TY - JOUR
AU - Urade, Yoshihiro
AB - Abstract Lipocalin-type prostaglandin D synthase is the key enzyme for the production of prostaglandin D2, a potent endogenous somnogen, in the brain. We cloned, produced, and crystallized the native enzyme and selenomethionyl Cys65Ala 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 P212121 with lattice constants a = 46.2, b = 66.8, and c = 105.3 Å. The selenomethionyl crystals obtained with citrate belong to orthorhombic space group C2221 with lattice constants a = 45.5, b = 66.8, and c = 104.5 Å. The native crystals diffracted beyond 2.1 Å resolution. Key words: crystallization, lipocalin, multiwavelength anomalous diffraction method, prostaglandin D2, selenomethionyl protein. Abbreviations: PG, prostaglandin; PGDS, prostaglandin D synthase; L-PGDS, lipocalin-type prostaglandin D synthase; MAD, multiwavelength anomalous diffraction. Received October 15, 2002; accepted November 18, 2002 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, yielding PGD2 with 9-hydroxy and 11-keto groups, in the presence of sulfhydryl compounds. PGD2 is actively produced in a variety of tissues as a major prostanoid, and is involved in numerous physiological and pathological events. For example, PGD2 is known as a potent endogenous somnogen (1, 2), nociceptive modulator (3), anti-coagulant, vasodilator, bronchoconstrictor (4), and allergic and inflammatory mediator (5). PGD2 is further converted in vitro to 9α,11β-PGF2 and J series 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 of other prostanoids (6, 7). 15-Deoxy-Δ12,14-PGJ2 was recently identified as a ligand for a nuclear receptor, peroxisome proliferator-activated receptor γ (8, 9), which is involved in adipocyte differentiation, and in the regulation of macrophage and monocyte functions (10). There are two evolutionally different types of PGDS (11); one is the lipocalin-type PGDS (L-PGDS, 12), and the other is the hematopoietic one (13). L-PGDS is a member of the lipocalin superfamily, which is composed of various secretory lipid-transporter proteins (12); it contributes to the production of PGD2 in the brains of various mammals and in the human heart (14). We recently reported that transgenic mice that overexpressed the human L-PGDS gene exhibited excessive non-rapid eye movement sleep in response to the noxious stimulus of tail clipping, coupled with a significant increase in PGD2 production in the brain (15). Furthermore, we also recently showed that infusion of PGD2 into the lateral ventricle of the brain remarkably increased the amount of non-rapid eye movement sleep in wild-type mice but not at all in D type prostanoid receptor–deficient mice (16). Therefore, PGD2 produced by L-PGDS is considered to play an important role in the regulation of non-rapid eye movement sleep in a DP receptor–dependent manner. In this study, we expressed and crystallized the recombinant methionyl and selenomethionyl mouse L-PGDS to determine its crystal structure by means of the multiwavelength anomalous diffraction (MAD) method (17). Firstly, we cloned the L-PGDS gene (EMBL/GenBank/DDBJ™ accession number D83329) from mouse brains. The cDNA for mouse L-PGDS without its N-terminal signal peptide of 24 amino acids was isolated from a mouse brain cDNA library by means of the polymerase chain reaction (PCR) with sense primer 5′-AGCTGGATCCCAGGGCCATGACACAGTGCAGCCC-3′ and antisense primer 5′-AGCTGAATTCTTACTCTTGAATGCACTTATCCGG-3′. The cDNA was cloned into expression vector pGEX-2T (Amersham Pharmacia Biotech, Uppsala, Sweden). Escherichia coli DH5α cells (TOYOBO, Tokyo) were transformed with this vector, and then the recombinant L-PGDS was expressed as a glutathione (GSH) transferase-fusion protein. The recombinant L-PGDS was purified by GSH-Sepharose 4B (Amersham Pharmacia Biotech) column chromatography from the soluble fraction of E. coli cells. After incubation with thrombin (Sigma-Aldrich, WI, USA), the L-PGDS was further purified by column chromatography on Superdex75 in 5 mM Tris/HCl (pH 8.0), followed by Mono-S chromatography in 10 mM sodium citrate (pH 4.5), as reported previously (18, 19). The structural homogeneity of the purified wild-type L-PGDS was not sufficient for further structural studies, since the major product had an incorrect S-S linkage between Cys65 and Cys89/Cys189, and not the correct one between Cys89 and Cys189. As judged on SDS-PAGE (Fig. 1), two isoforms of the wild type enzyme were observed under non-reducing conditions at positions corresponding to Mr of 20, 000 and 19,000. These mis-S-S linked proteins made isolation difficult. To produce a sufficient amount of the correctly folded recombinant L-PGDS for further structural studies, we constructed two mutants of L-PGDS in which Cys was substituted by Ala, i.e., Cys65Ala and Cys89,186Ala, to avoid major incorrectly folded proteins due to wrong S-S cross-linking with Cys65. The Cys65Ala mutant with the correct disulfide bond between Cys89 and Cys186 had no PGDS activity due to mutation of putative catalytic residue Cys65, and gave a single band at a position corresponding to Mr of 21,000 or 19,000 on SDS-PAGE under reduced and non-reduced conditions, respectively. The Cys89,186Ala mutant without the disulfide bond showed PGDS activity as described below, and gave a single band at the same position, Mr 21,000, on SDS-PAGE in the presence and absence of a thiol reducing agent. For MAD phasing, the selenomethionyl Cys65Ala L-PGDS mutant was produced using E. coli B834 (DE3) cells (Novagene, WI, USA). We modified the LeMaster medium (20) to increase the growth of the E. coli cells, as reported previously (21). Purification of the selenomethionyl L-PGDS was performed in the same way as for the native one. The PGDS activity of the selenomethionyl Cys89,186Ala mutant (5.0 µmol/min/mg of protein) was almost the same as that of the wild-type Cys89,186Ala mutant (2.9 µmol/min/mg of protein, Ref. 18) or that of the purified L-PGDS from rat brain (4.0 µmol/min/mg of protein), human seminal plasma (4.5 µmol/min/mg of protein), or bovine seminal plasma (4.5 µmol/min/mg of protein, Ref. 12). Furthermore, the elution profiles of both the Cys89,186Ala and Cys65Ala mutants from the Superdex75 and Mono-S columns were essentially indistinguishable for the native and selenomethionyl proteins. These results suggest that the catalytically essential structure of L-PGDS is not affected by the presence or absence of the correctly linked S-S bridge between Cys89 and Cys186, and also indicate that the overall architecture of selenomethionyl L-PGDS is the same as that of the native enzyme. For crystallization trials, both the native and selenomethionyl L-PGDS mutant proteins were dialyzed against 5 mM Tris/HCl (pH 8; crystallization stock buffer) containing 10 µM all-trans retinoic acid (Sigma-Aldrich), and then concentrated to 10 mg/ml by ultrafiltration with a YM-3 membrane (Millipore, Bedford, MA). Initial screening for the crystallization conditions was carried out at 22.5°C by the hanging drop vapor-diffusion method using 24-well tissue culture trays (ICN, OH, USA) and a screening solution set (Hampton Research, Laguna, CA, USA; Ref. 22). After refinement of the crystallization conditions, a crystal of the native Cys65Ala L-PGDS suitable for the diffraction study was obtained by mixing the protein solution (2 µl) with an equal volume of a reservoir solution (1 ml) of 0.1 M Tris/HCl (pH 8.0) containing 2.0 M sodium malonate and 10% (v/v) 1,4-dioxane (Fig. 2a). The native Cys65Ala L-PGDS was also crystallized using the reservoir solution of 0.1 M Tris/HCl (pH 8) containing 1.25 M sodium citrate and 10% (v/v) 1,4-dioxane (Fig. 2b). The selenomethionyl Cys65Ala L-PGDS was crystallized by using a reservoir solution of 0.1 M Tris/HCl (pH 9.5) containing 1.25 M sodium citrate, 10% (v/v) 1,4-dioxane and 2% Triton X-405. However, the crystallization of the native enzyme and selenomethionyl Cys89,186Ala mutants was not successful. Diffraction studies on the crystals were carried out at BL45XU-PX, SPring-8 (23), with a Rigaku R-axis IV imaging plate system at a cryogenic temperature (100 K) using the flash frozen method. The cryoprotectant was the crystallization mother solution containing 0.5 M trehalose. A crystal of the native Cys65Ala mutant obtained with malonate belonged to primitive orthorhombic space group P212121, with unit-cell dimensions of a = 46.2, b = 66.8, and c = 105.3 Å. Assuming two L-PGDS molecules per asymmetric unit, the VM value and solvent content were calculated to be 2.0 Å3/Da and 0.728, respectively. We collected a diffraction data set up to 2.1 Å (Fig. 3). The crystallographic parameters of a selenomethionyl crystal were face-centered orthorhombic space group C2221 with unit-cell dimensions of a = 45.5, b = 66.8, and c = 104.5 Å; and the VM value and solvent content were 2.2 Å3/Da and 0.44, respectively, with one molecule per asymmetric unit, respectively. We collected a MAD data set up to 2.5 Å. Structural determination of L-PGDS is currently in progress. This study was supported in part by Grants-in-Aid for CREST project and the SPring-8 Joint Research Promotion Scheme From the Japan Science Technology Corporation (to Y.U.); a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (12558087 to Y.U.); and by grants from the Ground Research Announcement for Space Utilization Promotion by Japan Space Forum (to Y.U.), the Special Coordination Fund for Promoting Science and Technology (to Y.U.), and Osaka City. We with to thank Prof. T. Tsukihara, Osaka Univ., for his help with the protein crystallization method. We also thank Prof. S. Uemura, Kyoto Univ., for the chemical synthesis of selenomethionine. We are grateful to Dr. B.K. Kubota (Osaka Bioscience Institute) for the guidance and technical assistance during the early stages of cultures with the amino acid–enriched medium. + Present address: Department of Medicine, Harvard Medical School, Division of Rheumatology, Immunology, and Allergy, Brigham and Women’s Hospital, Boston, MA 02115, USA. § Present address: Department of Life Science, Tokyo Insititute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501. ¶ To whom correspondence should be addressed at: Department of Molecular Behavioral Biology, Osaka Bioscience Institute, 6-2-4 Furuedai, Suita, Osaka, 565-0874. Tel: +81-6-6872-4851, Fax: +81-6-6872-2841, E-mail: uradey@obi.or.jp View largeDownload slide Fig. 1. SDS-PAGEoftherecombinantmouseL-PGDS. The samples for SDS-PAGE were prepared in the presence or absence of 10 mM β-mercaptoethanol. The positions of Mr marker proteins are indicated on the left. View largeDownload slide Fig. 1. SDS-PAGEoftherecombinantmouseL-PGDS. The samples for SDS-PAGE were prepared in the presence or absence of 10 mM β-mercaptoethanol. The positions of Mr marker proteins are indicated on the left. View largeDownload slide Fig. 2. CrystalsofthenativeCys65AlamutantofmouseL-PGDS. The crystals were prepared by the hanging drop vapor-diffusion method with malonate (a) or citrate (b) as the precipitant. View largeDownload slide Fig. 2. CrystalsofthenativeCys65AlamutantofmouseL-PGDS. The crystals were prepared by the hanging drop vapor-diffusion method with malonate (a) or citrate (b) as the precipitant. View largeDownload slide Fig. 3. DiffractionpatternofacrystalofthenativeCys65AlaL-PGDS. An oscillation photograph of a crystal of the native L-PGDS exposed using a synchrotron X-ray source. The diffraction was up to 2.1 Å resolution. View largeDownload slide Fig. 3. DiffractionpatternofacrystalofthenativeCys65AlaL-PGDS. An oscillation photograph of a crystal of the native L-PGDS exposed using a synchrotron X-ray source. 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Synchrotron Rad.  5, 222–225 Google Scholar Author notes 1Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation, 6-2-4 Furuedai, Suita, Osaka 565-0874; 2Department of Molecular Behavioral Biology, Osaka Bioscience Institute, 6-2-4 Furuedai, Suita, Osaka 565-0874; 3RIKEN Harima Institute at SPring-8, 1-1-1 Kohto, Mikazuki-cho, Sayo, Hyogo 679-5148; and 4Institute for Protein Research, Osaka University, Yamadaoka, Suita, Osaka 565-0871
TI - Cloning, Expression, Crystallization, and Preliminary X-Ray Analysis of Recombinant Mouse Lipocalin-type Prostaglandin D Synthase, a Somnogen-Producing Enzyme
JF - The Journal of Biochemistry
DO - 10.1093/jb/mvg006
DA - 2003-01-01
UR - https://www.deepdyve.com/lp/oxford-university-press/cloning-expression-crystallization-and-preliminary-x-ray-analysis-of-YcG9PyM6uY
SP - 29
EP - 32
VL - 133
IS - 1
DP - DeepDyve
ER -