Molecular cloning of goat 20alpha-hydroxysteroid dehydrogenase cDNA.
20Alpha-hydroxysteroid dehydrogenase (20alpha-HSD), which catalyzes the conversion of progesterone to its inactive form 20alpha-dihydroprogesterone, is expressed in murine placenta and has been suggested to play roles in maintaining pregnancy. To understand the role of 20alpha-HSD during pregnancy in the goat, as a first step, cloning and sequencing of 20alpha-HSD cDNA were performed. The full nucleotide sequence of 20alpha-HSD cDNA was determined on samples obtained from the corpus luteum at the luteal phase of the estrous cycle and the placenta in late pregnancy by RT-PCR and 3' and 5' RACE systems. Cloned 20alpha-HSD cDNA consisted of 1124 bp and belonged to the aldo-keto reductase superfamily. From the start codon to stop codon there were 323 amino acids, the same as in other species. To verify whether the protein derived from goat 20alpha-HSD cDNA had 20alpha-HSD activity, the cDNA was expressed by bacteria. Bacterially expressed goat 20alpha-HSD protein showed 20alpha-HSD enzyme activity. A tissue distribution study demonstrated that 20alpha-HSD was expressed in the placenta, but not in the adrenal gland, liver and spleen during pregnancy. The present study suggests that goat 20alpha-HSD is another member of the aldo-keto reductase superfamily and that it plays a role in the placenta during pregnancy.
Jayasekara
Walimuni Samantha Nilanthi
WS
Department of Veterinary Physiology, Veterinary Medical Science, The University of Tokyo, Japan.
Yonezawa
Tomohiro
T
Ishida
Maho
M
Yamanouchi
Keitaro
K
Nishihara
Masugi
M
D043607:20-alpha-Hydroxysteroid Dehydrogenase / Q000096:biosynthesis* / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002384:Catalysis; D003001:Cloning, Molecular; D018387:Codon, Initiator; D018388:Codon, Terminator; D003338:Corpus Luteum / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005260:Female; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000378:metabolism; D006041:Goats; D008969:Molecular Sequence Data; D010053:Ovary / Q000378:metabolism; D010920:Placenta / Q000378:metabolism; D011247:Pregnancy; D011270:Pregnancy, Animal; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D013997:Time Factors; D014018:Tissue Distribution
D018387:Codon, Initiator; D018388:Codon, Terminator; D018076:DNA, Complementary; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D043607:20-alpha-Hydroxysteroid Dehydrogenase; D005982:Glutathione Transferase