Isolation and expression of an isoform of rat estrogen sulfotransferase

~. Steroid Biochem. Molec. Biol. Vol. 52, No. 1, pp. 35-44, 1995

Pergamon

0960-0760(94)00147-2

Copyright © 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0960-0760/95 $9.50 + 0.00

I s o l a t i o n and E x p r e s s i o n of an I s o f o r m of Rat Estrogen Sulfotransferase Josie L. Falany, Victor Krasnykh, Galina Mikheeva and Charles N. Falany* Department of Pharmacology and Toxicology and Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, A L 35294, U.S.A.

A n e w i s o f o r m o f r a t l i v e r e s t r o g e n s u l f o t r a n s f e r a s e ( E S T ) , r E S T - 6 , w h i c h is d i s t i n c t f r o m t h e p r e v i o u s l y r e p o r t e d r a t E S T [ D e m y a n et al., Molec. Endocrinol. 6 (1992) 589], h a s b e e n c l o n e d , expressed, purified and characterized. A PCR procedure using oligonucleotide primers synthesized to t h e 5 ' - n o n t r a n s l a t e d a n d 3 ' - n o n t r a n s l a t e d r e g i o n s o f t h e p u b l i s h e d r E S T s e q u e n c e was u s e d to i s o l a t e r E S T - 6 c D N A . T h e c l o n e d D N A is 1000 b p in l e n g t h a n d e n c o d e s a p r o t e i n o f 295 a m i n o a c i d s w i t h a c a l c u l a t e d m o l e c u l a r m a s s o f 35,300Da. r E S T - 6 is s e l e c t i v e l y e x p r e s s e d in m a l e r a t s , as confirmed by Northern blot and immunoblot analyses. Northern blot analysis of male and female r a t l i v e r R N A w i t h t h e r E S T - 6 c D N A as a p r o b e shows a b a n d w i t h m a l e R N A b u t n o t w i t h f e m a l e RNA. S i m i l a r l y , i m m u n o b l o t a n a l y s i s o f m a l e a n d f e m a l e r a t l i v e r c y t o s o l s w i t h a n a n t i b o d y to r a t E S T y i e l d s a s t r o n g i m m u n o r e a c t i v e b a n d in r a t l i v e r c y t o s o l f r o m m a l e r a t s b u t n o t f r o m f e m a l e s . S u b s e q u e n t to b a c t e r i a l e x p r e s s i o n a n d p u r i f i c a t i o n o f r E S T - 6 , t h e e n z y m e was a n a l y z e d k i n e t i c a l l y a n d s h o w n to s u l f a t e e s t r o g e n s b u t n o t d e h y d r o e p i a n d r o s t e r o n e , p r e g n e n o l o n e , c o r t i s o l o r t e s t o s t e r one. M a x i m a l s u l f a t i o n a c t i v i t y t o w a r d s b o t h f l - e s t r a d i o l a n d e s t r o n e o c c u r r e d a t a c o n c e n t r a t i o n of lpM with substrate inhibition at higher concentrations. These results indicate that multiple, c l o s e l y r e l a t e d f o r m s o f E S T a r e p r e s e n t in r a t liver. A n a l y s i s o f t h e a c t i v i t y a n d r e g u l a t i o n o f t h e s e d i f f e r e n t E S T e n z y m e s is i m p o r t a n t in u n d e r s t a n d i n g e s t r o g e n m e t a b o l i s m in r a t s .

J. Steroid Biochem. Molec. Biol., Vol. 52, No. 1, pp. 35-44, 1995

INTRODUCTION

droxysteroids is catalyzed by at least three different classes of cytosolic STs. Purified rat and human hydroxysteroid STs have been shown to conjugate both estrogens and hydroxysteroids such as D H E A and pregnenolone [4, 5]. H u m a n phenol-sulfating phenol S T ( h P - P S T ) has been reported to sulfate estrogens but not hydroxysteroids [6, 7]. Separate estrogen STs ( E S T ) have been purified from several species [8, 9] and distinct forms of E S T have been cloned from bovine [10], rat [11] and guinea pig tissues [12]. T h e evaluation of the roles of the different forms of S T involved in steroid sulfation requires an understanding of the multiplicity and kinetic properties of the individual enzymes present in a given tissue. This manuscript reports the cloning and bacterial expression of a new isoform of rat liver E S T which is distinct from the previously reported rat E S T [11]. T h e enzyme is selectively expressed in male rats and has been purified and kinetically characterized following expression in E. coli. Multiple isoforms of the cytosolic hydroxysteroid STs and phenol STs have been reported in rat liver [1, 13]. This report

Sulfation is an important process in modulating the activity, transport and excretion of hydroxysteroids and estrogens [ 1, 2]. Sulfate-conjugated steroids cannot activate steroid receptors and, due to the addition of the charged sulfonate group, are more water soluble and therefore more readily excreted than are the unconjugated forms. Steroid sulfates in the brain, such as dehydroepiandrosterone ( D H E A ) sulfate and pregnenolone sulfate, may function in modulating the activity of GABA receptors [3]. In addition, steroid sulfates may serve as transport forms for steroids; the sulfate moiety can be removed by sulfatases in the target tissue to generate the active fbrm of the steroid [4, 5]. Multiple isoforms of cystolic sulfotransferase (ST) have been shown to conjugate steroids using 3'phosphoadenosine 5'-phosphosulfate (PAPS) as the sulfonate donor. T h e sulfation of estrogens and hy*Correspondence to C. N. Falany. Received 2 June 1994; accepted 24 Aug. 1994. 35

36

Josie L. Falany et al.

confirms that multiple isoforms of E S T are also present. Understanding the physical and kinetic properties of the different isoforms of E S T is important in analyzing the roles of these enzymes in steroid metabolism. EXPERIMENTAL

the Sequagel system. T h e gels were transferred to Whatman 3 M M paper, dried under vacuum and exposed to autoradiograph film for 24 h. T h e sequences were read manually and analyzed using the University of Wisconsin Genetic Computer group's program and the MacVector D N A Analysis program (Kodak/ IBI).

Materials

Immunological analysis

Oligonucleotide primers were synthesized in the Molecular Biology Core Facility of the Comprehensive Cancer Center at UAB. p-Nitrophenol, fi-estradiol (E2) , e s t r o n e (El), D H E A , testosterone, pregnenolone, dopamine, DEAE-Sepharose CL-6B and 3',5'diphosphoadenosine (PAP) agarose were obtained from Sigma Chemical Co. (St Louis, MO). [1,2,6,7-3H] D H E A (79Ci/mmol), [6,7-3H]fi-E 2 (45 Ci/mmol), [2,4,6,7-3H]E1 (96 Ci/mmol), [1,2,3-3H(N)]cortisol (54 Ci/mmol), [7-3H]testosterone (27.7 Ci/mmol), [7-3H]pregnenolone (25Ci/mmol) and [35S]3'-phosphoadenosine-5'-phosphosulfate (PAPS) (2 Ci/mmol) were purchased from New England Nuclear (Boston, MA). PAPS was purchased from D r Sanford Singer (University of Dayton, Dayton, OH). Minoxidil was a generous gift from the Upjohn Co. (Kalamazoo, MI). p K K 2 3 3 - 2 was obtained from Pharmacia (Piscataway, NJ). Sequagel and Protogel were purchased from National Diagnostics (Atlanta, GA). ,Mtinity-purified goat anti-rabbit horseradish peroxidase conjugate was purchased from Southern Biotechnology Associates (Birmingham, AL). T h e Lumiglo Chemiluminescence Substrate kit was obtained from Kirkegaard and Perry Labs (Gaithersburg, MD). All other chemicals were of reagent grade quality.

In order to generate a specific antibody to the rat E S T s , the r E S T - 3 c D N A was expressed in the p M A L - c 2 expression system for the generation of fusion protein. Antibodies were raised to r E S T - 3 rather than to r E S T - 6 because the antibody production was done simultaneously with the complete sequencing of both r E S T - 3 and r E S T - 6 , prior to sequence analysis which revealed that r E S T - 3 was an allele but that r E S T - 6 was an isoform of the previously reported r E S T . T h e p M A L - c 2 expression system generates a maltose binding protein (MBP) fusion protein which can be purified by affinity chromatography on an amylose affinity resin (New England Biolabs). For insertion into p M A L - c 2 , the r E S T - 3 c D N A in p C R I I was digested with Nco I, which cuts the c D N A at base 72. T h e c D N A was treated with the Klenow fragment of D N A polymerase I and digested with Hind III. This procedure generated a c D N A fragment which was in-frame for translation but lacked 15 amino acids at the amino-end of the translated region. This fragment was inserted into the X m m I and Hind III sites of p M A L - c 2 . E. coli XL1-Blue cells were transformed with the p M A L - r E S T - 3 vector (New England Biolabs). T o express the M B P - r E S T - 3 fusion protein, XL1-Blue cells containing p M A L r E S T - 3 were grown in Luria broth containing 50 #g/ml ampicillin to an O.D.550 of 0.5, then induced for 2 h with 0.3 m M isopropyl-/%D-galactopyranoside. Cells were pelleted and resuspended in bacterial lysis buffer (75 m M Tris, p H 8.0, 0.25 M sucrose, 0.25 m M E D T A and 0.02 mg/ml lysozyme) and incubated on ice for 20 min. T h e cells were re-pelleted, resuspended in 5 m M phosphate, p H 7 . 4 , containing 1 . 5 m M dithiothreitol and 10 #g/ml phenolmethylsulfonylfluoride, and sonicated 3 x on ice in 10s bursts with 30 s cooling between each burst. T h e cytosolic fraction was recovered following centrifugation at 100,000g for l h . T h e expressed M B P r - E S T - 3 fusion protein was purified by amylose affinity chromatography. T h e cytosolic fraction was diluted to 3 mg protein/ml and applied to an amylose affinity resin (New England Biolabs) equilibrated in 5 m M phosphate, p H 7.4. T h e amylose column (1.75 x 5 cm) was washed with 75 ml of 5 m M phosphate, p H 7.4, then the fusion protein was eluted with 10 ml of 10 m M maltose in the same buffer. T h e yield was approx. 20 mg fusion protein/ liter bacterial culture. T h e truncated r E S T - 3 fusion protein was not enzymatically active.

Isolation of rat E S T c D N A s

A polymerase chain reaction (PCR) procedure was used to isolate the rat E S T cDNAs. A pair of oligonucleotides ( 5 ' - C A G G A G C A T C T G G A C A G T A C - 3 ' and 5 ' - C T T C T A C T T C T A C T G A A T T C - 3 ' ) were synthesized to the 5'-nontranslated and 3'-nontranslated regions of the published rat E S T sequence [11], respectively. T h e template for the P C R reactions was obtained from a male rat 2 g t l l c D N A library (Stratagene). Approximately 10 7 pfu were amplified in E. coli Y1090 and total phage D N A was purified from plate lysates using a commercial kit (Promega). Phage D N A (0.1 #g) was denatured by incubation in boiling water for 5 min and used as the template for PCR. T h e PCR products were isolated from an agarose gel and subcloned into the p C R I I vector (Invitrogen) for sequence analysis and characterization. T h e complete nucleotide sequences of the E S T cDNAs were determined by dideoxynucleotide chain termination D N A sequencing using [3SS]dATP to label the D N A fragments as described previously [14]. Electrophoresis of the D N A fragments was carried out in 8% polyacrylamide wedge gels in 8 M urea using

Isoforms of Rat E S T T o raise a n t i b o d i e s 1:o r E S T - 3 , t h e M B P - r E S T - 3 f u s i o n p r o t e i n (500 p g ) was m i x e d w i t h F r e u n d ' s c o m p l e t e a d j u v a n t a n d i n j e c t e d s u b c u t a n e o u s l y at several

37

sites a l o n g t h e b a c k o f a female N e w E n g l a n d w h i t e r a b b i t as d e s c r i b e d b y V a i t u k a i t u s et al. [15]. T w o weeks later, t h e r a b b i t r e c e i v e d a b o o s t e r i n j e c t i o n o f

rEST-3 CAGGAGCATCTGGACAGTACACCACTTGTGATGGAGACTTCTATGCCTGAATACTATGA( MetGluThrSerMetProGluTyrTyrAsp

60

GTTTTTGGTGATTTCCATGGATTTTTAATGGATAAACGGTTCACCAAATATTGGGAAGA( ValPheGlyAspPheHisGlyPheLeuMetAspLysArgPheThrLysTyrTrpGluAsp

120 30

GTTGAAACATTCTTGGCAAGGCCAGATGACCTTCTCATTGTTACTTATCCTAAATCTGGC ValGluThrPheLeuAlaArgProAspAspLeuLeuIleValThrTyrProLysSerGly

180 50

AGCACATGGATTAGTGAAATTGTGGATATGATCTATAAAGAAGGTGATGTGGAAAAATG( SerThrTrpIleSerGluIleValAspMetIleTyrLysGluGlyAspValGluLysCys

240 70

AAGGAGGATGCACTTTTTAACAGAATACCTGACCTGGAGTGCAGAAATGAAGATCTAATA LysGluAspAlaLeuPheAsnArgIleProAspLeuGluCysArgAsnGluAspLeuIle

300 90

AACGGAATAAAACAACTAAAAGAAAAGGAATCGCCTAGAATAGTGAAAACTCACCTGCCA AsnGlyIleLysGlnLeuLysGluLysGluSerProArgIleValLysThrHisLeuPro

360 ii0

GCTAAGCTCCTTCCAGCATCATTTTGGGAAAAGAATTGCAAGATAATCTATCTTTGCCGA AlaLysLeuLeuProAlaSerPheTrpGluLysAsnCysLysIleIleTyrLeuCysArg

420 130

AATGCCAAAGATGTCGTCGTTTCTTATTACTACTTTTTCCTGATCATGAAAAGTTATCAA AsnAlaLysAspValValValSerTyrTyrTyrPhePheLeuIleMetLysSerTyrGln

480 150

AATCCTAAATCTTTTTCTGAATTTGTGGAGAAATTTATGGAAGGGCAAGTTCCGTATGGT AsnProLysSerPheSerGluPheValGluLysPheMetGluGlyGlnValProTyrGly

540 170

TCCTGGTATGATCATGTAAAATCTTGGTGGGAGAAGAGTAAGAATTCACGTGTTTTGTTT SerTrpTyrAspHisValLysSerTrpTrpGluLysSerLysAsnSerArgValLeuPhe

600 190

ATGTTCTATGAGGACATGAAAGAGGATATCCGAAGAGAAGTTGTGAAGCTGATAGAGTTC MetPheTyr31uAspMetLysGluAspIleArgArgGluValValLysLeuIleGluPhe

660 210

CTGGAGAGA3ACCCATCAGCAGAGCTAGTAGACAGAATCATTCAACATACATCATTCCAG LeuGluArgAspProSerAlaGluLeuValAspArgIleIleGlnHisThrSerPheGln

720 230

GAGATGAAGAACAATCCATGCATCAATTATTCAATGCTGCCAGAGACCATGATAGATCTA GluMetLysAsnAsnProCysIleAsnTyrSerMetLeuProGluThrMetIleAspLeu

780 250

AAAGTATCGCCTTTCATGAGAAAGGGAATTGTAGGAGACTGGAAGAACCACTTCCCTGAA LysValSerProPheMetArgLysGlyIleValGlyAspTrpLysAsnHisPheProGlu

840 270

GCCCTGAGGGAGAGATTTGAGGAGCACTACCAGCAGCAAATGAAGGACTGCCCTGTGAAA AlaLeuArgGluArgPheGluGluHisTyrGlnGlnGlnMetLysAspCysProValLys

900 290

TTTAGAGCAGAGCCCTGAGACAATTCCTTGTGTCTGAAATTGGAGTAGTCTCCAATTTAT PheArgAlaGluPro***

960 295

CCTTCAGTTTTTCTTGTTTTGAATTCAGTAGAAGTAGAAG

i0

i000

Fig. 1. Nucleotide sequence and translation of rEST-3. The nucleotide sequence and the derived translation of rEST-3 are numbered on the right. Nucleotides 1-20 and 981-1000 are derived from PCR primers. The asterisk represents the stop codon.

38

Josie L. Falany

5 0 0 p g M B P - r E S T - 3 in a similar manner. After 2 weeks, the rabbits were bled and the serum was tested by i m m u n o b l o t analysis for the presence of antibodies to the r E S T s .

et al.

I m m u n o b l o t analysis of the expressed rat E S T s and rat liver cytosolic fractions were carried out as described previously [16]. Briefly, after resolution of proteins by S D S - P A G E , proteins were electrotrans-

rEST-6 CAGGAGCATCTGGACAGTACACCACTTGAAATGGAGACTTCTATGCCTGAATACTATGAA MetGluThrSerMetProGluTyrTyrGlu

60 I0

GTTTTTGGTGATTTCCATGGAGTTTTAGTGGATAAACTGTTCACCAAATATTGGGAAGAT ValPheGlyAspPheHisGlyValLeuValAspLysLeuPheThrLysTyrTrpGluAsp

120 30

GTTGAAACATTCTCAGCAAGGCCAGATGACCTTCTCGTTGTTACTTATCCTAAATCTGGC ValGluThrPheSerAlaArgProAspAspLeuLeuValValThrTyrProLysSerGly

180 50

AGCACATGGATTGGTGAAATTGTGGATAT~ ~TCTATAAAGAAGGTGATGTGGAAAAATGC SerThrTrpIleGlyGluIleValAspMetIleTyrLysGluGlyAspValGluLysCys

240 70

AAGGAGGATGCAATTTTTAACAGAATACCTTACCTGGAGTGCAGAAATGAAGATCTAATA LysGluAspAlaIlePheAsnArgIleProTyrLeuGluCysArgAsnGluAspLeuIle

300 90

AATGGAATAAAACAACTAAAGGAAAAGGAATCGCCTAGAATAGTGAAAACTCACCTGCCA AsnGlyIleLysGlnLeuLysGluLysGluSerProArgIleValLysThrHisLeuPro

360 ii0

GCTAAGCTCCTTCCAGCATCATTTTGGGAAAAGAATTGCAAGATAATCTATCTTTGCCGA AlaLysLeuLeuProAlaSerPheTrpGluLysAsnCysLysIleIleTyrLeuCysArg

420 130

AATGCCAAAGATGTCGTCGTTTCTTATTA(]TACTTTTTCCTGATCATAAAAAGTTATCCA AsnAlaLysAspValValValSerTyrTyrTyrPhePheLeuIleIleLysSerTyrPro

480 150

AATCCTAAATCTTTTTCTGAATTTGTGGAGAAATTTATGGAAGGGCAAGTTCCGTATGGT AsnProLysSerPheSerGluPheValGluLysPheMetGluGlyGlnValProTyrGly

540 170

TCCTGGTATGATCATGTAAAATCTTGGTGGGAAAAGAGTAAGAACTCACGTGTTTTGTTT SerTrpTyrAspHisValLysSerTrpTrpGluLysSerLysAsnSerArgValLeuPhe

600 190

ATGTTCTATGAGGATATGAAAGAGGATATACGAAGAGAAGTTGTGAAGCTGATAGAGTTC MetPheTyrGluAspMetLysGluAspIleArgArgGluValValLysLeuIleGluPhe

660 210

CTGGAGAGAGACCCATTAGCAGAGCTAGTAGACAAAATCATTCAACATACGTCATTCCAG LeuGluArgAspProLeuAlaGluLeuValAspLysIleIleGlnHisThrSerPheGln

720 230

GAGATGAAGAACAATCCATGCACCAATTATTCAATGCTGCCAGAGACCATGATAGATCTA GluMetLysAsnAsnProCysThrAsnTyrSerMetLeuProGluThrMetIleAspLeu

780 250

AAAGTATCGCCTTTCATGAGAAAGGGAATTGTAGGAGACTGGAGGAACCACTTCCCTGAA LysValSerProPheMetArgLysGlyIleValGlyAspTrpArgAsnHisPheProGlu

840 270

GCCCTGAGGGAGAGATTTGAGGAGCACTACCAGCGGCATATGAAGGACTGCCCTGTGACG AlaLeuArgGluArgPheGluGluHisTyrGlnArgHisMetLysAspCysProValThr

900 290

TTTAGAGCAGAGCTCTGAGACACTTCCTTGTGTCTGAAATTGGAGTAGTCTCCAATTTAT PheArgAlaGluLeu***

960 295

CCTTCAGTTTTTCTTGTTTTG~ATTCAGTAGAAGTAGAAG

i000

Fig. 2. N u c l e o t i d e s e q u e n c e a n d t r a n s l a t i o n o f r E S T - 6 . T h e n u c l e o t i d e s e q u e n c e a n d t h e d e r i v e d t r a n s l a t i o n o f r E S T - 6 a r e n u m b e r e d on t h e right. N u c l e o t i d e s 1-20 a n d 981-1000 a r e d e r i v e d f r o m P C R p r i m e r s . T h e asterisk represents the stop codon.

Isoforms of Rat EST ferred to nitrocellulose paper. Primary rabbit antiM B P - r E S T - 3 antibodies were diluted 1:20,000, and incubated with the blots for 1 h. Goat anti-rabbit IgG horseradish peroxidase conjugate was used as the secondary antibody and immunoconjugates were visualized by chemiluminescence (Kirkegaard and Perry Labs).

Expression of r E S T - 6 in E. coli In order to characterize the enzymatic properties of r E S T - 6 , the active enzyme was expressed in bacteria and then highly purified as has been described previously for the expression of human cytosolic STs [7]. For the expression of active r E S T - 6 , the r E S T - 6 c D N A was inserted into the bacterial expression vector pKK233-3. T o subclone r E S T - 6 into pKK233-2, an oligonucleotide prime]: ( 5 ' - C C A C T T G A C C A T G G A G A C T T C - 3 ' ) was synthesized which incorporated two base changes to create an Nco I restriction site at the initiating methionine of the r E S T - 6 cDNA. T h e r E S T - 6 c D N A was amplified by P C R using the M13 universal primer as the antisense primer and p C R I I r E S T - 6 as a template. T h e amplified D N A was completely digested with Hind III and then partially digested with Nco I. T h e digestion products were resolved by electrophoresis in low melting agarose and the appropriately-sized D N A fragment was isolated and subcloned into the Nco I and Hind III sites of pKK233-3. E. coli XL1-Blue cells made competent using a calcium chloride procedure [17] were transformed with the P K K - : r E S T - 6 vector. Colonies were selected by ampicillin resistance, and those colonies containing the full-lenj~h Nco I - H i n d III r E S T - 6 D N A fragment were :identified by D N A sequence analysis.

EST-3 EST-6

39

Cytosol was prepared from induced E. coli X L 1-Blue cells transformed with p K K - r E S T - 6 using the same method described for the preparation of the M B P r E S T - 3 cytosol. Enzymatically active r E S T - 6 was purified from the bacterial cytosol prior to kinetic characterization because E. coli XL1-Blue cytosol contains enzymes that rapidly degrade PAPS. Bacterial cytosol was applied to a DEAE-Sepharose CL-6B column (1.5 × 5cm) equilibrated in T E A buffer (10 m M triethanolamine, p H 7.5, 1.5 m M dithiothreitol, 10% glycerol). T h e column was rinsed with 20 ml of T E A buffer, then with 30 ml of T E A buffer containing 100 m M NaC1. T h e E S T activity was eluted with a gradient of 100-225 m M NaC1 in T E A buffer, r E S T 6 was further purified by affinity chromatography on a 3',5' PAP-agarose column. T h e fractions from the anion-exchange column containing high levels of E S T activity were concentrated and desalted by ultrafiltration. T h e concentrated E S T activity was then applied to a 5 ml PAP-agarose affinity column and the column was washed with T E A buffer. E S T activity was eluted from the column with 10/~M PAPS in T E A buffer.

Sulfotransferase assays Steroid sulfation was assayed as described previously [18] using the tritiated steroids, fl-E2, E~, D H E A , testosterone, pregnenolone and cortisol, as the sulfate acceptors. Reactions contained 5 0 m M Tris-HC1, p H 7.4, 7 m M MgC12, and varying concentrations of steroid substrates (0.1-30/~M). T h e reactions were started by the addition of PAPS to a final concentration of 10/~M, in a final volume of 0.125 ml. Reactions were terminated by the addition of 4.0 ml of chloroform, followed by the addition of 0.375 ml 0.5 M Tris-HC1,

METSMPEYYDVFGDFHGFLMDKRFTKYWEDVETFLARPDDLLIVTYPKSGSTWISEIVDM lllllIlll IIIIIII I II IIIIIIIIIII lllllll IIIIIIIIIII IIIII METSMPEYYEVFGDFHGVLVDKLFTKYWEDVETFSARPDDLLVVTYPKSGSTWIGEIVDM

60

IYKEGDVEKCKEDALFNRIPDLECRNEDLINGIKQLKEKESPRIVKTHLPAKLLPASFWE IIIIIIIIIIIIII IIIII llllllllllIlllllllllllllllillllllllllll IYKEGDVEKCKEDAIFNRIPYLECRNEDLINGIKQLKEKESPRIVKTHLPAKLLPASFWE

120

KNCKIIYLCRNAKDVVVSYYYFFLIMKSYQNPKSFSEFVEKFMEGQVPYGSWYDHVKSWW IIIIIIIIIIIIIIIIIIIIIIIII III IIIIIIIIIIIIIIIIIIIIIIIIIIIIII KNCKIIYLCRNAKDVVVSYYYFFLIIKSYPNPKSFSEFVEKFMEGQVPYGSWYDHVKSWW

180

EKSKNSRVLFMFYEDMKEDIRREVVKLIEFLERDPSAELVDRIIQHTSFQEMKNNPCINY IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII IIIII IIIIIIIIIIIIIII II EKSKNSRVLFMFYEDMKEDIRREVVKLIEFLERDPLAELVDKIIQHTSFQEMKNNPCTNY

240

SMLPETMIDLKVSPFMRKGIVGDWKNHFPEALRERFEEHYQQQMKDCPVKFRAEP* IIIIIIIIIIIIIIIIIIIIIIII IIIIIIIIIIIIIIII IIIIII IIII I SMLPETMIDLKVSPFMRKGIVGDWRNHFPEALRERFEEHYQRHMKDCPVTFRAEL*

296

Fig. 3. Comparison of the amino acid sequences of rEST-3 and rEST-6. The derived amino acid s e q u e n c e s of rEST-3 and rEST-6 a r e c o m p a r e d a n d identical amino a c i d s a r e s h o w n by a vertical line. The a s t e r i s k s denote stop codons.

40

Josie L. Falany et al.

p H 8.7, to alkalinize the solution. T h e reaction mixtures were then vortexed for 30 s and centrifuged at 600 g for 5 min to separate the aqueous and organic phases. Synthesis of the tritiated steroid sulfates was determined by scintillation counting of the aqueous phase. Sulfation assays using non-radiolabeled steroids and small phenols, such as p-nitrophenol, dopamine and minoxidil, were performed as described previously [19,20].

phoresis in the presence of formaldehyde, visualized by brief staining in ethidium bromide, and then transferred to nitrocellulose paper. T h e nitrocellulose paper was then heated in vacuo and prehybridized in 50% deionized formamide with 0.8 M NaC1, 10 m M Tris-HC1, p H 7.2, 1 m M E D T A , 0.5% SDS, 50 # g / m l poly A + , 100/~g/ml yeast t R N A and 10 x D e n h a r d t ' s solution. [32p]-Labeled r E S T - 6 c D N A was generated by an oligolabeling procedure (Pharmacia). T h e nitrocellulose was hybridized at 42°C in fresh prehybridization buffer containing 2 × 106 d p m / m l of [32P]DNA. T h e filter was washed 2 x for 30 min at 65°C in 3 x SSC containing 0.5% SDS, then for 3 0 m i n in 3 × SSC alone. Autoradiography was p e r f o r m e d at - 70°C with an intensifying screen.

N o r t h e r n blot analysis

Total R N A was isolated from liver samples of 125-150 g male and female S p r a g u e - D a w l e y rats using an acid guanidinium thiocyanate extraction method [21]. T h e R N A was resolved by agarose gel electro-

5O rEST-6

METSMPEYY

rEST

EVFGDFHGVL

VDKLFTKYWE

D

M--R

F-

DVETFSARPD

DLLVVTYPKS I ......

L .......

gpEST

M-DS-EHD--

-Y-DE-R-I-

LY-Q-I'--D

N--A-Q

VIAA.

bEST

-SS-K-SFS

DY--KLG-IP

MY-K-IEQFH

N--E-E

VI ......

rEST-6

GSTWIGEIVD

MIYKEGDVEK

CKEDAIFNRI

PYLECRNEDL

i00 rEST

..... S ...................

gpEST

-T---S-V-C

---A .... K-

bEST

-T--LS--IC

---NN

rEST-6

ESPRIVKTHL

PAKLLPASFW

L ..... -RQ ...... V .V. . . . V

INGIKQLKEK

D -F ..... DKM

M--V---E-M

..... ST-HVMK-V---N-M 150

EKNCKIIYLC

RNAKDVVVSY

YYFFLIIKSY

rEST gpEST bEST

N .... I ..... A ...... S--

PR

MVb2q-H

-A

R--M-CI-

-V .... V ............

S

FLI-MVTAI 200

rEST-6

PNPKSFSEFV

EKFMEGQVPY

GSWYDHVKSW

WEKSKNSRVL

FMFYEDMKED

rEST gpEST bEST

-D-G--P -D-D--QD

....... ......

Q ................... D-E ......

rEST-6

IRREVVKLIE

FLERDPLAEL

rEST gpEST

--K--L---H

...... S ..... --G-K-SE

bEST

--K--M--L-

--G-KASD

rEST-6

LKVSPFMRKG

IVGDWRNHFP

gpEST

Q

bEST

Q

FE-T

VDKIIQHTSF

TDP-IPQ--

QEMKNNPCTN

-I -L

N

25O YSMLPETMID

R, K

S--

K

....... S--

-TT--DEVMN

-T .... EIMN

EALRERFEEH

YQRHMKDCPV

TFRAEL*

-S---K---T

V--N-S-DK-

--QQ--GSTL

QL-T-I*

D .... K---T

V--N-K-DM-

-EQQ--GSTL

K--T-I*

296 rEST

QQ ......

K,

K ..... *

Fig. 4. C o m p a r i s o n o f a m i n o a c i d s e q u e n c e o f r E S T - 6 w i t h t h e p u b l i s h e d a m i n o a c i d s e q u e n c e s o f t h e g u i n e a p i g [12], b o v i n e [10], a n d rat [11] ESTs. T h e a m i n o a c i d s e q u e n c e s o f t h e E S T s w e r e a l i g n e d a n d t h e initial m e t h i o n i n e o f t h e g u i n e a p i g s e q u e n c e w a s d e n o t e d as t h e first a m i n o acid. O n l y t h e a m i n o a c i d s w h i c h differ f r o m t h e s e q u e n c e of rEST-6 a r e s h o w n . I d e n t i c a l a m i n o a c i d s to t h e s e q u e n c e of rEST-6 a r e r e p r e s e n t e d by a dash. The a s t e r i s k s d e n o t e s t o p codons.

Isoforms of Rat EST RESULTS

A

A male rat liver 2gtl 1 c D N A library was amplified by P C R with oligonucleotide primers designed to amplify the c D N A of a previously published isoform of rat liver E S T [11]. Following amplification, two different but related cDNAs were identified by sequence analysis. T h e two different rat E S T c D N A sequences were termed E S T - 3 and E S T - 6 . Figures 1 and 2 show the nucleotide sequence and translation of r E S T - 3 and r E S T - 6 , respectively, r E S T - 3 was 1000 bp in length and possessed an open-reading frame of 885 nucleotides encoding a protein of 295 amino acids with a calculated molecular mass of 35,440 Da. T h e translated r E S T - 3 sequence contained three amino acid differences when compared to the published sequence of a previously reported male rat liver E S T [11]. These differences were at amino acids 150, Q for P; amino acid 238, I :for T ; and amino acid 295, P for L. r E S T - 6 was 1000bp in length and also encoded a 295 amino acid protein (Fig. 2). T h e calculated

A

41

B

C

D

94 6645--

31--

21-'

B

94

45-

31-

21-

Fig. 6. I m m u n o b l o t o f r E S T - 6 with a n t i - M R P - r E S T - 3 antibody. P r o t e i n s were r e s o l v e d by S D S - P A G E in a 12.5% gel a n d t r a n s f e r r e d to nitrocellulose. A f t e r i n c u b a t i o n with a 1/20,000 dilution of a n t i - M B P - r E S T - 3 antibody, the blot was developed as d e s c r i b e d in E x p e r i m e n t a l . Lane (A), 50pg f e m a l e r a t liver cytosol; lane (B), 0.5 p g r E S T - 6 P A P - a g a r o s e f r a c t i o n 3; lane (C), 2 #g r E S T - 6 P A P - a g a r o s e f r a c t i o n 4; lane (D), 50/lg m a l e r a t liver cytosol.

molecular mass of the translated protein encoded by r E S T - 6 was 35,300Da. T h e nucleotide sequence of r E S T - 6 was 96.7% identical to the nucleotide sequence of r E S T - 3 . A comparison of the translated proteins showed they were 93.5% identical and 96.0% similar in amino acid sequence (Fig. 3). Nine of the 19 amino acid differences between the two sequences were conservative substitutions. Figure 4 shows a comparison of the sequence of r E S T - 6 with the published E S T amino acid sequences of the guinea pig [12], bovine [10] and rat enzymes [11]. r E S T - 6 is 67.9% identical and 84.5% similar to the guinea pig E S T sequence and 63.7% identical and 80.1% similar to the bovine E S T , r E S T - 6 was 97.3% similar and 94.5% identical to a previously reported rat E S T sequence. Expression of rES T- 6

Fig. 5. S D S - p o l y a c r y l a m i d e gel o f rEST-6. P r o t e i n s w e r e r e s o l v e d by S D S - P A G E in a 12.5% gel, s t a i n e d with C o o m a s s i e blue a n d d e s t a i n e d . Lane (A), m o l e c u l a r weight m a r k e r s ; lane (B), 5 pg r E S T - 6 a f t e r P A P - a g a r o s e c o l u m n chromatography.

Enzymatically active r E S T - 6 was purified by DEAE-Sepharose C L - 6 B and PAP-agarose affinity chromatography after expression in E. eoli XL1-Blue cells using r E S T - 6 inserted into the p K K 2 3 3 - 2

42

]osie L. Falany et al.

bacterial expression vector. E S T activity co-eluted through both purification procedures with a protein which migrated with a molecular mass of approx. 31,000Da during S D S - P A G E (Fig. 5). T h e discrepancy between the molecular mass of the protein translated from r E S T - 6 and the estimation of its molecular mass by S D S - P A G E is most likely an artifact of the electrophoretic system, as similar discrepancies have been reported for other STs [14,22, 23]. T h e purified r E S T - 6 protein reacted with the rabbit antiM B P - r E S T - 3 antibody (Fig. 6); however, r E S T - 6 did not react with rabbit anti-rat minoxidil ( P S T ) - S T antibodies or rabbit anti-rat h y d r o x y s t e r o i d - S T antibodies (data not shown). Figure 6 also shows that the rabbit a n t i - M B P - r E S T - 3 antibody readily detected a protein in male rat liver cytosol which migrated with the same moleculr mass as r E S T - 6 . In contrast, no immunoreactive protein was detectable in female rat liver cytosol.

1

2

- 28S

-

18S

Northern blot analysis of rat liver R N A T o further investigate the expression of E S T in male and female rat liver, Northern blot analysis of R N A isolated from both sexes was carried out. Total R N A was isolated from the livers of male and female rats and analyzed by N o r t h e r n blot analysis using the r E S T - 6 c D N A as a probe. Figure 7 shows that the r E S T - 6 c D N A readily hybridized to a band of approx. 1400 nucleotides in R N A prepared from male rat liver. No detectabale hybridization was observed with the R N A prepared from female rat liver. T h e male-specific expression of E S T in rat liver is in agreement with the previously reported sexual dimorphism of S T expression in rodents [1, 11, 13,24]. T h e multiple bands may be due to the presence of multiple forms of r E S T or different sites of polyadenylation in the r E S T messages [14, 22, 23].

Fig. 7. N o r t h e r n b l o t a n a l y s i s o f m a l e a n d f e m a l e r a t l i v e r R N A w i t h r E S T - 6 c D N A as a p r o b e . T o t a l R N A s a m p l e s f r o m m a l e a n d f e m a l e a d u l t r a t l i v e r w e r e r e s o l v e d in a 1.5% agarose-formaldehyde gel. T h e R N A w a s t r a n s f e r r e d to n i t r o c e l l u l o s e a n d h y b r i d i z e d w i t h [32Pl-labeled r E S T - 6 . T h e a u t o r a d i o g r a p h o f t h e b l o t is s h o w n . T h e R N A f r a c t i o n s w e r e as follows: 30/~g f e m a l e r a t l i v e r t o t a l R N A ( l a n e 1) a n d 30 p g m a l e r a t l i v e r t o t a l R N A ( l a n e 2). T h e m i g r a t i o n o f r i b o s o m a l R N A is i n d i c a t e d o n t h e r i g h t .

Kinetic analysis of expressed r E S T - 6 T h e ability to express and purify r E S T - 6 from bacterial cytosol provided active enzyme for the initial characterization of its reactivity with steroids and of its kinetic properties, r E S T - 6 was capable of sulfating both f l - E 2 and El but no activity was observed with D H E A , testosterone, pregnenolone or cortisol. T h e effect of varying the concentrations of fl-E 2 and E, on E S T activity was tested. Figure 8 shows that maximal activity was observed with a f l - E 2 concentration of approx. 1/~M and above that concentration substrate inhibition was observed. E1 sulfation by r E S T - 6 also showed maximal activity at a concentration of 1 #M. T h e specific activity of r E S T - 6 was approx. 2-fold greater with fl-E 2 a s substrate than with E l . Other STs which are capable of conjugating estrogens have shown a partial dependence on M g 2+ ions for maximal activity [6, 18]. However, varying the M g 2÷ concentration (0-10 mM) in the f l - E 2 sulfation reactions did not affect the sulfation activity of r E S T - 6 .

5 E

4 3

t~

0 0

!

i

i

|

i

1

2

3

4

5

Estrogen (~M) Fig. 8. S u l f a t i o n o f fl-E 2 a n d E 1 b y e x p r e s s e d r E S T - 6 . T h e effect o f i n c r e a s i n g ~ - E 2 a n d E t c o n c e n t r a t i o n o n r E S T - 6 a c t i v i t y is s h o w n , r E S T - 6 w a s e x p r e s s e d in b a c t e r i a and partially purified by DEAE-Sepharose CL-6B chromatography, rEST-6 activity was assayed with varying concentrations o f ~ - E 2 a n d E 1 ( 0 . 0 2 5 - 5 / i M ) i n t h e p r e s e n c e o f 300/~ M PAPS.

Isoforms of Rat EST DISCUSSION This paper describes the cloning, bacterial expression, purification and immunological and kinetic characterization of a new isoform of rat liver E S T , r E S T - 6 . Although three other mammalian E S T s have been expressed in mammalian cells, to date there has been no kinetic characterization of the expressed E S T activities other than to report that they conjugate estrogens [11, 12]. Expression of high levels of r E S T - 6 activity in a bacterial expression system and subsequent purification of r E S T - 6 have allowed us to begin the examination of the kinetic characteristics of this important enzyme. T h e enzymatic characterization of r E S T - 6 demonstrates that this S T sulfates only estrogens, such as fl-E2 and E l , and does not sulfate D H E A , testosterone, pregnenolone or cortisol. Thus, r E S T - 6 is more specific for estrogen sulfation than are the P S T s and hydroxysteroid STs which may sulfate estrogens but only as part of a much broader spectrum of substrates [7]. Maximal sulfation activity towards both f l - E 2 and E 1 occurred at a substrate concentration of about 1 # M . T h e maximal sulfation activity of f l - E 2 by r E S T - 6 occurs at a lower concentration (1 # M ) than the maximal activities for either h P - P S T (6 # M ) or h D H E A - S T (20 # M ) , which also sulfate estrogens [7]. Substrate inhibition has been reported for steroid sulfation by other purified and expressed STs [7, 18] and was observed for both f l - E 2 and E1 sulfation by r E S T - 6 at concentrations greater than 1/~M. Unlike other STs capable of sulfating estrogens, r E S T - 6 showed no dependence upon the presence of M g 2+ ions in the reaction mixture,. T h u s , r E S T - 6 sulfates estrogens in a manner different from other STs and, due to its higher affinity for es'Erogens, may play a distinct role in estrogen metabolism. Sequence comparison of r E S T - 6 to r E S T - 3 , as well as to the rat liver E S T cloned and sequenced by D e m y a n et al. [11], indicates that r E S T - 3 and r E S T - 6 are isoforms rather than alleles of rat E S T ; however, r E S T - 3 and the E S T :reported by D e m y a n et al. [11] are apparently allelic forms of the same enzyme. T h r e e amino acid differences were noted between these sequences. These differences may have arisen during the P C R amplification of r E S T - 3 or represent minor allelic differences between different sources of rat liver m R N A . Immunological comparison of r E S T - 6 to r E S T - 3 confirms that these isoforms are cross-reactive, as r E S T - 6 reacts strongly with a polyclonal antibody raised to r E S T - 3 . T h e characl:erization of these rat E S T s confirms that there is a multiplicity of forms of E S T present in rats, similar 1:o the pattern described by Oeda et al. [12] for the guinea pig in which four individual but immunologically cross-reactive isoforms of E S T occur. T h e multiplicity of closely related STs is apparently the norm in rats, which have multiple forms of phenol-sulfating STs as well as hydroxysteroid STs.

43

T h e rat E S T described by D e m y a n et al. [11] shows very distinct sex differences, occurring in liver cytosol of young adult male rats but not at all in cytosol prepared from female rat liver, as determined by immunoblot analysis. Similarly, immunoblot analysis of male and female rat liver cytosols with the antibody to r E S T - 3 yields a strong immunoreactive band of about 31 kDa in male rat liver cytosol but not in female rat liver cytosol. These results confirm that there is selective sexual expression of E S T activity in male vs female rats [1, 11, 13]. It cannot be determined from immunoblot analysis which isoform or isoforms of E S T are present in male rat liver, as they are immunologically cross-reactive. N o r t h e r n blot analysis of R N A isolated from male and female rat livers with r E S T - 6 c D N A as a probe correlates with the immunoblot analysis; the r E S T - 6 c D N A also hybridizes with the other rat E S T c D N A s . T h e r E S T - 6 c D N A hybridizes to a band in male rat liver R N A but does not hybridize to female rat liver RNA. Both immunoblot and N o r t h ern blot analyses confirm that there are sexual differences in the levels of E S T expression in rat liver. Whether the different E S T activities are selectively expressed in other tissues, and whether one isoform is selectively expressed over another, has not yet been determined. T h e identification of r E S T - 6 , an S T which is relatively specific for the sulfation of estrogens, adds to the n u m b e r of isoforms of cytosolic S T reportedly present in rat liver. Characterization of the physical, regulatory and kinetic properties of the individual S T isoforms is important in determining the functions of these enzymes in steroid and drug metabolism. T o this end, the ability to express and purify the individual S T isoforms will be a powerful tool in their characterization, as has been demonstrated with rEST-6. Acknowledgement--The work presented in this manuscript was supported in part by NIH grant GM 38953 to CNF.

REFERENCES

1. Mulder G. J. and Jakoby W. B.: Sulfation. In Conjugation Reactions in Drug Metabolism (Edited by G. J. Mulder). Taylor and Francis, London (1990) pp. 107-161. 2. Hobkirk R.: Steroid Sulfation. Trends Endocr. Metab. 4 (1993) 69-74. 3. Baulieu E.: Neurosteroids: a new function in the brain. Biol. Cell 71 (1991) 3-10. 4. Mortola J. F. and Yen S. S. C.: The effects of oral dehydroepiandrosterone on endocrine-metabolic parameters in postmenopausal women,ft. Clin. Endocr. Metab. 71 (1990) 696-704. 5. Kalimi M. and Regelson W.: The Biologic Role for Dehydroepiandrosterone. Walter de Gruyter, New York, NY (1990). 6. Hernandez J. S., William R., Watson G., Wood T. C. and Weinshilboum R. M.: Sulfation of estrone and 17fl-estradiol in human liver. Drug Metab. Disp. 20 (1992) 413-422. 7. Falany C. N., Wheeler J., Oh T. S. and Falany J. L.: Steroid sulfation by expressed human cytosolic sulfotransferases. J. Steroid Biochem. Molec. Biol. 48 (1994) 369-375. 8. Hobkirk R., Glasier M. A. and Brown L. Y.: Purification and characteristics of an oestrogen sulphotransferase from guinea pig

44

9. 10.

11.

12.

13.

14. 15.

Josie L. F a l a n y et al. adrenal gland and its nonidentity with adrenal pregnenolone sulphotransferase. Biochem. J. 268 (1990) 759-764. Moore S. S., Thompson E. O. P. and Nash A. R.: Oestrogen sulfotransferase: isolation of a high specific activity species from bovine placenta. Aust. J. Biol. Sci. 41 (1988) 333-341. Nash A. R., Glenn W. K., Moore S. S., Kerr J., Thompson A. R. and Thompson E. O. P.: Oestrogen sulfotransferase: molecular cloning and sequencing of cDNA for the bovine placental enzyme. Aust. J. Biol. Sci. 41 (1988) 507-516. Demyan W. F., Song C. S., Kim D. S., Her S., Gallwitz W., Rao T. R., Slomczynska M., Chatterjee B. and Roy A. K.: Estrogen sulfotransferase of the rat liver: complementary DNA cloning age- and sex-specific regulation of messenger RNA. Molec. Endocrinol. 6 (1992) 589-597. Oeda T., Lee Y. C., Driscoll W. J., Chen H.-C. and Stroot C. A.: Molecular cloning and expression of a full-length complementary DNA encoding the guinea pig adrenocortical estrogen sulfotransferase. Molec. Endocrinol. 6 (1992) 1216-1226. Jakoby W. B., Sekura R. D., Lyon E. S., Marcus C. J. and Wang J. L.: Sulfotransferases. In Enzymatic Basis of Detoxication (Edited by W. B. Jakoby). Academic Press, New York (1980) pp. 199-227. Comer K. A., Falany J. L. and Falany C. N.: Cloning and expression of human liver dehydroepiandrosterone sulfotransferase. Biochem. J. 289 (1993) 233-240. Vaitukaitus J., Robbins J. B., Nieschlag E. and Ross G. T.: A method for producing specific antisera with small doses of immunogens. J. Clin. Endocr. Metab. 33 (1971) 988-991.

16. Comer K. A. and Falany C. N.: Immunological characterization of dehydroepiandrosterone sulfotransferase from human liver and adrenals. Molec. Pharmac. 41 (1992) 645-651. 17. Davis L. G., Dobner M. D. and Battery J. F.: Basic Methods in Molecular Biology. Elsevier Science, New York (1986). 18. Falany C. N., Vazquez M. E. and Kalb J. M.: Purification and characterization of human liver dehydroepiandrosterone sulfotransferase. Biochem. J. 260 (1989) 641-646. 19. Falany C. N., Vazquez M. E., Heroux J. A. and Roth J. A.: Purification and characterization of human liver phenol-sulfating phenol sulfotransferase. Arch. Biochem. Biophys. 278 (1990) 312-318. 20. Falany C. N. and Kerl E. A.: Sulfation of minoxidil by human liver phenol sulfotransferase. Biochem. Pharmac. 40 (1990) 1027-1032. 21. Chomczynski P. and Sacchi N.: Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162 (1987) 156-159. 22. Hirshey S. J., Dooley T. P., Reardon I. M., Heinrikson R. L. and Falany C. N.: Sequence analysis, in vitro translation and expression of the cDNA for rat liver minoxidil sulfotransferase. Molec. Pharmac. 40 (1992) 1027-1032. 23. Wilborn T. W., Comer K. A., Dooley T. P., Reardon I. M., Heinrikson R. L. and Falany C. N.: Sequence analysis and expression of the cDNA for the phenol-sulfating form of human liver phenol sulfotransferase. Molec. Pharmac. 43 (1993) 70-77. 24. Leiter E. H., Chapman H. D. and Falany C. N.: Synergism of obesity genes with hepatic steroid sulfotransferases to mediate diabetes in mice. Diabetes 40 (1991) 1360-1363.