(Received for publication, October 19, 1994)
From the
Glutathione-independent prostaglandin D synthase in rat brain is
composed of 189 amino acid residues and catalyzes the isomerization of
prostaglandin H to prostaglandin D
, an
endogenous sleep-promoting substance. This enzyme is the only enzyme
among members of the lipocalin superfamily composed of various
secretory lipophilic ligand-carrier proteins and is recently identified
to be a
-trace protein, a major constituent of human cerebrospinal
fluid. We expressed the active enzyme in Escherichia coli and
then systematically substituted all cysteine residues of the
1-29 enzyme at positions of 65, 89, and 186 with alanine or
serine. The parent and mutant enzymes were purified to apparent
homogeneity with a recovery of
30% by chromatography with Sephadex
G-50 and S-Sepharose, by which all the enzymes showed identical elution
profiles. The purified enzymes, irrespective of the mutation, showed
almost the same circular dichroism spectral characteristics as
displayed by a highly ordered
-structure. The recombinant enzymes
containing Cys
showed the activity comparable with that of
the enzyme purified from rat brain (
3 µmol/min/mg of protein)
in the presence, but not in the absence, of sulfhydryl compounds.
However, all of the single, double, and triple mutants without
Cys
lost the enzyme activity. The purified
1-29
Ala
enzyme was inactivated reversibly by conjugation
with glutathione at Cys
and irreversibly by the
stoichiometric chemical modification with N-ethylmaleimide.
These results indicate that Cys
is an essential thiol of
the enzyme and that both the intrinsic and extrinsic sulfhydryl groups
are necessary for nonoxidative rearrangement of 9,11-endoperoxide of
prostaglandin H
to produce prostaglandin D
catalyzed by the enzyme.
Prostaglandin (PG) ()D
is a major PG
produced in the brain of rats and humans and functions as a
neuromodulator of several central actions such as sleep-wake cycles,
body temperature, luteinizing hormone release, and odor responses (for
review see (1) ). PGD
induces natural sleep in
rodents and monkeys after the intracerebroventricular administration
(for review, see (2) ). Also in humans, accumulation of
PGD
has been observed in the cerebrospinal fluid of
patients with African sleeping sickness(3) . Therefore,
PGD
is proposed to be an endogenous sleep-promoting
substance in mammals.
Among several enzymes catalyzing the
conversion of PGH to PGD
(for review, see (4) ), GSH-independent PGD synthase,
(5Z,13E)-(15S)-9
,11
-epidioxy-15-hydroxyprosta-5,13-dienoate D-isomerase (EC 5.3.99.2)(5) , is responsible for
biosynthesis of PGD
in the brain. This enzyme is localized
in the choroid plexus, leptomeninges, and oligodendrocytes of the
central nervous system(6, 7) . It has been recently
demonstrated that a major protein constituent of human cerebrospinal
fluid, classically termed
-trace, is identical to PGD
synthase(8, 9) . Concurrently, sleep of unrestrained
rats has been found to be inhibited by the intracerebroventricular
administration of selenocompounds(10) , which inhibit the rat
brain PGD synthase activity without affecting the activities of three
other enzymes in the arachidonate cascade, GSH-requiring PGD synthase,
PGF synthase, and 11-keto PGD
reductase(11) .
Furthermore, when PGD
was infused into the subarachnoid
space near the ventral surface of the rostral basal forebrain, the
sleep-promoting effect of PGD
in rats was found to be more
remarkable than the intracerebroventricular administration or the
infusion into the brain parenchyma(12) . Thus, the enzyme is
considered to play a key role in the regulation of sleep-wake
activities in mammals(13) , probably via the cerebrospinal
fluid.
We have already reported the cloning and sequence analyses of
the rat and human cDNAs (14, 15) and the rat gene (16) for the enzyme. White et al. (17) have
isolated the human gene for the enzyme and mapped the gene to
chromosome 9, bands q34.2-34.3. A homology search in data bases
of protein primary structure revealed that the enzyme is a member of
the lipocalin superfamily(15, 18, 19) , a
group of proteins comprising a variety of secretory proteins that bind
and transport small lipophilic molecules(20) . PGD synthase is
the only known exception, being an enzyme rather than a lipophilic
ligand-carrier protein. Based on the observed sequence homology, the
enzyme was deduced to form an eight- or nine-stranded anti-parallel
-barrel structure with a hydrophobic pocket in which Cys
is located(15) . Among members of the lipocalin family,
the cysteine residue is conserved only in rat and human brain PGD
synthases and in a major secretory protein from amphibian choroid
plexus(21) , the latter of which has the highest homology to
the enzyme. Therefore, Cys
has been assigned to be a
putative active site of this enzyme(15, 18) .
In
this study, we investigated the structural and functional significance
of cysteine residues of GSH-independent PGD synthase by site-specific
mutagenesis and chemical modification. Our results demonstrate that
Cys is an essential thiol of the enzyme and that both the
intrinsic and extrinsic sulfhydryl groups are necessary for the
catalysis effected by this enzyme. The reaction mechanism of this
enzyme is also proposed.
For reversible
modification of the cysteine residue, the enzyme was incubated with a
mixture of each 1 mM of GSH and the oxidized form of GSH in
0.1 M Tris-HCl (pH 9.0) and 0.1 M KCl containing 1
mM EDTA. After the incubation at 4 °C overnight, the
cysteine residue was autooxidized to form a GS-adduct. The modification
was confirmed by the decrease in the free sulfhydryl content of the
enzyme, and the increase in M of the enzyme was
determined by time of flight mass spectrometry after matrix-assisted
laser-desorption/ionization (Maldi-Tof mass monitor LDI-1700, Jeol,
Tokyo, Japan). The modification was removed by incubation at 4 °C
overnight with excess DTT.
Figure 1:
SDS-PAGE of E. coli lysates
containing various sizes of recombinant rat brain PGD synthase after
sequential truncation. The amino acid residues of PGD synthase were
unidirectionally deleted from the N terminus (A) and the C
terminus (B). E. coli transfected with the mutated
plasmid were sonicated at 4 °C in a 10-fold volume of 20 mM Tris-HCl (pH 7.5) and 1 mM EDTA and centrifuged at 20,000
g for 30 min at 4 °C. The precipitated (P) (each leftlane) and soluble (S) (each rightlane) fractions were
subjected to SDS-PAGE under reducing conditions. Proteins in the gel
were stained with Coomassie Blue. M
values in
thousands of marker proteins are indicated on the left.
We then
constructed C-terminal truncated mutants by introducing the stop codon
into the positions of the amino acid residues of 154, 171, and 183 of
the 1-29 enzyme. The
(1-29, 183-189) enzyme
was efficiently, but partly, expressed into the soluble fraction and
associated with the enzyme activity (0.42 µmol/min/mg of protein).
However, the shorter mutants, the
(1-29, 171-189) and
(1-29, 154-189) enzymes, were completely recovered in
the insoluble fraction (Fig. 1B) and had no enzyme
activity (<0.01 µmol/min/mg of protein). Thus, the
(1-29, 183-189) mutant was an active enzyme of the
smallest size produced by terminal truncation, being composed of 153
amino acid residues from the cDNA plus 7 residues from the plasmid with
a calculated M
of 18,136. The core region from
Val
to Gln
, essential for stable expression
of the recombinant enzyme with the catalytic activity, ranges from
upstream of the first
-strand (A-strand) to downstream of the last
-strand (I-strand) assigned in the multiple alignment with other
members of the lipocalin family (Fig. 2).
Figure 2:
Multiple alignment of amino acid sequences
of rat and human brain PGD synthases, a major secretory protein from
toad choroid plexus, bovine -lactoglobulin, and human
retinol-binding protein. Cleavage sites of the signal sequences of
these proteins (arrowheads) and N-glycosylation sites
of PGD synthase (asterisks) are shown on the top of
each sequence. Vertical boxes represent the cysteine residue
conserved among PGD synthases and the toad protein (open box),
whose residue was identified as an active thiol of rat brain PGD
synthase, and 2 cysteine residues constructing an intramolecular
disulfide bond highly conserved among members of the lipocalin
superfamily (patched boxes). The positions of secondary
structure elements of
-lactoglobulin (33) and
retinol-binding protein (34) identified by x-ray
crystallography are shown at the bottom of each sequence:
3
helix, a hatched box;
-helix, open
boxes; and
-strand, closed boxes. In both proteins,
-strands B, C, and D, and F, G, and H construct two anti-parallel
-sheets forming a
-barrel
structure(33, 34) .
Recently the
N-terminal amino acid sequence of rat brain PGD synthase was determined
to be Gly-His-Asp-Thr-Val-Gln-Pro after digestion with pyroglutamyl
aminopeptidase, indicating that Gln is the N terminus of
the mature enzyme. (
)PGD synthase in human cerebrospinal
fluid,
-trace, is also processed posttranslationally, with the
first N-terminal 22 amino acid residues being
cleaved(7, 8, 15) . Therefore, the N-terminal
sequence is considered to function as a signal peptide for its sorting
into the membrane (5) and the secretion into the cerebrospinal
fluid. These results agree with the finding that the N-terminal
sequence upstream of Val
was not essential for the
activity of the enzyme. PGD synthases purified from rat brain (14) and human cerebrospinal fluid (7) are N-glycosylated at two positions, Asn
and
Asn
. However, since the recombinant enzyme displayed the
enzyme activity, the glycosyl residues are also not considered to be
essential for the enzyme activity.
Figure 3:
Western blot analysis after SDS-PAGE of E. coli lysates expressing recombinant rat brain PGD synthase
after Cys-Ala substitution. Samples are the lysates of transformed E. coli expressing the recombinant 1-29 PGD
synthase and the mutant enzymes after Cys-Ala substitution at three
positions. The samples were denatured in the presence (+) or the
absence(-) of
-mercaptoethanol (SH). The gel was
stained with silver (each rightlane), and the
transfer blot was immunostained with antibodies against rat brain PGD
synthase (each leftlane). Symbols represent the
recombinant enzymes of a monomer with (open arrowheads) and
without (closed arrowheads) intramolecular disulfide linkage,
a monomer after endogenous sulfhydryl modification (asterisks), dimers (small arrows), and oligomers (dotted lines). Positions of the M
marker
proteins are shown by horizontal bars at the left.
Major
components of nonreduced isoforms of the parent, Ala,
Ser
, Ala
, and Ser
enzymes
migrated in the gel faster than the reduced forms. These isoforms
possessed intramolecular disulfide bonds: a
Cys
-Cys
linkage in the Ala
and Ser
enzymes, a
Cys
-Cys
linkage in the Ala
and Ser
enzymes, and both linkages in the parent
enzyme. These results suggest that Cys
is accessible to
both Cys
and Cys
probably due to the location
of Cys
in a flexible C-terminal sequence of the enzyme.
In Western blot, the enzymes with the
Cys
-Cys
bond were less immunoreactive
than those with the Cys
-Cys
bond,
suggesting that the antigenic epitopes of the enzyme were masked by the
formation of the Cys
-Cys
disulfide
bond. Alternatively, the intramolecular disulfide linkage was not found
between Cys
and Cys
of both Ala
and Ser
mutants, indicating that these 2 residues
are kept at a distance from one another in the enzyme molecule. This
finding is also consistent with the model structure of the enzyme (14) in which three
-strands, the
Cys
-containing B-strand, the Cys
-containing
D-strand, and the C-strand, construct a
-sheet structure, and the
former two
-strands are separated by the C-strand in the same
-sheet structure.
For all mutants without Cys,
the major component existed as an isoform without disulfide bridge and
migrated to a position identical to that seen under reducing
conditions. The Cys
-possessing enzymes, but not other
mutants without Cys
, showed an additional major band at a
position of slightly higher M
. Since the
additional band disappeared after incubation with excess DTT, it was
considered to be due to sulfhydryl modification of Cys
that endogenously occurred during expression of the enzyme in E. coli. The modification was not found at other 2 cysteine
residues, Cys
and Cys
. Thus, Cys
is an active thiol of the enzyme in terms of the endogenous
modification.
Intermolecular disulfide formation was observed in all
enzymes except for the Ala enzyme without cysteine
residues. The parent and all single mutants formed dimers, trimers, and
oligomers via intermolecular disulfide bonds among the available
cysteine residues. The double mutants formed only dimers via an
intermolecular disulfide bond at a unique cysteine residue of each
mutant. These dimers and oligomers were completely dissociated into the
corresponding monomers after incubation with excess DTT. Among the
double mutants, the ratio of a dimer to a monomer was higher in the
Ala
and Ala
enzymes than in the
Ala
enzyme, suggesting that the disulfide bond is
formed more preferentially at Cys
and Cys
than at Cys
. Therefore, Cys
and
Cys
may be exposed on the surface of the enzyme molecule
to a greater extent than Cys
. It is in agreement with the
predicted tertiary structure of the enzyme(14) , in which
Cys
exists in the hydrophobic pocket of the enzyme and an
intramolecular disulfide bridge between Cys
and
Cys
occurs at the outside of the barrel structure.
Figure 4:
Purification of the reduced form of the
1-29 Ala
mutant (upper panels) and
the nonreduced forms of the
1-29 Ala
mutant (lower panels). Elution profiles of protein (solid
lines) and the PGD synthase activity (open circles) were
monitored during column chromatographies with Sephadex G-50 (A) and S-Sepharose (B). The PGD synthase activity of
the nonreduced isoforms was also determined after incubation of the
eluate at 4 °C overnight with excess DTT in 0.1 M Tris-HCl
(pH 8.0) (closed circles in lowerpanels).
The position of the void volume (V
in A) and the gradient of NaCl concentrations (dotted line in B) are also indicated. Horizontal bars represent the collected fractions.
The oxidized isoforms of
the Ala and Ser
enzymes with the
intramolecular Cys
-Cys
linkage were
also purified to apparent homogeneity in the absence of DTT, giving
almost the same elution profiles and recoveries as those of the reduced
isoforms in the presence of 1 mM DTT. The findings suggest
that the tertiary structure of the enzyme was unchanged by formation of
the intramolecular disulfide linkage.
Alternatively, in the absence
of DTT, other mutants showed heterogeneous peaks of various isoforms in
the two chromatograms. The elution profiles of the Ala enzyme purified in the absence of DTT are shown in Fig. 4B. The dimeric and oligomeric isoforms with the
intermolecular disulfide bond were eluted at positions of higher M
from Sephadex G-50 column. The monomeric
isoforms with the endogenously modified Cys
and the
intramolecular Cys
-Cys
linkage were
eluted from S-Sepharose column with peaks at NaCl concentrations of
about 0.2 and 0.6 M, respectively. These results revealed that
those isoforms were distict from others in terms of the net charge at
pH 4.5; the former isoform was charged less positively and the latter
more positively than other isoforms. From the difference of M
between the modified and unmodified enzymes, M
of the endogenous modifier was calculated to be
about 800, although the chemical structure remains unidentified.
Typical results of SDS-PAGE of the monomeric isoforms purified under
the nonreducing conditions are shown in Fig. 5.
Figure 5:
SDS-PAGE of the monomeric isoforms of the
1-29 Ala
, Ala
, and Ala
enzymes purified under nonreducing conditions. Each sample was
subjected to SDS-PAGE under nonreducing conditions before (A)
and after (B) incubation with excess DTT and then stained with
Coomassie Blue. Samples are the
1-29 Ala
enzyme without endogenous sulfhydryl modification at Cys
(lane 1), the
1-29 Ala
enzyme
with an intramolecular Cys
-Cys
disulfide bond (lane 2), the
1-29 Ala
enzyme with an intramolecular Cys
-Cys
disulfide bond (lane 3), the
1-29 Ala
enzyme without the disulfide bond and the endogenous modification
at Cys
(lane 4), and the
1-29
Ala
enzyme with the endogenous modified Cys
(lane 5). The marker proteins are shown at the left.
Fig. 6A shows the CD spectrum of the purified
1-29 Ala
enzyme, which revealed
characteristics of a
-sheet structure (35) with a broad
minimum peak at around 214 nm, and was remarkably similar to that of
-lactoglobulin(36) . The content of secondary structural
components of the enzyme was calculated to be 17%
-helix, 47%
-strand, and 36% unordered structure. The parent and all of the
other mutant enzymes showed the same CD spectra as that of
1-29 Ala
enzyme, in which the oxidized
isoforms of the Ala
and Ala
enzymes with the
intramolecular Cys
-Cys
and
Cys
-Cys
linkage, respectively, were
also included. Furthermore, the CD spectrum remained unchanged when the
disulfide bond of these isoforms was cleaved by incubation with excess
DTT. These results also indicate that the tertiary structure of the
enzyme is unchanged by the formation of the disulfide linkage.
Figure 6:
Circular dichroism spectra of the purified
recombinant rat brain PGD synthase. The samples are the 1-29
Ala
mutant purified in the presence of 1 mM DTT (A), and the
1-29 Ala
mutant with an endogenously modified Cys
purified in
the absence of DTT (B). The spectrum of the modified enzyme
was also measured after removing the modification by the incubation
with excess DTT (dottedline in B). The
spectrum was obtained as the mean of 10 recordings with the protein
solution at a concentration of 15 µM in 5 mM potassium phosphate (pH 7.5).
However, as shown in Fig. 6B, the CD spectrum of the
isoform with the endogenously modified Cys was clearly
different from that of other isoforms, suggesting that the modifier has
the minimum
value at about 220 nm. The CD spectrum of the
modified enzyme returned to that of the unmodified enzyme after
incubation with excess DTT, which removed the modification to produce
the free sulfhydryl group.
When the
purified 1-29 Ala
enzyme was incubated with
various amounts of NEM, the enzyme activity and the content of free
sulfhydryl group of the enzyme decreased dose-dependently in a parallel
fashion and disappeared almost completely after incubation with excess
amounts of NEM (Fig. 7). These results indicate that the
inactivation of this enzyme was due to stoichiometric chemical
modification of Cys
by NEM and that Cys
is an
essential thiol of the enzyme.
Figure 7:
Effects of chemical modification with NEM
on the enzyme activity and the free sulfhydryl content of the purified
1-29 Ala
PGD synthase. The enzyme (7 nmol)
was incubated at 25 °C for 5 min in the presence or absence of
various amounts of NEM in 240 µl of 25 mM Tris-HCl (pH
7.0) and 1 mM EDTA. After incubation, the residual enzyme
activity (open circles) and the free sulfhydryl content of the
enzyme (closed circles) were determined as described under
``Materials and Methods.'' The free sulfhydryl content of the
enzyme before incubation was determined to be 0.8 mol/mol
enzyme.
The Cys residue is
conserved among rat and human brain PGD synthases and a major secretory
protein from cane toad choroid plexus (21) but is not present
in any of the other members of the lipocalin
superfamily(15, 18, 19) . The toad secretory
protein has the highest homology with PGD synthase among lipocalins
identified to date; the identity and similarity are 41.4 and 66.1% to
the rat enzyme and 39.8 and 64.5% to the human enzyme, respectively (Fig. 2). The toad protein and PGD synthase also share several
common features for the tissue distribution; both the protein and the
enzyme are selectively expressed in the central nervous system,
actively produced in the choroid plexus, and secreted into the
cerebrospinal fluid(6, 7, 8, 21) .
The toad protein may, therefore, be an amphibian counterpart of the
mammalian enzyme and may also be associated with the PGD synthase
activity, acting as the enzyme in the central nervous system of
amphibians.
In conclusion, our results have shown that Cys is an essential thiol for the activity of PGD synthase and that
both intrinsic and extrinsic sulfhydryl groups are necessary for
catalyzing the conversion of PGH
to PGD
. The
extrinsic sulfhydryl group was required for the reaction but not
consumed stoichiometrically during the reaction. After the conversion
of PGH
to PGD
, configuration of chiral carbon
at C-9 is retained. Therefore, as shown in Fig. 8, PGD
formation is considered to be initiated by the transient
formation of an S-O linkage between the active intrinsic thiol of
Cys
and the oxygen at C-11 of PGH
. The
extrinsic sulfhydryl group then withdraws the hydrogen at C-11 of the
intermediate to produce PGD
. PGD synthase is known to be
rapidly inactivated during reaction(3) . The suicidal property
of this enzyme might be due to nucleophilic attack of the extrinsic
sulfhydryl group toward the carbon at C-11 instead of the hydrogen, as
shown by dottedarrows in Fig. 8. In
consequence, PGD synthase may be inactivated by oxidation of the
intrinsic thiol of Cys
.
Figure 8:
Proposed mechanism of a nonoxidative
rearrangement of PGHto PGD
catalyzed by PGD
synthase in the presence of exogenous sulfhydryl compounds. The
cyclopentane ring structure of PGs is shown. Dotted arrows show the putative inactivation process of PGD synthase during the
reaction. RSH represents exogenous sulfhydryl
compounds.
The reaction mechanism may also
be applicable to PGE synthase because this enzyme shows several
enzymatic characteristics identical to PGD
synthase(37, 38, 39) . For example, both of
the enzymes are sensitive against sulfhydryl modifiers, require the
extrinsic sulfhydryl compound as an essential cofactor, and are rapidly
inactivated during the reaction. The extrinsic sulfhydryl compound is
not oxidized stoichiometrically during the reactions catalyzed by these
two enzymes. Thus, the reaction mechanism of PGE synthase may be the
same as that of PGD synthase and different from the previously
speculated mechanism analogous to the glyoxylase I
reaction(40) . PGE, PGI, and thromboxane A synthases in the
arachidonic acid cascade also catalyze the nonoxidative rearrangement
of 9,11-endoperoxide of PGH. Since the reaction mechanisms
of these enzymes are still a matter of speculation, the proposed
mechanism in this study for PGD synthase is useful to predict their
reaction mechanisms.
Note Added in Proof-Ullrich and Brugger (41) have recently proposed the models for the reaction mechanism of PGI and thromboxane A synthases.