cDNA cloning, expression and characterization of human prostaglandin F synthase¹

Abstract

A cDNA clone of prostaglandin F synthase (PGFS) was isolated from human lung by using cDNA of bovine lungtype PGFS as a probe and its protein expressed in Escherichia coli was purified to apparent homogeneity. The human PGFS catalyzed the reduction of prostaglandin (PG) D 2 , PGH 2 and phenanthrenequinone (PQ), and the oxidation of 9K K,11L L-PGF 2 to PGD 2 . The k cat /K m values for PGD 2 and 9K K,11L L-PGF 2 were 21 000 and 1800 min 31 mM 31 , respectively, indicating that the catalytic efficiency for PGD 2 and 9K K,11L L-PGF 2 was the highest among the various substrates, except for PQ. The PGFS activity in the cytosol of human lung was completely absorbed with antihuman PGFS antiserum. Moreover, mRNA of PGFS was expressed in peripheral blood lymphocytes and the expression in lymphocytes was markedly suppressed by the T cell mitogen concanavalin A. These results support the notion that human PGFS plays an important role in the pathogenesis of allergic diseases such as asthma.z 1999 Federation of European Biochemical Societies.

Close kinship of human 20α‐hydroxysteroid dehydrogenase gene with three aldo‐keto reductase genes

“…Eighty positive clones were isolated from 8.0´10 5 l phages, and analysed by Southern blot hybridization. All the clones hybridized not only with rat 20a-HSD cDNA but also with human PGFS cDNA (Suzuki-Yamamoto et al 1999), suggesting that at least the 20a-HSD and PGFS genes might be included in the positive phage clones. However, restriction mapping in combination with Southern blot hybridization did not lead to an identi®cation of the genes.…”

“…We had cloned a cDNA encoding PGFS (Suzuki-Yamamoto et al 1999), and its sequence was exactly the same as the exon sequences of our PGFS genomic clones. In the present study, cDNAs encoding 20a-HSD, BABP and DD4 were cloned.…”

“…This 323 amino acid residue-protein has 80% and 69% amino acid identity to rabbit and rat 20a-HSDs, respectively. Surprisingly, it has a much higher amino acid identity to other human liver AKRs: 98% to bile acid-binding protein (BABP)/DD2 [AKR 1C2] ; 88% to prostaglandin F synthase (PGFS)/3a-HSD type 2 [AKR 1C3] (Khanna et al 1995b;Nagase et al 1995;Suzuki-Yamamoto et al 1999); and 83% to DD4/chlordecone reductase/3a-HSD type 1 [AKR 1C4] (Winters et al 1990;Qin et al 1993;Deyashiki et al 1994). Due to the extremely high homology among these AKR members, cDNA probes and antibodies can hardly discriminate 20a-HSD from the others by Northern and Western blot analyses.…”

“…Kinetic constants kcat, Km and kcat/Km are expressed in mM, min À1 and min À1 mM À1 , respectively, and the values represent means of duplicate or triplicate determinations. Kinetic data using PGFS are reported by Suzuki-Yamamoto et al (1999).…”

An indomethacin analogue, N-(4-chlorobenzoyl)-melatonin, is a selective inhibitor of aldo-keto reductase 1C3 (type 2 3α-HSD, type 5 17β-HSD, and prostaglandin F synthase), a potential target for the treatment of hormone dependent and hormone independent malignancies

“…AKR1C3 also plays an important role in the biosynthesis of prostaglandins, catalyzing the formation of prostaglandin (PG) F 2α and 9α,11β-PGF 2 from PGH 2 and PGD 2 , respectively [9,10]. Of its endogenously relevant substrates, AKR1C3 has the highest catalytic activity for the reduction of PGD 2 [9;10].…”