Glutathione-independent Prostaglandin Research Articles

Prostaglandin D2 synthase related to estrogen in the female reproductive tract

Prostaglandin D2 synthase (PTGDS), also known as a glutathione-independent prostaglandin D synthase, catalyzes prostaglandin H2 to prostaglandin D2 that exhibits functions that include regulation of the central nervous system, contraction/relaxation of smooth muscle and inhibition of platelet aggregation. Gene profiling data based on our previous study indicated that PTGDS is significantly increased during development, differentiation and remodeling of the oviduct in chickens in response to estrogen. Therefore, the aims of the present study were to investigate expression of PTGDS in the oviduct and examine if the relationship between PTGDS and estrogen is conserved during development and remodeling of the oviduct. Results of our study indicate d that PTGDS expression is specifically localized to the luminal (LE) and glandular epithelial (GE) cells of the chicken oviduct in response to diethylstilbestrol, a synthetic estrogen. In addition, PTGDS expression increased during the regeneration phase of the oviduct in concert with increasing concentrations of estrogen in the circulation of laying hens during induced molting. Moreover, PTGDS mRNA and protein were expressed abundantly in GE of ovarian carcinoma, but not in normal ovaries. These results provide the first evidence that PTGDS is a novel estrogen-stimulated gene in oviductal epithelial cells, as well as a candidate biomarker for diagnosis of ovarian carcinoma.

Prostaglandin D Synthase Isoforms from Cerebrospinal Fluid Vary with Brain Pathology

Glutathione independent prostaglandin D synthase (Swissprot P41222, PTGDS) has been identified in human cerebrospinal fluid and some changes in PTGDS in relation to disease have been reported. However, little is known of the extent that PTGDS isoforms fluctuate across a large range of congenital and acquired diseases. The purpose of this study was to examine changes in PTGDS isoforms in such a population. Spinal fluid from 22 healthy study participants (normal controls) with no classifiable neurological or psychiatric diagnosis was obtained and PTGDS isoforms were identified by specific immunostaining and mass spectrometry after denaturing 2D gel electrophoresis. The PTGDS isoforms in controls consisted of five charge isoforms that were always present and a small number of occasional, low abundance isoforms. A qualitative survey of 98 different people with a wide range of congenital and acquired diseases revealed striking changes. Loss of the control isoforms occurred in congenital malformations of the nervous system. Gain of additional isoforms occurred in some degenerative, most demyelinating and vasculitic diseases, as well as in Creutzfeldt-Jakob disease. A retrospective analysis of published data that quantified relative amounts of PTGDS in multiple sclerosis, schizophrenia and Parkinson’s disease compared to controls revealed significant dysregulation. It is concluded that qualitative and quantitative fluctuations of cerebrospinal fluid PTGDS isoforms reflect both major and subtle brain pathophysiology.

Rat prostaglandin D2 synthetase: its tissue distribution, changes during maturation, and regulation in the testis and epididymis.

The changes in glutathione-independent prostaglandin D2 synthetase (PGD-S) during maturation in the rat were determined in selected organs by an RIA using PGD-S purified from rat cerebrospinal fluid and a monospecific anti-rat PGD-S polyclonal antibody. In a survey of its tissue distribution in various organ extracts and biological fluids, it was found that the concentration of PGD-S was highest in the epididymis-about 6- and 80-fold greater than that in the brain and testis, respectively. During maturation, PGD-S concentration increased steadily in the testis and epididymis; this is in contrast to the pattern of changes in the brain and liver, which showed a general trend of decline. Reverse transcription-polymerase chain reaction and Southern blotting were used to demonstrate the presence of PGD-S mRNA transcript in the testis and in Sertoli and germ cells. In the epididymis, the steady-state PGD-S mRNA level was highest in the caput, followed by the cauda and corpus. Orchiectomy induced a drastic reduction of PGD-S concentration in all three epididymal compartments. Administration of dihydrotestosterone (DHT) failed to restore the reduced epididymal PGD-S level except in the caput epididymis, where 4 days after DHT treatment the level of PGD-S was restored to about 50% of the pre-orchiectomized level; this suggests that the epididymal PGD-S level is not entirely regulated by androgen and that another yet to be identified testicular factor(s) is likely to be involved in its regulation. Germ cell-conditioned medium was also shown to stimulate PGD-S expression in the Sertoli cell. These results illustrate that PGD-S is an important molecule in testicular and epididymal function and that it is likely involved in spermatogenesis and sperm maturation.

Prostaglandin D Synthase (β-Trace) in Human Arachnoid and Meningioma Cells: Roles as a Cell Marker or in Cerebrospinal Fluid Absorption, Tumorigenesis, and Calcification Process

Glutathione-independent prostaglandin D synthase (PGDS) is an enzyme responsible for biosynthesis of prostaglandin D2 in the CNS and is identical to a major cerebrospinal fluid protein, beta-trace. Although PGDS has been identified recently in rat leptomeninges, little information is available about human meninges or meningiomas. Here, we report PGDS to be expressed consistently in 10 human arachnoid and arachnoid villi and in 21 meningiomas by immunohistochemistry, Western blot, and reverse transcription (RT)-PCR analyses. In arachnoid, PGDS immunoreactivity was seen in arachnoid barrier cells but was negligible in arachnoid trabecula and pia mater. In contrast, in arachnoid villi, PGDS was seen in core arachnoid cells rather than in the cap cell cluster or arachnoid cell layer. Meningioma cells also showed intense immunoreactivity in the perinuclear region, and it was often concentrated within meningocytic whorls and around calcifying psammoma bodies. Immunoelectron microscopic data, when compared with the ultrastructure, showed that PGDS was localized at rough endoplasmatic reticulum of arachnoid and meningioma cells. Western blot showed a 29 kDa immunoreactive band indicating PGDS, but the extent of expression was variable from case to case, which was compatible with immunohistochemical data. RT-PCR revealed PGDS gene expression in all meningiomas studied, regardless of histological subtypes, and also in human arachnoid villi. Because human arachnoid and meningioma cells exclusively express PGDS, it can be considered their specific cell marker. These results show functional differences in various types of meningeal cells attributable to differences in PGDS expression.

Lipocalin-Type Prostaglandin D Synthase (β-Trace) Is Located in Pigment Epithelial Cells of Rat Retina and Accumulates within Interphotoreceptor Matrix

Glutathione-Independent prostaglandin D synthase, identical to beta-trace, (a major CSF protein), is localized in the CNS. This enzyme, lipocalin-type prostaglandin D synthase, is a member of the lipocalin family of secretory proteins that transport small lipophilic substances. This enzyme's activity in adult rat retina was enriched sixfold in retinal pigment epithelium (RPE) and even more in interphotoreceptor matrix (IPM), all higher than brain. Western blots with anti-lipocalin-type prostaglandin D synthase showed three distinct immunoreactive bands. In the retinal cytosolic fraction, only one band was observed (M(r) 25,000); in IPM, the larger component occurred (M(r), 26,000). The RPE membrane-bound fraction showed two bands (M(r) 20,000 and 23,000), indicating synthesis, and the cytosolic fraction contained two bands (M(r) 23,000 and 26,000), indicating modification for release into IPM. At least two glycosylation sites occurred on the prostaglandin D synthase moiety, explaining the three immunoreactive bands in Western blots. Immunohistochemistry with polyclonal antibodies against this lipocalin-type enzyme showed intense localization in RPE, but less in photoreceptor outer and inner segments. In situ hybridization showed mRNA specifically expressed in RPE. Thus, lipocalin-type prostaglandin D synthase is predominantly expressed in RPE and actively accumulated in IPM. This may demonstrate gene sharing because, while catalyzing prostaglandin D2 synthesis, it may perform an additional, unrelated role in IPM. This enzyme is secreted from the RPE into IPM from which it is then taken up by photoreceptors. However, the nature of its ligand(s) is not known; they may be retinoids and/or docosahexanoic acid.

On Arachnoid Villi and Meningiomas: Functional Implication of Ultrastructure, Cell Adhesion Mechanisms, and Extracellular Matrix Composition.

Arachnoid villi or granulations are small projections of the arachnoid barrier layer into the venous sinus and its major tributaries. They are closely related to the absorption of cerebrospinal fluid, and are widely accepted to be the origin of human meningiomas. Arachnoid villi and meningiomas show a number of similarities in ultrastructure, cell adhesion mechanisms, and extracellular matrix composition. Ultrastructurally, both arachnoid and meningioma cells are characterized by interdigitations connected with junctional complexes, and extracellular cisterns related to the fluid transport. Extracellular cisterns and the intercellular space reveal abundant membrane-derived multilamellar phospholipids when a conventional ultrastructural fixative supplemented with tannic acid is used. Both arachnoid and meningioma cells are connected by Ca2+-dependent adhesion molecules: epithelial-cadherins which are concentrated at the adherens junctions. Membrane-cytoskeleton interactions by means of merlin and a-catenin molecules are thought to be crucial in signal transduction resulting in contact inhibition of cell growth in normal arachnoid cells. Impairment of these molecules might be related to meningioma-genesis. Glutathione-independent prostaglandin D2 synthase [EC 5.3.99.2] responsible for the biosynthesis of prostaglandin D2 in the central nervous system is also consistently expressed in human arachnoid villi and meningiomas. The multilamellar phospholipids are conceivably related to this arachidonate metabolism.

Quantitative Sandwich Enzyme-linked Immunosorbent Assay for Human Secretory Prostaglandin D Synthase (.BETA.-Trace).

Glutathione-independent prostaglandin D synthase is responsible for the biosynthesis of prostaglandin D2, an endogenous sleep-promoting substance, in the central nervous system of various mammals including humans. This enzyme is localized in the choroid plexus, leptomeninges, and oligodendrocytes of the central nervous system and is secreted into the cerebrospinal fluid as β-trace protein, a major constituent of human cerebrospinal fluid. Two monoclonal antibodies against human β-trace (1B7 and 10A3) were prepared by immunization of BALB/c mice with the recombinant protein expressed in Escherichia coli. Western blot analysis with human cerebrospinal fluid revealed that both 1B7 and 10A3 antibodies were immunoreactive toward a single protein at the same position as that of the purified β-trace (Mr=27, 000). A quantitative sandwich enzyme-linked immunosorbent assay was constructed with these two monoclonal antibodies. The assay system showed a linearity in the range from 0.06 to 4.00ng β-trace/well (100μl). The β-trace concentration was determined by the immunoassay to be 11.30±2.54μg/ml in human cerebrospinal fluid, 1.25±0.37μg/ml in the urine, and 0.27±0.01μg/ml in the serum.

Glutathione-independent prostaglandin D synthase as a lead molecule for designing new functional proteins.

Glutathione-independent prostaglandin D synthase as a lead molecule for designing new functional proteins.

Biosynthesis of prostaglandin D2 by motoneurons and dorsal horn microneurons: a biochemical and high resolution immunocytochemical study in chick spinal cord.

Prostaglandin D2 is one of the major prostanoids formed from [14C]arachidonic acid by the central nervous system. The aim of the present study is to specify the prostaglandin D2 biosynthetic capacity in the chick spinal cord and to identify the cell type involved in this synthesis. A highly specific and sensitive enzyme immunoassay allowed us to demonstrate that the amount of newly formed prostaglandin D2 increases proportionally with the concentration of free arachidonic acid of either exogenous or endogenous origin and reaches concentration values ranging from 10(-9) to 10(-6) M. The sites of prostaglandin D2 synthesis were localized in Vibratome sections of spinal cord after incubation with antibodies raised against glutathione-independent prostaglandin D synthase; controls were performed with anti-glutathione-dependent prostaglandin D synthase antibodies and non-immune rabbit or goat serum. After immunoprocessing, electron microscope examination revealed that the specific immunoreactivity was confined to small neurons of laminae II and III in the dorsal horn and to motoneurons in the ventral horn of the spinal cord. The immunodeposits were associated with rough endoplasmic reticulum profiles distributed throughout the dorsal horn neurons or restricted to limited subsurface areas of perikarya and dendrites in motoneurons. Since the immunoreactive neurons in the dorsal horn were closely related to blood capillaries, prostaglandin D2 may be suspected to play a role in the regulation of the microcirculation. The accumulation of prostaglandin D synthase in motoneuron areas facing astrocytic membrane stacks suggests that prostaglandin D2 could interact with astrocytic functions.

Structural and Functional Significance of Cysteine Residues of Glutathione-independent Prostaglandin D Synthase: IDENTIFICATION OF CYS65 AS AN ESSENTIAL THIOL

Glutathione-independent prostaglandin D synthase in rat brain is composed of 189 amino acid residues and catalyzes the isomerization of prostaglandin H2 to prostaglandin D2, 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 beta-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 delta 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 approximately 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 beta-structure. The recombinant enzymes containing Cys65 showed the activity comparable with that of the enzyme purified from rat brain (approximately 3 mumol/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 Cys65 lost the enzyme activity. The purified delta 1-29 Ala89,186 enzyme was inactivated reversibly by conjugation with glutathione at Cys65 and irreversibly by the stoichiometric chemical modification with N-ethylmaleimide. These results indicate that Cys65 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 H2 to produce prostaglandin D2 catalyzed by the enzyme.

Dominant expression of mRNA for prostaglandin D synthase in leptomeninges, choroid plexus, and oligodendrocytes of the adult rat brain.

Glutathione-independent prostaglandin D synthase [prostaglandin-H2 D-isomerase; (5Z,13E)-(15S)-9 alpha,11 alpha-epidioxy-15-hydroxyprosta-5,13-dienoate D-isomerase, EC 5.3.99.2] is an enzyme responsible for biosynthesis of prostaglandin D2 in the central nervous system. In situ hybridization with antisense RNA for the enzyme indicated that mRNA for the enzyme was predominantly expressed in the leptomeninges, choroid plexus, and oligodendrocytes of the adult rat brain. The findings agree with those obtained by immunohistochemical staining with antibodies against the enzyme. It was further revealed that prostaglandin D synthase activity was considerably greater in the isolated leptomeninges (14.2 nmol per min per mg of protein) and choroid plexus (7.0 nmol per min per mg of protein) than the activity in the whole brain (2.0 nmol per min per mg of protein). These results, taken together, indicate that the enzyme is mainly synthesized and located in the leptomeninges, choroid plexus, and oligodendrocytes in the brain.

Human brain prostaglandin D synthase has been evolutionarily differentiated from lipophilic-ligand carrier proteins.

cDNAs for glutathione-independent prostaglandin D synthase were isolated from cDNA libraries of human brain. The longest cDNA insert was 837 base pairs long and contained a coding region of 570 base pairs corresponding to 190 amino acid residues with a calculated Mr of 21,016. Between two cDNA inserts isolated from the two different libraries, nucleotide substitutions were observed at 16 positions, including conservative amino acid substitutions at 2 positions and nonconservative substitutions at 5 positions, indicating genetic heterogeneity of this enzyme in humans. The computer-assisted homology search revealed that the enzyme is a member of the lipocalin superfamily, comprising secretory hydrophobic molecule transporters, showing the greatest homology (28.8-29.4% identity; 51.3-53.1% similarity) to alpha 1-microglobulin among the members of this superfamily. In a phylogenetic tree of the superfamily, this enzyme, alpha 1-microglobulin, and the gamma chain of the complement component C8 form a cluster separate from the other 14 members. The two distinctive characteristics of glutathione-independent prostaglandin D synthase, as compared to the other members of this superfamily, are its enzymatic properties and its association with membranes that were probably acquired after evolutionary divergence of the two lipocalins. Based on the observed sequence homology, the tertiary structure of the enzyme was deduced to consist of an eight-stranded anti-parallel beta-barrel forming a hydrophobic pocket. Furthermore, the Cys-65 residue in the pocket, which is conserved only in the human and rat enzymes but not in other lipocalins, was considered to be a putative active site of the enzyme.

Prostaglandin D 2 formation and characterization of its synthetases in various tissues of adult rats

When the amounts of primary prostaglandins formed from endogenous arachidonic acid were determined in homogenates of various tissues of adult rats, prostaglandin D 2 was the major prostaglandin found in most tissues. It was formed actively in the spleen (3100 ng/g tissue/5 min at 25 °C), intestine (2600), bone marrow (2400), lung (1100), and stomach (630); moderately in the epididymis, skin, thymus, and brain (140–340); and weakly in other tissues (<100). Addition of exogenous arachidonic acid (1 m m) accelerated the formation of prostaglandin D 2 in all tissues as follows: spleen (15,000); bone marrow, intestine, thymus, liver, and lung (1600–5200); stomach, adrenal gland, epididymis, brain, salivary gland, skin, spinal cord, and seminal vesicle (380–1000); and other tissues (80–310). The activity of prostaglandin D synthetase (prostaglandin-H 2 D-isomerase) was detected in 100,000 g supernatants of almost all tissues. As judged by glutathione requirement for the reaction, inhibition of the activity by 1-chloro-2,4-dinitrobenzene, and immunotitration or immunoabsorption analyses with specific antibodies, the enzyme in the epididymis, brain, and spinal cord (1.8–9.2 nmol/min/mg protein) was glutathione-independent prostaglandin D synthetase ( Y. Urade, N. Fuji-moto, and O. Hayaishi (1985) J. Biol. Chem. 260, 12410–12415 ). The enzyme in the spleen, thymus, bone marrow, intestine, skin, and stomach (2.0–57.1) was glutathione-requiring prostaglandin D synthetase ( Y. Urade, N. Fujimoto, M. Ujihara, and O. Hayaishi (1987) J. Biol. Chem. 262, 3820–3825 ). The activity in the kidney and testis (3.7–4.5) was catalyzed by glutathione S-transferase. The activity in the liver, lung, adrenal gland, salivary gland, heart, pancreas, and muscle (0.6–5.1) was due to both the glutathione-requiring synthetase and the transferase.