Báo cáo Y học: Isolation and characterization of a thioredoxin-dependent peroxidase from Chlamydomonas reinhardtii doc

Isolation and characterization of a thioredoxin-dependent peroxidase from Chlamydomonas reinhardtii Aymeric Goyer 1 , Camilla Hasleka Ê s 2 , Myroslawa Miginiac-Maslow 1 , Uwe Klein 2 , Pierre Le Marechal 3 , Jean-Pierre Jacquot 4 and Paulette Decottignies 3 1 Institut de Biotechnologie des Plantes, Universite  Paris-Sud, Orsay Cedex, France; 2 Department of Biology, Division of Molecular Biology, University of Oslo, Blindern, Oslo, Norway; 3 IBBMC, Universite  Paris-Sud, Orsay Cedex, France; 4 Interaction Arbres)Microorganismes INRA/Universite  Nancy, Vandoeuvre Cedex, France All living o rganisms contain redox systems i nvolving thior- edoxins (Trx), proteins featuring an extremely conserved and reactive active site that perform thiol-disul®de interch anges with disul®de bridges o f target p roteins. In photosynthetic organisms, numerous isoforms of Trx coexist, as revealed b y sequencing of Arabidopsis genome. The speci®c functions of many o f them are s till unknown. In an attempt to ®nd new molecular targets of Trx in Chlamydomonas reinhardtii,an anity column carrying a cytosolic Trx h mutated at the less reactive cysteine of its active site was used to trap Chlamydomonas proteins that form mixed disul®des with Trx. T he major protein bound to the column w as identi®ed by amino-acid sequencing and mass spectrometry as a thioredoxin-dependent 2Cys peroxidase. Isolation and sequencing of its gene revealed t hat this peroxidase is most likely a c hloroplast protein with a high homology to plant 2Cys peroxiredoxins. It is shown that t he Chlamydomonas peroxiredoxin ( Ch-Prx1) is active with various thioredoxin isoforms, functions as an antioxidant toward r eactive oxy- gen species (ROS), and protects DNA against ROS-induced degradation. Expression of the p eroxidase gene i n Chlamydomonas was found to be regulated b y light, oxygen concentration, and redox state. The data suggest a role for t he Chlamydomonas P rx in R OS detoxi®cation i n the chloroplast. Keywords: Chlamydomonas; peroxiredoxin; thioredoxin; redox signaling; oxidative stress. 1 Peroxiredoxins (Prx) form a ubiquitous group of peroxid- ases found in bacteria [1], yeast [2,3], animals [4], and, more recently, in higher plants [5±7]. Prx can be classi®ed according to the number of conserved cysteine residues: the 2Cys-Prx subgroup, and 1Cys-Prx subgroup contain two and one conserved cysteines, respectively. 2Cys-Prx proteins are reduced by the AhpF protein in bacteria, and by the thioredoxin/thioredoxin reductase system in yeast and animals, w hile 1Cys-Prx may be r educed by a small thiol molecule such as glutathione. R ecently, 1Cys-Prx has been identi®ed i n y east and Arabi dopsis, and has been shown to be thioredoxin-dependent and function in a similar manne r to 2 Cys-Prx [ 8,9]. 2Cys-Prx catalyzes, in vitro, the red uction of alkyl hydroperoxide and h ydrogen peroxide. These enzymes exist as homodimers. Each subunit contains the two conserved cysteines that are essential residues for the reduction of peroxides. The N-terminal cysteine is ®rst oxidized by a peroxide to sulfenic acid (Cys-SOH), which rapidly reacts with the C-terminal cysteine of the other s ubunit to form an intermolecular disul®de [10]. In animals, yeast, a nd plants, the disul®de is reduced via a thiol/disul®de redox inter- change with reduced thioredoxin (Trx), thus regenerating an active peroxidase. The present study was aimed at setting up an af®nity chromatography column for speci®c trapping of proteins that react w ith Trx, b ased on their a bility to f orm mixed disul®de-linked adducts with a single cysteine mutant thioredoxin. The system allowed u s to purify and identify for the ®rst time a 2Cys-Prx p rotein (Ch- Prx1 ) from t he green a lga Chlamydomonas reinhardtii. The puri®ed protein was characterized by its antioxidant properties towards reactive oxygen species (ROS), p rotection of DNA against degradation, its p eroxidase a ctivity, and its ability t o u se different thioredoxin isoforms as hydrogen donors. To better understand the function of Ch-Prx1 in vivo, we isolated the cDNA and the Ch-Prx1 gene, and examined the regulation of its expression by different cultur e conditions. EXPERIMENTAL PROCEDURES Algal strains and culture conditions The C. reinhardtii strain CW15 (137c, mt+, cw15, no cell wall present) and strain CC 125 were obtained from the Chlamydomonas Genetics Center at Duke University, NC, USA. Cells were grown in a pho toautotrophic minimal medium (HSM 2 ; [ 11]). CW15 cultures were grow n in ¯asks at 25 °C under c ontinuous stirring and bub bling with 5 % CO 2 enriched air. Light intensity was 3 00 lmolám )2 ás )1 at the level of the culture ¯asks. CC125 cells were grown in 200-mL Correspondence to M. Miginiac-Maslow, Institut de Bio t echnologie des P lantes, UMR CNRS 8618, Baà t. 630, Universite  Paris-Sud, 91405 Orsay Cedex, France. Fax: + 33 1 69 15 34 23, E-mail: miginiac@ibp.u-psud.fr Abbreviations:Nbs 2 ,5,5¢-dithiobis(2-nitrobenzoic acid); Prx, perox- iredoxin; t-BOOH, tertiobutyl hydroperoxide; Trx, thioredoxin; ROS, reactive oxygen species; NTR, NADPH-dependent thioredoxin reductase; DCMU, 3-(3¢,4¢-dichlorophenyl)-1,1-dimethyl urea; DBMIB, 3-methyl-6-isopropyl-p-benzoquinone. (Received 13 July 2001, revised 29 October 2001, accepted 31 October 2001) Eur. J. Biochem. 269, 272±282 (2002) Ó FEBS 2002 tubes at 32 °C. Cultures were kept in a 12-h light/dark regime (light intensity 150 lmolám )2 ás )1 ) and bubbled with 2% CO 2 -enriched air. Cell density was adjusted daily at the onset of light with fresh medium to  2 ´ 10 6 cellsámL )1 . Puri®cation of Ch-Prx1 Puri®cation of the native protein from Chlamydomonas cells. Chlamydomonas CW15 ce lls were grown to a cell density > 5 ´ 10 6 cells pe r mL. Ce lls were pelleted, resuspended in 30 m M Tris/HCl pH 7.9, and broken by two cycles o f freeze-thawing i n liquid n itrogen. Broken cells were centrifuged for 30 min at 15 000 g and the supernatant was adjusted to 2% (w/v) streptomycin sulfate b y addition of a 20% solution. After incubation for 20 min at 4 °C, the suspension was centrifuged at 15 000 g for 30 m in to pellet precipitated nucleic acids. The supernatant was adjusted to 95% (w/v) a mmonium sulfate a nd incubated for 20 min at 4 °C. The suspension was centrifuged at 15 000 g for 30minandthepelletwasresuspendedin10mL30m M Tris/HCl, pH 7.9, and dialyzed against 5 L 30 m M Tris/ HCl, pH 7.9. The protein solution was loaded onto an af®nity column m ade of a mutated Chlamydomonas cytosolic h-type thioredoxin (C39S mutant) grafted on a CNBr activated sepharose support and equilibrated with 30 m M Tris/HCl, p H 7.9. Covalent coupling of this thio- redoxin to activated Sepharose was carried out essentially as recommended b y the supplier of the Sepharo se (Amersham Pharmacia). The C39S mutant thioredoxin lacks one cysteine residue and has only its most reactive a ctive-site cysteine left (Cys36). Site-directed mutagenesis of Trx h and recombinant protein puri®cation was carried out as described previously [12]. T o avoid the formation of thio- redoxin dimers, the single-cysteine mutant thioredoxin was pretreated with a 40-fold excess of 5,5¢-dithiobis(2-nitro- benzoic acid) (Nbs 2 ; Pierce) before coupling. The deriv- atized thioredoxin was treated with dithiothreitol to eliminate t he 5-mercapto-2-nitrobenzoate 3 adduct, and was washed extensively to remove the dithiothreitol. After loading on the column, the proteins were eluted with 15 mL of 1 m M dithiothreitol in 30 m M Tris/HCl, pH 7.9. The e luted p roteins w ere d ialyzed against 30 m M Tris/HCl, pH 7.9 and re-applied on the af®nity column. P roteins were eluted with 15 mL of 1 m M dithiothreitol in 30 m M Tris/ HCl, pH 7.9, and analyzed by SDS/PAGE. Puri®cation of the recombinant Ch-Prx1 expressed in Escherichia coli. BL21 (DE3) E. coli strain was trans- formed with a pET-8c vector (Stratagene) containing a 600-bp fragment obtained by RT-PCR (see below) corresponding to the coding sequence of C h-Prx1 without the chloroplast transit peptide. Cells were grown in 5 L of M9 medium up to D 600  0.5, and recombinant protein expression was induced by the addition of 100 l M isopropyl t hio-b- D -galactoside. A fter induction, cells were grown for 18 h at 37 °C, pelleted, and r esuspended in 10 mL 30 m M Tris/HCl, pH 7.9, 1m M EDTA, 500 l M phenylmethanesulfonyl ¯uoride, and 14 m M 2-mercapto- ethanol. Cells were broken in a French press a t 60 MPa 4 . Broken cells were centrifuged for 30 min at 20 000 g and the supernatant was adjusted to 2% (w/v) streptomycin sulfate by a ddition of a 20% solution. After incubation for 20 min at 4 °C, the s uspension w as centrifuged at 20 000 g for 3 0 min to precipitate the nucleic acids. The supernatant was subjected to 35±80% (w/v) ammonium sulfate frac- tionation. After centrifugation at 20 000 g for 30 min, the pellet w as re su spend ed i n 10 mL 3 0 m M Tris/HCl, pH 7.9, dialyzed against 5 L o f 30 m M Tris/HCl, pH 7 .9, and the Ch-Prx1 protein was puri®ed as described above on a C39S Trx h af®nity column equilibrated with 3 0 m M Tris/HCl, pH 7.9. Polyacrylamide gel electrophoresis Denaturating [4% ( w/v) SDS] electrophoresis was carried out on 10% polyacrylamide gels. G els were st ained w ith CoomassieBlue(2.5gáL )1 ). Tryptic digestion, separation of the tryptic peptides, analysis by sequencing and MALDI-TOF mass spectrometry Further puri®cation of Ch-Prx1 was achieved, prior to digestion, by RP-HPLC o n a 4.6 ´ 25 cm Vydac C 4 (30 A Ê ) column. C h-Prx1 was eluted with a linear gradient from 2 8 to 70% acetonitrile in 0.1% tri¯uoroacetic acid over 30 min at a ¯ow rate of 1 mLámin )1 . Tryptic digestion was performed for 20 h at 3 7 °Cin0.1 M NH 4 HCO 3 with sequence grade trypsin (Boehringer). The peptides were separated b y RP-HPLC on a 0 .21 ´ 25 cm Vydac C18 (300 A Ê ) column. Peptides were eluted with a linear g radient from 0 to 70% acetonitrile in 0.1% tri¯uoroacetic acid, over 90 min, at a ¯ow rate of 0.3 m Lámin )1 . A bsorbance w as recorded at 215 nm and 280 nm. Peptides were sequenced using an Applied Biosystems model 476 A sequencer equipped with an online phenylthiohydantoin amino-acid analyser. F or MALDI-TOF analysis, 1 lL of tryptic digest or HPLC-puri®ed peptides was mixed with 1 lLof saturated solution of a-cyano-4-hydroxycinnamic acid in 50% acetonitrile, 0.3% tri¯uoroacetic a cid. Samples were loaded into MALDI-TOF spectrometer (Perseptive B io- systems, Voyager STR-DE) equipped with a nitrogen laser (337 nm). Spectr a were obtained in re¯ectron mode using delayed extraction. RT-PCR Ch-Prx1 cDNA was isolated by RT-PCR. In this approach, primers used in cDNA synthesis w ere designed based on the amino-acid sequence of N- and C-terminal peptides, a s determined by the p rocedure described above. First-strand cDNA was ®rst synthesized from total RNA with M-MuLV reverse t ranscriptase (Life T echnologies). The reaction mixture c ontained i n a volume of 20 lL: 0.9 lg heat-denatured Chlamydomonas total RNA, 1 ´ RT buffer, 1m M dNTPs, 20 m M dithiothreitol, 100 U reverse tran- scriptase (RT), and 1 l M of the degenerated reverse primer 5¢-GCGGATCCTTA(G/C)ACGGCGGCGAAGTAC TCC-3¢. After reverse transcription for 30 min at 42 °C, the ®rst-strand cDNA was ampli®ed in a PCR performed under the following conditions: 5 min at 96 °C; 39 cycles (94 °C for 1 min, 64 °C for 2 min, and 72 °Cfor2min).In addition to the above degenerated reverse primer two direct primers 5¢-AACCATGGCCTCCCACGCCGAGA AGCC(G/C)CTG-3¢ and 5¢-AACCATGGCCAGCCAC Ó FEBS 2002 Peroxiredoxin from Chlamydomonas (Eur. J. Biochem. 269) 273 GCCGAGAAGCC(G/C)CTG-3¢ were used. PCR products were separated b y electrophoresis on a 0.8% agarose g el. A 600-bp fragment was puri®ed by using the nucleospin extract k it (Macherey±N agel), then digested by NcoIand BamHI, and cloned i nto the pET-8c vector. Screening of a cDNA library A kgt11 cDNA library of Chlamydomonas was a gift from M. Goldschmidt-Clermont (Universite  de Gene Á ve, S witzer- land). The Ch-Prx1 coding sequence obtained b y R T-PCR was used to screen 200 000 plaque forming units of the kgt11 library. Colony hybridization was performed at 64 °C in buffer containing 0.5 M NaHPO 4 ,pH7.2,1m M Na 2 EDTA, 7% SDS, and 1% BSA. After p uri®cation of positive clones, k DNA was extracted and puri®ed as described in [ 13]. cDNA inserts were excised by a digestion with EcoRIandclonedintotheEcoR I site of the SK + Bluescript plasmid (Stratagene). Screening of a gene library A B AC library of genomic C. reinhardtii DNA (Genome Systems Inc., St Louis, USA, product FBAC-8417) spotted at high density on a nylon ® lter was used to isolate a clone containing the c omplete Ch-Prx1 gene. Two heterologous primers, (5 ¢-GACTTCACCTTCGTGTGCC CCACCGAG-3¢ and 5¢-GGGGTCGATGATGAACAG GCCGCG-3¢), designed from the conserved 5¢ and 3¢ sequences of known 2Cys peroxiredoxin genes and optimized for the codon usage of Chlamydomonas,were used to amplify by PCR from genomic Chlamydomonas DNA a fragment (  780 bp) of the Chlamydomonas peroxiredoxin gene. The PCR fragment was cloned, sequenced, and used as a probe to identify a Chlamydo- monas BAC clone that contains the Ch -Prx1 gene sequence. Two BAC clones that strongly hybridized to the probe on the ® lter were am pli®ed and used to i solate the complete Ch-Prx1 gene sequence using conventional S outhern ana- lyses, subcloning, and sequencing techniques [ 13]. Sequencing of cDNA and genomic DNA The BigDye T erminator Cycle Sequencing 5 Kit (Perkin- Elmer) or the Thermo Sequenase Radiolab eled Terminator Cycle Seq uencing Kit (United States Biochemicals) were used to sequence the Ch-Prx1 cDNA and t he Ch-Prx1 gene, respectiv ely. RNA isolations RNA for RT-PCR and for northern a nalyses was islolated by alternative m ethods. I n t he ®rst method,  30 million cells were pelleted by centrifugation (3000 g,5min).The pellet was immediately resuspended in 1 mL TRIzol reagent (Gibco BRL) and polysaccharides, membranes, and unlysed cells were eliminated by centrifugation (12 000 g,10min). The supernatant was treated as instructed by the supplier. The dried pellet was resuspended in 20 lL of milliQ (Millipore) puri®ed sterile water. The second method followed e ssentially a protocol described previously [14,15]. PolyA + RNA was isolated with magnetic oligo d(T) b eads (Dynal) f ollowing the protocol of t he supplier. Southern and Northern blot analysis For Southern analyses, genomic DNA was prepared following the protocol described previously [16]. DNA was digeste d w ith a ppropriate restri ction e nzymes, s ize- fractionated on a 0.8% a garose g el, and transferred t o a Hybond N + (Amersham Pharmacia) o r Z etaProbe (Bio- Rad) nylon membrane. Hybridization with t he 32 P-radio- labeled cprx probe (4 ´ 10 6 c.p.m.) and washin g of t he gel blot at 65 °C was carried out as described previo usly [ 17]. The membrane was exposed to X-ray ® lm at )20 °Cor, when using an intensifying s creen, at )80 °C. RNA for Northern analyses was isolated as described above and separated in a 1.3% agarose/formaldehyde gel. The RNA was b lotted to a nylon membrane (ZetaProbe, Bio-Rad) by alkaline t ransfer, U V-crosslinked, and hybrid- ized to the random primer radiolabeled Ch-Prx1 cDNA probe for 2 4 h. T he cDNA p robe was g enerated using biotinylated single stranded template bound to magnetic streptavidin-coated beads (Dynal) in a speci®c priming reaction [18]. The speci®c primer used for the reaction was the downstream primer used in the RT-PCR reaction. After washing [17], the membrane was exposed t o X-ray ®lm with an intensifying screen at )80 °Cfor 2days. Antioxidant activity of the Ch-Prx1 protein The antioxidant activity of the Ch-Prx1 protein was tested in a DNA-cleavage assay modi®ed after [19,20]. Bluescript plasmid DNA (2 lg) was exposed to a mixed function oxidation system containing 0.32 m M dithiothreitol and 3 l M FeCl 3 . The reaction contained various amounts of concentrated Ch-Prx1 p rotein (5±20 l M ), and was initiated 30 min before addition of the DNA. Control reactions were performed without Ch-Prx1 protein and with BSA (400 lgámL )1 ). The reactions were stopped by adding 3.3 m M EDTA and analyzed on an agarose gel. Thioredoxin-dependent peroxidase activity of Ch-Prx1 Peroxidase activity assays were initiated by the addition of H 2 O 2 (500 l M )ort-b utyl hydroper oxide (t-BOOH) (500 l M )to1mL30m M Tris/HCl, pH 7.9, reaction medium containing 197 n M A. thaliana NADPH Trx reductase (NTR), 180 l M NADPH, 5.7 l M Trx and 2.4 l M Prx. The reaction was monitored s pectrophotomet- rically by following the decrease in absorbance at 340 nm at 30 °C. Recombinant A. thaliana NTR was puri®ed as described p reviously [21]. C hloroplastic thioredoxins m and ffromspinachwereakindgiftofP.Schu È rmann (University of Neuchaà t el, Switzerland). Thioredoxins m and h from Chlamydomonas were puri®ed as described p reviously [22]. An alternative assay, avoiding t he need of a t hioredoxin reductase, was also used, based on colorimetric determina- tion of hydrogen peroxides or alkyl hydroperoxides with the PeroXOquant kit (Pierce) following the supplier's recom- mendations. Ch-Prx1 (43.8 l M ) was incubated w ith 400 l M dithiothreitol, 16.6 l M Trx a nd 500 l M t-butyl hydroper- oxide in 50 lLof30m M Tris/HCl, pH 7.9, buffer. The quantity o f t-BOOH was measured on 5 lL a liquots added to a spectrophotometer cuvette containing 500 lLof PeroXOquant medium. The activity was estimated from the decrease in absorbance at 595 nm. 274 A. Goyer et al. (Eur. J. Biochem. 269) Ó FEBS 2002 RESULTS Isolation of a 2Cys-Prx by using a single cysteine mutant of Chlamydomonas Trx h In an attempt to isolate new T rx targets in Chlamydomonas, a s trategy w as used based o n the formation o f stable mixed disul®des between a Trx, mutated in its less reactive active- site cysteine, and its potential targets. The approach has previously been used successfully to characterize in vivo complexes o f thioredoxins w ith s ome of its target proteins [8,23] and to identify in vitro the most reactive internal cysteine (Cys207) of Sorghum NADP-MDH [12]. The cytosolic Trx h of Chlamydomonas has only two cysteines in its primary sequence, both belonging to the active site disul®de. T herefore, t his excludes the poss ibility of Trx forming artefactual disul®des 6 with th e a dditional cysteines present in t he chloroplastic Trx m a nd f sequences. Many Trx targets, s uch a s NADP-MDH o r phosphoribulokinase can be a ctivated in vitro by various thioredoxin isoforms: chloroplastic Trx f or m [24,25] but also cytosolic Trx h [22,26]. We took advantage of this lack of speci®city t o try to isolate various putative Trx targets. AmutatedChlamydomonas cytosolic Trx h, in which only the most reactive cysteine (Cys36) remained, was coupled to an activated CNBr Sepharose column (see Materials and methods for details on preparation of the Trx af®nity c olumn). Among the Chlamydomonas proteins that were retained on the column after loading of a protein extract and extensive washing, was a major protein of  21 kDa (Fig. 1). This p rotein was further puri®ed to homogeneity by HPLC and digested with t rypsin. The tryptic peptides were separated by HPLC and some of them were totally or partially analyzed by Edman sequencing and/or by MALDI-TOF mass spectrometry ( Fig. 2). Computer database searches based on the amino-acid sequences of sequenced peptides revealed 75% identity with a thioredoxin-dependent peroxidase (TPx), also named peroxiredoxin, of barley (the BAS1 protein) and Arab idop- sis. These proteins belong to the 2Cys-Prx group, because of the p resence of two conserved cysteines [5,6]. Arabidopsis BAS1 was s hown to be a chloroplastic protein. The identity of our peptides with BAS1 and the presence of two cysteines in alignment with the conserved cysteines of barley and Arabidopsis BAS1 suggested that our 21-kDa protein also belongs to this protein family, and could b e chlo roplastic. We called the Chlamydomonas 21-kDa protein Ch-Prx1. Cloning and sequences of Ch-Prx1 cDNA and the Ch-Prx1 gene In order to complete the sequence data for this new protein, and to be able to make a thorough characterization of its biochemical properties, we isolated the cDNA and expressed it in E. coli to produce a pure recombinant protein. We also isolated and sequenced the gene e ncod- ing the 21-kDa polypeptide. Degenerate oligonucleotides designed from the s equences of N- and C-terminal peptides (P1 and P10) were us ed as primers to s ynthesize t he cDNA of the coding sequence of t he mature Prx. The direct P CR primers were synthesized with a 6-bp extension at their 5¢ ends (ATGGCC, encoding methionine and alanine) for translation i nitiation and in frame cloning. The ampli®ed product, cloned into the pET-8c vector, was sequenced showing that the 597-bp product encod ed the putative Ch-Prx1 (accession number AJ304857). To isolate t he full-length cDN A, the RT-PCR product was u sed to s creen a cDNA library. A clone carrying a 1244- bp fragment was isolated and sequenced. Unfortunately, the cDNA sequence in this clone was not complete. It contained the 591 bp sequence c oding for the mature protein, a 647-bp sequence corresponding to the 3¢ region including the polyA tail, plus 6 bp in the 5 ¢ region. Screening of a Chlamydo- monas genomic BAC library (Genome Systems Inc., St Louis, USA) resulted in the isolation of a clone that contained a 1946-bp sequence corresponding to the g ene coding for t he 21-kDa protein of Chlamydomonas.We named this gene Ch-Prx1 (accession number AJ304856). The 3¢ end of the gene was not complete but could be deduced from the sequence of t he cDNA. The gene contains two introns and three exons. A 12-bp exon separates both introns (data not shown). When the amino-acid sequence deduced from the gene s equence w as compared to the sequence of the N-terminal peptide of the mature protein, codons for 3 8 a dditional amino acids, which were not present i n the mature protein, were discovered at the 5 ¢ end LMW Elution product s kDa 1 4 . 4 2 0 . 1 3 0 4 3 6 7 9 7 21 kDa Fig. 1. An alysis of elution products f rom the thioredoxin anity column by red ucing SDS/PAGE. Protein extracts o f Chlamydomonas cu ltures were app lied on a cytosolic Trx h C39S mutant anity column. The elution was performed with d ithioth reitol. Ó FEBS 2002 Peroxiredoxin from Chlamydomonas (Eur. J. Biochem. 269) 275 of the c oding region (Fig. 2). A chloroplast transit peptide prediction program ( CHLOROP ) [27], predicted a putative cleavage site between arginine 37 an d alanine 38, but the mature protein starts at Ser39, as indicated by the peptide sequencing (Fig. 2). It is possible that the protein c leaved between positions 37 and 38 is further processed in the chloroplast to the native form. Southern blot analys is on genomic DNA digested with ApaI ( an enzyme known t o c leave w ithin the Ch-Prx1 gene sequence), or EcoRI (an enzyme t hat does not cleave within Ch-Prx1 gene), produced four and two fragments, respec- tively, that h ybridized to the radiolabeled Ch-Prx1 coding sequence (Fig. 3). These results indicate that an additional gene homologous to the Ch-Prx1 gene exists in C. rein- hardtii genome. In this respect, it c an be noted t hat a sequence o f a putative cytosolic Chla mydomonas Prx is available in d ata b anks. Amino-acid sequence comparisons We compared the C h-Prx1 amino-acid se quence t o other similar proteins. Three groups could be distinguished (Fig. 4 ). The ® rst group contains Prx with one conserved cysteine but these p roteins s eem to be functionally closer to 2Cys-Prx than to 1Cys-Prx. Members of the second group contain t wo con served c ysteines. C h-Prx1 belongs to this group and i s close to the higher p lant 2Cys-Prx pr oteins, which have been described as nuclear-encoded chloroplastic proteins. This suggests, in addition to the presence of a transit peptide, that our 2Cys-Prx is a chloroplastic protein. The third group contains Prx with one conserved c ysteine. Biochemical characterization of recombinant Ch-Prx1 Under o xidizing conditions, 2Cys-Prx e xists p redominantly as dimers linked by two identical d isul®de bonds between the ®rst Cys of one subunit and the s econd Cys of the other subunit [10]. Ch-Prx1 also shares this feature: under reducing conditions it is a monomer o f 21 kDa, which is converted to a dimer under nonreducing c onditions (Fig. 5). The antioxidant activity of Ch-Prx1 was characterized in a mixed function oxidation system ( Fig. 6). Prx proteins are Fig. 2. BLAST of peptide sequences of 21 kDa proteins in databases. Tryptic peptides were puri®ed by RP-HPLC and some of them were to tally or partially analyzed by Edman sequencing (ááá) and/or by MALDI-TOF mass spectrometry (±±). The experimental masses (M + H) + were compared with the c alculated masses ( indicated in brackets): P1, 1 681.71 (1681.89); P2 , 1853.42 (1853.93); P4, 297 2.26 (2972.51); P5, 163 1.07 (1630.87); P 6, 1393.66 (1393.73); P7, 2512.52 (2512.36). Accession numbers: barley BAS1, Z34917; Arabidopsis BAS1, X97910. The missing amino-acid stretches and the transit peptide sequence were deduced from the cloned c DNA and gene sequences (accession numbe rs: AJ304856 for t he ge ne and AJ304857 f or the cDNA). T he conserved r esidues are shade d in grey and the sequence of the putative t ransit peptide i s in i talics. ApaI EcoRI 12 10 4 1.7 1.4 kb Fig. 3. Sou thern blot analysis of the Ch-prx1 gene. Ge nomic DNA from CW15 Chlamydomonas strain was digested with ApaIorEcoRI, size-fractionated on a 0.8% agar ose gel, and transferred to a Hybond N + nylon m embrane. Th e ® lter was hybridized with the 32 P-radiola- beled Ch-Prx1 c oding reg ion probe. M olecular siz e markers a re indi- cated on t he left. 276 A. Goyer et al. (Eur. J. Biochem. 269) Ó FEBS 2002 known t o p revent damage of DNA against R OS. ROS can be produced by incubating dithiothreitol with Fe 3+ ,which catalyzes the reduction of O 2 to H 2 O 2 . The latte r is further converted by the Fenton reaction to hydroxyl radicals [19, 20]. The radicals produced by incubating dithiothreitol with Fe 3+ caused complete degradation of 2 lg pBluescript DNA within 1 h . Ch-Prx1 protected the pBluescript DNA against degradation, while BSA, even a t a concentration of Fig. 4. Phy logenetic tree of peroxiredoxins. CLUSTAL X was used t o generate the tree . Accession numbers: Trypanosoma-mpx, AJ006226; Try- panosoma, u26666; yeast: Type I T Px, N P013684, type II TPx, u53878, 1Cys-Prx, ybl0524; Chinese cabbage C2C-Prx, AF052 202; human: A OE37-2, u25182, AOP1, P30048, pag, q06830, NKEFB, l19185; barley: BAS1, Z34917, PER1, X96551; Arabidopsis: BAS1, X97910, TPx2, AF121356, MHF15.19, AF326871, Per1, O04005; Chlamydomonas Ch-Prx1, AJ304857; spinach B AS1, ´ 94219; mouse TPx, u20611; Bromus pbs128, p52571; Tortula, u40818; Dro sophila DPx-2540±2, AF311880; Rattus Prdx3, NM022540; Oryza, AF203879; Phaseolus, AJ288896; Thermus aquaticus, AF276071; Br assica Per1, A F139817, BAS1, A F311863. Ó FEBS 2002 Peroxiredoxin from Chlamydomonas (Eur. J. Biochem. 269) 277 20 l M , h ad no effect (not shown). T he degree of protection correlated with the a mount of Ch-Prx1 a dded to the a ssay. Thioredoxin-dependent peroxidase activity of Ch-Prx1 towards H 2 O 2 or t-BOOH was e xamined indirectly by measuring the oxidation r ate of NADPH (followe d by the decrease in A 340 ) in t he presence of NTR and thioredoxin. Pure recombinant proteins expressed i n Escherichia coli were used in this test: NTR from A. thaliana,Trxhfrom Chlamydomonas and C h-Prx1. Figure 7A shows a tim e- course of NADPH oxidation with either H 2 O 2 or t-BOOH as substrates. C learly, C h-Prx1 was equally ef®cient with both, and the reaction required all three protein components (Ch-Prx1, Trx h and NTR). The speci®c activity of the recombinant e nzyme w as identical to the speci®c activity of the native protein puri®ed fro m Chlamydomonas (dat a not shown). The rate of NADPH oxidation was very weak when Trx m from Chlamydomonas was used (data not shown), probably b ecause o f t he weaker af® n ity of Ar abid- opsis NTR for Trx m [21]. Therefore, the ability of Trx m from Chla mydomonas to donate protons to Ch-Prx1 w as measured directly by following the degradation of t-BOOH, A A340nm 0 0,2 0,4 0,6 0,8 1 1,2 0 5 10 15 20 25 Time (min ) B 0 20 40 60 80 100 120 0 2 4 6 8 10 Time (min) % t-BOOH initial Fig. 7. Per oxidase activity of recombinant Ch-Prx1 toward H 2 O 2 or t-butyl hydr operoxide. Dependence o f the re ac tion on re duce d thiore- doxin. (A) The r eaction was followed spectr ophotometrically by NADPH oxidation in a cou pled assay with N TR from Ar ab idopsis and thioredoxin h from Chlamydomonas. C omplete assays with H 2 O 2 (e) or t-BOOH (j). Controls: m in us T RX ( m), minus PRX (r), minus peroxide (s), minus NTR (q). (B) Peroxidase activity w ith various thioredoxins. T he concentration of t-BOOH was measured colori- metrically and expressed a s a percentage of the i nitial c oncentration. Thioredoxins were reduced with dithiothreitol. Chlamydomonas Trx h (m)orTrxm(j), spinach T rx f (d)orTrxm(r). Control w ithout Trx (n). SK DNA 20 µ M Ch -Prx1 5 µ M Ch -Prx1 C OC S Fig. 6. Prote ction of DNA against free r adical attac k by the recombi- nant Ch-Prx1 protein. Plasmid Bluescript SK DNA was incub ated for 30 min in a thiol-MFO system containing 3.0 l M Fe 3+ .Lane1is untreated SK DNA, open circle (OC) and superc oiled (S) plasmid DNA are indicated; lanes 2±3 containing 20 or 5 l M Ch-Prx1 protein show that protection of DNA against nic king increases with increasing amounts of prote in; lane 4 ( C ) contro l without pro tein s hows maxi- mum DNA degradat ion. LMW + ββ ββ - Mercapto - ββ ββ - Mercapto 1 4 . 4 2 0 . 1 3 0 4 3 6 7 9 7 k D a Fig. 5. Ana lysis by SDS/PAGE of the monomer/dimer shift of recom- binant Ch-Prx1. The protein was e ith er r educed with 2-mercapto- ethanol, or not, a s indicated. 278 A. Goyer et al. (Eur. J. Biochem. 269) Ó FEBS 2002 using the peroXOquant kit (see Experimental procedures), in the presence of dithiothreitol as an electron donor to Trx. The r ate of d isappearance of t-BOOH was i dentical with TrxmandTrxhfromChlamydomonas used at the same concentration ( Fig. 7B). When Trx was omitted, the r ate of disappearance of t-BOOH was negligible, proving that dithiothreitol alone, used at low concentration, cannot signi®cantly act ivate Ch-Prx1. In order to d etermine whether d ifferent chloroplastic T rx differ in their abilities to function with Ch-Prx1, we compared the e f®ciencies of two Trx isoforms f and m from spinach, b ecause no T rx f from Chlamydomonas has been isolated until now. Both were active with Ch-Prx1, but while Trx f was as ef®cient as Trx h and m from Chlam ydomonas, T rx m from spinach showed a lower ef®ciency. Regulation of Ch-Prx1 expression To study possi ble roles of Ch-Prx1 in vivo , the expression of the Ch-Prx1 gene was monitored under different culture conditions. A number of genes have been reported to be regulated by light, among them the Trx m and Trx h genes from Chlamydomonas [28]. As Prx are Trx-dependent proteins, we investigated a possible regulation of the Ch-Prx1 gene expression by light. Under dark conditions, levels of Ch-Prx1 gene transcripts are relatively low (Fig. 8A). Ch-Prx1 gene transcript levels increased in illuminated cells reaching a maximum after  6 h of light. In the dark, Ch-Prx1 mRNAs returned to the basal level in less than 2 h. These results show that the pool size of Ch-Prx1 transcripts is affected by light/dark conditions. The role o f noncyclic photosynthetic electron transport in regulating the expression of the Ch-Prx1 gene was s tudied by blocking electron transfer w ith 3-(3¢,4¢-dichlorophenyl)- 1,1,-dimethyl urea ( DCMU) or 2 ,5-dibromo-3-methyl-6- isopropyl-p-benzoquinone (DBMIB) 8 . Light induction of Ch-Prx1 gene expression was a ffected by bo th inhibitors (Fig. 8B, lanes 3 and 4), indicating that photosynthetic electron transport is required for upregulating Ch-Prx1 gene expression. This regulation does not imply the redox state of plastoquinone, suggesting that another element situated downstream the plastoquinone is responsible for this regulation. The highest levels of Ch-Prx1 transcripts were f ound in cells bubbled with pure o xygen for 90 min in the dark (Fig. 8C, lane 2). This shows that oxidative s tress, directly or indirectly, affects Ch-Prx1 gene expression. Taken together, the results support the notion that the redox state and/or the concentration of reactive oxygen species in the chloroplast play a role in regulating the level of transcripts of the Ch-Prx1 gene in Chlam ydomonas. DISCUSSION The mixed disul®de approach as a tool to isolate new thioredoxin targets The formation of stable mixed-disul®de cross -linked com- plexes has been used p reviously to determine interactions between target enzymes, such as phosphoribulokinase or NADP-malate dehydrogenase, and thioredoxins [12,29]. This approach also provid ed evidence for conformational changes occurring in the structure of thioredoxin reductase upon interaction with its substrate thioredoxin [30]. U sing the mixed disul®de approach for isolation of Trx target proteins in vivo is dif®cult because of the limited stability o f Trx-target complexes in c ells in which reductants t hat split disul®des, are abundant. To o vercome this dif®culty, thioredoxin-de®cient yeast or E. coli mutant strains have been used to express a single-cysteine m utant thioredoxin allowing the i solation of thioredoxin targets in y east [8] and Fig. 8. Ch -Prx1 gene expression. (A) Levels of Ch-Prx1 transcripts in cells growing in 12-h light/dark cycles. Total RNA was extracted in two hours intervals from division-synchronized Chlamydomonas cells kept in a 12- h light/dark regime. RNA samples were processed as described in Materials and methods. RNA gel blots were hybridized to the radiolabeled Ch-Prx1 cDNA probe. Numbers ab ove the lan es indicate the time a t which the RNA samples were take n. (B) I nduction of Ch-Prx1 gene expression in Chlamydomonas by light in the abse nce an d in th e prese nce of DCMU or DBMIB. Cultures were grown in continuous light and R NA samples were analyzed for Ch-Prx1 transcript levels by northern analysis. Lane 1, cells taken after 16 h in the dark; lane 2, cells taken 3 h a fter the start of the light period; lane 3, cells taken a fter 3 h in the light in the presence of 20 lM DCMU (added at 0 h light); lane 4, cells taken after 3 h in the light in the p resence of 1 lM DBMIB (added at 0 h light). (C) Induction of Ch-Prx1 gene expression in the dark by bubbling with oxygen. Cultures were grown in 12-h light/dark cycles. Lane 1, cells taken after 11 h in the d ark; lane 2, cells taken a fter 11 h in the dark after 90 min bubbling with 100% O 2 . Ó FEBS 2002 Peroxiredoxin from Chlamydomonas (Eur. J. Biochem. 269) 279 in E. c ol i [23]. B ecause T rx-de®cient mutants o f Chlamydo- monas are not available, we combined the mixed-disul®de method with af®nity chromatography, u sing an af®nity column m ade of Trx h (C39S Trx h) mutated at the less reactive Cys of i t s active site. T he major protein retained on the af®nity column was a thioredoxin-dependent peroxidase (Prx) that b elongs to t he same family as a peroxidase isolated in vivo in yeast using mutated Trx of Arabidopsis [8]. The predominant formation of mixed disul®des between Prx and C39S Trx h in the presence of a number of well- known T rx-dependent enzymes appears surprising but may be explained by t he natural abundance of t he peroxidase that might compete with other targets, but also by structural features of the r egulatory sites of the various target proteins. Extensively studied enzymes, such as NADP-MDH [12] and fructose bisphosphatase [31], can be slowly activated by a single cysteine mutant thioredoxin while almost no complex between the mutant thioredoxin and wild-type MDH is formed [12]. Oxidized Prx, o n the other hand, is linked by intermolecular disul®de bonds. Upon cleavage by Trx the subunits separate leaving no proximal Cys that could attack the mixed disul®de formed between Trx and its target. Thus, the mixed disul®de approach seems to favour the isolation of targets bearing an intermolecular disul®de bridge. Structural and functional characteristics of Ch-PRX1 The amino-acid sequence of Ch-Prx1 shares highest identity with the BAS1 protein of Brassica, spinach, barley and A. thaliana,PR1ofPhaseolus and MHF 9 of A. thaliana. These proteins b elong to the 2Cys-Prx subfamily. A ll plan t 2Cys-Prx proteins, except BAS1 of barley, the complete cDNA of which has not been isolated, contain putative chloroplast-targeting sequences. C h-Prx1 is likely to be a chloroplastic protein because it is synthesized as a precursor protein containing a short transit peptide that is predicted to be cleaved at a conserved s ite. The homologous BAS1 protein of Ara bidopsis wasshowntobeimportedinto isolated plastids after post-translational modi®cation [6]. Like other 2Cys-Prx enzymes previously described i n yeast, mammals and plants, Ch-Prx1 d isplayed antioxidant and peroxidase activities. The enzyme could reduce hydro- gen peroxide as well as alkyl hydrogen peroxide and exerted a s trong protective effect against DNA degradation by f ree radicals of oxygen. More extensive biochemical character- izations, including K m determinations, are needed to better de®ne the substrate speci®city of Ch-Prx1. Peroxiredoxin and thioredoxin speci®city There is i ncreasing evidence for a role o f Trx in coping with oxidative stress. A m utant strain o f yeast Sacc haromyces cerevisiae in which both Trx genes were disrupted has been found to be particularly sensitive to hydrogen peroxide [8] and to heavy-metals [32]. Heterologous complementation of this yeast mutant with Arabidopsis type h Trx3 or type m Trx1, 2, or 4, or type x Trx conferred hydrogen peroxide tolerance [8,33]. The fact that the thioredoxin-dependent Ch-Prx1 of Ch lamydomonas is able to detoxify hydrop er- oxides provides additional s upport f or a f unction of Trx in response to oxidative s tress. All types of plant T rx, whether cytosolic or chloroplas tic, were ab le to serve a s hydrogen donors for Ch-Prx1 in vitro. Interestingly, the spinach f-type chloroplastic isoform was more ef®cient with Ch-Prx1 than the m-type chloroplastic Trx suggesting t hat T rx f is t he preferred electron donor to Prx in vivo. This result differs from the results of yeast complementation experiments in which several m-type Arabidopsis Trx proteins have been shown to confer hydrogen peroxide tolerance, while complementation with f-type Trx d id not [33]. H owever, it cannot be e xcluded that the yeast cytosolic NTR is unable to r educe Trx f. The lack o f s peci®city of T rx toward Trx-dependent proteins can explain why a presumably chloroplastic P rx could be isolated with c ytosolic Trx h as bait even though Southern blot analysis and genome sequencing indicate that a cytosolic Prx exists in Chlamydomonas. It is also possible that the putative cytosolic Prx was present i n our protein extracts at much lower concentration than the chloroplast Prx. It can b e noted that a s imilar loss of speci®city w as recently reported b y Motohashi et al. [ 34] who trapped targets o f t hioredoxin f on an af®nity column comprised of thioredoxin m. Regulation of peroxiredoxin gene expression and defence against oxidative stress Light is an important environmental factor inducing, directly and indirectly, the production of ROS. ROS is known to impair photosynthesis by damaging chloroplast structures such as the D1 protein, LHCII, the chloroplast ATPase an d r ibulose 1 ,5-bisphosphate carboxylase/oxygen- ase (RubisCO) [35]. In Arabidopsis, peroxiredoxins have been found to protect chloroplast structures from damage by ROS [35]. Regulation of Ch-Prx1 gene expression may be controlled either d irectly by ROS, e.g. by H 2 O 2 ,whichis known for its role in signal transduction [36], or indirectly by sensors of redox conditions i n the chloroplast, e.g. ascorbate [37]. We f ound that transcript levels of the Ch-Prx1 gene markedly increased in illuminated cells (Fig. 8A,B) but a lso upon bubb ling cultures w ith 100% oxygen in t he dark (Fig. 8 C), conditions in which p roduction of ROS is likely to be high. Blocking noncyclic photosynthetic e lectron ¯ow with DCMU or DBMI B 10 inhibited the accu mulation of Ch-Prx1 transcripts in the light (Fig. 8B), suggesting an in¯uence o f the photosynthetic electron ¯ow on Ch-Prx1 gene expression. The redox state of plastoquinone, known to regulate the expression of s ome genes [38,39], does not seem to be the r esponsible for this r egulation, because both DCMU and DBMIB exert an inhibitory effect. The regulation of Ch lamydomonas Trx m gene expression was also shown t o b e d ependent on the photosynthetic electron ¯ow [28] but independent of the redox state of the plastoquinone pool. The expressions of other chloroplastic genes, such as those encoding phosphoribulokinas e and ferredoxin-NADP-reductase, follow the same pattern [40]. These results are in favor of a coordinated regulation o f both Trx and Prx in the c hloroplast a nd fully support the hypothesis that Ch-Prx1 is a c hloroplastic protein. 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Isolation and characterization of a thioredoxin-dependent peroxidase from Chlamydomonas reinhardtii Aymeric Goyer 1 , Camilla Hasleka Ê s 2 , Myroslawa. 2002 RESULTS Isolation of a 2Cys-Prx by using a single cysteine mutant of Chlamydomonas Trx h In an attempt to isolate new T rx targets in Chlamydomonas, a s trategy