Characterization of expressed human phenol-sulfating phenol sulfotransferase: effect of mutating cys70 on activity and thermostability

Chemico-Biological Interactions ELSEVIER

Chemico-Biological Interactions 92 (1994) 57-66

Characterization of expressed human phenol-sulfating phenol sulfotransferase: effect of mutating cys70 on activity and thermostability Charles N. Falany*, Wei Zhuang, Josie L. Falany Department of Pharmacology and Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, AL 35294, USA

Received 8 October 1993; revision received 23 January 1994; accepted 15 February 1994

Abstract

The cDNA for human liver phenol-sulfating phenol sulfotransferase (P-PST) has been cloned and the active enzyme expressed in Cos cells and bacteria. Analysis of the sequence identified two cysteine residues, one of which is highly conserved in the phenol sulfotransferase gene family. Previous studies with the pure human liver enzyme suggested that the conserved cysteine may be involved in binding substrates. Bacterial expression of P-PST with the cysteine converted to a serine indicates that the cysteine is not essential for activity or substrate binding, however, the mutant enzyme is significantly more sensitive to thermal inactivation. Keywords." Phenol sulfotransferase; Expression; Mutagenesis

The phenol sulfotransferases (PSTs) are m e m b e r s o f an i m p o r t a n t family o f enzymes is responsible for the c o n j u g a t i o n o f drugs, xenobiotics and e n d o g e n o u s c o m p o u n d s with a sulfonate moiety in h u m a n liver as well as m a n y other tissues [1,2]. To date two forms o f PST have been identified and purified from h u m a n tissues: the phenol-sulfating form o f PST (P-PST) [3] and the m o n o a m i n e - s u l f a t i n g form o f PST ( M - P S T ) [4]. P - P S T is c a p a b l e o f conjugating phenols, estrogens, aro* Corresponding author, Department of Pharmacology, 101 Volker Hall, University of Alabama at Birmingham, Birmingham, AL 35294, USA. Tel.: (205) 934-9848; Fax: (205) 934-8240. 0009-2797/94/$07.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved SSDI 0009-2797(94)03290-0

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C.N, Falany et al. / Chem.-Biol, Interact. 92 (1994) 57-66

2.0

1.5 P-PST

1.5 1.0 I-

¢"

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i,1.1 --r o r~E

~E

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Fig. 1. DEAE-Sepharose CL-6B chromatography of PFPST, P2-PST and DHEA-ST activities in cytosol from different human livers. Cytosol (300 mg) prepared from different human livers (HL2: 6-year-old female, and HL4:55 yr old male) were applied to DEAE-Sepharose CI-6B columns (2.5 x 10 cm) equilibrated in 10 mM triethanolamine, pH 7.4, 10% glycerol and 5% B,mercaptoethanol. The columns were washed with the same buffer containing 100 mM NaCI and the sulfotransferase activities were eluted with 100-225 mM NaC1 gradients. P-PST activity was assayed with 10 #M PNP as a substrate and DHEA-ST was assayed with 3 gM DHEA as described previously [3,12], The upper graph shows the elution of P2-PST which occurs after the elution of most of the DHEA-ST activity. The lower graph shows the elution of PFPST which occurs before the elution of the majority of the DHEA-ST activity,

C.N. Falany et al. / Chem.-Biol. Interact. 92 (1994) 57-66

59

matic amines, thyroid hormones, N-oxides in compounds such as minoxidil, and tyrosines in small peptides. M-PST is primarily responsible for the sulfation of monoamine neurotransmitters such as dopamine and epinephrine. P-PST is the more abundant form of PST in human liver cytosol and is responsible for most of the sulfate conjugation of drugs and xenobiotics which occurs in the liver. Understanding the structure and activity of the PSTs may therefore provide valuable information concerning the role of sulfation in drug and xenobiotic metabolism. P-PST has been purified to homogeneity from human liver cytosol. The purified enzyme migrates with a molecular mass of approximately 32 000 Da during SDSpolyacrylamide gel electrophoresis (SDS-PAGE) [3,51. In comparison, M-PST in human liver and platelet cytosol migrates with a molecular mass of approximately 34 000 Da [4,5]. Rabbit antihuman platelet M-PST polyclonal antibodies cross-react strongly with P-PST suggesting a significant degree of structural similarity between the proteins; however, because of the size difference in the two proteins they can be distinguished in tissues by immunoblot analysis [3-5]. The observation that the active forms of P-PST and M-PST elute with apparent molecular sizes of 66-72 000 Da during gel exclusion chromatography suggests that the enzymes exist in vivo as dimers [3,41. Although P-PST is relatively abundant in human liver, P-PST activity and immunoreactivity have been identified in many different human tissues including platelets, adrenals, brain and kidney [2,6-9]. Characterization of the enzyme in these different tissues has suggested that the extrahepatic forms of P-PST are very similar, if not identical, to the liver form of the enzyme; however, the PSTs may have specific tissue localizations in other tissues such as in neurons in human brain [9]. P-PST isolated from human adrenal or platelet cytosol has been shown to possess the same molecular mass, kinetic properties and immunologic reactivity as the liver form of the enzyme [3-5,7,9]. Characterization of the PST activities in human liver and platelet cytosol has demonstrated that there are several different forms of both P-PST and M-PST activities based on separation by anion-exchange chromatography, thermal stability and response to certain inhibitors such as dichloronitrophenol (DCNP) [3-5,10,11]. Our laboratory has previously reported that two forms of P-PST can be purified from different human livers by anion-exchange chromatography [3]. The two P-PST activities are termed PI-PST and P2-PST based on their elution profiles with respect to the elution of dehydroepiandrosterone (DHEA)-ST activity (Fig. 1). DHEA-ST is the major form of sterol/bile acid ST present in human liver cytosol [12,13]. These two forms of P-PST activity display many similar characteristics including substrate reactivities, migration with the same molecular mass during SDS-PAGE and reaction with a polyclonal rabbit antihuman PST antibody. In addition to differential elution patterns during anion-exchange chromatography, the two P-PST activities also differ in thermal stability when liver cytosol is prepared in phosphate buffer but not when cytosol is prepared in triethanolamine buffer. In phosphate buffer, P2PST was significantly more sensitive to thermal inactivation than P1-PST [3]. The amino acid sequence or structural differences between these forms of P-PST are unknown; however, analysis of the pharmacogenetic properties of the different forms of thermostable (TS) P-PST in human tissues suggests that they are allelic forms [14].

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C N . Falany et ak / Chem.-BioL Interact. 92 (1994) 57-66

Our laboratory has previously investigated the effects of the thio-specific reagent, N-ethylmaleimide (NEM), on the activity of the two different forms of P-PST activity resolved by anion-exchange chromatography of human liver cytosols to determine whether the forms of P-PST would respond differently to NEM inhibition [3]. Both the P1-PST and P2-PST forms of human liver PST were equally sensitive to inhibition by micromolar concentrations of NEM. Studies were performed to determine whether preincubation of P-PST with substrates protected from inactivation by NEM. Preincubation of P-PST with saturating levels of PAPS prior to the addition of NEM totally protected P-PST activity from inactivation, whereas preincubation with the substrate p-nitrophenol (PNP) resulted in partial protection. These results suggested that a cysteine residue was located at or near the PAPS binding site of the enzyme and that its binding with NEM resulted in the inactivation of the enzyme. To understand further the relationship between the different forms of P-PST activity, we have recently reported the cloning and sequencing of the cDNA for human liver P-PST and expressed the active enzyme in Cos-7 cells [15,16]. To characterize further P-PST in the absence of other mammalian ST activities, the cDNA for P-PST was inserted into the bacterial expression vector pKK 233-2 and expressed in E. coli XL1-Blue cells without alteration of the amino acid sequence of P-PST. Characterization of P-PST expressed in bacteria has demonstrated that it possesses kinetic, immunologic and physical properties very similar to the liver enzyme [17]. Also, use of the bacterial expression system has allowed our laboratory to begin the characterization of the relationship between the structure and kinetic properties of P-PST by site-directed mutagenesis. Sequence analysis of P-PST has shown that the enzyme contains two cysteine residues at positions 70 and 287 which may be sites for the binding of NEM (Fig. 2). Comparison of the amino acid sequence of P-PST with other members of the PST family, which includes the PSTs and estrogen STs, shows that the cysteine residue located at position 70 in P-PST is conserved in all of the sequences (Fig. 3), whereas the cysteine at position 287 is not as well conserved. We therefore investigated the possibility that Cys70 may function in the catalytic activity of P-PST by being involved with the binding of PAPS. The inhibition of P-PST activity by NEM treatment would therefore result in part from the interference with the binding of PAPS. Consistent with this idea is the observation that preincubation of P-PST with PAPS before the addition of NEM prevents the inactivation of the enzyme [3]. To in-

hP-PST MELIQDTSRP TTWVSQILDM APRLLKTHLP EPGTWDSFLE KREIQKILEF SISPFMRKGM

PLEYVKGVPL IYQGGDLEK~ LALLPQTLLD KFMVGEVSYG VGRSLPEETV AGDWKTTFTV

IKYFAEALGP HRAPIFMRVP QKVKWYVAR SWYQHVQEWW DFMVQHTSFK AQNERFDADY

LQSFQARPDD FLEFKAPGIP NAKDVAVSYY ELSRTHPVLY EMKKNPMTNY AKKMAG~SLT

LLISTYPKSG SGMETLKDTP HFYHMAKVHP LFYEDMKENP TTVPQEFMDH FRSEL*

50 i00 150 200 250 295

Fig. 2. The deduced amino acid sequence of h u m a n liver P-PST obtained ~ o m translation of the P-PST-1 cDNA [15]. The cysteine residues are at positions 70 and 287 are in bold type and underlined, The asterix denotes the stop codon in the translation.

61

C.N. Falany et al. ~Chem.-Biol. Interact. 92 (1994) 57-66

84

55 hP-PST rMnx-ST mSTP-I gpEST bEST rEST hDHEA-ST rSTa

SQILDMIYQG SEILDMIYQG SEIMDMIYQG SEVVCMIYAE SEIICMIYNN SEIVDMIYKE AEILCLMHSK IEIVCLIQTK

GDLEKCHRAP GKLEKCGRAP GKLDKCGRAP GDVKKCRQDA GDVEKCKEDV GDVEKCKEDA GDAKWIQSVP GDPKWIQSVT

IFMRVPFLEF IYARVPFLEF VYARIPFLEF IFNRVPFLEC IFNRVPYLEC LFNRIPDLEC IWERSPWVES IWDRSPWIET

Fig. 3. Comparison of the sequence surrounding the CYST0 residue of hP-PST with other sulfotransferases. The amino acid sequence of hP-PST from positions 55-84 is compared to the amino acid sequences of other sulfotransferases. The sequences are rMNX-ST, rat minoxidil-ST [18]; mSTP-I, mouse phenol ST-I [19]; gpEST, quinea pig estrogen-ST [201; bEST~ bovine estrogen-ST [21]; rEST. rat estrogen-ST [22]; hDHEA-ST, human dehydroepiandrosterone-ST [23]; rSTa, rat hydroxysteroid-STa [24]. The sequences were aligned using the Pileup program of the University of Wisconsin Genetics Computer Group.

vestigate this possibility, the Cys70 in P-PST was converted to a serine by oligonucleotide-directed mutagenesis (Fig. 4). The mutated Ser70-P-PST was expressed in E. coli XL1-Blue cells for comparison to normal P-PST possessing the Cys70 r e s i d u e .

Fig. 5 shows that the enzymatic activity of P-PST was essentially unaffected by the conversion of Cys70 to a Ser. Since the XLI-Blue bacterial cytosol contains a PAPS-degrading activity, the Cys70 and Ser70 cytosols were partially purified by DEAE-Sepharose CL-6B chromatography to remove this activity and allow for kinetic analysis. Both P-PST activities eluted from the anion-exchange column in extremely similar patterns under identical conditions indicating that the net charge on the protein was not greatly affected and that the Cys70 residue is not essential for enzymatic activity. The Cys70 residue is also apparently not involved in the formation of an essential cystine linkage between the subunits of P-PST since the enzyme is active when the CysTo has been mutated. Antisense 3' - C T C

Con%rQ1 ~¥$-P-PST TTC ACA GTG GCT CGA

GGG

TAG

- 5'

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GGG

TAG

- 5'

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Fig. 4. Mutagenesis of the cysteine at position 70 of P-PST to a serine. The upper sequence is the antisense nucleotide sequence of the P-PST-I cDNA from positions 272-295 [15]. The lower sequence is the antisense sequence of the oligonucleotide used to incorporate a serine in place of the cysteine at position 70. The A in the control sequence which was replaced with a T in the mutant sequence are both in bold type and underlined. A Sacl site is located at positions 285-290. The mutant oligonucleotide and the SP6 sequencing primer were used to amplify the 5-end of P-PST-I by PCR using pKK-233-2-P-PST-I as a template [17]. The PCR products were digested with Ncol and SacI and ligated into pKK-233-2-P-PST-I digested with Ncol and SacI. E. coli XLI-Blue cells were transformed and mutant P-PST identified by sequence analysis.

62

C.N.

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al. / Chem.-BioL Interact. 92 (1994) 57-66

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Fraction Fig. 5. DEAE-Sepharose CL-6B chromatography of control and mutant P-PST activities. XLI-Blue cells containing p K K 233-2 (Pharmacia) with either the control (Cys) or mutant (Ser) P-PST cDNA were grown to log phase (O.D.600 = 0.5) in Luria broth with ampicillin (200 ~g/ml), then induced with 0.3 mM IPTG and allowed to incubate for 2 h. The cells were pelleted at 3700 × g and resuspended in bacterial lysis buffer (10 mM TEA, pH 7.5, 10% glycerol, 1.5 mM DTT, 10/~g/ml phenolmethylsulfonylfluoride), and sonicated 4 x with 10 s bursts and 30 s cooling between each burst. A final centrifugation at 100 000 x g for 1 h was performed and the supernatant fractions from control and mutant cells were applied to separate identical DEAE-Sepharose CL-6B columns (1 × 10 cm). The columns were washed with 10 mM triethanolamine, pH 7.4 and 10% glycerol containing 100 m M NaCI. To elute the columns as similarly as possible, the P-PST activities were eluted with a NaCI gradient of 100 to 225 mM and a single gradient maker was used. The flow from the gradient maker was split to the two DEAE-Sepharose columns and identical size fractions (2 ml) were collected at a constant flow rate. The elution of the control (1"1) and mutant (@) P-PST activities are plotted on the same graph. Enzyme activities were assayed with minoxidil as a substrate as described previously [25].

Table 1 Kinetic analysis of expressed control and mutant P-PST Enzyme

Cys70-P-PST Ser70-P-PST

Substrates PAPS (txM)

PNP (/~M)

Mnx (#M)

1.6 ± 0.8 1.6, 1.7

0.6 ± 0.1 0.6 ± 0.1

46, 37 86, 89

The Km values for PAPS, PNP and Mnx were determined using DEAE-purified Cys70-P-PST and Ser70P-PST. The K m values represent the values of duplicate determinations or the mean ± S.D. (n = 3). Kinetic values were computed using the EnzymeKinetics programs (Trinity Software) .

C N . Falany et al. ~Chem.-Biol. Interact. 92 (1994) 57-66

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NEM (tiM) Fig. 6. Effect of N-ethylmaleimide (NEM) on the activity of the Cys70 and SerT0-P-PST activities. The DEAE-purified Cys70-P-PST (dots) and SerT0-P-PST(stripes) activities were preincubated at 37°C with 20 #M or 50 #M NEM for 10 min and the reaction terminated by the addition of 1 mM dithiothreitol. Control reactions were preincubated without NEM. Minoxidil sulfation activity was then assayed with both enzymes as described previously [25]. Reactions were run in quadruplicate and expressed as percent control reaction rates ± S.D.

To investigate whether the Ser70 mutant demonstrated kinetic properties different from those of the Cys70 enzyme, the K m values for PNP and minoxidil, P-PST specific substrates, and PAPS were determined with both forms of the enzyme. The K m values for PAPS obtained with both forms of the enzyme were very similar (Table 1). The Km for minoxidil was approximately two-fold higher using the SerTo-P-PST as compared to the Cys70 form of the enzyme. To investigate further whether the sulfate acceptor substrate binding is affected in the Ser70 mutant, the Km for PNP was also determined using both enzymes. The Km for PNP was approximately equal with both the SerTo enzyme and the control Cys70 enzyme. The lack of an obvious effect on the kinetic properties of P-PST in the Ser70 enzyme indicates that the CySTo residue probably does not have an important function in the reaction mechanism. The Cys residue, however, may still be located in or near the active site of PPST which would account for the inhibition by NEM and the protection from inhibition by PAPS observed previously [3]. Both the Cys70 and SerTo forms of P-PST were tested to determine whether the inhibition of activity by NEM was related to the presence of the Cys residue. The control CySToenzyme was more sensitive to loss of activity by increasing concentrations of NEM in the micomolar range (0-1000 #M) than the Ser70 form of P-PST. Fig. 6 shows that the addition of 20 and 50 #M NEM was significantly more effective at inhibiting the activity of CysTo-P-PST than Ser70-P-PST. This result indicates that

64

C.N. Falany et al. ~Chem.-Biol. Interact. 92 (1994) 57-66

125-

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Min Fig. 7. Temperature sensitivity of Cys70and Ser70-P-PST activities. The DEAE-purified Cys70-P-PST (dots) and Ser7o-P-PST(stripes) activities were incubated at 45°C for 4 or 8 min then placed on ice. Samples for control activities remained on ice. Minoxidil sulfation activity was then assayed with both enzymesas described previously [25]. Reactions were run in quadruplicate and expressed as percent control reaction rates + S.D.

the Cys70 residue is apparently involved in the binding and inactivation of P-PST by NEM since its removal decreased the sensitivity of enzyme activity to inhibition. To investigate whether the conversion of Cys70 to a Ser altered the structural properties of P-PST, the effects of this change on the thermostability properties of the enzyme were investigated. Thermostability has been used to distinguish between different types of PST activity in human liver and platelets. In human liver multiple forms of P-PST have been isolated which have differential responses to thermal inactivation and these have been proposed to be allelic forms of the enzyme (3,1 1,14). When the partially purified preparations of Cys70 and Ser70-P-PST were heated to 45°C, significant differences were observed between the loss of Sery0-P-PST activity as compared to CysT0-P-PST activity. When heated, the Serv0-P-PST lost activity more rapidly than the control Cysv0-P-PST (Fig. 7). The increased sensitivity of SerT0-P-PST to heat inactivation suggests that the presence of the Set70 alters the stability of the enzyme even though it does not affect its enzymatic activity. These data indicate that single amino acid changes may greatly alter the thermal stability of the P-PSTs and probably also the M-PSTs. This observation supports the conclusion that the different forms of P-PST isolated from human liver which display differences in thermal stability are allelic forms of the same enzyme (3,1 1,14). In summary, the Cys residue located at position 70 in the sequence of human PPST does not appear to be involved in the catalytic activity of the enzyme even though it is conserved in many members of the PST gene family. Mutation of the Cys codon to a Ser codon did not greatly alter the enzymatic properties of the

C N . Falany et al. ~Chem.-Biol. Interact. 92 (1994) 57-66

65

enzyme when expressed in bacteria. The Cys residue did appear to be involved in the inactivation of the enzyme by NEM suggesting that the Cys residue may be located in or near to the active site. Also, the Cys residue does not appear to be involved in an essential intermolecular or intramolecular cystine linkage since the Serv0-P-PST is enzymatically active. The conversion of the Cys to a Ser residue did have an effect on the structural properties of the enzyme in that it increased the sensitivity of enzyme activity to heat inactivation. The SerT0-P-PST was significantly more sensitive to thermal inactivation than the control enzyme. This result indicates that at least some single amino acid changes in the sequence of P-PST may be detected by thermal inactivation studies. These data support the previous reports that the differential sensitivity of different forms of P-PST isolated from human tissues may be a sensitive indicator of the expression of allelic forms of these enzymes (10,11,14). Whether the half-life of the active enzymes in human tissues in vivo is related to differences in their sensitivity to heat inactivation is not known.

Acknowledgements This research was supported by USPHS grant GM38953 to CNF.

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14 R. Weinshilboum, Sulfotransferase pharmacogenetics, Pharmacol. Ther., 45 (1990) 93107. 15 T.W. Wilborn, K.A. Comer, T.P. Dooley, I.M. Reardon, R.L. Heinrikson and C.N. Falany, Sequence analysis and expression of the cDNA for the phenol-sulfating form of human liver phenol sulfotransferase, Mol. Pharmacol., 43 (1993) 70-77. 16 J.L. Falany, L. Lawing and C.N. Falany, Identification and characterization of cytosolic sulfotransferase activities in MCF-7 human breast carcinoma cells, J. Steroid Biochem. Mol. Biol., 46 (1993) 481-487. 17 C.N. Falany, J. Wheeler, T.S. Oh and J.L. Falany, Steroid sulfation by expressed human cytosolic sulfotransferases, J. Steroid Biochem. Mol. Biol., 48 (1994) 369-375. 18 S.J. Hirshey, T.P. Dooley, I.M. Reardon, R.L. Heinrikson and C.N. Falany, Sequence analysis, in vitro translation and expression of the cDNA for rat liver minoxidil sulfotransferase, Mol. Pharmacol., 40 (1992) 1027-1032. 19 A.N. Kong, M. Ma, D. Tao and L. Yang, Molecular cloning of cDNA encoding the phenol/aryl form of sulfotransferase (mSTp 1) from mouse liver, Biochim. Biophys. Acta, 1171 (1993) 315-318. 20 K. Komatsu, T. Oeda and C.A. Strott, Cloning and sequence analysis of the 5'-flanking region of the estrogen sulfotransferase gene: steroid response elements and cell-specific nuclear DNA-binding proteins, Biochem. Biophys. Res. Commun., 194 (1993) 1297-1304. 21 A.R. Nash, W.K. Glenn, S.S. Moore, J. Kerr, A.R. Thompson and E.O.P. Thompson, Oestrogen sulfotransferase: molecular cloning and sequencing of cDNA for the bovine placental enzyme, Aust. J. Biol. Sci., 41 (1988) 507-516. 22 W.F. Demyan, C.S. Song, D.S. Kim, S. Her, W. Gallwitz, T.R. Rao, M. Slomczynska, B. Chatterjee and A.K. Roy, Estrogen sulfotransferase of the rat liver: complementary DNA cloning age- and sexspecific regulation of messenger RNA, Mol. Endocrinol., 6 (1992) 589-597. 23 K.A. Comer, J.L. Falany and C.N. Falany, Cloning and expression of human liver dehydroepiandrosterone, Biochem. J., 289 (1993) 233-240. 24 K. Ogura, J. Kajita, H. Narihata, T. Watabe, S. Ozawa, K. Nagata, Y. Yamazoe and R.Kato, Cloning and sequence analysis of a rat liver cDNA encoding hydroxysteroid sulfotransferase, Biochem. Biophys. Res. Commun., 165 (1989) 168-174. 25 C.N. Falany and E.A. Kerl, Sulfation ofminoxidil by human liver phenol sulfotransferase, Biochem. Pharmacol., 40 (1990) 1027-1032.