Thermal unfolding mechanism of lipocalin-type prostaglandin D synthase

Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) is a dual-functioning protein in the lipocalin family, acting as a PGD(2)-synthesizing enzyme and as an extracellular transporter for small lipophilic molecules. We earlier reported that denaturant-induced unfolding of L-PGDS follows a four-state pathway, including an activity-enhanced state and an inactive intermediate state. In this study, we investigated the thermal unfolding mechanism of L-PGDS by using differential scanning calorimetry (DSC) and CD spectroscopy. DSC measurements revealed that the thermal unfolding of L-PGDS was a completely reversible process at pH 4.0. The DSC curves showed no concentration dependency, demonstrating that the thermal unfolding of L-PGDS involved neither intermolecular interaction nor aggregation. On the basis of a simple two-state unfolding mechanism, the ratio of van't Hoff enthalpy (DeltaH(vH)) to calorimetric enthalpy (DeltaH(cal)) was below 1, indicating the presence of an intermediate state (I) between the native state (N) and unfolded state (U). Then, statistical thermodynamic analyses of a three-state unfolding process were performed. The heat capacity curves fit well with a three-state process; and the estimated transition temperature (T(m)) and enthalpy change (DeltaH(cal)) of the N<-->I and I<-->U transitions were 48.2 degrees C and 190 kJ.mol(-1), and 60.3 degrees C and 144 kJ.mol(-1), respectively. Correspondingly, the thermal unfolding monitored by CD spectroscopy at 200, 235 and 290 nm revealed that L-PGDS unfolded through the intermediate state, where its main chain retained the characteristic beta-sheet structure without side-chain interactions.     

Unexpected role of lipocalin-type prostaglandin D synthase in brain

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.     

Characterization of the unfolding process of lipocalin-type prostaglandin D synthase

We found that low concentrations of guanidine hydrochloride (GdnHCl, <0.75 M) or urea (<1.5 M) enhanced the enzyme activity of lipocalin-type prostaglandin (PG) D synthase (L-PGDS) maximally 2.5- and 1.6-fold at 0.5 M GdnHCl and 1 M urea, respectively. The catalytic constants in the absence of denaturant and in the presence of 0.5 M GdnHCl or 1 m urea were 22, 57, and 30 min(-1), respectively, and the K(m) values for the substrate, PGH(2), were 2.8, 8.3, and 2.3 microm, respectively, suggesting that the increase in the catalytic constant was mainly responsible for the activation of L-PGDS. The intensity of the circular dichroism (CD) spectrum at 218 nm, reflecting the beta-sheet content, was also increased by either denaturant in a concentration-dependent manner, with the maximum at 0.5 M GdnHCl or 1 M urea. By plotting the enzyme activities against the ellipticities at 218 nm of the CD spectra of L-PGDS in the presence or absence of GdnHCl or urea, we found two states in the reversible folding process of L-PGDS: one is an activity-enhanced state and the other, an inactive state. The NMR analysis of L-PGDS revealed that the hydrogen-bond network was reorganized to be increased in the activity-enhanced state formed in the presence of 0.5 M GdnHCl or 1 m urea and to be decreased but still remain in the inactive intermediate observed in the presence of 2 M GdnHCl or 4 M urea. Furthermore, binding of the nonsubstrate ligands, bilirubin or 13-cis-retinal, to L-PGDS changed from a multistate mode in the native form of L-PGDS to a simple two-state mode in the activity-enhanced form, as monitored by CD spectra of the bound ligands. Therefore, L-PGDS is a unique protein whose enzyme activity and ligand-binding property are biphasically altered during the unfolding process by denaturants.     

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.     

Enhancement of lipocalin-type prostaglandin D synthase enzyme activity by guanidine hydrochloride

The characterization of unfolding of mouse recombinant lipocalin-type prostaglandin D synthase (L-PGDS) by guanidine hydrochloride (GdnHCl) was carried out. In the presence of low concentrations of GdnHCl (up to 0.75 M), enhancement of the enzyme activity was observed. However, above a 1 M concentration of GdnHCl, the enzyme activity was reduced in a concentration-dependent manner. The maximum enzyme activity induced by GdnHCl was approximately 1. 5-fold compared with the activity under physiological conditions without GdnHCl. The ellipticity in circular dichroism (CD) spectrum of the L-PGDS at 218 nm, reflecting the beta-sheet content, was decreased by GdnHCl (up to 0.75 M), and the minimum ellipticity was observed at 0.5 M GdnHCl. The fluorescence quenching of the intrinsic tryptophan of L-PGDS due to the binding of bilirubin in the presence or absence of GdnHCl was measured. The K(d) values obtained in the presence and absence of 0.5 M GdnHCl were 447 and 115 nM, respectively, indicating lower affinity of the L-PGDS for bilirubin with GdnHCl than without it. Further, an NMR study revealed that the reorganization of hydrogen-bond network in the L-PGDS was observed in the presence of 0.5 M GdnHCl. These results, taken together, indicate that the enzyme activity of L-PGDS is enhanced by the conformational change, especially by the change in the secondary structure.     

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.     

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.     

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.     

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.     

Prevention of paraquat-induced apoptosis in human neuronal SH-SY5Y cells by lipocalin-type prostaglandin D synthase

Paraquat is a widely used herbicide that is structurally similar to the known dopaminergic neurotoxicant 1-methyl-4-phenyl-pyridine and acts as a potential etiologic factor for the development of Parkinson's disease. In this study, we investigated the protective roles of lipocalin-type prostaglandin (PG) D synthase (L-PGDS) against paraquat-mediated apoptosis of human neuronal SH-SY5Y cells. The treatment of SH-SY5Y cells with paraquat decreased the intracellular GSH level, and enhanced the cell death with elevation of the caspase activities. L-PGDS was expressed in SH-SY5Y cells, and its expression was enhanced with the peak at 2?h after the initiation of the treatment with paraquat. Inhibition of PGD? synthesis and exogenously added PGs showed no effects regarding the paraquat-mediated apoptosis. SiRNA-mediated suppression of L-PGDS expression in the paraquat-treated cells increased the cell death and caspase activities. Moreover, over-expression of L-PGDS suppressed the cell death and caspase activities in the paraquat-treated cells. The results of a promoter-luciferase assay demonstrated that paraquat-mediated elevation of L-PGDS gene expression occurred through the NF-κB element in the proximal promoter region of the L-PGDS gene in SH-SY5Y cells. These results indicate that L-PGDS protected against the apoptosis in the paraquat-treated SH-SY5Y cells through the up-regulation of L-PGDS expression via the NF-κB element. Thus, L-PGDS might potentially serve as an agent for prevention of human neurodegenerative diseases caused by oxidative stress and apoptosis.     

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.     

Fine-tuned broad binding capability of human lipocalin-type prostaglandin D synthase for various small lipophilic ligands

The hydrophobic cavity of lipocalin-type prostaglandin D synthase (L-PGDS) has been suggested to accommodate various lipophilic ligands through hydrophobic effects, but its energetic origin remains unknown. We characterized 18 buffer-independent binding systems between human L-PGDS and lipophilic ligands using isothermal titration calorimetry. Although the classical hydrophobic effect was mostly detected, all complex formations were driven by favorable enthalpic gains. Gibbs energy changes strongly correlated with the number of hydrogen bond acceptors of ligand. Thus, the broad binding capability of L-PGDS for ligands should be viewed as hydrophilic interactions delicately tuned by enthalpy–entropy compensation using combined effects of hydrophilic and hydrophobic interactions.     

Effects of removing a conserved disulfide bond on the biological characteristics of rat lipocalin-type prostaglandin D synthase

Three cysteine residues, Cys(65), Cys(89), and Cys(186) in lipocalin-type prostaglandin D synthase (L-PGDS), are conserved among all species and the disulfide bond between Cys(89) and Cys(186) is highly conserved among most, but not all, lipocalins. In this study, four rat L-PGDS variants were constructed by site-directed mutagenesis, and the conserved disulfide bond in several variants was removed by substituting cysteine with alanine. The effects of removing this disulfide bond on their biological characteristics were investigated. The NMR experiments indicated that the removal of disulfide did not change their conformations significantly. However, both thermal-induced and urea-induced unfolding experiments showed that the stabilities of enzymes without the disulfide bond decreased significantly. Moreover, the ligand-binding affinities of these variants were assessed by fluorescence experiments. Dissociation constants (K(d)) of 0.668, 0.689, 0.543 and 0.571 microM were obtained for ANS binding to wild-type rat L-PGDS, C(65)A, C(186)A, and C(89,186)A variants, respectively, and 71.2 and 62.3 nM for retinoic acid binding to wild-type rat L-PGDS and the C(186)A variant, respectively. These results suggested that the removal of the disulfide bond slightly increased the affinities for ligand binding by changing the hydrophobic regions. This study may offer valuable information for further studies on other rat lipocalins.     

Relationship between contents of lipocalin-type prostaglandin D synthase on the surface of infertility sperm and in seminal plasma

Lipocalin-type prostaglandin D synthase (L-PGDS) is localized in Leydig cells, sperm, and epithelial cells of the epididymis. The present study was to determine the correlation between content of this enzyme in seminal plasma and on the surface of sperm. We analyzed 90 semen samples. L-PGDS in seminal plasma was analyzed by an ELISA procedure. L-PGDS on sperm was analyzed by flow cytometry. The semen donors were categorized in three groups: normal, oligospermic, and azoospermic. According to results obtained, L-PGDS may have the ability to improve progressive motility of sperm, and L-PGDS in seminal plasma and on sperm surface may impact male fertility in the female reproductive tract. Published in Russian in Biokhimiya, 2007, Vol. 72, No. 2, pp. 255–259.     

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.