In an attempt to find new molecular targets of Trx in Chlamydomonas reinhardtii, an affinity column carrying a cytosolic Trx h mutated at the less reactive cysteine of its active site wa
Trang 1Isolation and characterization of a thioredoxin-dependent
peroxidase from Chlamydomonas reinhardtii
Aymeric Goyer’, Camilla Haslekas”, Myroslawa Miginiac-Maslow’, Uwe Klein’, Pierre Le Marechal’, Jean-Pierre Jacquot* and Paulette Decottignies®
‘Institut de Biotechnologie des Plantes, Université Paris-Sud, Orsay Cedex, France; *Department of Biology,
Division of Molecular Biology, University of Oslo, Blindern, Oslo, Norway; *IBBMC, Université Paris-Sud, Orsay Cedex,
France; “Interaction Arbres—Microorganismes INRA/Université Nancy, Vandoeuvre Cedex, France
All living organisms contain redox systems involving thior-
edoxins (Trx), proteins featuring an extremely conserved and
reactive active site that perform thiol-disulfide interchanges
with disulfide bridges of target proteins In photosynthetic
organisms, numerous isoforms of Trx coexist, as revealed by
sequencing of Arabidopsis genome The specific functions of
many of them are still unknown In an attempt to find new
molecular targets of Trx in Chlamydomonas reinhardtii, an
affinity 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 disulfides with
Trx The major protein bound to the column was identified
by amino-acid sequencing and mass spectrometry as a
thioredoxin-dependent 2Cys peroxidase Isolation and
sequencing of its gene revealed that this peroxidase is most likely a chloroplast protein with a high homology to plant 2Cys peroxiredoxins It is shown that the Chlamydomonas peroxiredoxin (Ch-Prx1) is active with various thioredoxin isoforms, functions as an antioxidant toward reactive oxy- gen species (ROS), and protects DNA against ROS-induced degradation Expression of the peroxidase gene in Chlamydomonas was found to be regulated by light, oxygen concentration, and redox state The data suggest a role for the Chlamydomonas Prx in ROS detoxification in the chloroplast
Keywords: Chlamydomonas; peroxiredoxin; thioredoxin; redox signaling; oxidative stress
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 classified
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, while 1Cys-Prx may be reduced by a small
thiol molecule such as glutathione Recently, 1Cys-Prx has
been identified in yeast and Arabidopsis, and has been
shown to be thioredoxin-dependent and function in a
similar manner to 2Cys-Prx [8,9]
2Cys-Prx catalyzes, in vitro, the reduction of alkyl
hydroperoxide and hydrogen 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 first oxidized by a
peroxide to sulfenic acid (Cys-SOH), which rapidly reacts
Correspondence to M Miginiac-Maslow, Institut de Biotechnologie
des Plantes, UMR CNRS 8618, Bat 630, Université Paris-Sud, 91405
Orsay Cedex, France Fax: + 33 1 69 15 34 23,
E-mail: miginiac@ibp.u-psud.fr
Abbreviations: Nbs», 5,5’-dithiobis(2-nitrobenzoic acid); Prx, perox-
iredoxin; -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)
with the C-terminal cysteine of the other subunit to form an
intermolecular disulfide [10] In animals, yeast, and plants,
the disulfide is reduced via a thiol/disulfide redox inter- change with reduced thioredoxin (Trx), thus regenerating an active peroxidase
The present study was aimed at setting up an affinity chromatography column for specific trapping of proteins that react with Trx, based on their ability to form mixed disulfide-linked adducts with a single cysteine mutant thioredoxin The system allowed us to purify and identify for the first time a 2Cys-Prx protein (Ch-Prx1) from the green alga Chlamydomonas reinhardtii The purified protein was characterized by its antioxidant properties towards reactive oxygen species (ROS), protection of DNA against degradation, its peroxidase activity, and its ability to use different thioredoxin isoforms as hydrogen donors To better understand the function of Ch-Prxl in vivo, we isolated the cDNA and the Ch-Prx/ gene, and examined the regulation of its expression by different culture conditions
EXPERIMENTAL PROCEDURES
Algal strains and culture conditions
The C reinhardtii strain CW15 (137c, mt+, cwl5, 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 photoautotrophic minimal medium (HSM; [11]) CW15 cultures were grown in flasks at
25 °C under continuous stirring and bubbling with 5% CO, enriched air Light intensity was 300 wmolm~s' at the level of the culture flasks CC125 cells were grown in 200-mL
Trang 2tubes at 32 °C Cultures were kept in a 12-h light/dark
regime (light intensity 150 pmolm™~s~') and bubbled
with 2% CQO>-enriched air Cell density was adjusted daily
at the onset of light with fresh medium to ~2 x 10°
cellsmL”
Purification of Ch-Prx1
Purification of the native protein from Chlamydomonas
cells Chlamydomonas CW15 cells were grown to a cell
density >5.x 10° cells per mL Cells were pelleted,
resuspended in 30 mm Tris/HCl pH 7.9, and broken by
two cycles of freeze-thawing in liquid nitrogen Broken cells
were centrifuged for 30 min at 15 000 g and the supernatant
was adjusted to 2% (w/v) streptomycin sulfate by addition
of a 20% solution After incubation for 20 min at 4 °C, the
suspension was centrifuged at 15 000 g for 30 min to pellet
precipitated nucleic acids The supernatant was adjusted to
95% (w/v) ammonium sulfate and incubated for 20 min at
4 °C The suspension was centrifuged at 15 000 ¢ for
30 min and the pellet was resuspended in 10 mL 30 mm
Tris/HCl, pH 7.9, and dialyzed against 5 L 30 mm Tris/
HCl, pH 7.9 The protein solution was loaded onto an
affinity column made of a mutated Chlamydomonas
cytosolic h-type thioredoxin (C39S mutant) grafted on a
CNBr activated sepharose support and equilibrated with
30 mmo Tris/HCl, pH 7.9 Covalent coupling of this thio-
redoxin to activated Sepharose was carried out essentially as
recommended by the supplier of the Sepharose (Amersham
Pharmacia) The C39S mutant thioredoxin lacks one
cysteine residue and has only its most reactive active-site
cysteine left (Cys36) Site-directed mutagenesis of Trx h and
recombinant protein purification was carried out as
described previously [12] To 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;; Pierce) before coupling The deriv-
atized thioredoxin was treated with dithiothreitol to
eliminate the 5-mercapto-2-nitrobenzoate adduct, and was
washed extensively to remove the dithiothreitol After
loading on the column, the proteins were eluted with
15 mL of | mm dithiothreitol in 30 mm Tris/HCl, pH 7.9
The eluted proteins were dialyzed against 30 mm Tris/HCl,
pH 7.9 and re-applied on the affinity column Proteins were
eluted with 15 mL of 1 mm dithiothreitol in 30 mm Tris/
HCl, pH 7.9, and analyzed by SDS/PAGE
Purification of the recombinant Ch-Prx1I expressed in
Escherichia coli BL21 (DE3) £ 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 Ch-Prx1 without
the chloroplast transit peptide Cells were grown in 5 L of
M9 medium up to Đạo = 0.5, and recombinant protein
expression was induced by the addition of 100 um
isopropyl thio-B-p-galactoside After induction, cells were
grown for 18h at 37°C, pelleted, and resuspended in
10 mL 30 mm Tris/HCl, pH 7.9, 1 mm EDTA, 500 um
phenylmethanesulfonyl fluoride, and 14 mm 2-mercapto-
ethanol Cells were broken in a French press at 60 MPa
Broken cells were centrifuged for 30 min at 20 000 g and
the supernatant was adjusted to 2% (w/v) streptomycin
sulfate by addition of a 20% solution After incubation for
20 min at 4 °C, the suspension was centrifuged at 20 000 g for 30 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 was resuspended in 10 mL 30 mm Tris/HCl, pH 7.9, dialyzed against 5 L of 30 mm Tris/HCl, pH 7.9, and the Ch-Prx1 protein was purified as described above on a C39S Trx h affinity column equilibrated with 30 mm Tris/HCl,
pH 7.9
Polyacrylamide gel electrophoresis Denaturating [4% (w/v) SDS] electrophoresis was carried out on 10% polyacrylamide gels Gels were stained with Coomassie Blue (2.5 g-L”)
Tryptic digestion, separation of the tryptic peptides, analysis by sequencing and MALDI-TOF mass
spectrometry
Further purification of Ch-Prxl was achieved, prior to digestion, by RP-HPLC on a 4.6 x 25 cm Vydac C4 (30 A) column Ch-Prx1 was eluted with a linear gradient from 28
to 70% acetonitrile in 0.1% trifluoroacetic acid over 30 min
at a flow rate of 1 mL-min™' Tryptic digestion was
performed for 20 h at 37°C in 0.1 mM NH4HCO; with
sequence grade trypsin (Boehringer) The peptides were separated by RP-HPLC on a 0.21 x 25cm Vydac C18 (300 A) column Peptides were eluted with a linear gradient
from 0 to 70% acetonitrile in 0.1% trifluoroacetic acid, over
90 min, at a flow rate of 0.3 mL-min! Absorbance was
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 For MALDI-TOF analysis, 1 wL of tryptic digest
or HPLC-purified peptides was mixed with | pL of saturated solution of o-cyano-4-hydroxycinnamic acid in 50% acetonitrile, 0.3% trifluoroacetic acid Samples were loaded into MALDI-TOF spectrometer (Perseptive Bio- systems, Voyager STR-DE) equipped with a nitrogen laser (337 nm) Spectra were obtained in reflectron mode using delayed extraction
RT-PCR Ch-Prx1 cDNA was isolated by RT-PCR In this approach, primers used in cDNA synthesis were designed based on the amino-acid sequence of N- and C-terminal peptides, as determined by the procedure described above First-strand cDNA was first synthesized from total RNA with M-MuLYV reverse transcriptase (Life Technologies) The reaction mixture contained in a volume of 20 uwL: 0.9 ug heat-denatured Chlamydomonas total RNA, 1 x RT buffer,
1 mm dNTPs, 20 mm dithiothreitol, 100 U reverse tran-
scriptase (RT), and 1 um of the degenerated reverse primer 5’-GCGGATCCTTA(G/C)ACGGCGGCGAAGTAC TCC-3’ After reverse transcription for 30 min at 42 °C, the first-strand cDNA was amplified 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 °C for 2 min) In
addition to the above degenerated reverse primer two direct primers 5-AACCATGGCCTCCCACGCCGAGA AGCC(G/C)CTG-3’ and 5’-AACCATGGCCAGCCAC
Trang 3GCCGAGAAGCC(G/C)CTG-3’ were used PCR products
were separated by electrophoresis on a 0.8% agarose gel
A 600-bp fragment was purified by using the nucleospin
extract kit (Macherey—Nagel), then digested by NcoI and
BamHI, and cloned into the pET-8c vector
Screening of a cDNA library
A Agtl1 cDNA library of Chlamydomonas was a gift from
M Goldschmidt-Clermont (Universite de Geneve, Switzer-
land) The Ch-Prx1 coding sequence obtained by RT-PCR
was used to screen 200 000 plaque forming units of the
Agtl1 library Colony hybridization was performed at 64 °C
in buffer containing 0.5m NaHPO,, pH 7.2, 1 mm
Na,EDTA, 7% SDS, and 1% BSA After purification of
positive clones, 4 DNA was extracted and purified as
described in [13] cDNA inserts were excised by a digestion
with EcoRI and cloned into the EcoRI site of the SK”
Bluescript plasmid (Stratagene)
Screening of a gene library
A BAC library of genomic C reinhardtii DNA (Genome
Systems Inc., St Louis, USA, product FBAC-8417)
spotted at high density on a nylon filter was used to
isolate a clone containing the complete Ch-Prx/1 gene Two
heterologous primers, (5’-GACTTCACCTTCGTGTGCC
CCACCGAG-3ˆ and 5-GGGŒTCGATGATGAACAG
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-Prx/] gene sequence
Two BAC clones that strongly hybridized to the probe on
the filter were amplified and used to isolate the complete
Ch-Prx1 gene sequence using conventional Southern ana-
lyses, subcloning, and sequencing techniques [13]
Sequencing of cDNA and genomic DNA
The BigDye Terminator Cycle Sequencing Kit (Perkin-
Elmer) or the Thermo Sequenase Radiolabeled Terminator
Cycle Sequencing Kit (United States Biochemicals) were
used to sequence the Ch-Prx] cDNA and the Ch-Prx/ gene,
respectively
RNA isolations
RNA for RT-PCR and for northern analyses was islolated
by alternative methods In the first method, ~ 30 million
cells were pelleted by centrifugation (3000 g, 5 min) 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, 10 min)
The supernatant was treated as instructed by the supplier
The dried pellet was resuspended in 20 HL of milliQ
(Millipore) purified sterile water The second method
followed essentially a protocol described previously [14,15]
PolyA ” RNA was isolated with magnetic oligo d(T) beads
(Dynal) following the protocol of the supplier
Southern and Northern blot analysis For Southern analyses, genomic DNA was prepared following the protocol described previously [16] DNA was digested with appropriate restriction enzymes, size- fractionated on a 0.8% agarose gel, and transferred to a Hybond N* (Amersham Pharmacia) or ZetaProbe (Bio- Rad) nylon membrane Hybridization with the P-radio- labeled cprx probe (4 x 10° c.p.m.) and washing of the gel blot at 65 °C was carried out as described previously [17] The membrane was exposed to X-ray film at —20 °C or, when using an intensifying screen, at —80 °C
RNA for Northern analyses was isolated as described above and separated in a 1.3% agarose/formaldehyde gel The RNA was blotted to a nylon membrane (ZetaProbe, Bio-Rad) by alkaline transfer, UV-crosslinked, and hybrid- ized to the random primer radiolabeled Ch-Prx] cDNA probe for 24h The cDNA probe was generated using biotinylated single stranded template bound to magnetic streptavidin-coated beads (Dynal) in a specific priming reaction [18] The specific primer used for the reaction was the downstream primer used in the RT-PCR reaction After washing [17], the membrane was exposed to X-ray film with
an intensifying screen at —80 °C for ~2 days
Antioxidant activity of the Ch-Prx1 protein The antioxidant activity of the Ch-Prx1 protein was tested in
a DNA-cleavage assay modified after [19,20] Bluescript plasmid DNA (2 ug) was exposed to a mixed function oxidation system containing 0.32 mm dithiothreitol and
3 um FeCl; The reaction contained various amounts of concentrated Ch-Prx1 protein (5—20 um), and was initiated
30 min before addition of the DNA Control reactions were performed without Ch-Prxl protein and with BSA (400 ugmL™') The reactions were stopped by adding 3.3 mm EDTA and analyzed on an agarose gel
Thioredoxin-dependent peroxidase activity of Ch-Prx1 Peroxidase activity assays were initiated by the addition of HO; (500 um) or fbutyl hydroperoxide (t-BOOH)
(500 um) to 1mL 30mm Tris/HCl, pH 7.9, reaction
medium containing 197 nm _ A thaliana NADPH Trx reductase (NTR), 180 um NADPH, 5.7 um Trx and 2.4 um Prx The reaction was monitored spectrophotomet- rically by following the decrease in absorbance at 340 nm at
30 °C Recombinant A thaliana NTR was purified as described previously [21] Chloroplastic thioredoxins m and
f from spinach were a kind gift of P Schiirmann (University
of Neuchatel, Switzerland) Thioredoxins m and h from Chlamydomonas were purified as described previously [22]
An alternative assay, avoiding the need of a thioredoxin
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 tm) was incubated with 400 um dithiothreitol, 16.6 HM Trx and 500 um ¢-butyl hydroper- oxide in 50 uL of 30 mm Tris/HCl, pH 7.9, buffer The quantity of t-BOOH was measured on 5 wL aliquots added
to a spectrophotometer cuvette containing 500 nL of PeroXOquant medium The activity was estimated from the decrease in absorbance at 595 nm
Trang 4RESULTS
Isolation of a 2Cys-Prx by using a single cysteine
mutant of Chlamydomonas Trx h
In an attempt to isolate new Trx targets in Chlamydomonas,
a strategy was used based on the formation of stable mixed
disulfides 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 of thioredoxins with some of its target proteins
[8,23] and to identify im 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
disulfide Therefore, this excludes the possibility of Trx
forming artefactual disulfides with the additional cysteines
present in the chloroplastic Trx m and f sequences Many
Trx targets, such as NADP-MDH or phosphoribulokinase
can be activated 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 specificity to try
to isolate various putative Trx targets
A mutated Chlamydomonas 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
affinity column) 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 protein was further purified to
homogeneity by HPLC and digested with trypsin 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 Arabidop-
sis These proteins belong to the 2Cys-Prx group, because of
the presence of two conserved cysteines [5,6] Arabidopsis
BASI was shown to be a chloroplastic protein The identity
of our peptides with BAS] 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 be chloroplastic
We called the Chlamydomonas 21-kDa protein Ch-Prx1
Cloning and sequences of Ch-Prx7 cDNA
and the Ch-Prx7 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 encod-
ing the 21-kDa polypeptide Degenerate oligonucleotides
designed from the sequences of N- and C-terminal peptides
(P1 and P10) were used as primers to synthesize the cDNA
of the coding sequence of the mature Prx The direct PCR
primers were synthesized with a 6-bp extension at their
5’ ends (ATGGCC, encoding methionine and alanine) for
translation initiation and in frame cloning The amplified
ow
2 2 Lp,
“py ay,
kDa
=| f
07—»
67—>
43—»
30—>
20.1—>
Sate
ti
14.4»
Fig 1 Analysis of elution products from the thioredoxin affinity column
by reducing SDS/PAGE Protein extracts of Chlamydomonas cultures were applied on a cytosolic Trx h C39S mutant affinity column The elution was performed with dithiothreitol
product, cloned into the pET-8c vector, was sequenced showing that the 597-bp product encoded the putative Ch-Prx1 (accession number AJ304857)
To isolate the full-length cDNA, the RT-PCR product was used to screen 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 coding for the mature protein, a 647-bp sequence corresponding to the 3’ region including the polyA tail, plus 6 bp in the ’ 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 gene coding for the 21-kDa protein of Chlamydomonas We named this gene Ch-Prx/ (accession number AJ304856) The 3’ end of the gene was not complete but could be deduced from the sequence of the 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 sequence was compared to the sequence of the N-terminal peptide of the mature protein, codons for 38 additional amino acids, which were not present in the mature protein, were discovered at the 5’ end
Trang 521 kDa protein _._._. _.———~—~—-============~
Barley BAS] -
~ MAALQSASRSSAVAFSRQARVAPRVAAS VARRSLVVRA
c======~~~~=~~~~~~~~~~~~~~~~~~ DARAEFVARS
Arabidopsis BAS] MASVASSTTLISSPSSRVFPAKSSLSSPSVSFLRTLSSPSASASLRSGFARRSSLSSTSRRSFAVKA
Pl
21 kDa protein KEGVVQH?
Barley BAS1 eae
Fig 2 BLast of peptide sequences of 21 kDa proteins in databases Tryptic peptides were purified by RP-HPLC and some of them were totally or partially analyzed by Edman sequencing (-) and/or by MALDI-TOF mass spectrometry (—) The experimental masses (M + H)* were compared with the calculated masses (indicated in brackets): P1, 1681.71 (1681.89); P2, 1853.42 (1853.93); P4, 2972.26 (2972.51); P5, 1631.07 (1630.87); P6, 1393.66 (1393.73); P7, 2512.52 (2512.36) Accession numbers: barley BAS1, 734917; Arabidopsis BAS1, X97910 The missing amino-acid stretches and the transit peptide sequence were deduced from the cloned cDNA and gene sequences (accession numbers: AJ304856 for the gene and AJ304857 for the cDNA) The conserved residues are shaded in grey and the sequence of the putative transit peptide is in italics
of the coding region (Fig 2) A chloroplast transit peptide
prediction program (CHLOROP) [27], predicted a putative
cleavage site between arginine 37 and alanine 38, but the
mature protein starts at Ser39, as indicated by the peptide
sequencing (Fig 2) It is possible that the protein cleaved
between positions 37 and 38 is further processed in the
chloroplast to the native form
Southern blot analysis on genomic DNA digested with
Apal (an enzyme known to cleave within the Ch-Prx/ gene
sequence), or EcoRI (an enzyme that does not cleave within
Ch-Prx1 gene), produced four and two fragments, respec-
tively, that hybridized to the radiolabeled Ch-Prx/ coding
sequence (Fig 3) These results indicate that an additional
gene homologous to the Ch-Prx/ gene exists in C rein-
hardtii genome In this respect, it can be noted that a
sequence of a putative cytosolic Chlamydomonas Prx is
available in data banks
Amino-acid sequence comparisons
We compared the Ch-Prxl amino-acid sequence to other
similar proteins Three groups could be distinguished
(Fig 4) The first group contains Prx with one conserved
cysteine but these proteins seem to be functionally closer to
2Cys-Prx than to 1Cys-Prx Members of the second group
contain two conserved cysteines Ch-Prx1 belongs to this
group and is close to the higher plant 2Cys-Prx proteins,
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 cysteine
Biochemical characterization of recombinant Ch-Prx1
Under oxidizing conditions, 2Cys-Prx exists predominantly
as dimers linked by two identical disulfide bonds between
the first Cys of one subunit and the second Cys of the other
subunit [10] Ch-Prxl also shares this feature: under
Apal EcoRI
kb
12- 10-
1.7- 1.4-
Fig 3 Southern blot analysis of the C#-prx7ƒ gene CGenomic DNA from CW15 Chlamydomonas strain was digested with Apal or EcoRI, size-fractionated on a 0.8% agarose gel, and transferred to a Hybond N* nylon membrane The filter was hybridized with the *’P-radiola- beled Ch-Prx] coding region probe Molecular size markers are indi- cated on the left
reducing conditions it is a monomer of 21 kDa, which is converted to a dimer under nonreducing conditions (Fig 5) The antioxidant activity of Ch-Prx1 was characterized in a mixed function oxidation system (Fig 6) Prx proteins are
Trang 6
Oryza
I Conserved Cys but
y-typell trypanosoma
2 Conserved Cys AOE37-2human
paghuman mouse
ma
RattusPrdx3
——— AOPI-human
y-type I
Trypanosoma-mpx
Ch-Prx1
Phaseolus
——AtMHF Chloroplastic proteins
~BrassicaBAS1
barleyBAS1 [Chinese cabbage spinachBAS1 AtBAS1
T aquaticus
| FT Tortula
— AtPer1
—— BrassicaPer1
pm barleyPER1
Fig 4 Phylogenetic tree of peroxiredoxins CLUSTAL x was used to generate the tree Accession numbers: Trypanosoma-mpx, AJ006226; Try- panosoma, 126666; yeast: Type ITPx, NP013684, type IT TPx, u53878, 1Cys-Prx, ybl0524; Chinese cabbage C2C-Prx, AF052202; human: AOE37-2, u25182, AOPI, P30048, pag, q06830, NKEFB, 119185; barley: BAS1, 734917, PER1, X96551; Arabidopsis: BAS1, X97910, TPx2, AF121356, MHF1S5.19, AF326871, Perl, 004005; Chlamydomonas Ch-Prx1, AJ304857; spinach BAS1, x 94219; mouse TPx, u20611; Bromus pbs128, p52571; Tortula, u40818; Drosophila DPx-2540—2, AF311880; Rattus Prdx3, NM022540; Oryza, AF203879; Phaseolus, AJ288896; Thermus aquaticus, AF276071; Brassica Perl, AF139817, BASI, AF311863
known to prevent damage of DNA against ROS ROS can
be produced by incubating dithiothreitol with Fe’ *, which
catalyzes the reduction of Oz to HO The latter is further
converted by the Fenton reaction to hydroxyl radicals
[19, 20] The radicals produced by incubating dithiothreitol with Fe** caused complete degradation of 2 ug pBluescript DNA within | h Ch-Prx1 protected the pBluescript DNA against degradation, while BSA, even at a concentration of
Trang 7kDa
97->
67 —>
43—*
30 —*>
20.1—>
14.4—»
Fig 5 Analysis by SDS/PAGE of the monomer/dimer shift of recom-
binant Ch-Prx1 The protein was either reduced with 2-mercapto-
ethanol, or not, as indicated
Fig 6 Protection of DNA against free radical attack by the recombi-
nant Ch-Prx1 protein Plasmid Bluescript SK DNA was incubated for
30 min in a thiol-MFO system containing 3.0 um Fe** Lane 1 is
untreated SK DNA, open circle (OC) and supercoiled (S) plasmid
DNA are indicated; lanes 2-3 containing 20 or 5 HM Ch-Prx1 protein
show that protection of DNA against nicking increases with increasing
amounts of protein; lane 4 (C) control without protein shows maxi-
mum DNA degradation
20 um, had no effect (not shown) The degree of protection
correlated with the amount of Ch-Prx1 added to the assay
Thioredoxin-dependent peroxidase activity of Ch-Prxl
towards HO, or t-BOOH was examined indirectly by
measuring the oxidation rate of NADPH (followed by the
A 12
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Time (min)
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20)
Time (min)
Fig 7 Peroxidase activity of recombinant Ch-Prx1 toward H,O, or t-butyl hydroperoxide Dependence of the reaction on reduced thiore- doxin (A) The reaction was followed spectrophotometrically by NADPH oxidation in a coupled assay with NTR from Arabidopsis and thioredoxin h from Chlamydomonas Complete assays with HạO; (©)
or t-BOOH (WM) Controls: minus TRX (4), minus PRX (), minus peroxide (O), minus NTR (5%) (B) Peroxidase activity with various thioredoxins The concentration of t-BOOH was measured colori- metrically and expressed as a percentage of the initial concentration Thioredoxins were reduced with dithiothreitol Chlamydomonas Trx h (A) or Trx m (), spinach Trx f (@) or Trx m () Control without Trx (A)
decrease in A349) in the presence of NTR and thioredoxin
Pure recombinant proteins expressed in Escherichia coli
were used in this test: NTR from A thaliana, Trx h from
Chlamydomonas and Ch-Prx1 Figure 7A shows a time-
course of NADPH oxidation with either H»O> or t-BOOH
as substrates Clearly, Ch-Prxl was equally efficient with both, and the reaction required all three protein components (Ch-Prxl, Trx h and NTR) The specific activity of the recombinant enzyme was identical to the specific activity of the native protein purified from Chlamydomonas (data not shown) The rate of NADPH oxidation was very weak when Trx m from Chlamydomonas was used (data not shown), probably because of the weaker affinity of Arabid- opsis NTR for Trx m [21] Therefore, the ability of Trx m from Chlamydomonas to donate protons to Ch-Prxl was measured directly by following the degradation of t-BOOH,
Trang 8using the peroXOquant kit (see Experimental procedures),
in the presence of dithiothreitol as an electron donor to Trx
The rate of disappearance of t-BOOH was identical with
Trx m and Trx h from Chlamydomonas used at the same
concentration (Fig 7B) When Trx was omitted, the rate of
disappearance of t-BOOH was negligible, proving that
dithiothreitol alone, used at low concentration, cannot
significantly activate Ch-Prxl In order to determine
whether different chloroplastic Trx differ in their abilities
to function with Ch-Prxl, we compared the efficiencies of
two Trx isoforms f and m from spinach, because no Trx f
from Chlamydomonas has been isolated until now Both
were active with Ch-Prx1, but while Trx f was as efficient as
Trx h and m from Chlamydomonas, Trx m from spinach
showed a lower efficiency
Regulation of Ch-Prx7 expression
To study possible roles of Ch-Prx1 in vivo, the expression of
the Ch-Prx] gene was monitored under different culture
conditions
A number of genes have been reported to be regulated by
light, among them the 7rxm and Trxh genes from
Chlamydomonas [28] As Prx are Trx-dependent proteins,
we investigated a possible regulation of the Ch-Prx/ gene
expression by light Under dark conditions, levels of
Ch-Prx1l gene transcripts are relatively low (Fig 8A)
Ch-Prx1 gene transcript levels increased in illuminated cells
reaching a maximum after ~6h 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-Prx/ transcripts
is affected by light/dark conditions
The role of noncyclic photosynthetic electron transport in
regulating the expression of the Ch-Prx/ gene was studied
by blocking electron transfer with 3-(3/,4-dichlorophenyl)-
1,1,-dimethyl urea (DCMU) or 2,5-dibromo-3-methyl-6-
isopropyl-p-benzoquinone (DBMIB) Light induction of Ch-Prx1 gene expression was affected by both inhibitors (Fig 8B, lanes 3 and 4), indicating that photosynthetic electron transport is required for upregulating Ch-Prx/ 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-Prx/ transcripts were found in cells bubbled with pure oxygen for 90 min in the dark (Fig 8C, lane 2) This shows that oxidative stress, directly or indirectly, affects Ch-Prx] 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-Prx/ gene in Chlamydomonas
DISCUSSION
The mixed disulfide approach as a tool to isolate new thioredoxin targets
The formation of stable mixed-disulfide cross-linked com- plexes has been used previously to determine interactions between target enzymes, such as phosphoribulokinase or NADP-malate dehydrogenase, and thioredoxins [12,29] This approach also provided evidence for conformational changes occurring in the structure of thioredoxin reductase upon interaction with its substrate thioredoxin [30] Using the mixed disulfide approach for isolation of Trx target proteins in vivo is difficult because of the limited stability of Trx-target complexes in cells in which reductants that split disulfides, are abundant To overcome this difficulty, thioredoxin-deficient yeast or EF coli mutant strains have been used to express a single-cysteine mutant thioredoxin allowing the isolation of thioredoxin targets in yeast [8] and
0 2 4 6 8 10 12 14 16 18 20 22
a=
Fig 8 Ch-Prx1 gene expression (A) Levels of Ch-Prx/ 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-Prx] cDNA probe Numbers above the lanes indicate the time at which the RNA samples were taken (B) Induction of Ch-Prx] gene expression in Chlamydomonas by light in the absence and in the presence of DCMU or DBMIB Cultures were grown in continuous light and RNA samples were analyzed for Ch-Prx/ transcript levels by northern analysis Lane 1, cells taken after 16 h in the dark; lane 2, cells taken 3 h after the start of the light period; lane 3, cells taken after 3 h in the light in the presence of 20 uM DCMU (added at 0 h light); lane 4, cells taken after 3 h in the light in the presence of 1 uM 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 dark; lane 2, cells taken after 11 h in the dark after 90 min bubbling with 100% Os.
Trang 9in E coli [23] Because Trx-deficient mutants of Chlamydo-
monas are not available, we combined the mixed-disulfide
method with affinity chromatography, using an affinity
column made of Trx h (C39S Trx h) mutated at the less
reactive Cys of its active site The major protein retained on
the affinity column was a thioredoxin-dependent peroxidase
(Prx) that belongs to the same family as a peroxidase
isolated in vivo in yeast using mutated Trx of Arabidopsis [8]
The predominant formation of mixed disulfides between
Prx and C39S Trx h in the presence of a number of well-
known Trx-dependent enzymes appears surprising but may
be explained by the natural abundance of the peroxidase
that might compete with other targets, but also by structural
features of the regulatory 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, on the other hand, is linked by
intermolecular disulfide bonds Upon cleavage by Trx the
subunits separate leaving no proximal Cys that could attack
the mixed disulfide formed between Trx and its target Thus,
the mixed disulfide approach seems to favour the isolation
of targets bearing an intermolecular disulfide bridge
Structural and functional characteristics of Ch-PRX1
The amino-acid sequence of Ch-Prx1 shares highest identity
with the BASI protein of Brassica, spinach, barley and
A thaliana, PR1 of Phaseolus and MHF of A thaliana
These proteins belong to the 2Cys-Prx subfamily All plant
2Cys-Prx proteins, except BASI of barley, the complete
cDNA of which has not been isolated, contain putative
chloroplast-targeting sequences Ch-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 site The homologous BASI
protein of Arabidopsis was shown to be imported into
isolated plastids after post-translational modification [6]
Like other 2Cys-Prx enzymes previously described in
yeast, mammals and plants, Ch-Prx1 displayed antioxidant
and peroxidase activities The enzyme could reduce hydro-
gen peroxide as well as alkyl hydrogen peroxide and exerted
a strong protective effect against DNA degradation by free
radicals of oxygen More extensive biochemical character-
izations, including K,, determinations, are needed to better
define the substrate specificity of Ch-Prx1
Peroxiredoxin and thioredoxin specificity
There is increasing evidence for a role of Trx in coping with
oxidative stress A mutant strain of yeast Saccharomyces
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
Trxl, 2, or 4, or type x Trx conferred hydrogen peroxide
tolerance [8,33] The fact that the thioredoxin-dependent
Ch-Prxl of Chlamydomonas is able to detoxify hydroper-
oxides provides additional support for a function of Trx in
response to oxidative stress
All types of plant Trx, whether cytosolic or chloroplastic,
were able to serve as hydrogen donors for Ch-Prx1 in vitro
Interestingly, the spinach f-type chloroplastic isoform was more efficient with Ch-Prx! than the m-type chloroplastic Trx suggesting that Trx fis the 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 did not [33] However, it cannot be excluded that the yeast cytosolic NTR is unable to reduce Trx f
The lack of specificity of Trx toward Trx-dependent proteins can explain why a presumably chloroplastic Prx could be isolated with cytosolic 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 in our protein extracts at much lower concentration than the chloroplast Prx It can be noted that a similar loss of specificity was recently reported by Motohashi ef al [34] who trapped targets of thioredoxin f on an affinity 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, LHCH, the chloroplast ATPase and ribulose 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-Prx/ gene expression may
be controlled either directly by ROS, e.g by H2O>, which is known for its role in signal transduction [36], or indirectly by sensors of redox conditions in the chloroplast, e.g ascorbate [37] We found that transcript levels of the Ch-Prx/ gene markedly increased in illuminated cells (Fig 8A,B) but also upon bubbling cultures with 100% oxygen in the dark (Fig 8C), conditions in which production of ROS is likely
to be high Blocking noncyclic photosynthetic electron flow with DCMU or DBMIB inhibited the accumulation of Ch-Prx1 transcripts in the light (Fig 8B), suggesting an influence of the photosynthetic electron flow on Ch-Prx1 gene expression The redox state of plastoquinone, known
to regulate the expression of some genes [38,39], does not seem to be the responsible for this regulation, because both DCMU and DBMIB exert an inhibitory effect The regulation of Chlamydomonas Trx m gene expression was also shown to be dependent on the photosynthetic electron
flow [28] but independent of the redox state of the
plastoquinone pool The expressions of other chloroplastic genes, such as those encoding phosphoribulokinase and ferredoxin-NADP-reductase, follow the same pattern [40] These results are in favor of a coordinated regulation of both Trx and Prx in the chloroplast and fully support the hypothesis that Ch-Prx1 is a chloroplastic protein
Because Ch-Prx] gene expression correlates with pro-
duction of ROS, and because Ch-Prx! is able to remove
peroxides, it is likely that Ch-Prx1 is involved in detoxifi- cation of ROS in the Chlamydomonas chloroplast It should
be of interest to over- and under-express Ch-Prxl in Chlamydomonas to better understand its importance in protection against oxidative stress
Trang 10ACKNOWLEDGEMENTS
We would like to thank Prof Peter Schiirmann for a generous gift of
thioredoxins m and f from spinach and Dr E Issakidis-Bourguet for
stimulating discussions
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