Isolation and partial characterization of a full-length cDNA clone for 3α-hydroxysteroid dehydrogenase: A potential target enzyme for nonsteroidal anti-inflammatory drugs

Abstract

Homogeneous 3α-hydroxysteroid dehydrogenase (3α-HSD; EC 1.1.1.50) of rat liver cytosol is a monomeric (MR 34000) NAD(P)⁺ dependent oxidoreductase which displays 9-, 11- & 15-hydroxyprostaglandin dehydrogenase activity. The enzyme is potently inhibited by nonsteroidal anti-inflammatory drugs (NSAIDs), suggesting that 3α-HSD may be a target enzyme for NSAIDs. A monospecific, polyclonal anti-sera raised against the purified enzyme was used to screen a λgt11 expression library and oligonucleotide probes complementary to the 5′ and 3′ ends of immunopositive clones were used to isolate a 2.1 kb full-length cDNA. Digestion of the full-length cDNA with Eco RI generated two fragments of 1.1 and 1.0 kb in length. Both fragments were subcloned into pGEM3 and partially sequenced. The 1.1. kb fragment contains the C-terminus of 3α-HSD which was confirmed by an in-frame stop codon and comparison of the predicted amino acid sequence to peptide sequence obtained from two endo lys-C peptides of 3α-HSD. The 1.0 kb fragment is 5′ to the 1.1 kb fragment and is sufficient in length to contain the remainder of the entire open reading frame for 3α-HSD. Dideoxysequencing reveals significant sequence homology with bovine lung prostaglandin PGF_2α synthase. These findings support the role 3α-HSD in inflammation and suggest that hydroxysteroid dehydrogenases, hydroxyprostaglandin dehydrogenases and prostaglandin F_2α synthase may be members of a common gene family.