Immunochemical characterisation of a dehydroepiandrosterone sulfotransferase in rats and humans

Eur. J. Biochem. 211, 539-548 (1993) 0 FEBS 1993

Immunochemical characterisation of a dehydroepiandrosterone sulfotransferase in rats and humans

. Sheila SHARP‘, Emma V. BARKER‘, Michael W. H. COUGHTRIE’, Pedro R. LOWENSTEIN’ and Robert HUME3 ’ Department of Biochemical Medicine, University of Dundee, Ninewells Hospital and Medical School, Scotland

Department of Anatomy and Physiology, University of Dundee, Scotland Department of Child Life and Health, University of Edinburgh, Scotland

(Received October 6, 1992) - EJB 92 1410

A member of the rat liver hydroxysteroid sulfotransferase (ST) enzyme family metabolising dehydroepiandrosterone (DHEA) was purified from female rats and used to raise rabbit polyclonal antibodies. Characterisation of this antibody preparation demonstrated that it was specific for DHEA ST, and recognised a single 30-kDa protein on immunoblot analysis of rat liver cytosol which was expressed preferentially in female rat liver, and immunohistochemical localisation of the protein in female rat liver determined that DHEA ST was distributed homogeneously in the cytoplasm of hepatocytes. Examination of the extrahepatic expression of this protein showed it to be located predominantly in the liver, although a small amount of enzyme activity was found in the kidney which was not apparently subject to the same sex difference as the hepatic activity. Immunological analysis suggested that this activity was not due to the action of DHEA ST, but to another, unidenti- fied ST isozyme. The antibody cross-reacted strongly with adult human liver DHEA ST, recognising a protein of 35 kDa on immunoblotting. Using this antibody preparation, the distribution of DHEA ST in mid-trimester human fetal tissues was examined, and it was shown that the enzyme is expressed in the adrenal and liver, but not to any significant extent in the kidney or lung. This antibody therefore provides a powerful tool for investigating the function of DHEA ST.

Sulfation is an important pathway of metabolism for a host of xenobiotics (e.g. drugs, carcinogens, environmental chemicals) and endogenous compounds (e.g. steroid hormones, bile acids, neurotransmitters and small peptides j, which in general (though not always) reduces the biological activity of compounds through the attachment of a polar sulfate group to various hydroxyl and amine groups [ l , 21. These reactions are catalysed by a family of sulfotransferase isozymes (ST), present in the cytosolic fraction of the liver and other tissues. In rats, a number of subfamilies of ST have been discovered, which exhibit enzyme activities towards different classes of substrate, including phenol ST, hydroxysteroid ST and estrone ST, and it is believed, based on protein-purification experiments and the sex differences in the expression of the different activities, that each of these subfamilies comprises a number of different ST enzymes with dis- tinct but overlapping substrate specificities [3 - 121. Recently, cDNA-cloning work has provided the nucleotide sequences of two closely related hydroxysteroid ST [13, 141, a phenol ST [lS] and an estrogen ST [16] in rat liver, and a DHEA ST in human liver [17], providing further evidence for this subdivision of ST isozymes.

Correspondence to M. W. H. Coughtrie, Department of Bio- chemical Medicine, University of Dundee, Ninewells Hospital and Medical School, Dundee, Scotland DD1 9SY

Fax: +44 382 644620. Abbreviations. DHEA, dehydroepiandrosterone ; DHEA-S, de-

hydroepiandrosterone sulfate ; ST, sulfotransferase; PAdoPS, 3’- phosphoadenosine 5‘-phosphosulfate; FITC, fluorescein isothiocyanate; PAdoP, adenosine 3’,5’-diphosphate.

Dehydroepiandrosterone (DHEA) is an important steroid hormone, and its sulfate ester (DHEA-S) is the major circu- lating steroid. The exact physiological role(s) of DHEA (and DHEA-S) still remains unclear, although it has been established that DHEA-S is a good substrate for steroid biosynthesis [18-201, and much speculation has appeared regard- ing the observed anticancer [21], anti-obesity [22] and anti- diabetic [23-25] effects of DHEA and DHEA-S. In order to improve our understanding of the processes governing the regulation of the levels of DHEA and DHEA-S, we have prepared antibodies against purified rat liver DHEA ST and characterised these antibodies in rats and humans by enzyme inhibition and by immunoblotting. This antibody preparation, by virtue of its strong cross-reactivity with the corresponding human DHEA ST, will prove of great value in investigating the function of this enzyme in man.

MATERIALS AND METHODS Chemicals

[ 1 ,2,3,6,7-1H(N)]-Dehydroepiandrosterone (11 5 Ci/mmol) was purchased from Du PontJNew England Nuclear, Steven- age, UK. Dehydroepiandrosterone, Freund’s adjuvant (complete and incomplete), alkaline-phosphatase-conjugated goat anti-(rabbit IgG) serum (adsorbed with human serum proteins), nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (p-toluidine salt) were obtained from Sigma Chemical Company Ltd., Poole, UK, and 5’-phosphoadenosine 3’-phosphosulphate (PAdoPS) and all protein purification

540

meda were from Pharmacia Ltd., Milton Keynes, UK. Elec- trophoresis chemicals were obtained from Merck/BDH Ltd., Glasgow, UK, and scintillation fluid (Emulsifier Safe) was from Canberra Packard, Pangbourne, UK. Paraformaldehyde was purchased from Agar Scientific Ltd (Essex, UK), and biotinylated goat anti-(rabbit IgG) IgG and normal goat serum were obtained from Vector Laboratories, Peterbor- ough, UK. Streptavidin-fluorescein-isothiocyanate (FITC) conjugate was obtained from Pierce and Warriner, Cheshire, UK and optimal-cutting-temperature compound-embedding medium was purchased from Miles Laboratories, USA. All other reagents were of analytical grade and purchased from commonly used local suppliers.

Tissue preparation and enzyme assay

Adult Wistar rats (4 months of age) were obtained from the colony maintained in this institute, and adult human liver and kidney samples were obtained from organ donors (no further clinical details available). Tissue cytosols were prepared by differential centrifugation : homogenates (30 % mass/vol.) were prepared in 10 mM triethanolamine/Cl-, pH 7.4, 250 mM sucrose, 3 mM 2-mercaptoethanol (buffer A) and centrifuged at 10000 g for 15 min, whereupon the resulting supernatants were subjected to further centrifugation at 105000 g for 60 min. The supernatants were aspi- rated carefully, avoiding the lipid layer at the surface, divided into 1-ml aliquots and stored at - 70°C until used (within 3 months). Human fetal tissues, from a 16-week fetus, were homogenised as above, centrifuged at 10000 g for 15 min and the supernatants stored at - 70°C for transport to the laboratory. Cytosols were prepared from the thawed 10000 g supernatants as described above. The use of fetal tissues was approved by the Paediatric-Reproductive Medicine Ethics of Medical Research Sub-committee, Lothian Health Board.

DHEA ST enzyme activity was assayed essentially as described [26]. Briefly, incubations (final volume, 190 pl) contained cytosolic protein (150 pg), DHEA (75 pM), PAdoPS (60 pM) and buffer (0.1 M Tris/Cl-, pH 7.4,20 mM MgCl,), and after incubation for 20 min at 37°C reactions were stopped by the addition of 2 ml water-saturated dichlor- oethane. 250 pl water was added and, following brief shak- ing, the phases were separated by centrifugation at 3000g for 3 min, 250 pl aqueous phase were mixed with 3 ml scintillation fluid and the radioactivity determined by liquid scintillation spectrometry. Blank incubations contained no PA- doPS. Other ST enzyme assays have been previously described : estrogens, testosterone, pregnenolone [27] ; 1 -naphthol in rat (pH 6.6) [26] and man [28]; androsterone [lo] and dopamine [291.

Purification of DHEA ST Livers from three female Wistar rats (total mass, 25-

30 g) were homogenised in 2 vol. buffer A, and the cytosolic fraction prepared as described above. The cytosol was applied directly to a column of DEAE-Sepharose Fast Flow (2.6 cm X 60 cm), previously equilibrated in buffer A, at a flow rate of 80mVh. Following the elution of unbound material, a linear gradient between buffer A and buffer A containing 200 mM NaCl in a total volume of 1 1 at a flow rate of 40 mVh was developed. Fractions of 6 ml were collected and assayed for DHEA ST activity. Fractions having high DHEA ST and low 1-naphthol ST activity were pooled, concentrated to approximately 10 ml by ultrafiltration (Ami- con, Stonehouse, UK) and applied to a column (2.5 cm X 85 cm) of Sephacryl S-200 HR equilibrated in buffer A containing 0.1 M NaCl at a flow rate of 20 mVh. Fractions of 3 ml were collected and assayed for DHEA ST activity. Active fractions were again pooled, concentrated to 5 ml and subjected to affinity chromatography on PAdoP-agarose (1.6 cmX 4 cm) in buffer A. Following elution of unbound material, the column was washed extensively with buffer A containing 50 mM KCI, and DHEA ST was eluted with 4 ml 100 pM PAdoPS in buffer A containing 50 mM KCl. Active fractions were pooled, concentrated to 2 ml and subjected to a final clean-up step by anion-exchange FPLC on MonoQ HR5/5 (Pharmacia). 1-ml aliquots of the affinity-purified material were injected onto the MonoQ column which had been previously equilibrated in buffer A at a flow rate of 1 mumin, and following elution of unbound material in 5 ml, a gradient between buffer A and buffer A containing 200mM NaCl in a total volume of 30 ml was established. Fractions of 1 ml were collected and assayed for DHEA ST activity, and active fractions were pooled, concentrated and used for antibody production. Purity of the final preparations was monitored by SDSFAGE, followed by Coomassie blue and silver staining.

Antibody production Pure DHEA ST (150pg in l m l ) was mixed with

Freund's complete adjuvant and used to immunise an adult female New Zealand white rabbit by a combination of intra- muscular and subcutaneous injections. Four weeks later, an additional 150 pg DHEA ST was mixed with Freund's incomplete adjuvant, and the immunisation procedure repeated. A further two sets of booster injections, each of 150 pg DHEA ST, performed four weeks apart, were administered and the antiserum collected 14 days after the fourth immunisation by cardiac puncture under terminal barbiturate anaes- thesia. The success of the immunisation procedure was monitored at four-week intervals using immunoblot analysis with

Table 1. Purification of rat liver DHEA ST. Specific activity was determined with DHEA as substrate, as described in Materials and Methods.

Step Protein Specific activity Purification Yield

mg pmol . min-' . mg protein-' -fold %

Cytosol 1344 170 1 100

PAdoP-agarose 1.83 15 215 89.5 12.2

DEAE-Sepharose 23 8 848 5.0 88 Sephacryl S-200 33 1490 8.8 21.5

F P L C /M o n o Q 1.11 31 040 183 15

541

'*0° F A

Fig. 1. SDWAGE analysis of purified DHEA ST. FPLC-purified female rat liver DHEA ST was resolved on a SDS/polyacrylamide gel (11 % monomer) and stained with Coomassie blue. Lanes 1 and 6 contain molecular mass standards (Sigma) molecular masses ( m a ) are identified in the left margin. Lanes 2-5 contain the purified enzyme (0.5, 1, 2 and 5 pg, respectively).

purified DHEA ST and rat liver cytosol. The IgG fraction of the antiserum was enriched by precipitation with saturated ammonium sulfate to achieve 50% saturation, followed by extensive dialysis against 0.1 M sodium phosphate, pH 7.4. This crude IgG preparation was divided into 0.5-ml aliquots and stored at - 70°C. For immunoblotting experiments involving human tissue, anti-(DHEA ST)IgG was adsorbed against human serum proteins immobilised on cyanogen-bro- mide-activated Sepharose 4B, since some background staining was observed against unrelated human proteins. This pro- cess did not affect the interaction of the antibody with human DHEA ST.

SDS/PAGE and immunoblotting Proteins were resolved on denaturing SDS/polyacrylam-

ide gels (11% acrylamide monomer), according to the method f i s t described by Laemmli [30], and stained with Coomassie blue. For immunoblotting experiments, the staining was omitted, and proteins were electrophoretically transferred to nitrocellulose sheets (Schleicher and Schiill) as described by Towbin et al. [31], and immunochemical localisation of DHEA ST was performed by the alkaline phosphatase method as previously described [32] with the exception that the pH of the buffer used for all incubation and washing steps was 9.0. All electrophoresis and blotting apparatus was from Hoefer Scientific Instruments, Newcastle-under-Lyme, UK.

Immunological quantification of DHEA ST The amount of DHEA immunoreactive protein in tissue

cytosols was estimated using immuno-dot blots. Briefly, a nitrocellulose sheet was placed within the dot-blot apparatus (Bio-Rad, Heme1 Hempstead, UK) and the wells were rinsed with 10Opl Tris/NaCl (1OmM Tris/Cl-, pH9, 154mM NaCI) and the cytosol samples applied to the wells. The

0 0.2 0.4 0.6 0.8 1

AntibodylCytosol Protein Ratio (mglmg)

140.0

120 0

lw.o 2,

> c .- e a 80.0 e - + 0 ,

60.0

z

40.0

--)- FEMALE PREIMMUNE - FEMALE ANTIBODY

-t MALE PREIMMUNE

MALE ANTIBODY

0.0 1 ~ 1 ~ ~ ' ~ ~ ~ ~ ~ ~ ~ ' ~ ~ ~ ~ ' ~ ~ ~ ~ ' ~ ~ ~ ~ ' 1 ' ' ~ ' ~ ~ ~ ~ ' ~ ~ ~ ~ ' ~ ~ ~ ~

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1

AntibodylCytosol Protein Ratio (mglmg)

Fig. 2. Inhibition of rat liver steroid ST activities by anti-(DHEA ST) serum. Rat liver cytosols were incubated, in duplicate, with increasing amounts of IgG prepared either from preimmune or immune serum on ice for 1 h. The samples were then centrifuged for 10 min at 15000 g (4°C) and the supernatants assayed for ST activity. (A) DHEA ST activity: female preimmune (H); male preimmune (+); female immune (0); male immune (0). (B) Estrone ST activity: symbols as for A.

nitrocellulose was then soaked in Tris/NaCl for 10 min, blocked for 30 min with 1 % (masdvol.) bovine serum albumin in Tris/NaCl containing 0.05% (mass/vol.) Tween 20, and exposed to anti-(DHEA ST) IgG. Antibodyhtigen interactions were visualised as described above for immunoblot analysis.

Following air drying of the nitrocellulose filter, the amount of DHEA ST enzyme protein was quantitated by densitometric analysis. A scanned image of the filters was made on an IBM-compatible personal computer (MJN

542

Table 2. Inhibition of rat and human liver cytosolic ST activities by anti-(rat liver DHEA ST) serum. Inhibition of ST activity at a ratio of 1 mg antibody/mg cytosolic protein, relative to controls containing an equal amount of non-immune IgG. Figures in parentheses are specific activities for control incubations. All measurements were made in duplicate.

Substrate Inhibition of ST activity (specific activity)

female Rat male Rat

1-Naphthol DHEA Testosterone Androsterone Pregnenolone Estrone P-Estradiol Estriol Dopamine

30 (390) 99 (173) 94 (33.7) 99 (227) 99 (115) 95 (3.37) 90 (24.4) 84 (10.6) 0 (16.9)

26 (290) 96 (11.5) 77 (1.67) 90 (18.5) 99 (17.2) 0 (51) 0 (61.7) 0 (45.8) 0 (89.9)

human A human B

% (pmol . min-' . mg-')

19 (51.2) 32 (29.9) 59 (24.2) 49 (74.3) 0 (10.1) 0 (2.0) 0 (13.9) 0 (28.2) 0 (119)

19 (101) 0 (40.8)

41 (24.7) 36 (97.1) 0 (13.8) 0 (5.2) 0 (7.3) 0 (28.8) 0 (88.9)

Fig. 3. Characterisation of anti-(DHEA ST) serum by immunoblot analysis. Cytosols were subjected to SDSPAGE in 11 % monomer gels, transferred to nitrocellulose and probed with anti-(DHEA ST) IgG at a concentration of 8 pg/ml (A) and 4 pg/ml (B). Antigen/ antibody complexes were visualised with alkaline-phosphatase-conjugated anti-(rabbit IgG) IgG at a dilution of 1 : 2000, as described in Materials and Methods. (A) Lane 1, female rat liver cytosol (5 pg); lane 2, male rat liver cytosol (5 pg); lanes 3-6, purified female rat liver DHEA ST (1.5, 0.75 and 0.25 pg, respectively). (B) Lanes 1-3, female rat liver cytosol at 2, 5 and 10 pg, respectively; lanes 4-6, human liver cytosol at 5, 10 and 20 pg, respectively.

Fig. 4. Immunoblot analysis of DHEA ST expression in various rat tissues. Immunoblot analysis was performed as described in Materials and Methods. The anti-(DHEA ST) IgG was used at a concentration of 4 pg/ml and the alkaline-phosphatase-conjugated anti-(rabbit IgG) IgG was at a 1:2000 dilution. Lanes 1-10 contained 50 pg cytosolic protein and lanes 11 and 12 contained 10 pg. Lanes 1 and 2, female and male adrenal respectively; lanes 3 and 4, female and male intestine; lanes 5 and 6, female and male brain; lanes 7 and 8, female and male lung; lanes 9 and 10, female and male kidney; lanes 11 and 12, female and male liver.

Table 3. DHEA ST activities in various rat tissues. Enzyme activity was determined, as described in Materials and Methods, with the tissue cytosol samples used for immunoblot analysis in Fig. 5. n.d., enzyme activity not detectable. i.e. below the limit of sensitivity of the assay method, which under the conditions employed was approximately 1 pmol . min-' . mg-'.

Tissue DHEA ST activity

male female

pmol . min-I . mg-'

Liver 11.5 173

Lung n.d. n.d. Brain n.d. n.d. Adrenal n.d. n.d. Intestine n.d. n.d.

Kidney 1.3 4.4

PC486, MJN Technology Ltd, Cheltenham, UK) using a ScanJet IIC desktop scanner (Hewlett Packard), and analysed using the QuantiScan densitometric computer software pack- age (Biosoft, Cambridge, UK). For quantitation of human cytosolic DHEA ST, the filters contained a set of dilutions of female rat liver cytosol (0.0625-2 pg) which allowed construction of a standard curve against which the relative amount of DHEA ST protein in the samples could be estimated. The human cytosol samples were applied to nitrocellulose filters as a series of dilutions, and different samples were compared using data points which were within the linear portion of the curves constructed from the analysed scanned images.

Immunohistochemistry

An adult female Wistar rat (approximately 300 g) was terminally anaesthetised with Equithesin and perfused via the left ventricle with Tyrode's solution followed by perfusion for 15 min with 4% paraformaldehyde in buffer B (100 mM sodium phosphate, pH 7.4 containing 150 mM NaCl and 5 mM KCl). The liver was then dissected, sectioned into approximately 1-cm3 blocks and cryoprotected with 10% and 30% sucrose. Tissue was then frozen at - 20°C and 10-pm-

543

Fig.5. Immunohistochemical localisation of DHEA ST in rat liver. The figure shows a 10-pm cyrostat section of female rat liver immunostained with anti-(DHEA ST) IgG. Liver is shown at low (A) and high (C) magnification. B and D show adjacent liver sections in which the primary antibody was replaced by non-immune rabbit serum. Scale bars: (B) = 28 pm; (D) = 18.8 pm.

thick sections were cut with a cryostat and directly mounted onto gelatidchrome alum-coated slides and air dried. Sec- tions were washed clean with buffer B, incubated for 5 min in 50 mM ammonium chloride in buffer B, and incubated in 10% normal goat serum, 0.2% Triton X-100 in buffer B for 1 h. After washing in 1% normal goat serum in buffer B, they were incubated in primary antibody or preimmune rabbit serum diluted 1 : 500 in 1 % normal goat serum in buffer B overnight at room temperature. Following several washes in 1 % normal goat serum in buffer B, the sections were incubated with biotinylated goat anti-(rabbit IgG) serum diluted 1: 300 in 1 % normal goat IgG in buffer B for 2 h at room temperature. After washing with buffer B, they were sub- sequently incubated with HTC-labelled streptavidin diluted

1 : 50 in buffer B for 2 h at room temperature, rinsed with buffer B and mounted and cover-slipped with 2.5% propyl- gallate in 85% glycerol. Sections were then examined and photographed using an Olympus AH2-FGL-T light micro- scope equipped with epifluorescence attachments.

Partial purification of human liver DHEA ST Human liver cytosol (10 mg protein) was applied to a

MonoQ HR5/5 FPLC anion-exchange column in buffer C (20 mM triethanolamine/Cl-, pH 7.4, 3 mM 2-mercaptoethanol), at a flow rate of 1 mumin and, following elution for 10 ml, buffer C (30 rnl) containing 100 mM NaCl was applied to the column. DHEA ST was eluted using a linear

Correlation Coefficient = 0.909 p = 0.005

/ * 0.0 1/

2 0 4 0 0 0 8 0 100 120

DHEA ST ENZYME ACTIVITY (pmol.min-1 .mg-I)

Fig. 6. Correlation of DHEA ST activity and immunoreactive enzyme protein in human liver cytosol. DHEA ST activity was measured in six different human liver cytosols, and the same cytosols were subjected to immuno-dot-blot analysis as described in Materials and Methods. Dot-blot data from human cytosols were normalised against a standard curve comprising female rat liver cytosol (total protein range, 0.0625 -2 pg), hence the rat equivalent units. The correlation was assessed using least-squares linear-regression analysis. Assays and dot-blot analysis were performed in duplicate on each sample, and data points represent the mean values.

gradient of 100-225 mh4 NaCl in buffer B over 45 ml. The gradient elution was extended to 1 M NaCl over a further 55 ml. 1-ml fractions were collected, assayed for DHEA and 1-naphthol ST activities and subjected to immuno-dot-blot analysis and quantitation as described above.

Protein determination Protein was estimated by the method of Lowry et al. [33]

with bovine serum albumin as standard, with the exception of highly purified preparations of DHEA ST, where sensitivity considerations necessitated the use of the method of Bradford [34], again with bovine serum albumin as standard.

RESULTS Purification of DHEA ST

The purification of DHEA ST from female rat liver is summarised in Table 1, which demonstrates the 183-fold en- richment of the purified enzyme with respect to the cytosolic fraction. The calculated yield of the final preparation appeared rather high (i.e. higher than the yield following the affinity-purification step), but this could be due to the fact that it was necessary to employ the dye-binding method [34] of protein determination for these highly purified fractions. The procedure followed differed from that reported by other authors [8, 35, 361, but we found that the introduction of a final clean-up step involving FPLC chromatography on MonoQ was necessary in order to remove all contaminating proteins and so produce a preparation of sufficient purity for antibody production. Electrophoresis of the purified protein on an SDS/polyacrylamide gel (Fig. 1) revealed a single

Coomassie-blue-stained band with a subunit mokmlar mass corresponding to 30 kDa (when calculated wing Cnear-regression analysis following construction of a standad curve from the molecular mass standards). The purified protein re- tained a small amount of activity towards the prototypical xenobiotic substrate 1 -naphthol, but this adiyity probably represents the overlapping substrate specificity of the purified enzyme.

Production and characterisation of anti-(DHEA ST) serum

The purified DHEA ST protein was used b immunise a female New Zealand white rabbit and, following the adminis- tration of a total of 600 pg protein over the mume of four immunisations, the resulting antiserum was characterised by examining its inhibition of various ST activities in rat liver cytosol, and by immunoblot analysis of the purified enzyme and of rat liver cytosol. Its specificity for human D€€EA ST was also extensively assessed.

Fig. 2A shows the inhibition of DHEA ST in male and female rat liver cytosol by the anti-(DHEA ST) IgG preparation, and demonstrates that the activities were almost completely inhibited at a ratio of 1 mg IgGlmg cytosolic protein, that the enzyme was inhibited in both female and male rats, although the activity is considerably higher (approximately 15-fold) in females, and that approximately 50% less antibody was required to inhibit the enzyme activity in male rats. The antibody was also able to inhibit estrone ST activity in female rat liver, but no inhibition of this enzyme activity was observed with male rat liver cytosol (Fig. 2B). The data confirm the view that there is a separate and specific estrogen ST expressed solely in male rat liver 19, 27, 37, 381, and indicate that the small residual estrone ST activity in female rat liver is in fact the result of the action of DHEA ST, since this enzyme activity was inhibited by the anti-DHEA serum. Indeed, we have recently prepared an antibody against the male rat liver estrogen ST, which does not detect the presence of any corresponding estrogen ST protein in female rat liver on immunoblot analysis and which, while inhibiting estrogen ST activity, is not able to inhibit DHEA ST in either male or female rat liver cytosol [27]. This has also been confirmed at the RNA level [ 161.

Further examination of the inhibition of additional ST substrates in rat and human liver resulted in the data pre- sented in Table 2. Testosterone, androsterone and pregnenolone ST activities were inhibited almost 100% in both male and female rats at an antibody/protein ration of 1: 1 (by mass), whereas sulfation of the three estrogens (estrone, p- estradiol and estriol) was inhibited only in female liver cytosol. Dopamine ST was not inhibited in either male or female rat liver cytosol, and a slight inhibition of 1-naphthol ST activity in rat liver cytosol was observed ( S 30 %), an observation which agrees with the residual ST activity towards this substrate observed with the purified DHEA ST preparation. When the effect on human liver cytosolic sulfation was examined, there was some (variable) inhibition (up to 60% for testosterone) of the ST activity directed towards the androgens DHEA, testosterone and androsterone, whereas pregnenolone sulfation was not inhibited. Estrogen and dopamine ST activities were not inhibited by the antibody, although there was a small inhibition of l-naphthol ST. These data support the conclusion that the antibody interacts specifically with androgen-metabolising ST (hydroxysteroid ST) in both rats and humans.

545

5000 I

20000

18000 - 16000 - 14000

12000

loo00

8000 - 6000 - 4000

2000

-

- - -

- -

A P

+ NAPHTHOLST

-Y ANTIBODY

0 60 65 70 75 80 85 90 95

0 5000 10000 15000 20000 DHEA ST ACTIVITY (cpm)

5000 I i

FRACTION No.

0 2000 4000 6000 8000 10000 1-NAPHTHOL ST ACTIVITY (cpm)

Fig. 7. Anti-(rat liver DHEA ST) IgG recognises partially purified human liver DHEA ST. Human liver cytosol was subjected to FPLC anion exchange chromatography as described (A). Fractions were assayed for DHEA (0) and I-naphthol (B) ST activities (cpm incorporated20 min), and were also subjected to immuno-dot-blot analysis with anti-(rat liver DHEA ST) IgG (A); data are arbitrary units. The correlations between immunoreactive protein, as determined by dot-blot analysis, and DHEA ST enzyme activity, and immunoreactive protein and I-naphthol ST enzyme activity are shown in B and C , respectively.

Table 4. DHEA ST activities in human adult and fetal tissue samples. Enzyme activities were determined (in duplicate) in the same cytosol samples which were used for immunoblot analysis as shown in Fig. 6. n.d., no enzyme activity detectable.

Tissue DHEA ST activity ~ ~

pmol . min-' . mg-' Adult liver 219 Adult kidney n.d. Fetal liver 68 Fetal kidney 23 Fetal lung 9.6 Fetal adrenal 1860

Further characterisation of the antibody preparation was performed using immunoblot analysis, and Fig. 3 shows the results of these experiments. The antibody recognised a single immunoreactive polypeptide of subunit molecular mass 30 kDa when purified DHEA ST was immunoblotted, and a corresponding protein of identical subunit molecular mass was also detected in rat liver cytosol (Fig. 3A). The observed sex difference in DHEA ST activity was confirmed at the

enzyme protein level, with much stronger immunoreactivity observed in the lane containing female liver cytosol (Fig. 3A, lane 1). The antibody also cross-reacted strongly with a polypeptide present in human liver cytosol (Fig. 3B). This protein had a subunit molecular mass of 35 kDa, which is identical to the size of human liver DHEA ST purified by Falany's group [39], and indeed the anti-(DHEA ST) IgG showed strong immunoreactivity with partially purified human liver DHEA ST (see below).

Tissue distribution of DHEA ST in rats In order to assess the tissue distribution of DHEA ST in

the rat, cytosolic fractions were prepared from a range of tissues from a male and female rat, subjected to immunoblot analysis with anti-(DHEA ST) IgG, and were assayed for DHEA ST enzyme activity. Fig. 4 shows the results of immunoblot analysis of DHEA ST in various rat tissues, and demonstrates that, of the tissues examined, the immunoreactive polypeptide of 30 kDa was only detected to any significant extent in the liver, an observation confirmed by the enzyme activity measurements (Table 3) which showed only a small residual activity in the kidney. The antibody was not able to inhibit this residual DHEA ST enzyme activity in either male or female kidney cytosol (not shown), suggesting that this

546

Fig. 8. Distribution of DHEA ST in human adult and fetal tissues. Immunoblot analysis was performed as described in the legend to Fig. 4. 20 pg total cytosolic protein was loaded onto each lane. Lane 1, adult human liver; lane 2, adult human kidney; lane 3, fetal human liver; lane 4, fetal human kidney; lane 5, fetal human lung; lane 5. fetal human adrenal.

enzyme reaction is actually carried out by another, unidenti- fied ST isozyme, and indeed anti-(DHEA ST) IgG did immunostain a protein of approximately 36 kDa in rat kidney cytosol, which may represent such an enzyme. Thus, in adult rats significant DHEA sulfation capacity appears to be re- stricted to the liver.

Immunohistochemical localisation of DHEA ST in rat liver

The immunoreactive DHEA ST protein was distributed widely throughout the liver, and the immunofluorescence staining of hepatocytes was homogeneous (Fig. 5A). Exam- ination of the sections at higher magnification demonstrated the delicate reticular intracellular pattern of immunostaining, which was apparently excluded from the cellular organelles, including the nucleus (Fig. 5B). These features are consistent with a cytoplasmic localisation for DHEA ST.

Immunochemical characterisation of human DHEA ST As a result of the strong cross-reactivity on immunoblot

analysis of the anti-(rat DHEA ST) IgG preparation with a polypeptide which appeared to correspond to DHEA ST in human liver cytosol (Fig. 3B), we carried out further investi- gations in order to confirm that this interaction was with the human DHEA ST. Immuno-dot-blot analysis followed by quantitation by densitometry showed that there was a highly significant correlation between the DHEA ST enzyme activity and the amount of DHEA ST immunoreactive protein measured in human liver cytosol (Fig. 6). Additional evidence came from chromatographic analysis of human liver cytosol DHEA ST on FPLCMonoQ chromatography, followed by analysis by immuno-dot blotting. As shown in Fig. 7, the DHEA ST enzyme activity peaks corresponded with the immunochemically detected DHEA ST protein, as measured with the anti-(rat liver DHEA ST) IgG preparation (Fig. 7A). The fiist main peak of 1-naphthol ST activity (fraction 76) contained no DHEA ST activity, and correspondingly the antibody preparation did not recognise any immunoreactive protein in this peak. The correlation coef- ficients, calculated by least-squares linear-regression analysis, for DHEA ST activity and anti-(DHEA ST) immunoreactive protein and 1-naphthol ST activity and anti-(DHEA ST) immunoreactive protein, over fractions 68 -92 from the FPLC chromatography, were 0.984 ( P < 0.0001) and 0.235

( P > 0.4), respectively (Fig. 7B and C). This extensive evidence strongly supports the assumption that this antibody preparation specifically recognises human DHEA ST.

We then investigated the distribution of DHEA ST in adult and fetal human tissues. Fig. 8 shows that DHEA ST is present in adult human liver, but is not present in the kidney. In the fetus, the 35-kDa immunoreactive DHEA ST was not detectable in the kidney and lung, but was present in the liver, and to a much greater extent in the adrenal. These data agreed with the enzyme activity measurements as listed in Table 3. The high level of expression of DHEA ST in the fetal adrenal was as expected, considering the major role that the human fetal adrenal gland plays in the production of DHEA-S during development, and the high level of DHEA ST enzyme activity in fetal adrenal cytosol (Table 4).

DISCUSSION

Sulfation of steroid hormones is an important metabolic reaction, and as such contributes significantly to the ho- meostasis of these compounds. In order to understand further the function of the sulfation of one of the major body steroids, DHEA, we have purified the enzyme responsible for this reaction from rat liver and used this purified preparation to prepare rabbit antibodies specific for DHEA ST. Charac- terisation of this antibody preparation by enzyme-inhibition studies and immunoblot analysis has confirmed that these antibodies will be of significant benefit in studying the properties and function of DHEA ST in rats and man.

Analysis of the distribution of DHEA ST enzyme activity in rats determined that the enzyme was located primarily in the liver, with little or no activity present in the lungs, adrenal, small intestine, or brain. A small amount of DHEA ST activity was detected in rat kidney, but this was towards the limit of sensitivity of the assay method especially in the male sample, and there was still a female predominance in the expression of DHEA ST activity in kidney, although to a much lesser extent than that observed for the liver. Confir- mation of this enzyme activity data was obtained by per- forming immunoblot analysis of cytosols prepared from these various tissues, the results of which demonstrated the presence of the 30-kDa immunoreactive polypeptide only in the liver. The small amount of DHEA ST found in the kidney was not. reflected in the immunostaining of a 30-kDa polypeptide in kidney cytosol, but the level of expression of the protein may be too low to detect by this method or possibly this activity is the result of the action of another ST isozyme, which seems the most likely explanation, when one considers that the antibody did not inhibit this residual renal activity. The role of the liver in the sulfation of DHEA is not clear, although the presence of such large amounts of enzyme activity and enzyme protein suggests that this organ is a major contributor to the metabolism of DHEA (and other androgens) in adult rats, since the adrenal is devoid of this activity. Immunohistochemical determination of DHEA ST in rat liver demonstrated that the enzyme is widely distributed in hepatocytes throughout the liver, and this is similar to the distribution of androsterone ST in female rat liver, as determined by immunohistochemical analysis by anti-(androsterone ST) serum [40].

The DHEA ST purified in this study is likely to correspond to hydroxysteroid ST I, as classified by Jakoby et al. [41], since it was contained within the earliest eluting peak of DHEA ST activity recovered from the DEAE-Sepharose

547

column (not shown) and therefore is also likely to represent the cDNA (STa) isolated by Ogura et al. [13]. It is possible that the enzyme purified here corresponds to the androsterone ST purified recently by Matsui’s group [lo], since our antibody preparation strongly inhibited the sulfation of androsterone. The substrate specificity of this enzyme is not limited to DHEA, since it has also been reported that it can sulfate testosterone, P-estradiol, certain alcohols such as isoamyl alcohol [41], and it has been implicated in the bioac- tivation of methylated polycyclic aromatic hydrocarbons [B]. Our data would confirm this wide-ranging substrate specificity, since the antibody preparation inhibited the sulfation of pregnenolone, androsterone and testosterone, to a similar extent to the DHEA activity (Table 2). Therefore, when considering the function of this enzyme in vivo, it is important to take into account this rather broad substrate specificity.

The anti-(DHEA ST) IgG cross-reacted strongly with a 35-kDa polypeptide in human liver cytosol, which we con- clude represents human DHEA ST. This conclusion is supported by the following evidence: 35 kDa is the molecular mass of the human enzyme [39] ; there was very high staining intensity observed on immunoblot analysis of human fetal adrenal cytosol (Fig. 8), a tissue which also has extremely high DHEA ST activity (Table 3); human hepatic DHEA ST enzyme activity showed a high degree of correlation with the intensity of immunostaining on dot-blot analysis (Fig. 6) ; our antibody recognised this protein in partially purified human hepatic DHEA ST (Fig. 7). This strong reaction with human DKEA ST means that this antibody represents an important molecular tool for studying the location and function of DHEA ST in man, as illustrated by the data reported here.

The enzyme protein is present in high levels in adult human liver, but is virtually absent from adult kidney, an observation backed up by enzyme activity measurements. Simi- larly, fetal human liver possessed DHEA ST activity, and the 35-kDa human DHEA ST protein was observed on immunoblot analysis (Fig. S), however the fetal adrenal exhibited a very large DHEA ST activity and also the levels of the corresponding enzyme protein, as determined by immunoblot analysis, were extremely high. This is consistent with the role of the fetal adrenal as the major production source of DHEA-S during pregnancy, which in turn is the major substrate for androgen and estrogen biosynthesis by the placenta. Enzyme activity was very low in the fetal kidney, little enzyme protein was detected by immunoblot analysis, and, as previously demonstrated, DHEA ST activity was extremely low in the fetal lung [42] (Table 4); correspondingly, no DHEA ST enzyme was detectable by immunoblotting (Fig. 8).

We have therefore produced an antibody which has per- mitted the examination of DHEA ST expression in both rats and humans, and using this antibody we have demonstrated the differential distribution of this important enzyme in rats and adult and fetal human tissues. The use of this antibody preparation for future studies will significantly enhance our understanding of the function and regulation of DHEA ST, and therefore our appreciation of the role of steroid sulfation as a means of modulating steroid-hormone function.

We are grateful to the Sir Jules Thorn Charitable Trust (M. W. H. C.) and Birthright-Royal College of Obstetricians and Gynae- cologists (R. H.) for financial support for this work. M. W. H. C. is a Caledonian Research FoundatiodRoyal Society of Edinburgh Research Fellow, R. H. is a Welcome Trust Senior Clinical Research Fellow, and E. V. B. was supported by an Intercalated Degree Grant from the Medical Research Council. We thank Professor G. B. Ode11

and Dr J. Dick for their assistance in obtaining human liver and kidney samples, respectively, and Hazel Brewerton for skilled tech- nical assistance.

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Immunochemical characterisation of a dehydroepiandrosterone sulfotransferase in rats and humans

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