Lipocalin-type Prostaglandin (PG) D Synthase Research Articles

NMR resonance assignments of mouse lipocalin-type prostaglandin D synthase/prostaglandin J2 complex

Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) catalyzes the isomerization of PGH2 to produce PGD2, an endogenous somenogen, in the brains of various mammalians. We recently reported that various other PGs also bind to L-PGDS, suggesting that it could serve as an extracellular carrier for PGs. Although the solution and crystal structure of L-PGDS has been determined, as has the structure of L-PGDS complexed PGH2 analog, a structural analysis of L-PGDS complexed with other PGs is needed in order to understand the mechanism responsible for the PG trapping. Here, we report the nearly complete 1H, 13C, and 15N backbone and side chain resonance assignments of the L-PGDS/PGJ2 complex and the binding site for PGJ2 on L-PGDS.

Lipocalin-type prostaglandin D synthase regulates light-induced phase advance of the central circadian rhythm in mice

We previously showed that mice lacking pituitary adenylate cyclase-activating polypeptide (PACAP) exhibit attenuated light-induced phase shift. To explore the underlying mechanisms, we performed gene expression analysis of laser capture microdissected suprachiasmatic nuclei (SCNs) and found that lipocalin-type prostaglandin (PG) D synthase (L-PGDS) is involved in the impaired response to light stimulation in the late subjective night in PACAP-deficient mice. L-PGDS-deficient mice also showed impaired light-induced phase advance, but normal phase delay and nonvisual light responses. Then, we examined the receptors involved in the response and observed that mice deficient for type 2 PGD2 receptor DP2/CRTH2 (chemoattractant receptor homologous molecule expressed on Th2 cells) show impaired light-induced phase advance. Concordant results were observed using the selective DP2/CRTH2 antagonist CAY10471. These results indicate that L-PGDS is involved in a mechanism of light-induced phase advance via DP2/CRTH2 signaling.

L-PGDS-produced PGD2 in premature, but not in mature, adipocytes increases obesity and insulin resistance

Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) is responsible for the production of PGD2 in adipocytes and is selectively induced by a high-fat diet (HFD) in adipose tissue. In this study, we investigated the effects of HFD on obesity and insulin resistance in two distinct types of adipose-specific L-PGDS gene knockout (KO) mice: fatty acid binding protein 4 (fabp4, aP2)-Cre/L-PGDSflox/flox and adiponectin (AdipoQ)-Cre/L-PGDSflox/flox mice. The L-PGDS gene was deleted in adipocytes in the premature stage of the former strain and after maturation of the latter strain. The L-PGDS expression and PGD2 production levels decreased in white adipose tissue (WAT) under HFD conditions only in the aP2-Cre/L-PGDSflox/flox mice, but were unchanged in the AdipoQ-Cre/L-PGDSflox/flox mice. When fed an HFD, aP2-Cre/L-PGDSflox/flox mice significantly reduced body weight gain, adipocyte size, and serum cholesterol and triglyceride levels. In WAT of the HFD-fed aP2-Cre/L-PGDSflox/flox mice, the expression levels of the adipogenic, lipogenic, and M1 macrophage marker genes were decreased, whereas those of the lipolytic and M2 macrophage marker genes were enhanced or unchanged. Insulin sensitivity was improved in the HFD-fed aP2-Cre/L-PGDSflox/flox mice. These results indicate that PGD2 produced by L-PGDS in premature adipocytes is involved in the regulation of body weight gain and insulin resistance under nutrient-dense conditions.

Lipocalin-Type Prostaglandin D Synthase Possesses Two Binding Sites for its Product

Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) belongs to the lipocalin superfamily which consists of transporter proteins for lipophilic ligands in the extracellular space, and is referred to as the PGD2-synthesizing enzyme, which is responsible for sleep regulation. Recently, we found that L-PGDS binds, not only to its substrate, PGH2, but also to its product, PGD2. However, the mechanism responsible for the binding of L-PGDS to its product, PGD2, remains unclear. In the present study, to address this issue, we investigated the interactions between L-PGDS and its ligand, such as PGD2, PGF2α and PGE2.

Isothermal Analysis of Interaction between Lipocalin-Type Prostaglandin D Synthase and Prostanoids

Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) catalyzes the isomerization of PGH2 to produce PGD2, which acts as a endogenous somnogen in the brain. This enzyme belongs to the lipocalin superfamily which consists of transporter proteins for lipophilic substances in the extracellular space. Our previous studies suggested that L-PGDS consists of a β-barrel structure with a hydrophobic pocket where the active thiol group of the Cys65 residue is located.Numerous studies of L-PGDS, as a drug target for sleep disorders, have been reported in attempts to understand its catalytic mechanism, and several substrate recognition models of L-PGDS have been proposed. However, details of the mechanism by which L-PGDS recognizes its substrates and he production of products have not been determined yet. In the present study, we report on an investigation of the interaction between L-PGDS and prostanoids using isothermal titration calorimetry (ITC) and propose the mechanism responsible for the substrate recognition of L-PGDS.An ITC analysis of the binding of L-PGDS and its substrates suggested that all prostanoids are able to bind to L-PGDS and that the 9-hydroxy group of prostanoids play a significant role in the binding to the catalytic site of L-PGDS. In addition, the results also indicate that L-PGDS acts as a extracellular transporter of PGF2α. These results will be discussed in this presenation.

Structural Analysis of Lipocalin-Type Prostaglandin D Synthase Complexed with Prostaglandin J2

Lipocalin-type prostaglandin (PG) D synthase (L-PGDS), a member of the lipocalin superfamily, is present in the brain. L-PGDS catalyzes the isomerization of PGH2 to produce PGD2, a potent endogenous somnogen. L-PGDS-produced PGD2 is sleep-inducing and accumulates in the brain during prolonged periods of wakefulness. We recently found that PGJ2, a metabolite of PGD2, inhibits the enzyme activity of L-PGDS and displays a much stronger inhibitory activity than other reported inhibitors, such as retinoids and AT-56. Therefore, information regarding the binding of the L-PGDS/PGJ2 complex should provide useful information for the development of drugs for treating sleep disorders. In this study, we report on an investigation of the three-dimensional structure of recombinant mouse L-PGDS complexed with PGJ2 by NMR and a proposed binding mechanism from the deduced molecular structure.Chemical shift assignments of the backbone and side-chain protons of L-PGDS/PGJ2 complex were done using two-dimensional 1H-15N HSQC, three-dimensional HNCACB, CBCA(CO)NH, HBHA(CO)NH, CCH-TOCSY, and HCCH-TOCSY. NOEs were collected from three-dimensional 13C-edited and 15N-edited NOESY experiments. In addition, to investigate the binding site of PGJ2, we performed NMR titration experiments and compared the HSQC of L-PGDS/PGJ2 complex with that of apo-L-PGDS. Significant changes in the chemical shifts of the catalytic Cys65 residue and other residues located in the bottom area of the binding pocket of L-PGDS were observed upon PGJ2 binding, indicating that PGJ2 competitively binds to the substrate binding site. NMR titration experiments further revealed that the exchange rate between the PGJ2 bound and unbound state is slow on the NMR time scale, suggesting that PGJ2 binds tightly to the L-PGDS with a dissociation constant in the nanomolar (nM) range.

Structural Analysis of Lipocalin-Type Prostaglandin D Synthase Complexed with Prostaglandin J2

Lipocalin-type prostaglandin (PG) D synthase (L-PGDS), a member of the lipocalin superfamily, is present in the brain. L-PGDS catalyzes the isomerization of PGH2 to produce PGD2, a potent endogenous somnogen. L-PGDS-produced PGD2 is sleep-inducing and accumulates in the brain during prolonged periods of wakefulness. We recently found that PGJ2, a metabolite of PGD2, inhibits the enzyme activity of L-PGDS and displays a much stronger inhibitory activity than other reported inhibitors, such as retinoids and AT-56. Therefore, information regarding the binding of the L-PGDS/PGJ2 complex should provide useful information for the development of drugs for treating sleep disorders. In this study, we report on an investigation of the three-dimensional structure of recombinant mouse L-PGDS complexed with PGJ2 by NMR and a proposed binding mechanism from the deduced molecular structure.Chemical shift assignments of the backbone and side-chain protons of L-PGDS/PGJ2 complex were done using two-dimensional 1H-15N HSQC, three-dimensional HNCACB, CBCA(CO)NH, HBHA(CO)NH, CCH-TOCSY, and HCCH-TOCSY. NOEs were collected from three-dimensional 13C-edited and 15N-edited NOESY experiments. In addition, to investigate the binding site of PGJ2, we performed NMR titration experiments and compared the HSQC of L-PGDS/PGJ2 complex with that of apo-L-PGDS. Significant changes in the chemical shifts of the catalytic Cys65 residue and other residues located in the bottom area of the binding pocket of L-PGDS were observed upon PGJ2 binding, indicating that PGJ2 competitively binds to the substrate binding site. NMR titration experiments further revealed that the exchange rate between the PGJ2 bound and unbound state is slow on the NMR time scale, suggesting that PGJ2 binds tightly to the L-PGDS with a dissociation constant in the nanomolar (nM) range.

Structural and Dynamic Analysis of Lipocalin Type Prostaglandin D Synthase in its APO form and Substrate Analog Complex Form

Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) is endowed with dual functions as a PGD2-synthesizing enzyme and a transporter for lipophilic ligands in the central nervous system. The structure of apo-L-PGDS has already been reported, and substrate docking models and mutational studies has revealed the mechanism of activation of Cys65 and the important residues for substrate recognition at the barrel retion. However, the information of residues involved in the substrate recognition has been almost limited to the region around the catalytic center Cys65. Here, we present the first structure of mouse L-PGDS/substrate analog (U-46619) complex determined by using NMR. Structural and dynamic comparison with apo and complex form revealed that apo-L-PGDS has large open state with overall slow motions and becomes rigid closed state upon substrate analog binding. This conformational change induced the interaction of residues, which are located at the entrance region (H2-helix, CD-loop and EF-loop) with substrate analog. Especially, Phe55 and Pro110 cover the ω chain of the substrate via hydrophobic contacts and fix it in the cavity. Further mutational and kinetic studies demonstrated that the mutation of these residues causes the decrease of affinity for substrate, indicating that the entrance region of L-PGDS is important for the substrate recognition as well as the catalytic region in the barrel.

Activation of adipogenesis by lipocalin-type prostaglandin D synthase-generated Δ12-PGJ2 acting through PPARγ-dependent and independent pathways

Lipocalin-type prostaglandin (PG) D synthase (L-PGDS)-produced PGD2 accelerates adipogenesis. In this study, we investigated the molecular mechanism of PGD2-mediated activation of adipogenesis in mouse adipocytic 3T3-L1 cells. LC/MS analysis showed that Δ12-PGJ2, one of the PGD2 metabolites, was predominantly produced in the differentiated 3T3-L1 cells. Δ12-PGJ2 enhanced the expression of adipogenic genes in a Δ12-PGJ2-concentration-dependent manner. Suppression of the expression of the adipogenic genes by L-PGDS siRNA or AT-56, an L-PGDS inhibitor, was cleared by the addition of Δ12-PGJ2. Moreover, the production of adiponectin and leptin was increased by treatment with Δ12-PGJ2. Furthermore, the results of a mammalian two-hybrid assay demonstrated that Δ12-PGJ2 enhanced the PPARγ-mediated transcription activity. However, Δ12-PGJ2-activated expression of adipogenic genes such as fatty acid binding protein 4 (aP2) and stearoyl-CoA desaturase was inhibited only at 38% and 42%, respectively, by treatment with GW9662, a PPARγ antagonist in 3T3-L1 cells, although Troglitazone-mediated activation of the expression of these adipogenic genes was completely suppressed by GW9662, suggesting the existence of a PPARγ-independent mechanism for Δ12-PGJ2-activated adipogenesis. These results, taken together, indicate that Δ12-PGJ2 is a dominant metabolite of L-PGDS-produced PGD2 during adipogenesis and acts as an activator for adipogenesis through both PPARγ-dependent and -independent mechanisms in 3T3-L1 cells.

ChemInform Abstract: Ligand Recognition Mechanism of Lipocalin‐Type Prostaglandin D Synthase

AbstractReview: 22 refs.

Ligand Recognition of Lipocalin-Type Prostaglandin D Synthase

Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) is a multi functional protein that functions as a PGD2 synthesizing enzyme, a scavenger of various lipophilic ligands, and an amyloid β chaperone in the brain. L-PGDS is a member of the lipocalin superfamily and has the ability to bind various lipophilic molecules, such as prostanoid, retinoid, bile pigment, and amyloid β peptide. However, the molecular mechanism for this wide variety of ligand binding is not fully understood. In this study, we determined the NMR structures of recombinant mouse L-PGDS and a L-PGDS/PGH2 analog. L-PGDS has a typical lipocalin fold, consisting of an eight-stranded β-barrel and a single α-helix. The interior of the barrel forms a hydrophobic cavity and the upper end of the barrel is open. The size of the barrel was found to be larger than those of other lipocalins and the cavity contained two pockets. The results of NMR titration, kinetic and molecular docking experiments revealed that PGH2 and retinoic acid occupied the hydrophilic pockets 1 and 2, respectively. A structural comparison of the L-PGDS/PGH2 analog complex with apo-L-PGDS showed that the H2-helix, CD-loop, and EF-loop located at the upper end of the β-barrel undergo a conformational change and cover the entrance of the cavity upon U-46619 binding. These results indicate that the two binding sites in the large cavity are responsible for the broad ligand specificity of L-PGDS in its induced-fit mechanism.

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 PGD₂ 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.

Ligand Recognition Mechanism of Lipocalin-type Prostaglandin D Synthase

Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) is a multi functional protein acting as a PGD(2) synthesizing enzyme, a transporter or scavenger of various lipophilic ligands, and an amyloid β chaperon in the brain. L-PGDS is a member of the lipocalin superfamily and has the ability to bind various lipophilic molecules such as prostanoid, retinoid, bile pigment, and amyloid β peptide. However, the molecular mechanism for a wide variety of ligand binding has not been well understood. In this study, we determined by NMR the structure of recombinant mouse L-PGDS and L-PGDS/PGH(2) analog complex. L-PGDS has the typical lipocalin fold, consisting of an eight-stranded β-barrel and a long α-helix. The interior of the barrel formed a hydrophobic cavity opening to the upper end of the barrel, the size of which was larger than those of other lipocalins and the cavity contained two pockets. Kinetic studies and molecular docking studies based on the result of NMR titration experiments provide the direct evidence for two binding sites for PGH(2) and retinoic acid in the large cavity of L-PGDS. Structural comparison of L-PGDS/U-46619 complex with apo-L-PGDS showed that the H2-helix, CD-loop, and EF-loop located at the upper end of the β-barrel change the conformation to cover the entry of the cavity upon U-46619 binding. These results indicated that the two binding sites in the large cavity and induced fit mechanism were responsible for the broad ligand specificity of L-PGDS.

Sustained expression of lipocalin-type prostaglandin D synthase in the antisense direction positively regulates adipogenesis in cloned cultured preadipocytes

Adipocytes express preferentially lipocalin-type prostaglandin (PG)D synthase (L-PGDS) that is responsible for the biosynthesis of PGD2 and other related prostanoids with pro-adipogenic or anti-adipogenic effects. To evaluate the role of L-PGDS in cultured adipocytes and the precursor cells, we attempted to interfere the intracellular expression of L-PGDS in cultured 3T3-L1 preadipocytes by stable transfection with a mammalian expression vector having the full-length cDNA of L-PGDS oriented in the antisense direction. The cloned transfectants with antisense L-PGDS exhibited the reduction in the transcript and protein levels of L-PGDS, resulting in the significant inhibition of the PGD2 synthesis from exogenous and endogenous arachidonic acid. By contrast, the synthesis of PGE2 was not influenced appreciably, indicating no interfering effects on cyclooxygenases and PGE synthases. The stable transfection with antisense L-PGDS induced markedly the stimulation of fat storage in cultured adipocytes during the maturation phase. In addition, the spontaneous accumulation of fats occurred in the transfectants with antisense L-PGDS without undergoing the stimulation with inducing factors. The gene expression studies revealed the enhanced expression of adipocyte-specific markers in the transfectants with antisense L-PGDS, indicating the up-regulation of adipogenesis program. The stimulated adipogenesis was significantly reversed by anti-adipogenic prostanoids including PGE2 and PGF2α, while the storage of fats was additionally enhanced by pro-adipogenic 15-deoxy- Δ12,14-prostaglandin J2. These results suggest that the stably reduced expression levels of L-PGDS regulates positively adipogenesis program in a cellular mechanism independent of pro-adipogenic action of PGJ2 series.

High-Quality Protein Crystal Growth of Mouse Lipocalin-Type Prostaglandin D Synthase in Microgravity

Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) catalyzes the isomerization of PGH2 to PGD2 and is involved in the regulation of pain and of nonrapid eye movement sleep and the differentiation of male genital organs and adipocytes, etc. L-PGDS is secreted into various body fluids and binds various lipophilic compounds with high affinities, acting also as an extracellular transporter. Mouse L-PGDS with a C65A mutation was previously crystallized with citrate or malonate as a precipitant, and the X-ray crystallographic structure was determined at 2.0 Å resolution. To obtain high-quality crystals, we tried, unsuccessfully, to crystallize the C65A mutant in microgravity under the same conditions used in the previous study. After further purifying the protein and changing the precipitant to polyethylene glycol (PEG) 8000, high-quality crystals were grown in microgravity. The precipitant solution was 40% (w/v) PEG 8000, 100 mM sodium chloride, and 100 mM HEPES-NaOH (pH 7.0). Crystals grew on board the International Space Station for 11 weeks in 2007, yielding single crystals of the wild-type L-PGDS and the C65A mutant, both of which diffracted at around 1.0 Å resolution. The crystal quality was markedly improved through the use of a high-viscosity precipitant solution in microgravity, in combination with the use of a highly purified protein.

Biochemical, Functional, and Pharmacological Characterization of AT-56, an Orally Active and Selective Inhibitor of Lipocalin-type Prostaglandin D Synthase

We report here that 4-dibenzo[a,d]cyclohepten-5-ylidene-1-[4-(2H-tetrazol-5-yl)-butyl]-piperidine (AT-56) is an orally active and selective inhibitor of lipocalin-type prostaglandin (PG) D synthase (L-PGDS). AT-56 inhibited human and mouse L-PGDSs in a concentration (3–250 μm)-dependent manner but did not affect the activities of hematopoietic PGD synthase (H-PGDS), cyclooxygenase-1 and -2, and microsomal PGE synthase-1. AT-56 inhibited the L-PGDS activity in a competitive manner against the substrate PGH2 (Km = 14 μm) with a Ki value of 75 μm but did not inhibit the binding of 13-cis-retinoic acid, a nonsubstrate lipophilic ligand, to L-PGDS. NMR titration analysis revealed that AT-56 occupied the catalytic pocket, but not the retinoid-binding pocket, of L-PGDS. AT-56 inhibited the production of PGD2 by L-PGDS-expressing human TE-671 cells after stimulation with Ca2+ ionophore (5 μm A23187) with an IC50 value of about 3 μm without affecting their production of PGE2 and PGF2α but had no effect on the PGD2 production by H-PGDS-expressing human megakaryocytes. Orally administered AT-56 (<30 mg/kg body weight) decreased the PGD2 production to 40% in the brain of H-PGDS-deficient mice after a stab wound injury in a dose-dependent manner without affecting the production of PGE2 and PGF2α and also suppressed the accumulation of eosinophils and monocytes in the bronco-alveolar lavage fluid from the antigen-induced lung inflammation model of human L-PGDS-transgenic mice.

Compact Packing of Lipocalin-type Prostaglandin D Synthase Induced by Binding of Lipophilic Ligands

Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) is a multi-functioning protein belonging to the lipocalin family, acting as a PGD(2)-synthesizing enzyme and as an extracellular transporter for small lipophilic molecules. In the present study, to clarify the conformational changes of lipocalin proteins induced by binding of lipophilic ligands, such as all-trans-retinoic acid (RA), bilirubin (BR) and biliverdin (BV), we measured small-angle X-ray scattering (SAXS) of L-PGDS and that of two other lipocalins, beta-lactoglobulin (betaLG) and retinol-binding protein (RBP). L-PGDS bound all three ligands with high affinity, while betaLG and RBP could bind only RA. The radius of gyration was estimated to be 19.4 A for L-PGDS, and 18.8 A for L-PGDS/RA, 17.3 A for L-PGDS/BR and 17.8 A for L-PGDS/BV complexes, indicating that L-PGDS became compact after binding of these ligands. Alternatively, the radius of gyration of betaLG and RBP was 20.3 and 26.2 A, respectively, and was almost the same before and after RA binding. Based on the SAXS data, we found that the compact packing upon binding ligands is a special feature of L-PGDS and it may be ascribed to the conformational flexibility of L-PGDS molecule itself, which underlies the high-affinity for its ligands.

Association of Serum Lipocalin-Type Prostaglandin D Synthase Levels with Subclinical Atherosclerosis in Untreated Asymptomatic Subjects

Recent studies suggest that lipocalin-type prostaglandin (PG) D synthase (L-PGDS), which converts PGH2 to PGD2, is implicated in the pathogenesis of atherosclerosis. However, clinical evidence for the association between serum L-PGDS levels and atherosclerosis has not been reported. In this study, we measured the serum L-PGDS concentration using sandwich enzyme-linked immunosorbent assay (ELISA) and investigated the association with traditional cardiovascular risk factors and surrogate atherosclerotic indices, such as the maximum score of the intima-media complex thickness of the carotid artery (C-IMT(max)) and the brachial-ankle pulse wave velocity (ba-PWV), in 500 non-treated asymptomatic subjects. The serum concentration of L-PGDS was 0.56+/-0.01 (mean+/-SEM, range 0.25-1.27, median 0.54) mg/L. Serum L-PGDS levels increased with age and were higher in men than in women. Serum L-PGDS was higher in subjects with hypertension and increased with increasing numbers of the traditional atherosclerotic risk factors. When the subjects were divided into four groups according to the levels of serum L-PGDS, the age-adjusted values of C-IMT(max) and ba-PWV were significantly increased in subjects with higher serum L-PGDS levels (quartile 3 and quartile 4) compared to those in the lowest quartile (quartile 1), for both genders. Multiple regression analysis including risk factors revealed that serum L-PGDS was an independent determinant for ba-PWV (beta=0.130, p<0.001). Serum L-PGDS tended to associate with C-IMT(max) but was not statistically significant (beta=0.084, p=0.075). In conclusion, our results suggest that an increase in serum L-PGDS concentration is associated with the progression of atherosclerosis.

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.

Pharmacokinetics of recombinant human lipocalin-type prostaglandin D synthase/β-trace in canine

Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) is identical to β-trace, a major protein in human cerebrospinal fluid (CSF), and acts as both a PGD 2-producing enzyme and as an extracellular transporter for lipophilic ligands. In this study, we investigated the pharmacokinetics of recombinant human L-PGDS (rh-L-PGDS) in canines. After an intravenous bolus injection of rh-L-PGDS, the serum concentration decreased bi-exponentially with a half-life of the terminal line phase of 0.77 h, which was markedly shorter than that of other proteins with the same molecular weight as that of rh-L-PGDS. The distribution volume was 55.4 ml/kg, which was close to the volume of canine circulation plasma, indicating that the administrated rh-L-PGDS was distributed mainly in the blood. Only 10.3% of the administered rh-L-PGDS was excreted to the urine, suggesting that rh-L-PGDS was actively degraded within the body. After an intrathecal injection, the peak serum concentration of rh-L-PGDS was observed at 4–5 h. The area under the plasma concentration–time curve obtained for 12 h after the intrathecal injection was one third of the value for 3 h after the intravenous injection, suggesting that at least one third of the intrathecally injected rh-L-PGDS shifted to the blood.