Bacterial expression and characterization of a cDNA for human liver estrogen sulfotransferase

doi:10.1016/0960-0760(95)00015-R

The Journal of Steroid Biochemistry and Molecular Biology

Volume 52, Issue 6, June 1995, Pages 529-539

https://doi.org/10.1016/0960-0760(95)00015-R Get rights and content

Abstract

A distinct human estrogen sulfotransferase (hEST-1) cDNA has been isolated from a human liver γZap cDNA library using a PCR procedure. The enzymatically active protein has been expressed in two bacterial expression systems and the kinetic and immunologic properties of the enzyme have been characterized. The full-length cDNA for hEST-1 is 994 base pairs in length and encodes a 294 amino acid protein with a calculated molecular mass of 35,123 Da. Purified hEST-1 migrated with an apparent molecular mass of 35,000 Da during SDS-polyacrylamide gel electrophoresis. Immunoblot analysis of hEST-1 expressed in E. coli with a rabbit anti-hEST-1 antibody yields a band of approximately 35,000 Da. The anti-hEST-1 antibody also detects a single band in human liver and jejunum cytosol which migrates with the same molecular mass as expressed hEST-1. There was also no cross-reactivity of hEST-1 with rabbit anti-hP-PST or rabbit anti-hDHEA-ST antibodies upon immunoblot analysis. hEST-1 was expressed in bacteria and purified to homogeneity. Expressed hEST-1 activity has a significantly greater affinity for estrogen sulfation than that found for the other human STs which conjugate estrogens. hEST-1 maximally sulfates β-estradiol and estrone at concentrations of 20 nM. hEST-1 also sulfates dehydroepiandrosterone, pregnenolone, ethinylestradiol, and 1-naphthol, at significantly higher concentrations; however, cortisol, testosterone and dopamine are not sulfated. The results presented in this paper describe the expression and characterization of a human EST distinct from other human STs which sulfate estrogens. The high affinity of hEST-1 for estrogens indicates that this ST may be important in both the metabolism of estrogens and in the regulation of their activities.

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Conjugation of steroids and sterol compounds with a sulfonate group is a major pathway in the regulation of their activity, synthesis and excretion. Three human cytosolic sulfotransferases are highly involved in the sulfonation of sterol compounds. SULT1E1 has a low nM affinity for estrogen sulfonation and also conjugates non-aromatic steroids with a significantly lower affinity. SULT2A1 is responsible for the high levels of fetal and adult dehydroepiandrosterone (DHEA) sulfate synthesis in the adrenal gland as well as many 3α and 3ß-hydroxysteroids and bile acids. SULT2B1b is responsible for the majority of cholesterol sulfation in tissues as well as conjugating 3ß-hydroxysteroids. Although there are multiple methods for assaying cytosolic SULT activity, two relatively simple, rapid and versatile assays for steroid sulfonation are described. The first method utilizes radiolabeled substrates and organic solvent extraction to isolate the radiolabeled product from the aqueous phase. The second assay utilizes ³⁵S-3′-phosphoadenosine 5′-phosphosulfate (PAPS) to generate ³⁵S-conjugated products that are resolved by thin layer chromatography. Both assays useful in situations requiring measurement of SULT activity in a timely manner.
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Xian-Ling-Gu-Bao capsule (XLGB) is a widely prescribed traditional Chinese medicine used for the treatment of osteoporosis. However, it significantly elevates levels of serum estrogens. Here we aimed to assess the dominant contributors of sulfotransferase (SULT) enzymes to the sulfation of estrogens and identify the effective inhibitors of this pathway in XLGB. First, estrone, 17β-estradiol, and estriol underwent sulfation in human liver S9 extracts. Phenotyping reactions and enzyme kinetics assays revealed that SULT1A1, 1A2, 1A3, 1C4, 1E1, and 2A1 all participated in estrogen sulfation, with SULT1E1 and 1A1 as the most important contributors. The incubation system for these two active enzymes were optimized with Tris-HCl buffer, DL-Dithiothreitol (DTT), MgCl₂, adenosine 3'-phosphate 5'-phosphosulfate (PAPS), protein concentration, and incubation time. Then, 29 compounds in XLGB were selected to investigate their inhibitory effects and mechanisms against SULT1E1 and 1A1 through kinetic modelling. Moreover, in silico molecular docking was used to validate the obtained results. And finally, the prenylated flavonoids (isobavachin, neobavaisoflavone, etc.) from Psoralea corylifolia L., prenylated flavanols (icariside II) from Epimedium brevicornu Maxim., tanshinones (dihydrotanshinone, tanshinone II-A,) from Salvia miltiorrhiza Bge., and others (corylifol A, corylin) were identified as the most potent inhibitors of estrogen sulfation. Taken together, these findings provide insights into the understanding regioselectivity of estrogen sulfation and identify the effective components of XLGB responsible for the promotion of estrogen levels.
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Citation Excerpt :
Indeed, SULT1E1 is not only crucial for regulating low estrogen concentrations in target tissues (sulfated estrogens being inactive on estrogen receptors), but might be involved in the regulation of estrogen-dependent carcinogenesis or in the mechanisms of endocrine disruption.84 SULT1E1 can also sulfate other steroids and their analogs, including dehydroepiandrosterone, pregnenolone, or diethylstilbestrol (DES).85,86 Other substrates are flavonoids, fulvestrant (a drug used for breast cancer treatment), the active metabolites of toremifene and tamoxifen, troglitazone (an oral antidiabetic), or tibolone.9,40,87
Sulfotransferases (SULTs) are Phase II drug-metabolizing enzymes (DMEs) catalyzing the sulfation of a variety of endogenous compounds, natural products, and drugs. Various drugs, such as nonsteroidal anti-inflammatory drugs (NSAIDS) can inhibit SULTs, affecting drug–drug interactions. Several polymorphisms have been identified for SULTs that might be crucial for interindividual variability in drug response and toxicity or for increased disease risk. Here, we review current knowledge on non-synonymous single nucleotide polymorphisms (nsSNPs) of human SULTs, focusing on the coded SULT allozymes and molecular mechanisms explaining their variable activity, which is essential for personalized medicine. We discuss the structural and dynamic bases of key amino acid (AA) variants implicated in the impacts on drug metabolism in the case of SULT1A1, as revealed by molecular modeling approaches.
Biomimetic estrogen sensor based on soft colloidal probes
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An increasing number of reports substantiate the link between emerging estrogenic pollutants and a variety of adverse effects including developmental disorders, infertility, cancer and neurological disorders, threatening public health as well as environment. The detection of the diverse classes of estrogenic and antiestrogenic substances is still challenging due to analytics which needs to cover the whole range of compounds acting on estrogen receptors and the complex estrogen pathways. In this proof-of-concept study, we report a novel biomimetic detection scheme based on the specific recognition of estrogenic ligands by estrogen sulfotransferase 1E1 (SULT1E1), which acts as one of the key enzymes in estrogen homeostasis. SULT1E1 was site-specifically immobilized on transparent glass slides via a hexahistidine-tag in a multi-step procedure. Soft colloidal probes (SCPs) covalently functionalized with ligands of SULT1E1, namely estrone and estradiol 17-(β-D-glucuronide), served as adhesion probes. The various functionalization steps were analyzed and optimized using epifluorescence, confocal laser scanning as well as reflection interference contrast microscopy (RICM). A competitive SCP binding assay probing the elastic SCP deformation driven by the specific interaction between SCPs and the SULT1E1 decorated glass slides was employed in conjunction with an optical readout by RICM and automated image analysis to detect estrogenic compounds by their inhibition of SCP adhesion. This sensing concept has demonstrated exceptional specificity for estrogenic steroid compounds compared to structurally related substance classes and provides promising options for multiplexed assays and incorporation of other proteins of the endocrine system to fully capture the whole ensemble of hormonally active substances.
Comparative pharmacokinetic estimates of drospirenone alone and in combination with ethinyl estradiol after single and repeated oral administration in healthy females
2020, Contraception
Citation Excerpt :
Rohn et al. [16] and Rohn-Glowacki [17] recently explored the potent inhibition by EE of human sulfotransferase 1A1 (SULT1A1), the major xenobiotic sulfating isoform in the liver. The isoforms of greatest interest in these studies were SULT1E1, known to sulfate EE at nanomolar levels (Falany et al., [18], Falany and Falany, [19]), and SULT1A2, which has the most similar loop 1 aa sequence including Ile89 identical to SULT1A1. The inhibition of SULT1E1 sulfation activity by EE would be competitive since it is known to be a substrate.
To determine the pharmacokinetics (PK) of drospirenone (DRSP), alone versus in combination with ethinyl estradiol (EE), after single and repeated administration.
We conducted a single-centre, open-label, crossover, 2-treatment, 2-period, 2-sequence study in which non-micronized DRSP 4 mg or a combination of DRSP 3 mg and EE 0.02 mg were administered to healthy female subjects on day 1 to obtain a single-dose kinetic profile, and from day 4 to day 15 to obtain a repeated-dose kinetic profile. The maximum observed concentration (C_max) and area under the concentration/time curve (AUC) were determined in a model-independent way using non dose corrected data. Statistical analysis was based on a parametric method (ANOVA-log).
A total of 24 healthy female subjects were randomized 1:1 into the study. The mean relative, non-dose-corrected PK estimates after single-dose administration for the endpoints AUC_(0-72h), AUC_(0-24h) and C_max were 543.5 ng*h/mL, 296.1 ng*h/mL and 27.3 ng/mL for DRSP alone, and 442.0 ng*h/mL, 264.7 ng*h/mL and 37.5 ng/mL for the DRSP/EE combination; p < 0.001. The mean relative, non-dose-corrected PK estimates after repeated dose administration for the endpoints AUC_(0-72h), AUC_(0-24h) and C_max were 1066.8 ng*h/ml, 570.2 ng*h/mL and 41.0 ng/mL for DRSP alone, and 1394.5 ng*h/mL, 732.8 ng*h/mL and 61.4 ng/mL for the DRSP/EE combination; p < 0.001.
DRSP alone exhibits a lower accumulation ratio than together with EE. The extent of systemic exposure at steady-state is about 32% less with the new formulation (AUC_(0-24h), steady-state geometric mean ratio: 77.8%; 90% confidence interval: 74.6%–81.1%). This PK profile may be caused by EE.
Our results suggest that metabolic pathways of DRSP can be inhibited by EE resulting in higher DRSP plasma concentrations in DRSP/EE formulations than in a DRSP-alone formulation. The enzymes CYP3A4 and SULT1A1 may play a role. Additional drug-drug-interaction studies are needed to better understand these metabolic pathways and their future clinical implications.
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2019, Journal of Steroid Biochemistry and Molecular Biology
Advances in technology have allowed for the sensitive, specific, and simultaneous quantitative profiling of steroid precursors, bioactive steroids and inactive metabolites, facilitating comprehensive characterization of the serum and urine steroid metabolomes. The quantification of steroid panels is therefore gaining favor over quantification of single marker metabolites in the clinical and research laboratories. However, although the biochemical pathways for the biosynthesis and metabolism of steroid hormones are now well defined, a gulf still exists between this knowledge and its application to the measured steroid profiles. In this review, we present an overview of steroid hormone biosynthesis and metabolism by the liver and peripheral tissues, specifically highlighting the pathways linking and differentiating the serum and urine steroid metabolomes. A brief overview of the methodology used in steroid profiling is also provided.

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Bacterial expression and characterization of a cDNA for human liver estrogen sulfotransferase

Abstract

J. Steroid Biochem. Molec. Biol.

Biochem. Biophys. Res. Commun.

Arch. Biochem. Biophys.

Biochem. Pharmac.

Biochem. Biophys. Res. Commun.

Molec. Cell. Endocr.

Biochem. Biophys. Res. Commun.

The effects of oral dehydroepiandrosterone on endocrine-metabolic parameters in postmenopausal women

J. Clin. Endocr. Metab.

Sulfation

Human liver steroid sulfotransferase sulfates bile acids

Biochem. J.

Sulfation of estrone and 17β-estradiol in human liver

Drug Metab. Disp.

Purification and characterization of human liver dehydroepiandrosterone sulfotransferase

Biochem. J.