Journal of Experimental Botany

JXB Advance Access originally published online on April 25, 2005
Journal of Experimental Botany 2005 56(416):1463-1468; doi:10.1093/jxb/eri170

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FOCUS PAPER

Thioredoxin affinity chromatography: a useful method for further understanding the thioredoxin network

Toru Hisabori^1,2,*, Satoshi Hara¹, Tetsufumi Fujii¹, Daisuke Yamazaki¹, Naomi Hosoya-Matsuda¹ and Ken Motohashi^1,2

¹Chemical Resources Laboratory, Tokyo Institute of Technology, Nagatsuta 4259, Midori-ku, Yokohama 226-8503, Japan
²ATP System Project, Exploratory Research for Advanced Technology (ERATO), Japan Science and Technology Agency (JST), 5800-3 Nagatsuta-cho, Midori-ku, Yokohama 226-0026, Japan

^* To whom correspondence should be addressed. Fax: +81 45 924 5277. E-mail: thisabor{at}res.titech.ac.jp

Received 1 February 2005; Accepted 22 March 2005

	Abstract

Top Abstract Introduction Contribution of Trx affinity... Limitations of the Trx... How to confirm the... Possible Trx target proteins Trx targets in the... Conclusion References

 
Thioredoxin affinity chromatography can be used to recognize the target proteins of thioredoxin or thioredoxin-related proteins in whole cells or certain cellular compartments. In the last couple of years, many potential target proteins have been identified from various organelles and organisms by this method. Based on the information on the target proteins provided by these studies, the complete thioredoxin-related redox networks can now be efficiently described.

Key words: Disulphide bond, protein–protein interaction, redox regulation, thioredoxin, thioredoxin affinity chromatography

	Introduction

Top Abstract Introduction Contribution of Trx affinity... Limitations of the Trx... How to confirm the... Possible Trx target proteins Trx targets in the... Conclusion References

 
Thioredoxin (Trx) is a small ubiquitous protein, which mediates dithiol-disulphide exchange in all living cells. Hence, this protein can regulate the activity of enzymes through the formation or the reduction of a disulphide bridge in the target enzyme (Buchanan, 1991; Schürmann, 1995; Jacquot et al., 1997). In higher plants, Trx is especially important for transferring the reducing equivalent produced by the photosynthetic electron transport chain to the so-called thiol enzymes, thereby regulating the activity of these enzymes. Consequently, various reactions in the chloroplasts are indirectly controlled by the photosynthetic electron transport system. The common active site sequence (-Trp-Cys-Gly-Pro-Cys-) of Trx is conserved in most of the Trxs and their homologues. These proteins share a common structure known as the Trx motif consisting of four -helices and five ß-sheets (Eklund et al., 1984; Qin et al., 1994; Katti et al., 1995; Capitani et al., 2000). Although the significance of Trx in photosynthetic organisms is widely accepted, detailed information on the Trx target proteins was limited for a long time. Historically, the enzymes whose activity is controlled by Trx have been identified in biochemical studies. So far, four Calvin cycle enzymes (glyceraldehyde 3-phosphate dehydrogenase (GAPDH), fructose 1,6-bisphosphatase, sedoheptulose 1,7-bisphosphatase, and phosphoriburokinase) were known as thiol enzymes in the chloroplasts (Jacquot et al., 1997). NADP-dependent malate dehydrogenase (MDH), and glucose 6-phosphate dehydrogenase were also reported as enzymes that were controlled by thioredoxin (Scheibe and Anderson, 1981). The activation of the chloroplast ATP synthase due to the reduction of the subunit under reducing conditions was studied in depth (Mills et al., 1980; Nalin and McCarty, 1984), and redox-regulation of the rotation of the subunit was recently clarified (Bald et al., 2001; Ueoka-Nakanishi et al., 2004). In 1997, Sasaki and Kozaki found that acetyl CoA carboxylase, the key enzyme for lipid synthesis in the chloroplasts, is also under the control of the Trx redox regulation network (Sasaki et al., 1997; Kozaki et al., 2000, 2001). In addition, Zhang et al. (2002) reported that the activity of rubisco activase is regulated not only by the chloroplast ATP/ADP ratio but also by its redox state controlled by Trx (Zhang and Portis, 1999; Zhang et al., 2002). These studies suggested that there might be more target proteins of Trx in the chloroplasts which have not yet been identified. Indeed, recent progress in the genome sequencing projects of various organisms made it possible to survey potential Trx target protein without further biochemical purification. Hence, a number of studies have attempted to elucidate the variety of the target proteins in plant cells or in chloroplasts by two-dimensional gel electrophoresis analysis (Yano et al., 2001) and Trx-affinity chromatography using an immobilized single cysteine Trx-mutant (Motohashi et al., 2001; Balmer et al., 2003; Wong et al., 2003; Yamazaki et al., 2004). These approaches may be useful to describe the whole profile of Trx target proteins in any organelle or in the cytoplasm.

	Contribution of Trx affinity chromatography

Top Abstract Introduction Contribution of Trx affinity... Limitations of the Trx... How to confirm the... Possible Trx target proteins Trx targets in the... Conclusion References

 
Trx mediates the thiol-disulphide exchange reaction, and can transfer the reducing equivalents to other proteins. First, Trx induces the conformational change of the target protein and facilitates the reduction of the disulphide bridge in the molecule (Brandes et al., 1993; Stumpp et al., 1999). Then Trx and its target protein form a mixed-disulphide intermediate at the first reactive cysteine which is the cysteinyl residue nearest the N-terminus of the Trx molecule (Brandes et al., 1993). This intermolecular disulphide bond is attacked by the second cysteine of Trx, hereby the target protein is reduced and the reduced form Trx is oxidized. The substitution of the second cysteine to serine can interrupt this reduction process at the stage of the formation of the mixed-disulphide intermediate. This basic concept of Trx affinity chromatography was first applied by Verdoucq et al. (1999) to capture the interacting partner protein of Trx in yeast. They first prepared the mutant C35S (based on E. coli numbering), and transformed the mutant into the Trx-disrupted yeast cells. In their experiments they obtained the mixed disulphide intermediate and finally identified peroxidase YLR109 as the counterpart protein in the mixed disulphide complex. The strategy that was applied was conceptually similar to this work. However, in this study the Trx mutant was immobilized on the gel surface and the gel was used for the affinity chromatography (Motohashi et al., 2001). When the protein pool of the chloroplast stroma proteins or cytoplasm proteins were incubated with this gel, potential Trx target proteins having an accessible disulphide bond should form a mixed-disulphide intermediate with the immobilized Trx mutant on the resin. This intermediate complex should be stable under the experimental conditions, i.e. in the absence of any reductants. In order to remove non-specifically-bound proteins the gel was washed with either high concentrations of NaCl, 0.1% (w/v) SDS, or both compounds. After repeated washing, the captured proteins were then eluted from the gel using 10 mM dithiothreitol (DTT), a concentration sufficient to reduce the mixed disulphide bond formed (Fig. 1). By this procedure several potential Trx target proteins could be captured and it was possible to identify some of these proteins by N-terminal sequencing (Motohashi et al., 2001). Following this work, several groups applied a similar method to identify further potential target proteins of Trx and its related proteins (Table 1). Consequently, a number of potential Trx target proteins have been identified, but no functional analysis has been carried out.

View larger version (48K): [in this window] [in a new window]   Fig. 1. Trx affinity chromatography. The second cysteine of Trx was substituted by serine. The mutant protein was immobilized on CNBr-activated Sepharose (Amersham) according to the manufacturer's instruction. The prepared resin was then incubated with the protein mixture for 30 min to 1 h at room temperature. After repeated washing captured proteins were eluted from the resin using 10 mM DTT. For details, see Motohashi et al., 2001.

 

View this table: [in this window] [in a new window]   Table 1. Reports on the comprehensive survey on the target proteins of Trx and its related proteins

 

	Limitations of the Trx affinity chromatography

Top Abstract Introduction Contribution of Trx affinity... Limitations of the Trx... How to confirm the... Possible Trx target proteins Trx targets in the... Conclusion References

 
Accumulation of the genome information of the various organisms and the progress of the hypersensitive analysis of the proteins using MS-spectrometry made it possible to identify very small amounts of proteins, which were detected on the gel as weakly silver-stained bands. These advances are highly useful to analyse a certain protein–protein interaction comprehensively. Trx affinity chromatography used here simply follows the reaction process between Trx and its targets and intends to capture the target proteins. Indeed, it was possible to capture several well-known target proteins of Trx like GAPDH, sedoheptulose 1,7-bisphosphatase, and 2-Cys type peroxiredoxin in the chloroplast stroma (Motohashi et al., 2001). Furthermore, several novel potential Trx target proteins were obtained. Based on the proteins identified in these studies, recombinant proteins have been prepared of a new peroxiredoxin homologue (Prx-Q) and the chloroplast cyclophilin. Using these recombinant proteins the Trx-dependent reduction of the disulphide bond has been confirmed in these proteins.

However, it was noticed that this approach might also capture proteins which are not specific interaction partners of Trx in vivo. This is related to the fact that the cysteinyl residue on the Trx molecule is highly reactive to interact with disulphide bonds on the protein molecule surface of the target enzymes, even if the protein molecule is actually not redox-regulated. Although it was thought that there must be a strict rule based on the protein–protein interaction in order to establish the correct formation of the mixed-disulphide intermediate, this rule might not work efficiently enough when the Trx mutant was incubated with their target proteins for a long period (for 30 min or 1 h), which seemed to be sufficient to establish the formation of the intermolecular disulphide bond and also sufficient to obtain large enough amounts of proteins for further analysis. In this case the abundant proteins in the target protein pool may interfere to capture the rare target proteins effectively.

In addition, the pseudo-positive target proteins might be captured by this method. Recently, the interaction between Trx and type II peroxiredoxin in cyanobacteria was reported (Hosoya-Matsuda et al., 2005). This protein was easily captured by the immobilized TrxA mutant from the cytoslic proteins of Synechocystis sp. PCC6803. The captured peroxiredoxin is an abundant protein in Synechocystis and it was possible to confirm the in vitro reduction of the internal peroxiredoxin disulphide bond by the reduced form of Trx. However, we found that the peroxiredoxin might use reduced glutathione as the reducing equivalent for its function. Nonetheless, it could not be definitely concluded that the cyanobacterial type II peroxiredoxin is not a target of Trx, because a Trx-dependent reduction of the disulphide bond was also confirmed. Thus further interaction studies are needed to clarify the reducing partner of type II peroxiredoxin under physiological conditions.

However, these results may suggest that the immobilized cysteine Trx mutant may interact with a pseudo-positive target protein during the incubation process. Therefore careful biochemical research is required for each of the captured proteins.

	How to confirm the actual Trx target proteins

Top Abstract Introduction Contribution of Trx affinity... Limitations of the Trx... How to confirm the... Possible Trx target proteins Trx targets in the... Conclusion References

 
To confirm the validity of a Trx target protein, it is necessary to clarify the following three points: (i) Trx-dependent reduction of the oxidized form of a potential target protein, (ii) oxidation/reduction-dependent change in the activity, and (iii) the crucial cysteine pair for the redox regulation. In the authors' first paper on Trx affinity chromatography, it was reported that the chloroplast cyclophilin is a novel candidate protein for the chloroplast Trx-m (Motohashi et al., 2001). The redox-dependent switch of peptidyl-prolyl cis-trans isomerase activity of this protein has also been confirmed (Motohashi et al., 2003). In addition, the crucial cysteine pairs, which are related to the redox change of the enzyme activity, were identified by analysing the protease digestion profiles of the protein under reducing and oxidizing conditions. Very interestingly, this enzyme has two possible candidate cysteine pairs and both of them seem to affect the redox change of the enzyme activity. Oxidation and reduction of these disulphide bonds under physiological conditions are now being examined using intact chloroplasts and a polyclonal antibody raised against this protein.

	Possible Trx target proteins

Top Abstract Introduction Contribution of Trx affinity... Limitations of the Trx... How to confirm the... Possible Trx target proteins Trx targets in the... Conclusion References

 
The molecular chaperone in chloroplasts, chaperonin-60 (Cpn60) was one of the most abundant potential target protein captured by Trx affinity chromatography in the chloroplasts of higher plants (Balmer et al., 2003; Yamazaki et al., 2004). To assess whether Cpn60 is a real target for Trx, the redox response of this protein complex was studied using recombinant subunits obtained from Arabidopsis thaliana. Chloroplast chaperonin homologues are rather complex compared with the bacterial system (Martel et al., 1990; Koumoto et al., 1999). There are two GroEL isoforms, Cpn60- and Cpn60-ß, and two GroES isoforms, Cpn20 and Cpn10. The authentic combination of these subunits in the chloroplast chaperonin complex has not yet been confirmed. Therefore, the recombinant proteins of these four subunits were prepared and the formation of internal disulphide bond(s) was examined in the complex. Consequently, the formation and the Trx-dependent reduction of the disulphide bond on the oxidized form Cpn-60 subcomplex could be confirmed (data not shown). However, no change in the activity of the complex to assist protein folding could be observed (T Fujii and T Hisabori, unpublished results). Hence it has to be investigated further whether the interaction between Trx and Cpn60 has a real physiological meaning.

	Trx targets in the cytosol

Top Abstract Introduction Contribution of Trx affinity... Limitations of the Trx... How to confirm the... Possible Trx target proteins Trx targets in the... Conclusion References

 
When the possible target proteins in the Arabidopsis cytosol were analysed using the Trx affinity chromatography method, several metabolic enzymes such as cytosolic GAPDH, cytosolic MDH, and alcohol dehydrogenase (ADH) were captured (Yamazaki et al., 2004). Their homologues, chloroplast GAPDH (Scheibe and Anderson, 1981; Baalmann et al., 1995) and MDH (Miginiac-Maslow et al., 1997; Johansson et al., 1999) are well characterized thiol enzymes. In chloroplasts, these thiol enzymes have extended amino acid region(s) containing multiple cysteine residues and the relevance between their reduction/oxidation and modulation of the enzyme activity has already been clarified. However, nothing is yet known of the change in their activity in the cytosol under redox controlled conditions, although several candidate cysteines in each of these molecules could be assigned (Yamazaki et al., 2004). The redox dependent changes of the activities of ADH, GAPDH, and MDH were therefore examined using recombinant proteins. For this purpose, the genes for ADH (AT1G77120), GAPDH (AT3G041209), and MDH (AT1G04410) were cloned using the RT-PCR method with the total RNA of A. thaliana as templates. The desired proteins were expressed in E. coli and the redox-dependent changes of their activities were measured. The results indicate that three enzymes are redox-sensitive; their activities were suppressed by oxidation and recovered by reduction. Of these enzymes, ADH and MDH were easily reduced by the reduced Trx (Fig. 2). The Trx-dependent reduction of the internal or intra-molecular disulphide bond in ADH and MDH molecules were also confirmed (S Hara and T Hisabori, unpublished results).

View larger version (16K): [in this window] [in a new window]   Fig. 2. Trx-dependent reduction and activation of the metabolic enzymes in the cytosol. The genes for ADH (AT1G77120), GAPDH (AT3G041209), and MDH (AT1G04410) were cloned and ligated into the pET23c expression vector and the desired proteins were individually expressed in E. coli strain BL21(DE3). As the target protein was expressed in high amounts in each host cell, their enzyme activities could be directly assessed using the cell lysates. The activity of ADH was measured according to the method described in Chinnawirotpisan et al. (2003) by monitoring NADH oxidation at 340 nm. The method for the measurement of the MDH activity was taken from Pastore et al. (2003). Before the assay the cell lysate was incubated with 20 µM CuCl2 for 1 h for oxidation. It was then incubated with the indicated concentrations of DTT for 1 h at 30 °C in the presence (open circles) or the absence (closed circles) of 1 µM Trx-h1 from A. thaliana.

 
NADPH is the reducing equivalent in the cytosol to reduce the cytosolic Trx via NADPH-Trx reductase. Thus, the remaining question is whether the change of the NADP⁺/NADPH balance in the cytosol is sufficient to affect the formation of the disulphide bond and their reduction on these enzymes or not. Although the reported concentration of NADPH and NADP⁺ in the cytoplasm is more than several hundred µM, it seems difficult to estimate the correct change in the redox potential due to the existence of large amounts of other redox-related substances like glutathione. Another question is what are the conditions where complete oxidation of these metabolic enzymes take place. Low concentrations of CuCl₂ were used here to catalyse the formation of the disulphide bond on the enzyme. Since the cupper-catalysed oxidation of cysteines is much harder than the corresponding oxidation under physiological conditions, a more suitable oxidant should be applied to confirm the inactivation of the enzymes by oxidation. Hence, further studies on the redox dynamics of the potential target proteins are necessary to reveal the whole redox network system.

	Conclusion

Top Abstract Introduction Contribution of Trx affinity... Limitations of the Trx... How to confirm the... Possible Trx target proteins Trx targets in the... Conclusion References

 
As indicated in Table 1, Trx affinity chromatography and its expanded method were applied to the various proteomics projects to identify Trx target proteins. In the past this method has became extremely useful to identify the various potential target proteins. The data obtained from these studies strongly suggest that the redox network is highly important in the control of various metabolic processes in the cell. On the other hand, these studies indicate a large number of the possible, but not confirmed, protein–protein interactions between Trx and their target proteins. Based on the data obtained from these experiments, it is necessary to clarify the actual interaction between Trx and these proteins. The final aim of this is to elucidate the extent and role of the redox-networks in the cell, which is an important physiological regulatory system.

	Acknowledgements

 
We thank Dr G Groth (Heinrich-Heine University) for his critical reading of the manuscript.

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