Abstract
A novel stereoselective hydroxysteroid sulfotransferase (HST) that acts on neutral steroids having the 3-hydroxyl group in the alpha orientation but not on steroids where the 3-hydroxyl group is oriented in the beta position has been cloned and expressed. Primary screening of a guinea pig adrenal cDNA library was performed by colony hybridization using an oligonucleotide probe based on a highly homologous region among known steroid sulfotransferases. Selected clones consisted of overlapping sequences but were incomplete. The rapid amplification of cDNA ends procedure was used to construct a full-length guinea pig HST cDNA. The guinea pig HST cDNA transiently transfected into Chinese hamster ovary K1 cells expressed a protein identical in size to that of purified guinea pig HST specific for 3 alpha-hydroxylated neutral steroids that was recently reported (Driscoll, W. J., Martin, B. M., Chen, H.-C., and Strott, C. A. (1993) J. Biol. Chem. 268, 23496-23503). The expressed HST likewise exhibited sulfotransferase activity that was directed specifically toward steroid substrates containing a 3-hydroxyl group in the alpha orientation; on the other hand, steroids with a 3 beta-hydroxyl group were not sulfonated by the expressed HST. Thus, the cloned HST cDNA clearly coded for a steroid sulfotransferase with chiral specificity for 3 alpha-hydroxylated neutral steroids and was, therefore, given the designation of guinea pig 3 alpha-hydroxysteroid sulfotransferase (gp3 alpha-HST). The full-length gp3 alpha-HST cDNA consisted of 1182 base pairs and coded for a protein containing 287 amino acids. The deduced amino acid sequence of the protein shares 65/79, 65/80, and 62/76% identity/similarity with rat, mouse, and human HST, respectively. Northern blot analysis of guinea pig tissues revealed no apparent gender differences in either mRNA species or distribution; a single 1.4-kilobase HST mRNA species was present in adrenal and liver tissue. Interestingly, the adrenal mRNA content was considerably more abundant than that found in the liver. Evidence for 3 alpha-HST mRNA was not detected in kidney, heart, lung, muscle, spleen, or uterus.
Highlights
3-hydroxylgroup in thea orientation but not on steroids where the 3-hydroxylgroup is oriented in the p position has been clonedand expressed
The guinea pig hydroxysteroid sulfotransferases (HSTs) cDNAtransiently transfected into Chinese hamster ovary K1 cells expressed a protein identical in size to that of purified guinea pig HST specific for 3a-hydroxylatedneutral steroids that was recently reported
Determination of Full-length cDNA for gpHST-A pcDNA I1 library generated using random hexampreimr ing of guinea pig adrenal poly(A)+RNA was screened with an oligonucleotide that hadbeen synthesized based on a sequence of the rat liver HST cDNA (Table I, S1) that hisighly conserved among known sulfotransferases
Summary
3-hydroxylgroup in thea orientation but not on steroids where the 3-hydroxylgroup is oriented in the p position has been clonedand expressed. Determination of Full-length cDNA for gpHST-A pcDNA I1 library generated using random hexampreimr ing of guinea pig adrenal poly(A)+RNA was screened with an oligonucleotide that hadbeen synthesized based on a sequence of the rat liver HST cDNA (Table I, S1) that hisighly conserved among known sulfotransferases.
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