(Received for publication, December 17, 1996, and in revised form, March 21, 1997)
From Protein Engineering Research Institute, 6-2-3 Furuedai, Suita, Osaka 565, Japan, the § Biomolecular
Engineering Research Institute, 6-2-3 Furuedai, Suita, Osaka 565, Japan,
Osaka Bioscience Institute, 6-2-4 Furuedai, Suita, Osaka
565, Japan, and ** PRESTO, Japan Science and Technology Corporation,
6-2-4 Furuedai, Suita, Osaka 565, Japan
Lipocalin-type prostaglandin D synthase is
responsible for the biosynthesis of prostaglandin D2
in the central nervous system and the genital organs and is secreted
into the cerebrospinal fluid and the seminal plasma as -trace. Here
we analyzed retinoids binding of the enzyme by monitoring the
fluorescence quenching of an intrinsic tryptophan residue, and
appearance of circular dichroism around 330 nm, and a red shift of the
UV absorption spectra of retinoids. We found that the enzyme binds
all-trans- or 9-cis-retinoic acid and
all-trans- or 13-cis-retinal, but not all-trans-retinol, with affinities (Kd
of 70-80 nM) sufficient for function as a retinoid
transporter. All-trans-retinoic acid inhibited the enzyme
activity in a noncompetitive manner, suggesting that it binds to the
same hydrophobic pocket as prostaglandin H2, the substrate
for prostaglandin D synthase, but at a different site in this pocket.
It is likely that this enzyme is a bifunctional protein that acts as
both retinoid transporter and prostaglandin D2-producing
enzyme.
Retinoids play an important role in regulating a variety of
biological processes, including differentiation, morphogenesis, and
cell proliferation. The process is initiated by retinoid binding to the
nuclear receptor for retinoic acid
(RAR/RXR)1; RAR binds both
all-trans- and 9-cis-retinoic acids, whereas RXR
is specific for the 9-cis-isomer (1-3). The binding of
retinoic acids to the dimerized receptor, RAR-RXR or RXR-RXR, activates or inhibits the transcription of retinoid-responsive genes. The proteins that transport retinoids are divided into two distinct families based on their sequence, structure, and function: the secretory transporters and the intracellular transporters (4). Secretory retinoid transporters, such as plasma retinol-binding protein
(RBP) and -lactoglobulin, circulate retinoids in a variety of body
fluids and transport them to the intracellular retinoid transporters,
cellular RBP (CRBP), and cellular retinoic acid-binding protein
(CRABP), which finally transfer the retinoids to RAR or RXR.
Retinoids also regulate a number of genes expressed in the central
nervous system and thus play a variety of important roles, particularly
in development (5, 6). In the brain, mRNAs for RAR 1 and
3
(5, 7) and for RXR
and
(5, 7) were found as well as CRBP and
CRABP (8, 9). However, secretory retinoid transporters in the central
nervous system have not been identified, although retinoids would need
to change transporters at the blood-brain barrier as the barrier is
impermeable to the secretory transporter.
Recently, a major protein in human cerebrospinal fluid, classically
termed -trace (10), was identified as prostaglandin (PG) D synthase
(11-14). The enzyme is responsible for biosynthesis of
PGD2, which is a major PG in the brain of various mammals, including humans, and is proposed to be an endogenous sleep-promoting substance (15, 16), as well as a modulator of several central actions,
such as the regulation of body temperature, luteinizing hormone
release, and odor responses (17, 18). PGD synthase is produced in the
choroid plexus, leptomeninges, and oligodendrocytes of the central
nervous system (19, 20) and secreted into the cerebrospinal fluid. It
is also localized in the pigmented epithelial cells of the rat retina
(21) and in the epithelial cells of human male genital organs (22) and
secreted into the interphotoreceptor matrix and seminal plasma,
respectively, both of which are a closed compartment isolated by the
respective blood-retina and blood-testicular barriers.
The results of cloning and sequence analyses of the rat and human cDNAs (23, 24) for the enzyme have already been reported. A homology search in data bases of protein primary structure revealed that the enzyme is a new member of the lipocalin superfamily (24-26), a group of proteins comprising a variety of secretory proteins that bind and transport small lipophilic molecules (27, 28). The gene structures for the rat and human enzymes are also comparable with those of other members of the lipocalin superfamily, in terms of the numbers and sizes of the exons and the phasing patterns of the introns (29, 30). Furthermore, the gene for the enzyme has been mapped within the lipocalin gene cluster in human chromosome 9 (30) and mouse chromosome 2 (31).
Secretory retinoid transporters, such as plasma RBP and
-lactoglobulin, are also members of the lipocalin family and show weak homology (20% identity) toward PGD synthase (24, 26). Thus, we
considered that the enzyme may be involved in the transport of
bioactive lipophilic substances in the central nervous system via the
cerebrospinal fluid, analogous to the functions of other lipocalins,
such as plasma RBP in the systemic circulation and
-lactoglobulin in
milk. In this study, by measuring the fluorescence, UV, and circular
dichroism (CD) spectra after incubation of the recombinant rat brain
PGD synthase (32) with various isoforms of retinoid, we found that the
enzyme binds all-trans- or 9-cis-retinoic acid
and all-trans- or 13-cis-retinal with affinities
comparable with those of other secretory retinoid transporters.
All-trans-retinoic acid, all-trans-retinal, all-trans-retinol, and N-acetyl-L-tryptophanamide were purchased from Sigma. 9-cis-Retinoic acid was purchased from Wako Junyaku (Osaka, Japan), and 13-cis-retinal was provided by Dr. K. Yoshihara, Suntory Institute for Molecular Biology. [3H]Retinoids (2.20 GBq/mmol) and [1-14C]arachidonic acid (2.20 GBq/mmol) were from DuPont NEN.
Expression and Purification of Recombinant Rat Brain PGD SynthaseThe full-length cDNA for rat brain PGD synthase,
which is composed of 189 amino acid residues (GenBankTM accession
number [GenBank]) (24), was ligated into the
EcoRI-HindIII site of pUC119. The N-terminal 29 amino acid residues containing the signal peptide were deleted, and the
Cys residue was replaced by Ala, as reported previously (32). The
recombinant enzyme and the Cys Ala-substituted mutants were
expressed in Escherichia coli JM109 and were purified to
apparent homogeneity by Sephadex G-50 and S-Sepharose column chromatography (32). The Ala89,186 enzyme with a GS adduct
at Cys65 was prepared as reported previously (32).
Retinoids were dissolved in ethanol
to give a stock solution of 1 mM. The concentrations of
retinoic acid, retinal, and retinol were determined
spectrophotometrically in an ethanol solution based on their respective
molar absorption coefficients of 336 of 45,000 M
1 cm
1,
383 of 42,800 M
1 cm
1, and
325 of 46,000 M
1 cm
1, respectively (33). The
retinoid solution (10 µl), which also contained a trace amount of
[3H]retinoids (10,000 dpm), was added to 1 ml of 5 mM Tris-HCl, pH 8.0, containing 1.5 nmol of the purified
enzyme. After incubation at 22 °C for 30 min, the fluorescence of
the tryptophan residue was measured with a Shimazu
Spectrofluorophotometer RF-5000 (Kyoto, Japan) with the excitation
wavelength at 282 nm and emission wavelength at 338 nm. The loss of
ligand, due to nonspecific adsorption to the incubation tubes, was
corrected for by measuring the radioactivity of the
[3H]retinoids in the incubation mixture after the
measurements. The titration of
N-acetyl-L-tryptophanamide was used to correct for the quenching of the tryptophan fluorescence due to nonspecific interactions with retinoids.
The apparent dissociation constant (Kd) of a single binding site was calculated by the method of Cogan et al. (34),
![]() |
(Eq. 1) |
![]() |
(Eq. 2) |
Absorption spectra were recorded at 25 °C after the addition of retinoids (20 µM, final concentration) or an equal volume of buffer (5 mM Tris-HCl, pH 8.0) to the enzyme (80 µM, final concentration) for a final volume of 1 ml in a cuvette (10-mm path length) in a DU 64 spectrophotometer (Beckman).
Circular DichroismCD spectra were measured after incubation of the protein (80 µM) with retinoids (20 µM) in 5 mM Tris-HCl, pH 8.0 (2 ml). They were recorded on a J-720 spectropolarimeter (Japan Spectroscope, Tokyo, Japan) with the sample in a 10-mm path length cuvette at 10 °C. The spectra were recorded five times for each sample in the near-UV range from 300 to 400 nm, with a bandwidth of 1 nm and a resolution of 1 nm.
Enzyme AssayThe PGD synthase activity was measured by incubation at 25 °C for 1 min with [1-14C]PGH2 (final 40 µM) in 50 µl of 0.1 M Tris-HCl, pH 8.0, in the presence of 1 mM GSH, unless otherwise stated (35). [1-14C]PGH2 was prepared from [1-14C]arachidonic acid (35).
Modeling of Interaction between PGD Synthase and Retinoic AcidThe model of PGD synthase was built with a homology modeling software HOMOLOGY (Molecular Simulations, Inc., San Diego, CA) as reported previously (36). Retinoic acid was built and manually docked into the cavity of PGD synthase by using Insight II molecular modeling software (Molecular Simulations, Inc.). During the process, we referred to the binding structure between rat epididymal retinoic acid-binding protein and retinoic acid (Protein Data Bank entry 1EPB), because this protein shows a sequence similarity of 19.7% identity to PGD synthase, and the amino acids lining the bottom of the hydrophobic cavity well correspond to those of PGD synthase. The model was further refined by manual modification of side chains to circumvent unfavorable atom-atom bumping. After the manual operation, molecular mechanics calculations were performed to relax side chains with the main chain fixed, by use of DISCOVER (Molecular Simulations, Inc.).
Several members of the lipocalin family, such as plasma
RBP (37), -lactoglobulin (38-40), and epididymal retinoic
acid-binding protein (2), function as secretory retinoid transporters
in a variety of body fluids. Since the binding of retinoids to those proteins is known to quench their intrinsic tryptophan fluorescence, we
monitored the tryptophan fluorescence of recombinant PGD synthase after
incubation with retinoids. For this purpose, we used recombinant
1-29 PGD synthase (32) from which the signal sequence had been deleted.
The 1-29 PGD synthase showed fluorescence quenching after addition
of all-trans-retinoic acid in a dose-dependent
manner (Fig. 1A). The fluorescence intensity
decreased to 20% in the presence of excess amounts of
all-trans-retinoic acid (Fig. 1B). The quenching
was not observed after denaturation of the protein in the presence of 6 M guanidine hydrochloride. 9-cis-Retinoic acid
resulted in fluorescence quenching, giving an almost identical titration curve to that of all-trans-retinoic acid (Fig.
1B). All-trans-retinal and
13-cis-retinal also quenched the fluorescence to about 40%;
however, all-trans-retinol led to only 20% quenching under
conditions of ligand excess.
When P0 was plotted against
R0
(1
) by the method of Cogan
et al. (34), a positive regression line was obtained for the
fluorescence quenching of the
1-29 enzyme with
all-trans-retinoic acid (Fig. 1C). The number of
apparent binding sites of the
1-29 enzyme was calculated from the
slope to be 1.1 mol/mol, and the Kd value was found
to be 80 nM. The Kd values for
9-cis-retinoic acid, all-trans-retinal, and
13-cis-retinal were 80, 70, and 70 nM,
respectively, and the stoichiometries of the retinoid-protein complexes
were about 1.1 for the three retinoids (Table I). The
affinities of the enzyme for all-trans- and
9-cis-retinoic acid were comparable with or somewhat higher than those of other retinoid transporters. The Kd
value for all-trans-retinol was not determined.
|
In the presence of a 5-fold molar excess of all-trans-retinol, all-trans-retinoic acid and all-trans-retinal still exhibited fluorescence quenching and gave titration curves essentially identical to those in the absence of retinol (data not shown). These results indicate that retinol does not displace retinoic acid or retinal from the protein.
Spectrophotometric and CD Analyses of Retinoid Binding to PGD SynthaseChanges in the UV and CD spectra of retinoids have been
reported to occur when retinoids bind to their transporter proteins, such as plasma RBP (41), -lactoglobulin (42), CRBP (43), and CRABP
(44). Therefore, we also examined the UV and CD spectra of retinoids
after incubation with PGD synthase.
Retinoic acid, retinal, and retinol yielded absorption spectra with
peaks at 340, 380, and 317 nm, respectively. After forming the complex
with PGD synthase, the spectra of all-trans-retinoic acid
and all-trans-retinal were red-shifted approximately 30 nm, with peaks at 373 and 409 nm, respectively (Fig. 2,
A and B). On the other hand,
all-trans-retinol did not induce a significant change in the
spectrum and instead produced a small shoulder around 373 nm (Fig.
2C).
Incubation of the protein with all-trans-retinoic acid and
all-trans-retinal induced changes in the CD spectra, with
minima at 345 and 369 nm, respectively (Fig. 3,
A and B). However, when the protein was incubated
with all-trans-retinol, a weak positive change in the CD
spectra was observed around 300 nm (Fig. 3C). These changes
in the UV and CD spectra indicate that retinoic acid and retinal occupy
fixed positions in the protein.
Effect of Cys Residues on Retinoid Binding to PGD Synthase
We
constructed three types of Cys Ala-substituted mutants of PGD
synthase, which contains three Cys residues at 65, 89, and 186: the
Ala65 mutant without the Cys65 active thiol
that is essential for the catalytic reaction, the Ala89,186
mutants lacking the intramolecular disulfide linkage between Cys89 and Cys186, and the
Ala65,89,186 mutant lacking all of the Cys residues
(32).
The titration curves of fluorescence quenching with
all-trans-retinoic acid were almost identical between the
1-29 enzyme and the Ala65 mutant, indicating that the
active thiol of Cys65 is not necessary for the retinoic
acid binding (Fig. 4). The Cys65 residue is
considered to be located in the hydrophobic pocket of the enzyme, and
it is reactive with a sulfhydryl modifier and reduced glutathione (GSH)
(32). When the Cys65 residue of the parent and
Ala89,186 enzymes was modified with GSH to form a
glutathione adduct, the fluorescence quenching was inhibited by about
90% under conditions of ligand excess (Fig. 4). The Cys65
residue of the parent and Ala89,186 enzymes is also
endogenously modified during expression in E. coli (32). The
endogenously modified enzymes displayed only 10% of the fluorescence
quenching after incubation with excess amounts of
all-trans-retinoic acid (data not shown). Once the exogenous
or endogenous modifiers were removed from Cys65 by
treatment with dithiothreitol as reported previously (32), the retinoic
acid binding activity was again recovered. These results suggest that
retinoic acid binds in the hydrophobic pocket of PGD synthase, in which
the active site of the enzyme is located.
When the disulfide linkage in the parent and Ala65 enzymes was cleaved by incubation with 1 mM dithiothreitol for 15 h, the fluorescence quenching was attenuated by about 40% in the presence of excess all-trans-retinoic acid (Fig. 4). Both the Ala89,186 and Ala65,89,186 mutants without the disulfide bond also exhibited weak fluorescence quenching with all-trans-retinoic acid, similar to the reduced forms of the parent and Ala65 enzymes. The Kd values for the single binding sites of the parent and Ala65 enzymes of the reduced form, and the Ala89,186 and Ala65,89,186 mutants, were about 160 nM. These results suggest that the formation of an intramolecular disulfide bond between Cys89 and Cys186 strengthens the binding affinity for retinoic acid by 1.5-2-fold.
Inhibition of PGD Synthase Activity by RetinoidsWe then
examined the effects of retinoids on the PGD synthase activity. Fig.
5A shows the inhibition of enzyme activity by all-trans-retinoic acid and retinol. Retinol did not inhibit
the enzyme activity, whereas the addition of
all-trans-retinoic acid reduced the enzyme activity. This
result suggests that all-trans-retinoic acid binds in the
active site of the enzyme but that retinol does not bind there.
The mode of inhibition by retinoic acid was analyzed at various substrate and retinoic acid concentrations. The results of this study are shown in the Lineweaver-Burk plot in Fig. 5B. The series of lines at different retinoic acid concentrations intersects at the 1/[substrate] axis, indicating that retinoic acid inhibits the enzyme activity in a noncompetitive manner. The individual binding sites for retinoic acid and PGH2 are occupied independently. The Km value of PGD synthase with respect to PGH2 was calculated to be 5 µM, which is a similar value to that obtained for PGD synthase isolated from rat brain (14 µM) (35), and the Ki value for retinoic acid was 5 µM.
The retinoid binding by PGD synthase was demonstrated by the measurement of fluorescence quenching of Trp residues of the enzyme (Figs. 1 and 4) and the red shift of the UV spectra (Fig. 2) and the appearance of CD spectral changes around 330 nm (Fig. 3) of retinoids. Among all-trans-retinoids, PGD synthase is specifically bound to retinoic acid and retinal, but not retinol (Figs. 1, 2, 3), suggesting that the functional groups, such as amino or hydroxy group exist in the ligand-binding pocket to interact with the carbonyl group of retinoic acid or retinal. We found that 9-cis-retinoic acid also bound PGD synthase with the same affinity as that of all-trans-retinoic acid (Fig. 1B). Both all-trans- and 9-cis-retinoic acids are ligands for RXR and RAR. All-trans-retinoic acid may be bound as a horseshoe conformation twisted at the C-8-C-9 bond, similar to that of the 9-cis-isoform. This is also found in the case of the epididymal retinoic acid-binding protein (2).
PGD synthase possesses three Cys residues: Cys65 is an
essential thiol for the catalytic activity, and Cys89 and
Cys186 form an intramolecular disulfide bridge. Our results
indicate that Cys65 is crucial for the enzyme activity, but
not for retinoid binding, because the substitution of Cys65
to Ala did not any affect retinoid binding (Fig. 4). However, when
Cys65 was modified with GSH, the retinoid binding was
abolished (Fig. 4). Modification by GSH is a mild chemical
modification, and therefore, it should not alter the conformation of
the enzyme. We confirmed that CD spectrum of the 1-29
Ala89,186 enzyme was unchanged by the GSH modification of
Cys65 (data not shown). It is, therefore, considered that
all-trans-retinoic acid cannot enter the hydrophobic pocket
of the modified enzyme, due to the steric hindrance by GSH.
Furthermore, we speculated that retinoic acid binds in the same cavity
as the substrate binds. In fact, all-trans-retinoic acid,
but not all-trans-retinol, inhibited the enzyme activity in
a noncompetitive manner (Fig. 5). Our results suggest that retinoic
acid binds in the same cavity, but at a different site from where the
substrate binds. Two types of
-lactoglobulin-retinol complex have
been reported: one in which retinol binds in the cavity (38) and the
other where retinol binds to the external surface of
-lactoglobulin
(39). Retinoid binding by PGD synthase corresponds to the former
case.
We constructed a computer graphic model of PGD synthase and
successfully placed retinoic acid within the hydrophobic cavity of the
enzyme (Fig. 6). Based on the observed sequence
homology, the enzyme is predicted to form an eight-stranded
antiparallel -barrel structure with a hydrophobic pocket, similar to
other lipocalins (24, 36). Retinoids usually bind in the hydrophobic pocket of their transporters with the
-ionone ring of the retinoid toward the inside and the tail portion outside. In the model, retinoic
acid binds at the bottom of the cavity with the distance from
Trp43 short enough to cause the fluorescence quenching. The
Trp residue is highly conserved among members of the lipocalin family.
The Cys65 residue is located near the edge of the cavity,
and PGH2 binds relatively outside of the cavity. Binding of
retinoic acid interferes with the interaction between PGH2
and Cys65, which is in good agreement with the fact that
retinoic acid inhibited the enzyme activity.
PGD synthase was originally isolated as an enzyme converting PGH2 to PGD2. In the arachidonic acid cascade, arachidonic acid is converted to PGH2 by PGH synthase, a membrane protein associated with both cyclooxygenase and peroxidase activities. Since PGH2 is an unstable, highly reactive precursor, PGD synthase should exist close to PGH synthase. As soon as PGH2 is formed by PGH synthase from arachidonic acid, PGD synthase takes PGH2 and converts it to the stable PGD2. This reaction should be carried out near the membranes. In rat oligodendrocytes (19) and human arachnoid barrier cells (45), the enzyme was found by electron microscopy to be localized in the rough endoplasmic reticulum and outer nuclear membrane. The fact that retinoic acid inhibits production of PGD2 (Fig. 5) suggests that retinoids may endogenously regulate the synthesis of PGD2.
On the other hand, PGD synthase is also actively secreted into
cerebrospinal fluid, interphotoreceptor matrix, and seminal plasma, in
which a continuous supply of the substrate is unlikely. PGD synthase
would thus have another function as a secretory protein. In fact, as
shown in this study, the enzyme possesses high affinities for retinoids
(Kd < 100 nM), sufficient for it to
function as a secretory retinoid transporter (Table I). In the
cerebrospinal fluid, the enzyme (-trace) is found to be the second
major protein following albumin. Moreover, different from albumin,
which penetrates from the systemic circulation, PGD synthase is
synthesized at the arachnoid membrane and choroid plexus in the brain,
which form the blood-cerebrospinal fluid barrier. Transthyretin is also produced in the choroid plexus and mediates the transport of thyroxine from the blood stream to the thyroxine receptors in the brain (46). As
judged by the affinities for retinoids, the content in the
cerebrospinal fluid, and the sites of production, we propose that one
of the functions of the secretory PGD synthase (
-trace) is to
transport retinoids in the brain.
PGD synthase seems to be distributed where retinoids are required. For example, PGD synthase is detected in the neurons of infant rat brains, despite the absence of the corresponding mRNA. After the neurons have completely developed, PGD synthase is not detectable in them (19). There should thus be a mechanism that allows PGD synthase to be taken into the immature nerve cell but not into the completely developed cell. This suggests that PGD synthase plays a critical role in regulating the development of the neurons by, for example, the transfer of all-trans- or 9-cis-retinoic acid to RAR or RXR in the immature nerve cell. PGD synthase is produced in the retinal pigment epithelium and secreted into the interphotoreceptor matrix (21). The function there may be to supply retinal to the photoreceptor. PGD synthase showed lower binding affinity for retinoid when the disulfide bond between Cys89 and Cys186 was reduced (Fig. 4). Such reduction might facilitate the release of retinoids in the cell.
Since PGD synthase binds all-trans- and
9-cis-retinoic acid, and all-trans- and
13-cis-retinal with the same affinity as reported for other
retinoid transporters (Table I), the secreted PGD synthase (-trace)
is considered to be a powerful newly recognized secretory retinoid
transporter in the brain and retina. Proteins that may function as
transporters of retinoids in the central nervous system have not been
identified yet, although many genes in the brain and various
photoreceptor functions are regulated by retinoids. Thus, our results
suggest that, as well as functioning as the PGD2-producing
enzyme, PGD synthase may play important roles in regulating retinoid
metabolism as a secretory retinoid transporter.
We are grateful to Dr. H. Kubodera, Mitsubishi Chemical Corporation, for the modeling of a binding structure between PGD synthase and retinoic acid. We also thank Y. Ono and S. Matsumoto, Osaka Bioscience Institute, for technical and secretarial assistance, respectively.