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.     

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.     

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.     

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.     

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.     

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.     

The model plant Medicago truncatula exhibits biparental plastid inheritance

The plastid, which originated from the endosymbiosis of a cyanobacterium, contains its own plastid DNA (ptDNA) that exhibits a unique mode of inheritance. Approximately 80% of angiosperms show maternal inheritance, whereas the remainder exhibit biparental inheritance of ptDNA. Here we studied ptDNA inheritance in the model legume, Medicago truncatula. Cytological analysis of mature pollen with DNA-specific fluorescent dyes suggested that M. truncatula is one of the few model plants potentially showing biparental inheritance of ptDNA. We further examined pollen by electron microscopy and revealed that the generative cell (a mother of sperm cells) indeed has many DNA-containing plastids. To confirm biparental inheritance genetically, we crossed two ecotypes (Jemalong A17 and A20), and the transmission mode of ptDNA was investigated by a PCR-assisted polymorphism. Consistent with the cytological observations, the majority of F(1) plants possessed ptDNAs from both parents. Interestingly, cotyledons of F(1) plants tended to retain a biparental ptDNA population, while later emergent leaves tended to be uniparental with either one of the parental plastid genotypes. Biparental transmission was obvious in the F(2) population, in which all plants showed homoplasmy with either a paternal or a maternal plastid genotype. Collectively, these data demonstrated that M. truncatula is biparental for ptDNA transmission and thus can be an excellent model to study plastid genetics in angiosperms.     

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.     

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.     

Medicago truncatula as a model for nonhost resistance in legume-parasitic plant interactions

Crenate broomrape (Orobanche crenata) is a root parasitic weed that represents a major constraint for grain legume production in Mediterranean and West Asian countries. Medicago truncatula has emerged as an important model plant species for structural and functional genomics. The close phylogenic relationship of M. truncatula with crop legumes increases its value as a resource for understanding resistance against Orobanche spp. Different cytological methods were used to study the mechanisms of resistance against crenate broomrape of two accessions of M. truncatula, showing early and late acting resistance. In the early resistance accession (SA27774) we found that the parasite died before a tubercle had formed. In the late resistance accession (SA4327) the parasite became attached without apparent problems to the host roots but most of the established tubercles turned dark and died before emergence. The results suggest that there are defensive mechanisms acting in both accessions but with a time gap that is crucial for a higher success avoiding parasite infection.     

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.     

Identification and characterization of nucleotide-binding site-leucine-rich repeat genes in the model plant Medicago truncatula

The nucleotide-binding site (NBS)-Leucine-rich repeat (LRR) gene family accounts for the largest number of known disease resistance genes, and is one of the largest gene families in plant genomes. We have identified 333 nonredundant NBS-LRRs in the current Medicago truncatula draft genome (Mt1.0), likely representing 400 to 500 NBS-LRRs in the full genome, or roughly 3 times the number present in Arabidopsis (Arabidopsis thaliana). Although many characteristics of the gene family are similar to those described on other plant genomes, several evolutionary features are particularly pronounced in M. truncatula, including a high degree of clustering, evidence of significant numbers of ectopic translocations from clusters to other parts of the genome, a small number of more evolutionarily stable NBS-LRRs, and numerous truncations and fusions leading to novel domain compositions. The gene family clearly has had a large impact on the structure of the genome, both through ectopic translocations (potentially, a means of seeding new NBS-LRR clusters), and through two extraordinarily large superclusters. Chromosome 6 encodes approximately 34% of all TIR-NBS-LRRs, while chromosome 3 encodes approximately 40% of all coiled-coil-NBS-LRRs. Almost all atypical domain combinations are in the TIR-NBS-LRR subfamily, with many occurring within one genomic cluster. This analysis shows the gene family not only is important functionally and agronomically, but also plays a structural role in the genome.     

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.     

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.