TY - JOUR
T1 - Human dehydroepiandrosterone sulfotransferase pharmacogenetics
T2 - Quantitative Western analysis and gene sequence polymorphisms
AU - Wood, Thomas C.
AU - Her, Chengtao
AU - Aksoy, Ibrahim
AU - Otterness, Diane M.
AU - Weinshilboum, Richard M.
N1 - Funding Information:
The two polymorphic sequences that we discovered were transiently expressed, both individually and together, in COS-1 cells. Although these alterations in amino acid sequence, as anticipated, changed enzyme thermal stability (Table 4), they did not result in striking changes in either inhibitor sensitivity (Table 4) or substrate kinetics (Table 5). However, the presence of the nucleotide 557 polymorphism resulted in decreased expression of both DHEA ST enzymatic activity and immunoreactive protein (see Fig. 5 and Fig. 6), but neither of the polymorphisms was systematically related to level of either DHEA ST enzymatic activity or immunoreactive protein in the human liver biopsy samples studied here (Table 3). Obviously, a variety of mechanisms other than genetic polymorphism within the open reading frame of the cDNA could potentially alter the level of enzyme activity in human tissue. Those mechanisms include altered regulation of transcription, variation in mRNA stability, and alteration in translation, among others. Epidemiological studies have raised the possibility of a relationship between levels of plasma DHEA sulfate and a variety of human diseases, including cardiovascular disease and neoplasia \[7-9\]. DHEA has also generated both scientific and popular interest as a possible "anti-aging" drug \[44-47\]. Those reports increase the importance of understanding factors involved in the regulation of DHEA ST, an enzyme that catalyzes a major pathway in the metabolism of DHEA, a pathway that generates DHEA sulfate. Our discovery and characterization of common genetic polymorphisms for DHEA ST will make it possible, in the future, to determine whether those polymorphisms might be one factor involved in individual variation in the function or expression of this important enzyme in humans. \[22, 23, 37-40\] Acknowledgements--This work was supported in part by NIH grants RO1 GM 28157 and RO1. GM 35720, as well as a supplement to RO1 GM 35720 supported by the Office of Research on Women's Health. We thank Luarme Wussow for her assistance with the preparation of this manuscript.
PY - 1996/12
Y1 - 1996/12
N2 - Dehydroepiandrosterone sulfotransferase (DHEA ST) catalyzes the sulfation of DHEA and other hydroxysteroids. DHEA ST enzymatic activity in individual human liver biopsy samples has been shown to vary over a five-fold range, and frequency distribution histograms are bimodal, with approximately 25% of subjects included in a high activity subgroup. We set out to characterize the molecular basis for variation in human liver DHEA ST activity. The first step involved performing quantitative Western analysis of cytosol preparations from 92 human liver samples that had been phenotyped with regard to level of DHEA ST enzymatic activity. There was a highly significant correlation (r(s) = 0.635, P < 0.0001) between levels of DHEA ST activity and immunoreactive protein. We next attempted to determine whether the expression of DHEA ST might be controlled, in part, by a genetic polymorphism. DNA was isolated from three 'low' and three 'high' DHEA ST activity liver samples. Exons and the 5'-flanking region of the DHEA ST gene (STD) were amplified for each of these samples with the polymerase chain reaction (PCR). When compared with ('wild type' STD sequence, some of the samples contained a T→C transition at DHEA ST cDNA nucleotide 170, located within exon 2, resulting in a Met 57→Thr change in amino acid. Other samples contained an A→T transversion at nucleotide 557 within STD exon 4 that resulted in a Glu 186→Val change. STD exons 2 and 4 were then sequenced for DNA isolated from an additional 87 liver samples that had been phenotyped with regard to level of DHEA ST enzymatic activity. The allele frequency for the exon 2 polymorphism in these samples was 0.027, whereas that for the exon 4 polymorphism was 0.038, but neither polymorphism was systematically related to the level of enzyme activity in these samples. Transient expression in COS-1 cells of cDNA that contained the nucleotide 170 and 557 polymorphisms, either separately or together, resulted in decreased expression of both DHEA ST enzymatic activity and level of immunoreactive protein, but only when the nucleotide 557 variant was present. Identification of common genetic polymorphisms within STD will now make it possible to test the hypothesis that those polymorphisms might alter in vivo expression and/or function of this important human steroid-metabolizing enzyme.
AB - Dehydroepiandrosterone sulfotransferase (DHEA ST) catalyzes the sulfation of DHEA and other hydroxysteroids. DHEA ST enzymatic activity in individual human liver biopsy samples has been shown to vary over a five-fold range, and frequency distribution histograms are bimodal, with approximately 25% of subjects included in a high activity subgroup. We set out to characterize the molecular basis for variation in human liver DHEA ST activity. The first step involved performing quantitative Western analysis of cytosol preparations from 92 human liver samples that had been phenotyped with regard to level of DHEA ST enzymatic activity. There was a highly significant correlation (r(s) = 0.635, P < 0.0001) between levels of DHEA ST activity and immunoreactive protein. We next attempted to determine whether the expression of DHEA ST might be controlled, in part, by a genetic polymorphism. DNA was isolated from three 'low' and three 'high' DHEA ST activity liver samples. Exons and the 5'-flanking region of the DHEA ST gene (STD) were amplified for each of these samples with the polymerase chain reaction (PCR). When compared with ('wild type' STD sequence, some of the samples contained a T→C transition at DHEA ST cDNA nucleotide 170, located within exon 2, resulting in a Met 57→Thr change in amino acid. Other samples contained an A→T transversion at nucleotide 557 within STD exon 4 that resulted in a Glu 186→Val change. STD exons 2 and 4 were then sequenced for DNA isolated from an additional 87 liver samples that had been phenotyped with regard to level of DHEA ST enzymatic activity. The allele frequency for the exon 2 polymorphism in these samples was 0.027, whereas that for the exon 4 polymorphism was 0.038, but neither polymorphism was systematically related to the level of enzyme activity in these samples. Transient expression in COS-1 cells of cDNA that contained the nucleotide 170 and 557 polymorphisms, either separately or together, resulted in decreased expression of both DHEA ST enzymatic activity and level of immunoreactive protein, but only when the nucleotide 557 variant was present. Identification of common genetic polymorphisms within STD will now make it possible to test the hypothesis that those polymorphisms might alter in vivo expression and/or function of this important human steroid-metabolizing enzyme.
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U2 - 10.1016/S0960-0760(96)00142-2
DO - 10.1016/S0960-0760(96)00142-2
M3 - Article
C2 - 9010352
AN - SCOPUS:0030461435
VL - 59
SP - 467
EP - 478
JO - Journal of Steroid Biochemistry and Molecular Biology
JF - Journal of Steroid Biochemistry and Molecular Biology
SN - 0960-0760
IS - 5-6
ER -