©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Independence of the Chaperone Activity of Protein Disulfide Isomerase from Its Thioredoxin-like Active Site (*)

(Received for publication, March 29, 1995; and in revised form, May 22, 1995)

Hui Quan , Guibao Fan , Chih-chen Wang (§)

From the National Laboratory of Biomacromolecules, Institute of Biophysics, Academia Sinica, 15 Datun Road, Beijing 100101, People's Republic of China

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Protein disulfide isomerase (PDI) alkylated at thiols of the thioredoxin-like -CHC- active sites is devoid of isomerase activity, but its chaperone-like activity to increase the reactivation yield and prevent the aggregation of guanidine hydrochloride-denatured D-glyceraldehyde-3-phosphate dehydrogenase upon dilution is unimpaired. A peptide of 28 amino acids markedly inhibits both the enzyme and the chaperone activities of PDI. The above results indicate that the -CGHC- active site is necessary for the isomerase activity but not required for the chaperone activity of PDI, whereas the peptide binding site is essential for both activities.


INTRODUCTION

It is now generally accepted that, in most cases, the folding and assembly of nascent peptides to functional proteins require the assistance of other proteins, i.e. molecular chaperones and foldases(1) . Protein disulfide isomerase (PDI),() one of the two foldases so far characterized, promotes protein folding by catalyzing the formation and rearrangement of disulfide bonds(2) . The two thioredoxin (Tdx)-like -CGHC- sequences of the PDI protomer are putative active sites involved in its catalytic activity(3, 4) . The enzyme is capable of binding peptides with low specificity(5) , and peptide binding inhibits its isomerase activity(6) . We have hypothesized that PDI is not only an enzyme but also functions as a molecular chaperone during protein folding(7) , and we have presented evidence to show that PDI displays chaperone activity independent of the isomerase activity by increasing reactivation yield and decreasing aggregation of guanidine hydrochloride (GdnHCl) denatured D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a protein containing no disulfide bond, during its refolding upon dilution(8) . Recently, several authors have also suggested the chaperone function of PDI(9, 10, 11) , and La Mantia and Lennarz (12) have indicated that the isomerase activity is not essential for cell viability which may require instead some other functions of PDI. A study by Noiva et al.(13) on the relationship between the putative active site and the peptide binding activity showed that the cysteines in the -CGHC- active sites are not required for the peptide binding activity. However, using a disulfide-containing protein, lysozyme, as the target protein, Puig et al.(14) reported recently that PDI showed chaperone activity, which requires both the -CGHC- active site and the peptide binding site by site-directed mutagenesis at the active site cysteine residues.

In order to examine further the structural basis for the isomerase and chaperone activities of PDI, we have studied directly the relationship between the -CGHC- active sites and chaperone activity of PDI and the effects of peptide binding on the chaperone function of PDI displayed during the refolding of GAPDH. The results presented in this report provided evidence to show that -CGHC- active site is necessary for the isomerase activity but not required for the chaperone activity of PDI and the peptide binding site is essential for both activities.


EXPERIMENTAL PROCEDURES

Materials

Purification and activity determination of rabbit muscle GAPDH and PDI were as before(8) . D-Glyceraldehyde 3-phosphate, glutathione (GSH), glutathione reductase, NADPH, Tdx, and GdnHCl were from Sigma. NAD (98%) was from Boehringer Mannheim. Dithiothreitol (DTT) from Promega. 5,5`-dithiobis(2-nitrobenzoic acid) (DTNB) was from Fluka and iodoacetic acid from Merck. The N-terminal segment of staphylococcal nuclease with the sequence ATSTK KLHKE PATLI KAIDG DTVKL MYK (A-K) was synthesized on Applied Biosystem 431A synthesizer using standard Fmoc method and purified by pepRPC 10/10 column, and its Fourier transformed infrared spectrum was measured with a Bio-Rad model FTS-65A infrared spectrophotometer. Porcine insulin was from Sigma and treated by EDTA to remove zinc ion before use. Staphylococcal nuclease was prepared according to Shortle(15) . All other chemicals were local products of analytical grade. In all experiments 0.1 M sodium phosphate buffer, pH 7.5, containing 1 mM EDTA was employed.

The concentrations of PDI, GAPDH, Tdx, and insulin were determined spectrophotometrically at 280 nm (A = 0.9 for PDI, and A = 0.98 for GAPDH, and A = 1.14 for Tdx, and A= 1.03 for insulin). For the convenience of comparison, both GAPDH and PDI are considered protomers in the calculation of molar ratios.

Modification of PDI

PDI (200 µM) was reduced by incubation with DTT (4 mM) in 0.1 M Tris/HCl buffer, pH 8.0, at 30 °C for 30 min, and then modified by adding iodoacetic acid or CHI to final concentrations of 25 mM and 40 mM, respectively, at 30 °C for 2 h with stirring. In the case of modification with iodoacetic acid, the pH of the reaction mixture was readjusted to 8.0 with 0.2 M NaOH. The reaction mixture was then dialyzed thoroughly at 4 °C to remove the excess of the alkylating reagents. The number of free thiols of PDI was determined by DTNB (16) in 6 M GdnHCl.

Denaturation and Reactivation of GAPDH

GAPDH (140 µM) was completely denatured in 3 M GdnHCl overnight at 4 °C in presence of 5 mM DTT. Reactivation was carried out by 50-fold dilution of the denatured enzyme into phosphate buffer containing various concentrations of PDI or alkylated PDI. The reactivation mixture was first kept at 4 °C for 30 min and then for an additional 3-h period at 25 °C (17) before aliquots containing 2 µg of GAPDH were taken for activity assay at 25 °C. Aggregation, monitored at 20 °C by 90° light scattering at 488 nm continuously in a Hitachi model F-4010 spectrofluorometer, approached completion in 40 min.

Enzyme Activity Assay

The isomerase activity and thiol-protein oxidoreductase (TPOR) activity of PDI were determined according to Lambert and Freedman(18) . Tdx activity was assayed by turbidity due to the reduction of insulin by DTT(19) . Inhibition of the TPOR activity by the peptide A-K was determined in presence of different concentrations of the inhibitor at four substrate concentrations and the competitive inhibition constant, K, was calculated according to Morjana and Gilbert (6) .


RESULTS

Effect of Alkylation of PDI on Its Isomerase Activity

As shown in Table 1, newly prepared PDI contains two free thiols and after reduction by DTT and removal of the reductant in excess by dialysis about 5.8 thiols as determined with DTNB consistent with 6 cysteine residues as deduced from the cDNA sequence of PDI(20) . CHI reacts with all six cysteine residues, and PDI thus modified is devoid of isomerase activity, whereas iodoacetic acid modifies only two cysteines but the enzyme modified at each N-terminal cysteine residue of the two -CGHC- active sites (3) shows likewise very little isomerase activity.



Effects of Alkylated PDI on Refolding of Denatured GAPDH

GdnHCl-denatured GAPDH shows only a limited extent of reactivation after dilution, especially at high concentrations, and has a marked propensity to aggregate during refolding(8) . The presence of either native or the two alkylated derivatives of PDI in the refolding system increases reactivation of GdnHCl denatured GAPDH from 5% to about 20% as shown in Fig. 1A. At the same time, both native and the alkylated enzymes markedly suppress the aggregation of GAPDH during refolding to the same extent as shown by light scattering measurements (Fig. 1B). The above results indicate that only the isomerase but not the chaperone activity of the enzyme is abolished by blockage of the -CGHC- active site thiols, suggesting that the chaperone activity of PDI is independent of its -CGHC- active site. Tdx, containing a similar -CGPC- active site, is devoid of peptide binding property (13) and shows no effect on the refolding of GAPDH even at 30-fold molar excess to GAPDH (data not shown).


Figure 1: Effects of concentrations of native and alkylated PDI on the reactivation (A) and aggregation (B) presented by the final level of light scattering, of 2.8 µM denatured GAPDH upon dilution. The dilution buffer contained various concentrations of native PDI (), S-methylated PDI (), and S-carboxymethylated PDI (), respectively.



Effects of Concentrations of the Peptide A-K on the Refolding of GAPDH

At least one peptide binding site in PDI protomer has been characterized, which might be responsible for its chaperone-type function(13) . In order to confirm the role of the peptide binding site on the chaperone activity of PDI, a peptide of 28 amino acid residues (A-K), the N-terminal segment of staphylococcal nuclease, was added to the refolding buffer to compete with GAPDH at the peptide binding sites of PDI(6) . As shown in Fig. 2A, the reactivation yield of GAPDH is indeed markedly reduced by increasing concentrations of the peptide, with half of the chaperone activity of PDI suppressed at a peptide concentration of 20-fold molar excess to PDI in the refolding buffer. The presence of A-K alone has no effect on the spontaneous refolding of GAPDH. Furthermore, the presence of A-K in the dilution mixture of denatured GAPDH also results in the decrease of protective effect of PDI on the aggregation of GAPDH, which increased with increasing concentrations of the peptide (Fig. 2B). Without PDI, A-K has little effect on the aggregation of GAPDH. A-K apparently competes with GAPDH for binding to PDI, and the binding of A-K to PDI suppressed the chaperone activity of PDI as shown by both the decrease of reactivation and the increase of aggregation of GAPDH. In contrast, native staphylococcal nuclease has little effect on both the spontaneous and PDI-assisted refolding of GAPDH at the same concentrations (data not shown), indicating that PDI cannot recognize and therefore bind with the same peptide segment in the native protein. A Fourier transformed infrared spectra analysis of A-K indicates that it is not completely disordered but contains some residual structure (data not shown), which could contribute to its binding to PDI.


Figure 2: Effects of concentrations of the peptide A-K on the reactivation (A) and aggregation (B) of 2.8 µM denatured GAPDH upon dilution. The experiments for reactivation were carried out as in Fig. 1. except A-K of different concentrations were also present in the dilution buffer. , no PDI; , in the presence of 5.2 µM PDI. The time courses of light scattering change with concentrations of A-K and PDI as indicated.



Inhibition of the TPOR Activity of PDI by Peptide A-K

PDI is a thiol-protein oxidoreductase (TPOR) and under reducing conditions catalyzes the reduction of the disulfide bridges in proteins(21) . Peptide A-K inhibits the TPOR activity of PDI as measured by the reduction of insulin (Fig. 3) with a competitive inhibition constant, K, of about 200 µM. In contrast, this peptide has no effect on the TPOR activity of Tdx (data not shown), indicating further the lack of a peptide binding site in the Tdx molecule. This indicates that, even though Tdx has a very similar -CGPC- active site, it has no chaperone activity and a much lower isomerase activity as compared to PDI(2, 22) , as suggested previously in our hypothesis(7) .


Figure 3: Inhibition of the TPOR activity of PDI by peptide A-K. The reaction mixture contained 2.8 µM insulin, 8 mM GSH, 100 µM NADPH, and 1 unit of glutathione reductase. Native staphylococcal nuclease () or peptide A-K (▾) were also present. The absorption decrease at 340 nm of the coupled reaction was followed.




DISCUSSION

The chaperone activity of PDI in assisting refolding of other proteins has been discussed recently(14, 23, 24) . However, the target proteins employed are invariably disulfide-containing proteins, such as lysozyme (14, 23) and immunoglobulin(24) , and thus it is difficult to distinguish the chaperone from the isomerase activity unambiguously. With a protein containing no disulfide bonds (like GAPDH) as the target protein, it is possible to analyze the chaperone and isomerase activities of PDI independently. The suppression of its chaperone-like activity by the peptide A-K, but not by native staphylococcal nuclease, suggests that PDI binds with unfolded or partially folded peptides by recognizing nonnative conformation and thus prevents these peptides from nonproductive aggregation so as to facilitate their correct folding. This is consistent with the mechanism proposed for chaperones function through interaction with partially folded intermediates in the folding process to prevent their aggregation and promote correct folding. As we suggested previously(7) , in its action as a foldase (isomerase), the first step is most likely the binding of the peptide, which provides an opportunity for the peptide to fold correctly without aggregation so as to facilitate correct pairing of thiols and thus assists the second step of oxidative formation of native disulfide bonds. Modification of the thiols at the -CGHC- active sites, which are essential for the catalysis of disulfide formation, results in the inactivation of the isomerase, but has no effect on its chaperone function in the refolding of GAPDH. In cases where target proteins containing disulfide bonds, such as lysozyme (14) and immunoglobulin(24) , were used for the study of the chaperone function of PDI and the final reactivation yield of target proteins was taken as a measure of the chaperone activity by PDI, the yield would depend on both the first chaperone assisted step of folding and the second catalytic thiol-disulfide exchange step for the formation of the correct disulfide bonds. Thus, both the peptide binding site and the Tdx-like active site are involved in the correct folding to form the native structure and the formation of the native disulfides. Binding of denatured and reduced lysozyme to PDI is necessary but not sufficient for the formation of correct disulfide bonds and, thus, the native conformation of this enzyme, which requires in addition a thiol-disulfide exchange step for the formation of the native disulfides. Mutation at the -CGHC active site would result in the loss of isomerase activity and failure of reactivation of lysozyme or antibody but does not necessarily mean any damage to the chaperone function of PDI. The thioredoxin-like active sites cannot therefore be considered essential for its chaperone activity. As mentioned above, with GAPDH as the target protein for the examination of the chaperone activity of PDI, the refolding and reactivation has nothing to do with disulfide formation, and depends on the chaperone activity of PDI alone. Peptide binding is essential for the chaperone activity of PDI and blockage of the peptide binding sites results in the loss of the chaperone function of PDI, as shown by decreased productive reactivation and increased aggregation of GAPDH in presence of the peptide A-K.


FOOTNOTES

*
This work was supported in part by the Pandeng Project of the Chinese Commission of Science and Technology. 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. Tel.: 86-10-2022027; Fax: 86-10-2022026.

The abbreviations used are: PDI, protein disulfide isomerase; DTT, dithiothreitol; DTNB, 5,5`-dithiobis(2-nitrobenzoic acid); GAPDH, D-glyceraldehyde-3-phosphate dehydrogenase; GdnHCl, guanidine hydrochloride; Tdx, thioredoxin; TPOR, thiol-protein oxidoreductase.


ACKNOWLEDGEMENTS

We sincerely thank Prof. C. L. Tsou for continuous encouragement and helpful advice and Prof. M. Y. Jiang for a gift of staphylococcal nuclease.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.