From the Core Research for Evolutional Science and
Technology, Japan Science and Technology Corporation and
§ Department of Molecular Behavioral Biology, Osaka
Bioscience Institute, 6-2-4 Furuedai, Suita, Osaka 565-0874, Japan and
the
Faculty of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan
Received for publication, September 27, 2002, and in revised form, November 18, 2002
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ABSTRACT |
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We found that low concentrations of
guanidine hydrochloride (GdnHCl, <0.75 M) or urea
(<1.5 M) enhanced the enzyme activity of lipocalin-type
prostaglandin (PG) D synthase (L-PGDS) maximally 2.5- and 1.6-fold at
0.5 M GdnHCl and 1 M urea, respectively. The
catalytic constants in the absence of denaturant and in the presence of
0.5 M GdnHCl or 1 M urea were 22, 57, and 30 min Lipocalin-type prostaglandin
(PG)1 D synthase (L-PGDS,
prostaglandin-H2 D-isomerase, EC 5.3.99.2) is
abundantly expressed in the central nervous system and male genitals of
various mammals and in the human heart (1, 2). In these tissues, L-PGDS catalyzes the isomerization of PGH2, a common precursor of
various prostanoids, to PGD2 in the presence of sulfhydryl
compounds (3). PGD2 acts as a neuromodulator in the central
nervous system, where it induces sleep and regulates body temperature,
luteinizing hormone release, and pain responses (1, 2). In the
peripheral tissues, PGD2 functions as a mediator of allergy
and inflammatory responses (2, 4). Sequence analyses and a homology
search in data bases of protein primary structure have revealed that
L-PGDS is a member of the lipocalin superfamily (1, 5), which is
composed of various secretory lipid-transporter proteins, such as
Despite their low mutual sequence homology, members of the lipocalin
family share a conserved barrel of 8 antiparallel We recently found that the L-PGDS activity was enhanced in the presence
of low concentrations of guanidine hydrochloride (GdnHCl) (17). In most
cases, an enzyme is inactivated by denaturants at concentrations lower
than those required for the complete unfolding (18). However, a few
enzymes have been reported to be activated instead. For example,
adenylate kinase was reported to be activated in the presence of urea
up to 1 M or up to 0.25 M GdnHCl (19). Dihydrofolate reductase also showed enhanced enzyme activity in the
presence of urea up to 7 M (20, 21). By measuring the change in the fluorescence of 8-anilino-1-naphthalenesulfonic acid
binding to adenylate kinase during denaturation and the activity change
in dihydrofolate reductase during trypsin digestion in the presence of
GdnHCl or urea, both groups of investigators suggested that the enzyme
activation was caused by increased flexibility of the conformation at
the enzyme active site. On the other hand, the mechanism of L-PGDS
activation by denaturants has remained unclear.
In this study, we investigated the unfolding process of L-PGDS in the
presence of GdnHCl and urea by CD and NMR analyses combined with
measurements of its synthase and ligand-binding activities. We found
that two states were formed during the unfolding process, an
activity-enhanced state at a low concentration of denaturants and an
inactive intermediate at a higher concentration of denaturants. The
enzyme activity was in good correlation with the amount of secondary
structure, especially the amount of Materials--
GdnHCl, urea, SDS, and dithiothreitol were
purchased from WAKO (Tokyo, Japan). Thrombin, bilirubin, and
13-cis-retinal were obtained from Sigma. All other chemicals
were of analytical grade.
Expression of Recombinant Mouse L-PGDS--
The full-length
cDNA for mouse L-PGDS, which is composed of 189 amino acid residues
(GenBankTM accession number X89222 (22)), was ligated into
the BamHI-EcoRI sites of the expression vector
pGEX-2T plasmid (Amersham Biosciences). The N-terminal 22-amino
acid residues of the signal peptide were deleted, and the
C89A/C186A-, W43F-, and W54F-substituted recombinant enzymes
were expressed in Escherichia coli DH5 Purification of Human L-PGDS in Cerebrospinal Fluid (CSF) after
Aneurysmal Subarachnoid Hemorrhage (SAH)--
Patients with aneurysmal
SAH, who were consecutively admitted to the Department of Neurosurgery,
Nagoya City University Hospital, were treated with intravascular
embolization of the aneurysmal dome by using detachable coils or
surgically clipping of the dome within 24 h after the initial SAH
attack. After the treatment of the aneurysms, a silicon drainage tube
was placed in the lumbar CSF space for continuous draining of the
subarachnoid clots into the CSF. Approximately 2 months after SAH, the
normal CSF of patients (which does not contain blood) who developed
communicating hydrocephalus was collected over 3 days. Informed consent
was obtained from all patients to perform the lumbar puncture, which
was part of the diagnostic workup. Human L-PGDS was purified from the
CSF by immunoaffinity chromatography with the monoclonal antibody 1B7-conjugated column, as reported previously (23). Human L-PGDS was
further purified to apparent homogeneity by Superdex 75 column chromatography (Amersham Biosciences).
Enzyme Assays--
The L-PGDS activity was measured by
incubating the enzyme at 25 °C for 1 min with
[1-14C]PGH2 (final concentration of 40 µM) in 50 µl of 0.1 M Tris/HCl (pH 8.0)
containing 1 mg/ml IgG and 1 mM dithiothreitol (16). [1-14C]PGH2 was prepared from
[1-14C]arachidonic acid (2.20 GBq/mmol, PerkinElmer Life
Sciences) as described previously (3).
CD Measurements--
The CD spectrum of L-PGDS was
measured with a spectropolarimeter model J-700 (Jasco, Tokyo, Japan).
The temperature of the solution in the cuvette was controlled at
4.0 ± 0.5 or 25.0 ± 0.5 °C by circulating water. The CD
spectra were essentially the same between these two temperatures. The
path length of the optical quartz cuvette was 1.0 mm for far-UV range
CD measurements at 200 to 250 nm, and 10 mm for near-UV to visible
range CD measurements at 250 to 600 nm. The protein solutions contained
enzyme at 200 µg/ml, 5 mM Tris/HCl (pH 8.0), and the
appropriate amount of denaturant, bilirubin, or
13-cis-retinal. The data were expressed as molar residue
ellipticity ( Fluorescence Quenching Assays--
Various concentrations of
bilirubin or 13-cis-retinal (10 µl) were added to L-PGDS
in 990 µl of 5 mM Tris/HCl (pH 8.0) to give a final
concentration of 1.5 µM in the presence or absence of
denaturant. Bilirubin was dissolved in Me2SO to give a 2 mM stock solution, and 13-cis-retinal in ethanol
to give a stock solution of 1 mM. The concentrations of
hydrophobic ligands were determined spectroscopically based on their
respective molar absorption coefficients, i.e.
NMR Spectroscopy--
Uniformly 15N-labeled protein
was obtained by growing the transformed E. coli in M9
minimal medium containing 15NH4Cl as the sole
nitrogen source. Samples for NMR spectroscopy were dissolved in 90%
H2O, 10% D2O-containing 50 mM sodium phosphate, pH 8.0, and the protein concentrations
were set at 2 mg/ml in 250 µl in a 5-mm microcell NMR tube (Shigemi
Inc., Tokyo, Japan). 15N-1H heteronuclear
multiple quantum coherence spectra (27) were measured with a Varian
Unity Inova 500 spectrometer (Varian Instruments, Palo Alto, CA)
equipped with a triple resonance probe. Water suppression was achieved
by solvent presaturation. Under the condition of pH 8.0, the time
constant for intrinsic amide hydrogen exchange was about 10 ms, and so
presaturation of the water signal obliterated the signals of rapidly
exchanging protons in unstructured parts of the protein (28).
One-dimensional 1H NMR spectra of L-PGDS were obtained in
the absence or presence of 0.5, 2, or 6 M GdnHCl.
Considering the protein solubility during the GdnHCl titration, we set
the protein concentrations at 250 µg/ml. The temperature for NMR
experiments was 30 °C. Phase-shifted sine-bell window functions,
solvent-suppression filter on the time-domain data, and zero filling
were applied before Fourier transformation. 1H chemical
shifts were referenced to 2,2-dimethyl-2-silapentane-5-sulfonate and
were measured with respect to acetone as an internal standard. The
15N chemical shifts were measured relative to an external
standard, NH4Cl.
Statistical Analysis--
Data were expressed as the mean ± S.E. The statistical significance between the control and the
experimental group was assessed by Student's t test.
p < 0.05 was considered to be significant.
Changes in Enzyme Activity and Secondary Structure of L-PGDS
Induced by Denaturants--
We investigated first the effects of
denaturants, such as GdnHCl, urea, and SDS, on the enzyme activity of
the C89A/C186A-substituted recombinant mouse L-PGDS. In the
presence of 0.10-0.75 M GdnHCl or 0.25-1.50 M
urea, the PGDS activity was significantly enhanced (Fig.
1A). At 0.5 M
GdnHCl and 1 M urea, the enzyme activity reached its
maximum of 2.5 ± 0.1 and 1.6 ± 0.1 µmol
min
The PGDS activity of human L-PGDS purified from CSF after SAH also was
significantly enhanced in the presence of 0.1-1 M GdnHCl or 0.25-2 M urea. At 0.5 M GdnHCl and 1 M urea, the enzyme activity reached its maximum, which was
3- and 1.7-fold, respectively, higher than the activity in the absence
of denaturants.2 The enzyme
activity was reduced above 0.5 M GdnHCl or 1 M
urea in a concentration-dependent manner and became less
than 5% of the control at 3 M GdnHCl or 4 M
urea. These results showed that the activation of L-PGDS occurred in
the presence of low concentrations of denaturants not only on the
recombinant protein but on the native one.
Next we determined the kinetic properties of the recombinant mouse
L-PGDS in the absence or presence of denaturants (Table I). In the absence of denaturants, the
apparent Km for PGH2 was 2.8 µM, and the catalytic constant
(kcat) was calculated to be 22 min
The conformational changes in the recombinant mouse L-PGDS in the
presence of denaturants were investigated by measuring the CD spectra.
Fig. 1B shows the far-UV equilibrium CD spectra of L-PGDS in
the absence or presence of 0.25, 1, 2.5, 4, and 6 M urea.
The far-UV CD in the native state of L-PGDS showed a spectrum with an
abundance of
Fig. 1C shows the equilibrium transition curves of L-PGDS
obtained followed by treatment with GdnHCl and urea monitored at the
wavelength of 218 nm, which reflects the content of Two Equilibrium States of L-PGDS in the Unfolding Process--
To
investigate the correlation between the enzyme activity and the content
of
Table II summarizes the contents of the
secondary structure of L-PGDS of the native state in the absence of
denaturants and those of the activity-enhanced state in the presence of
low concentrations of denaturants calculated from the CD spectra by the
method of Chen et al. (29). The native L-PGDS was composed
of 17% Structural Changes in the Activity-enhanced and Inactive States of
L-PGDS by Denaturants--
We then recorded the
15N-1H heteronuclear multiple quantum coherence
spectra of the recombinant mouse L-PGDS in the absence or presence of
0.5 and 2 M GdnHCl or 1 and 4 M urea to detect
the signals of amide protons slowly exchanging with the hydrogen
bond (Figs. 3,
A-E). In the 15N-1H
heteronuclear multiple quantum coherence spectrum, the positions of
several cross-peaks were changed in the activity-enhanced state of
L-PGDS formed in the presence of 0.5 M GdnHCl or 1 M urea (Fig. 3, B and C), whereas
their intensities were close to those observed in the spectrum without
denaturants. In addition, new cross-peaks were observed in the spectrum
of the activity-enhanced state (arrowheads in Fig. 3,
B and C), indicating that the hydrogen-bond
network in the L-PGDS molecule was reorganized by these denaturants.
The number of observed cross-peaks was drastically decreased but still remained in the inactive state in the presence of 2 M
GdnHCl (Fig. 3D) or 4 M urea (Fig.
3E). This phenomenon is interpreted as the loss of hydrogen
bonds, and is typical of the denaturation process.
We also determined one-dimensional proton NMR spectra to obtain useful
gross information on the conformational status of the two equilibrium
states of L-PGDS in the absence and presence of 0.5 to 6 M
GdnHCl (Fig. 4). One-dimensional
1H NMR spectra of L-PGDS in the absence or presence of 0.5 M GdnHCl were well dispersed, which reflect the compact
folded states of the protein. These features were notably evident in
the amide and aromatic (7.5 to 10.0 ppm, Fig. 4A) and the
aliphatic (0.5 to 3.0 ppm, Fig. 4B) regions of the
one-dimensional 1H NMR spectrum of the protein.
One-dimensional 1H NMR spectra of L-PGDS in the presence of
2 M GdnHCl showed limited chemical shift dispersion in the
1H dimension, which reflects the overall similarity to an
unfolded state in the presence of 6 M GdnHCl (Fig. 4,
A and B).
Binding Affinities of L-PGDS for Lipophilic Ligands in the Presence
of Denaturants--
To investigate the binding affinity of the
activity-enhanced state of L-PGDS for small lipophilic ligands, we
measured the fluorescence quenching of the intrinsic tryptophan residue
of L-PGDS in the presence or absence of 1 M urea or 0.5 M GdnHCl after incubating the enzyme with various
concentrations of bilirubin or 13-cis-retinal. The
recombinant mouse L-PGDS in the absence of these denaturants showed
fluorescence quenching in a concentration-dependent manner
after addition of bilirubin or 13-cis-retinal (Fig.
5A). The fluorescence
intensity in the presence of an excess amount of bilirubin (above 2 µM) or 13-cis-retinal (above 6 µM) decreased below 10 and 30% of the fluorescence
intensity in the absence of lipophilic ligands, respectively. In the
presence of 1 M urea, bilirubin and
13-cis-retinal showed fluorescence quenching curves almost
identical to those in the absence of urea (Fig. 5B).
However, in the presence of 0.5 M GdnHCl (Fig.
5C), L-PGDS required higher concentrations of bilirubin to
quench the fluorescence by an amount comparable with that in the
absence of GdnHCl, whereas the fluorescence quenching curve with
13-cis-retinal was almost identical to that in the absence
of denaturants.
The calculated Kd values obtained by the method of
Levine (26) are also summarized in Table I. In the presence of 1 M urea, the binding affinity of L-PGDS for bilirubin
(Kd = 120 nM) was almost identical to
that in the absence of denaturant (Kd = 110 nM). However, the affinity in the presence of 0.5 M GdnHCl (Kd = 450 nM) was
significantly lower than that in the absence of denaturants. In the
case of 13-cis-retinal, the Kd values
were estimated to be 1.0 µM in the absence of
denaturants, 1.1 µM in the presence of 1 M
urea, and 1.2 µM in the presence of 0.5 M
GdnHCl. The fluorescence quenching was not observed in the inactive
state of L-PGDS after denaturation in the presence of 4 M
urea or 2 M GdnHCl (data not shown). The binding affinity
for bilirubin was lower in the presence of 0.5 M GdnHCl
than in the absence of denaturants, but unchanged in the presence of 1 M urea, suggesting that 0.5 M GdnHCl appears to
have affected more effectively the conformation of the
bilirubin-binding site of L-PGDS than 1 M urea.
Mouse L-PGDS contains two tryptophan residues at positions 43 and 54. To investigate which tryptophan residue contributes to the fluorescence
quenching by the small lipophilic ligands, we generated two new mutants
of L-PGDS, in which each of these two tryptophan residues was
substituted by phenylalanine. Although both mutants of L-PGDS showed
fluorescence quenching in a concentration-dependent manner
after addition of bilirubin (Fig.
6A) and
13-cis-retinal (Fig. 6B), the W54F mutant showed
the more intense tryptophan fluorescence than that of the W43F mutant.
The calculated Kd values of W54F mutant and W43F
mutant for bilirubin were 109 and 150 nM, respectively, and
those for 13-cis-retinal were 0.9 and 1.1 µM,
respectively. These results indicated that the Trp43
residue is located in the more hydrophobic region within a molecule of
L-PGDS than the Trp54 residue.
Changes in the Binding Mode of L-PGDS for Lipophilic Ligands
from Multistate Mode in the Native Form to Simple Two-state Mode in the
Activity-enhanced Form--
We then compared the binding mode of
ligands to the recombinant mouse L-PGDS between the native and the
activity-enhanced forms by measuring CD spectra in near-UV and visible
ranges. In the absence of denaturants, within the range of the molar
ratio of bilirubin/L-PGDS from 0.2 to 0.6, bilirubin bound to L-PGDS showed both negative and positive CD Cotton effects at peaks of 394 and
509 nm, respectively, giving an isosbestic point at 450 nm (open
arrow in Fig. 7A). At the
molar ratio of 0.8, new negative and positive peaks appeared at
wavelengths of 282 and 461 nm, respectively. Above this molar ratio,
the intensity of the positive peak at 509 nm was decreased and that of
the other positive peak at 461 nm was increased; and the negative peak
at 394 nm was decreased and blue-shifted to 389 nm. The CD spectral
changes of bilirubin bound to L-PGDS did not show any isosbestic point
above the molar ratio of 0.8. These results suggest that the bilirubin
binding mode for the native L-PGDS in the absence of denaturants was
not a simple two-state but a multistate one.
On the other hand, in a case of the activity-enhanced form produced in
the presence of 1 M urea, the intensities of both negative (284 and 411 nm) and positive (506 nm) CD Cotton effects increased in a
molar ratio-dependent manner with an isosbestic point at 460 nm (open arrow in Fig. 7B). At the molar
ratio of 1.6, the intensity of the negative Cotton effect at 411 nm was
~1.7-fold higher than that in the absence of urea (Fig. 7,
A and B). In the presence of 0.5 M
GdnHCl, in which the activity-enhanced form also exists, the negative
Cotton effects with peaks at 282 and 401 nm and a positive effect with
a peak at 515 nm were also found to be increased with a tight
isosbestic point at 474 nm (open arrow in Fig.
7C) similar to that in the presence of 1 M urea. However, the intensity of the negative Cotton effect at 401 nm was
almost identical to that in the absence of GdnHCl. These results suggest that the bilirubin binding mode of the activity-enhanced form
of L-PGDS conformed to a simple two-state model.
When 13-cis-retinal was used as a ligand bound to L-PGDS,
negative CD Cotton effects were observed at wavelengths of 260 and 355 nm. In the absence of denaturants, the Cotton effect was increased in a
molar ratio-dependent manner up to the molar ratio of 2 without any isosbestic point (Fig. 7D). In the presence of 1 M urea, the negative Cotton effects at 260 and 370 nm also
increased in a molar ratio-dependent manner without any
isosbestic point (Fig. 7E). At the molar ratio of 2.0, the
intensity of the negative Cotton effect at 370 nm was ~1.3-fold
higher than that in the absence of urea. In the presence of 0.5 M GdnHCl, the negative Cotton effects at 260 and 375 nm
increased in a molar ratio-dependent manner with a tight
isosbestic point at 290 nm (open arrow in Fig.
7F). These results indicate that in the presence of low
concentrations of GdnHCl the binding mode between
13-cis-retinal and the activity-enhanced form of L-PGDS was
a simple two-state one.
Two Equilibrium States of L-PGDS in the Reversible Folding
Process--
We showed that two states were formed during the process
of unfolding of L-PGDS induced by GdnHCl or urea. Based on these findings, we propose the triangle and pole model for L-PGDS (Fig. 8). In this scheme, both A and
I are equilibrium states from the native (N)
state to the unfolded (U) state, which are associated with
the changes in the enzyme activity representing both activation and
inactivation, respectively. The A state is the activity-enhanced form
with a rigid native-like tertiary structure enriched in
To the best of our knowledge, L-PGDS is the first example of
multiphasic equilibrium states with the A and I states in the unfolding
process of a Correlation between Enzyme Activity and Conformational Changes in
the Presence of Denaturants--
We showed that the L-PGDS activity
was increased 2.5- and 1.6-fold in the presence of 0.5 M
GdnHCl or 1 M urea, respectively (Fig. 1), mainly because
of an increase in kcat of the A state (Table I).
Moreover, an increase in the binding affinity of the A state for the
substrate, as shown by the decreased Km value, may
also contribute to the enhancement of L-PGDS activity by urea.
Activation of an enzyme by denaturants has been reported in the case of
only a few enzymes, such as adenylate kinase and dihydrofolate
reductase. The adenylate kinase activity was enhanced 1.6-fold in the
presence of 1 M urea or 0.25 M GdnHCl as
compared with that in the absence of denaturants (19). Dihydrofolate reductase was also activated about 2-fold in the presence of 0.5 M GdnHCl and about 5-fold in the presence of 4 M urea (20, 21), the latter of which was associated with an
increase in the kcat value (20). However, the
overall structures of adenylate kinase and dihydrofolate reductase were
not significantly altered within the indicated concentration ranges of
denaturants as monitored by CD and UV spectra (19-21). The change in
the enzyme activity of adenylate kinase in the presence of the low
concentration of denaturants coincided with that in the binding rate of
8-anilino-1-naphthalenesulfonic acid (19). The activation of
dihydrofolate reductase was accompanied by an increase in the rate of
digestion by trypsin (20, 21). Therefore, the activation of these
enzymes was proposed to be caused by an increase in the conformational
flexibility locally at the active site. On the other hand, L-PGDS
clearly changed its secondary and tertiary structures in the presence
of low concentrations of denaturants to be highly correlated with the
Correlation between Ligand-binding Activity and Conformational
Changes in the Presence of Denaturants--
The fluorescence quenching
study on the intrinsic tryptophan residue of L-PGDS (Fig. 5 and Table
I) revealed that the Kd value of L-PGDS for
bilirubin was almost unchanged from the N state (110 nM) to
the A state in the presence of 1 M urea (120 nM) but remarkably increased in the A state in the presence
of 0.5 M GdnHCl (450 nM). On the other hand,
the Kd value for 13-cis-retinal was
almost identical either in the absence or presence of denaturants (Fig.
5 and Table I), suggesting that the N and A states of L-PGDS possess
distinct binding modes against these 2 ligands within the hydrophobic
pocket. The CD spectra of bilirubin and 13-cis-retinal bound
to the A form of L-PGDS showed well defined isosbestic points (Fig. 6),
demonstrating that the binding mode of ligands to L-PGDS changed from a
multistate binding mode in the N state to a simple two-state mode in
the A state. The simple two-state binding mode at the A state may also
reflect the effective interaction between PGH2 and L-PGDS, whose activity was enhanced by denaturants. The Km
value of L-PGDS for PGH2 was also almost unchanged from the
N state (2.8 mM) to the A state in the presence of 1 M urea (2.3 mM) but clearly increased in the A
state in the presence of 0.5 M GdnHCl (8.3 mM,
Table I). The structural change near the active thiol of
Cys65 at the entrance of the hydrophobic pocket in the A
state of L-PGDS is considered to be induced by 0.5 M GdnHCl
more significantly than by 1 M urea, as was shown by the
slight difference in the curves of the activity-enhanced L-PGDS
obtained with the 2 kinds of denaturants (Figs. 1 and 2).
Most recently, the x-ray crystallographic analysis of L-PGDS revealed
that the Trp43 residue is located at the bottom of the
hydrophobic pocket, whereas the Trp54 residue locates on
the loop position and faces the outside of the hydrophobic
pocket.4 Therefore, it is
likely that the Trp43 residue is considered to be
responsible for the major portion of fluorescence quenching by the
binding of ligands. In this study, we showed that both tryptophan
residues contributed to the fluorescence quenching of L-PGDS by the
ligand binding, although the Trp43 residue showed the more
intense fluorescence than that of the Trp54 residue. The
Trp54 residue might move to the lipophilic ligand entered
at the hydrophobic pocket of L-PGDS and weakly contribute to the
fluorescence quenching.
L-PGDS binds various hydrophobic ligands, such as retinoids, bilirubin,
biliverdin, and thyroid hormones, with high affinities (Kd = 30 ~ 200 nM, Refs. 9, 10,
and 17). This broad selectivity may reflect a general transport role as
a lipocalin for the clearance of unwanted endogenous or exogenous
lipophilic molecules. 1, respectively, and the Km values
for the substrate, PGH2, were 2.8, 8.3, and 2.3 µM, respectively, suggesting that the increase in the
catalytic constant was mainly responsible for the activation of L-PGDS.
The intensity of the circular dichroism (CD) spectrum at 218 nm,
reflecting the
-sheet content, was also increased by either
denaturant in a concentration-dependent manner, with the
maximum at 0.5 M GdnHCl or 1 M urea. By
plotting the enzyme activities against the ellipticities at 218 nm of
the CD spectra of L-PGDS in the presence or absence of GdnHCl or urea, we found two states in the reversible folding process of L-PGDS: one is
an activity-enhanced state and the other, an inactive state. The NMR
analysis of L-PGDS revealed that the hydrogen-bond network was
reorganized to be increased in the activity-enhanced state formed in
the presence of 0.5 M GdnHCl or 1 M urea and to
be decreased but still remain in the inactive intermediate observed in
the presence of 2 M GdnHCl or 4 M urea.
Furthermore, binding of the nonsubstrate ligands, bilirubin or
13-cis-retinal, to L-PGDS changed from a multistate mode in
the native form of L-PGDS to a simple two-state mode in the
activity-enhanced form, as monitored by CD spectra of the bound
ligands. Therefore, L-PGDS is a unique protein whose enzyme activity
and ligand-binding property are biphasically altered during the
unfolding process by denaturants.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
-lactoglobulin, retinol-binding protein, major urinary protein, and
bilin-binding protein (6). Lipocalin is a family of diverse proteins
that normally serve for the storage or transport of physiologically important lipophilic ligands (6-8). L-PGDS also has the ability to
bind retinoids, bile pigments such as bilirubin and biliverdin, and
thyroid hormones (9, 10). Therefore, L-PGDS is a unique dual functional
protein in the lipocalin family, acting as a
PGD2-synthesizing enzyme and also as an extracellular
transporter protein for lipophilic ligands.
-strands as their
central folding motif; and the large cup-shaped hydrophobic cavity
within the
-barrel and a loop scaffold at its entrance are well
adapted to the task of ligand binding (11-15). The preliminary crystal
structure analysis revealed that the tertiary structure of mouse
recombinant L-PGDS also showed an 8-stranded
-barrel structure, and
one free SH group because of Cys65 faced the inside of the
hydrophobic cavity of the barrel structure (1). The enzyme activity of
L-PGDS disappeared completely by chemical modification or replacement
of the Cys65 residue with serine or alanine by
site-directed mutagenesis (16). Therefore, Cys65 is
considered to play an important role as an active center for the
catalytic function of L-PGDS.
-sheet structure. The ligand
binding mode was also changed from a multistate mode for the native
form of L-PGDS in the absence of denaturants to a two-state mode for
the activity-enhanced form. The inactive intermediate still retained a
secondary structure to maintain the folded core despite nearly complete
loss of activity. Based on our findings, we propose a four-state
equilibrium folding model for L-PGDS composed of the activity-enhanced
and inactive states.
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
(TOYOBO, Tokyo, Japan). Site-directed mutagenesis was performed using the QuikChange site-directed mutagenesis kit (Stratagene, Heidelberg, Germany). The DNA sequence were confirmed with a LI-COR model 4000L automated DNA
sequencer (LI-COR Inc., Lincoln, NE) after cycle sequencing with a
SequiTherm cycle sequencing kit (Epicentre Technologies, Madison, WI).
The recombinant enzymes retained enzymatic activity comparable with
that of the wild-type L-PGDS and is stable for long-term use. The
mutated L-PGDS was expressed as a glutathione transferase fusion
protein. The fusion protein was bound to glutathione-Sepharose 4B
(Amersham Biosciences) and incubated with thrombin (100 units/100 µl)
to release the L-PGDS. The recombinant protein was further purified to
apparent homogeneity by Superdex 75 and Mono-Q column chromatographies.
The purified protein was dialyzed against 5 mM Tris/HCl (pH
8.0).
).
453 in chloroform for bilirubin = 61,700 M
1 cm
1 (24) and
383 for 13-cis-retinal = 42,800 M
1 cm
1 (25). After incubation
at 25 °C for 60 min, the intrinsic tryptophan fluorescence was
measured by an FP-750 spectrofluorometer (Jasco) with an excitation
wavelength at 282 nm and an emission wavelength at 338 nm. The
quenching of tryptophan fluorescence caused by nonspecific interactions
with each ligand was corrected with 1.5 µM
N-acetyl-L-tryptophanamide. The dissociation constant
(Kd) value for binding between ligand and L-PGDS was
calculated by the method as described previously (26).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 mg
1, respectively, which was 2.5- and
1.6-fold, respectively, higher than the activity in the absence of
denaturants (1.0 ± 0.1 µmol min
1
mg
1). The enzyme activity was reduced above 0.5 M GdnHCl or 1 M urea in a
concentration-dependent manner and became less than 5% of the control at 2 M GdnHCl or 4 M urea.
Furthermore, the enzyme activity was completely recovered by the
depletion of denaturants (data not shown), showing the reversibility of
the denaturant effect on folding. On the other hand, the enzyme
activity of L-PGDS was reduced by SDS in a
concentration-dependent manner without any enhancement of
the enzyme activity and reached less than 5% at 0.3% SDS (data not
shown). These results show that the L-PGDS activity was enhanced by low
concentrations of urea or GdnHCl, but not by SDS.
View larger version (16K):
[in a new window]
Fig. 1.
Changes in enzyme activity and secondary
structure of L-PGDS induced by denaturants.
A, the curves show the changes in the enzyme
activity of L-PGDS in the presence or absence of various concentrations
of urea ( ) or GdnHCl (
). The reaction was carried out at 25 °C
for 1 min in the presence of 40 µM PGH2 and 1 mM dithiothreitol, pH 8.0. Data are expressed as the
mean ± S.E. of three independent experiments. Significant
difference was based on Student's t test, **,
p < 0.01; *, p < 0.05 versus in the absence of denaturants. B, CD
spectra of L-PGDS in the far-UV region. The CD spectra of L-PGDS (200 µg/ml) were obtained at 4 °C, pH 8.0, in the absence (
) or
presence of 0.25 (
), 1 (
), 2.5 (
), 4 (
), or 6 (
)
M urea. An arrow shows the isosbestic point.
C, transition curves of L-PGDS unfolding induced by
denaturants. The curves show the changes in the CD
ellipticities (
) at the wavelength of 218 nm, reflecting the content
of
-sheet structure of L-PGDS, in the presence or absence of various
concentrations of urea (
) or GdnHCl (
).
1. In the presence of 0.5 M GdnHCl, the
Km was 8.3 µM and
kcat was 57 min
1 and in the
presence of 1 M urea, the Km dropped to
2.3 µM and kcat rose to 30 min
1. These results suggest that the enhancement of
L-PGDS activity in the presence of the low concentrations of
denaturants was mainly because of the increase in the catalytic
constant.
Kinetic parameters and binding affinities of L-PGDS for lipophilic
small ligands in the absence or presence of denaturants
-sheets (closed circle). By the treatment with 0.25 (open circle) or 1 (closed square)
M urea, the absolute CD intensity was increased over a
range of wavelengths from 206 to 230 nm, showing the augmentation of
the secondary structure of L-PGDS. The secondary structure gradually
disappeared by an increase in the urea concentration (2.5 M, open square; 4 M, closed triangle) and was completely lost at 6 M urea
(open triangle). A tight isosbestic point was observed at
230 nm on the CD spectra (arrow in Fig. 1B) over
the concentration range of urea up to 4 M, but not above 4 M, thus showing 2 phases of the structural changes in
L-PGDS. The CD spectra in the unfolding state of L-PGDS induced by
GdnHCl (17) also showed an isosbestic point over the range up to 2 M, but not above 2 M, giving almost the same propensity of the 2 phases of the unfolding process as those induced by
urea. Furthermore, the structural reversibility was confirmed in the CD
spectra by the depletion of GdnHCl or urea (data not shown).
-sheet structure. The absolute intensity of CD at 218 nm was increased by
0.2-0.8 M GdnHCl and 0.25-1.75 M urea. The
absolute intensity reached a maximum at 0.5 M GdnHCl or 1 M urea, and decreased above 0.5 M GdnHCl or 1 M urea in a concentration-dependent manner. Above 1 M GdnHCl or 2 M urea, the transition of
L-PGDS was shown to occur in a biphasic manner, having a shoulder
around 2 M GdnHCl or 3.5 M urea with respect to
the formation of non-native state, as described later.
-sheet structure, we plotted the PGDS activities against the
ellipticities of the CD spectra of L-PGDS at 218 nm in the presence or
absence of GdnHCl or urea (Fig. 2).
Nonlinear but ellipticity-dependent activation was observed
in the presence of GdnHCl up to 0.5 M or urea up to 1 M (solid lines). After showing the maximum
activity at the ellipticities of
9.2 and
8.7 × 103 deg cm2 dmol
1 in the presence
of 0.5 M GdnHCl and 1 M urea, respectively, the PGDS activity decreased linearly in an
ellipticity-dependent manner with a horizontal intercept at
approximately
5 × 103 deg cm2
dmol
1 ellipticity in the presence of 2 M
GdnHCl and 4 M urea (dotted lines). Although the
slopes of the linear phase were different between GdnHCl and urea, the
enzyme activity was highly dependent on the ellipticity at 218 nm. When
the ellipticity was higher than
5.0 × 103 deg
cm2 dmol
1, however, the enzyme activity was
below the detection limit. These results, taken together, show that the
unfolding of L-PGDS in the presence of denaturants proceeded via two
states, i.e. an activity-enhanced one and an inactive
one.
View larger version (17K):
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Fig. 2.
Correlation between the PGDS activity and the
secondary structure. The enzyme activities were plotted against
the CD ellipticities ( ) at 218 nm in the presence and absence of
various concentrations of urea (
) or GdnHCl (
). The correlation
factors (r2) of dotted lines with
GdnHCl and urea were 0.984 and 0.974, respectively.
-helix, 45%
-sheet, and 38% coil. The secondary
structure contents of L-PGDS are similar to those of
-lactoglobulin,
a member of the lipocalin family, estimated from both CD spectra and
x-ray crystallography (30). In the presence of 0.5 M GdnHCl
or 1 M urea, the
-sheet content increased from 45 to 51 or 50%, respectively, whereas the coil contents decreased from 38 to
32 or 32%, respectively, without any changes in the
-helical
structures. The secondary structure contents of the inactive
intermediate in the presence of 2 M GdnHCl or 4 M urea could not be calculated, because of the highly
deviated CD spectra.
Contents of secondary structure of L-PGDS calculated from the CD
spectra
View larger version (22K):
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Fig. 3.
15N-1H heteronuclear
multiple quantum coherence spectra of L-PGDS in the presence or absence
of denaturants. The spectra were obtained in the absence
(A) or presence of 0.5 M (B) or 2 M (C) GdnHCl, and 1 M (D)
or 4 M (E) urea. Arrowheads show the
new peaks that appeared in the presence of denaturants.
View larger version (26K):
[in a new window]
Fig. 4.
Structural changes in the equilibrium states
of L-PGDS monitored by NMR spectra. Amide and aromatic regions
(A) and aliphatic regions (B) of one-dimensional
1H NMR spectra of L-PGDS in the presence or absence of
GdnHCl. The spectra were obtained in the absence or presence of 0.5, 2, or 6 M GdnHCl. The concentration of the protein used was
250 µg/ml.
View larger version (10K):
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Fig. 5.
Tryptophan fluorescence quenching by
lipophilic small ligands. The relative fluorescence intensities of
L-PGDS in the presence of various concentrations of bilirubin ( ) or
13-cis-retinal (
) were obtained in the absence
(A) and presence of 1 M urea (B) or
0.5 M GdnHCl (C). Data are expressed as
mean ± S.E. of three independent experiments.
View larger version (14K):
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Fig. 6.
Tryptophan fluorescence quenching of the W43F
and W54F mutants by lipophilic small ligands. The relative
fluorescence intensities of W43F ( ) and W54F (
)-substituted
L-PGDS were measured in the presence of bilirubin (A) or
13-cis-retinal (B). Data were expressed as
percentages of those with the wild-type enzyme (mean ± S.E.,
n = 3).
View larger version (31K):
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Fig. 7.
CD spectra in near-UV and visible range of
lipophilic small ligands bound to L-PGDS. CD spectra of bilirubin
bound to L-PGDS (10 µM) without denaturant
(A), in the presence of 1 M urea (B),
and in the presence of 0.5 M GdnHCl (C) were
recorded. CD spectra of 13-cis-retinal bound to L-PGDS (20 µM) without denaturant (D), in the presence of
1 M urea (E), and in the presence of 0.5 M GdnHCl (F) were also prepared.
Numbers indicate the molar ratios of ligand to L-PGDS.
Closed arrows indicate the peaks of the Cotton effects; and
open arrows, the isosbestic points.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-sheet conformation formed maximally in the presence of 0.5 M
GdnHCl or 1 M urea, giving the ellipticities of
9.2 or
8.7 × 103 deg cm2 dmol
1,
respectively (Figs. 1 and 2). On the other hand, the I state is the
inactive state without the enzyme activity and the ligand-binding activity but possessing the core secondary structure maximally formed
in the presence of 2 M GdnHCl or 4 M urea,
giving the ellipticity of
4.8 × 103 deg
cm2 dmol
1 (Figs. 1-4). The U state is found
in the presence of excess amounts of denaturants, exhibits the
ellipticity above
4.8 × 103 deg cm2
dmol
1, and is nonrelevant to enzyme activity and ligand
binding. In the present study, we proposed that both A and I
equilibrium states were located on a sequential pathway from the N to U
state. However, it is still unclear whether the A state is an
on-pathway intermediate from the N to I state. The N and A state each
may convert directly to the I state. As a minor possibility, the A
state must convert back to the N state before converting to the I
state.
View larger version (10K):
[in a new window]
Fig. 8.
A model of the reversible folding and
unfolding process of L-PGDS. N, A,
I, and U represent the native,
activity-enhanced, inactive, and unfolded states of L-PGDS,
respectively.
-barrel protein; whereas a number of other
-barrel
proteins, such as
-lactoglobulin, cellular retinoic acid-binding
protein, and human acidic fibroblast growth factor, have been shown to
fold in a two-state transition (31). For example, human acidic
fibroblast growth factor forms a stable equilibrium intermediate
accumulated maximally at 0.96 M GdnHCl resembling a molten
globule state as examined by CD, fluorescence, and NMR (32). The molten
globule is found in various proteins, such as
-lactoglobulin,
cytochrome c, and
-lactalbumin, and is proposed to exist
in the partially folded equilibrium state of the protein with
pronounced secondary structure but no rigid tertiary structure
(33-35). Thus, we consider the I state of L-PGDS to be a molten
globule-like state because it contained pronounced secondary structure
without rigid tertiary structure and apparent L-PGDS function. The
molten globule has been proposed to be involved in a number of
physiological processes, such as protein recognition by chaperones,
release of protein ligands, and protein translocation across
biomembranes (36, 37). L-PGDS is secreted into various body fluids such
as cerebrospinal fluid, interphotoreceptor matrix, seminal plasma, and
plasma, after cleavage of its N-terminal signal peptide to bind and
transfer various small lipophilic ligands such as retinoids, bile
pigments, and thyroid hormones (1, 2). The L-PGDS concentration is
increased in the cerebrospinal fluid of patients after subarachnoid
hemorrhage (38, 39), and the enzyme is produced by leptomeningeal cells
of adult rats, secreted in the cerebrospinal fluid, and taken up by
macrophages to be accumulated in their lysosome, as concluded from
examination by confocal immunofluorescence microscopy (40). Therefore,
the I state of L-PGDS may also be involved in protein translocation across biomembranes of the leptomeningeal cells and in the release of
the bound ligands within the lysosome.
-sheet content, as monitored by the CD spectra at 218 nm (Fig. 2).
The reorganization of the hydrogen-bond network was also associated
with the activation of L-PGDS in the presence of urea or GdnHCl, as
revealed by the NMR spectra (Figs. 3 and 4). As judged from our recent
x-ray crystallographic analyses, L-PGDS contains a well ordered
hydrophobic coil structure near the
-sheet.3 In the presence
of low concentrations of the denaturants, the hydrophobic interaction
is weakened to transform a part of the coil structure to the
-sheet,
which is stabilized by a newly formed hydrogen bonding network. Such a
rearrangement of the L-PGDS structure might occur in vivo by
binding of metal ions or other substances.
-Lactoglobulin, a member of the lipocalin
family, also exhibits a broad selectivity of ligand binding (41), but it does not bind bilirubin or biliverdin (data not shown). In previous
unfolding studies on
-lactoglobulin by CD analysis (30, 42), the
ellipticities at 219 and 222 nm were shown to be increased in the
presence of GdnHCl up to 2.5 M, as compared with the value in the absence of GdnHCl. Although the increase in the secondary structure of
-lactoglobulin was not examined in those
studies, such results indicate that the
-sheet content in
-lactoglobulin was increased by low concentrations of GdnHCl and
suggest that the activity enhanced-like equilibrium state was also
formed during the unfolding of
-lactoglobulin. On the other hand,
neither retinoic acid- nor retinol-binding proteins showed any increase
in ellipticity at 218 nm on CD spectra in the presence of urea up to 9 M (43). The increase in the secondary structure by low
concentrations of denaturants may not be a feature of all molecules of
the lipocalin family. Therefore, L-PGDS is a unique protein that
possesses two equilibrium states: the activity-enhanced and the
inactive states, in a reversible folding process and shows a high
correlation between structural changes in itself and its dual functions
of enzyme activity and ligand binding.
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ACKNOWLEDGEMENTS |
---|
We thank N. Uodome, Y. Hoshikawa, and M. Inui for technical assistance and S. Sakae, T. Nishimoto, and M. Yamaguchi for secretarial assistance. We also thank Dr. M. Mase (Department of Neurosurgery, Nagoya City University Medical School) for providing human CSF.
![]() |
FOOTNOTES |
---|
* This work was supported in part by grants from the Core Research for Evolutional Science and Technology of the Japan Science and Technology Corporation (to Y. U.), Grant 12558078 (to Y. U.) from the Grants-in-aid for Scientific Research (B) of the Ministry of Education, Culture, Sports, Science and Technology of Japan, and Ministry of Health and Welfare of Japan Grant 100107 (to O. H.), the Takeda Science Foundation (to Y. U.), and Osaka City.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Present address: Dept. of Food and Nutrition, Tsu City College, 157, Ishinden, Tsu City, Mie 514-0112, Japan.
** Present address: Dept. of Pharmacology, Ehime University School of Medicine, Shigenobu-cho, Onsen-gun, Ehime 791-0295, Japan.
To whom correspondence should be addressed: Dept. of Molecular
Behavioral Biology, Osaka Bioscience Institute, 6-2-4 Furuedai, Suita,
Osaka 565-0874, Japan. Tel.: 81-6-6872-4851; Fax: 81-6-6872-2841; E-mail: uradey@obi.or.jp.
Published, JBC Papers in Press, November 18, 2002, DOI 10.1074/jbc.M209934200
2 M. Emi, D. Irikura, T. Inui, and Y. Urade, unpublished observation.
3 D. Irikura and Y. Urade, unpublished results.
4 D. Irikura and Y. Urade, unpublished results.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: PG, prostaglandin; L-PGDS, lipocalin-type prostaglandin D synthase; GdnHCl, guanidine hydrochloride; kcat, catalytic constant; Kd, dissociation constant; CSF, cerebrospinal fluid; SAH, aneurysmal subarachnoid hemorrhage; deg, degree.
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