Sequence of pig 17beta-hydroxysteroid dehydrogenase Type3 cDNA and its expression in mammalian cells.
17beta-Hydroxysteroid dehydrogenase (17beta-HSD) Type3 is an NADPH-dependent membrane-bound enzyme that is specifically expressed in testis and catalyzes the conversion of androstenedione to testosterone. To date, the sequence of Type3 enzymes has been clarified in humans, mice and rats; however, the sequence of the pig enzyme remains unknown. In this study, we determined the cDNA sequence of pig testicular 17beta-HSD Type3. PCR primers for partial pig testicular 17beta-HSD Type3 were designed from rat and human enzyme consensus sequences. Full-length cDNA was obtained by 3'- and 5'-RACE based on partial PCR products. The cDNA coding region was 933 bp in length, which is the same as the human enzyme, and shared 84.7% sequence identity with the human cDNA coding region. The monomer was estimated to have a molecular weight of 34,855 and to contain 310 amino acid residues. The predicted pig amino acid sequence showed 81.9, 75.5 and 72.9% sequence identity with the human, rat and mouse sequences, respectively. To elucidate 17beta-HSD Type3 activity, the expression vector pCMV/pig17beta-HSD3 was established and transfected into human embryo kidney 293 cells. Subsequently, 17beta-HSD activity (androstenedione conversion to testosterone) was strongly detected in cell lysates.
Ohno
Shuji
S
Department of Biochemistry, Hoshi University College of Pharmacy and Pharmaceutical Sciences, 2-4-41 Ebara, Shinagawa-ku, Tokyo 142-8501, Japan. ohno@hoshi.ac.jp
Honda
Yoko
Y
Nakajima
Yonako
Y
Nakajin
Shizuo
S
D015067:17-Hydroxysteroid Dehydrogenases / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D018076:DNA, Complementary; D015870:Gene Expression; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000378:metabolism; D013552:Swine / Q000378:metabolism*; D013737:Testis / Q000201:enzymology*; D014162:Transfection
D018076:DNA, Complementary; D012333:RNA, Messenger; D015067:17-Hydroxysteroid Dehydrogenases; C476565:17beta-hydroxysteroid dehydrogenase type 3