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.. Deve
Trang 1cytosolic 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-T3), the major thyroid hormone, loses its affinity for thyroid hormone receptors upon sulfation, and sulfated l-T3 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: ming.liu@uthct.edu
(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-T3) 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-T3-sulfating activity of SULT1 ST5 revealed that Fe2+, Hg2+, Co2+, Zn2+, Cu2+, Cd2+ and Pb2+exhibited dramatic inhibitory effects, whereas Mn2+ 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 -T3, 3,3¢,5-triiodo- L -thyronine; L -T4, L -thyroxine; PAPS, 3¢-phosphoadenosine-5¢-phosphosulfate; SULT, sulfotransferase.
Trang 2inner 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
Trang 3new 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].
Trang 4amino 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-T3as 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-T3
as substrates are due to their structural differences
(b-naphthol being a neutral compound and l-T3, 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-T3, 3,3¢,5-triiodo-d-thyronine (d-T3),
3,3¢,5¢-triiodo-l-thyronine (l-rT3), l-Thyroxine (l-T4), 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-T3and l-T4 [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
pH
0 20 40 60 80 100
pH
B A
Fig 3 pH dependence of the sulfating activity of zebrafish SULT1 ST5 with (A) b-naphthol and (B) L - T3as 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.
Trang 5excel 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 Km 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
Vmax values showed smaller variations, with the lowest
activity for l-T4 Catalytic efficiency of the enzyme, as
reflected by Vmax⁄ Km, appeared to be comparable with
l-T3, d-T3, l-rT3or l-Thyronine as substrates, and
sig-nificantly lower with l-T4 as substrate It is worth
mentioning that human and rat thyroid
hormone-sulfating SULT1B1 also display Km values
(approxi-mately 40 lm) for l-T3 [18,19] comparable with that
(38.7 lm) of zebrafish SULT1 ST5 With b-naphthol
as substrate, zebrafish SULT1 ST5 showed Vmax and
Km 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-T3as the substrate were carried out in the absence or presence of various divalent metal cations at a concentration of
1 mm 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, Fe2+, Hg2+, Co2+, Zn2+, Cu2+,
Cd2+ and Pb2+ 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)1Æmg)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)1Æmg protein)1).
Endogenous compounds Specific activity (nmolÆmin)1Æmg)1) Xenobiotic compounds Specific activity (nmolÆmin)1Æmg)1)
3,3¢,5-Triiodo- D -Thyronine ( D -T3) 24.32 ± 1.47 Chlorogenic acid 23.52 ± 0.51
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 -rT3) 11.62 ± 0.48 Genistein 12.71 ± 0.17
Table 2 Kinetic constants of zebrafish SULT1 ST5 with L -T3, D -T3,
L -rT3, L -thyronine, and b-naphthol as substrates Data shown
repre-sent means ± SD derived from three determinations.
Substrate V max (min)1) K m (l M ) V max ⁄ K m
L -Thyronine 41.9 ± 2.3 114.2 ± 12.8 0.37
Trang 6on the sulfating activity of the zebrafish SULT, whereas
Mn2+ showed a significant stimulation Addition of
EDTA (at 2 mm concentration) restored the sulfating
activity of SULT1 ST5 in the cases of Zn2+, Cd2+, and
Pb2+ In contrast, the inhibition by Fe2+, Hg2+, Co2+,
and Cu2+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 -T3as 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)1Æmg)1)
Divalent cation
only (1 m M )
Divalent cation + EDTA (1 m M cations + 2 m M EDTA) Control 27.4 ± 2.3 (100%) 29.0 ± 0.4 (106%)
FeCl2 1.9 ± 0.1 (6.9%) 1.9 ± 0.1 (6.9%)
HgCl2 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%)
ZnCl2 1.7 ± 0.5 (6.2%) 31.5 ± 0.2 (115%)
CuCl2 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%)
MnCl2 30.9 ± 3.4 (113%) 32.1 ± 1.2 (117%)
CaCl2 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 m M 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).
Trang 7(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-T3,
d-T3, l-rT3, l-T4, 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) T4
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 [35S]sulfate, Ecolume scintillation cocktail, cortisone, corti-costerone, 4-androstene-3,17-dione, hydrocortisone, prog-esterone, pregnenolone, 17a-OH progprog-esterone, 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 94C and 25 cycles of 30 s at 94 C, 30 s at
59C, and 5 min at 72 C, followed by a 5 min extension
at 72C 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
Trang 8AGGACA-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 condiAmplifica-tions were 2 min at 94C and 20 cycles of 94 C
for 35 s, 60C 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 D600 0.6 in 1 L Luria–Bertani
med-ium supplemented with 60 lgÆmL)1ampicillin, 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 4C 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 CaCl2) containing
5 unitÆmL)1bovine thrombin Following 20 min incubation
at room temperature with constant agitation, the
prepar-ation was subjected to centrifugprepar-ation 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 [35S]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 [35S]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 28C, and terminated by heating at
100C for 2 min The precipitates formed were cleared by centrifugation, and the supernatant was subjected to the analysis of [35S]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-T3as substrate For the kinetic studies on the sulfation of l-T3, d-T3, l-rT3, l-T4, 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-T3as 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
Trang 9SULTs 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 94C
fol-lowed by 40 cycles of 30 s at 94C, 40 s at 60 C, and
1 min at 72C 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
[35S]PAPS was synthesized from ATP and carrier-free
[35S]sulfate using the bifunctional human ATP
sulfury-lase⁄ APS kinase and its purity determined as previously
described [27,28] The [35S]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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