The formation of native disulfide bonds is a rate-limiting process in the folding of many nascent proteins in the endoplasmic reticulum (ER) ()(Creighton, 1986; Gething and Sambrook, 1992). According to present views, oxidized glutathione (GSSG) is the oxidant of cysteine thiols of a newly synthesized and reduced protein in the ER lumen. The ratio of GSH to GSSG is lower in the ER than in the cytosol (3:1 to 1:1 and 100:1, respectively) and should correspond to a redoxpotential (E`) that is compatible with protein disulfide formation (Hwang et al., 1992). Oxidative folding reactions invitro have been performed in redox buffers of GSH and GSSG with ratios of about 10, typically 3-10 mM GSH and 0.3-1 mM GSSG (Wetlaufer et al., 1987). The folding of RNase with four disulfide bonds in the native, enzymatically active state (RNase(S)) and with eight free SH groups in the reduced and unfolded state RNase-(SH) has been extensively characterized by Scheraga and co-workers (Konishi et al., 1981; Konishi et al., 1982a, 1982b, 1982c; Scheraga et al., 1987; Rothwarf and Scheraga, 1993a, 1993b, 1993c, 1993d) in glutathione redox buffers.

The folding process of RNase proceeds via three reversible reactions (Fig. R1Fig. R2Fig. R3), where SH, SSG, and SS represent a free cysteine residue, a cysteine residue involved in a mixed disulfide with GSH and a disulfide bond in the protein, respectively. Fig. R3represents the intramolecular reshuffling of disulfide bonds. Since RNase-(SH) has 8 cysteines, the total number of theoretically possible species in the pathway is 7193 (Scheraga et al., 1987) (7191 intermediates plus the fully reduced and native forms of RNase). Several folding pathways were deduced depending on the physiochemical conditions and the E` value of the redox buffer. It has been experimentally shown that GSH can form a large number of intermediate mixed disulfides with RNase (Konishi et al., 1981). The reaction can take multiple pathways dictated by the GSH-buffer, leading to native RNase-(S) (Rothwarf and Scheraga, 1993c).

Figure R1:

Figure R2:

Figure R3:

Glutaredoxin (Grx), was discovered as a hydrogen donor for ribonucleotide reductase in Escherichiacoli and in mammalian cells together with GSH, NADPH, and glutathione reductase (the glutaredoxin system) (Holmgren, 1979a; Holmgren, 1989). Grx is also a general GSH-disulfide-oxidoreductase-catalyzing NADPH-dependent reduction of disulfides like 2-hydroxyethyldisulfide in a complete system with GSH and glutathione reductase. In particular, glutaredoxin is an efficient reductant of mixed disulfides with glutathione. Together with thioredoxin and protein disulfide-isomerase (PDI), Grx is a member of a growing superfamily of well characterized proteins that share a common fold and an exposed active site dithiol/disulfide. This consists of a 14-member disulfide ring in the oxidized form that is located at the end of a -strand and followed by an -helix. The N-terminal active site cysteine sulfur is exposed and surrounded by a hydrophobic surface area. Glutaredoxin from E. coli consists of 85 amino acids residues (M 10,000) with the active site sequence CPYC. The structure in solution of both the oxidized form (Grx-S) and the reduced form (Grx-(SH)) have been determined by NMR (Xia et al., 1992, Sodano et al., 1991). Recently, the mutant protein C14S Grx (Bushweller et al., 1992) was used to generate a complex with GSH as a mixed disulfide (GrxSSG), the solution structure of which has also been determined by NMR, demonstrating a binding site for GSH in the structure (Bushweller et al. 1993, 1994). C14S Grx, which retains only 1 active site Cys residue cannot catalyze protein disulfide oxido reductions like reduced thioredoxin, but it has activity as a GSH-disulfide oxidoreductase with small disulfides.

PDI catalyzes disulfide bond formation in vitro and in vivo (LaMantia and Lennarz, 1993; Bulleid and Freedman, 1988) and contains two domains with clear sequence homology to thioredoxin (Edman et al., 1985; Eklund et al., 1991; Freedman, 1989). The well known structure of thioredoxin shows no binding site for GSH, and the enzyme functions with NADPH and thioredoxin reductase to reduce a protein disulfide via a dithiol reaction mechanism (Holmgren, 1979b, 1985; Jeng et al., 1994). Previously, Lyles and Gilbert (1991a, 1991b) observed that the complete folding of RNase A with a GSH/GSSG redox buffer was enhanced up to 23-fold by PDI. A much greater rate enhancement by PDI (5000-fold) was observed by Weissman and Kim (1993) in specific oxidation and isomerization steps during the oxidative folding of reduced bovine pancreatic trypsin inhibitor. These results imply that PDI is enzymatically active only in certain steps of disulfide bond formation.

While the identity and mode of action of the ultimate oxidant of protein thiols in the ER of eukaryotic cells is unknown, a pathway of electron transport has been elucidated for disulfide bond formation in E. coli. A reduced polypeptide is oxidized by DsbA, a 21-kDa oxidoreductase, which in turn transfers electrons to a second protein, DsbB (Akiyama and Ito, 1993; Akiyama et al., 1992; Bardwell et al., 1991, 1993; Kamitani et al., 1992). The activity of DsbB can partly be rescued by GSSG or cystine in vivo and in vitro, but the requirement for DsbA or functionally equivalent proteins (Schevchik et al., 1994; Missiakas et al., 1994) seems to be absolute.

This study was undertaken to study the effect of glutaredoxin on glutathione-mediated folding of ribonuclease. Mixed disulfides between proteins and GSH may be important and necessary folding intermediates in vitro. Our results demonstrate that Grx and C14S Grx showed strong synergistic effects with PDI during the first few minutes of the folding reaction.

EXPERIMENTAL PROCEDURES

Materials

Preparation of Fully Reduced RNase

Preparation of Scrambled RNase

Enzyme-catalyzed Disulfide Formation of RNase

The concentration of active RNase at any time was calculated from the A/min and converted to the actual v in µM/min by using the extinction coefficients of cCMP and CMP (0.19 mM cm and 0.38 mM cm) and the known total concentration of nucleotide. With correction for the competitive inhibition of RNase by CMP, the concentration of active RNase (E) at any time point was calculated according to .

where k is the turnover number of fully active RNase (196 µmol cCMP (minµmol of RNase)), K is the K for cCMP under these conditions (8.0 mM), and K is the inhibition constant for CMP (2.1 mM) (Lyles and Gilbert, 1991a, 1991b). ()

A standard curve of RNase A resulted in good proportionality in the concentration range used (0-8 µM) and with a CMP concentration below 1 mM. The calculated RNase A concentration was slightly lower, (85%) than the expected concentration. In our hands, RNase A standards gave even lower calculated RNase activity at other total cCMP plus CMP concentrations lower than 4.5 mM. All results presented in graphs represent mean values of duplicate experiments run in parallel.

Determination of Final Yield of RNase Activity

Protein Concentration Determinations

Assay for Transhyrogenase Activity

RESULTS

Enzymatic catalysis of native disulfide bond formation in fully reduced, inactive RNase-(SH) was followed as the rate of hydrolysis of 2`,3`-cCMP catalyzed by the product, which is native RNase-(S). This method is useful to study early events in disulfide formation and gives a fast measure of the rate order. A second step of mathematical processing is required to account for inhibition by the product CMP and to convert hydrolysis of cCMP to the actual concentration of active RNase-(S) (Lyles and Gilbert, 1991a).

Synergy Effect between Glutaredoxin and PDI in GSH-dependent Refolding of RNase-(SH)

Figure 1: Effects of Grx and PDI on refolding of reduced RNase A in the presence of a glutathione redox buffer. RNase-(SH), 8 µM, was incubated with 1 mM GSH, 0.2 mM GSSG, 4.5 mM cCMP in 0.055 mM Tris acetate buffer, pH 8.0, and 1 mM EDTA in the presence of 1 µM PDI () and without PDI (). The absorbance at 296 nm was followed (upper panels A-C) and converted to active RNase-(S) concentration (lower panels A-C). A, without Grx; B, in the presence of 0.5 µM wild-type Grx; and C in the presence of 0.5 µM C14S Grx. Each data point represents a mean value of two parallel experiments.

In order to understand the mechanism by which Grx acts together with PDI, we tried the mutant protein C14S Grx (Fig. 1C). This had the same effect together with PDI as its wild-type counterpart, which strongly suggests a monothiol reaction mechanism as the mode of action of Grx in this system.

The recovery of RNase activity after a 90-min incubation was analyzed separately (Table 1). The maximal yield of active RNase was 5.2 µM or 65% of the theoretical using only PDI. The result was almost the same in the presence also of Grx. We conclude that Grx acts in a synergistic manner together with PDI during the first few minutes of refolding of RNase-(SH) but has much less influence on the rate (µM RNase formed/min) in the linear phase of the reaction or on the overall yield of native RNase. Thus, glutaredoxin and PDI may act by different mechanisms; glutaredoxin does not appear to catalyze formation of native RNase on its own part, but rather it takes part in early events of thiol disulfide interchange between RNase-(SH) and GSH/GSSG.

The synergistic effect of glutaredoxin and PDI was analyzed in three different redox buffers of glutathione (E` of -160, -181, and -208 mV). In these experiments, we applied a higher concentration of PDI, 2 µM, in order to detect catalysis under nonoptimal conditions, resulting in higher yield of active RNase. As expected, PDI catalyzed activation of RNase-(SH) at all three redoxpotentials, albeit with different rates (Fig. 2). Grx enhanced the activity of PDI only in the two more reducing redox buffers (-181 and -208 mV, respectively). When PDI catalyzes formation of disulfide bonds in RNase-(SH) under oxidizing conditions using GSSG, there is a quick buildup of a small population of active RNase molecules. However, this activation does not continue because nonnative redox-isomers cannot be rescued in the absence of GSH (Lyles and Gilbert, 1991a, 1991b). The presence of Grx does not change this behavior in agreement with the redox potential of its active site dithiol/disulfide (see ``Discussion'').

Figure 2: Effects of Grx and PDI on refolding of RNase-(SH) in glutathione redox buffers with different redox potentials. RNase-(SH) was mixed with 0.4 mM GSH and 1 mM GSSG defining a redox potential (E` value) of -160 mV (A), 1 mM GSH and 0.2 mM GSSG or E` = -181 mV (B), and 2 mM GSH and 0.1 mM GSSG or E` = -208 mV (C). The amount of active RNase A-(S) is plotted against time of incubation. Control (), in the presence of 2 µM PDI only (), and in the presence 2 µM PDI and 0.5 µM G ().

PDI and Glutaredoxin in Refolding Systems Containing Cystamine/Cysteamine or Selenite

Figure 3: Effects of glutaredoxin and PDI in refolding systems containing cystamine/cysteamine or selenite. RNase-(SH) , 8 µM was mixed with 1 mM GSH and 0.2 mM GSSG (A); 2 mM CSH and 0.1 mM CSSC (B); and 2 µM SeO (C). The conditions were as in Fig. 1, but GSH and GSSG were left out in B and C. Control without PDI or Grx (), 1 µM PDI only (), and 1 µM PDI and 0.5 µM Grx ().

Grx showed no synergy effects with PDI in a refolding system with only selenite as oxidizing agent, Fig. 3C. Selenium compounds like SeO and selenodiglutathione (GSSeSG) oxidize the active site of thioredoxin in a nonstoichiometric manner (Björnstedt et al., 1992; Kumar et al., 1992). We have shown previously that 1 µM selenite together with PDI or P34H Trx catalyzes efficient refolding of 25 µM reduced RNase and that selenite oxidizes the active-site dithiols of PDI (Lundström et al., 1992; Lundström and Holmgren, 1993). Thus, the synergy effect of glutaredoxin together with PDI appeared exclusive for glutathione in keeping with glutaredoxin being a specific glutathionyl-mixed disulfide reductase (Bushweller et al., 1992, 1994; Gravina and Mieyal, 1993).

Glutaredoxin and PDI in Refolding of Scrambled Ribonuclease

In principle, refolding of scrambled RNase-(S) could proceed either by disulfide bond isomerization or by complete reduction and disulfide bond formation. In the presence of 1 mM GSH and 0.2 mM GSSG, refolding of scrambled RNase A-(S) probably mainly involve disulfide isomerization catalyzed by PDI. This may not result in a sufficient buildup of mixed disulfides between glutathione and RNase and allows no role for glutaredoxin.

Refolding in the Presence of E. coli P34H Trx

Figure 4: Refolding of RNase-(SH) dependent on P34H Trx in the presence and absence of glutaredoxin. RNase-(SH), 8 µM was mixed with a redox buffer of 1 mM GSH and 0.2 mM GSSG and activity was followed. The additions were as follows: 5 µM P34H Trx (), 10 µM P34H Trx (), 5 µM P34H Trx and 0.5 µM Grx (), 10 µM P34H Trx and 0.5 µM Grx (). The conditions were as in Fig. 1.

Refolding in the Presence of DsbA

Figure 5: Refolding of RNase-(SH) dependent on DsbA in the presence and absence of glutaredoxin. RNase-(SH), 8 µM was mixed with a redox buffer of 1 mM GSH and 0.2 mM GSSG and activity was followed. The additions were as follows: 5 µM DsbA (), 10 µM DsbA (), 5 µM DsbA and 0,5 µM Grx (), 10 µM DsbA and 0.5 µM Grx (). The conditions were as in Fig. 1.

PDI or DsbA Do Not Display GSH-disulfide Oxidoreductase Activity

Glutaredoxin exhibited activity but neither PDI, nor DsbA showed any activity (Table 2).

DISCUSSION

Disulfide formation in unfolded and inactive RNase-(SH) was followed continuously as the rate of hydrolysis of 2`,3`-cCMP catalyzed by native folded and active RNase-(S). PDI catalyzed efficient refolding and activation of RNase-(SH) in the presence of 1 mM GSH and 0.2 mM GSSG, in agreement with results of Lyles and Gilbert (1991a, 1991b). We observed a lower activity with the E. coli mutant protein P34H Trx or with the E. coli periplasmic protein DsbA under these conditions. Glutaredoxin showed marked synergistic effects with PDI, P34H Trx, and DsbA.

Disulfide formation in proteins is a multiple-step process that involves intramolecular disulfide interchange or shuffling. Using bovine pancreatic trypsin inhibitor, a concept of folding was first established stating that proteins with nonnative disulfides are important folding intermediates (Creighton, 1988). This has later been questioned and reevaluated indicating that nonnative intermediates and conformations do not play a major role in folding (Weissman and Kim, 1991). A lag phase preceding a steady-state refolding of RNase-(SH) in GSH/GSSG redox buffers has been attributed to a prerequisite for a buildup of redox-isomers that can be converted to the native protein (Fig. R1Fig. R2Fig. R3). During the early phase of the reaction, reduced ribonuclease partitions between GSH-mixed disulfide redox-isomers that are good and poor substrates for PDI. The formation of both types of redoxisomers is promoted by the presence of GSSG, but molecules that once have been generated as poor substrates for PDI can only be rescued by GSH (Lyles and Gilbert, 1991a, 1991b; Shaffer et al., 1975).

Glutaredoxin significantly shortened or eliminated the lag phase. This effect was obvious in the more reducing glutathione redox buffers (E`, -181 and -208 mV), but almost absent under more oxidizing conditions (E`, -160 mV). The redox potential of glutaredoxin has been determined from a change in tyrosine fluorescence in glutathione redox buffers and resulted in an E` value of -240 mV. ()Using an E` value of glutathione of -260 mV (Scott et al., 1963), 15% of glutaredoxin would be in the reduced state at -208 mV, 5% at -181, and only 0.05% at -160 mV. The similar activity of the mutant protein C14S Grx glutaredoxin containing only 1 active-site cysteine residue, demonstrated that reduced glutaredoxin must act via a monothiol reaction mechanism on the mixed disulfides between RNase A and GSSG (see Fig. R1Fig. R2Fig. R3for explanations) since C14S Grx has been shown not to reduce a protein disulfide.

The synergistic effects between Grx and PDI during the early phase of RNase A-(SH) refolding can be ascribed to one of the following.

1) GSSG forms mixed disulfide with RNase that are either no or poor substrates for PDI. In the absence of Grx, these mixed disulfides are reduced by a relatively slow intramolecular displacement of the glutathione moitey that leads to formation of a protein disulfide. Grx catalyzes rapid turnover of the RNase-SSG molecules.

2) PDI does reduce RNase-SSG intermediates, but the subsequent chemical reduction of PDI containing GSH to one of the active site cysteines by GSH is a slow step in which glutaredoxin could take part. In this case, PDI would operate via a monothiol mechanism. However, PDI showed no glutaredoxin-like activity with 2-hydroxyethyldisulfide. Furthermore, this alternative does not explain why a synergy effect with Grx was not observed when scrambled RNase-(S) was the substrate for the reaction or why the effect of Grx was limited to the first minutes. Moreover, the Trx-like properties of PDI make this unlikely, since Trx has no tendency to form a stable mixed disulfide (Holmgren, 1985).

3) The 2 extra cysteine residues in bovine PDI, outside the two active sites can form mixed disulfides with GSSG, and this leads to inactivation of PDI. Grx would reactivate PDI. This may be true, but it does not explain why Grx acts in synergy with P34H Trx and with DsbA.

Of these three alternatives, the first seems most likely. The strong synergistic effect of Grx and the mutant protein C14S Grx with PDI in regeneration of RNase activity in the glutathione redox buffer is reason to question whether mixed disulfides between unfolded or partially folded proteins and GSSG are efficient substrates for PDI. However, Darby et al.(1994) have investigated the effects of substoiciometric amounts of PDI during disulfide bond formation in the presence of glutathione in an unstructured model peptide. In this system, two steps involving mixed disulfides and their subsequent rearrangement to form a peptide disulfide bond accounted for most of the catalytic effect of PDI. Although PDI thus can act via a monothiol mechanism, the moderate rate enhancement (10-fold by 1.6 µM PDI) does not necessarily describe the whole reaction mechanism during folding and disulfide bond formation of native proteins. Since multiple pathways of folding have been suggested, the presence of Grx may indeed influence the distribution of folding intermediates or mechanism by making certain intermediates kinetically available. We suggest that a major role of Grx in the RNase refolding reaction is to catalyze reduction of redox isomers of RNase and GSSG that are inefficient substrates for PDI as depicted in Fig. 6.

Figure 6: Mechanism for formation of active RNase in the presence of PDI and glutaredoxin. Modified from Lyles and Gilbert (1991b).

In the ER of eukaryotes, there are several proteins related to PDI by sequence and by activity in the ERp72 (CaBP2) (Nguyen Van et al., 1993; Mazzarella et al., 1990), ERp61 (Bennet et al., 1988), and CaBP1 (Chauduri et al., 1992; Füllekrüg et al., 1994). Like PDI, they all contain two or three (ERp72) domains with strong homology to thioredoxin (Holmgren, 1989). All of these proteins belonging to the thioredoxin superfamily have similar activity as substrates for thioredoxin reductase and as protein disulfides isomerases (Lundström et al., 1994; Rupp et al., 1994). Since the concentration of PDI is very high or in the mM range and the other thioredoxin-like proteins are present at comparable concentrations, the catalytically active thiols in the thioredoxin-like proteins should exist at a concentration similar, if not higher than that of glutathione, the dominating low molecular weight redox buffer (Hwang et al., 1992). Thus, the system of thiols in the endoplasmic reticulum can hardly be mimicked in vitro experiments.

Whether mixed disulfides between glutathione and reduced polypeptides are indeed important folding intermediates in vivo is thus still an open question. We postulate that there are two alternatives. 1) The rate by which PDI and other thioredoxin-like proteins react with reduced polypeptides and disulfide bond intermediates is much greater than the rate of formation of mixed disulfides with glutathione. Such mixed disulfides are only formed to a limited extent and are not important folding intermediates. Glutathione is present in the ER merely as a redox buffer and is not kinetically important. This requires a highly efficient unknown electron transport from reduced PDI.

2) Nascent reduced polypeptide chains do react with glutathione the ER, and this is kinetically important. If this explanation is true, there must exist an hitherto unidentified glutaredoxin or a protein with glutaredoxin-like activity in the ER. A possible candidate is a protein EUG1, which was recently identified in yeast. This protein contains only 1 active site cysteine residue, but allows yeast cells to grow in the absence of the PDI1 gene product and partly restores the wild-type phenotype (Tachibana and Stevens, 1992). The catalytic properties of this protein have not yet been studied nor has its presence in other eukaryotic cells been reported.

In conclusion, glutaredoxin acts synergistically with PDI to promote oxidative folding of RNase-(SH) in glutathione redox buffers. Since GSH/GSSG are used to refold many proteins expressed in procaryotic systems, the addition of Grx has important potential advantages with proteins that are difficult to refold in vitro with good yields. Further studies regarding the pathways of refolding by observation of the folding intermediates may shed light on mechanistic aspects of the complex reaction of RNase reactivation.

FOOTNOTES

*

This work was supported by grants from the Swedish Medical Reseach Council (13X-3529, 13Y-11213), The Karolinska Institute, the Knut and Alice Wallenberg Stiftelse, the Inga-Britt and Arne Lundbergs Stiftelse, and Beng Lundquists minne. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

To whom correspondence should be addressed: Medical Nobel Inst. for Biochemistry, Dept. of Medical Biochemistry and Biophysics, Karolinska Inst., S-171 77 Stockholm, Sweden. Tel.: 46 8 728 7686; Fax: 46 8 728 4716.

()

The abbreviations used are: ER, endoplasmic reticulum; PDI, protein disulfide-isomerase; RNase, ribonuclease A; Grx, E. coli glutaredoxin; Grx C14S, E. coli glutaredoxin with Cys-14 mutated to Ser; CSH, cysteamine; CSSC, cystamine.

()

H. F. Gilbert, personal communication.

()

O. Björnberg and A. Holmgren, submitted for publication.

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