Characterization of a major secretory protein in the cane toad (Bufo marinus) choroid plexus as an amphibian lipocalin-type prostaglandin D synthase.
Here we report the enzymatic and ligand-binding properties of a major secretory protein in the choroid plexus of cane toad, Bufo marinus, whose protein is homologous with lipocalin-type prostaglandin (PG) D synthase (L-PGDS) and is recombinantly expressed in Xenopus A6 cells and Escherichia coli. The toad protein bound all-trans retinal, bile pigment, and thyroid hormones with high affinities (K(d)=0.17 to 2.00 microM). The toad protein also catalysed the L-PGDS activity, which was accelerated in the presence of GSH or DTT, similar to the mammalian enzyme. The K(m) value for PGH(2) (17 microM) of the toad protein was almost the same as that of rat L-PGDS (14 microM), 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 Cys(59) and Thr(61) residues were crucial for the PGDS activity. The quadruple Gly(39)Ser/Ala(75)Ser/Ser(140)Thr/Phe(142)Tyr 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.
Irikura
Daisuke
D
Department of Molecular Behavioral Biology, Osaka Bioscience Institute, 6-2-4 Furuedai, Suita, Osaka 565-0874, Japan.
Inui
Takashi
T
Beuckmann
Carsten T
CT
Aritake
Kousuke
K
Schreiber
Gerhard
G
Miyano
Masashi
M
Inoue
Tsuyoshi
T
Urade
Yoshihiro
Y
D000595:Amino Acid Sequence; D000818:Animals; D001654:Bile Pigments / Q000378:metabolism; D002024:Bufo marinus / Q000378:metabolism*; D002831:Choroid Plexus / Q000201:enzymology*; D018076:DNA, Complementary; D019746:Intramolecular Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D054834:Lipocalins; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012176:Retinoids / Q000378:metabolism; D013963:Thyroid Hormones / Q000378:metabolism; D014981:Xenopus
D001654:Bile Pigments; D018076:DNA, Complementary; D054834:Lipocalins; D011994:Recombinant Proteins; D012176:Retinoids; D013963:Thyroid Hormones; D019746:Intramolecular Oxidoreductases; C022466:prostaglandin R2 D-isomerase