Identification of novel hydroxysteroid-sulfating cytosolic SULTs, SULT2 ST2 and SULT2 ST3, from zebrafish: Cloning, expression, characterization, and developmental expression

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Identi

Wcation of novel hydroxysteroid-sulfating cytosolic

SULTs, SULT2 ST2 and SULT2 ST3, from zebra

Wsh:

Cloning, expression, characterization, and developmental expression

Shin Yasuda

a

, Ming-Yih Liu

b

, Yuh-Shyong Yang

c

, Rhodora Snow

d

,

Saki Takahashi

a

, Ming-Cheh Liu

a,¤

a _{Biomedical Research Center, The University of Texas Health Center, Tyler, TX 75708, USA} b _{National Synchrotron Radiation Research Center, Hsinchu, Taiwan, ROC}

c _{Department of Biological Science and Technology, College of Biological Science and Technology, National Chiao Tung University, Hsinchu, Taiwan, ROC} d _{Department of Chemistry, Jarvis Christian College, Hawkins, TX 75765, USA}

Received 2 June 2006, and in revised form 18 August 2006 Available online 25 September 2006

Abstract

By searching the expressed sequence tag database, two zebra

Wsh cDNAs encoding putative cytosolic sulfotransferases (SULTs) were

identi

Wed. Sequence analysis indicated that these two zebraWsh SULTs belong to the cytosolic SULT2 gene family. The recombinant form

of these two novel zebra

Wsh SULTs, designated SULT2 ST2 and SULT2 ST3, were expressed using the pGEX-2TK glutathione

S-trans-ferase (GST) gene fusion system and puri

Wed from transformed BL21 (DE3) Escherichia coli cells. PuriWed GST-fusion protein form of

SULT2 ST2 and SULT2 ST3 exhibited strong sulfating activities toward dehydroepiandrosterone (DHEA) and corticosterone,

respec-tively, among various endogenous compounds tested as substrates. Both enzymes displayed pH optima at »6.5. Kinetic constants of the

two enzymes, as well as the GST-fusion protein form of the previously identi

Wed SULT2 ST1, with DHEA and corticosterone as

sub-strates were determined. Developmental stage-dependent expression experiments revealed distinct patterns of expression of SULT2 ST2

and SULT2 ST3, as well as the previously identi

Wed SULT2 ST1, during embryonic development and throughout the larval stage onto

maturity.

© 2006 Elsevier Inc. All rights reserved.

Keywords: Sulfotransferase; SULT; Sulfation; Dehydroepiandrosterone; Corticosterone; Molecular cloning; Developmental expression; ZebraWsh

The cytosolic sulfotransferases (SULTs) in mammals

and other vertebrates constitute a group of enzymes that

catalyze the transfer of a sulfonate group from the active

sulfate, 3

⬘-phosphoadenosine 5⬘-phosphosulfate (PAPS)

[1]

, to substrate compounds containing hydroxyl or amino

groups

[2–5]

. Such sulfation reactions are generally thought

to serve for detoxi

Wcation of xenobiotics, as well as

bio-transformation of endogenous compounds such as steroid

and thyroid hormones, catecholamines, cholesterol, and

bile acids

[2–5]

. Based on amino acid sequence homology,

all cytosolic SULTs from vertebrate animals are proposed

to constitute a gene superfamily, and distinct gene families

have been further categorized

[6]

. Two major gene families

among them are the phenol SULT

1

family (designated

SULT1) and hydroxysteroid SULT family (designated

SULT2)

[6–8]

. The hydroxysteroid SULT (SULT2) family

presently comprises two sub-families,

dehydroepiandroster-one (DHEA) SULT (SULT2A) and cholesterol/pregnenoldehydroepiandroster-one

* _{Corresponding author. Fax: +1 903 877 2863.}

E-mail address: [email protected] (M.-C. Liu).

1 _{Abbreviations used: SULT, sulfotransferase; PAPS, 3} ⬘-phosphoadeno-sine 5⬘ phosphosulfate; RT-PCR, reverse transcription-polymerase chain reaction; 3⬘-RACE, 3⬘-rapid ampliWcation of cDNA ends; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis.

SULT (SULT2B). Members of the hydroxysteroid SULT

family are involved in the sulfation of both endogenous and

exogenous steroids, which has been suggested to be an

important mechanism for the homeostasis, bioactivation/

inactivation, or transport of these compounds in vivo

[9]

.

How the hydroxysteroid-sulfating cytosolic SULT enzymes

function to regulate the activity of steroids, as well as their

metabolism and homeostasis, however, remains to be fully

elucidated. Moreover, only fragmentary information is

available concerning the cell type/tissue/organ-speci

Wc

expression of these hydroxysteroid-sulfating SULTs, and

very little is known with regard to the ontogeny of these

enzymes.

ZebraWsh has in recent years emerged as a popular

ani-mal model for a wide range of studies

[10,11]

. Its

advanta-ges, compared with mouse, rat, or other vertebrate animal

models, include the small size, availability of a relatively

large number of eggs, rapid development externally of

vir-tually transparent embryos, and short generation time.

These unique characteristics make the zebraWsh an

excel-lent model for a systematic investigation of the ontogeny,

cell type/tissue/organ-speciWc expression, and physiological

involvement of individual cytosolic SULTs. A prerequisite

for using the zebra

Wsh in these studies, however, is the

iden-ti

Wcation of the various cytosolic SULTs and their

func-tional characterization. We have recently embarked on the

molecular cloning of zebra

Wsh cytosolic SULTs

[12–18]

.

Sequence analysis via BLAST search revealed that the

zebra

Wsh cytosolic SULTs we have cloned

[12–18]

display

sequence homology to mammalian cytosolic SULTs. Of the

eight zebra

Wsh cytosolic SULTs that have been cloned, six

fall within the SULT1 gene family

[12,13,16–18]

, one

belongs to the SULT2 gene family

[14]

, and one appears to

be independent from all known SULT gene families

[15]

.

The zebra

Wsh SULT2 enzyme (now designated SULT2

ST1) previously cloned and expressed displayed sulfating

activities toward several steroids including DHEA,

preg-nenolone, allopregnanolone, 4-androstene 3,17-dione, and

17-hydroxypregnenolone

[14]

. Whether additional SULT2

enzymes, dedicated to the sulfation of other

hydroxyster-oids, exist in zebraWsh remained an open question.

We report here the identiWcation of two novel zebraWsh

cytosolic SULT2 enzymes, designated SULT2 ST2 and

SULT2 ST3. Their enzymatic activities toward a variety of

endogenous compounds and xenobiotics were examined.

Kinetic parameters of the two enzymes in catalyzing the

sulfation of DHEA and corticosterone were determined.

Moreover, their developmental stage-dependent expression

during embryogenesis onto maturity was investigated.

Materials and methods

Materials

DHEA, 17-estradiol, estrone, acetaminophen, bisphenol A, butylated hydroxyanisole, butylated hydroxytoluene, caVeic acid, catechin, chloro-genic acid, daidzein, diethylstilbestrol, L-3,4-dihydroxyphenylalanine (L-Dopa), D-Dopa, dopamine, epicatechin, epigallocatechin gallate, 17

-eth-ynylestradiol, gallic acid, genistein, hydrocortisone, minoxidil, myricetin, -naphthylamine, -naphthol, p-nitrophenol, n-nonylphenol, n-octylphe-nol, n-propyl gallate, quercetin, L-thyronine, 3,3⬘,5-triiodo-L-thyronine, aprotinin, thrombin, adenosine 5⬘-triphosphate (ATP), sodium dodecyl sulfate (SDS), sodium acetate, 2-morpholinoethanesulfonic acid (MES), 3-(N-morpholino)propanesulfonic acid (MOPS),

N-2-hydroxylpiperazine-N⬘-2-ethanesulfonic acid (HEPES), 3-[N-tris-(hydroxymethyl)methyla-mino]-propanesulfonic acid (TAPS), 2-(cyclohexylamino)ethanesulfonic acid (CHES), 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS), Tri-zma base, dithiothreitol (DTT), and isopropyl -D-thiogalactopyranoside (IPTG) were products of Sigma Chemical Company. TRI Reagent was from Molecular Research Center, Inc. Unfertilized zebraWsh eggs, embryos and larvae at diVerent developmental stages were prepared by ScientiWc Hatcheries. Total RNA from a 3-month-old zebraWsh was pre-pared as described previously [13]. Taq DNA polymerase was a product of Promega Corporation. Takara Ex Taq DNA polymerase was purchased from PanVera Corporation. T₄ DNA ligase and BamHI restriction endo-nuclease were from New England Biolabs. Oligonucleotide primers were synthesized by MWG Biotech. pSTBlue-1 AccepTor Vector Kit and BL21 (DE3) competent cells were from Novagen. Prestained protein molecular weight standard were from Life Technologies. pGEX-2TK glutathione S-transferase (GST) gene fusion vector, GEX-5⬘ and GEX-3⬘ sequencing primers, and glutathione–Sepharose 4B were products of Amersham Bio-sciences. GST-fusion protein form of the previously identiWed zebraWsh SULT2 ST1 [14] was expressed and puriWed based on the same procedure

described below for SULT2 ST2 and ST3. Recombinant human bifunc-tional ATP sulfurylase/adenosine 5⬘-phosphosulfate kinase was prepared as described previously [19]. Cellulose thin-layer chromatography (TLC) plates were products of EM Science. Carrier-free sodium [35_S]sulfate, Eco-lume scintillation cocktail, corticosterone, pregnenolone, 4-androsterone-3,17-dione, hydrocortisone, progesterone, 17-hydroxyprogesterone, and 17-hydroxypregnenolone were from ICN Biomedicals. Allopregnanolone was from Calbiochem. All other reagents were of the highest grades commercially available.

Cloning, bacterial expression, and puri

Wcation of recombinant

zebra

Wsh cytosolic SULT2 ST2 and ST3

By searching the expressed sequence tag database, two zebraWsh cDNAs (GenBank Accession Nos. CD014163 (SULT2 ST2) and BQ132464 (SULT2 ST3)) encoding putative cytosolic SULTs were identi-Wed. These two cDNAs, obtained from Open Biosystems, were subjected to nucleotide sequencing [20]. To subclone the two cDNAs for expression, sense and antisense oligonucleotide primers designed based on 5⬘- and 3⬘-coding regions of the nucleotide sequences determined were synthesized with BamHI restriction site incorporated at the end (see Table 1). Using these primer sets, PCRs were carried out under the action of EX Taq DNA polymerase, with the two commercially obtained cDNAs as templates. AmpliWcation conditions were 2 min at 94 °C and 20 cycles of 94 °C for 35 s, 60 °C for 40 s, 72 °C for 1 min. The Wnal reaction mixtures were applied onto a 1.2% agarose gel, separated by electrophoresis, and visual-ized by ethidium bromide staining. The PCR product bands detected were excised from the gel, and the DNAs therein were isolated by spin Wltration. PuriWed PCR products were subjected to BamHI restriction and cloned into BamHI-restricted pGEX-2TK vector, and veriWed for authenticity by nucleotide sequencing [20]. To express the recombinant zebraWsh SULT2

ST2 and ST3, competent Escherichia coli BL21 (DE3) cells, transformed with pGEX-2TK harboring the cDNA encoding SULT2 ST2 or ST3, were grown in 1 L LB medium supplemented with 60g/ml ampicillin. After the cell density reached 0.6 OD600 nm, IPTG at a Wnal concentration of 0.1 mM was added to induce the production of recombinant zebraWsh SULT. After an overnight induction at room temperature, the cells were collected by centrifugation and homogenized in 25 ml ice-cold lysis buVer (10 mM Tris–HCl, pH 8.0, 150 mM NaCl, and 1 mM EDTA) using an Aminco French Press. Twenty microliters of 10 mg/ml aprotinin (a protease inhibi-tor) was added to the crude homogenate. The crude homogenate was sub-jected to centrifugation at 10,000g for 15 min at 4 °C. The supernatant collected was fractionated using 2.5 ml of glutathione–Sepharose, and the

bound GST-fusion protein was either eluted by an elution buVer (50 mM Tris–HCl, pH 8.0, plus 10 mM reduced glutathione) at 4 °C or treated with 3 ml of a thrombin digestion buVer (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, and 2.5 mM CaCl2) containing 5 U/ml bovine thrombin at room temperature. Following a 10 to 15-min incubation with constant agitation, the preparation was subjected to centrifugation. The recombinant zebraWsh SULT (in GST-fusion protein form or free (thrombin-cleaved) form) present in the supernatant collected was analyzed for purity by SDS–polyacrylamide gel electrophoresis (SDS–PAGE) and subjected to enzymatic characterization.

Enzymatic assay

The sulfating activity of the recombinant zebraWsh cytosolic SULTs was assayed using radioactive PAP[35_{S] as the sulfate donor. The standard} assay mixture, with a Wnal volume of 25 l, contained 50 mM Mops buVer at pH 7.0 (for SULT2 ST2 and ST3) or Ches at pH 9.5 (for SULT2 ST1), 14M PAP[35S] (15 Ci/mmol), 1 mM DTT, and 50M substrate. Controls with DMSO or water, in place of substrate, were also prepared. The reac-tion was started by the addireac-tion of the enzyme, allowed to proceed for 5 min at 28 °C, and terminated by heating at 100 °C for 2 min. The precipi-tates formed were cleared by centrifugation, and the supernatant was sub-jected to the analysis of [35_{S]sulfated product using the previously} developed TLC procedure [21], with n-butanol/isopropanol/88% formic acid/water (3:1:1:1; by volume) as the solvent system. To examine the pH-dependence of the sulfation of DHEA or corticosterone, diVerent buVers (50 mM sodium acetate at 4.5, 5.0, or 5.5; Mes at 5.5, 6.0, or 6.5; Mops at 6.5, 7.0, or 7.5; Hepes at 7.0, 7.5 or 8.0; Taps at 8.0, 8.5 or 9.0; Ches at 9.0, 9.5, or 10.0; and Caps at 10.0, 10.5, 11.0 or 11.5), instead of 50 mM Mops (pH 7.0), were used in the reactions. For the kinetic studies on the sulfation of DHEA and corticosterone, varying concentrations of these substrate compounds and 50 mM Mops buVer at pH 7.0 (for SULT2 ST2 and ST3) or Ches at pH 9.5 (for SULT2 ST1) were used.

Analysis of the developmental stage-dependent expression of the

zebra

Wsh cytosolic SULT2 STs

RT-PCR was employed to investigate the developmental stage-depen-dent expression of the zebraWsh cytosolic SULT2 ST2 and ST3, as well as the previously identiWed SULT2 ST1. Total RNAs from zebraWsh embryos, larvae, and adult (male or female) Wsh at diVerent developmental stages were isolated using TRI Reagent, based on manufacturer’s

instruc-tions. Aliquots containing 5g each of the total RNA preparations were used for the synthesis of the Wrst-strand cDNA using the First-Strand cDNA Synthesis Kit (Amersham Biosciences). One microliter aliquots of the 33l Wrst-strand cDNA solutions prepared were used as the template for the subsequent PCR ampliWcation. PCRs were carried out in 25 l reaction mixtures using EX Taq DNA polymerase, in conjunction with gene-speciWc sense and antisense oligonucleotide primers (see Table 1). AmpliWcation conditions were 2 min at 94 °C followed by 40 cycles of 30 s at 94 °C, 40 s at 60 °C, and 1 min at 72 °C. The Wnal reaction mixtures were applied onto a 1.2% agarose gel, separated by electrophoresis, and visual-ized by ethidium bromide staining. As a control, PCR ampliWcation of the sequence encoding zebraWsh -actin was concomitantly performed using the above-mentioned Wrst-strand cDNAs as templates, in conjunction with gene-speciWc sense and antisense oligonucleotide primers (Table 1) designed based on reported zebraWsh -actin nucleotide sequence (GenBank Accession No. AF057040).

Miscellaneous methods

PAP[35_{S] was synthesized from ATP and carrier-free [}35_{S]sulfate using} the bifunctional human ATP sulfurylase/APS kinase and its purity deter-mined as previously described [22,23]. The PAP[35_{S] synthesized was} adjusted to the required concentration and speciWc activity by the addition of cold PAPS. SDS–PAGE was performed on 12% polyacrylamide gels using the method of Laemmli [24]. Protein determination was based on the method of Bradford [25] with bovine serum albumin as the standard.

Results and discussion

In vertebrates, the sulfation of steroids by the cytosolic

hydroxysteroid SULTs, belonging to the SULT2 gene

fam-ily

[6]

, is recognized as an important regulatory pathway for

the homeostasis as well as bioactivation/inactivation of

these compounds

[9]

. As a part of an e

Vort to develop a

zebra

Wsh model for investigating in greater detail the

func-tional involvement of the hydroxysteroid SULTs, we had

previously cloned, expressed and characterized a zebra

Wsh

DHEA-sulfating SULT2 (now designated SULT2 ST1)

[14]

. In view of the diversity of SULT2 enzymes in other

vertebrates including human and mouse

[6,26]

, we

specu-Table 1

Oligonucleotide primers used for the cDNA cloning of zebraWsh SULT2 ST2 and ST3 and for the RT-PCR analysis of the developmental stage-dependent expression of the SULT2 STs

a _{Recognition sites of BamHI restriction endonuclease in the oligonucleotides are underlined. Initiation and termination codons for translation are in} bold type.

b _{The sense and antisense oligonucleotide primer sets listed were veriWed by BLAST Search to be speciWc for the target zebraWsh SULT2 or -actin} nucleotide sequence.

Target sequence Sense and antisense oligonucleotide primers used

I. For cDNA cloninga:

SULT2 ST2 Sense: 5⬘-CGCGGATCCATGACTGAATCGGAGCTGTA-3⬘

Antisense: 5⬘-CGCGGATCCTCAGTCCCATGGAAACTTGAA-3⬘

SULT2 ST3 Sense: 5⬘-CGCGGATCCATGGAGGTCAGCGAATTCAAT-3⬘

Antisense: 5⬘-CGCGGATCCTTATTCCTCATCCCAGGGGAATTT-3⬘

II. For RT-PCR analysisb_:

SULT2 ST1 Sense: 5⬘-ACAAAACCGCAGTCGCTGCACAGAACCGGT-3⬘

Antisense: 5⬘-TGATACAGATAAACATCACTATTACTGTGG-3⬘

SULT2 ST2 Sense: 5⬘-TGCAGCTGCTCTCTAGATTAATTCTTCATT-3⬘

Antisense: 5⬘-TTTTTGGCTGTGCACCAAATGTTTATTAGAA-3⬘

SULT2 ST3 Sense: 5⬘-GAACTCTTTGCACAAAACTACCTAGTTTTCCCA-3⬘

Antisense: 5⬘-GGACAGACTGAAGAACTCCACCTTAACCAA-3⬘

-Actin Sense: 5⬘-ATGGATGAGGAAATCGCTGCCCTGGTC-3⬘

lated that additional SULT2 enzymes may be present in

zebra

Wsh. Here, we report the identiWcation,

characteriza-tion and ontogeny of two novel zebra

Wsh cytosolic SULT2

enzmes, designated SULT2 ST2 and ST3.

Molecular cloning of the zebra

Wsh cytosolic SULT2 ST2 and

ST3

By searching the expressed sequence tag database, two

zebra

Wsh cDNAs (GenBank Accession No. CD014163

(SULT2 ST2) and BQ132464 (SULT2 ST3)) encoding

putative cytosolic SULTs were identi

Wed. These two

cDNAs, obtained commercially, were subjected to

nucleo-tide sequencing

[20]

. The nucleotide sequences obtained

were submitted to the GenBank database under the

Acces-sion No. DQ640387 for SULT2 ST2 and DQ640388 for

SULT2 ST3.

Fig. 1

shows the alignment of the deduced

amino acid sequence of the two newly cloned zebraWsh

SULT2 STs. The open reading frame of the SULT2 ST2

encompasses 864 nucleotides and codes for a 287-amino

acid polypeptide, and that of the SULT2 ST3 contains 867

nucleotides and encodes a 288-amino acid polypeptide.

Similar to other cytosolic SULTs, these two new zebraWsh

SULTs contains sequences resembling the so-called

“signa-ture sequences” (YPKSGTxW in the N-terminal region

and RKGxxGDWKNxFT in the C-terminal region; as

underlined) characteristic of SULT enzymes

[8]

. Of these

two sequences, YPKSGTxW has been demonstrated by

X-ray crystallography to be responsible for binding to the

5

⬘-phosphosulfate group of PAPS, a co-substrate for

SULT-catalyzed sulfation reactions

[27]

, and thus

desig-nated the “5

⬘-phosphosulfate binding (5⬘-PSB) motif”

[28]

.

The cloned zebra

Wsh SULT also contains the

“3⬘-phos-phate binding (3

⬘-PB) motif” (amino acid residues 187–197;

as underlined) responsible for the binding to the 3

⬘-phos-phate group of PAPS

[28]

. Sequence analysis based on a

BLAST Search revealed that the deduced amino acid

sequence of the zebra

Wsh SULT2 ST2 displays 43 and 40%

identity to human SULT2B1a and SULT2A1, and lower %

identity to other known SULTs. The deduced amino acid

sequence of the zebra

Wsh SULT2 ST3 displays 49% identity

to human SULT2B1a and SULT2B1b, and lower %

iden-tity to other known SULTs. It is generally accepted that

members of the same SULT gene family share at least 45%

amino acid sequence identity, and members of subfamilies

further divided in each SULT gene family are greater than

60% identical in amino acid sequence

[6–8]

. Based on these

criteria, both the zebra

Wsh SULT2 STs appear to belong to

the SULT2 gene family, and are designated the zebra

Wsh

SULT2 ST2 and ST3 in accordance with the nomenclature

used in ZFIN database (cf. the dendrogram shown in

Fig. 2

). It is interesting to note that the newly cloned

zebraWsh SULT2 ST2 and ST3 displays, respectively, 87.5

and 52.6% amino acid sequence identity to the previously

identiWed zebraWsh SULT2 ST1

[14]

.

Expression, puriWcation, and characterization of recombinant

zebraWsh cytosolic SULT2 ST2 and ST3

The coding region of the zebraWsh SULT2 ST2 or ST3

cDNA was subcloned into pGEX-2TK, a prokaryotic

expression vector, for the expression of recombinant

enzyme in E. coli. As shown in

Fig. 3

, the GST-fusion

pro-tein form of the recombinant zebraWsh SULT2 ST2 (lane 1)

and ST3 (lane 3), puriWed from the E. coli extract, migrated

at »58 kDa position upon SDS–PAGE. Upon thrombin

digestion, the free form of SULT ST2 (lane 2) and ST3

(lane 4) migrated as »33 kDa proteins (

Fig. 3

). This is in

agreement with the molecular weight (32,000–35,000)

gen-erally found for cytosolic SULTs

[1–3]

. It was subsequently

noted that the thrombin-digested zebra

Wsh SULT2 ST2

and ST3 exhibited lower and unstable sulfating activity in

comparison with the GST-fusion protein form of these two

enzymes (data not shown). The GST-fusion protein form of

both zebra

Wsh SULT2 STs, therefore, was used for the

enzymatic characterization. (The speci

Wc activities

deter-Fig. 1. Alignment of the deduced amino acid sequences of the zebraWsh SULT2 ST1, ST2, and ST3. Residues conserved between the two enzymes are boxed. Two “signature sequences,” respectively, located in the N- and C-terminal regions, as well as a conserved sequence in the middle region are under-lined. The percent numbers in parentheses refer to the percent amino acid identities to the SULT2 ST1 sequence.

mined in the following studies were corrected for the

molec-ular mass of the GST moiety in the fusion protein form of

the enzymes.) A pilot experiment Wrst revealed that the

SULT2 ST2 and ST3 exhibited strong activity toward

DHEA and corticosterone, respectively. A pH-dependence

experiment subsequently performed showed that the

SULT2 ST2 with DHEA as substrate (

Fig. 4

A) and ST3

with corticosterone as substrate (

Fig. 4

B) both exhibited

optimum activity at pH 6.5. It is interesting to note that the

previously identiWed SULT2 ST1, while sharing a high

degree of sequence homology (87.5% amino acid identity)

with the SULT2 ST2, displayed a pH optimum at 9.5

[14]

.

Representative endogenous and xenobiotic compounds

were tested as substrates for these enzymes, as well as the

GST-fusion protein form of the previously identiWed

SULT2 ST1. The activity data obtained are compiled in

Table 2

. Among the compounds we tested, the zebraWsh

SULT2 ST2 and ST3 displayed strongest sulfating activities

toward DHEA (at 579 pmol/min/mg enzyme) and

cortico-sterone (at 510 pmol/min/mg enzyme), respectively. In

addi-tion, SULT2 ST2 also showed sulfating activities toward

pregnenolone, 17

-estradiol and estrone; and SULT2 ST3

displayed activities toward pregnenolone, 17

-estradiol,

DHEA and allopregnanolone. Neither enzyme, however,

exhibited detectable activities toward other endogenous

compounds including

L

-Dopa, dopamine,

4-androstene-3,17-dione, hydrocortisone, 17

-hydroxypregnenolone,

17

-hydroxyprogesterone, progesterone,

L

-thyroxine, and

L

-triiodothyronine, and exogenous compounds including

acetaminophen, bisphenol A, n-nonylphenol,

n-octylphe-nol, p-nitrophen-octylphe-nol, -naphthol, -naphthylamine, butylated

hydroxyanisole, caVeic acid, catechin, epicatechin, gallic

acid, chlorogenic acid, daidzein, genistein, myricetin,

quer-cetin, n-propyl gallate, and minoxidil. That both SULT2

ST2 and ST3 exhibited sulfating activities toward

exclu-sively hydroxysteroids is in line with these enzymes being

Fig. 2. ClassiWcation of the zebraWsh SULT2 ST2 and ST3 on the basis of their amino acid sequences. The dendrogram shows the degree of amino acid sequence homology among cytosolic SULTs. For references for individual SULTs, see the review by Blanchard et al. [6]. h, human; m, mouse; and zf, zebraWsh. The dendrogram was generated based on Greedy algorithm [42,43].

mSULT2B1 hSULT2B1a mSULT2A1 mSULT2A2 mSULT1C1 hSULT2A1 mSULT1E1 hSULT1A3 hSULT1A2 hSULT1A1 hSULT1C3 hSULT1C2 hSULT1E1 mSULT1B1 hSULT1B1 mSULT1D1 mSULT1A1

SULT1

SULT2

zfSULT2 ST3 zfSULT2 ST2 zfSULT1 ST4 zfSULT1 ST3 zfSULT1 ST2 zfSULT1 ST1 zfSULT1 ST6 zfSULT1 ST5 zfSULT2 ST1

Fig. 3. SDS gel electrophoretic pattern of the puriWed recombinant zebraWsh SULT2 ST2 and ST3. PuriWed zebraWsh SULT samples were subjected to SDS–PAGE on a 12% gel, followed by Coomassie blue stain-ing. Samples analyzed in lanes 1 and 2 were, respectively, GST-fusion pro-tein and thrombin-digested forms of SULT2 ST2. Samples in lanes 3 and 4 were GST-fusion protein and thrombin-digested forms of SULT2 ST3. Protein molecular weight markers co-electrophoresed are: lysozyme (MrD14,300), -lactoglobulin (MrD18,400), carbonic anhydrase (MrD29,000), ovalbumin (MrD43,000), bovine serum albumin (MrD68,000), phosphorylase b (MrD97,400), and myosin (H-chain) (MrD200,000).

1

2

M

- 97.4 kDa - 68 kDa - 43 kDa - 29 kDa - 18.4 kDa - 14.3 kDa - 200 kDa

3

4

members of the SULT2 gene family. Compared with the

highly homologous SULT2 ST2, the previously identiWed

SULT2 ST1 exhibited sulfating activities toward DHEA,

pregnenolone, 17

-estradiol, allopregnanolone,

4-andro-stene-3,17-dione and 17

-hydroxypregnenolone, but not

estrone (

Table 2

). The three zebra

Wsh SULT2 enzymes

identi

Wed to date therefore appeared to sulfate distinct,

albeit overlapping, groups of hydroxysteroids. Whether

their distinct substrate speci

Wcity may reXect the diVerential

functional involvement of the three enzymes in vivo remains

to be clari

Wed.

The kinetics of the sulfation of DHEA by SULT2 ST1

and ST2 and the sulfation of corticosterone by SULT2

ST3 were further examined. Data obtained were processed

using the Excel program to generate the best

Wtting

trend-lines for the Lineweaver–Burk double-reciprocal plots.

Table 3

shows the kinetic constants determined for the

sulfation of DHEA by SULT2 ST1 and SULT2 ST2, as

well as the sulfation of corticosterone by ST3. The K

_m

val-ues of SULT2 ST1 and SULT2 ST2 with DHEA and ST3

with corticosterone were 102, 177, and 101

M,

respec-tively. TheseK

_m

values are considerably higher than those

previously determined for the sulfation of DHEA,

choles-terol or pregnenolone by human SULT2A1, SULT2B1a

or SULT2B1b

[29–31]

. It is possible that the high K

_m

val-ues of SULT2 ST1, ST2, and ST3 may represent

adapta-tion to high steroid concentraadapta-tions that might be present

systematically or locally in zebra

Wsh. Previous studies

have revealed circulating levels of some hydroxysteroids

in

Wsh to be in M concentration ranges

[32,33]

, which are

considerably higher than those found in humans (sub-nM

to nM)

[34]

. Moreover, it has been demonstrated that

physiological levels of hydroxysteroids may increase

dra-matically in response to aquatic environmental stimuli

[32,35,36]

. Another possibility is that certain

yet-unidenti-Wed co-factors or mechanisms may exist for the regulation

Fig. 4. pH dependency of the sulfating activity of the zebraWsh SULT2 ST2 with DHEA (A) and ST3 with corticosterone (B) as a substrate. The enzymatic assays were carried out under standard assay conditions as described under Materials and methods, using diVerent buVer systems as indicated. The data represent calculated mean § standard deviation derived from three experiments.

0 100 200 300 400 500 600 700 0 100 200 300 400 500 600

A

B

4 5 6 7 8 9 10 11 12 4 5 6 7 8 9 10 11 12 pH Specif ic Acti vity (pmol/min/mg) Specif ic Acti vity (pmol/min/mg) pH Table 2

SpeciWc activities of the zebraWsh SULT2 ST1, ST2, and ST3 with endogenous compounds as substratesa

a _{SpeciWc activity refers to pmol substrate sulfated/min/mg puriWed enzyme. Data represent means § SD derived from three experiments.} b _{SpeciWc activity determined is lower than the detection limit (estimated to be »10 pmol/min/mg protein).}

SpeciWc activity (pmol/min/mg)

SULT2 ST1 SULT2 ST2 SULT2 ST3

Dehydroepiandrosterone (DHEA) 554 § 48 579 § 24 40.9 § 2.5 Corticosterone NDb _{ND 510 § 23} Pregnenolone 628 § 59 47.4 § 5.9 68.8 § 4.0 17-Estradiol 63.2 § 0.5 23.6 § 2.7 51.7 § 3.3 Allopregnanolone 245 § 18 ND 37.8 § 2.1 Estrone ND 17.9 § 0.4 ND 4-Androstene-3,17-dione 122 § 10 ND ND 17-Hydroxypregnenolone 44.8 § 9.7 ND ND Table 3

Kinetic constants of the zebraWsh SULT2 ST1 and ST2 with DHEA, and SULT2 ST3 with corticosterone as substratea

a _{Data shown represent means § SD derived from three experiments.}

V_max (nmol/min/mg) K_m (M) V_max/K_m

SULT2 ST1 (with DHEA) 2.10 § 0.20 102 § 13 0.021

SULT2 ST2 (with DHEA) 2.64 § 0.21 177 § 26 0.015

of the kinetic properties of SULT2 ST2 and ST3 for their

functioning in the homeostasis of hydroxysteroids in

zebra

Wsh. Based on calculated V

_max

/K

_m

shown in

Table 3

the catalytic e

Yciency of SULT2 ST1 or ST2 with DHEA

as substrate appeared to be comparable to that of ST3

with corticosterone as substrate.

Developmental stage-dependent expression of the zebra

Wsh

cytosolic SULT2 STs

In vertebrates, steroid hormones, including

mineralo-corticoids, glucomineralo-corticoids, and sex steroids, are known to

play critical roles in the regulation of mineral balance,

glucose homeostasis, and sexual di

Verentiation

[37]

. In

view of their hydroxysteroid-sulfating activity, an

impor-tant question is whether the expression of the newly

iden-tiWed SULT2 ST2 and ST3 correlates with the

development of endocrine system of the zebraWsh. To gain

insight into this issue, RT-PCR was employed to examine

the expression of mRNAs encoding SULT2 ST2 and ST3,

as well as the previously identiWed SULT2 ST1, at

diVer-ent developmdiVer-ental stages. As shown in

Fig. 5

A, the

mRNA encoding SULT2 ST1 was not present in

unfertil-ized eggs and was not expressed until the embryos reached

the neurula/segmentation period (12-hour pf). Thereafter,

the expression of the SULT2 ST1 mRNA continued into

the larval stage onto maturity. For SULT2 ST2, a signi

W-cant level of its coding mRNA was detected in unfertilized

eggs, indicating clearly its maternal origin. Upon

fertiliza-tion, however, no SULT2 ST2 mRNA was detected until

the neurula/segmentation period (12-h pf), which then

increased dramatically when reaching the larval stage

(1-week pf) onto maturity. Both SULT2 ST1 and ST2

mRNAs were detected in adult male and female zebra

Wsh.

It is worthwhile pointing out that DHEA, a major

substrate for both SULT2 ST1 and ST2, occupies an

important position as the biosynthetic precursor for

tes-tosterone and 17-estradiol, as well as other sex steroids

[38]

. It has been reported that DHEA and some other

ste-roids are also produced de novo in the nervous system

where these so-called “neurosteroids” may play important

roles in the regulation of the activity of genes and protein

synthesis, cellular development, and the functioning of

neuroendocrine system and behavioral pattern

[39]

. That

the initiation of the expression of both SULT2 ST1 and

ST2 coincided with the beginning of the development of

the nervous system (at the neurula/segmentation stage)

poses an interesting question whether these two enzymes

are involved in the regulation and/or homeostasis of

DHEA and other neurosteroids. Moreover, since the

expression of SULT2 ST1 and ST2 continues into the

lar-val stage onto maturity, it is also possible that they may

Fig. 5. Developmental stage-dependent expression of the zebraWsh SULT2 STs. (A) RT-PCR analysis of the expression of mRNAs encoding SULT2 ST2 and ST3, as well as the previously identiWed zebraWsh SULT2 ST1, at diVerent stages during embryogenesis and larval development onto maturity. Final PCR mixtures were subjected to 2% agarose electrophoresis. Samples analyzed in lanes 1 through 15 correspond to unfertilized zebraWsh eggs, zebraWsh embryos during the zygote period (0-h post-fertilization (pf)), cleavage period (1-h pf), blastula period (3-h pf), gastrula period (6-h pf), neurula/segmenta-tion period (12-h pf), pharyngula period (24-h pf), and hatching period (48- and 72-h pf), 1, 2, 3, 4-week-old zebraWsh larvae, and 3-month-old male (lane 14) or female (lane 15) zebraWsh. The PCR products corresponding to diVerent zebraWsh SULT2 ST cDNAs, visualized by ethidium bromide staining, are marked by arrows. (B) RT-PCR analysis of the expression of the zebraWsh -actin at the same developmental stages as those described in (A).

3,000 bp 2,000 bp 1,500 bp 1,200 bp 1,031 bp 900 bp

1 2 3 4 5 6 7 8 9 10 11 12

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

A

B

SULT2 ST1

SULT2 ST2

SULT2 ST3

β-Actin

M

13 14 15 M

be involved in the regulation of sex steroids. The

physio-logical relevance of the expression of SULT2 ST2, but not

SULT2 ST1, as a maternal transcript in unfertilized

zebra

Wsh egges remains unclear. For SULT2 ST3, a

sig-ni

Wcant level of its coding message was detected in

unfer-tilized eggs, indicating its maternal origin. During the

embryonic development, there appeared to be an initial

decrease in expression, followed by disappearance during

the blastula (3-h pf) and gastrula period (6-h pf).

Thereaf-ter, the expression of SULT2 ST3 mRNA resumed in the

neurula/segmentation period (12-h pf) and continued into

the larval stage onto maturity. SULT2 ST3 mRNA was

detected in both male and female zebraWsh. The

physio-logical relevance of this unique pattern of developmental

stage-dependent expression of SULT2 ST3 remains to be

clariWed. It is to be noted, however, that corticosterone, a

favorite substrate for SULT2 ST3, is a major

glucocorti-coid in rodents, and the precursor of aldosterone, the

major mineralocorticoid in man

[37]

. In Wsh,

corticoste-rone can also be converted to cortisol

[40]

, which is known

to play an important role in the osmoregulation

[41]

. It is

plausible that SULT2 ST3 may be critical to the

homeo-stasis of corticosterone and therefore the ability of Wsh to

face osmotic challenges, as well as glucose homeostasis.

The expression of SULT2 ST3 in unfertilized eggs and

embryos during early developmental stages may serve to

regulate the corticosteroids of maternal origin, prior to

the development of endocrine organs. In contrast to the

developmental stage-dependent expression of the SULT2

STs,

-actin, housekeeping protein, was found to be

expressed throughout the entire developmental process

(

Fig. 5

B).

To summarize, we have identi

Wed two novel DHEA/

corticosterone-sulfating cytosolic SULT2 STs, designated

as SULT2 ST2 and ST3, which may be involved in

metab-olism and homeostasis of DHEA and cortoicosterone,

respectively, in zebraWsh. Our goal is to obtain a complete

repertoire of the cytosolic SULT enzymes present in

zebraWsh, and this study is part of an overall eVort. As

pointed out earlier, the identiWcation of the various

cyto-solic SULTs and their biochemical characterization is a

prerequisite for using the zebraWsh as a model for a

sys-tematic investigation on fundamental issues regarding

cytosolic SULTs. More works is warranted in order to

achieve this goal.

Acknowledgments

This work was supported in part by a Grant-in-Aid from

the American Heart Association (Texas AYliate) and a

grant (#0542235) from National Science Foundation.

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