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Journal of Biochemistry Advance Access published online on December 13, 2006
Journal of Biochemistry, doi:10.1093/jb/mvm016
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© 2006 The Japanese Biochemical Society

Characterization of a Major Secretory Protein in the Cane Toad (Bufo marinus) Choroid Plexus as an Amphibian Lipocalin-type Prostaglandin D Synthase *

Daisuke Irikura1,2, Takashi Inui1, Carsten T Beuckmann1, Kousuke Aritake1, Gerhard Schreiber3, Masashi Miyano2, Tsuyoshi Inoue4 and Yoshihiro Urade1,{ddagger}

From the 1Department of Molecular Behavioral Biology, Osaka Bioscience Institute, 6-2-4 Furuedai, Suita, Osaka 565-0874, Japan; 2Structural Biophysics Laboratory, RIKEN Harima Institute, 1-1-1 Kouto, Sayo, Hyougo, 679-5148, Japan; 3Russell Grimwade School of Biochemistry, University of Melbourne, Parkville, Victoria 3052, Australia; and the 4 Department of Engineering Osaka University, Yamadaoka, Suita, Osaka 565-0871, Japan

{ddagger}To whom correspondence should be addressed: Dept. of Molecular Behavioral Biology, Osaka Bioscience Institute, 6-2-4 Furuedai, Suita, Osaka 565-0874, Japan. Tel. +81-6-6872-4851; Fax: +81-6-6872-2841; e-mail: uradey{at}obi.or.jp

Received October 16, 2006; Accepted November 23, 2006


  Abstract

Here we report the enzymatic and ligand-binding properties of a major secretory protein in the choroid plexus of cane toad, Bufo marinus, which protein was homologous with lipocalin-type prostaglandin (PG) D synthase (L-PGDS) and recombinantly expressed in Xenopus A6 cells and Escherichia coli. The toad protein bound all-trans retinal, bile pigment, and thyroid hormones with high affinities (Kd= 0.17 to 2.00 µM). The toad protein also catalyzed the L-PGDS activity, which was accelerated in the presence of GSH or DTT, similar to the mammalian enzyme. The Km value for PGH2 (17 µM) of the toad protein was almost the same as that of rat L-PGDS (14 µM), whereas the turnover number (6 min-1) was approximately 28-fold lower than that of rat L-PGDS. Site-directed mutagenesis based on a modeled structure of the toad protein revealed that Cys59 and Thr61 residues were crucial for the PGDS activity. The quadruple Gly39Ser/Ala75Ser/Ser140Thr/Phe142Tyr mutant of the toad protein, resembling mouse L-PGDS, showed a 1.6-fold increase in the turnover number and a shift in the optimum pH for the PGDS activity from 9.0 to 8.5. Our results suggest that the toad protein is a prototype of L-PGDS with a highly functional ligand-binding pocket and yet with a primitive catalytic pocket.

Key Words: amphibian, lipocalin, lipocalin-type prostaglandin D synthase, prostaglandin D2, site-directed mutagenesis


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