Human dehydroepiandrosterone sulfotransferase: molecular cloning of cDNA and genomic DNA

Chemico-Biological Interactions ELSEVIER

Chemico-Biological Interactions 92 (1994) 145-159

Human dehydroepiandrosterone sulfotransferase: molecular cloning of cDNA and genomic DNA Diane M. Otterness, Richard Weinshilboum* Department of Pharmacology, Mayo Medical School, Mayo Clinic, Mayo Foundation, Rochester, MN 55905, USA Received 16 August 1993; revision received 3 January 1994; accepted 5 January 1994

Abstract

Human tissues contain at least three well-characterized cytoplasmic sulfotransferase (ST) enzymes, dehydroepiandrosterone (DHEA) ST and two of phenol ST (PST). DHEA ST catalyzes the sulfation of DHEA and other steroids. We cloned and expressed two cDNAs for human liver DHEA ST. The cloning strategy involved the design of PCR primers directed against two conserved domains in ST proteins. These primers were used to generate a specific PCR product that was then used successfully to clone cDNAs for DHEA ST from a human liver cDNA library. Two cDNAs were isolated that were approximately 1.1 and 1.8 kb in length. These two clones had identical open reading frames. Both cDNAs produced enzymatically active DHEA ST protein in a mammalian expression system. Northern blot analysis confirmed the presence of 1.1 and 1.8 kb transcripts in human liver, cDNAs for a number of eukaryotic enzymes have now been cloned, and they share significant sequence homology. These ST cDNAs appear to fall into distinct groups on the basis of amino acid sequences of the proteins that they encode, thus demonstrating that the enzymes comprise a gene superfamily. We have also isolated a genomic clone for human DHEA ST that contains approximately 3 kb of 5'-flanking sequence, exon 1 and 1.7 kb of intron 1. Characterization of the structure and regulatory elements of this gene should help to elucidate mechanisms involved in the regulation of DHEA ST in humans.

Keywords: Dehydroepiandrosterone sulfotransferase; Dehydroepiandrosterone; Sulfation; Sulfate conjugation

* Corresponding author. 0009-2797/94/$07.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved SSDI 0009-2797(94)03297-L

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1. Introduction Dehydroepiandrosterone sulfotransferase (DHEA ST) is one of three wellcharacterized cytosolic ST enzymes known to be present in human liver [1]. DHEA ST catalyses the sulfate conjugation of DHEA, steroid hormones such as estrone, bile acids and cholesterol [2-5]. The other two well-characterized human hepatic ST enzymes are phenol sulfotransferases (PSTs). One form of PST is thermostable (TS); it preferentially catalyses the sulfation of 'simple' planar phenols and is sensitive to inhibition by 2,6-dichloro-4-nitrophenol (DCNP). The other form is thermolabile (TL); it preferentially utilizes phenolic or catechol monoamines as substrates and is relatively resistant to DCNP inhibition [1,6]. Levels of activity for DHEA ST and the two forms of PST vary widely among individuals, and all three activities are regulated independently [3,5]. The subsequent discussion summarizes current understanding of the biochemistry and molecular biology of DHEA ST, the first human ST for which a cDNA was cloned and expressed. An attempt is also made to place this information within the context of our evolving knowledge of the molecular biology of the ST enzyme gene superfamily. 2. Biochemical characteristics and regulation of human DHEA ST DHEA ST catalyses the sulfation of 3-hydroxysteroids such as DHEA, steroid hormones such as estrone, bile acids such as lithocholic acid, the cardiac glycoside digitoxin and even cholesterol itself [2-5,7]. Therefore, DHEA ST is one member of a group of mammalian ST enzymes that are most frequently referred to as hydroxysteroid STs (HSSTs) [1]. DHEA is the most abundant circulating steroid hormone in both women and men, and this steroid, as well as its sulfate conjugate, plays an important role in the hormonal regulation of pregnancy [8]. Epidemiologic data also suggest that plasma levels of DHEA sulfate might be related to risk for the occurrence of cardiovascular disease or neoplasia [9-11]. Therefore, knowledge of the properties and regulation of DHEA ST has potential implications for human health. DHEA ST has been well characterized in two human tissues, liver and adrenal cortex [2,12]. In the adrenal cortex, immunohistochemical studies have localized the enzyme to the zona reticularis [13]. The enzymes present in human liver and adrenal glands have very similar or identical physical and biochemical properties [12]. DHEA ST is a homodimer with a monomer Mr value of approximately 35 kDa [2]. The activity of the enzyme is maximal in the presence of 0.3-10 mM Mg ~+ [2,3]. There are similarities between human and rat HSSTs. Purified human liver DHEA ST and purified rat liver HSST can each be resolved by isoelectric focussing into at least three charged species [14-16]. In addition, three separate HSSTs with differing amino acid sequences are known to be present in rat liver [17-19]. Rat liver HSST also shows striking gender-dependent regulation. Mature female rats have levels of activity approximately 15-fold higher than males have [16]. However, hepatic enzyme activity in male rats rises to levels found in female animals during senescence [16]. These sex-related differences in enzyme activity are paralleled by changes in mRNA levels [20]. Regulation of DHEA ST activity in human liver

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contrasts sharply with the situation in rats. There were no significant age or genderdependent differences in DHEA ST activity in 94 samples of human liver tissue obtained during clinically-indicated hepatic biopsy, even though there was a 4.6-fold range of enzyme activity [21]. However, 20-25% of these samples were included in a high enzyme activity subgroup [21] - - raising the possibility that DHEA ST activity, like the two forms of PST found in human tissue [22-24], might be controlled by a genetic polymorphism. As an important step toward understanding possible molecular mechanisms involved in the regulation of DHEA ST in humans, we set out to clone a cDNA for human liver DHEA ST, with the ultimate goal of cloning the human DHEA ST gene or genes. 3. Molecular cloning of human liver DHEA ST cDNA The strategy employed in cloning DHEA ST cDNA began with purification of the human liver enzyme. DHEA ST was purified approximately 600-fold by sequential DEAE-Sepharose CL-6B ion exchange, Affi-Gel Blue and heparin Sepharose CL-6B chromatography [14]. This purified enzyme preparation was then photoaffinity labeled with 35S-3'-phosphoadenosine-5'-phosphosulfate (PAPS), the sulfate donor for the enzyme reaction. We had demonstrated previously that aSS-PAPS could be used as a photoactive ligand for ST enzymes [25]. When photoaffinity labeled DHEA ST was subjected to two-dimensional SDS-polyacrylamide gel electrophoresis, three distinct photoaffinity labeled proteins, all with molecular mass values of 35 kDa, were present [14]. Unfortunately, the N-termini of all three proteins were blocked to Edman degradation, but it was possible to obtain partial amino acid sequence information from peptide fragments generated by limited proteolysis. The partial amino acid sequence data obtained were identical for all three of the pI variants [14]. This amino acid sequence data helped to verify the identity of the cDNA that was cloned. The cloning strategy that proved successful involved use of the polymerase chain reaction (PCR) performed with primers designed on the basis of two highly homologous sequences found in the three ST cDNAs, all from non-human mammalian species, that had been cloned at the time that the experiments were performed. These two areas of sequence homology will be discussed in greater detail subsequently when a molecular classification of ST enzymes is described. With human liver cDNA as template, the primers were used to amplify a specific PCR product approximately 650 nucleotides in length. This amplification product encoded the amino acid sequences determined after limited proteolysis of DHEA ST. The PCR amplification product was then used to screen a human liver cDNA library. Thirteen positive clones were isolated, ten of which were at least partially sequenced. Two clones, designated 'A' and 'G', were completely characterized and were found to be approximately 1.8 and 1. l kb in length, respectively (Fig. 1). Both clones had identical open reading frames of 855 nucleotides that encoded a protein of 285 amino acids with a predicted Mr of 33.78 kDa [14]. The deduced amino acid sequence included the 28 and 23 amino acid sequences obtained after limited proteolysis of the native protein (underlined amino acid sequences in Fig. 1). Clone G had 44 more nucleotides

148

D.M. Otterness, R. Weinshilboum / Chem.-Biol. Interact. 92 (1994) 145-159

HUMAN -60

CLONE A CLONE G

CLONE A CLONE G

LIVER

DHEA

ST c D N A

-40

-20 0 20 CGCAGGAAGACGTCATCATCATGTCOCACGATTTCTTATOGTTTOAAGGCATAGCT GCTGCCACAGCTCCAGCGTCGGTACAGTTGAAACCCTCACACCA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . M S D D F L W F E G I A 40 60 80 i00 120 TT~CCTACTATGGGTTT~AGATCCGAAACCTT~G~AAGTACGTGATGAGTTCGTGATAAGGGAT~AA~ATGT~T~TATTGACTTAC~CC~ATCAG .................................................................................................... F P T M G F R S E T L R K V R D E F V I R D E D V I I L T Y P K S G 140 160 180 200 220

CLONE A CLONE G

GAA~ACTGGTTG~CTGAGATTCTCTGCCTGATG~ACTCCAAG~GGGATGCCAA~T~GAT~C~TCTGTGCCCAT~TGGGAGCGATCACC~TGGGTAGA

CLONE A

GAGTGAGATTGG~TATACAG~ACTCAGTG~ACGGAGAGT~ACGTTTATTCT~CTCCCACCT~C~ATC~AGTTATTCC~GTCTTTCTTCAGTTCC

CLONE A CLONE O

AAGGCCAAGGTGATTTAT~TCATGAGA~T~CCAGAGATGTTTTGGTGT~TGGTTATTTTTTCTGGAA~A~ATGAAGTTTATTAA~A~CA~GTCAT

.................................................................................................... T N W L A E I L C L M H S K G D A K W I Q S V P I W E R S P W V E 240 260 280 300 320

340

CLONE A CLONE G

CLONE A CLONE O

CLONE A CLONE O

CLONE A CLONE G

CLONE A CLONE G

CLONE A CLONE O

360

380

400

420

.................................................................................................... K A K V I Y L M R N P R D V L V S G Y F E W K N M K F I K M P K S W 440 460 480 500 520 GGCAAGAATATTTT~AATGGTTTTGTC~GGAA~TGTGCTATATGG~TCATGGTTTGACCACATTCATG~CTGGATGCCCATGAGAGAGGAGA~AA~TT .................................................................................................... E E Y F E W F C Q G T V ( ~ ) Y G S W F D H I H G W M P M R E E K N F 540 560 580 600 620 ~CT~TTA~TGAGTTATGA~GAGCTGAAACAGGACACAGGAAGAACCATAGAG~GATCTGTCAATTCCTGGGAAAGACGTTAGAACC~GAAGAACT~AA~ .................................................................................................... L L L S Y E E L K Q D T G R T I E K I C Q F L G K T L E P E E L N 640 660 680 ?00 720

TT~TTCTCAAGAACA~CT~CTTTCAGAGCATGAAAGA~A~AAGATGT~CAATTATTC~CTCCTGAGTGTTGATTATGTAGTGGA~A~G~ACAA~TTC .................................................................................................... L I L K N S S F Q S M K E N K M S N Y S L L S V D Y V V D K A Q L L 740 760 780 800 820 TGAO~AAGGTGTAT~TGGGGACTGGA~TCACTTCA~AGTGG~C~AAG~TG~GACTTTGAT~ATTGTTCCAAGAGAAGATGGCAGATCTTCCTCG .................................................................................................... R K G V S G D W K N H F T V A Q A E D F O K L F Q E K M A D L P R 840 860 880 900 920 AGAGCTGTTCCCATGGGAATAACGTCC~CACTCTGGATCTTATATGGAG~TGACATTGATTCTCCTGT~CTTGTA~ATGTA~CTGACTGGGGTCAT .................................................................................................... E L F P W £ * 9~0 960 980 IQ00 1020 TGT~TAA~ACTTATTATTTTAT~CTGA~CCTTAAATATC~ACCTCTGCATCTCTGATC~CTTCCTTGTT~AA~TTAC~ACGGTTCGCCAGGCG~GGT .................................................. (A)n 1040 1060 1080 ]100 1120

CLONE A

G~TT~AT~CCTGT~TC~CAGCA~TATGGGAGG~CGAGA~GG~GGATCACGAGGTCAGGAGA~TGAGACCAT~CTGG~TAA~ACGGT~A~C~C~ATCT

CLONE A

CTACTAA~ATAC~A~A~A~A~AATTAG~A~GCATTG~CTCATGTCTGTAAT~CA~A~TTTGGGA~TCGGGGGG~TGGGGGAGGATCA~GG

CLONE A

GGTCAGGAGATCGAGACCATC~TGG~CATGATGAAACC~TATCTCTACT~ATAC~A~TTA~CCGGGCATGGTGGTGCA~C~TATA~TCCCA

CLONE A

GCTA~TCGGGGG~CTGAGGTAGGAGAATCGTTTGAACTCACGAG~CA~AGGTT~AATGAGCC~ATCCCGCCACTGCA~TC~A~CTG~GTGACAGA~

CLONE A

~GAGA~CGTCTCA~AAG~A~AAGTGACTAGGTTCAGAG~CCA~TTCA~CCAGGGATGCAAAGGTTG~A~TGAGTTGAGTCAT~GGATCC~AG

CLONE A

A~TTTTTTAAATGTTTGC~TGTTTCCCGTTTA~AGAAT~CTA~AAGAATAATGTAC~TACTA~CTAA~G~ATGTCTA~TGTTT~TTAATA~AATAA

CLONE A

1640 1660 1680 GA~TAGCTACAGTGACAGATTTTAGAGC~A~TTAGTAATA~AATAAGA~TAA~TT(A)28

1140

1240

1340

1440

1540

1160

1260

1360

1460

1560

1180

1280

1380

1480

1580

1200

1300

1400

1500

1600

1220

1320

1420

1520

1620

Fig. 1. Human liver D H E A ST c D N A clones A and G nucleotide sequences [14]. The dotted line represents nucleotides in clone G that were identical to those present in clone A. Underlined amino acid sequences represent areas identical to amino acid sequences in human liver DHEA ST determined after limited proteolysis. Underlined nucleotide sequences represent areas used to design PCR primers (see text for details). Nucleotides 1149, 1156, and 1166 were sites of polyadenylation for truncated clones, as was one site located eight nucleotides 3' to the polyadenylation site for clone A. Circled amino acids represent those that would be changed on the basis of nucleotide differences reported by other laboratories [26,27]. See text for details. Reproduced with permission of the American Society for Pharmacology and Experimental Therapeutics from D.M. Otterness, E.D. Wieben, T.C. Wood, R.W.G. Watson, B.J. Madden, D.J. McCormick and R.M. Weinshilboum, Human liver dehydroepiandrosterone sulfotransferase: molecular cloning and expression of c D N A , Moh Pharmacol., 41 (1992) 865-872.

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at the 5'-end than did clone A. The 3 '-untranslated region (UTR) of clone A was approximately 840 nucleotides in length, while that of clone G was approximately 130 nucleotides long. However, both 3'-UTRs terminated with poly(A) tails. Two other laboratories subsequently reported cloning cDNAs for human liver DHEA ST [26,27]. Both of those clones were approximately 1.1 kb in length. The most important differences among the clones isolated by the three laboratories were present within the open reading frame and resulted in alterations in amino acid sequence. One change involved the substitution of GC for CG at nucleotides 268 and 269 in Fig. 1, resulting in serine rather than threonine at amino acid 90 [27]. The other difference involved the substitution of CG for GC at nucleotides 473 and 474, resulting in valine rather than leucine at amino acid 159 [26]. The longest 5'-UTRs were very similar in the cDNA clones reported by all 3 laboratories. However, when all of the reported cDNA clones were compared, including clones truncated at the 5'-end, they could be separated into three groups based on length of the 3'-UTR. One group of cDNAs contained 3'-UTRs approximately 130-145 nucleotides in length, a second contained 3 '-UTRs approximately 300 nucleotides long (corresponding to polyadenylation at nucleotides 1149, 1156 or 1166) [14], and a third group contained 3'-UTRs approximately 800-840 nucleotides in length. These three groups would correspond to mRNA transcripts approximately 1.1, 1.3, or 1.8 kb in length, respectively. Although the 1.8 kb clone, clone A, contained a consensus polyadenylation sequence, there was not a consensus polyadenylation sequence prior to the poly(A) tails in either the 1.1- or 1.3-kb clone [14]. Whether the apparent existence of differential processing of the 3'-ends of DHEA ST transcripts has functional implications remains to be determined. As described subsequently, the existence of three different length DHEA ST mRNA species was supported by the results of Northern blot analyses. 4. Expression of human liver DHEA ST cDNA Human liver DHEA ST cDNA was the first ST cDNA to be expressed in a mammalian system. To perform those experiments, clones A and G were subcloned into the eukaryotic expression vector p91023(B). Constructs with the cDNAs present in either the sense or antisense orientations were then transfected into COS-1 cells. DHEA ST, TS PST and TL PST activities were measured in homogenates of these cells. The proteins encoded by both clones A and G were capable of catalysing the sulfation of DHEA when the cDNA was placed in the expression vector in the proper orientation (designated 11A and 17G in Table 1) [14]. However, no DHEA ST enzymatic activity was present when transfection was performed with either vector alone or with constructs in which the cDNA was in the antisense orientation (designated 3A and 15G in Table 1). Transfection did not increase either TS or TL PST activities above the low basal levels present in the COS-1 cells. In addition, the DCNP inhibition profile for the DHEA ST activity expressed in COS-1 cells was virtually identical with that of the human liver enzyme. A subsequent report of the cloning of DHEA ST cDNA confirmed these transient expression results [27].

150

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Table 1 Expression of human liver DHEA ST cDNA in COS-1 cells DHEA ST enzymatic activity, counts/min

Control, No DNA Control, No DNA Control, p91023(B) Control, 3A antisense Control, 15G antisense 11A sense 17G sense

Experiment I

Experiment 2

0 0 28 0 0 58 882 59 435

11 0 0 16 106 29 864 28 805

cDNA clones A and G in either sense (1 IA and 17G, respectively) or antisense (3A and 15G, respectively) orientations were used in these studies. The results of two separate experiments are shown. Cytosol preparations were assayed for ST activities by the method of Foldes and Meek [30] as modified by Hern~indez et al. [3] for the measurement of human liver DHEA ST activity. No changes in the low basal levels of TS PST or TL PST activities were detected after transfection. Details of experimental procedures were described by Otterness et al. [14]. Reproduced with permission of the American Society for Pharmacology and Experimental Therapeutics from D.M. Otterness, E.D. Wieben, T.C. Wood, R.W.G. Watson, B.J. Madden, D.J. McCormick and R.M. Weinshilboum, Human liver dehydroepiandrosterone sulfotransferase: molecular cloning and expression of cDNA, Mol. Pharmacol., 41 (1992) 865-872.

5. Northern and Southern analyses of human DHEA ST W h e n N o r t h e r n blot analysis o f m R N A from eight different h u m a n tissues was p e r f o r m e d with D H E A ST c D N A as a p r o b e , two transcripts, a p p r o x i m a t e l y 1.1 and 1.8 kb in length, were detected in h u m a n liver p r e p a r a t i o n s (Fig. 2). N o r t h e r n analysis also showed that the relative expression o f D H E A ST m R N A was much greater in liver than in any o f the other tissues studied (Fig. 2). The results for h u m a n liver shown in Fig. 2 were identical with those r e p o r t e d in one other study o f h u m a n liver D H E A ST m R N A [26], but a s e p a r a t e study reported the existence o f a 1.3-kb transcript in a d d i t i o n to the two shown in Fig. 2 [27]. These three m R N A species, 1.1, 1.3 and 1.8 kb in length, c o r r e s p o n d e d closely with sizes o f the cloned c D N A s , sizes that d e p e n d e d p r i m a r i l y on differences in length o f 3 ' - U T R s . Finally, the resuits o f Southern analyses p e r f o r m e d with D H E A ST c D N A were c o m p a t i b l e with the conclusion that either a single o r very few genes for D H E A ST were present in the h u m a n genome (Fig. 3). These results confirmed a previous report o f Southern analysis d a t a for h u m a n D H E A ST [27].

6. Preliminary cloning of human DHEA ST genomic DNA Preliminary attempts to isolate genomic D N A clones for h u m a n D H E A ST resulted in the isolation o f a single clone from a h u m a n leukocyte genomic D N A lib r a r y (Clontech L a b o r a t o r i e s , Palo Alto, CA). A 7-kb p o r t i o n o f this clone was subcloned into pBluescript a n d was partially sequenced. This genomic clone contain-

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151

HUMAN TISSUE DHEA ST NORTHERN ANALYS~S Skeletal Liver

Heart

Brain

Placenta

Lung L i v e r

Muscle Kidney Pancreas

9.5

7.5

et

4.4

2.4 1.35

I HR

EXPOSURE

7 HR EXPOSURE

Fig. 2. Northern analysis of human DHEA ST. A human multiple tissue Northern blot (2/~g of poly A*RNA per lane, Clontech Laboratories, Palo Alto, CA) was probed with DHEA ST cDNA clone G~

ed exon 1, approximately 1700 nucteotides of intron 1 and approximately 3 kb of sequence 5'-upstream of the translation initiation codon, No prototypic TATA or CAAT sequences were identified. The exon 1/intron 1 junction was located at nucleotide + 136, exactly the position at which a similar junction is located in the rat SMP2 gene [28], a gene that encodes a protein homologous to DHEA ST (see below). Further characterization of this and other human DHEA ST genomic DNA clones should make it possible to perform studies designed to investigate regulation of the transcription of the DHEA ST gene. 7. Human liver DHEA ST and the molecular classification of ST enzymes

cDNAs for thirteen eukaryotic ST enzymes have been reported. "We compared the predicted amino acid sequence of DHEA ST with those of proteins encoded by the other twelve ST cDNAs to determine possible relationships among these proteins. Because of the diverse nomenclature used for ST enzyznes in different laboratories,

152

D.M. Otterness, R. Weinshilboum! Chem.-Biol. Interact. 92 (1994) 145-159

HUMAN DHEA ST SOUTHERN ANALYSIS

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"-

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~,~j~iiiijii~,i!~

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Fig. 3. Southern analysis of human D H E A ST. Five micrograms of human lymphocyte genomic D N A was exhaustively digested with BamHl, EcoRl, Pstl or HindllI; separated by electrophoresis on a 0.8% agarose gel; transferred to an MSI nylon membrane and probed with D H E A ST c D N A clone G.

D.M. Otterness, R. Weinshilboum /Chem.-Biol. Interact. 92 (1994) 145-159

153

A

hTSPST rPST mPST rEST bEST gpEST hDHEAST rHSST1 rHSST2 rSMP2 mHSST fcFST3 fcFST4"

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LIQDTSRPPL L. F . . . . SRPPL L. SNMEPLRKPL L. TSMPEYYDVF M. SSKPSFSDYF M. SSEHDYYEYF L. MSDDF FPT MPDY FHA MPDY FPA MMSDY FPA MMMSDY FPA HTCSFLKHRF TLYKYKDAWN HTCSSLKGRI TLYKYQDFWG

51

10 F~ FL~FK APGIPS~ET Y# iFL~K CPGVPS~ET Y# iFL~S CPGVPP~ET F~ TEHVMK~VKQ F~ F~ JViF L~C R NDKMMN~VKQ WE

150 QTI

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FGIP~LQN ISYQ~ILED ISYQ~ILED HQEFL~RIL LQNNI~AIL 100 CHRAPI . . . . CGRAPI . . . . CGRAPV . . . . CKEDAL . . . . CKEDVI . . . . CRQDAI . . . . IQSVPI . . . . IQSVTI . . . . IQSVTI . . . . IQSCPF . . . . IQTVPI . . . . STSPLLTTMP FTSPLLTNIP

hTSPST rPST mPST rEST bEST gpEST hDHEAST rHSST1 rHSST2 rSMP2 mHSST fcFST3 fcFST4"

hTSPST rPST mPST rEST bEST gpEST hDHEAST rHSST1 rHSST2 rSMP2 mHSST fcFST3 fcFST4"

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_ QRNSLYTPI QNNSCFTPM

Fig. 4. Amino acid sequence alignment of cytoplasmic ST enzymes for which cDNA clones have been reported. Sequences were aligned with the PILEUP program [41] from the Genetics Computer Group package (Madison, W1) [40]. White type on a black background indicates that the amino acid at that position was identical in at least 9 of the 13 proteins. Abbreviations for enzyme names are those listed in Table 2.

D.

154

Fig.4 (continue

B hTSPST rPST mPST rEST bEST gpEST hDHEAST rHSST1 rHSST2 rSMP2 mHSST fcFST3 fcFST4'

IVKLSVEEAP ITKLPLEDAP

hTSPST rPST mPST rEST bEST wEST ?DHEAST rHSST1 rHSST2 rSMP2 mHSST fcFST3 fcFST4' ‘

c

250 . . . ..RSLPE . . . ..RSLPE . . . ..RSLPE . . . ..RDPSA . . . . . RKASD . . . ..RKPSE . . . ..KTLEP . . . ..KKLEP . . . ..KKLEP . . . ..KNLGP . . . ..KNLGP HPFTPKEEEA YPFTFEEEKE

SRTHP RHTHP RRTHP SKNSR SKNPQ STDPR REEKN QEWDN RELDN REWDN REWDN EKPEI ERPEV

hTSPST rPST mPST rEST bEST gpEST t-IDHEAST rHSST1 rHSST2 rSMP2 mHSST fcFST3 fcFST4' 25 ET ET ET :: EL EE DE DE :: :i

hTSPST rPST mPST rEST bEST #ST hDHEAST rHSST1 rHSST2 rSMP2 mHSST fcFST3 fcFST4

V.PQE I.PTE I.PTE M L.PET C L.PDE S L.PEE S L.SVD L.MKK ! LM.EKE M LI.KED LI.KED f: MHRPEE V V NKSG..NSKG

FMDHSISP IMDHNVSP VMDHTIYP MIDLKVSP VMNQKVSP IMNQKVSP YV.VDKAQ SI.FTGTG LI. LPGFT PI.LTGLK OVTNGL.K AHSIENRL FLPIENRLYF

STLQLRTEI.

TGLVLK..,. IDEKLSA TGLVLK....

PWE. PWE. PWD. PWE. PWE.

D.M. Otterness, R. Weinshilboum / Chem.-BioL Interact. 92 (1994) 145-159

155

for purposes of this discussion, the species from which the cDNA was cloned is indicated by a small letter or letters in which 'h' represents human, 'r' rat, 'm' mouse, 'gp' guinea pig, 'b' bovine, and 'fc' Flaveria chloraefolia. The names of the enzymes are abbreviated PST for phenol ST, EST for estrogen ST, HSST for hydroxysteroid ST, FST for flavonol ST, and SMP for senescence marker protein. Alignment of the amino acid sequences of all thirteen proteins showed many areas of sequence homology (Fig. 4). The presence of such a high degree of structural similarity indicated that it might be useful to determine the degree of homology of the complete amino acid sequences, and, as anticipated, they demonstrated significant amino acid sequence homology (Table 2). The existence of this sequence homology made it possible to develop a tentative classification for cytoplasmic ST enzymes analogous to the classification scheme that has been developed for the cytochrome P450 gene superfamily [29]. In the case of the cytochromes P450, an arbitrary value of 40% identity of amino acid sequence was chosen for inclusion in an enzyme family, and 60% sequence identity was chosen for inclusion in a subfamily [29]. If an identical, and equally arbitrary set of criteria were used to classify the proteins encoded by cDNAs for the ST enzymes reported thus far, STs could be separated into three families: the PSTs, HSSTs and FSTs (Fig. 5). The PST family would consist, at present, of two subfamilies, the PSTs and the ESTs. This provisional classification scheme is intended to be neither final nor all inclusive. It should also be emphasized that,

CLUSTER ANALYSIS

I I

EST SUBFAM LY

PST SUBFAM LY

I

I

I I m

~

m

~

~

z

m

m

m

m

m

Fig. 5. Provisional molecular classification scheme for cytoplasmic ST enzymes. Enzymes were included in the same family if their amino acid sequences were more than 40% identical, and they were included in the same subfamily if their amino acid sequences were more than 60% identical. Abbreviations for enzyme names are those listed in Table 2. See text for details. Cluster analysis was performed with the PILEUP program [41] from the Genetics Computer Group package (Madison, WI) [40].

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although functional designations have been retained in the names assigned to the ST families and subfamilies depicted in Fig. 5, the classification is based entirely on amino acid sequence, not on function. Therefore, the scheme outlined in Fig. 5 represents only a 'way-station' on a path leading eventually to a nomenclature that would be entirely neutral with regard to function. Comparison of the deduced amino acid sequences for these thirteen ST enzymes showed many areas of sequence homology (Fig. 4), including at least two highly conserved regions. One of those sequences, YPKSGTXW, was located toward the amino terminus between the positions numbered 67 and 74 in the multiple alignment shown in Fig. 4, and the other, R K G X X G D W K N X F T , was located toward the carboxy terminus between positions 291 and 303 in the multiple alignment. 'X' in these sequences represents any amino acid. It is tempting to speculate that these regions, conserved across phylogeny from plants to vertebrates, might be related to the binding site(s) for PAPS, the high energy sulfate donor for the reaction catalysed by these enzymes. That hypothesis could be tested directly by utilizing 35S-PAPS as a photoaffinity ligand [25], followed by sequencing of the protein to determine which amino acids were radioactively labeled. These two sequences also bring this discussion 'full circle', since the PCR primers used to clone human liver DHEA ST cDNA [14] were designed on the basis of these two highly conserved sequences sequences that were known to be present in only three proteins at the time that those primers were synthesized.

8. Conclusions DHEA ST plays an important role in the sulfation of steroid compounds [2-5]. There are large individual differences in DHEA ST activity in human liver [21], and factors responsible for the regulation of this variation have not yet been determined. Cloning of human liver DHEA ST cDNA was required to make it possible to study molecular mechanisms regulating DHEA ST in humans. The human liver DHEA ST cDNAs that have been cloned differ within the open reading frame for two different amino acids, one at position 90 and the other at position 159. Whether those amino acid differences might have implications with regard to individual variations in level of enzyme activity remains to be determined. The presence of 3'-UTRs of three different lengths raises the possibility of differential processing of the 3'-ends of DHEA ST transcripts [14]. The possible functional implications of such differential processing are also unknown. Finally, comparison of the amino acid sequence of DHEA ST with those of other cytosolic ST enzymes for which cDNAs have been cloned demonstrated that these enzymes comprise a gene superfamily, and that DHEA ST is a member of the HSST family of ST proteins. The cloning of cDNA for human liver DHEA ST represents an important step in our advancing understanding of the properties and regulation of ST enzymes in humans.

9. Acknowledgements This work was supported in part by National Institutes of Health grants GM

158

D.M. Otterness, R. Weinshilboum/ Chem.-Biol. Interact. 92 (1994) 145-159

28157 a n d G M 35720. W e t h a n k preparation of this manuscript.

Luanne

Wussow

for her assistance with the

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