Human liver dehydroepiandrosterone sulfotransferase: Molecular cloning and expression of cDNA

Diane M. Otterness, Eric D Wieben, Thomas C. Wood, R. William G Watson, Benjamin J. Madden, Daniel J Mc Cormick, Richard M Weinshilboum

Research output: Contribution to journalArticle

153 Citations (Scopus)

Abstract

Sulfation is an important pathway in the metabolism of many hormones and drugs. Human liver contains at least three well characterized sulfotransferase (ST) enzymes, i.e., dehydroepiandrosterone (DHEA) ST and two forms of phenol sulfotransferase (PST). Our goal was to purify, to obtain partial amino acid sequence for, and to clone and express cDNA for human liver DHEA ST. Polymerase chain reaction primers were designed on the basis of homology among rat liver hydroxysteroid ST, rat liver PST, and bovine estrogen ST. These primers amplified a unique sequence from human liver cDNA, and this polymerase chain reaction product was used to screen a human liver cDNA library. Two clones were isolated that contained identical open reading frames, of 855 nucleotides, that encoded a protein of 285 amino acids. The deduced amino acid sequence of the encoded protein included two separate 27- and 23-amino acid sequences that were identical to those obtained by microsequencing of proteolytic fragments from purified human liver DHEA ST. Translation, in a rabbit reticulocyte lysate system, of mRNA transcribed in vitro from the two cDNA clones resulted in a 35-kDa translation product that comigrated with purified human liver DHEA ST during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This translation product also catalyzed the sulfation of DHEA but not the sulfation of model substrates for the two forms of PST found in human liver. The two cDNA clones were also used to create expression constructs with the eukaryotic expression vector P91023(B), and these constructs were used to transfect COS-1 cells. The transfected cells expressed a high level of DHEA ST activity, and this enzyme activity displayed a pattern of inhibition by the ST inhibitor 2,6-dichloro-4-nitrophenol identical to that of human liver DHEA ST. Cloning of cDNA for this important human sulfate-conjugating enzyme will enhance understanding of the relationship between DHEA ST and other human liver STs, as well as ST enzymes in other species.

Original languageEnglish (US)
Pages (from-to)865-872
Number of pages8
JournalMolecular Pharmacology
Volume41
Issue number5
StatePublished - May 1992

Fingerprint

Molecular Cloning
Complementary DNA
Liver
Arylsulfotransferase
Sulfotransferases
Clone Cells
Amino Acid Sequence
Enzymes
dehydroepiandrosterone sulfotransferase
Polymerase Chain Reaction
Dehydroepiandrosterone
COS Cells
Reticulocytes
Gene Library
Sodium Dodecyl Sulfate
Open Reading Frames
Sulfates
Organism Cloning
Polyacrylamide Gel Electrophoresis
Proteins

ASJC Scopus subject areas

  • Pharmacology

Cite this

Human liver dehydroepiandrosterone sulfotransferase : Molecular cloning and expression of cDNA. / Otterness, Diane M.; Wieben, Eric D; Wood, Thomas C.; Watson, R. William G; Madden, Benjamin J.; Mc Cormick, Daniel J; Weinshilboum, Richard M.

In: Molecular Pharmacology, Vol. 41, No. 5, 05.1992, p. 865-872.

Research output: Contribution to journalArticle

Otterness, Diane M. ; Wieben, Eric D ; Wood, Thomas C. ; Watson, R. William G ; Madden, Benjamin J. ; Mc Cormick, Daniel J ; Weinshilboum, Richard M. / Human liver dehydroepiandrosterone sulfotransferase : Molecular cloning and expression of cDNA. In: Molecular Pharmacology. 1992 ; Vol. 41, No. 5. pp. 865-872.
@article{4462091083ef49c1b7d089b6dec0a14d,
title = "Human liver dehydroepiandrosterone sulfotransferase: Molecular cloning and expression of cDNA",
abstract = "Sulfation is an important pathway in the metabolism of many hormones and drugs. Human liver contains at least three well characterized sulfotransferase (ST) enzymes, i.e., dehydroepiandrosterone (DHEA) ST and two forms of phenol sulfotransferase (PST). Our goal was to purify, to obtain partial amino acid sequence for, and to clone and express cDNA for human liver DHEA ST. Polymerase chain reaction primers were designed on the basis of homology among rat liver hydroxysteroid ST, rat liver PST, and bovine estrogen ST. These primers amplified a unique sequence from human liver cDNA, and this polymerase chain reaction product was used to screen a human liver cDNA library. Two clones were isolated that contained identical open reading frames, of 855 nucleotides, that encoded a protein of 285 amino acids. The deduced amino acid sequence of the encoded protein included two separate 27- and 23-amino acid sequences that were identical to those obtained by microsequencing of proteolytic fragments from purified human liver DHEA ST. Translation, in a rabbit reticulocyte lysate system, of mRNA transcribed in vitro from the two cDNA clones resulted in a 35-kDa translation product that comigrated with purified human liver DHEA ST during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This translation product also catalyzed the sulfation of DHEA but not the sulfation of model substrates for the two forms of PST found in human liver. The two cDNA clones were also used to create expression constructs with the eukaryotic expression vector P91023(B), and these constructs were used to transfect COS-1 cells. The transfected cells expressed a high level of DHEA ST activity, and this enzyme activity displayed a pattern of inhibition by the ST inhibitor 2,6-dichloro-4-nitrophenol identical to that of human liver DHEA ST. Cloning of cDNA for this important human sulfate-conjugating enzyme will enhance understanding of the relationship between DHEA ST and other human liver STs, as well as ST enzymes in other species.",
author = "Otterness, {Diane M.} and Wieben, {Eric D} and Wood, {Thomas C.} and Watson, {R. William G} and Madden, {Benjamin J.} and {Mc Cormick}, {Daniel J} and Weinshilboum, {Richard M}",
year = "1992",
month = "5",
language = "English (US)",
volume = "41",
pages = "865--872",
journal = "Molecular Pharmacology",
issn = "0026-895X",
publisher = "American Society for Pharmacology and Experimental Therapeutics",
number = "5",

}

TY - JOUR

T1 - Human liver dehydroepiandrosterone sulfotransferase

T2 - Molecular cloning and expression of cDNA

AU - Otterness, Diane M.

AU - Wieben, Eric D

AU - Wood, Thomas C.

AU - Watson, R. William G

AU - Madden, Benjamin J.

AU - Mc Cormick, Daniel J

AU - Weinshilboum, Richard M

PY - 1992/5

Y1 - 1992/5

N2 - Sulfation is an important pathway in the metabolism of many hormones and drugs. Human liver contains at least three well characterized sulfotransferase (ST) enzymes, i.e., dehydroepiandrosterone (DHEA) ST and two forms of phenol sulfotransferase (PST). Our goal was to purify, to obtain partial amino acid sequence for, and to clone and express cDNA for human liver DHEA ST. Polymerase chain reaction primers were designed on the basis of homology among rat liver hydroxysteroid ST, rat liver PST, and bovine estrogen ST. These primers amplified a unique sequence from human liver cDNA, and this polymerase chain reaction product was used to screen a human liver cDNA library. Two clones were isolated that contained identical open reading frames, of 855 nucleotides, that encoded a protein of 285 amino acids. The deduced amino acid sequence of the encoded protein included two separate 27- and 23-amino acid sequences that were identical to those obtained by microsequencing of proteolytic fragments from purified human liver DHEA ST. Translation, in a rabbit reticulocyte lysate system, of mRNA transcribed in vitro from the two cDNA clones resulted in a 35-kDa translation product that comigrated with purified human liver DHEA ST during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This translation product also catalyzed the sulfation of DHEA but not the sulfation of model substrates for the two forms of PST found in human liver. The two cDNA clones were also used to create expression constructs with the eukaryotic expression vector P91023(B), and these constructs were used to transfect COS-1 cells. The transfected cells expressed a high level of DHEA ST activity, and this enzyme activity displayed a pattern of inhibition by the ST inhibitor 2,6-dichloro-4-nitrophenol identical to that of human liver DHEA ST. Cloning of cDNA for this important human sulfate-conjugating enzyme will enhance understanding of the relationship between DHEA ST and other human liver STs, as well as ST enzymes in other species.

AB - Sulfation is an important pathway in the metabolism of many hormones and drugs. Human liver contains at least three well characterized sulfotransferase (ST) enzymes, i.e., dehydroepiandrosterone (DHEA) ST and two forms of phenol sulfotransferase (PST). Our goal was to purify, to obtain partial amino acid sequence for, and to clone and express cDNA for human liver DHEA ST. Polymerase chain reaction primers were designed on the basis of homology among rat liver hydroxysteroid ST, rat liver PST, and bovine estrogen ST. These primers amplified a unique sequence from human liver cDNA, and this polymerase chain reaction product was used to screen a human liver cDNA library. Two clones were isolated that contained identical open reading frames, of 855 nucleotides, that encoded a protein of 285 amino acids. The deduced amino acid sequence of the encoded protein included two separate 27- and 23-amino acid sequences that were identical to those obtained by microsequencing of proteolytic fragments from purified human liver DHEA ST. Translation, in a rabbit reticulocyte lysate system, of mRNA transcribed in vitro from the two cDNA clones resulted in a 35-kDa translation product that comigrated with purified human liver DHEA ST during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This translation product also catalyzed the sulfation of DHEA but not the sulfation of model substrates for the two forms of PST found in human liver. The two cDNA clones were also used to create expression constructs with the eukaryotic expression vector P91023(B), and these constructs were used to transfect COS-1 cells. The transfected cells expressed a high level of DHEA ST activity, and this enzyme activity displayed a pattern of inhibition by the ST inhibitor 2,6-dichloro-4-nitrophenol identical to that of human liver DHEA ST. Cloning of cDNA for this important human sulfate-conjugating enzyme will enhance understanding of the relationship between DHEA ST and other human liver STs, as well as ST enzymes in other species.

UR - http://www.scopus.com/inward/record.url?scp=0027100441&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0027100441&partnerID=8YFLogxK

M3 - Article

C2 - 1588921

AN - SCOPUS:0027100441

VL - 41

SP - 865

EP - 872

JO - Molecular Pharmacology

JF - Molecular Pharmacology

SN - 0026-895X

IS - 5

ER -