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

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Abstract

Lipocalin-type prostaglandin (PG) D synthase (PGDS) catalyzes the isomerization of PGH2, a common precursor of various prostanoids, to produce PGD2, a potent endogenous somnogen and nociceptive modulator, in the presence of sulfhydryl compounds. PGDS is an N-glycosylated monomeric protein with an Mr of 20 000–31 000 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 β-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 PGD2-producing enzyme and also as a lipophilic ligand-binding protein, because the enzyme binds biliverdin, bilirubin (Kd=30 nM), retinaldehyde, retinoic acid (Kd=80 nM) with high affinities. X-ray crystallographic analyses revealed that PGDS possesses a β-barrel structure with a hydrophobic pocket in which an active thiol, Cys65, 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.

Introduction

Prostaglandin (PG) D synthase (PGDS, EC 5.3.99.2) catalyzes the isomerization of the 9,11-endoperoxide group of PGH2, a common precursor of various prostanoids, to produce PGD2 with 9-hydroxy and 11-keto groups, in the presence of sulfhydryl compounds (Fig. 1). PGD2 is actively produced in a variety of tissues as a major prostanoid and is involved in numerous physiological and pathological functions. For example, PGD2 is known as a potent endogenous somnogen [1], [2], nociceptive modulator [3], anti-coagulant, vasodilator, bronchoconstrictor, and also as an allergic and inflammatory mediator [4] released from mast cells [5]. PGD2 is further converted in vitro to 9α,11β-PGF2 and the J series of PGs, such as PGJ2, Δ12-PGJ2, and 15-deoxy-Δ12,14-PGJ2, although the natural occurrence of these PGs in vivo remains to be clarified. These more recently recognized types of PGs exert various pharmacological actions different from those induced by other prostanoids [6], [7]. 15-Deoxy-Δ12,14-PGJ2 was recently identified to be an endogenous ligand for a nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) [8], [9] which is involved in adipocyte differentiation and in the regulation of macrophage and monocyte functions [10]. The J series of PGs promote neurite outgrowth of PC 12 cells induced by nerve growth factor [11]. The neurite outgrowth-promoting effect is recently demonstrated to be mediated by immunoglobulin heavy chain binding protein/glucose-regulated protein 78 (Bip/GRP78), but not PPARγ [12].

Two distinct types of PGDS have been purified and characterized [13], [14]; i.e. one is the lipocalin-type PGDS (L-PGDS) that was previously known as the brain-type enzyme or glutathione (GSH)-independent enzyme [15], and the other is hematopoietic PGDS, the spleen-type enzyme or GSH-requiring enzyme [16], [17]. The hematopoietic enzyme is quite different from L-PGDS in terms of catalytic properties [17], amino acid sequence, tertiary structure, evolutional origin [18], gene structure, chromosomal localization [19], cellular localization, tissue distribution, and also functional relevance, as we indicated in an earlier review [14]. L-PGDS is the first member of the lipocalin family to be recognized as an enzyme and a highly glycosylated protein [20], [21]. In this paper, we summarize the structural and functional properties of L-PGDS as a unique member of the lipocalin family. A part of these findings was also reviewed elsewhere [2], [13], [14].

Section snippets

Enzymatic properties of L-PGDS and identification as β-trace

We purified L-PGDS from rat brain as an acidic, monomeric soluble protein with a Mr of 26 000 [15]. L-PGDS was recently identified to be the same protein as β-trace [22], [23], [24], [25], which was originally discovered in 1961 as a major protein of human cerebrospinal fluid (CSF; [26], [27]). Since the identification of β-trace as L-PGDS, the enzyme has also been purified from various body fluids, such as the CSF [25] and seminal plasma [28], [29], [30], of humans and several mammals. The

Binding of non-substrate lipophilic ligands by L-PGDS

L-PGDS shares the characteristic as lipocalin, i.e. it is secreted into various body fluids and binds small lipophilic-ligands. By monitoring the quenching of the intrinsic tryptophan fluorescence of the recombinant protein and by circular dichroism spectroscopy of the bound ligands, we showed that L-PGDS binds all-trans- or 9-cis-retinoic acid and all-trans- or 13-cis-retinaldehyde, but not all-trans-retinol, at a molar ratio of 1:1 [40]. The affinities of L-PGDS for retinoids (Kd=70–80 nM)

cDNA cloning of L-PGDS

The cDNA for L-PGDS was first isolated from a rat brain cDNA library [43] and subsequently from many other mammalian species, including human [20], mouse [44], pig [45], bull [29], horse [30], sheep [30], bear, cat, dog, and also from two amphibians [42], [46]. Fig. 2 shows multiple alignment of amino acid sequences of the enzymes of various species with a zebrafish lipocalin which is highly homologous to L-PGDS. The cDNA for L-PGDS encoded a protein composed of 180–190 amino acid residues. A

Tertiary structure of L-PGDS

Circular dichroism spectroscopy of the recombinant rat L-PGDS revealed that the enzyme is composed of mainly β-strands [37], similar to other lipocalins. We previously modeled the tertiary structure of rat L-PGDS based on the crystal structure of other lipocalins to be the typical β-sheet barrel composed of two sets of 4-stranded anti-parallel β-sheets and a three-turn α-helix [40], [41], [47]. In the model structure, the Cys65 residue was located in the hydrophobic pocket with a size

Genomic structure and chromosomal localization of L-PGDS

The genes for L-PGDS have been cloned from rat [50], human [51], and mouse [52] sources. The human, rat, and mouse genes for L-PGDS span about 3 kb and contain seven exons split by six introns. The gene organization of L-PGDS was shown to be remarkably analogous to that of other lipocalins, in terms of number and size of exons and phase of splicing of introns [50], [51], as reviewed in the article by Salier. The mouse and human genes were mapped to mouse chromosome 2 B-C1 [53] and human

Tissue distribution and cellular localization

The tissue distribution profiles of L-PGDS and its mRNA were determined in various animal species by measuring the enzyme activity [61] and Western and Northern blot analyses. L-PGDS is localized in the CNS and related organs, such as the cochlea and ocular tissues, and in the male genital organs of various mammals and in the human and monkey heart. L-PGDS produced in these tissues is secreted into various body fluids, such as the CSF, eye fluids, seminal plasma, and plasma. The cellular

Generation of gene-manipulated mice

We generated gene-knockout (KO) mice for L-PGDS by homologous recombination to produce the null mutation of the gene [52] and also transgenic (TG) mice [85] that over-expressed the human L-PGDS under the control of the β-actin promoter. These mice showed abnormalities in the regulation of nociception and sleep.

The PGDS-KO mice did not exhibit allodynia (touch-evoked pain), which is a typical phenomenon of neuropathic pain, after an intrathecal administration of PGE2 or of the γ-aminobutyric

Diagnostic marker

Due to the expression of L-PGDS specific for the CNS, genital organs, and heart and its secretion into several body fluids, the L-PGDS concentration may be a useful clinical marker for various diseases. We developed a highly sensitive immunoassay method for L-PGDS with monoclonal antibodies against the recombinant human enzyme [86] and used it to determine the content of the enzyme in several human body fluids [86], [87]. The L-PGDS in human CSF was also determined by capillary electrophoresis

Future studies

The three-dimensional coordinates of L-PGDS determined by X-ray crystallographic analysis will be useful for designing inhibitors for the enzyme. Further investigation to examine the functional abnormality in the KO- and TG-mice for L-PGDS mice should provide us with new insight into the physiological relevance of L-PGDS, PGD2 and its metabolites. These gene-manipulated mice should also be useful for development of a novel animal model to study sleep, nociception, allergic reaction, and

Acknowledgements

We are grateful to Drs. N. Eguchi, C.T. Beuckmann, Y. Kanaoka, D.Y. Gerashchenko, E. Pinzar, K.B. Kubata, T. Inui, Y. Fujitani, K. Fujimori, T. Mochizuki, and M. Lazarus of Osaka Bioscience Institute, to Drs. T. Kumasaka and M. Yamamoto of RIKEN Harima Institute, and to Drs. H. Toh and T. Tanaka of Biomolecular Engineering Research Institute, for valuable discussions. We also thank Y. Ono, D. Irikura, Y. Kuwahata, S. Matsumoto, N. Uodome, R. Matsumoto, M. Emi, K. Okazaki for technical

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