Isolation and characterization of a stereospecific 3 beta-hydroxysteroid 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 3 alpha-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 3 beta-hydroxysteroid substrate, It converts pregnenolone to pregnenolone sulfate most efficiently, whereas dehydroepiandrosterone and epiandrosterone were-transformed at a much lower rate, and androsterone, a 3 alpha-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 3 beta-hydroxysteroid sulfotransferase is thus different from that in humans, who possess two mRNA species of 1.3 and 1.8 kb.