Báo cáo khoa học: Identification of a novel thyroid hormone-sulfating cytosolic sulfotransferase, SULT1 ST5, from zebrafish Molecular cloning, expression, characterization and ontogenic study ppt

Identification of a novel thyroid hormone-sulfating
cytosolic sulfotransferase, SULT1 ST5, from zebrafish
Molecular cloning, expression, characterization and ontogenic
study
Shin Yasuda, A. Pavan Kumar, Ming-Yih Liu, Yoichi Sakakibara, Masahito Suiko, Lanzhuang Chen
and Ming-Cheh Liu
Biomedical Research Center, The University of Texas Health Center, Tyler, USA
Sulfation is an important pathway in vivo for the bio-
transformation of low molecular mass xenobiotics as
well as endogenous compounds [1–3]. Upon sulfation,
xenobiotic compounds become more water soluble and
can be excreted from the body more easily. For endo-
genous compounds such as steroid and thyroid
hormones, catecholamines, cholesterol and bile acids,
sulfation may be involved in their regulation and
homeostasis. In the case of thyroid hormones, sulfa-
tion may increase their water solubility and subsequent
biliary and urinary excretion [4]. Moreover, 3,3¢,5-tri-
iodo-l-Thyronine (l-T
3
), the major thyroid hormone,
loses its affinity for thyroid hormone receptors upon
sulfation, and sulfated l-T
3
is subject to accelerated
Keywords
molecular cloning; sulfotransferase; SULT1;
thyroid hormone; zebrafish
Correspondence
M C. Liu, Biomedical Research Center,
The University of Texas Health Center,

11937 U.S. Highway 271, Tyler, TX 75708,
USA
Fax: +1 903 877 2863
Tel: +1 903 877 2862
E-mail:
(Received 7 February 2005, revised 20 April
2005, accepted 25 May 2005)
doi:10.1111/j.1742-4658.2005.04791.x
By employing RT-PCR in conjunction with 3¢-RACE, a full-length cDNA
encoding a novel zebrafish cytosolic sulfotransferase (SULT) was cloned
and sequenced. Sequence analysis revealed that this zebrafish SULT (desig-
nated SULT1 ST5) is, at the amino acid sequence level, close to 50%
identical to human and dog SULT1B1 (thyroid hormone SULT). A
recombinant form of zebrafish SULT1 ST5 was expressed using the
pGEX-2TK bacterial expression system and purified from transformed
BL21 (DE3) cells. Purified zebrafish SULT1 ST5 migrated as a 34 kDa
protein and displayed substrate specificity for thyroid hormones and their
metabolites among various endogenous compounds tested. The enzyme
also exhibited sulfating activities toward some xenobiotic phenolic com-
pounds. Its pH optima were 6.0 and 9.0 with 3,3¢,5-triiodo-l-thyronine
(l-T
3
) as substrate and 6.0 with b-naphthol as substrate. Kinetic constants
of the enzyme with thyroid hormones and their metabolites as substrates
were determined. Quantitative evaluation of the regulatory effects of diva-
lent metal cations on the l-T
3
-sulfating activity of SULT1 ST5 revealed
that Fe
2+

,Hg
2+
,Co
2+
,Zn
2+
,Cu
2+
,Cd
2+
and Pb
2+
exhibited dramatic
inhibitory effects, whereas Mn
2+
showed a significant stimulation. Devel-
opmental stage-dependent expression experiments revealed a significant
level of expression of this novel zebrafish thyroid hormone-sulfating SULT
at the beginning of the hatching period during embryogenesis, which
gradually increased to a high level of expression throughout the larval stage
into maturity.
Abbreviations
D-T
3
, 3,3¢,5-triiodo-D-thyronine; DTT, dithiothreitol; estrone, 1,3,5[10]-estratrinen-3-ol-17-one; IPTG, isopropyl thio-b-D-galactoside; L-Dopa,
L-3,4-dihydroxyphenylalanine; L-rT3, 3,3¢,5¢-triiodo-L-thyronine; L-T
3
, 3,3¢,5-triiodo-L-thyronine; L-T
4
, L-thyroxine; PAPS, 3¢-phosphoadenosine-5¢-

phosphosulfate; SULT, sulfotransferase.
3828 FEBS Journal 272 (2005) 3828–3837 ª 2005 FEBS
inner ring deiodination, followed by subsequent degra-
dation reactions [5].
The enzymes responsible for the sulfation reactions,
the cytosolic sulfotransferases (SULTs), catalyze the
transfer of a sulfonate group from 3¢-phosphoadeno-
sine-5¢-phosphosulfate (PAPS) to the hydroxyl group
or amino group of substrate compounds [1–3]. Since
the early 1990s, increasing numbers of cytosolic
SULTs from different vertebrates have been cloned
and sequenced [6,7]. It is now known that all cytosolic
SULTs from vertebrates constitute a gene superfamily
and, based on amino acid sequence homology, distinct
gene families have been further categorized [8]. Two
major gene families among them are the phenol SULT
family (designated SULT1) and the hydroxysteroid
SULT family (designated SULT2) [6–8]. The phenol
SULT family consists of at least five subfamilies,
phenol SULT (SULT1A), Dopa ⁄ tyrosine (or thyroid
hormone) SULT (SULT1B), hydroxyarylamine (or
acetylaminofluorene) SULT (SULT1C), tyrosine ester
SULT (SULT1D), and estrogen SULT (SULT1E).
The hydroxysteroid SULT family currently compri-
ses two subfamilies, dehydroepiandrosterone SULT
(SULT2A) and cholesterol ⁄ pregnenolone SULT
(SULT2B).
Despite a considerable amount of work carried out
in the past two decades, to a large extent the physio-
logical involvement of the various cytosolic SULTs

remains unclear. Moreover, only fragmentary informa-
tion is available concerning the cell type ⁄ tissue ⁄ organ-
specific expression of the different cytosolic SULTs,
and very little is known with regard to the ontogeny of
these enzymes. To resolve these outstanding issues, a
suitable animal model is required. Zebrafish has
recently emerged as a popular animal model for a wide
range of studies [9,10]. Its advantages, compared with
mouse, rat or other vertebrate models, include its small
size, the availability of a relatively large number of
eggs, rapid development externally of virtually trans-
parent embryo, and short generation time. These
unique features make the zebrafish an excellent model
for systematic studies on the ontogeny of cytosolic
SULTs and their tissue- and cell-Type-specific distribu-
tion, as well as the physiological relevance of individ-
ual cytosolic SULTs. A prerequisite for using zebrafish
in these studies, however, is the identification of the
various cytosolic SULTs and their biochemical charac-
terization. We have recently embarked on the mole-
cular cloning of zebrafish cytosolic SULTs [11–14].
Sequence analysis via blast search revealed that the
zebrafish cytosolic SULTs we have cloned [11–14] dis-
play sequence homology to mammalian cytosolic
SULTs. Of the six zebrafish cytosolic SULTs cloned,
four fall within the SULT1 gene family [11,12], one
belongs to the SULT2 gene family [13], and one
appears to be independent of all known SULT gene
families [14].
We report here the molecular cloning, expression

and characterization of a novel thyroid hormone-sulf-
ating cytosolic SULT from zebrafish. Its enzymatic
activities toward a variety of endogenous and xeno-
biotic compounds including some flavonoids, isoflavo-
noids, and other phenolic compounds were examined.
Kinetic parameters of the enzyme with thyroid hor-
mones and their metabolites as substrates were deter-
mined. Moreover, its developmental stage-dependent
expression was investigated.
Results and Discussion
As part of an effort to develop a zebrafish model for
investigating, in greater detail, the role of sulfation in
the metabolism and homeostasis of thyroid hormones,
we identified and characterized a novel zebrafish thy-
roid hormone-sulfating SULT in this study.
Molecular cloning of the zebrafish cytosolic
SULT1 ST5
By employing RT-PCR in conjunction with 3¢-RACE,
a full-length cDNA encoding a novel zebrafish cyto-
solic SULT was cloned and sequenced. The nucleotide
sequence obtained was submitted to the GenBank
database under accession no. AY879099. Figure 1
shows the alignment of the deduced amino acid
sequence of the newly cloned zebrafish SULT with
those of the other four zebrafish SULT1 STs previ-
ously identified [11,12]. The open reading frame of the
newly cloned SULT encompasses 882 nucleotides and
encodes a 293-amino acid polypeptide. Similar to other
cytosolic SULTs, the new zebrafish SULT contains
sequences resembling the so-called ‘signature seq-

uences’ (YPKSGTxW in the N-terminal region and
RKGxxGDWKNxFT in the C-terminal region; as
underlined in Fig. 1) characteristic of SULT enzymes
[7]. Of these two sequences, YPKSGTxW has been
demonstrated by X-ray crystallography to be respon-
sible for binding to the 5¢-phosphosulfate group of
PAPS, a cosubstrate for SULT-catalyzed sulfation
reactions [15], and thus designated the 5¢-phosphosul-
fate binding (5¢-PSB) motif [16]. The cloned zebrafish
SULT also contains the 3¢-phosphate binding (3¢-PB)
motif (amino acid residues 187–197; as underlined)
responsible for the binding to the 3¢-phosphate group
of PAPS [16]. Sequence analysis based on blast search
revealed that the deduced amino acid sequence of the
S. Yasuda et al. A novel zebrafish cytosolic sulfotransferase
FEBS Journal 272 (2005) 3828–3837 ª 2005 FEBS 3829
new zebrafish SULT displays 48 and 46% identity to
dog and human SULT1B1, and lower percentage 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 fam-
ily are > 60% identical in amino acid sequence [6–8].
Based on these criteria, the newly cloned zebrafish
SULT appears to belong to the SULT1 gene family,
and is tentatively designated zebrafish SULT1 ST5 in
accordance with the nomenclature used in the ZFIN
database (cf. the dendrogram shown in Fig. 2). Com-
pared with known zebrafish SULTs, the newly cloned
zebrafish SULT1 ST5 displays 44, 45, 43, and 46%

Fig. 1. Alignment of the deduced amino acid sequences of SULT1 ST5 and four known zebrafish SULT1 STs. Two ‘signature sequences’,
respectively located in the N- and C-terminal regions, as well as a conserved sequence in the middle region, are underlined.
Fig. 2. Classification of zebrafish SULT1 ST5
on the basis of deduced amino acid sequen-
ce. The dendrogram shows the degree of
amino acid sequence homology among
cytosolic SULTs. References for individual
SULTs are given in [18]. h, Human; m, mouse;
zf, zebrafish. The dendrogram was gen-
erated based on the Greedy algorithm [31,
32].
A novel zebrafish cytosolic sulfotransferase S. Yasuda et al.
3830 FEBS Journal 272 (2005) 3828–3837 ª 2005 FEBS
amino acid sequence identity to, respectively, zebrafish
SULT1 ST1, 2, 3, and 4 previously reported [11,12].
Expression, purification, and characterization of
recombinant zebrafish cytosolic SULT1 ST5
The coding region of zebrafish SULT1 ST5 cDNA was
subcloned into pGEX-2TK, a prokaryotic expression
vector, for the expression of recombinant enzyme in
Escherichia coli. Recombinant zebrafish SULT1 ST5,
purified from the E. coli extract, migrated at  34 kDa
position upon SDS ⁄ PAGE (not shown). This is in
agreement with the calculated molecular mass
(34 452 Da) based on its deduced amino acid sequence.
Purified zebrafish SULT1 ST5 was subjected to func-
tional characterization with respect to its enzymatic
properties. A pilot experiment showed that the enzyme
exhibited strong activity toward b-naphthol. A pH-
dependence experiment subsequently performed

showed that the enzyme displayed maximum activity at
pH 6.0, with b-naphthol as substrate (Fig. 3A). With
l-T
3
as substrate, zebrafish SULT1 ST5 was active over
a broader pH range, with optimal activities observed at,
respectively, 6.0 and 9.0 (Fig. 3B). Whether the differ-
ent pH-dependence profiles with b-naphthol and l-T
3
as substrates are due to their structural differences (b-
naphthol being a neutral compound and l-T
3
, with its
amino and carboxyl groups, being a charged molecule)
or, in fact, reflect distinct catalytic mechanisms remains
to be clarified. Several endogenous and xenobiotic
compounds were tested as substrates for the enzyme,
and the activity data obtained are given in Table 1.
Interestingly, among the endogenous substrates, zebra-
fish SULT1 ST5 showed sulfating activities toward
only thyroid hormones and their metabolites, including
l-T
3
, 3,3¢,5-triiodo-d-thyronine (d-T
3
), 3,3¢ ,5¢-triiodo-
l-thyronine (l-rT
3
), l-Thyroxine (l-T
4

), and l-Thyro-
nine. The enzyme also exhibited activities toward some
of the xenobiotic compounds tested, including chloro-
genic acid, kaempferol, gallic acid, genistein, b-naph-
thol, catechin, caffeic acid, daidzein, butylated hydroxy
anisole, quercetin, myricetin, n-propyl gallate, and
p-nitrophenol. These latter activities are in line with this
new enzyme being a member of the SULT1 (phenol
SULT) gene family. It is worthwhile pointing out that
human and dog thyroid hormone SULTs (SULT1B1)
have also been shown to display activities toward xeno-
biotic phenolic compounds such as b-naphthol, and
p-nitrophenol [17–19]. It should also be pointed out
that, of the five zebrafish SULTs previously reported
[11–14], SULT1 ST1, 2, and 3 also exhibited consider-
able activities toward l-T
3
and l-T
4
[11,12]. Unlike the
SULT1 ST5 identified in this study, however, zebrafish
SULT1 ST1, 2, and 3 were also found to be active
toward several other endogenous compounds including
dopamine, 1,3,5[10]-estratrinen-3-ol-17-one (estrone),
l-3,4-dihydroxyphenylalanine (l-Dopa), and dehydro-
epiandrosterone [11,12]. SULT1 ST5 therefore appears
to be the only zebrafish enzyme known, to date, that
displays substrate specificity exclusively for thyroid
hormones and their metabolites. It will be interesting
to investigate whether SULT1 ST5 plays a unique and

important role in the metabolism and homeostasis of
thyroid hormones in vivo.
To investigate in more detail the sulfation of thyroid
hormones and their metabolites, the kinetics of sulfa-
tion of these compounds by zebrafish SULT1 ST5 was
examined. Data obtained were processed using the
0
10
20
30
40
3456789101112
pH
n( ytivitcA cificepS)gm/nim/lom
0
20
40
60
80
100
3456789101112
pH
n( ytivitcA cificepS)gm/nim/lom
B
A
Fig. 3. pH dependence of the sulfating activity of zebrafish
SULT1 ST5 with (A) b-naphthol and (B)
L-T
3
as substrates. The enzy-

matic assays were carried out under standard assay conditions as
described under Experimental procedures, using different buffer
systems as indicated. The data represent calculated mean values
derived from three experiments.
S. Yasuda et al. A novel zebrafish cytosolic sulfotransferase
FEBS Journal 272 (2005) 3828–3837 ª 2005 FEBS 3831
excel program to generate the best fitting trendlines
for the Lineweaver–Burk double-reciprocal plots.
Table 2 shows the kinetic constants determined for the
sulfation of thyroid hormones and their metabolites, as
well as b-naphthol. It appeared that the K
m
values for
the thyroid hormone⁄ metabolites, except l-Thyronine,
were of the same order of magnitude, indicating com-
parable affinities of the enzyme for these substrates.
V
max
values showed smaller variations, with the lowest
activity for l-T
4
. Catalytic efficiency of the enzyme, as
reflected by V
max
⁄ K
m
, appeared to be comparable with
l-T
3
, d-T

3
, l-rT
3
or l-Thyronine as substrates, and sig-
nificantly lower with l-T
4
as substrate. It is worth
mentioning that human and rat thyroid hormone-
sulfating SULT1B1 also display K
m
values (approxi-
mately 40 lm) for l-T
3
[18,19] comparable with that
(38.7 lm) of zebrafish SULT1 ST5. With b-naphthol
as substrate, zebrafish SULT1 ST5 showed V
max
and
K
m
values comparable with those determined for
l-Thyronine. Despite these similar kinetic parameters,
however, whether the same catalytic mechanism is
involved with b-naphthol and thyroid hormones as
substrates remains to be clarified. As discussed earlier,
b-naphthol is a neutral compound and thyroid hor-
mones, by contrast, are charged molecules. Moreover,
the enzyme showed distinct pH-dependence profiles
with these two kinds of substrates (Fig. 3).
Previous studies performed in our laboratory revealed

that the sulfating activity of human cytosolic SULTs
could be dramatically inhibited by certain divalent
metal cations [20–22]. As an aquatic animal, zebrafish is
a good model for studying the effects of divalent metal
cations. To investigate the inhibitory ⁄ stimulatory effects
of divalent metal cations on the sulfating activity of ze-
brafish SULT1 ST5, enzymatic assays using l-T
3
as the
substrate were carried out in the absence or presence of
various divalent metal cations at a concentration of
1mm. As a control for the counter ion, Cl
–
, parallel
assays in the presence 2 mm NaCl were also performed.
Results obtained are compiled in Table 3. The degrees
of inhibition or stimulation were calculated by compar-
ing the activities determined in the presence of metal
cations with the activities determined in the absence of
metal cations. It was noted that, among the 10 divalent
metal cations tested, Fe
2+
,Hg
2+
,Co
2+
,Zn
2+
,Cu
2+

,
Cd
2+
and Pb
2+
exhibited profound inhibitory effects
Table 1. Specific activities of zebrafish SULT1 ST5 with endogenous and xenobiotic compounds as substrates. Specific activity refers to
nmol substrate sulfatedÆmin
)1
Æmg
)1
purified enzyme. Data represent means ± SD derived from three experiments. ND, Specific activity
determined is lower than the detection limit (estimated to be  0.01 nmolÆmin
)1
Æmg protein
)1
).
Endogenous compounds Specific activity (nmolÆmin
)1
Æmg
)1
) Xenobiotic compounds Specific activity (nmolÆmin
)1
Æmg
)1
)
3,3¢,5-Triiodo-
D-Thyronine (D-T
3
) 24.32 ± 1.47 Chlorogenic acid 23.52 ± 0.51

L-Thyronine 17.74 ± 1.20 Kaempferol 14.52 ± 0.73
3,3¢,5-Triiodo-
L-Thyronine (L-T
3
) 17.39 ± 0.78 Gallic acid 14.26 ± 0.25
3,3¢,5¢-Triiodo-
L-Thyronine (L-rT
3
) 11.62 ± 0.48 Genistein 12.71 ± 0.17
L-Thyroxine (L-T
4
) 4.31 ± 0.14 b-Naphthol 12.26 ± 0.16
17b-Estradiol ND
b
Catechin 11.47 ± 0.41
Estrone ND Caffeic acid 9.95 ± 0.17
4-Androstene-3,17-dione ND Daidzein 9.87 ± 0.58
Cholesterol ND Butylated hydroxy anisole 8.38 ± 0.31
Corticosterone ND Quercetin 7.88 ± 0.33
Cortisone ND Myricetin 5.38 ± 0.19
Dehydroepiandrosterone ND n-Propyl gallate 5.32 ± 0.03
D-Dopa ND p-Nitrophenol 4.76 ± 0.11
L-Dopa ND b-Napthylamine 0.44 ± 0.03
Dopamine ND Bisphenol A 0.37 ± 0.02
Hydrocortisone ND p-Octylphenol 0.27 ± 0.03
17a-Hydroxy progesterone ND Epigallocatechin gallate ND
17a-Hydroxy pregnenolone ND Butylated hydroxy toluene ND
Pregnenolone ND Diethylstilbestrol ND
Progesterone ND Epicatechin ND
Table 2. Kinetic constants of zebrafish SULT1 ST5 with L-T

3
, D-T
3
,
L-rT
3
, L-thyronine, and b-naphthol as substrates. Data shown repre-
sent means ± SD derived from three determinations.
Substrate V
max
(min
)1
) K
m
(lM) V
max
⁄ K
m
L-T
3
28.8 ± 2.5 38.7 ± 5.9 0.74
D-T
3
35.6 ± 3.4 27.7 ± 2.8 1.29
L-rT
3
14.6 ± 0.6 17.1 ± 0.7 0.85
L-T
4
6.6 ± 0.6 44.5 ± 6.7 0.15

L-Thyronine 41.9 ± 2.3 114.2 ± 12.8 0.37
b-Naphthol 32.3 ± 3.7 97.7 ± 8.9 0.33
A novel zebrafish cytosolic sulfotransferase S. Yasuda et al.
3832 FEBS Journal 272 (2005) 3828–3837 ª 2005 FEBS
on the sulfating activity of the zebrafish SULT, whereas
Mn
2+
showed a significant stimulation. Addition of
EDTA (at 2 mm concentration) restored the sulfating
activity of SULT1 ST5 in the cases of Zn
2+
,Cd
2+
, and
Pb
2+
. In contrast, the inhibition by Fe
2+
,Hg
2+
,Co
2+
,
and Cu
2+
appeared to be irreversible. It should be poin-
ted out, however, that these divalent metal cations were
tested at a 1 mm concentration. Whether the divalent
metal cations as environmental pollutants may enter
zebrafish and accumulate to high enough levels to exert

inhibitory or stimulatory effects on the SULT, thereby
disrupting the homeostasis of thyroid hormones, remain
to be clarified.
Developmental stage-dependent expression of
zebrafish thyroid hormone-sulfating cytosolic
SULT1 ST5
In view of its thyroid hormone-sulfating activity, an
important question is whether expression of the newly
identified SULT1 ST5 correlates with the development
of the thyroid hormone endocrine system of the zebra-
fish. To gain insight into this, RT-PCR was used to
examine the expression of mRNA encoding the thyroid
hormone-sulfating SULT1 ST5 at different develop-
mental stages. As shown in Fig. 4A, no expression was
detected in unfertilized eggs and during the early phase
of embryonic development. A low level of expression of
zebrafish SULT1 ST5 was observed at the beginning
of the hatching period during embryogenesis, and this
gradually increased to a high level of expression
throughout the larval stage and into maturity. Interest-
ingly, previous studies have revealed that it is during
the hatching period when primary organ systems inclu-
ding the thyroid gland are formed [23]. The develop-
mental expression of the four zebrafish SULT1 STs
previously identified were also examined (Fig. 4A). For
SULT1 ST1, a low level of message was detected in
unfertilized eggs and in embryos immediately following
fertilization. Throughout the cleavage period, blastula
period, and the early part of gastrula period, however,
the message encoding SULT1 ST1 could not be detec-

ted. Thereafter, the expression started and increased to
a high level during the larval stage onto maturity. For
SULT1 ST2, no expression was detected in unfertilized
eggs and during embryonic development. The expres-
sion appeared in 1- to 4-week-old larvae, and, intrigu-
ingly, decreased considerably in adult zebrafish. For
SULT1 ST3, a significant level of its coding message
was detected in unfertilized eggs. During embryonic
development, there appeared to be an initial decrease
in expression until the end of the segmentation period
Table 3. Effects of divalent metal cations on the sulfating activity
of zebrafish SULT1 ST5 with
L-T
3
as the substrate. Data represent
means ± SD derived from three determinations. Data shown in par-
entheses are percentage of the activity determined for the control
without divalent cation or EDTA.
Divalent
cation tested
Specific activity (nmolÆmin
)1
Æmg
)1
)
Divalent cation
only (1 m
M)
Divalent cation + EDTA
(1 mM cations + 2 mM EDTA)

Control 27.4 ± 2.3 (100%) 29.0 ± 0.4 (106%)
FeCl
2
1.9 ± 0.1 (6.9%) 1.9 ± 0.1 (6.9%)
HgCl
2
2.0 ± 0.4 (7.3%) 3.8 ± 0.2 (13.9%)
CoCl
2
2.3 ± 0.7 (8.4%) 2.6 ± 0.2 (9.5%)
ZnCl
2
1.7 ± 0.5 (6.2%) 31.5 ± 0.2 (115%)
CuCl
2
3.0 ± 0.4 (10.9%) 1.9 ± 0.4 (6.9%)
CdCl
2
1.7 ± 0.1 (6.2%) 27.6 ± 1.5 (101%)
MnCl
2
30.9 ± 3.4 (113%) 32.1 ± 1.2 (117%)
CaCl
2
29.7 ± 1.3 (108%) 29.2 ± 1.1 (107%)
MgCl
2
31.9 ± 0.4 (116%) 25.9 ± 2.8 (94.5%)
Pb(CH
3

COO)
2
1.4 ± 0.1 (5.1%) 27.2 ± 1.0 (99.2%)
NaCl
a
29.8 ± 1.9 (109%) 31.0 ± 4.3 (113%)
a
Tested at a 2 mM concentration as a control for the counter ion,
Cl
–
.
A
B
Fig. 4. Developmental stage-dependent expression of zebrafish
SULTs. (A) RT-PCR analysis of the expression of SULT1 ST5 and
previously identified zebrafish SULT1 STs at different stages during
embryogenesis and larval development onto maturity. Final PCR
mixtures were subjected to 2% agarose electrophoresis. Samples
analyzed in lanes 1–14 correspond to unfertilized zebrafish eggs, 0,
1, 3, 6, 12, 24, 48, and 72-h zebrafish embryos, 1, 2, 3, 4-week-old
zebrafish larvae, and 3-month-old zebrafish. The PCR products cor-
responding to different zebrafish SULT1 ST cDNAs, visualized by
ethidium bromide staining, are marked by arrows. (B) RT-PCR ana-
lysis of the expression of zebrafish b-actin at the same develop-
mental stages as those described in (A).
S. Yasuda et al. A novel zebrafish cytosolic sulfotransferase
FEBS Journal 272 (2005) 3828–3837 ª 2005 FEBS 3833
(24 h post fertilization), which then increased to a high
level of expression throughout the rest of the embry-
onic development and the larval stage onto maturity.

A low level of message encoding SULT1 ST4 was
detected in unfertilized eggs, indicating its presence as
a maternal transcript. No SULT1 ST4 message, how-
ever, was detected in the embryos until the segmen-
tation period (12–24 h post fertilization); it then
gradually increased to a high level of expression
throughout the rest of the embryonic development and
the larval stage onto maturity. The physiological impli-
cations of the differential expression of the various
SULTs mentioned above remain to be clarified. In
contrast to the developmental stage-dependent expres-
sion of the SULT1 isoform 5, b-actin, a housekeeping
protein, was found to be expressed throughout the
entire developmental process (Fig. 4B).
To summarize, we identified a thyroid hormone-sulf-
ating cytosolic SULT that may be involved in meta-
bolism and homeostasis of thyroid hormones and their
metabolites in zebrafish. This study is part of an over-
all effort to obtain a complete repertoire of the cyto-
solic SULT enzymes present in zebrafish. As pointed
out earlier, the identification of the various cytosolic
SULTs and their biochemical characterization is a pre-
requisite for using the zebrafish as a model for a sys-
tematic investigation on fundamental issues regarding
cytosolic SULTs. More work is warranted in order to
achieve this goal.
Experimental procedures
Materials
p-Nitrophenol, dopamine, l-Dopa, d-Dopa, b-naphthol,
b-naphthylamine, aprotinin, thrombin, l-Thyronine, l-T

3
,
d-T
3
, l-rT
3
, l-T
4
, estrone, 17b-estradiol, bisphenol A,
4-octylphenol, daidzein, kaempferol, caffeic acid, genistein,
myricetin, quercetin, gallic acid, chlorogenic acid, catechin,
epicatechin, epigallocatechin gallate, n-propyl gallate, dehy-
droepiandrosterone, ATP, SDS, Mes, Mops, Hepes, Taps,
Ches, Caps, Trizma base, dithiothreitol (DTT), and isopro-
pyl thio-b-d-galactoside (IPTG) were products of Sigma
Chemical Company (St. Louis, MO, USA). TRI Reagent
was from Molecular Research Center, Inc, (Cincinnati, CH,
USA). Unfertilized zebrafish eggs and zebrafish embryos and
larvae at different developmental stages were prepared by
Scientific Hatcheries (Huntington Beach, CA, USA). Total
RNA from a 3-month-old zebrafish was prepared as des-
cribed previously [12]. Taq DNA polymerase was a product
of Promega (Madison, WI, USA). Takara Ex Taq DNA
polymerase and 3¢-Full RACE Core Kit were purchased
from PanVera Corp ⁄ Invitrogen (Carlsbad, CA, USA). T
4
DNA ligase and BamHI restriction endonuclease were from
New England Biolabs (Beverly, MA, USA). Oligonucleotide
primers were synthesized by MWG Biotech (Highpoint, NC,
USA). pSTBlue-1 AccepTor Vector Kit and BL21 (DE3)

competent cells were purchased from Novagen (Madison,
WI, USA). Prestained protein molecular mass standard was
from Life Technologies (Grand Island, NY, USA). First-
strand cDNA Synthesis Kit, pGEX-2TK glutathi-
one S-Transferase (GST) gene fusion vector, GEX-5¢-and
GEX-3¢ sequencing primers, and glutathione Sepharose 4B
were products of Amersham Biosciences (Piscataway, NJ,
USA). Recombinant human bifunctional ATP sulfurylase ⁄
adenosine 5¢-phosphosulfate kinase was prepared as des-
cribed previously [24]. Cellulose TLC plates were products of
EM Science (Gibbstown, NJ, USA). Carrier-free sodium
[
35
S]sulfate, Ecolume scintillation cocktail, cortisone, corti-
costerone, 4-androstene-3,17-dione, hydrocortisone, prog-
esterone, pregnenolone, 17a-OH progesterone, and 17a-OH
pregnenolone were from ICN Biomedicals (Costa Mesa, CA,
USA). All other reagents were of the highest grades commer-
cially available.
cDNA cloning of zebrafish cytosolic SULT1 ST5
By searching the EST database, a zebrafish cDNA (Gen-
Bank accession no. BI884567) encoding the 5¢-region of a
putative cytosolic SULT was identified. To obtain the
3¢-coding region and the untranslated sequence further
downstream, 3¢-RACE was performed using the Takara-3¢-
Full RACE Core Kit. First-strand cDNA was synthesized
using AMV reverse transcriptase with zebrafish total RNA
as the template in conjunction with an oligo(dT)-3 sites
adaptor primer. Afterwards, PCR was carried out using an
oligonucleotide (5¢-CCATGGAAACAGTATCTGGAGA

GG-3¢) designed based on the sequence determined for the
above-mentioned zebrafish SULT cDNA and a 3 sites
adaptor primer as the primer pair with the first-strand
cDNA as the template. Amplification conditions were
2 min at 94 °C and 25 cycles of 30 s at 94 °C, 30 s at
59 °C, and 5 min at 72 °C, followed by a 5 min extension
at 72 °C. The reaction mixture was analyzed by agarose
electrophoresis. A discrete PCR product detected was iso-
lated and subcloned into pSTBlue-1 cloning vector and sub-
jected to nucleotide sequencing [25]. The nucleotide and
deduced amino acid sequences of the cDNA were analyzed
using blast search for sequence homology to known cyto-
solic SULTs. RT-PCR was subsequently employed to
amplify the complete coding region of this novel zebrafish
SULT. With zebrafish total RNA as the template and
oligo(dT) as the primer, the first-strand cDNA was syn-
thesized using the First-Strand cDNA Synthesis Kit
(Amersham Biosciences). Using sense (5¢-CGCGGATCC
ATGAGCCGGAGAACAAGCGAAACT-3¢) and antisense
(5¢-CGCGGATCCTTATATAGTGAAGCGTATTGGAAG
A novel zebrafish cytosolic sulfotransferase S. Yasuda et al.
3834 FEBS Journal 272 (2005) 3828–3837 ª 2005 FEBS
AGGACA-3¢) oligonucleotide primers designed based on
5¢- and 3¢-coding sequences determined as mentioned
above, a PCR in a 100 lL reaction mixture was carried out
under the action of EX Taq DNA polymerase, with zebra-
fish first-strand cDNA prepared as the template. Amplifica-
tion 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 final reaction
mixture was applied onto a 1.2% agarose gel, separated by

electrophoresis, and visualized by ethidium bromide stain-
ing. The PCR product band detected was excised from the
gel, and the DNA therein was isolated by spin filtration.
Purified PCR product was subjected to BamHI restriction
and cloned into BamHI-restrictd pGEX-2TK vector, and
verified for autheticity by nucleotide sequencing [25].
Bacterial expression and purification of the
recombinant zebrafish cytosolic SULT1 ST5
pGEX-2TK harboring cloned zebrafish SULT cDNA was
transformed into competent BL21 (DE3) cells. Transformed
cells were grown to D
600
 0.6 in 1 L Luria–Bertani med-
ium supplemented with 60 lgÆmL
)1
ampicillin, and induced
with 0.1 mm IPTG. After an overnight induction at room
temperature, cells were collected by centrifugation and
homogenized in 25 mL ice-cold lysis buffer (10 mm
Tris ⁄ HCl, pH 8.0, 150 mm NaCl, 1 mm EDTA) using an
Aminco French Press. Twenty microliters of 10 mgÆmL
)1
aprotinin (a protease inhibitor) was added to the crude
homogenate. The crude homogenate was subjected to cen-
trifugation at 10 000 g 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 treated
with 3 mL of a thrombin digestion buffer (50 mm Tris ⁄ HCl,
pH 8.0, 150 mm NaCl, and 2.5 mm CaCl
2

) containing
5 unitÆmL
)1
bovine thrombin. Following 20 min incubation
at room temperature with constant agitation, the prepar-
ation was subjected to centrifugation. The recombinant
zebrafish SULT present in the supernatant collected was
analyzed with respect to its enzymatic properties.
Enzymatic assay
The sulfating activity of the purified zebrafish SULT was
assayed using [
35
S]PAPS as the sulfate donor. The standard
assay mixture, with a final volume of 25 lL, contained
50 mm Taps buffer (pH 8.0), 14 lm [
35
S]PAPS (15 CiÆ
mmol
)1
), 1 mm DTT, and 50 lm of substrate. The reaction
was started by the addition of the enzyme, allowed to pro-
ceed for 3 min at 28 °C, and terminated by heating at
100 °C for 2 min. The precipitates formed were cleared by
centrifugation, and the supernatant was subjected to the
analysis of [
35
S]sulfated product using the previously devel-
oped TLC procedure [26], with n-butanol ⁄ isopropanol ⁄ 88%
formic acid ⁄ water (3 : 1 : 1 : 1, v ⁄ v ⁄ v ⁄ v) as the solvent sys-
tem. To examine the pH dependence, different buffers

(50 mm Mes at 5.5, 6.0, or 6.5; Mops at 6.5, 7.0, or 7.5;
Taps at 7.5, 8.0 8.5 or 9.0; Ches at 9.0, 9.5, or 10.0; and
Caps at 10.0, 10.5, or 11.0), instead of 50 mm Taps
(pH 8.0), were used in the reactions, with 500 lm b-naph-
thol or 200 lm L-T
3
as substrate. For the kinetic studies on
the sulfation of l-T
3
, d-T
3
, l-rT
3
, l-T
4
, l-Thyronine, and
b-naphthol, varying concentrations of these substrate com-
pounds and 50 mm Taps buffer at pH 8.0 were used. To
determine the stimulatory ⁄ inhibitory effects of divalent
metal cations, enzymatic assays in the presence or absence
of different divalent metal cations, at 1 mm concentration,
were performed under standard conditions described above,
with 200 lm L-T
3
as substrate.
Analysis of the developmental stage-dependent
expression of zebrafish cytosolic SULT1 ST5
RT-PCR was employed to investigate the developmental
stage-dependent expression of the zebrafish cytosolic
Table 4. Oligonucleotide primers used for PCR amplifications in the analysis of developmental stage-dependent expression of zebrafish cyto-

solic SULT1 STs. The sense and antisense oligonucleotide primer sets listed were verified by
BLAST search to be specific for the target zebra-
fish SULT or b-actin nucleotide sequence.
Target
sequence
Sense and antisense
oligonucleotide primers
SULT1 ST1 Sense: 5¢-AGTTCAACAAGGAACTGCAGGACGTGTTTG-3¢
Antisense: 5¢-CACATGGCTATAAAATGGTTACATCTGTGT-3¢
SULT1 ST2 Sense: 5¢-TATGTAGGAGCTACAAGAAACATTGAAGGC-3¢
Antisense: 5¢-CAATTCTTACTAGCTGCAGGGAGGGTTGGT-3¢
SULT1 ST3 Sense: 5¢-GAATTGGCCCTAATTTGCACATTAAAGATA-3¢
Antisense: 5¢-GCCTGAAGTTTTTGGTTCACAGTGAAATTT-3¢
SULT1 ST4 Sense: 5¢-ACACTCTGAAGGGGAATTAGGATTAAGAAA-3¢
Antisense: 5¢-CTGACTATACAAGGCTGTGTGCCACAAAAC-3¢
SULT1 ST5 Sense: 5¢-GAAAACACATCACGTACCCTCCCTCTCTGCG-3¢
Antisense: 5¢-ACATCATGGTATATTATTCATTTAGCTGACACTTT-3¢
b-Actin Sense: 5¢-ATGGATGAGGAAATCGCTGCCCTGGTC-3¢
Antisense: 5¢-TTAGAAGCACTTCCTGTGAACGATGGA-3¢
S. Yasuda et al. A novel zebrafish cytosolic sulfotransferase
FEBS Journal 272 (2005) 3828–3837 ª 2005 FEBS 3835
SULTs. Total RNAs from zebrafish embryos and larvae at
different developmental stages were isolated using the TRI
Reagent based on the manufacturer’s instructions. Aliquots
containing 5 lg each of the total RNA preparations were
used for the synthesis of the first-strand cDNA using the
First-Strand cDNA Synthesis Kit (Amersham Biosciences),
according to manufacturer’s instructions. One-microliter
aliquots of the 33 lL first-strand cDNA solutions prepared
were used as the template for the subsequent PCR amplifi-

cation. PCRs were carried out in 25 lL reaction mixtures
using EX Taq DNA polymerase, in conjunction with gene-
specific sense and antisense oligonucleotide primers
(Table 4). Amplification conditions were 2 min at 94 °C fol-
lowed by 40 cycles of 30 s at 94 °C, 40 s at 60 °C, and
1 min at 72 °C. The final reaction mixtures were applied
onto a 1.2% agarose gel, separated by electrophoresis, and
visualized by ethidium bromide staining. As a control, PCR
amplification of the sequence encoding zebrafish b-actin
was concomitantly performed using the above-mentioned
first-strand cDNAs as templates, in conjunction with gene-
specific sense and antisense oligonucleotide primers
(Table 3) designed based on the reported zebrafish b-actin
nucleotide sequence (GenBank accession no. AF057040).
Miscellaneous methods
[
35
S]PAPS was synthesized from ATP and carrier-free
[
35
S]sulfate using the bifunctional human ATP sulfury-
lase ⁄ APS kinase and its purity determined as previously
described [27,28]. The [
35
S]PAPS synthesized was then
adjusted to the required concentration and specific activ-
ity by the addition of cold PAPS. SDS ⁄ PAGE was per-
formed on 12% polyacrylamide gels using the method of
Laemmli [29]. Protein determination was based on the
method of Bradford [30] with bovine serum albumin as

the standard.
Acknowledgements
This work was supported in part by a Grant-in-Aid
from the American Heart Association (Texas Affiliate)
and a UTHCT President’s Council Research Member-
ship Seed Grant.
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