Isolation and characterization of a stereospecific 3beta-hydroxysteriod sulfotransferase (pregnenolone sulfotransferase) cDNA.
In contrast to humans, who possess a hydroxysteroid sulfotransferase (HSST), namely, DHEA sulfotransferase (DHEA-ST), that displays broad substrate specificities, HSSTs of the guinea pig show a high substrate stereoselectivity, as shown by the recent cloning of a chiral-specific 3alpha-hydroxysteroid sulfotransferase. Herein, we report the cloning and expression of the substrate and chiral-specific pregnenolone sulfotransferase (PREG-ST). Transfection of the pCMV expression vector containing PREG-ST cDNA in transformed human embryonal kidney (293) cells showed that the expressed enzyme selectively catalyzes the 3beta-hydroxysteroid substrate. It converts pregnenolone to pregnenolone sulfate most efficiently, whereas dehydroepiandrosterone and epiandrosterone were transformed at a much lower rate, and androsterone, a 3alpha-hydroxysteroid, was not significantly metabolized (30-fold lower). Thus, the enzyme was identified as pregnenolone sulfotransferase. DNA analysis predicts a protein of 287 amino acids with a calculated molecular mass of 34,199 daltons. Alignment of the amino acid sequence with other sulfotransferases indicated that guinea pig pregnenolone sulfotransferase shares 75 and 80% homology with human DHEA sulfotransferase and rat hydroxysteroid dehydrogenase, respectively. RNA blot analysis using guinea pig liver, intestine, adrenal, kidney, epididymis, testis, and lung showed a single RNA species at 1.3 kb is expressed in liver, intestine, and kidney. Guinea pig 3beta-hydroxysteroid sulfotransferase is thus different from that in humans, who possess two mRNA species of 1.3 and 1.8 kb.
Dufort
I
I
Medical Research Council Group in Molecular Endocrinology, CHUL Research Center and Laval University, Quebec, Canada.
Tremblay
Y
Y
Bélanger
A
A
Labrie
F
F
Luu-The
V
V
D000595:Amino Acid Sequence; D000728:Androgens / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D002461:Cell Line, Transformed; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D006168:Guinea Pigs; D006801:Humans; D006914:Hydroxysteroids / Q000378:metabolism; D007668:Kidney; D007700:Kinetics; D008969:Molecular Sequence Data; D009928:Organ Specificity; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D015238:Sulfotransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D000728:Androgens; D018076:DNA, Complementary; D006914:Hydroxysteroids; D012333:RNA, Messenger; D015238:Sulfotransferases; C036994:pregnenolone sulfotransferase