously [25]. Quantificationof mRNA levels was carried out by using a real-time PCRsystem (LightCycler, Roche Diagnostics) and Fast SYBRGreen Master Mix (Roche Diagnostics) with gene-specificprimer sets: forward, 5′-CCAACTTCCAGCAGGACAAG-3′ andreverse, 5′-CCACAGACTTGCACATGGAC-3′ for L-PGDS,and forward, 5′-CCAACCGCGAGAAGATGA-3′ and reverse, 5′-CCAGAGGCGTACAGGGATAG-3′ for β-actin. All data wereobtained from three independent experiments and transcriptionlevels of all genes were normalized to the level of the β-actingene used as the internal control.

siRNA (small interfering RNA) study

The following StealthTM siRNAs for L-PGDS and StealthTM

NC (negative control) siRNA were purchased from In-vitrogen: L-PGDS siRNA#1, 5′-CAGGACUUCCGCAUG-GCCACCCUCU-3′ and L-PGDS siRNA#2, 5′-GACUUCCG-CAUGGCCACCCUCUACA-3′. At 1 day prior to the transfec-tion, SH-SY5Y cells were plated at a density of 2 × 104 cells/wellon a 96-well culture plate. The cells were transfected witheither siRNA or the NC siRNA (20 nM) by using X-tremeGENEsiRNA Transfection Reagent (Roche Diagnostics) according tothe manufacturer’s instructions. After 1 day of transfection, H2O2

(100 μM) was added and the cells were then cultured for a further1 day. RNA was extracted and mRNA levels were then measuredby quantitative PCR as described above.

Expression of L-PGDS in SH-SY5Y cells

The expression vector of human L-PGDS was constructedpreviously [25a]. Site-directed mutagenesis of Cys65 to alanine


ROS scavenging by L-PGDS 77

(C65A) was carried out as described above. SH-SY5Y cellswere transfected with FuGENE6 Transfection Reagent (RocheDiagnostics) according to the protocols prescribed by themanufacturer. At 1 day after transfection, the transfectants wereincubated in medium containing 100 μM H2O2 for 24 h. Cellviability and LDH activity were measured as described above.

Western blot analysis

Cells were lysed in RIPA buffer containing 50 mM Tris/HCl(pH 8.0), 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate,1% (v/v) Nonidet P40 and 1% (v/v) Triton X-100 along withprotease inhibitor cocktail (Nacalai Tesque), and then centrifugedat 12000 g for 20 min at 4 ◦C. The proteins were separated onan SDS/PAGE (12.5 % gel) and then transferred on to PVDFmembranes (Immobilon P, Millipore) for Western blot analysisby the use of the SNAP i.d. Protein Detection System (Millipore).Blots were incubated with anti-L-PGDS (1:2000 dilution; 1B7[26]), anti-FLAGM2 (1:5000; Sigma) and anti-actin monoclonalantibody (1:3000; AC-15; Sigma), and then incubated withHRP (horseradish peroxidase)-conjugated secondary antibody(1:1000; Santa Cruz Biotechnology). Immunoreactive signalswere detected by the use of a Luminata Forte Western HRPsubstrate (Millipore). Protein concentrations were measured witha Pierce BCA (bicinchoninic acid) protein assay kit (ThermoScientific).

Determination of free thiol groups

L-PGDS (5 μM) was incubated with 50 μM H2O2 for 0–24 h orH2O2 (0–100 μM) in PBS at 37 ◦C for 24 h. The reaction wasstopped by adding trichloroacetic acid to a final concentration of10% (v/v). The sample tubes were placed on ice for 30 min andthen centrifuged at 29000 g for 30 min at 4 ◦C. The supernatantwas removed, and the pellet was dissolved in 500 μl of 50 mMTris/HCl buffer (pH 8.0) containing 6 M guanidinium chlorideand incubated for 15 min at room temperature (25 ◦C). Freethiol groups were determined by the reaction with DTNB, whichwas added at 5-fold excess. The number of free thiol groups wascalculated from the absorbance of the released TNB (5-thiobenzoic acid) anion (ε412 = 13600 M− 1 · cm− 1 [27]).

Mass spectroscopy

The samples (200 μl) were desalted by using a C4 Zip Tip(Millipore), and eluted in 70% acetonitrile. MALDI–TOF(matrix-assisted laser-desorption ionization–time-of-flight)-MSanalysis was performed with an Ultraflex TOF/TOF (BrukerDaltonics) operating under FlexControl 2.0 (Bruker Daltonics)in the positive-ion linear mode. Acceleration voltages of 25 kVwere applied. Ions ranging from 10 to 22 kDa were registered.Approximately 300 single spectra were summarized. Dataanalysis was performed with FlexAnalysis 2.0. The MALDI–TOF mass spectrometer was equipped with a standard nitrogenlaser (337 nm), and data were acquired in the positive-ion modewith external calibration. Before MALDI–TOF-MS analysis,both C89A/C167A/C186A and C65A/C167A L-PGDS at 5 μMwere either treated or not with 50 μM H2O2 at 37 ◦C for 24 h.Then the proteins were mixed with saturated sinapinic acid inacetonitrile/0.1 % trifluoroacetic acid [1:3 (v/v)] and externallycalibrated. Prepared proteins were loaded on to a single spot ofan AnchorChip (Bruker Daltonics). The function of AnchorChipwas to condense each protein in a very small area so that a highersignal intensity could be obtained even with a low concentrationof analyte on the target plate.

Fluorescence-quenching assays

BV (biliverdin) and all-trans-RA (retinoic acid) were eachdissolved in DMSO to give a 2 mM stock solution. Theconcentrations were determined spectroscopically based on theirrespective molar absorption coefficients of ε377 in methanol forBV (51500 M− 1 · cm− 1) [28] and ε336 in ethanol for RA (45000M− 1 · cm− 1) [29]. C89A/C167A/C186A L-PGDS at 5 μM wasreacted with 50 μM H2O2 for 24 h at 37 ◦C in PBS. The reactionsample was dialysed against PBS, and the oxidized L-PGDSwas thus obtained. Various concentrations of BV or RA wereadded to untreated or oxidized L-PGDS in PBS for pH 7.4, andthe final concentration of each protein was adjusted to 1.5 μM.After incubation at 25 ◦C for 30 min, the intrinsic tryptophanfluorescence was measured as described previously [30]. Thequenching of tryptophan fluorescence caused by non-specificinteractions with each ligand was corrected by use of 1.5 μMN-acetyl-L-tryptophanamide. The dissociation constants (Kd) forbinding between the ligands and each L-PGDS were calculatedusing the method described previously [31].

Structural model of human L-PGDS

Homology modelling was performed by using the SWISS-MODEL workspace [32,33]. The homology model of WT humanL-PGDS was built by using the solution structure of the C65Amouse L-PGDS (PDB code 2RQ0 [13]) as a template to illustratethe environment around Cys65 in human L-PGDS. Human L-PGDS (168 residues) shows approximately 75% amino acidsequence identity with mouse L-PGDS. The model obtained wasevaluated by PROCHECK [34]. Graphical representations wereprepared using PyMOL (http://www.pymol.org).

Statistical analysis

Data are expressed as means +− S.D. The statistical significancebetween the control and the experimental group was assessedusing Student’s t test. P < 0.05 was considered to be significant.

RESULTS

Cytotoxicity in H2O2-treated human neuronal SH-SY5Y cells

The cytotoxicity of H2O2 towards human neuronal SH-SY5Ycells was investigated by performing the WST-8 assay, whichis a general measure of cellular metabolic activity. The resultsshow that H2O2 at 0 to 10 μM had a negligible effect on cellviability (Supplementary Figure S1A at http://www.BiochemJ.org/bj/443/bj4430075add.htm). However, at higher concentra-tions of 30–100 μM, H2O2 reduced the cell viability in aconcentration-dependent manner. These results were almostconsistent with the results reported previously [36–39]. The IC50

value was determined to be 49.5 +− 1.4 μM by using the curve-fitting program GraFit Version 3.0 (Erithacus Software).

Since we observed the H2O2-induced cell death of SH-SY5Ycells, we next investigated whether these cells had undergoneapoptosis. We examined the nuclear morphologies of dying SH-SY5Y cells by using a fluorescent DNA-binding agent, DAPI.When the cells were incubated with 100 μM H2O2 for 24 h,they displayed the typical morphological features of apoptosis,that is, chromatin condensation in their nuclei (SupplementaryFigure S1B). We also examined the ability of H2O2 to cause DNAfragmentation in SH-SY5Y cells and showed that a DNA ladderappeared upon electrophoresis when genomic DNA preparedfrom H2O2-treated SH-SY5Y cells, but not from untreated cells,


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Figure 1 Protective effect of human L-PGDS against H2O2-induced neuronal cell death

(A) Elevation of L-PGDS gene expression in H2O2-treated SH-SY5Y cells. Cells were treated with various concentrations of H2O2 (0–100 μM) for 24 h. RNA was then extracted, and mRNA levels weremeasured by quantitative PCR. Results are means +− S.D. from three independent experiments. *P < 0.01 compared with untreated cells. (B) Protein levels of L-PGDS and actin in H2O2-treated cells.Crude cell extracts were prepared and protein (20 μg) was loaded into each lane for SDS/PAGE and Western blot analysis by the use of anti-L-PGDS and anti-actin antibodies. Band intensity wasnormalized to actin levels. Results are means +− S.D. from three independent experiments. *P < 0.01 compared with untreated cells. (C) Mutants of human L-PGDS used in the present study. Threemutant proteins, i.e. C89A/C167A/C186A, C89A/C186A and C65A/C167A L-PGDS, were prepared as described in the Experimental section. (D) Protective effects of human L-PGDS mutants againstH2O2-induced neuronal cell death. SH-SY5Y cells were incubated with 50 μM H2O2 in the absence or presence of various concentrations of L-PGDS mutants for 24 h. Results are means +− S.D.from five independent experiments. *P < 0.01 compared with in the absence of L-PGDS.

was used (Supplementary Figure S1C). These results suggest thatH2O2 potentially induced apoptosis in the SH-SY5Y cells.

Protective effect of human L-PGDS against H2O2-induced neuronalcell death

We first investigated the gene expression of human L-PGDS inSH-SY5Y cells. After the cells had been treated with H2O2, RNAwas isolated and gene expression was measured by performingquantitative PCR. L-PGDS was expressed in the SH-SY5Y cells,and its expression level was enhanced in an H2O2 concentration-dependent manner (Figure 1A). These results were consistentwith the results obtained from Western blot analysis by use ofanti-L-PGDS and anti-actin antibodies (Figure 1B).

Next, in order to investigate the effect of human L-PGDSon H2O2-induced neuronal cell death, we prepared threekinds of mutants, i.e. C89A/C167A/C186A, C89A/C186A andC65A/C167A L-PGDS (Figure 1C). As shown in Figure 1(D), a50 μM concentration of H2O2 reduced the viability of the neuronalcells by 50 +− 1.7% as compared with that of the untreatedones. However, C89A/C167A/C186A L-PGDS, in which Cys65

was retained, protected against H2O2-induced cell death in aconcentration-dependent manner. That is, the cell viability wasrecovered to 77 +− 1.5% of wild-type by C89A/C167A/C186A L-PGDS (5 μM). In addition, C89A/C186A L-PGDS (5 μM) alsoprotected against the H2O2-induced cell death to almost the samedegree as C89A/C167A/C186A L-PGDS (78 +− 2.2%). On theother hand, C65A/C167A L-PGDS (5 μM) did not show anyprotective effect against H2O2-induced cell death. These results,

taken together, indicate that L-PGDS expression was enhancedin the neuronal cells by the oxidative stress and that L-PGDShad the ability to protect against the neuronal cell death.Furthermore, the essential role for Cys65 was demonstrated.

Association of human L-PGDS level with cell viability inH2O2-treated neuronal cells

Next, we examined the protective effect of endogenous L-PGDS in H2O2-treated SH-SY5Y cells by employing thesiRNA-knockdown method to reduce the level of endogenousL-PGDS. First, to investigate the knockdown efficiency of L-PGDS expression by siRNA, we transfected SH-SY5Y cells witheither of two siRNAs, i.e. siRNA#1 or siRNA#2, for human L-PGDS. The mRNA level of L-PGDS was significantly decreasedby more than 50% by either siRNA as compared with that whentreated with the NC siRNA (Figure 2A). Moreover, either L-PGDSsiRNA suppressed the level of L-PGDS protein as comparedwith that obtained with the NC siRNA, although the actin level,an internal control, was not altered in any sample (Figure 2B).We used the more suppressive siRNA#1 (L#1) for further study.The cells were transfected with either L#1 or NC siRNA, andthen cultured with 100 μM H2O2 for 24 h. The cell viabilityof the NC siRNA-treated cells was decreased approximately43% as compared with that of untreated cells (Figure 2C).Moreover, when the siRNA-transfected cells were treated withH2O2, their viability was decreased further, approximately 62%,as compared with that of the untreated ones (Figure 2C).Furthermore, the LDH activity of the NC siRNA-treated cells



Figure 2 Suppression of L-PGDS expression enhances H2O2-inducedneuronal cell death

(A) siRNA-mediated suppression of L-PGDS gene expression. SH-SY5Y cells were transfectedwith either of two siRNAs for the L-PGDS gene or with NC siRNA. At 1 day after transfection,cells were further incubated in medium containing 100 μM H2O2 for 24 h. RNA was extractedfor quantitative PCR analysis. Results are means +− S.D. from three independent experiments.*P < 0.01 compared with NC siRNA. (B) Suppression of L-PGDS protein levels by L-PGDSsiRNAs. SH-SY5Y cells were knocked down by either of the two L-PGDS siRNAs as describedabove. Crude cell extracts (30 μg for L-PGDS and 10 μg for actin) was loaded on to each lane,and protein levels of L-PGDS and actin as an internal control were detected by Western blotanalysis. (C) Cell viability and LDH activity in siRNA-transfected cells. SH-SY5Y cells weretransfected with L-PGDS siRNA (L#1) or NC siRNA. At 1 day after transfection, 100 μM H2O2

was added and the cells were then cultured for a further 1 day. Results are means +− S.D. fromthree independent experiments. *P < 0.01, as indicated by the brackets.

was increased approximately 158 % as compared with that inthe untreated cells (Figure 2C). When the cells were transfectedwith L#1 siRNA and incubated with H2O2, their LDH activitywas slightly enhanced, being approximately 184 %, as comparedwith that in the untreated cells (Figure 2C). When we utilizedL-PGDS siRNA#2 instead of L#1 (Supplementary Figure S2 athttp://www.BiochemJ.org/bj/443/bj4430075add.htm), almost thesame results were obtained.

Then, to confirm that L-PGDS was involved in the prevention ofH2O2-induced cell death of SH-SY5Y cells, we expressed FLAG-tagged WT or C65A L-PGDS (C65A) protein in SH-SY5Y cells,and then incubated the cells in medium containing H2O2. First, weused Western blot analysis to detect the heterologous expressionof FLAG-tagged L-PGDS WT or C65A mutant protein in thecells. When the cells were transfected with L-PGDS WT or C65Amutant vector, both L-PGDS WT and C65A mutant proteins weredetected by using anti-FLAG monoclonal antibody (Figure 3A).In contrast, no corresponding signal was observed in the emptyvector-transfected cells, although the actin level was not altered inany sample (Figure 3A). Moreover, L-PGDS was expressed in theempty vector-transfected cells, but a stronger signal was detectedwhen the cells were transfected with either L-PGDS WT or C65Amutant vector (Figure 3A).

Next, cells were transfected with either the L-PGDS WT orC65A mutant vector and then cultured with 100 μM H2O2 for24 h. The viability of the empty vector-transfected cells in thepresence of H2O2 was decreased approximately 48% compared

Figure 3 Expression of L-PGDS prevents H2O2-induced neuronal cell death

(A) Heterologous expression of FLAG-tagged L-PGDS proteins. SH-SY5Y cells were transfectedwith empty vector (pFLAG-CMV-5a; Sigma), FLAG-tagged L-PGDS WT or L-PGDS C65Avector. At 1 day after transfection, cells were cultured for an additional 24 h in mediumcontaining 100 μM H2O2. Crude extracts were prepared and analysed by Western blotting(20 μg of protein/lane) with anti-FLAG, anti-L-PGDS or anti-actin monoclonal antibody.(B) Cell viability and LDH activity of L-PGDS-expressing SH-SY5Y cells. SH-SY5Y cellswere transfected with empty, L-PGDS WT or L-PGDS C65A vectors. At 1 day after transfection,100 μM H2O2 was added and the cells were cultured for a further 1 day, after which the cellviability and LDH activity were measured. Results are means +− S.D. from three independentexperiments. *P < 0.01, as indicated by the brackets.

with that in its absence (Figure 3B). Moreover, when the L-PGDSWT vector-transfected cells were treated with H2O2, their viabilitywas approximately 66% of that of the empty vector-transfectedones (Figure 3B). In contrast, no apparent preventive effect wasobserved when the cells were transfected with the L-PGDS C65Amutant vector (Figure 3B). Moreover, LDH activity was elevatedapproximately 167 % when the empty vector-transfected cellswere incubated with H2O2 (Figure 3B). It is noteworthy that,when the cells were transfected with the L-PGDS WT vector,LDH activity was decreased to approximately 136% of the valuefor the empty vector-transfected cells (Figure 3B). In contrast,no repressive effect on LDH activity was observed when thecells were transfected with the L-PGDS C65A mutant vector andtreated with H2O2 (Figure 3B). These results indicate that humanL-PGDS plays a critical role in the prevention of H2O2-inducedcell death of SH-SY5Y cells.

Titration of free thiol in L-PGDS with DTNB

As we considered Cys65 to be a key amino acid to reducethe neuronal cell death induced by H2O2, we investigatedthe thiol modification of cysteine residues of L-PGDSby DTNB. Figure 4(A) shows the free thiol titration ofthree human L-PGDS mutants, C89A/C186A (open circle),C89A/C167A/C186A (closed circle) and C65A/C167A L-PGDS(open triangle), by various concentrations of H2O2. In theabsence of H2O2, the free thiol concentration of C89A/C186AL-PGDS was 8.13 +− 0.32 μM. This concentration was decreasedin a concentration-dependent manner by treatment with H2O2.Moreover, when the cells were treated with 50 μM H2O2,




Figure 4 Titration of free thiols in L-PGDS with DTNB

(A) H2O2 concentration-dependency of free thiol concentrations of three L-PGDS mutants, C89A/C186A (�), C89A/C167A/C186A (�) and C65A/C167A (�) L-PGDS. Human L-PGDS mutants(5 μM) were incubated with various concentrations of H2O2 (0–100 μM) in PBS at 37◦C for 24 h. Free thiols were determined by the reaction with DTNB, which was added at 5-fold excess, andthe number of free thiols was calculated by the absorbance of the released TNB anion, as described in the Experimental section. (B) Time courses of free thiol concentrations of L-PGDS mutantsC89A/C186A (�), C89A/C167A/C186A (�) and C65A/C167A (�) L-PGDS in the presence of 50 μM H2O2 in PBS at 37◦C for 24 h. Results are means +− S.D. from three independent experiments.

Figure 5 Linear positive-ion MALDI–TOF-MS spectra of human L-PGDS

(A) MALDI–TOF-MS spectra of C89A/C167A/C186A L-PGDS treated (grey line) or not (black line) with 50 μM H2O2. A double-headed arrow shows the �M, which indicates the difference in m/zof each peak. (B) MALDI–TOF-MS spectra of C65A/C167A L-PGDS treated (grey line) or not (black line) with 50 μM H2O2.

the free thiol concentration reached a plateau correspondingto approximately 40% of that by the non-treatment withH2O2. In case of C89A/C167A/C186A L-PGDS, the free thiolconcentration was estimated to be 3.96 +− 0.14 μM, and wasdecreased in an H2O2 concentration-dependent manner. Free thiolalmost disappeared by the treatment with 50 μM H2O2. In the caseof C65A/C167A L-PGDS, however, no free thiol was detected,even in the absence of H2O2. Figure 4(B) shows the time course ofchanges in the free thiol concentration of L PGDS mutants causedby treatment with 50 μM H2O2. The free thiol concentrationsof both C89A/C186A (open circle) and C89A/C167A/C186A(closed circle) L-PGDS mutants were decreased in a time-dependent manner after the initiation of treatment with H2O2, andreached a plateau at 12 h. These results show that H2O2 reactedwith the thiol of only Cys65 of human L-PGDS, but not with thatof Cys167.

Thiol modification of Cys65 of human L-PGDS by S-oxidation

To assess the oxidative status of Cys65 of L-PGDS, wesubjected C89A/C167A/C186A and C65A/C167A L-PGDS tolinear positive-ion MALDI–TOF-MS analysis. The MALDI–TOF-MS spectrum of C89A/C167A/C186A L-PGDS showed apeak at m/z 18746, whereas by the treatment with 50 μM H2O2,

C89A/C167A/C186A L-PGDS showed a peak at m/z 18778 whichcorresponded to an increase in the mass of 32 Da (Figure 5A).On the other hand, C65A/C167A L-PGDS, lacking the active-centre Cys65, showed a peak at m/z 18778 and did not showany change in molecular mass by treatment with 50 μM H2O2

(Figure 5B). These results demonstrate that Cys65 oxidized byH2O2 to its sulfinic acid (-SO2H) form, i.e. Cys65 of human L-PGDS is involved in the scavenging of ROS.

Tryptophan fluorescence quenching of oxidized L-PGDS by smalllipophilic molecules

We reported previously that L-PGDS is an extracellulartransporter protein for small lipophilic molecules such asretinoids, BV, bilirubin and thyroid hormones [11,40,41].So, to investigate whether the oxidized L-PGDS possessedthe ability to bind small lipophilic molecules, we measured thefluorescence quenching of intrinsic tryptophan residues of L-PGDS with various concentrations of BV or all-trans-RA.Human L-PGDS contains three tryptophan residues, locatedat positions 43, 54 and 112, and these tryptophan residuesare considered to contribute to the fluorescence quenchingof L-PGDS by binding with small lipophilic molecules.Both untreated and oxidized C89A/C167A/C186A L-PGDS



Figure 6 Tryptophan fluorescence quenching of oxidized L-PGDS by BV or all-trans-RA

The relative fluorescence intensities of C89A/C167A/C186A L-PGDS treated (�) or not (�) with 50 μM H2O2 were obtained against the various concentrations of BV (A) and RA (B). Results aremeans +− S.D. from three independent experiments.

showed BV concentration-dependent fluorescence quenching(Figure 6A). In the presence of 10 μM BV, both fluorescenceintensities decreased to less than 10% of that in the absence ofBV. From the quenching curves, the Kd values of BV of untreatedand oxidized C89A/C167A/C186A L-PGDS were calculated tobe 91 +− 10 μM and 110 +− 20 μM respectively. In the case ofRA, again both untreated and oxidized C89A/C167A/C186A L-PGDS showed fluorescence quenching, which occurred in an RAconcentration-dependent manner, and the Kd values calculatedwere 1520 +− 20 μM and 770 +− 320 μM respectively (Figure 6B).These results show that oxidized L-PGDS retained its ability tobind small lipophilic molecules.

Critical roles for amino acid residues around Cys65 of humanL-PGDS in scavenging ROS

To investigate the difference in the reactivity toward H2O2 betweenhuman and mouse L-PGDS, we constructed a structural model ofWT human L-PGDS (Figure 7A). The model obtained for humanL-PGDS had 97.9% of its amino acid residues within the favouredor allowed regions of the Ramachandran ϕ–ψ plots, indicating agood quality of the predicted model. Figures 7(B) and 7(C) showmagnifications of the area around Cys65, which is S-oxidized byROS, in human and mouse L-PGDS. In human L-PGDS, Ser63 andSer67 were respectively arranged above and below Cys65, whereasin the mouse L-PGDS, Tyr63 and Thr67 were respectively situatedabove and below Cys65. The other amino acid residues aroundCys65 in mouse L-PGDS, such as Trp43, Ser45, Leu48, Ser81, Phe83

and Arg85, were located in the same position as in human L-PGDS.So we prepared Y63S/T67S/C89A/C186A mouse L-PGDS, i.e.a human-type mouse L-PGDS, and compared the efficiency asa ROS scavenger of human, mouse and human-type mouse L-PGDSs. Figure 7(D) shows the changes in free thiol concentrationof C89A/C167A/C186A human (open circle), C89A/C186Amouse (closed circle) and Y63S/T67S/C89A/C186A mouse(open triangle) L-PGDS incubated with various concentrationsof H2O2. In all cases, the free thiol concentrations decreasedin a concentration-dependent manner by treatment with H2O2.From these curves, the IC50 values of H2O2 against human,mouse and human-type mouse L-PGDS were calculated to be12.1 +− 0.7, 38.4 +− 5.7 and 19.3 +− 1.9 μM respectively (GraFitVersion 3.0). These results indicate that human L-PGDS showedhigher reactivity towards H2O2 than mouse L-PGDS and thatthe amino acid modification around Cys65 in the mouse L-PGDScorresponding with human L-PGDS led to increased reactivity

towards H2O2. Thus we concluded that the environment aroundCys65 in L-PGDS is essential for the reaction with H2O2.

DISCUSSION

Apoptosis is the process of cell death characterized bycell shrinkage, nuclear condensation, DNA fragmentation andmembrane blabbing. Neurons are very delicate, being susceptibleto oxidative stress, and such stress is tightly associated withcellular damage in a variety of human diseases includingneurodegenerative disorders [42,43]. ROS, including H2O2,superoxide anion and hydroxyl radicals, damage biologicalmolecules such as lipids, proteins and DNA, which leads toapoptotic or necrotic cell death [44]. H2O2 has been extensivelyutilized as an inducer of oxidative stress in in vitro cell culturesystems [45]. The treatment of cells with H2O2 results in animbalance in energy metabolism and harmful effects of hydroxyland peroxyl radicals on membrane lipids and proteins. Muchexperimental evidence indicates the occurrence of oxidative stressin the pathogenesis of various neurodegenerative diseases [46].Thus the removal of excessive ROS or suppression of theirgeneration is critical for the prevention of oxidative cell deathin various diseases.

L-PGDS was first identified in the rat brain as a PGD2-synthesizing enzyme. L-PGDS was reported to be one of themost abundant proteins in human CSF and is expressed inthe mammalian central nervous system in locations such as thechoroid plexus and leptomeninges, and in oligodendrocytes[10, 21]. Interestingly, L-PGDS, as a protein of the lipocalinfamily, binds small lipophilic molecules, such as retinal andRA [11,40], BV, bilirubin [13,41,47] and gangliosides [48].Therefore L-PGDS has been considered as a unique bifunctional,i.e. multifunctional, protein acting as both a PGD2-synthesizingenzyme and a lipophilic molecule-binding protein, i.e. a lipocalin.In the present study, we found a novel property of L-PGDS asa ROS scavenger in vitro. L-PGDS expression was increasedin the H2O2-treated SH-SY5Y cells (Figures 1A and 1B), anda reduction in the L-PGDS level was seemed to correlatewith an increase in H2O2-induced cell death (Figure 2C).Prostaglandins are known to be involved in the regulation ofneuronal cell death. 15d-PGJ2 (15-deoxy-�12,14-prostaglandin J2),one of the PGD2 metabolites, has a potential to suppress H2O2-induced cell death of mouse neuronal N18D3 cells [49] and ratPC12 cells [50]. In contrast, Kondo et al. [51] reported that15d-PGJ2 induced the apoptotic cell death of SH-SY5Y cells.Therefore the roles for 15d-PGJ2 in the regulation of neuronal



Figure 7 Environment around Cys65 in human and mouse L-PGDS proteins

(A) Modelled structure of WT human L-PGDS. Ribbon representation of human L-PGDS showsthe typical lipocalin fold including a single eight-stranded antiparallel β-sheet (cyan) and a longα-helix (red) with two free thiol groups due to Cys65 and Cys167 and a disulfide bond betweenCys89 and Cys186 (yellow). Cys65 is considered to play an important role as an active centre forthe catalytic function of L-PGDS. The side chains of cysteine residues are shown as green sticks.(B) Environment around Cys65 in WT human L-PGDS. The side chain of the residues aroundCys65 is shown as a stick model. Residues are coloured as follows: tryptophan (beige), serine(blue), leucine (purple), phenylalanine (pink) and arginine (orange). (C) Environment aroundCys65 in C89A/C167A mouse L-PGDS [12]. The residues around Cys65 are shown as stickscoloured as in (B). The side chain of threonine and of tyrosine are shown as light green and darkgreen sticks respectively. (D) Changes in free thiol concentration of C89A/C167A/C186A human(�), C89A/C186A mouse (�) and Y63S/T67S/C89A/C186A mouse (�) L-PGDS incubatedwith various concentrations of H2O2 in PBS at 37◦C for 24 h. Results are means +− S.D. fromthree independent experiments.

cells death are controversial, indicating the requirement of furtherstudy for elucidation of the roles for PGs on the regulation ofneuronal cell death. In the present study, exogenously addedprostaglandins, including PGD2 which is non-enzymaticallymetabolized to 15d-PGJ2, had no effect on the viability ofH2O2-treated SH-SY5Y cells (Supplementary Figure S3 athttp://www.BiochemJ.org/bj/443/bj4430075add.htm). Moreover,the L-PGDS C65A mutant, lacking PGD2-synthesizing activity,could not decrease the H2O2-induced cell death of SH-SY5Y cells(Figures 1D and 3B). Thus the PGD2-synthesizing activity of L-PGDS was not related to the protection against H2O2-induced celldeath of SH-SY5Y cells, indicating that the ability of L-PGDSto afford this protection was due to some other feature of thisprotein. As shown in Figure 1(D), L-PGDS suppressed the H2O2-induced death of SH-SY5Y cells in a dose-dependent manner byscavenging ROS. So far, several studies have been reported relatedto the protective effects of lipocalin proteins against oxidativestress. Tear lipocalin has been shown to bind lipid-peroxidationproducts and also to be up-regulated in human NT2 neuronalprecursor cells during oxidative stress [52]. α1-Microglobulinbinds toxic haem [53] and has antioxidant catalytic activities [54].Apolipoprotein D is an acute-response protein with a protectiveeffect on the brain, and its expression is enhanced by oxidativestress in the brain [55]. However, the molecular mechanism of thescavenging effects of lipocalins remains unclear.

In the present study, the ROS-scavenging mechanism effectedby L-PGDS was clearly shown to be the oxidation of the thiolgroup of its Cys65, which resulted in the conversion of this cysteineresidue into cysteine sulfinic acid (-SO2H). This is the first reportto clarify the ROS-scavenging mechanism by lipocalin familyproteins. In addition, the S-oxidized L-PGDS still possessed thebinding property for small lipophilic molecules such as BV andRA (Figure 6), and, conversely, the complex of L-PGDS and RAalso possessed ROS-scavenging capability (Supplementary Fig-ure S4 at http://www.BiochemJ.org/bj/443/bj4430075add.htm).Furthermore, human L-PGDS exhibited higher reactivity towardsH2O2 than did mouse L-PGDS, and the amino acid modificationaround Cys65 in mouse L-PGDS, making it correspond tohuman L-PGDS, led to increased reactivity towards ROS(Figure 7). These results suggested to us that the specific aminoacid composition near the active site of human L-PGDS wasresponsible for the more prominent protective action against ROSby the human L-PGDS than by the mouse one. L-PGDS is not onlya multifunctional protein, but also a multitasking protein able tosimultaneously perform the crucial functions of both ROS scaven-ging and the binding of small lipophilic molecules. Thus L-PGDSis a unique multitasking protein in the lipocalin family proteins.

In summary, we found that L-PGDS plays protectively againstthe H2O2-mediated apoptosis in human neuronal SH-SY5Ycells by scavenging ROS. Free radicals produced from H2O2

bind to the active centre of L-PGDS, indicating that L-PGDSmight be useful for the suppression of oxidative stress-mediatedneurodegenerative diseases.

AUTHOR CONTRIBUTION

Mao Yamada, Ko Fujimori and Takashi Inui designed the research; Mao Yamada, AyanoFukuhara, Ko Fujimori, Hidemitsu Nakajima and Takashi Inui performed the biochemicalresearch; Yuya Miyamoto performed the structural modelling; Toshihide Kusumotoperformed the MS analysis; and Ko Fujimori and Takashi Inui wrote the paper.

ACKNOWLEDGEMENT

We thank Nobuko Uodome (Osaka Bioscience Institute, Osaka, Japan) for technicalassistance.





FUNDING

This work was supported in part by Grants-in-Aid for Scientific Research (B) and (C) [grantnumbers 17300165 and 21500428 (to T.I.)], by Grants-in-Aid for Scientific Research onInnovative Areas [grant number 21200076 (to T.I.)] and by a Special Research Grant (toT.I.) from Osaka Prefecture University.

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Received 24 October 2011/3 January 2012; accepted 16 January 2012Published as BJ Immediate Publication 16 January 2012, doi:10.1042/BJ20111889


Biochem. J. (2012) 443, 75–84 (Printed in Great Britain) doi:10.1042/BJ20111889

SUPPLEMENTARY ONLINE DATALipocalin-type prostaglandin D synthase protects against oxidativestress-induced neuronal cell deathAyano FUKUHARA*1, Mao YAMADA*1, Ko FUJIMORI†, Yuya MIYAMOTO*, Toshihide KUSUMOTO*, Hidemitsu NAKAJIMA‡ andTakashi INUI*2

*Laboratory of Biological Macromolecules, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan, †Laboratory of Biodefenseand Regulation, Osaka University of Pharmaceutical Sciences, Takatsuki, Osaka 569-1094, Japan, and ‡Laboratory of Veterinary Pharmacology, Graduate School of Life andEnvironmental Sciences, Osaka Prefecture University, Izumisano, Osaka 598-8531, Japan

Figure S1 H2O2 induces apoptosis in human neuronal SH-SY5Y cells

(A) Cell viability in the presence of various concentrations of H2O2 (3–100 μM) is shown as a percentage of that of untreated cells (0 μM). Results are means +− S.D. from five independentexperiments. *P < 0.01 compared with untreated cells. (B) H2O2-induced apoptosis of SH-SY5Y cells. The cells were cultured in the absence (upper panel) or presence of H2O2 (100 μM; lowerpanel), and assessed by DAPI staining. Arrowheads show the chromatin condensation within nuclei of SH-SY5Y cells. (C) Cells were incubated as indicated in (B). DNA was prepared and utilizedfor agarose gel electrophoresis. The DNA ladder was detected by staining with ethidium bromide.

1 These authors contributed equally to this work.2 To whom correspondence should be addressed (email [email protected]).


A. Fukuhara and others

Figure S2 Suppression of L-PGDS expression enhances H2O2-inducedneuronal cell death

Cell viability and LDH activity in siRNA-transfected cells. SH-SY5Y cells were transfected withL-PGDS siRNA (L#2) or NC siRNA. At 1 day after transfection, 100 μM H2O2 was added and thecells were then cultured for a further 1 day. Results are means +− S.D. from three independentexperiments. *P < 0.01, as indicated by the brackets.

Figure S3 Prostaglandin production was not associated with H2O2-inducedapoptotic death of SH-SY5Y cells

(A) Cells were cultured with each prostaglandin (PG; 10 μM; Cayman Chemical) and H2O2

(100 μM) for 24 h. Results are means +− S.D. from three independent experiments. V, vehicle.(B) SH-SY5Y cells were incubated with 100 μM H2O2 for 24 h together with each inhibitor, i.e.cytosolic phospholipase A2 inhibitor (Ci; 10 mM; Cayman Chemicals), indomethacin (I; 2 μM;Cayman Chemicals) and for cyclo-oxygenases SC-560 (SC; 100 pM; Cayman Chemicals) forCOX-1, NS398 (NS; 1 μM; Cayman Chemicals) for COX-2, and AT-56 (AT; 1 μM; CaymanChemicals) for L-PGDS. Results are means +− S.D. from three independent experiments. V,vehicle.

Received 24 October 2011/3 January 2012; accepted 16 January 2012Published as BJ Immediate Publication 16 January 2012, doi:10.1042/BJ20111889

Figure S4 Titration time courses of free thiols in C89A/C167A/C186A humanL-PGDS and C89A/C167A/C186A human L-PGDS–RA complexed with DTNB

C89A/C167A/C186 human L-PGDS (�; 10 μM) was incubated with 10 μM RA in PBS at37◦C for 24 h. After incubation, the C89A/C167A/C186A human L-PGDS–RA complex (�; 5μM) was reacted with 50 μM H2O2 in PBS at 37◦C. Results are means +− S.D. from threeindependent experiments.


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