In order to investigate whether chloroplast GAPDH may undergo glutathionylation and other redox modi-fications, we examined the effect of oxidized glutathi-one GSSG and other oxidizing mo
Trang 1glyceraldehyde-3-phosphate dehydrogenase is selectively regulated by glutathionylation
Mirko Zaffagnini1,2, Laure Michelet2, Christophe Marchand3, Francesca Sparla1,
Paulette Decottignies3, Pierre Le Mare´chal3, Myroslawa Miginiac-Maslow2,
Graham Noctor2, Paolo Trost1and Ste´phane D Lemaire2
1 Laboratory of Molecular Plant Physiology, University of Bologna, Italy
2 Institut de Biotechnologie des Plantes, UMR 8618, CNRS ⁄ Universite´ Paris-Sud, Orsay, France
3 Institut de Biochimie et Biophysique Mole´culaire et Cellulaire, CNRS ⁄ Universite´ Paris-Sud, Orsay, France
Glutathione represents the major low-molecular-weight
thiol in most cells In addition to its well-established
role in cellular defense against oxidative stress,
gluta-thione can also promote a reversible post-translational
modification, termed protein glutathionylation [1,2]
This modification consists of the formation of a mixed
disulfide between glutathione and cysteine residues of
proteins In mammals, glutathionylation occurs under
oxidative stress conditions and may protect cysteines from oxidation to cysteine sulfinic (-SO2H) or sulfonic (-SO3H) acids In fact, while oxidation into sulfinic or sulfonic groups is irreversible, 2-electron reduction of glutathionylated cysteines can regenerate protein thiols Glutathionylation has been shown to alter, either posi-tively or negaposi-tively, the activity of several proteins [3–8] Recent proteomic approaches allowed the
Keywords
Arabidopsis thaliana; Calvin cycle; GAPDH;
glutathionylation; oxidative stress
Correspondence
S D Lemaire, Institut de Biotechnologie
des Plantes, Baˆtiment 630, Universite´
Paris-Sud, F-91405 Orsay Cedex, France
Fax: +33 1 69153423
Tel +33 1 69153338
E-mail: stephane.lemaire@u-psud.fr
(Received 3 October 2006, revised 25
October 2006, accepted 7 November 2006)
doi:10.1111/j.1742-4658.2006.05577.x
In animal cells, many proteins have been shown to undergo glutathionyla-tion under condiglutathionyla-tions of oxidative stress By contrast, very little is known about this post-translational modification in plants In the present work,
we showed, using mass spectrometry, that the recombinant chloroplast
A4-glyceraldehyde-3-phosphate dehydrogenase (A4-GAPDH) from Arabid-opsis thaliana is glutathionylated with either oxidized glutathione or reduced glutathione and H2O2 The formation of a mixed disulfide between glutathione and A4-GAPDH resulted in the inhibition of enzyme activity
A4-GAPDH was also inhibited by oxidants such as H2O2 However, the effect of glutathionylation was reversed by reductants, whereas oxidation resulted in irreversible enzyme inactivation On the other hand, the major isoform of photosynthetic GAPDH of higher plants (i.e the AnBn-GAPDH isozyme in either A2B2 or A8B8 conformation) was sensitive to oxidants but did not seem to undergo glutathionylation significantly GAPDH cata-lysis is based on Cys149 forming a covalent intermediate with the substrate 1,3-bisphosphoglycerate In the presence of 1,3-bisphosphoglycerate, A4 -GAPDH was fully protected from either oxidation or glutathionylation Site-directed mutagenesis of Cys153, the only cysteine located in close proximity to the GAPDH active-site Cys149, did not affect enzyme inhibi-tion by glutathionylainhibi-tion or oxidainhibi-tion Catalytic Cys149 is thus suggested
to be the target of both glutathionylation and thiol oxidation Glutathiony-lation could be an important mechanism of reguGlutathiony-lation and protection of chloroplast A4-GAPDH from irreversible oxidation under stress
Abbreviations
BPGA, 1,3-bisphosphoglyceric acid; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GSH, reduced glutathione; GSSG, oxidized glutathione; ROS, reactive oxygen species; TRX, thioredoxin.
Trang 2identification of many proteins undergoing such
post-translational modifications in mammalian and yeast
cells [9–12] One of the prominent glutathionylated
pro-teins in mammalian cells under stress is the glycolytic
enzyme, glyceraldehyde-3-phosphate dehydrogenase
(GAPDH), which is inactivated by glutathionylation,
pre-sumably of its active-site Cys149 [13–17]
In addition to cytosolic GAPDHs, plants also contain
chloroplast GAPDH isoforms that participate in the
Calvin cycle by catalyzing the reduction of
1,3-bisphos-phoglyceric acid (BPGA) to glyceraldehyde-3-phosphate
using NAD(P)H as a reductant All GAPDHs, including
chloroplastic isoforms, share a common reaction
mech-anism based on a highly reactive cysteine (Cys149),
which is made acidic by an interaction with His176 [18]
During the catalytic cycle, the highly reactive thiolated
group of Cys149 (Cys-S–) forms a thioacylenzyme
inter-mediate by nucleophilic attack on the substrate [19,20]
As a side-effect, the acidic nature of Cys149 makes it
particularly prone to oxidation and to other redox
modi-fications of its thiol group [15,21,22] In glycolytic
mammalian GAPDH, these modifications include
S-glu-tathionylation, S-nitrosylation [15,23–26] and formation
of an intrasubunit disulfide with the neighboring Cys153
[27] However, although chloroplasts are a major site of
reactive oxygen species (ROS) production, particularly
under photoinhibitory conditions [28], it is not known
whether chloroplast GAPDH is also subject to thiol
oxi-dation or glutathionylation, or any other redox reaction
affecting catalytic Cys149
The major isoform of chloroplast GAPDHs in
higher plants displays the AnBnstructure and is
regula-ted by metabolites and thioredoxin f (TRX f), a small
disulfide oxidoreductase involved in the activation of
the Calvin cycle by light [29–33] B subunits are almost
identical to A subunits, except for the presence of a
C-terminal extension containing a pair of cysteines,
which is the target of TRX regulation [34] Oxidized
thioredoxin and low NADP(H)⁄ NAD(H) ratios
pro-mote the aggregation of fully active A2B2-GAPDH
into A8B8-GAPDH hexadecamers with partially
inhib-ited NADPH-dependent activity, whereas the
secon-dary activity with NADH as a coenzyme is
constitutively low and not regulated [35] A second
iso-form of chloroplast GAPDH is homotetrameric (A4
-GAPDH) and TRX-insensitive [36,37], but can be
regulated by the redox-sensitive peptide, CP12, which
promotes the formation of a supramolecular complex
with phosphoribulokinase [35,38–40] Besides
regula-tion of chloroplast GAPDH by photosynthetically
reduced TRX and pyridine nucleotides, a type of
regu-lation that is primarily linked to light⁄ dark conditions
in the chloroplast, post-translational modifications of
catalytic Cys149, such as glutathionylation, might con-stitute novel mechanisms of GAPDH regulation under oxidative stress
Compared with nonphotosynthetic organisms, very little is known about glutathionylation in plants, although recent studies have allowed the identification
of a number of plant proteins undergoing glutathiony-lation [41–46] We have recently shown that chloro-plast f-type TRXs are modified by glutathionylation, resulting in less efficient activation of TRX-sensitive GAPDH and NADP-malate dehydrogenase in the light [46] Fructose-1,6-bisphosphate aldolase, a mem-ber of the Calvin cycle, like GAPDH, has also been reported to undergo glutathionylation [44]
In order to investigate whether chloroplast GAPDH may undergo glutathionylation and other redox modi-fications, we examined the effect of oxidized glutathi-one (GSSG) and other oxidizing molecules, including
H2O2, on recombinant Arabidopsis thaliana A4-GAPDH and on native spinach A2B2-GAPDH and A8B8
-GAP-DH We examined the effect of these modifications on the enzyme activities, their reversibility in the presence
of reductants, and the protective effect of the substrate BPGA and cofactors Glutathionylation was investi-gated by MALDI-TOF mass spectrometry and site-directed mutagenesis The results indicate that glutathionylation might constitute a previously unde-scribed mechanism of redox regulation and⁄ or protection against oxidative damage of chloroplastic
A4-GAPDH
Results
Inactivation of A4-GAPDH by GSSG and other oxidants, and protection by substrate and cofactors
Incubation of recombinant Arabidopsis A4-GAPDH with 5 mm GSSG resulted in a rapid decrease in enzyme activity Indeed, the NADPH-dependent activ-ity decreased to less than 20% after 10 min of incuba-tion (Fig 1A) and identical results were obtained with NADH as the coenzyme (data not shown) The decrease in GAPDH activity displayed a linear rela-tionship with increasing GSSG concentrations in the 0–5 mm range (Fig 1B) A slow, reproducible loss of activity, observed when the enzyme was incubated in buffer alone or with reduced glutathione (GSH) as a control, suggested that A4-GAPDH underwent sponta-neous inactivation upon dilution The exposure of
A4-GAPDH to 1 mm H2O2, CuCl2, or diamide also caused a rapid loss of enzyme activity (Fig 2) Kinet-ics of inactivation were comparable for the three
Trang 3different oxidants and led to a complete loss of activity
within 2 min of incubation In the presence of a
10-fold lower concentration of H2O2(0.1 mm, Fig 2), the
inactivation kinetics was comparable to that obtained
in the presence of 5 mm GSSG (Fig 1) These results
are consistent with the extreme sensitivity to oxidants
described for glycolytic GAPDH [15,18]
In order to determine if substrate and cofactors
might protect A4-GAPDH from glutathionylation
and⁄ or oxidation, the enzyme was incubated with
GSSG or diamide in the presence of BPGA or
NADPH Both the substrate and the cofactor appeared
to protect efficiently the enzyme from the two
inactivat-ing treatments (Fig 3A) In contrast, protection against
the small H2O2molecule was only observed in the pres-ence of BPGA (Fig 3B) Moreover, in the prespres-ence of BPGA, but not in the presence of NADPH, the enzyme remained totally active during the time-course of incu-bation (Fig 3), suggesting protection from the slow spontaneous inactivation of A4-GAPDH observed in the control samples (Fig 1) Similar results were obtained with NADH as a cofactor (data not shown)
Glutathionylation of chloroplastic A4-GAPDH
by GSSG is reversible
In order to test the reversibility of A4-GAPDH inacti-vation, the enzyme was treated with 10 mm dithiothrei-tol after pre-incubation with 5 mm GSSG, 1 mm diamide or 1 mm H2O2. As shown in Fig 4, enzyme inactivation caused by 1 mm diamide or H2O2 could not be reversed by dithiothreitol, suggesting that these oxidants induced irreversible oxidation, possibly affect-ing the sulfhydryl group of the active site Cys149 Irre-versibly oxidized cysteine thiols are typically converted
to sulfinic (-SO2H) or sulfonic (-SO3H) acids [22] On the other hand, inactivation caused by GSSG was par-tially (60%) reversed by dithiothreitol, although the remaining 40% of the initial activity was still irrevers-ibly lost These results clearly indicate that oxidants, such as diamide or H2O2, led to A4-GAPDH inactiva-tion by irreversible oxidainactiva-tion, whereas GSSG, at least
in part, inhibited A4-GAPDH by a different and reversible mechanism, possibly involving glutathionyla-tion
A
B
GSSG Concentration (m M )
A 4
0
20
40
60
80
100
Time (min)
A 4
G-0
20
40
60
80
100
Buffer or GSH
GSSG
Fig 1 Inactivation of A 4 -GAPDH by GSSG (A) Time-dependent
inactivation A4-GAPDH was incubated with 5 m M GSSG (open
cir-cles), or with tricine buffer as a control (closed circles) Results
with 5 m M GSH were identical to those of controls (B)
Concentra-tion-dependent inactivation A 4 -GAPDH was incubated with various
concentrations of GSSG for 10 min at 25 C Aliquots of the
incuba-tion mixtures were withdrawn at the indicated time points and the
remaining NADPH-dependent activity was determined Activity is
given as a percentage of the initial activity.
Time (min)
A 4
G-0 20 40 60 80
100
Buffer
0.1 m M H 2 O 2 1m M oxidant
Fig 2 Inactivation of A4-GAPDH by oxidants Time-dependent inac-tivation by 1 m M H2O2, 1 m M diamide or 1 m M CuCl2 (open squares), 0.1 m M H 2 O 2 (closed squares) or tricine buffer as the control (closed circles) Aliquots of the incubation mixtures were withdrawn at the indicated time points and the remaining NADPH-dependent activity was determined Activity is given as a percent-age of the initial activity.
Trang 4In order to test this hypothesis, GSSG-treated A4
-GAPDH was analyzed by MALDI-TOF mass
spectro-metry (Fig 5) A clear shift in molecular mass was
observed after GSSG treatment The shift of the major
peak, corresponding to GAPDH subunit A (theoretical
molecular mass 36 346 Da), is consistent with the
pres-ence of one glutathione adduct per subunit (theoretical
additional mass¼ 305 Da) The other peaks observed
correspond to matrix adducts on A4-GAPDH
More-over, upon the addition of dithiothreitol, the molecular
mass of A4-GAPDH shifted back to the mass of the
untreated protein These results clearly demonstrate
that Arabidopsis A4-GAPDH can undergo
glutathiony-lation, and that dithiothreitol can reverse this
post-translational modification
Glutathionylation of chloroplastic A4-GAPDH by GSH in the presence of low concentrations of hydrogen peroxide is fully reversible
Besides thiol disulfide exchange mediated by GSSG, it has been reported that protein glutathionylation can also be achieved in the presence of GSH and oxidants, conditions that are believed to promote the conversion
of protein thiols into sulfenic acids which then react with GSH to give rise to mixed disulfides [47–49] Incubation of Arabidopsis A4-GAPDH with 0.1 mm
H2O2 and 0.5 mm GSH (Fig 6A) resulted in a rapid decrease of enzyme activity with kinetics comparable
to that obtained in the presence of 0.1 mm H2O2alone (Table 1) Moreover, as in the case of 0.1 mm H2O2 alone, BPGA appeared to provide full protection from inactivation, whereas almost no protection was observed in the presence of NADPH
The addition of dithiothreitol provided nearly 50% recovery of the initial activity of samples inactivated
by 0.1 mm H2O2 (Fig 6B) This partial recovery, not observed after treatment with 1 mm H2O2 (Fig 4), indicated that part of the A4-GAPDH molecules were reversibly oxidized, probably to sulfenic acid (-SOH)
or, by analogy to animal GAPDH [27], an intrasubunit disulfide with Cys153 (conserved in chloroplast GAPDH [50]) might have formed Indeed, both sulfenic acids and disulfides are generally reduced by dithiothreitol On the other hand, an almost complete recovery of the initial
A
B
Time (min)
A 4
G-0
20
40
60
80
100 BPGA then H 2 O 2 (0.1 m M )
NADPH then H 2 O 2 (0.1 m M )
H 2 O 2 (0.1 m M )
Time (min)
A 4
G-0
20
40
60
80
100 BPGA then (GSSG or Diamide)
NADPH then (GSSG or Diamide)
GSSG Diamide
Fig 3 Protection of A4-GAPDH by BPGA and NADPH A4-GAPDH
was incubated in the presence of (A) 5 m M GSSG or 1 m M diamide
in the presence of BPGA (open triangles) or 0.2 m M NADPH
(closed triangles) at 25 C, or (B) 0.1 m M H2O2in the presence of
BPGA (open diamonds) or 0.2 m M NADPH (closed diamonds)
Inac-tivation by 5 m M GSSG (open circles), 1 m M diamide (closed
circles), or 0.1 m M H2O2(open squares) are presented for
compar-ison NADPH-dependent activity is given as a percentage of the
initial activity.
A 4
G-0 20 40 60 80 100
+dithiothreitol +dithiothreitol +dithiothreitol GSSG
Diamide
Fig 4 Reversal, by dithiothreitol, of the inactivation of
pretreat-ed A 4 -GAPDH A 4 -GAPDH was incubated with 5 m M GSSG, 1 m M
diamide or 1 m M H2O2for 10 min and subsequently treated with
10 m M dithiothreitol for 10 min at 25 C The remaining NADPH-dependent activity was determined before (black bars) and after (white bars) treatment with dithiothreitol Activities are given as a percentage of the initial activity measured before the inactivation treatment.
Trang 5activity upon the addition of dithiothreitol was observed
for A4-GAPDH samples treated with 0.1 mm H2O2and
0.5 mm GSH, either in the presence or absence of
NADPH This suggested that, under the latter
condi-tions, the mechanism of inactivation might have been
different and may involve glutathionylation This
hypo-thesis was confirmed by MALDI-TOF mass
spectro-metry, which demonstrated a 305 Da mass increase for
50% of the A4-GAPDH subunits in the samples
trea-ted with 0.1 mm H2O2 and 0.5 mm GSH (Fig 7)
Hence, these results clearly demonstrate that A4-GAPDH
can undergo glutathionylation in the presence of low
concentrations of H2O2 and GSH Moreover, the
com-plete recovery of the initial activity after dithiothreitol
treatment indicates that glutathionylation is able to
protect A4-GAPDH from irreversible oxidation
Identification of the cysteine residue involved
in the inactivation of A4-GAPDH
The observation that BPGA fully protects GAPDH
from oxidation and⁄ or glutathionylation suggests
indeed that cysteines of the active site are the targets
of the redox modification Besides Cys149, which
forms a covalent thioacylenzyme with BPGA during
the catalytic cycle, Cys153 is also in proximity to the
active site, whereas the remaining three cysteines of
Arabidopsis A4-GAPDH reside in different regions of
the protein [51] In theory, inactivation of A4-GAPDH
by glutathione and⁄ or oxidants could thus depend on either Cys149 or Cys153 being redox modified High performance liquid chromatography and MALDI-TOF mass spectrometry analyses of tryptic peptides of
A4-GAPDH could not clarify this ambiguity because both Cys149 and Cys153 belong to a single long pep-tide which was very weakly desorbed⁄ ionized from the matrix This point was thus addressed by site-directed mutagenesis of Cys153 Mutation of Cys149 was not attempted, because this residue is known to be abso-lutely essential for catalysis [20] On the other hand, the activity of the purified recombinant C153S mutant was similar to that of the wild-type protein (data not shown), and the kinetics of inactivation in the presence
of 1 mm H2O2, 0.1 mm H2O2, or 0.1 mm H2O2 + 0.5 mm GSH were identical for both proteins (Fig 8A,B and Table 1) Similarly to wild-type GAPDH, BPGA (but not NADPH) protected the mutant from irreversible oxidation by H2O2 and glutathionylation
by H2O2 + GSH MALDI-TOF mass spectrometry confirmed that C153S A4-GAPDH underwent gluta-thionylation after treatment with 0.1 mm H2O2+ 0.5 mm GSH (Fig 9) The spectra are comparable to those obtained for the wild-type enzyme, showing a similar reversion of the 305 Da shift after dithiothreitol treatment Overall, these results rule out the possibility that Cys153 might be the target of glutathionylation or
B
36400 36353
36663
36000
36000
A
0
20
40
60
80
100
0
20
40
60
80
100
Mass (m/z)
Mass (m/z)
Fig 5 MALDI-TOF mass spectrometry indi-cates that A 4 -GAPDH undergoes glutath-ionylation Mass spectra of GSSG treated
A4-GAPDH were performed before and after treatment with 10 m M dithiothreitol (20 min) After dithiothreitol treatment (A),
A4-GAPDH was at its expected mass (36 346 Da) and shows a decrease of
310 Da in comparison with the spectrum before dithiothreitol treatment (B) Accuracy
of the measurement is ± 7 Da (0.02%).
Trang 6irreversible oxidation Although a conformational
change after glutathionylation of a cysteine far distant
from the active site cannot be completely excluded, the
results strongly suggest that Cys149 is the target of
chloroplast GAPDH glutathionylation or irreversible
oxidation
Nonetheless, Cys153 seemed to play some role in
GAPDH protection against irreversible oxidation In
fact, the activity of mutant C153S treated with 0.1 mm
H2O2+ 0.5 mm GSH could not be completely recov-ered by adding dithiothreitol, even when glutathionyla-tion was performed in the presence of NADPH (compare Figs 8C and 6B) This indicates that under mild oxidizing conditions (0.1 mm H2O2), mutant C153S was not completely protected by GSH and par-tially underwent irreversible oxidation Moreover, the recovery by dithiothreitol after treatment with 0.1 mm
H2O2 alone was also much lower in mutant C153S than in wild-type A4-GAPDH (Figs 8C and 6B, respectively) It is possible that the role played by Cys153 in preventing the irreversible oxidation of Cys149 might depend on the formation of a (transient) disulfide between Cys149 and Cys153, as observed in human GAPDH [27]
AnBn-GAPDH is sensitive to oxidants, but is not significantly prone to glutathionylation
Besides A4-GAPDH, the major chloroplast GAPDH isoform of higher plants comprises A and B subunits in
a stoichiometric ratio and is regulated by thioredoxins and metabolites [35] As attempts to produce recombin-ant Arabidopsis AnBn GAPDH were unsuccessful, we purified native AnBnGAPDH from spinach chloroplasts
in order to test its sensitivity to oxidants and its ability
to undergo glutathionylation AnBn-GAPDH exists in different conformations, and experiments were conduc-ted on either fully active A2B2-GAPDH (conformation prevailing in chloroplasts in the light) or A8B8-GAPDH (‘dark’ conformation with partially inhibited NADPH-dependent activity) Although both forms were inacti-vated by H2O2 treatment, with or without GSH (Fig 10A,B), hexadecameric A8B8-GAPDH was clearly less sensitive to H2O2 than A2B2-GAPDH (Table 1) Similar results were obtained with NADH as a cofactor (data not shown), indicating that treatment with H2O2 did not affect the redox regulation (mediated by C-ter-minal extensions) of the enzyme, a process which is thioredoxin-dependent and specific for the NADPH-linked activity of the enzyme [34] Furthermore, BPGA protected A2B2-GAPDH from inactivation, as in the case of A4-GAPDH, suggesting that catalytic Cys149 was the target of the redox modification (Fig 10A) Pro-tection by BPGA of the A8B8 isoform could not be tested because BPGA incubation is known to convert the hexadecamer into active tetramers [32]
In order to test the reversibility of AnBn inactiva-tion, the enzymes were treated with 10 mm dithiothrei-tol for 10 min after the oxidative treatment Similarly
to A4-GAPDH, dithiothreitol treatment of A2B2 GAPDH, after incubation with 0.1 mm H2O2, allowed
A 4
G-0
20
40
60
80
100
A
B
H 2 O 2 (0.1 m M )
H 2 O 2 (0.1 m M ) +GSH
H 2 O 2 (0.1 m M ) + GSH + NADPH
Time (min)
A 4
G-0
20
40
60
80
100
BPGA then H 2 O 2 (0.1 m M )+GSH
H 2 O 2 (0.1 m M )+GSH
NADPH then H 2 O 2 (0.1 m M )+GSH
Fig 6 Inactivation of A4-GAPDH in the presence of H2O2 and
GSH (A) Protective effect of NADPH and BPGA A4-GAPDH was
incubated with 0.1 m M H 2 O 2 and 0.5 m M GSH alone (open
trian-gles), or in the presence of 0.2 m M NADPH (closed circles) or
BPGA (closed triangles) NADPH-dependent activity is given as a
percentage of the initial activity (B) Reversal of A 4 -GAPDH
inactiva-tion by dithiothreitol A4-GAPDH was inactivated by incubation with
0.1 m M H2O2, alone or in the presence of 0.5 m M GSH, or 0.2 m M
NADPH and 0.5 m M GSH for 10 min at 25 C and subsequently
treated with 10 m M dithiothreitol for 10 min at 25 C The
NADPH-dependent activity was determined before (black bars) and after
(white bars) treatment with dithiothreitol Activities are given as a
percentage of the initial activity measured before the inactivation
treatment.
Trang 7a partial recovery of the initial enzyme activity
(Fig 10C) However, when dithiothreitol treatment
was performed after inactivation with 0.1 mm H2O2+
0.5 mm GSH, the recovery was only slightly improved
This result contrasts with the almost complete recovery
observed for A4-GAPDH in the same conditions
(Fig 6B) and suggests that A2B2 GAPDH might not
be significantly glutathionylated In the case of A8B8
GAPDH, the lower sensitivity of the enzyme to
oxi-dants led to a more complete recovery of the initial activity after dithiothreitol treatment of samples trea-ted either with 0.1 mm H2O2 alone or in the presence
of 0.5 mm GSH (Fig 10C) Therefore, AnBn-GAPDH samples incubated with 0.1 mm H2O2+ 0.5 mm GSH, with or without subsequent dithiothreitol treatment, were analyzed by MALDI-TOF mass spectrometry (Fig 11) No significant shift of the peak correspond-ing to B subunits (theoretical mass¼ 39 357 Da) was
Table 1 Half-time inactivation (in min) of wild-type A 4 -glyceraldehyde-3-phosphate dehydrogenase (A 4 -GAPDH), C153S A 4 -GAPDH,
A 2 B 2 -GAPDH and A 8 B 8 -GAPDH under different oxidative treatments Results are presented as mean ± standard deviation (SD), representa-tive of at least three independent experiments Because A2B2-GAPDH and A8B8-GAPDH were prepared by the addition of 0.2 m M NADP(H)
or NAD(H), respectively, the kinetics in the absence of cofactors could not be determined (ND).
Oxidative Treatment
0.1 m M H2O2
0.1 m M H2O2 + 0.2 m M NAD(P)H
H2O20.1 m M
+ 0.5 m M GSH
0.1 m M H2O2+ 0.5 m M GSH + 0.2 m M NAD(P)H Wild-type A4-GAPDH
half time of inactivation (min)
C153S A 4 -GAPDH
half time of inactivation (min)
A2B2-GAPDH
half time of inactivation (min)
A 8 B 8 -GAPDH
half time of inactivation (min)
0 5 3 3
0 4 6 3 2 1 3 3
35600
Mass (m/z)
35000
Mass (m/z)
0
20
40
60
80
100
0
20
40
60
80
100
Fig 7 MALDI-TOF mass spectrometry indi-cates that A4-GAPDH also undergoes glu-tathionylation in the presence of H2O2and GSH Mass spectra of A 4 -GAPDH treated with 0.1 m M H2O2and 0.5 m M GSH for 1 h
at 25 C were performed before and after a
10 m M dithiothreitol treatment (30 min at
25 C) Accuracy of the measurement is
± 7 Da (0.02%).
Trang 8observed after the glutathionylation treatments For A
subunits (theoretical mass¼ 36 141 Da), besides the
matrix adduct peak (sinapinic acid, theoretical mass
increase 205 Da), a very discrete peak, corresponding
to a 305 Da mass increase, was observed after
treat-ment with H2O2 and GSH, but disappeared after
sub-sequent treatment with dithiothreitol These results are
consistent with the recovery of activity observed after
dithiothreitol treatment (Fig 10C) Therefore, it can
be concluded that the B subunits of AnBn-GAPDH do
not undergo glutathionylation and that
glutathionyla-tion of the A subunits is very limited Overall, these
in vitroresults indicate that glutathionylation does not
seem to play a significant role in the protection of
AnBnGAPDH isoforms against oxidative stress
Discussion
The aims of the present study were to establish
whe-ther chloroplastic GAPDH isoforms undergo
gluta-thionylation and thiol oxidation, and to examine the
effect of these modifications on enzyme activity The
results demonstrate that Arabidopsis A4-GAPDH can
undergo glutathionylation and that this
post-transla-tional modification results in inhibition of the enzyme
activity MALDI-TOF mass spectrometry revealed the
presence of one glutathione adduct per A4-GAPDH
subunit, which could be removed by dithiothreitol with
a concomitant recovery of enzyme activity
Glutath-ionylation of the protein can occur either in the pres-ence of GSSG or in the prespres-ence of H2O2 and GSH
A4-GAPDH can also be irreversibly inactivated by oxi-dants, including H2O2 The substrate BPGA, forming
a covalent intermediate with catalytic Cys149, fully protects the enzyme from either oxidation or glutath-ionylation Mutant C153S, having no other cysteines than Cys149 in the catalytic site, was oxidized and
H 2 O 2 (0.1 m M )
H 2 O 2 (0.1 m M ) +GSH
H 2 O 2 (0.1 m M ) + GSH + NADPH
H 2 O 2 (1 m M )
A 4
G- 0
20 40 60 80 100
Time (min)
0 20 40 60 80
A 4
G-100
A
B
C
BPGA then H 2 O 2 (0.1 m M )+GSH
H 2 O 2 (0.1 m M )+GSH
NADPH then H 2 O 2 (0.1 m M )+GSH
Time (min)
A 4
G- 0 20 40 60 80
NADPH then H 2 O 2 (0.1 m M )
H 2 O 2 (0.1 m M )
H 2 O 2 (1 m M )
Fig 8 Inactivation of the C153S A 4 -GAPDH mutant and reversal by
dithiothreitol (A) Time-dependent inactivation of C153S A 4 -GAPDH
in the presence of H2O2 C153S A4-GAPDH was incubated with
1 m M H2O2(open circles), 0.1 m M H2O2alone (open squares) or in
the presence of 0.2 m M NADPH (closed squares), or in tricine
buf-fer as a control (closed circles) Aliquots of the incubation mixtures
were withdrawn at the indicated time points and the remaining
NADPH-dependent activity was determined Activity is given as a
percentage of the initial activity (B) Time-dependent inactivation of
C153S A4-GAPDH in the presence of H2O2and reduced glutathione
(GSH) C153S A 4 -GAPDH was incubated either with 0.1 m M H 2 O 2
and 0.5 m M GSH alone (open triangles) or in the presence of
0.2 m M NADPH (closed diamonds) or BPGA (open diamonds).
Aliquots of the incubation mixtures were withdrawn at the
indica-ted time points and the remaining NADPH-dependent activity was
determined Activity is given as a percentage of the initial activity.
(C) Reversal of C153S A 4 -GAPDH inactivation by dithiothreitol.
C153S A 4 -GAPDH was inactivated by incubation with 1 m M H 2 O 2 ,
0.1 m M H2O2, alone or in the presence of 0.5 m M GSH, or 0.2 m M
NADPH and 0.5 m M GSH for 10 min at 25 C, and subsequently
treated with 10 m M dithiothreitol for 10 min at 25 C The
NADPH-dependent activity was determined before (black bars) and after
(white bars) treatment with dithiothreitol Activities are given as a
percentage of the initial activity measured before the inactivation
treatment.
Trang 9glutathionylated similarly to wild-type A4-GAPDH.
Taken together, these results strongly suggest that
ArabidopsisA4-GAPDH can be reversibly
glutathionyl-ated and irreversibly oxidized on its catalytic Cys149
Moreover, analysis of the C153S mutant also suggests
that the formation of a disulfide between catalytically
essential Cys149 and the neighboring Cys153 could
contribute to the protection of A4-GAPDH from
irre-versible oxidation
By contrast, the results of the present study show
that AnBn GAPDH isoforms, representing the major
isoforms of photosynthetic GAPDH in chloroplasts of
higher plants, though being sensitive to oxidants, do
not undergo significant modification by
glutathionyla-tion As A and B subunits are almost identical, except
for the C-terminal extension of subunits B, it is very
likely that in AnBn-GAPDH isoforms, the C-terminal
extension of subunits B could partially protect catalytic
Cys149 from oxidation by H2O2 and effectively
pre-vent the attack of glutathione The C-terminal
exten-sion bears a pair of TRX-sensitive cysteines and allows
A2B2-GAPDH to associate into hexadecamers in the
presence of NAD(H) [35] The low sensitivity of A8B8
-GAPDH to H2O2 alone, or in the presence of GSH, is
thus likely to depend on partial steric protection of
active sites within the hexadecamer
Because the results presented here were obtained
in vitro, the question arises as to how important such modifications are in vivo In all the initial experiments described above, we performed A4-GAPDH glutath-ionylation assays in the presence of 5 mm GSSG This corresponds to classical conditions generally used to test protein glutathionylation in vitro However, this concentration of GSSG is significantly higher than the estimated concentration in chloroplasts Indeed, the concentration of the glutathione pool has been estima-ted to be between 1 and 4.5 mm in the stroma [52] and GSSG only represents 10% of this pool All these considerations suggest that glutathionylation of A4 -GAPDH, through nonenzymatic thiol disulfide exchange mediated by GSSG, is probably of limited physio-logical significance The low efficiency of GSSG, as a mediator of protein glutathionylation, has been repor-ted previously [8,48] Other possible mechanisms lead-ing to glutathionylation might involve more reactive oxidized forms of glutathione, such as S-nitrosoglu-tathione and gluS-nitrosoglu-tathione disulfide S-oxide [8], or the initial oxidation of a protein thiol that would subse-quently react with GSH [47–49,53] Which of these mechanisms determines glutathionylation of GAPDH
in vivo remains to be determined However, the results presented here clearly show that A4-GAPDH is
+dithiothreitol
Mass (m/z)
0
20
40
60
80
100
36324.5 36633.4 36326.0
Mass (m/z)
0
20
40
60
80
100
Fig 9 MALDI-TOF mass spectrometry indicates that C153S A 4 -GAPDH undergoes glutathionylation in the presence of H 2 O 2 and GSH Mass spectra of C153S A4-GAPDH treated with 0.1 m M H2O2and 0.5 m M GSH for 1 h at 25 C were performed before and after treatment with 10 m M dithiothreitol (30 min at 25 C) The accuracy of the measurement is ± 7 Da (0.02%).
Trang 10glutathionylated in the presence of physiologically
rele-vant concentrations of H2O2and GSH [52,54],
suggest-ing a mechanism of glutathionylation based on the
primary oxidation of the catalytic Cys149 followed by
reaction with GSH rather than GSSG In vivo, such a
mechanism would be favored under conditions of
oxi-dative stress, leading to enhanced ROS production in
the chloroplast, such as exposure to high light under
unfavorable conditions for photosynthetic metabolism
(e.g cold or water stress) Considering the high
sensi-tivity of A4-GAPDH to oxidation, the formation of a
mixed disulfide between GSH and the active site
cys-teine oxidized to sulfenic acid would prevent its
irre-versible oxidation The results we obtained in vitro
indeed confirmed that GSH-mediated glutathionylation
of A4-GAPDH can effectively protect the enzyme from
irreversible oxidation
However, besides protecting A4-GAPDH,
glutath-ionylation might play a more general role in the
regu-lation of photosynthesis under stress TRX f plays a
major role in the regulation of Calvin cycle enzymes
that are mostly inactive in the dark and are activated
by TRXs under illumination [55] Glutathionylation of
a conserved extra cysteine of TRX f, distinct from the
two active site cysteines, strongly decreases the ability
of TRX f to activate target enzymes, including
GAPDH isoforms containing B subunits [46] This suggests that, under conditions leading to protein glu-tathionylation in the chloroplast (e.g under conditions
of enhanced ROS production), the activity of TRX-dependent Calvin cycle enzymes would be decreased
In particular, AnBn-GAPDH would be down-regulated
A
B
C
Time (min)
A 8
B 8
G-0 20 40 60 80
H 2 O 2 (0.1 m M )
H 2 O 2 (1 m M )
H 2 O 2 (0.1 m M )+GSH
Time (min)
A 2
B 2
G-0 20 40 60 80
H 2 O 2 (0.1 m M )
H 2 O 2 (1 m M )
BPGA then H 2 O 2 (0.1 m M )+GSH
H 2 O 2 (0.1 m M )+GSH
0 20 40 60 80 100
H 2
O 2
H 2
O 2
H 2
O 2
H 2
O 2
H 2
O 2
H 2
O 2
+dithiothreitol +dithiothreitol
+dithiothreitol +dithiothreitol
Fig 10 Inactivation of A n B n -glyceraldehyde-3-phosphate
dehydroge-nase (A n B n -GAPDH) and reversal by dithiothreitol A time-dependent
inactivation of nonaggregated AnBn-GAPDH (tetrameric form) by
1 m M H2O2 (open circles), 0.1 m M H2O2 alone (open squares),
0.1 m M H 2 O 2 and 0.5 m M GSH, in the absence (open triangles)
or presence of BPGA (open diamonds), or in tricine buffer as a
con-trol (closed circles) Aliquots of the incubation mixtures were
withdrawn at the indicated time points, and the remaining
NADPH-dependent activity was determined Activity is given as a
percent-age of the initial activity (B) Time-dependent inactivation of
aggregated A n B n -GAPDH (hexadecameric form) by 1 m M H 2 O 2
(open circles), 0.1 m M H 2 O 2 alone (open squares) or in the
pres-ence of 0.5 m M GSH (open triangles), or in K-phosphate buffer as
the control (closed circles) Aliquots of the incubation mixtures
were withdrawn at the indicated time points and the remaining
NADPH-dependent activity was determined Activity is given as a
percentage of the initial activity Note that the specific activity of
A 8 B 8 -GAPDH with NADPH as the coenzyme is about fourfold lower
than that of A2B2-GAPDH (C) Reversal of AnBn-GAPDH inactivation
by dithiothreitol Nonaggregated and aggregated forms of GAPDH
were inactivated by incubation with either 1 m M H 2 O 2 or 0.1 m M
H2O2, with or without 0.5 m M GSH, for 10 min at 25 C, and
sub-sequently treated with 10 m M dithiothreitol for 10 min at 25 C.
The NADPH-dependent activity was determined before (black bars)
and after (white bars) treatment with dithiothreitol Activities are
given as a percentage of the initial activity measured before the
inactivation treatment.