(Received for publication, March 29, 1995; and in revised form, May 22, 1995)
From the
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.
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),(
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.
The concentrations of PDI, GAPDH, Tdx, and insulin were
determined spectrophotometrically at 280 nm (A
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 (
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.
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 (
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.
We sincerely thank Prof. C. L. Tsou for continuous
encouragement and helpful advice and Prof. M. Y. Jiang for a gift of
staphylococcal nuclease.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)
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.
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.
= 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) .
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).
), 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.
, 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) .
) or
peptide A-K (▾) were also present. The absorption decrease
at 340 nm of the coupled reaction was
followed.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.