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Studies on the reducing systems for plant and animal thioredoxin-independent methionine sulfoxide reductases B
Biochemical and Biophysical Research Communications 361 (2007) 629–633 www.elsevier.com/locate/ybbrc
Studies on the reducing systems for plant and animal thioredoxin-independent methionine sulfoxide reductases B Di Ding a, Daphna Sagher b, Edith Laugier c, Pascal Rey c, Herbert Weissbach b, Xing-Hai Zhang a,b,* a Department of Biological Sciences, Florida Atlantic University, Boca Raton, FL 33431, USA Center for Molecular Biology and Biotechnology, Florida Atlantic University, Boca Raton, FL 33431, USA CEA, DSV, IBEB, SBVME, LEMP, Laboratoire d’Ecophysiologie Mole´culaire des Plantes, UMR 6191 CNRS-CEA-Universite´ de la Me´diterrane´e, 13108 Saint-Paul-lez-Durance, Cedex, France b
c
Received 22 June 2007 Available online 25 July 2007
Abstract Two distinct stereospecific methionine sulfoxide reductases (Msr), MsrA and MsrB reduce the oxidized methionine (Met), methionine sulfoxide [Met(O)], back to Met. In this report, we examined the reducing systems required for the activities of two chloroplastic MsrB enzymes (NtMsrB1 and NtMsrB2) from tobacco (Nicotiana tabacum). We found that NtMrsB1, but not NtMsrB2, could use dithiothreitol as an efficient hydrogen donor. In contrast Escherichia coli thioredoxin (Trx) could serve as a reducing agent for NtMsrB2, but not for NtMsrB1. Similar to previously reported human Trx-independent hMsrB2 and hMsrB3, NtMsrB1 could also use bovine liver thionein and selenocysteamine as reducing agents. Furthermore, the unique plant Trx-like protein CDSP32 was shown to reduce NtMsrB1, hMsrB2 and hMsrB3. All these tested Trx-independent MsrB enzymes lack an additional cysteine (resolving cysteine) that is capable of forming a disulfide bond on the enzyme during the catalytic reaction. Our results indicate that plant and animal MsrB enzymes lacking a resolving cysteine likely share a similar reaction mechanism. 2007 Elsevier Inc. All rights reserved. Keywords: Methionine sulfoxide reductase (Msr); NtMsrB1 and NtMsrB2; Thioredoxin; Thionein; Selenocysteamine; Chloroplastic drought-induced stress protein of 32 kDa (CDSP32); Chloroplast; Nicotiana tabacum (tobacco); Resolving cysteine
Reactive oxygen species (ROS) can cause damage to macromolecules such as DNA, proteins and lipids, thereby impairing cellular functions. Oxidative damage to proteins has been implicated in many studies on oxidative stress in a wide variety of organisms [1–4]. The sulfur-containing amino acid, methionine (Met), is particularly susceptible to oxidation by ROS, forming methionine sulfoxide [Met(O)]. In response, most living organisms have a repair mechanism involving the action of the methionine sulfoxide reductase (Msr) system. Msr catalyzes the reduction of Met(O) to Met in proteins [5], repairing the oxidized * Corresponding author. Address: Department of Biological Sciences, Florida Atlantic University, Boca Raton, FL 33431, USA. Fax: +1 561 297 2749. E-mail address: [email protected] (X.-H. Zhang).
0006-291X/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.07.072
proteins. Because of the Msr system Met residues in proteins can also act as catalytic antioxidants by removing ROS [6], since one equivalent of ROS is destroyed for every Met residue repaired. Met oxidation in proteins by ROS leads to the formation of equal amounts of the two epimers of Met(O) called Met-S-(O) and Met-R-(O), which are reduced by two different enzymes, MsrA and MsrB, respectively [7–9]. During the Msr catalytic reaction one or more cysteine (Cys) residues on the Msr enzyme become oxidized and a reducing system is needed to regenerate the reduced form of the enzyme [9,10]. In vitro, dithiothreitol (DTT) has been a ubiquitous reducing agent for most of the Msr enzymes studied in detail [5,11]. In vivo, reduced thioredoxin (Trx) appears to be the major reducing system for all MsrA enzymes and most known MsrB enzymes in bacteria.
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All the plants examined so far possess multigene families of MsrA and MsrB. For example, Arabidopsis thaliana contains at least 5 MsrA genes including one for the plastidic enzyme and 9 MsrB genes including two plastidic forms [12]. Numerous studies have demonstrated the important role for MsrA in plant responses to oxidative stress [12]. However, very limited information is available for MsrB, even though many putative MsrB cDNAs from different plants can be identified from databases. In a recent report [13], two Arabidopsis chloroplastic MsrB proteins, named AtMsrB1 and AtMsrB2, were found to be induced by photooxidative stress; AtMsrB2, but not AtMsrB1, could use Trx as its reducing agent. Similarly, it has recently been shown that human hMsrB2 and hMsrB3, the former found in mitochondria and the latter in both mitochondria and endoplasmic reticulum [14], do not use Trx efficiently [11]. These three enzymes, AtMsrB1, hMsrB2 and hMsrB3, contain a single Cys residue at the catalytic site that can form a sulfenic acid derivative during the catalytic reaction, but they lack a ‘‘resolving’’ Cys residue that can form a disulfide intermediate, as seen with the MsrA enzymes and many bacterial MsrB enzymes [9,10,15]. A unique plastidic double-module Trx called CDSP32 was found to interact on an affinity column with AtMsrB1, raising the possibility that this protein is the endogenous reducing agent for chloroplast AtMsrB1 [16–18]. Recently thionein, the Cys-rich apoprotein of metallothionein, was shown to function as a reducing agent for hMsrB3 [11]. Thionein and Trx could also reduce oxidized selenocompounds such as selenocystamine and selenocystine to their respective sulfides, selenocysteamine and selenocysteine, which in turn served as very efficient reducing agents for both hMsrB2 and hMsrB3 [19]. In this report, we isolated tobacco chloroplast NtMsrB1 and NtMsrB2 and compared their reducing system requirements with those for hMsrB2 and hMsrB3. We found that the Trx-independent NtMsrB1, like hMSRB2 and hMSRB3, which lacks a resolving cysteine, can be reduced by thionein, by selenocysteamine and by the Trx-like CDSP32.
Materials and methods Cloning of methionine sulfoxide reductases (Msr) genes. Tobacco (Nicotiana tabacum L.) was grown at 24 C under 50–70 lE m 2 s 1 white fluorescent light (16 h daily). Total RNA was isolated from leaves using Qiagen Plant RNeasy kit (Valencia, CA). Synthesis of the first-stranded cDNA was carried out using oligo dT and SuperScript II RNase H reverse transcriptase kit (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. From the databases, we identified homologous expressed Msr cDNA sequences and designed primers. To clone NtMsrB1 and NtMsrB2, polymerase chain reaction (PCR) was performed using the high fidelity proof-reading DNA polymerases Pfx (Invitrogen). The amplified fragments (540–595 bp) were cloned into a pCR-Blunt vector (Invitrogen) and completely sequenced. DNA sequencing and analysis. DNA sequencing was done at the Northwestern University Genomic Center (Chicago, IL). Sequence analysis was carried out with Biology Workbench (San Diego Supercomputer
Center, CA) and NCBI/GenBank. The putative chloroplast transit peptide sequences were identified with ChloroP program. Overexpression and purification of recombinant proteins. The plasmids harboring NtMsrB1 and NtMsrB2 genes were cut with NdeI and XhoI and ligated into NdeI/XhoI digested vector pET-30c which was then introduced into the Escherichia coli strain BL21/DE3 (Novagen, La Jolla, CA). This resulted in a translation frame without a 6· His-tag, since preliminary studies indicated that the His-tag interfered with protein solubility and enzyme activity. The recombinant proteins were purified by (NH4)2SO4 precipitation and Superdex 75 sizing column followed by DE-52 anion-exchange chromatography. The elution fractions with highest specific activity were collected, analyzed by SDS– PAGE and used for enzyme assays. The enzymes were >85% pure. Recombinant hMsrB2 and hMsrB3 were obtained as described previously [11]. Mature CDSP32 protein from A. thaliana, fused to an N-terminal 6· His-tag, was produced in E. coli and purified as described elsewhere [17]. Tests confirmed that all the 5 cysteines of this protein as isolated were reduced. This reduced form of CDSP32 was used directly for assays without any regenerating system. Substrates. The dabsyl derivatives of two epimers of oxidized methionine, Met-S-(O) and Met-R-(O), E. coli thioredoxin (Trx) reductase and thionein were prepared as described [11]. The E. coli Trx was purchased from Promega (Madison, WI). The A. thaliana Trx f was kindly provided by Dr. E. Issakidis-Bourguet. Spinach leaf ferredoxin (Fd), ferredoxinNADP reductase, glutathione and selenocystamine were purchased from Sigma. Enzymatic activity assay of methionine sulfoxide reductases. Msr enzyme activity was assayed spectrophotometrically based on the amount of dabsyl-L-Met formed [11]. Incubations were at 37 C for 1 h. To test various reducing systems for NtMsrB1, the reactions contained 100 nmol of dabsyl-Met-R-(O) and one of the followings: (1) 15 mM DTT; (2) either 12.6 lg reduced Arabidopsis Trx f or 8 lg E. coli Trx plus 2 lg E. coli thioredoxin reductase and 0.2 mM NADPH; (3) reduced bovine thionein (30 lg, 5 nmol); (4) 50 lM selenocystamine with thionein (30 lg, 5 nmol) or 12 mM glutathione; (5) 20 lg spinach ferredoxin (Fd) with 2 lg spinach Fd-NADP reductase and 0.2 mM NADPH, and (6) various amounts of reduced CDSP32.
Results and discussion Analysis of tobacco chloroplast MsrB1 and MsrB2 sequences There has been no published report of any tobacco Msr enzymes. To obtain the tobacco chloroplast MsrB genes, we used the conserved sequences of MsrB identified in the database to search for available expressed cDNAs of Nicotiana species. Analysis of these sequences led to the cloning of the putative genes for NtMsrB1 (GenBank accession number: EF990629) and NtMsrB2 (EF990628) and expression of the proteins. The two NtMsrB cDNAs contain the typical conserved motifs for MsrB such as the catalytic Cys in RxCxN near the C-terminus (box IV, Fig. 1). Four conserved Cys residues in domains I and III (Fig. 1) constitute a zinc-binding motif [20]. It is noteworthy that NtMsrB2, as well as MsrBs from E. coli and rice, and eight of the nine Arabidopsis MsrBs including the secretory AtMsrB3 all have the conserved Cys at the GCGWP domain, whereas the MsrB1 from tobacco and Arabidopsis and hMsrB2 and hMsrB3 do not have a resolving Cys at this site (box II, Fig. 1). As noted above, hMsrB2 and hMsrB3 and AtMsrB1 all show low activity using Trx [11,13].
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Fig. 1. Sequence comparison of the tobacco MsrB1 (Nt1) and MsrB2 (Nt2) to representative MsrBs from A. thaliana (At1, NP_564640, At2, NP_567639; At3, NP_567271), O. sativa (Os, AY224463), H. sapiens (Hs2, NP_036360; Hs3, AAH40053), E. coli (Ec, NP_754077) and H. marismortui ATCC 43049 (Hm, YP_134890; a halophilic archaeon). Four putative MsrB signature motifs are boxed. The resolving Cys absent in AtMsr1, NtMsrB1, hMsrB2 and hMsrB3 is located in motif II with an arrow. The alanine underlined for NtMsrB1 and NtMsrB2 indicates the start of the mature proteins. The conserved residues are shown in bold.
NtMsrB1 and NtMsrB2 contain the putative N-terminal chloroplast transit peptide sequences, suggesting their chloroplast localization. In addition, RT-PCR using gene-specific primers revealed a higher mRNA level in green leaves than in etiolated shoots/roots, suspension culture or flowers for both NtMsrB1 and NtMsrB2 (data not shown) indicating a preferential expression in green tissues. Furthermore, sequence analysis shows that NtMsrB1 and NtMsrB2 are more similar to the plastidial AtMsrB1 and AtMsrB2, respectively, than to the cytosolic AtMsrB3 (Fig. 1). Considering the apparent lack of any Msr enzymes in plant mitochondria [21], the overall evidence indicates that these two NtMsrB enzymes are localized in the chloroplasts.
NtMsrB substrate specificity and reducing requirements The purified recombinant NtMsrB1 and NtMsrB2 had apparent molecular masses of ca 15.2 and 14.8 kDa, respectively. Both recombinant proteins showed a marked substrate preference for Met-R-(O), with very little activity towards Met-S-(O) (Fig. 2). However, whereas NtMsrB1 can use DTT as the reducing system, it was not active with an E. coli Trx reducing system (Trx–Trx reductaseNADPH; Fig. 2A). NtMsrB2, on the other hand, had almost no activity with DTT, but was highly active with E. coli Trx (Fig. 2B). The lack of activity with DTT has been observed previously with an E. coli membrane vesicle-associated Msr enzyme(s) [22]. These results indicate a
D. Ding et al. / Biochemical and Biophysical Research Communications 361 (2007) 629–633
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Protein (µg) Fig. 2. Substrate specificity and reducing agents for recombinant NtMsrB1 (A) and NtMsrB2 (B). Reactions were incubated at 37 C for 1 h. The values are the average taken from at least three experiments for each point.
distinct difference in the reducing systems for these two chloroplastic NtMsrB enzymes. Since NtMsrB2 was efficiently reduced by E. coli Trx (Fig. 2B), it suggests that Trx may be its in vivo reducing agent, similar to Arabidopsis AtMsrB2 [13]. However, NtMsrB1 is similar to AtMsrB1, hMsrB2 and hMsrB3 in that it cannot use E. coli Trx as its reducing system (Fig. 2A). Plants possess many types of Trxs including the chloroplastic forms of Trx f and Trx m [23]. Trx m was not available for this study, however the E. coli Trx is similar to plant Trx m (49.5% identity with Arabidopsis Trx m). As shown in Table 1, NtMsrB1 did not show activity with E. coli Trx or chloroplast Trx f from Arabidopsis, suggesting that chloroplast Trx cannot serve as a direct electron donor to NtMsrB1. Because of the known important role of ferredoxin (Fd) in electron transport in the chloroplast, reduced Fd was also tested as a possible reducing agent, but found to be inactive (Table 1). Human MsrB2 and Table 1 Activity of NtMsrB1 (2 lg) with various reducing reagents Reducing reagents
Dabsyl-L-Met (nmol)a
DTT E. coli Trx/Trx-NADPH reductase Reduced Arabidopsis Trx f Reduced Fd Thionein Selenocystamine + thionein Selenocystamine + GSH
64.7 1.7 <1 <1 5.9 32.4 57.1
a
See text for details.
MsrB3 are also unable to use E. coli Trx or mitochondrial Trx2, but can be reduced by bovine liver thionein and by selenocysteamine (produced by reduction of selenocystamine by thionein or Trx) [11,19]. As also shown in Table 1, bovine liver thionein and selenocysteamine, generated with either thionein or GSH, give significant activity with NtMsrB1. This is the first observation that thionein and selenocysteamine can serve as reducing agents, at least in vitro, for a plant Msr enzyme. Thionein is the apo form of metallothionein that contains 20 Cys residues in animals and 8–16 Cys in plants [24]. Plants contain a wide variety of metallothioneins that bind heavy metal ions such as Zn2+ or Cu2+ [24,25], but do not appear to contain any selenoprotein-encoding genes, based on a study of the Arabidopsis genome [26]. A unique plastidial Trx, CDSP32, composed of two Trx modules with one active site in the C-terminal domain has been identified [16]. CDSP32 participates in plant responses to oxidative stress [17,27]. In potato plant extracts, CDSP32 specifically binds through a disulfide bond two plastidial peroxiredoxins and the MsrB1 protein related to NtMsrB1 [18]. Preliminary results suggested that the CDSP32 Trx from Arabidopsisis could serve as a reducing agent for AtMsrB1. Thus, we tested whether this protein was able to reduce NtMsrB1 as well as hMsrB2 and hMsrB3. Very interestingly, all three enzymes can use CDSP32 as a reducing system (Fig. 3). It should again be stressed that a common feature of all the Trx independent Msr proteins is that they lack a resolving Cys that is present in Trx-dependent MsrB proteins in the conserved domain (box II in Fig. 1). It appears that the
D. Ding et al. / Biochemical and Biophysical Research Communications 361 (2007) 629–633
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20 hMsrB3 15 10 NtMsrB1 5
hMsrB2
0 0
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CDSP32 (µg) Fig. 3. MsrB enzyme activity using reduced CDSP32 (without a regeneration system) as the reducing agent and 100 nmol of dabsyl-Met-R-(O) as substrate. Approximately 1.1 lg of each enzyme was used. Reactions were incubated at 37 C for 1 h. The average was taken from at least two measurements for each point.
ability of an Msr protein to form a disulfide intermediate is required for the Trx-dependent Msr enzymes. In contrast, the group of MsrB enzymes lacking a resolving Cys, such as NtMsrB1, hMsrB2 and hMsrB3, may require distinct Trx structures, similar to that present in CDSP32. Although this unique CDSP32 protein may be the natural reducing agent for the chloroplast MsrB enzymes in plants, there is no known mammalian homologue of CDSP32 in several genomes examined. It seems reasonable that the natural reducing system for mammalian MsrB2 and MsrB3 will share some common biochemical and structural features with the plant CDSP32 thioredoxin. Acknowledgments We thank Dr. E. Issakidis-Bourguet for a gift of Arabidopsis Trx f, D. Brunell for assistance with HPLC protein purification and L. Tarrago for valuable discussion. Financial support from ANR-Ge´noplante (GNP05010G) to E.L. and P.R. is acknowledged. This work is supported in part by Florida Atlantic University startup fund to X.-H.Z. References [1] W. Vogt, Oxidation of methiononyl residues in proteins: tools, targets, and reversal, Free Rad. Biol. Med. 18 (1995) 93–105. [2] B.S. Berlett, E.R. Stadtman, Protein oxidation in aging, disease, and oxidative stress, J. Biol. Chem. 372 (1997) 20313–20316. [3] M.J. Davies, The oxidative environment and protein damage, Biochim. Biophys. Acta 1703 (2005) 93–109. [4] H. Weissbach, L. Resnick, N. Brot, Methionine sulfoxide reductases: history and cellular role in protecting against oxidative damage, Biochim. Biophys. Acta 1703 (2005) 203–212. [5] N. Brot, L. Weissbach, J. Werth, H. Weissbach, Enzymatic reduction of protein-bound methionine sulfoxide, Proc. Natl. Acad. Sci. USA 78 (1981) 2155–2158. [6] R.L. Levine, B.S. Berlett, E. Stadtman, Methionine residues as endogenous antioxidants in protein, Proc. Natl. Acad. Sci. USA 93 (1996) 15036–15040. [7] J. Moskovitz, J.M. Oistin, B.S. Berlett, N.J. Nosworthy, R. Szczepanowski, E.R. Stadtman, Identification and characterization of a putative active site for peptide methionine sulfoxide reductase (MsrA) and its substrate stereospecificity, J. Biol. Chem. 275 (2000) 14167–14172.
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[8] G. Grimaud, B. Ezraty, J.K. Mitchell, D. Lafitte, C. Briand, P.J. Derrick, F. Barras, Repair of oxidized proteins: identification of a new methionine sulfoxide reductase, J. Biol. Chem. 276 (2001) 48915–48920. [9] W.T. Lowther, N. Brot, H. Weissbach, B.W. Matthews, Structure and mechanism of peptide methionine sulfoxide reductase, an ‘‘antioxidation’’ enzyme, Biochemistry 39 (2000) 13307–13312. [10] S. Boschi-Muller, S. Azza, S. Sanglier-Cianferani, F. Talfournier, A. Van Dorsselear, G. Branlant, A sulfenic acid enzyme intermediate is involved in the catalytic mechanism of peptide methionine sulfoxide reductase from Escherichia coli, J. Biol. Chem. 275 (2000) 35908–35913. [11] D. Sagher, D. Brunell, J.F. Hejtmancik, M. Kantorow, N. Brot, H. Weissbach, Thionein can serve as a reducing agent for methionine sulfoxide reductases, Proc. Natl. Acad. Sci. USA 103 (2006) 8656–8661. [12] N. Rouhier, C. Vieira Dos Santos, L. Tarrago, P. Rey, Plant methionine sulfoxide reductase A and B multigenic families, Photosyn. Res. 89 (2006) 247–262. [13] C.V. Vieira Dos Santos, S. Cuine´, N. Rouhier, P. Rey, The Arabidopsis plastidic methionine sulfoxide reductase B proteins. Sequence and activity characteristics, comparison of the expression with plastidic methionine sulfoxide reductase A, and induction by photooxidative stress, Plant Physiol. 138 (2005) 909–922. [14] H.Y. Kim, V.N. Gladyshev, Methionine sulfoxide reduction in mammals: characterization of methionine-R-sulfoxide reductases, Mol. Biol. Cell 15 (2004) 1055–1064. [15] W.T. Lowther, H. Weissbach, F. Etienne, N. Brot, B.W. Matthews, The mirrored methionine sulfoxide reductases of Neisseria gonorrhoeae pilB, Nature Struct. Biol. 9 (2002) 348–352. [16] P. Rey, G. Pruvot, N. Becuwe, F. Eymery, D. Rumeau, G. Peltier, A novel thioredoxin-like protein located in the chloroplast is induced by water deficit in Solanum tuberosum L. plants, Plant J. 13 (1998) 97–107. [17] M. Broin, S. Cuine´, F. Eymery, P. Rey, The plastidic 2-cysteine peroxiredoxin is a target for a thioredoxin involved in the protection of the photosynthetic apparatus against oxidative damage, Plant Cell 14 (2002) 1417–1432. [18] P. Rey, S. Cuine´, F. Eymery, J. Garin, M. Court, J.-P. Jacquot, N. Rouhier, M. Broin, Analysis of the proteins targeted by CDSP32, a plastidic thioredoxin participating in oxidative stress responses, Plant J. 41 (2005) 31–42. [19] D. Sagher, D. Brunell, N. Brot, B.L. Vallee, H. Weissbach, Selenocompounds can serve as oxidoreductants with the methionine sulfoxide reductase enzymes, J. Biol. Chem. 281 (2006) 31184–31187. [20] R.A. Kumar, A. Koc, R.L. Cerny, V.N. Gladyshev, Reaction mechanism, evolutionary analysis, and role of zinc in Drosophila methionineR-sulfoxide reductase, J. Biol. Chem. 277 (2002) 37527–37535. [21] N. Navrot, N. Rouhier, E. Gelhaye, J.-P. Jacquot, Reactive oxygen species generation and antioxidant systems in plant mitochondria, Physiol. Plant 129 (2007) 185–195. [22] D. Spector, F. Etienne, N. Brot, H. Weissbach, New membraneassociated and soluble peptide methionine sulfoxide reductases in Escherichia coli, Biochem. Biophys. Res. Comm. 302 (2003) 284–289. [23] Y. Meyer, J.P. Reichheld, F. Vignols, Thioredoxins in Arabidopsis and other plants, Photosynth. Res. 86 (2005) 419–433. [24] J. Dome`nech, G. Mir, G. Huguet, M. Capdevila, M. Molinas, S. Atrian, Plant metallothionein domains: functional insight into physiological metal binding and protein folding, Biochimie 88 (2006) 583–593. [25] C. Cobbett, P.B. Goldsbrough, Phytochelatins and metallothioneins: roles in heavy metal detoxification and homeostasis, Annu. Rev. Plant Biol. 53 (2002) 159–182. [26] S.V. Novoselov, M. Rao, N.V. Onoshko, H. Zhi, G.V. Kryukov, Y. Xiang, D.P. Weeks, D.L. Hatfield, V.N. Gladyshev, Selenoproteins and selenocysteine insertion system in the model plant cell system, Chlamydomonas reinhardtii, EMBO J. 21 (2002) 3681–3693. [27] M. Broin, S. Cuine´, G. Peltier, P. Rey, Involvement of CDSP32, a drought-induced thioredoxin, in the response to oxidative stress in potato plants, FEBS Lett. 467 (2000) 245–248.
Studies on the reducing systems for plant and animal thioredoxin-independent methionine sulfoxide reductases B Di Ding a, Daphna Sagher b, Edith Laugier c, Pascal Rey c, Herbert Weissbach b, Xing-Hai Zhang a,b,* a Department of Biological Sciences, Florida Atlantic University, Boca Raton, FL 33431, USA Center for Molecular Biology and Biotechnology, Florida Atlantic University, Boca Raton, FL 33431, USA CEA, DSV, IBEB, SBVME, LEMP, Laboratoire d’Ecophysiologie Mole´culaire des Plantes, UMR 6191 CNRS-CEA-Universite´ de la Me´diterrane´e, 13108 Saint-Paul-lez-Durance, Cedex, France b
c
Received 22 June 2007 Available online 25 July 2007
Abstract Two distinct stereospecific methionine sulfoxide reductases (Msr), MsrA and MsrB reduce the oxidized methionine (Met), methionine sulfoxide [Met(O)], back to Met. In this report, we examined the reducing systems required for the activities of two chloroplastic MsrB enzymes (NtMsrB1 and NtMsrB2) from tobacco (Nicotiana tabacum). We found that NtMrsB1, but not NtMsrB2, could use dithiothreitol as an efficient hydrogen donor. In contrast Escherichia coli thioredoxin (Trx) could serve as a reducing agent for NtMsrB2, but not for NtMsrB1. Similar to previously reported human Trx-independent hMsrB2 and hMsrB3, NtMsrB1 could also use bovine liver thionein and selenocysteamine as reducing agents. Furthermore, the unique plant Trx-like protein CDSP32 was shown to reduce NtMsrB1, hMsrB2 and hMsrB3. All these tested Trx-independent MsrB enzymes lack an additional cysteine (resolving cysteine) that is capable of forming a disulfide bond on the enzyme during the catalytic reaction. Our results indicate that plant and animal MsrB enzymes lacking a resolving cysteine likely share a similar reaction mechanism. 2007 Elsevier Inc. All rights reserved. Keywords: Methionine sulfoxide reductase (Msr); NtMsrB1 and NtMsrB2; Thioredoxin; Thionein; Selenocysteamine; Chloroplastic drought-induced stress protein of 32 kDa (CDSP32); Chloroplast; Nicotiana tabacum (tobacco); Resolving cysteine
Reactive oxygen species (ROS) can cause damage to macromolecules such as DNA, proteins and lipids, thereby impairing cellular functions. Oxidative damage to proteins has been implicated in many studies on oxidative stress in a wide variety of organisms [1–4]. The sulfur-containing amino acid, methionine (Met), is particularly susceptible to oxidation by ROS, forming methionine sulfoxide [Met(O)]. In response, most living organisms have a repair mechanism involving the action of the methionine sulfoxide reductase (Msr) system. Msr catalyzes the reduction of Met(O) to Met in proteins [5], repairing the oxidized * Corresponding author. Address: Department of Biological Sciences, Florida Atlantic University, Boca Raton, FL 33431, USA. Fax: +1 561 297 2749. E-mail address: [email protected] (X.-H. Zhang).
0006-291X/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.07.072
proteins. Because of the Msr system Met residues in proteins can also act as catalytic antioxidants by removing ROS [6], since one equivalent of ROS is destroyed for every Met residue repaired. Met oxidation in proteins by ROS leads to the formation of equal amounts of the two epimers of Met(O) called Met-S-(O) and Met-R-(O), which are reduced by two different enzymes, MsrA and MsrB, respectively [7–9]. During the Msr catalytic reaction one or more cysteine (Cys) residues on the Msr enzyme become oxidized and a reducing system is needed to regenerate the reduced form of the enzyme [9,10]. In vitro, dithiothreitol (DTT) has been a ubiquitous reducing agent for most of the Msr enzymes studied in detail [5,11]. In vivo, reduced thioredoxin (Trx) appears to be the major reducing system for all MsrA enzymes and most known MsrB enzymes in bacteria.
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All the plants examined so far possess multigene families of MsrA and MsrB. For example, Arabidopsis thaliana contains at least 5 MsrA genes including one for the plastidic enzyme and 9 MsrB genes including two plastidic forms [12]. Numerous studies have demonstrated the important role for MsrA in plant responses to oxidative stress [12]. However, very limited information is available for MsrB, even though many putative MsrB cDNAs from different plants can be identified from databases. In a recent report [13], two Arabidopsis chloroplastic MsrB proteins, named AtMsrB1 and AtMsrB2, were found to be induced by photooxidative stress; AtMsrB2, but not AtMsrB1, could use Trx as its reducing agent. Similarly, it has recently been shown that human hMsrB2 and hMsrB3, the former found in mitochondria and the latter in both mitochondria and endoplasmic reticulum [14], do not use Trx efficiently [11]. These three enzymes, AtMsrB1, hMsrB2 and hMsrB3, contain a single Cys residue at the catalytic site that can form a sulfenic acid derivative during the catalytic reaction, but they lack a ‘‘resolving’’ Cys residue that can form a disulfide intermediate, as seen with the MsrA enzymes and many bacterial MsrB enzymes [9,10,15]. A unique plastidic double-module Trx called CDSP32 was found to interact on an affinity column with AtMsrB1, raising the possibility that this protein is the endogenous reducing agent for chloroplast AtMsrB1 [16–18]. Recently thionein, the Cys-rich apoprotein of metallothionein, was shown to function as a reducing agent for hMsrB3 [11]. Thionein and Trx could also reduce oxidized selenocompounds such as selenocystamine and selenocystine to their respective sulfides, selenocysteamine and selenocysteine, which in turn served as very efficient reducing agents for both hMsrB2 and hMsrB3 [19]. In this report, we isolated tobacco chloroplast NtMsrB1 and NtMsrB2 and compared their reducing system requirements with those for hMsrB2 and hMsrB3. We found that the Trx-independent NtMsrB1, like hMSRB2 and hMSRB3, which lacks a resolving cysteine, can be reduced by thionein, by selenocysteamine and by the Trx-like CDSP32.
Materials and methods Cloning of methionine sulfoxide reductases (Msr) genes. Tobacco (Nicotiana tabacum L.) was grown at 24 C under 50–70 lE m 2 s 1 white fluorescent light (16 h daily). Total RNA was isolated from leaves using Qiagen Plant RNeasy kit (Valencia, CA). Synthesis of the first-stranded cDNA was carried out using oligo dT and SuperScript II RNase H reverse transcriptase kit (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. From the databases, we identified homologous expressed Msr cDNA sequences and designed primers. To clone NtMsrB1 and NtMsrB2, polymerase chain reaction (PCR) was performed using the high fidelity proof-reading DNA polymerases Pfx (Invitrogen). The amplified fragments (540–595 bp) were cloned into a pCR-Blunt vector (Invitrogen) and completely sequenced. DNA sequencing and analysis. DNA sequencing was done at the Northwestern University Genomic Center (Chicago, IL). Sequence analysis was carried out with Biology Workbench (San Diego Supercomputer
Center, CA) and NCBI/GenBank. The putative chloroplast transit peptide sequences were identified with ChloroP program. Overexpression and purification of recombinant proteins. The plasmids harboring NtMsrB1 and NtMsrB2 genes were cut with NdeI and XhoI and ligated into NdeI/XhoI digested vector pET-30c which was then introduced into the Escherichia coli strain BL21/DE3 (Novagen, La Jolla, CA). This resulted in a translation frame without a 6· His-tag, since preliminary studies indicated that the His-tag interfered with protein solubility and enzyme activity. The recombinant proteins were purified by (NH4)2SO4 precipitation and Superdex 75 sizing column followed by DE-52 anion-exchange chromatography. The elution fractions with highest specific activity were collected, analyzed by SDS– PAGE and used for enzyme assays. The enzymes were >85% pure. Recombinant hMsrB2 and hMsrB3 were obtained as described previously [11]. Mature CDSP32 protein from A. thaliana, fused to an N-terminal 6· His-tag, was produced in E. coli and purified as described elsewhere [17]. Tests confirmed that all the 5 cysteines of this protein as isolated were reduced. This reduced form of CDSP32 was used directly for assays without any regenerating system. Substrates. The dabsyl derivatives of two epimers of oxidized methionine, Met-S-(O) and Met-R-(O), E. coli thioredoxin (Trx) reductase and thionein were prepared as described [11]. The E. coli Trx was purchased from Promega (Madison, WI). The A. thaliana Trx f was kindly provided by Dr. E. Issakidis-Bourguet. Spinach leaf ferredoxin (Fd), ferredoxinNADP reductase, glutathione and selenocystamine were purchased from Sigma. Enzymatic activity assay of methionine sulfoxide reductases. Msr enzyme activity was assayed spectrophotometrically based on the amount of dabsyl-L-Met formed [11]. Incubations were at 37 C for 1 h. To test various reducing systems for NtMsrB1, the reactions contained 100 nmol of dabsyl-Met-R-(O) and one of the followings: (1) 15 mM DTT; (2) either 12.6 lg reduced Arabidopsis Trx f or 8 lg E. coli Trx plus 2 lg E. coli thioredoxin reductase and 0.2 mM NADPH; (3) reduced bovine thionein (30 lg, 5 nmol); (4) 50 lM selenocystamine with thionein (30 lg, 5 nmol) or 12 mM glutathione; (5) 20 lg spinach ferredoxin (Fd) with 2 lg spinach Fd-NADP reductase and 0.2 mM NADPH, and (6) various amounts of reduced CDSP32.
Results and discussion Analysis of tobacco chloroplast MsrB1 and MsrB2 sequences There has been no published report of any tobacco Msr enzymes. To obtain the tobacco chloroplast MsrB genes, we used the conserved sequences of MsrB identified in the database to search for available expressed cDNAs of Nicotiana species. Analysis of these sequences led to the cloning of the putative genes for NtMsrB1 (GenBank accession number: EF990629) and NtMsrB2 (EF990628) and expression of the proteins. The two NtMsrB cDNAs contain the typical conserved motifs for MsrB such as the catalytic Cys in RxCxN near the C-terminus (box IV, Fig. 1). Four conserved Cys residues in domains I and III (Fig. 1) constitute a zinc-binding motif [20]. It is noteworthy that NtMsrB2, as well as MsrBs from E. coli and rice, and eight of the nine Arabidopsis MsrBs including the secretory AtMsrB3 all have the conserved Cys at the GCGWP domain, whereas the MsrB1 from tobacco and Arabidopsis and hMsrB2 and hMsrB3 do not have a resolving Cys at this site (box II, Fig. 1). As noted above, hMsrB2 and hMsrB3 and AtMsrB1 all show low activity using Trx [11,13].
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Fig. 1. Sequence comparison of the tobacco MsrB1 (Nt1) and MsrB2 (Nt2) to representative MsrBs from A. thaliana (At1, NP_564640, At2, NP_567639; At3, NP_567271), O. sativa (Os, AY224463), H. sapiens (Hs2, NP_036360; Hs3, AAH40053), E. coli (Ec, NP_754077) and H. marismortui ATCC 43049 (Hm, YP_134890; a halophilic archaeon). Four putative MsrB signature motifs are boxed. The resolving Cys absent in AtMsr1, NtMsrB1, hMsrB2 and hMsrB3 is located in motif II with an arrow. The alanine underlined for NtMsrB1 and NtMsrB2 indicates the start of the mature proteins. The conserved residues are shown in bold.
NtMsrB1 and NtMsrB2 contain the putative N-terminal chloroplast transit peptide sequences, suggesting their chloroplast localization. In addition, RT-PCR using gene-specific primers revealed a higher mRNA level in green leaves than in etiolated shoots/roots, suspension culture or flowers for both NtMsrB1 and NtMsrB2 (data not shown) indicating a preferential expression in green tissues. Furthermore, sequence analysis shows that NtMsrB1 and NtMsrB2 are more similar to the plastidial AtMsrB1 and AtMsrB2, respectively, than to the cytosolic AtMsrB3 (Fig. 1). Considering the apparent lack of any Msr enzymes in plant mitochondria [21], the overall evidence indicates that these two NtMsrB enzymes are localized in the chloroplasts.
NtMsrB substrate specificity and reducing requirements The purified recombinant NtMsrB1 and NtMsrB2 had apparent molecular masses of ca 15.2 and 14.8 kDa, respectively. Both recombinant proteins showed a marked substrate preference for Met-R-(O), with very little activity towards Met-S-(O) (Fig. 2). However, whereas NtMsrB1 can use DTT as the reducing system, it was not active with an E. coli Trx reducing system (Trx–Trx reductaseNADPH; Fig. 2A). NtMsrB2, on the other hand, had almost no activity with DTT, but was highly active with E. coli Trx (Fig. 2B). The lack of activity with DTT has been observed previously with an E. coli membrane vesicle-associated Msr enzyme(s) [22]. These results indicate a
D. Ding et al. / Biochemical and Biophysical Research Communications 361 (2007) 629–633
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Protein (µg) Fig. 2. Substrate specificity and reducing agents for recombinant NtMsrB1 (A) and NtMsrB2 (B). Reactions were incubated at 37 C for 1 h. The values are the average taken from at least three experiments for each point.
distinct difference in the reducing systems for these two chloroplastic NtMsrB enzymes. Since NtMsrB2 was efficiently reduced by E. coli Trx (Fig. 2B), it suggests that Trx may be its in vivo reducing agent, similar to Arabidopsis AtMsrB2 [13]. However, NtMsrB1 is similar to AtMsrB1, hMsrB2 and hMsrB3 in that it cannot use E. coli Trx as its reducing system (Fig. 2A). Plants possess many types of Trxs including the chloroplastic forms of Trx f and Trx m [23]. Trx m was not available for this study, however the E. coli Trx is similar to plant Trx m (49.5% identity with Arabidopsis Trx m). As shown in Table 1, NtMsrB1 did not show activity with E. coli Trx or chloroplast Trx f from Arabidopsis, suggesting that chloroplast Trx cannot serve as a direct electron donor to NtMsrB1. Because of the known important role of ferredoxin (Fd) in electron transport in the chloroplast, reduced Fd was also tested as a possible reducing agent, but found to be inactive (Table 1). Human MsrB2 and Table 1 Activity of NtMsrB1 (2 lg) with various reducing reagents Reducing reagents
Dabsyl-L-Met (nmol)a
DTT E. coli Trx/Trx-NADPH reductase Reduced Arabidopsis Trx f Reduced Fd Thionein Selenocystamine + thionein Selenocystamine + GSH
64.7 1.7 <1 <1 5.9 32.4 57.1
a
See text for details.
MsrB3 are also unable to use E. coli Trx or mitochondrial Trx2, but can be reduced by bovine liver thionein and by selenocysteamine (produced by reduction of selenocystamine by thionein or Trx) [11,19]. As also shown in Table 1, bovine liver thionein and selenocysteamine, generated with either thionein or GSH, give significant activity with NtMsrB1. This is the first observation that thionein and selenocysteamine can serve as reducing agents, at least in vitro, for a plant Msr enzyme. Thionein is the apo form of metallothionein that contains 20 Cys residues in animals and 8–16 Cys in plants [24]. Plants contain a wide variety of metallothioneins that bind heavy metal ions such as Zn2+ or Cu2+ [24,25], but do not appear to contain any selenoprotein-encoding genes, based on a study of the Arabidopsis genome [26]. A unique plastidial Trx, CDSP32, composed of two Trx modules with one active site in the C-terminal domain has been identified [16]. CDSP32 participates in plant responses to oxidative stress [17,27]. In potato plant extracts, CDSP32 specifically binds through a disulfide bond two plastidial peroxiredoxins and the MsrB1 protein related to NtMsrB1 [18]. Preliminary results suggested that the CDSP32 Trx from Arabidopsisis could serve as a reducing agent for AtMsrB1. Thus, we tested whether this protein was able to reduce NtMsrB1 as well as hMsrB2 and hMsrB3. Very interestingly, all three enzymes can use CDSP32 as a reducing system (Fig. 3). It should again be stressed that a common feature of all the Trx independent Msr proteins is that they lack a resolving Cys that is present in Trx-dependent MsrB proteins in the conserved domain (box II in Fig. 1). It appears that the
D. Ding et al. / Biochemical and Biophysical Research Communications 361 (2007) 629–633
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CDSP32 (µg) Fig. 3. MsrB enzyme activity using reduced CDSP32 (without a regeneration system) as the reducing agent and 100 nmol of dabsyl-Met-R-(O) as substrate. Approximately 1.1 lg of each enzyme was used. Reactions were incubated at 37 C for 1 h. The average was taken from at least two measurements for each point.
ability of an Msr protein to form a disulfide intermediate is required for the Trx-dependent Msr enzymes. In contrast, the group of MsrB enzymes lacking a resolving Cys, such as NtMsrB1, hMsrB2 and hMsrB3, may require distinct Trx structures, similar to that present in CDSP32. Although this unique CDSP32 protein may be the natural reducing agent for the chloroplast MsrB enzymes in plants, there is no known mammalian homologue of CDSP32 in several genomes examined. It seems reasonable that the natural reducing system for mammalian MsrB2 and MsrB3 will share some common biochemical and structural features with the plant CDSP32 thioredoxin. Acknowledgments We thank Dr. E. Issakidis-Bourguet for a gift of Arabidopsis Trx f, D. Brunell for assistance with HPLC protein purification and L. Tarrago for valuable discussion. Financial support from ANR-Ge´noplante (GNP05010G) to E.L. and P.R. is acknowledged. This work is supported in part by Florida Atlantic University startup fund to X.-H.Z. References [1] W. Vogt, Oxidation of methiononyl residues in proteins: tools, targets, and reversal, Free Rad. Biol. Med. 18 (1995) 93–105. [2] B.S. Berlett, E.R. Stadtman, Protein oxidation in aging, disease, and oxidative stress, J. Biol. Chem. 372 (1997) 20313–20316. [3] M.J. Davies, The oxidative environment and protein damage, Biochim. Biophys. Acta 1703 (2005) 93–109. [4] H. Weissbach, L. Resnick, N. Brot, Methionine sulfoxide reductases: history and cellular role in protecting against oxidative damage, Biochim. Biophys. Acta 1703 (2005) 203–212. [5] N. Brot, L. Weissbach, J. Werth, H. Weissbach, Enzymatic reduction of protein-bound methionine sulfoxide, Proc. Natl. Acad. Sci. USA 78 (1981) 2155–2158. [6] R.L. Levine, B.S. Berlett, E. Stadtman, Methionine residues as endogenous antioxidants in protein, Proc. Natl. Acad. Sci. USA 93 (1996) 15036–15040. [7] J. Moskovitz, J.M. Oistin, B.S. Berlett, N.J. Nosworthy, R. Szczepanowski, E.R. Stadtman, Identification and characterization of a putative active site for peptide methionine sulfoxide reductase (MsrA) and its substrate stereospecificity, J. Biol. Chem. 275 (2000) 14167–14172.
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