Different expression of lipocalin-type prostaglandin D synthase in rat epididymidis

This study was designed to explore the different expression of L-PGDS (lipocalin-type prostaglandin D synthase) in rat epididymidis and to gain further insight into the potential function of L-PGDS in male reproduction. The expression of L-PGDS in rat epididymidis was assessed using real-time quantitative PCR and immunoblotting. The distribution of L-PGDS in rat epididymidis was explored by immunohistochemical methods. The result of immunohistochemistry displayed that L-PGDS was mainly distributed in epididymidis and localized within the cytoplasm and the cilia of the epithelial cells. Real-time quantitative PCR and immunoblotting showed that L-PGDS was strikingly expressed in the caput epididymidis, while a moderate to weak expression was observed in the corpus and cauda epididymidis, the level of mRNA was 0.52+/-0.02 in the caput, 0.48+/-0.03 in the corpus and 0.32+/-0.01 in the cauda epididymidis, the level of protein expression in caput, corpus and the cauda groups was 1, 0.89+/-0.03 and 0.62+/-0.01, which suggested that L-PGDS may play certain kind of role during the process of the spermatozoa maturation.     

Induction of lipocalin-type prostaglandin D synthase in mouse heart under hypoxemia

Hypoxemia is a common manifestation of various disorders and generates pressure overload to the heart. Here we analyzed the expression of lipocalin-type prostaglandin D synthase (L-PGDS) in the heart of C57BL/6 mice kept under normobaric hypoxia (10% O₂) that generates hemodynamic stress. Northern and Western blot analyses revealed that the expression levels of L-PGDS mRNA and protein were significantly increased (>twofold) after 14 days of hypoxia, compared to the mice kept under normoxia. Immunohistochemical analysis indicated that L-PGDS was increased in the myocardium of auricles and ventricles and the pulmonary venous myocardium at 28 days of hypoxia. Moreover, using C57BL/6 mice lacking heme oxygenase-2 (HO-2^−/−), a model of chronic hypoxemia, we showed that the expression level of L-PGDS protein was twofold higher in the heart than that of wild-type mouse. L-PGDS expression is induced in the myocardium under hypoxemia, which may reflect the adaptation to the hemodynamic stress.     

Specific regulation of lipocalin-type prostaglandin D synthase in mouse heart by estrogen receptor beta

Estrogens have important physiological roles in the cardiovascular system. We use DNA microarray technology to study the molecular mechanism of estrogen action in the heart and to identify novel estrogen-regulated genes. In this investigation we identify genes that are regulated by chronic estrogen treatment of mouse heart. We present our detailed characterization of one of these genes, lipocalin-type prostaglandin D synthase (L-PGDS). Northern and Western blot analysis revealed that L-PGDS was induced both by acute and chronic estrogen treatment. Northern blot analysis, using estrogen receptor (ER)-disrupted mice, suggests that L-PGDS is specifically induced by ERbeta in vivo. In further support of ERbeta-selective regulation, we identify a functional estrogen-responsive element in the L-PGDS promoter, the activity of which is up-regulated by ERbeta, but not by ERalpha. We demonstrate that a one-nucleotide change (A to C) in the L-PGDS estrogen-responsive element affects receptor selectivity.     

Binding of biliverdin, bilirubin, and thyroid hormones to lipocalin-type prostaglandin D synthase.

Lipocalin-type prostaglandin D synthase is a major protein of the cerebrospinal fluid and was originally known as beta-trace. We investigated the binding ability of prostaglandin D synthase toward bile pigments, thyroid hormones, steroid hormones, and fatty acids in this present study. We found that the recombinant enzyme binds bile pigments and thyroid hormones, resulting in quenching of the intrinsic tryptophan fluorescence, the appearance of induced circular dichroism of the lipophilic ligands, and a red shift of the absorption spectra of bilirubin and biliverdin. The binding of prostaglandin D synthase to lipophilic ligands was also demonstrated by the resonant mirror technique and surface plasmon resonance detection. The dissociation constants were calculated to be 33 nM, 37 nM, 660 nM, 820 nM, and 2.08 microM for biliverdin, bilirubin, L-thyroxine, 3,3',5'-triiodo-L-thyronine, and 3,3', 5-triiodo-L-thyronine, respectively. Biliverdin and bilirubin underwent a shift in their absorption peaks from 375 to 380 nm and from 439 to 446 nm, respectively, after binding to prostaglandin D synthase. Bilirubin bound to the enzyme showed a bisignate CD spectrum with a (-) Cotton effect at 422 nm and a (+) Cotton effect at 472 nm, indicating a right-handed chirality. The ligands also inhibited prostaglandin D synthase activity noncompetitively in a concentration-dependent manner, with IC50 values between 3.9 and 10. 9 microM. Epididymal retinoic acid-binding protein and beta-lactoglobulin, two other lipocalin proteins that bind retinoids such as prostaglandin D synthase, did not show any significant interaction with bile pigments or thyroid hormones. These results show that prostaglandin D synthase binds small lipophilic ligands with a specificity distinct from that of other lipocalins.     

Immunocytochemical localization of lipocalin-type prostaglandin D synthase in the bull testis and epididymis and on ejaculated sperm

Previously, we identified a 26-kDa fertility-associated protein in bull seminal plasma as lipocalin-type prostaglandin D synthase. The objective of the present study was to immunohistochemically localize this enzyme to the various cell types within the bull testis and seven subsegments of the epididymis, and on ejaculated sperm in order to gain further insight into its potential function in male reproduction. In the testis, immunoperoxidase staining was localized within the elongating spermatids and Sertoli cells of the seminiferous tubules, varying with the stage of the spermatogenic cycle. The highest level of staining occurred during stages III-VII. The cuboidal epithelial cells of the rete testis and efferent ducts were also immunoreactive. Expression of lipocalin-type prostaglandin D synthase was not uniform in the seven epididymal subsegments, suggesting a possible role in sperm maturation. In all epididymal regions, expression was limited to the epithelial principal cells; no immunoreactivity was apparent in other cell types. Lipocalin-type prostaglandin D synthase was strikingly localized in the caput epididymidis, while moderate to weak staining was observed in the remainder of the epididymis. Droplets of reaction product observed within the lumen increased progressively from the caput to cauda. Using fluorescence microscopy, we also localized lipocalin-type prostaglandin D synthase to the apical ridge of the acrosome on ejaculated sperm.     

NMR assignment of 1H, 13C, and 15N resonances of rat lipocalin-type prostaglandin D synthase

Lipocalin-type prostaglandin D synthase (L-PGDS) acts as both a PGD₂-synthesizing enzyme and an extracellular transporter for small lipophilic molecules. Here we report the backbone and side-chain resonance assignments of uniformly ¹⁵N, ¹³C labeled rat L-PGDS.     

Pronounced eosinophilic lung inflammation and Th2 cytokine release in human lipocalin-type prostaglandin D synthase transgenic mice.

PGD(2) is a major lipid mediator released from mast cells, but little is known about its role in the development of allergic reactions. We used transgenic (TG) mice overexpressing human lipocalin-type PGD synthase to examine the effect of overproduction of PGD(2) in an OVA-induced murine asthma model. The sensitization of wild-type (WT) and TG mice was similar as judged by the content of OVA-specific IgE. After OVA challenge, PGD(2), but not PGE(2), substantially increased in the lungs of WT and TG mice with greater PGD(2) increment in TG mice compared with WT mice. The numbers of eosinophils and lymphocytes in the bronchoalveolar lavage (BAL) fluid were significantly greater in TG mice than in WT mice on days 1 and 3 post-OVA challenge, whereas the numbers of macrophages and neutrophils were the same in both WT and TG mice. The levels of IL-4, IL-5, and eotaxin in BAL fluid were also significantly higher in TG mice than in WT mice, although the level of IFN-gamma in the BAL fluid of TG mice was decreased compared with that in WT mice. Furthermore, lymphocytes isolated from the lungs of TG mice secreted less IFN-gamma than those from WT mice, whereas IL-4 production was unchanged between WT and TG mice. Thus, overproduction of PGD(2) caused an increase in the levels of Th2 cytokines and a chemokine, accompanied by the enhanced accumulation of eosinophils and lymphocytes in the lung. These results indicate that PGD(2) plays an important role in late phase allergic reactions in the pathophysiology of bronchial asthma.     

Cloning,expression, crystallization,and preliminary X-ray analysis of recombinant mouse lipocalin-type prostaglandin D synthase,a somnogen-producing enzyme

Lipocalin-type prostaglandin D synthase is the key enzyme for the production of prostaglandin D(2), a potent endogenous somnogen, in the brain. We cloned, produced, and crystallized the native enzyme and selenomethionyl Cys(65)Ala mutants of the recombinant mouse protein by the hanging drop vapor-diffusion method with both malonate and citrate as precipitants. The native crystals obtained with malonate belong to orthorhombic space group P2(1)2(1)2(1) with lattice constants a = 46.2, b = 66.8, and c = 105.3 A. The selenomethionyl crystals obtained with citrate belong to orthorhombic space group C222(1) with lattice constants a = 45.5, b = 66.8, and c = 104.5 A. The native crystals diffracted beyond 2.1 A resolution.     

Biochemical, structural, genetic, physiological, and pathophysiological features of lipocalin-type prostaglandin D synthase

Lipocalin-type prostaglandin (PG) D synthase (PGDS) catalyzes the isomerization of PGH(2), a common precursor of various prostanoids, to produce PGD(2), a potent endogenous somnogen and nociceptive modulator, in the presence of sulfhydryl compounds. PGDS is an N-glycosylated monomeric protein with an M(r) of 20000-31000 depending on the size of the glycosyl moiety. PGDS is localized in the central nervous system and male genital organs of various mammals and in the human heart and is secreted into the cerebrospinal fluid, seminal plasma, and plasma, respectively, as beta-trace. The PGDS concentrations in these body fluids are useful for the diagnosis of several neurological disorders, dysfunction of sperm formation, and cardiovascular and renal diseases. The cDNA and gene for PGDS have been isolated from several animal species, and the tissue distribution and cellular localization have also been determined. This enzyme is considered to be a dual functional protein; i.e. it acts as a PGD(2)-producing enzyme and also as a lipophilic ligand-binding protein, because the enzyme binds biliverdin, bilirubin (K(d)=30 nM), retinaldehyde, retinoic acid (K(d)=80 nM) with high affinities. X-ray crystallographic analyses revealed that PGDS possesses a beta-barrel structure with a hydrophobic pocket in which an active thiol, Cys(65), the active center for the catalytic reaction, was located facing to the inside of the pocket. Gene-knockout and transgenic mice for PGDS were generated and found to have abnormalities in the regulation of nociception and sleep.     

Expression and regulation of lipocalin-type prostaglandin d synthase in rat testis and epididymis

Lipocalin-type prostaglandin D synthase (L-PGDS), a bifunctional protein, is expressed in the male reproductive organs of many species. However, the expression and regulation of L-PGDS in rat are still uncertain. The present study investigated the regionalization and regulation of L-PGDS expression in rat testis and epididymis by in situ hybridization and immunohistochemistry under the conditions of sexual maturation, castration, and ethylene dimethane sulfonate (EDS) treatments. In sexually mature rats, L-PGDS mRNA was weakly expressed only in the testicular peritubular cells, whereas L-PGDS immunostaining was highly detected in the Leydig cells by Day 70 postpartum. During sexual maturation, L-PGDS mRNA expression was highly detected in the caput, corpus, and cauda of the epididymis 70 days after birth. Compared with normal L-PGDS expression in adult epididymis, both L-PGDS mRNA expression and protein immunostaining were significantly reduced in the caput, corpus, and cauda epididymis after castration. Testosterone propionate treatment induced a significant increase of L-PGDS expression in the epididymis of castrated rats. Compared with adult rat epididymis, L-PGDS mRNA and protein expression was down-regulated after EDS treatment. Testosterone propionate treatment could induce an increase of L-PGDS mRNA and protein expression in the epididymis of EDS-treated rats. In conclusion, both castration and EDS treatments caused a significant decrease of L-PGDS expression in the epididymis, whereas testosterone propionate treatment could induce an increase of L-PGDS expression in the epididymis of both castrated and EDS-treated rats, indicating that L-PGDS expression in the rat epididymis can be up-regulated by testosterone.     

Single-chain antibody against human lipocalin-type prostaglandin D synthase: Construction, expression, purification, and activity assay

An active form of single-chain antibody (ScFv) from murine monoclonal antibody 4A7, which is specific for lipocalin-type prostaglandin D synthase (L-PGDS), was produced in Escherichia coli. The complementary DNA fragments encoding the variable regions of heavy chain (VH) and light chain (VL), which amplified from hybridoma 4A7 producing a monoclonal antibody (IgG1) against L-PGDS, were connected by a (Gly4Ser)3 linker using an assembly polymerase chain reaction. The resultant ScFv were cloned into the vector pGEM and expressed in E. coli as inclusion bodies. The expressed ScFv fusion proteins were purified by Ni2+-nitrilotriacetic acid chromatography. The purity and activity of purified ScFv were confirmed by SDS-PAGE and ELISA. The result revealed that 4A7 ScFv conserved the same characteristics of specific recognition and binding to sperm as the parental 4A7 monoclonal antibody.     

1H, 13C,and 15N resonance assignments of mouse lipocalin-type prostaglandin D synthase/substrate analog complex

Lipocalin-type Prostaglandin D synthase (L-PGDS) acts as the PGD₂-synthesizing enzyme in the brain of various mammalian species. It belongs to the lipocalin superfamily and is the first member of this family to be recognized as an enzyme. Although the solution and crystal structure of L-PGDS has been determined to understand the molecular mechanism of catalytic reaction, the structural analysis of L-PGDS in complex with its substrate remains to be performed. Here, we present the nearly complete assignment of the backbone and side chain resonances of L-PGDS/substrate analog (U-46619) complex. This study lays the essential basis for further understanding the substrate recognition mechanism of L-PGDS.     

NMR and CD analysis of an intermediate state in the thermal unfolding process of mouse lipocalin-type prostaglandin D synthase

We previously reported that the thermal unfolding of mouse lipocalin-type prostaglandin D synthase (L-PGDS) is a completely reversible process under acidic conditions and follows a three-state pathway, including an intermediate state (I) between native state (N) and unfolded state. In the present study, we investigated the intermediate state of mouse C65A L-PGDS and clarified the local conformational changes in the upper and bottom regions by using NMR and CD spectroscopy. The (1)H-(15)N HSQC measurements revealed that the backbone conformation was disrupted in the upper region of the β-barrel at 45°C, which is around the T(m) value for the N ? I transition, but that the signals of the residues located at the bottom region of L-PGDS remained at 54°C, where the maximum accumulation of the intermediate state was found. (1)H-NMR and CD measurements showed that the T(m) values obtained by monitoring Trp54 at the upper region and Trp43 at the bottom region of the β-barrel were 41.4 and 47.5°C, respectively, suggesting that the conformational change in the upper region occurred at a lower temperature than that in the bottom region. These findings demonstrate that the backbone conformation of the bottom region is still maintained in the intermediate state.     

Expression and purification of cysteine mutation isoforms of rat lipocalin-type prostaglandin D synthase for nuclear magnetic resonance study

Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) is the only member of the lipocalin superfamily that displays enzymatic activity. It binds lipophilic ligands with high affinity and also can catalyze PGH₂ to produce PGD₂. Three cysteine residues, Cys⁶⁵, Cys⁸⁹, and Cys¹⁸⁶ in L-PGDS, are conserved among all species, of which Cys⁸⁹ and Cys¹⁸⁶ residues form a disulfide bridge. In this study, we clarified the effects of thiol groups on the structure of the protein and investigated the structural significance of Cys residues of rat L-PGDS by site-directed mutagenesis. Four mutants were constructed by substituting Cys residues with alanine to identify the correct formation of disulfide bonds among these three residues. The effects of thiol groups on the structure of rat L-PGDS were also identified by these mutants. Analysis of HSQC experiments indicated that these enzymes were all properly folded with well defined tertiary structures. As the first step towards the 3-D nuclear magnetic resonance solution structure, we optimized expression of recombinant rat L-PGDS in Escherichia coli and established an efficient and economic purification protocol yielding large amounts of pure isotopically labeled rat L-PGDS. The results of assignments indicated that the wild-type rat L-PGDS obtained using this expression system was suitable for determination of 3-D nuclear magnetic resonance solution structure.     

Unexpected role of lipocalin-type prostaglandin D synthase in brain: Regulation of glial cell migration and morphology

Lipocalin-type prostaglandin D synthase (L-PGDS) is one of the most abundant proteins in the cerebrospinal fluid. Nevertheless, its role in the central nervous system is far from clear. Here, we present evidence that L-PGDS induces glial cell migration and morphological changes in vitro and in vivo. We also identified myristoylated alanine-rich C-kinase substrate (MARCKS), heat shock proteins and actin as L-PGDS-binding proteins, demonstrating that MARCKS/Akt/Rho/Jnk pathways are involved in the L-PGDS actions in glia. We further show that the cell migration-promoting activity of L-PGDS is independent of PGD₂ production. The results suggest a novel non-enzymatic function of L-PGDS protein in brain inflammation, and may have an impact on glial cell biology and brain pathology related with reactive gliosis. L-PGDS is a potential drug target that can be exploited for therapeutic intervention of glia-driven neuroinflammation and related diseases.     

Metabolism of prostaglandin D2 in the monkey.

[3H7]Prostaglandin D2 was biosynthesized and infused into an unanesthetized monkey. The urinary metabolites were isolated and subsequently identified by gas chromatography-mass spectrometry. Two pathways of prostaglandin D2 metabolism were identified and resulted in metabolites with prostaglandin D (3-hydroxycyclopentanone) and prostaglandin F (cyclopentane-1,3-diol) ring structures. The major prostaglandin D ring metabolite was identified as 9,20-dihydroxy-11,15-dioxo-2,3-dinorprost-5-en-1-oic acid. Nine other prostaglandin D ring metabolites were identified reflecting various combinations of metabolism by beta and omega oxidation, 15 dehydrogenation, and 13-14 reduction. In greater abundance were those prostaglandin D2 metabolites which had the prostaglandin F ring structure. The major prostaglandin D2 metabolite which had the prostaglandin F ring structure was identified as 9,11,15-trihydroxy-2,3-dinorprosta-5,13-dien-1-oic acid (dinor prostaglandin F2 alpha). Nine other metabolites with the prostaglandin F ring structure were identified, including prostaglandin F2 alpha itself. These, for the most part, were the structural counterparts of the metabolites with the prostaglandin D ring. Since many prostaglandin D2 metabolites were found to be identical with the metabolites of prostaglandin F2 alpha, quantitative determinations of prostaglandin F ring metabolites may not be a specific indicator of prostaglandin F2 alpha biosynthesis. Likewise, data involving the measurement of a biological effect of prostaglandin D2 must be re-examined to account for the possible contribution of prostaglandin F2 alpha, a metabolite of prostaglandin D2, to the biological response.     

Cellular retinoic acid-binding protein messenger RNA: levels in rat tissues and localization in rat testis.

Studies were conducted to explore the tissue- and cell-specific regulation of cellular retinoic acid-binding protein (CRABP) expression in the rat. Two studies were carried out. The first explored the regulation of CRABP mRNA levels in selected rat tissues by dietary retinoid status, and the relationship between CRABP mRNA and protein levels in different tissues. The second examined the cellular localization of CRABP expression in the testis. In order to conduct these experiments, a cDNA encoding CRABP was isolated and characterized. The DNA sequence of the coding region had 96% identity with that of the mouse CRABP cDNA and encodes a protein identical to mouse and bovine CRABP. CRABP mRNA and protein levels were quantified in five tissues from normal, retinoid-deficient, and retinol-repleted rats. Tissue CRABP and CRABP mRNA levels were highly correlated (P less than 0.01) indicating that inter-tissue variability of CRABP levels mainly results from regulation of CRABP mRNA levels. Neither CRABP protein nor mRNA levels were affected by retinol deficiency, in marked contrast with results previously demonstrated with cellular retinol-binding protein (CRBP) (J. Lipid Res. 1990. 31: 821-829). 35S-labeled CRABP cRNA probes were used to localize CRABP mRNA within the testis of adult rats by in situ hybridization. CRABP mRNA was localized selectively in the periphery of the seminiferous tubules, primarily in type A spermatogonia. The localization of CRABP mRNA differs from that of CRABP protein, which is known to be enriched in maturing and more mature germinal cells. This difference suggests that CRABP in germ cells may be highly stable, remaining in the maturing germ cells without degradation long after CRABP mRNA levels have declined to very low levels. The specific localization of CRABP mRNA and protein presumably reflects the biological roles of retinoic acid in the development and/or later function of germinal cells.     

Comparative study of the asparagine-linked sugar chains of human lipocalin-type prostaglandin D synthase purified from urine and amniotic fluid, and recombinantly expressed in Chinese hamster ovary cells

Lipocalin-type prostaglandin D synthase (L-PGDS) is a highly glycosylated member of the lipocalin gene family and is secreted into various human body fluids. We comparatively analyzed the structures of asparagine-linked sugar chains of human L-PGDS produced by recombinant Chinese hamster ovary cells and naturally occurring human urine and amniotic fluid. After the sugar chains were liberated by hydrazinolysis followed by N-acetylation, they were derivatized with 2-aminobenzamide. All of the sugar chains of three L-PGDSs occur as biantennary complex-type sugar chains. Most of the sugar chains of three samples were fucosylated on the inner most N-acetylglucosamine residue. Although the sugar chains of the recombinant L-PGDS do not contain any bisecting N-acetylglucosamine residues, 58% and 34% of the fucosylated-sugar chains of amniotic fluid and urine L-PGDSs, respectively, contain bisecting N-acetylglucosamine residues. The sialic acid residues occur solely as Siaalpha2-->3Gal groups of the recombinant L-PGDS; the sialic acid residues of other L-PGDS occur as both Siaalpha2-->3Gal and Siaalpha2-->6Gal groups. Variations in L-PGDS glycosylation may prove useful as markers to further elucidate the role of L-PGDS glycoforms in different tissues.