Human dehydroepiandrosterone sulfotransferase: purification and characterization of a recombinant protein

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Abstract

Dehydroepiandrosterone sulfate is the most abundant sulfated steroid transformed in human tissues and serves as a precursor for steroid hormones. Recombinant human dehydroepiandrosterone sulfotransferase (DHEA-ST) expressed in glutathione sulfotransferase fusion form in E. coli was purified using glutathione sepharose 4B affinity adsorption chromatography, a Factor Xa cleavage step, and Q-sepharose fast flow column chromatography. The homogeneous preparation had an activity toward dehydroepiandrosterone (DHEA) of 150±40 nmol/min per mg of protein under the assay conditions at an overall yield of 38.4%. The recombinant human DHEA-ST was shown to have a subunit mass of 34 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis, while having a molecular mass of 67.2 kDa by Superose-12 gel filtration. Our results indicate that the active recombinant enzyme expressed in E. coli is a homodimer.

Biochemical properties for purified DHEA-ST were studied using DHEA as a substrate. The optimum pH ranged from pH 7 to 8, and the optimum temperature 40–45°C. Ninety percent of basal DHEA-ST activity remained even after the enzyme was treated at 45°C for 15 min. The 50% inactivation concentration of NaCl for DHEA-ST activity was determined to be around 500 mM. The Km value for DHEA was 1.9±0.3 μM and Vmax=190±18 nmol/min per mg of protein at 37°C, pH 7.5.

Introduction

Sulfate conjugation (or more precisely, sulfonate conjugation), the transfer of a sulfonate group (SO3) from 3′-phosphoadenosine-5′-phosphosulphate (PAPS) to a hydroxyl acceptor site has been considered a key pathway in the biotransformation of steroids, catecholamines, vitamin D, bile acids, and many xenobiotics. The sulfate group of PAPS has a pKa of approximately 2, so that the transfer of the charged sulfonate moiety results in an important increase in the hydrophilicity of the compound and a reduction in its biological activity, thereby enhancing its excretion into the urine and/or bile leading to detoxification [1], [2], [3], [4], [5].

In addition to inactivation of the compound, the sulfoconjugation reaction may also be a special system for maintaining steroid hormone levels since the steroid sulfates could play a key role as a precursor of steroid hormones such as active androgens and estrogens in peripheral tissues [6], [7], [8]. In other words, dehydroepiandrosterone sulfate (DHEAS) is the most abundant circulating steroid precursor and one of the most important secretory products of the adrenal cortex. Up to 200 mg/day of DHEAS is produced from the adrenal glands mainly by dehydroepiandrosterone sulfotransferase (DHEA-ST) in the fetal zone [9], [10], [11]. An important biological function of DHEAS is to serve as the main substrate for oestrogen biosynthesis and therefore to maintain pregnancy [12], [13].

In human tissues, a single isoform of DHEA-ST, the enzyme that catalyzes the sulfate conjugation reaction of dehydroepiandrosterone (DHEA), bile acids, and steroids, has been reported on the basis of protein purification [14], [15], [16] and molecular cloning studies [17], [18], [19], [20] in comparison to several rat isoforms [21], [22], [23], [24]. Human DHEA-ST has been partially purified or to apparent homogeneity from natural sources such as liver and adrenal tissues [14], [16] and recombinant DHEA-ST has recently been expressed and characterized in both bacterial and mammalian systems [25], [26].

To date, the mechanism of sulfate conjugation and the structure-function relationship of DHEA-ST have not been clearly elucidated. In order to make a detailed study of the structure–function, large quantities of homogeneous protein in a functionally active state are required.

Here we report a rapid and simple purification procedure and also characterization of homogeneous recombinant DHEA-ST expressed in E. coli. The entire purification process can be completed in one day in comparison to the several days previously required, when using preparations from natural sources. Homogeneity of our preparation was verified by crystallization of the enzyme. We characterized the dimeric nature of the active recombinant DHEA-ST and investigated the biochemical properties and steady-state kinetics of this enzyme.

Section snippets

Materials

Glutathione sepharose 4B, Q-sepharose fast flow, and superose 12 (HR 10/30) prepacked column were purchased from Amersham Pharmacia Biotech (Baie d'Urfe, Que., Canada). Protein standards for gel filtration chromatography and SDS-PAGE were obtained from Bio-Rad (Mississauga, Ont., Canada). 14C-radiolabelled DHEA were from New England Nuclear Dupont (Boston, MA). DHEA, lysozyme, and dithiothreitol were from Sigma (St. Louis, MO).

Dehydroepiandrosterone sulfotransferase activity assay

Prior to assay, the DHEA-ST protein was diluted with 20 mM Tris, pH

Purification of recombinant dehydroepiandrosterone sulfotransferase from E. coli

Following overnight cleavage of GST fused DHEA-ST by Factor Xa, approximately 90% of the fusion protein was cleaved into its constituent parts (Fig. 1).

The active DHEA-ST fractions from Glutathione sepharose 4B column were applied on to a Q-sepharose fast flow chromatography to remove trace amounts of Factor Xa and other contaminants. Elution of DHEA-ST with a NaCl linear gradient resulted in 6–7 mg of the active DHEA-ST (Table 1). The final purified DHEA-ST protein had an activity of 150±40

Discussion

In general, sulfotransferases play a key role in the sulfate conjugation (or more correctly, sulfonate conjugation) process resulting in an inactivation of compounds [1], [2], [3], [4], [5]. Additionally, due to the importance of steroid sulfates as precursors in the biotransformation of active steroid hormones in target tissues [6], [7], [8], [9], [10], [11], [12], [13], many studies have concentrated on the purification, cloning, and biochemical characterization of DHEA-ST [14], [15], [16],

Acknowledgements

The authors would like to thank Dr. F. Labrie for his interests in this work, and Dr. V. Luu-The for providing the E. coli strain overproducing the DHEA-ST.

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