Sulfation represents an important mechanism in vivo for
the biotransformation and excretion of a variety of
compounds(1, 2, 3) . Upon sulfation, some
peptides or proteins undergo changes in their biological activities to
fulfill particular biochemical/physiological needs(3) . For low
molecular weight xenobiotics or endogenous compounds such as steroid
hormones, catecholamines, and bile acids, sulfation may increase the
water solubility and facilitate their excretion from the body by
endowing them with (additional) charged
properties(1, 2) . In relation to this latter aspect,
the biochemistry and functional relevance of the excretion of free
tyrosine-O-sulfate (TyrS) (
)in mammalian urine have
remained intriguing questions for the past 40 years.
Free TyrS was
first reported to be present in human urine by Tallan et
al.(4) . Similar findings were made subsequently for other
mammalian species including rat, rabbit, and
mouse(5, 6) . Because none of the mammalian
arylsulfatases could effectively catalyze its
desulfation(7, 8) , the free TyrS produced by
mammalian cells has been generally considered a modified amino acid
destined to be excreted. Concerning the biochemical origin of the free
TyrS excreted, two distinct mechanisms for its generation have been
suggested: i) the enzymatic sulfation of L-tyrosine forming
free TyrS and ii) the turnover (degradation) of tyrosine sulfated
proteins, thereby releasing free TyrS. Aiming at demonstrating the
first of these two mechanisms, a great number of studies using various
cell homogenates or purified aryl sulfotransferases have, however,
persistently failed to reveal the enzymatic activity that catalyzes the
sulfation of free L-tyrosine(1, 9, 10, 11, 12) .
Among the different mammalian aryl sulfotransferases characterized,
only the rat liver type IV aryl sulfotransferase, also named the
``tyrosine-ester sulfotransferase,'' can use tyrosine esters
or N-terminally located tyrosine residues as substrates. This enzyme,
however, cannot catalyze the sulfation of unmodified L-tyrosine(13) . In view of the widespread occurrence
of the post-translational tyrosine sulfation among proteins of
multicellular eukaryotic organisms(14) , it has become
increasingly accepted that the free TyrS excreted in mammalian urine is
probably derived exclusively from the degradation of tyrosine sulfated
proteins (1, 6, 15, 16, 17) . In
support of this hypothesis, free Tyr[
S] was
shown to be generated when tyrosine
S-sulfated peptides or
proteins were either injected into rabbits (15) or added to the
medium of cultured cells(18) . However, to quantitatively
account for the excreted TyrS reported to be approximately 28
mg/day/normal adult human(4) , the turnover of a large amount
of tyrosine sulfated proteins would be needed. This has therefore
continued to raise the question whether the turnover of tyrosine
sulfated proteins is truly the sole source of the free TyrS produced
and released by mammalian animals.
By employing radioactive
3`-phosphoadenosine 5`-phospho[
S]sulfate
(PAP[
S]) as the sulfate donor, we have recently
obtained evidence showing the enzymatic sulfation of L-p-tyrosine in several mammalian cell
lines(19, 20) . We have further demonstrated that
3,4-dihydroxyphenyl-alanine (Dopa) and m-tyrosine can be
sulfated more efficiently by the sulfotransferase(s) present in the
cytosol of HepG2 human hepatoma cells. (
)An important
question therefore is whether, in mammalian cells, there is a single
enzyme catalyzing the sulfation of all Dopa and tyrosine isomers or if
there are distinct sulfotransferases responsible for the sulfation of
different Dopa and tyrosine isomers. Furthermore, is (are) the
enzyme(s) different from the previously reported sulfotransferase(s) (21, 22, 23, 24) ?
In this paper,
we report the purification, characterization, and molecular cloning of
a single species of the enzyme, designated the ``Dopa/tyrosine
sulfotransferase,'' from the rat liver. Comparison of the
nucleotide sequence and the deduced amino acid sequence of the cloned
cDNA with sequences of known aryl (phenol) sulfotransferases, as well
as the data from the biochemical characterization, demonstrated the
Dopa/tyrosine sulfotransferase to be a novel enzyme.
EXPERIMENTAL PROCEDURES
Materials
L-Dopa, D-Dopa, L-p-tyrosine, D-p-tyrosine, DL-m-tyrosine, DL-o-tyrosine, ninhydrin, aprotinin, antipain,
benzamidine, soybean trypsin inhibitor, phenylmethylsulfonyl fluoride,
2,6-dichloro-4-nitrophenol (DCNP), ATP, adipic acid
dihydrazide-agarose, 5`-AMP, Hepes, Ches, Taps,
3-[dimethyl(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic
acid (Ampso), and Caps were products of Sigma. PAPS was a generous gift
from Unitika, Ltd. (Japan). L-Tyrosine (disodium salt) was
purchased from Research Organics, Inc. Bio-Gel HTP hydroxylapatite and
DEAE Bio-Gel A were from Bio-Rad Laboratories. ATP-agarose was prepared
by coupling sodium periodate-oxidized ATP to adipic acid
dihydrazide-agarose using the procedure of Lamed et al.(25) . TyrS, dopamine-O-sulfate, and a mixture of L-Dopa 3-O-sulfate and L-Dopa
4-O-sulfate (collectively referred to as DopaS) were
synthesized according to the procedure developed by
Jevons(26) . Rat liver Lambda ZAP II cDNA library and XL1-Blue
MRF` Escherichia coli host strain were purchased from
Stratagene. SuperScript Preamplification System and LipofectAMINE were
from Life Technologies, Inc. Taq polymerase was purchased from
Perkin-Elmer. Cycle sequencing kits were products of Applied
Biosystems, Inc. The mammalian expression vector, pMSG
CMV, was
kindly provided by Dr. Nakayama at Miyazaki Medical College. BcaBEST
labeling kit, Exonuclease III, mung bean nuclease, and DNA ligation kit
were products of Takara Shuzo. All restriction endonucleases were from
New England Biolabs. Carrier-free sodium
[
S]sulfate and
[
-
P]dCTP (3,000 Ci/mM) were from
ICN Biomedicals. Chromatogram cellulose TLC plates were from Eastman
Kodak Company. COS-7 SV40 transformed African green monkey kidney cells
(ATCC CRL 1651) were obtained from the American Type Culture
Collection. Rabbit antiserum against the rat liver Dopa/tyrosine
sulfotransferase was prepared according to the procedure previously
described(27) . All other chemicals were of the highest grades
commercially available.
Preparation of the Rat Liver Cytosol
Rat
liver (190 g) rinsed thoroughly with ice-cold phosphate-buffered saline
was ground through a USA standard testing sieve (35 mesh) and made into
a 1:2 (w/v) suspension in a buffer A containing 10 mM Tris-HCl
(pH 7.4), 250 mM sucrose, 10 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride. The preparation was homogenized by
20 strokes in a tight fitting Teflon glass homogenizer placed on ice.
The crude homogenate was centrifuged at 10,000
g for
20 min at 4 °C, and the supernatant collected was further subjected
to ultracentrifugation at 140,000
g for 2 h at 4
°C. The supernatant containing cytosolic proteins was used for the
purification as described below.
Purification of the Rat Liver Dopa/Tyrosine
Sulfotransferase
Unless otherwise indicated, all buffer
solutions used in the purification were at pH 7.4 and supplemented with
1 mM dithiothreitol. All operations described below were
carried out at 4 °C.
First Bio-Gel HTP Hydroxylapatite Column
Chromatography
The rat liver cytosol (400 ml) was applied
onto a Bio-Gel HTP column (4.5
25 cm) pre-equilibrated with 10
mM Tris-HCl. After loading, 200 ml of 10 mM Tris-HCl
was passed through the column to remove unbound proteins. The proteins
bound on the column were eluted using a linear potassium phosphate
buffer gradient composed of 350 ml each of 10 mM and 400
mM potassium phosphate buffer. The eluted fractions (spanning
from 150 to 300 mM) containing the Dopa/tyrosine
sulfotransferase activity were combined and dialyzed overnight against
10 mM Tris-HCl.
DEAE Bio-Gel A Anion Exchange
Chromatography
The dialyzed fraction was applied onto a
DEAE Bio-Gel A column (4.5
25 cm) pre-equilibrated with 10
mM Tris-HCl. The bound proteins were eluted with an NaCl
gradient composed of 350 ml each of 0 mM and 350 mM
NaCl solution containing 10 mM Tris-HCl. The eluted fractions
(spanning from 130 to 250 mM) containing the Dopa/tyrosine
sulfotransferase activity were combined and dialyzed overnight against
10 mM Tris-HCl.
First ATP-Agarose Column
Chromatography
The dialyzed fraction was loaded onto an
ATP-agarose column (2.5
4 cm). After loading, the bound
proteins were eluted with an NaCl gradient composed of 120 ml each of 0
and 350 mM NaCl solution containing 10 mM Tris-HCl.
The eluted fractions (spanning from 125 to 200 mM) containing
the Dopa/tyrosine sulfotransferase activity were pooled.
Second Bio-Gel HTP Column
Chromatography
The Dopa/tyrosine sulfotransferase eluate
from the first ATP agarose column was directly applied through a
Bio-Gel HTP hydroxylapatite column (2.5
10 cm) pre-equilibrated
with 10 mM Tris-HCl. The bound proteins were eluted with a
linear gradient composed of 150 ml each of 10 and 350 mM of
potassium phosphate buffer. The fractions (spanning from 150 to 210
mM) containing the Dopa/tyrosine sulfotransferase activity
were combined and dialyzed overnight against 10 mM Tris-HCl.
Second ATP-Agarose Column
Chromatography
The eluate from the second Bio-Gel HTP
column was applied onto a second ATP-agarose column (1.5
5 cm)
pre-equilibrated with 10 mM Tris-HCl. The bound proteins were
eluted with a linear gradient composed of 100 ml each of 0 µM and 75 µM PAPS in 10 mM Tris-HCl. The
Dopa/tyrosine sulfotransferase eluted throughout the entire PAPS
concentration range was found to be electrophoretically homogeneous.
Enzymatic Assay
The activities of the
Dopa/tyrosine sulfotransferase were assayed using
PAP[
S] as the sulfate donor. The standard assay
mixture, with a final volume of 50 µl, contained 50 mM Ampso-NaOH (pH 9.25), 250 mM sucrose, 25 mM NaF,
1 mM 5`-AMP, protease inhibitors (30 µg/ml aprotinin, 30
µg/ml antipain, 300 µg/ml benzamidine, and 30 µg/ml soybean
trypsin inhibitor), 14 µM PAP[
S]
(4.4 Ci/mmol), and 1 mM substrate (Dopa, tyrosine, etc.). The
reaction was started by the addition of the enzyme preparation, allowed
to proceed for 60 min at 37 °C, and terminated by heating at 100
°C for 3 min. The precipitates formed were cleared by
centrifugation. The clear supernatant was subjected to the analysis of
S-sulfated product as described below. For the K
determination, the enzymatic assay was performed
at a fixed 14 µM concentration of
PAP[
S] with varying concentrations of the
substrate tested. To examine the pH dependence of the Dopa/tyrosine
sulfotransferase activity, different buffers (50 mM Hepes at
pH 7.0, 7.5, or 8.0; Taps at pH 8.0, 8.25, 8.5, 8.75, or 9.0; Ampso at
pH 8.5, 8.75, 9.0, 9.25, or 9.5; Ches at pH 9.0, 9.25, 9.5, 9.75, or
10.0; or Caps at pH 10, 10.25. 10.5, or 11) instead of 50 mM Ampso-NaOH (pH 9.25) were used in the reaction mixtures.
Analysis of
S-Sulfated
Compound
For the analysis of Tyr[
S],
Dopa[
S], dopamine[
S], or
other
S-sulfated products, 5 µl of the clear reaction
mixture were mixed with 10 µg of synthetic standard (TyrS, DopaS,
or dopamine-O-sulfate), spotted onto a 20
20-cm
cellulose TLC plate, and analyzed according to a two-dimensional
thin-layer separation procedure previously developed(28) .
Briefly, the plate was first subjected to high voltage electrophoresis
(1,000 V for 70 min) in 7.8% (v/v) acetic acid/2.5% (v/v) 88% formic
acid (pH 1.9). After electrophoresis, the plate was air-dried and
subjected in the second dimension to ascending chromatography in
n-butanol/88% formic acid/isopropanol/H
O (3:1:1:1,
v/v/v/v). Upon completion of the chromatography, the plate was sprayed
with ninhydrin solution (0.5% in acetone). The ninhydrin-stained spot
of the sulfated product was scraped off, suspended in 0.5-ml aliquots
of H
O, and mixed with 4 ml of scintillation mixture
(Ecolume, ICN Radiochemicals). The radioactivity associated with
Tyr[
S], Dopa[
S],
dopamine[
S], or other
S-sulfated
product was counted.
N-terminal and Internal Partial Amino Acid Sequence
Analysis
N-terminal amino acid sequence determination was
performed according to the method of Matsudaira(29) . Briefly,
the purified Dopa/tyrosine sulfotransferase was subjected to SDS-PAGE (30) and electrotransferred onto a Millipore Immobilon-P
membrane. The blotting was performed at a constant 200 mA for 6 h
in 10 mM Caps-NaOH (pH 11). The blotted membrane was briefly
stained with 0.1% Ponceau S in 5% acetic acid to reveal the protein
band. After extensive washing with water, the membrane piece containing
the bound Dopa/tyrosine sulfotransferase was used for the analysis of
the N-terminal sequence. To determine the internal amino acid
sequences, the Dopa/tyrosine sulfotransferase bound on the Immobilon-P
membrane was digested with endoproteinase Lys-C, and the peptide
fragments were purified using high performance liquid chromatography
(HPLC). Purified peptide fragments were subjected to the N-terminal
sequence determination according to the method of Matsudaira (29) . The amino acid sequencing described above was performed
using the sequencing facilities at The Rockefeller University.
Cloning of the Rat Liver Dopa/Tyrosine
Sulfotransferase cDNAs
The reverse
transcriptase-polymerase chain reaction (PCR) technique was employed to
prepare the DNA probe used for cDNA library screening. Total RNA was
isolated from the liver specimen of a 3-month-old male Sprague-Dawley
rat using the method of Chomczynski and Sacchi(31) . The first
strand cDNA was synthesized using the SuperScript Preamplification
system (Life Technologies, Inc.) with oligo(dT) as the primer. With the
first strand cDNA as the template, a PCR reaction performed in a
50-µl reaction mixture using the Taq polymerase was
carried out with 5`-TGGGA(T/C)AA(T/C)AA(A/G)TG(T/C)AA(A/G)ATG3` as the
sense primer and 5`-CATIGT(A/G)AA(A/G)TA(A/G)TT(T/C)TTCCA-3` as the
antisense primer. Amplification conditions were 30 cycles of 30 s at 94
°C, 30 s at 50 °C, and 1 min at 72 °C. The reaction mixture
was applied onto a 2% agarose gel, separated by electrophoresis, and
visualized by ethidium bromide staining. A major 452-nucleotide PCR
product was detected and excised from the gel, and the DNA was isolated
by spin filtration. Upon verification of the sequence by cycle
sequencing, the purified PCR product was subcloned into the EcoRV site of pBluescript SK(-) and transformed into E. coli XL1-Blue MRF`. The recombinant plasmid was purified
using alkali lysis method, and the PCR product insert was cut out by
digestion with EcoRI and HindIII. The insert DNA was
purified by agarose gel electrophoresis followed by spin filtration.
The purified insert DNA was labeled with
[
-
P]dCTP using random primers, and the
labeled DNA was used as the probe for screening the cDNA encoding the
Dopa/tyrosine sulfotransferase in a rat liver Lambda ZAP II cDNA
library. Approximately 3
10
plaques from the
library were screened with
P-labeled DNA probe by
hybridization on nylon membrane filters. Nylon membrane filter replicas
of plaques/XL1-Blue MRF` grown on 150-mm Petri dishes upon
prehybridization for 2 h at 65 °C were hybridized with
P-labeled DNA probe overnight at 65 °C. Hybridized
membranes were washed once with 2
SSC (8.765 g of NaCl and 4.41
g of sodium citrate) plus 0.1% SDS and twice with 0.1
SSC plus
0.1% SDS at 65 °C, followed by autoradiography to reveal the
positive cDNA clones.
DNA Sequence Determination and
Analysis
The positive cDNA clones were subjected to
double-stranded sequencing according to the cycle sequencing method
using Taq dye primer cycle sequencing kits (Applied
Biosystems, Inc.) with -21M13 or M13 reverse primer. Serial
deletional mutants were prepared by using Exonuclease III and mung bean
nuclease according to the method of Henikoff(32) . The
nucleotide sequence, as well as the deduced amino acid sequence, of the
full-length cDNA was analyzed using the E-mail servers at NCBI and EMBL
for sequence homology to other known aryl sulfotransferases.
Transient Expression of the Dopa/Tyrosine
Sulfotransferase in COS-7 Cells
COS-7 cells, normally
maintained in Dulbecco's modified Eagle's medium containing
10% fetal bovine serum, were used as the host cells for the expression
of the enzyme. Dishes (60 mm) of COS-7 cells were individually
transfected with 2 µg of pMSG
CMV vector only or a
pMSG
CMV derivative containing the sequence of D/TST-11 using the
LipofectAMINE-mediated procedure. Incubation was for 18 h at 37 °C,
according to the manufacturer's instructions. The transfected
cells were incubated at 37 °C in Dulbecco's modified
Eagle's medium containing 10% fetal bovine serum. At the end of a
48-h incubation, the cells were washed twice with phosphate-buffered
saline and homogenized in buffer A containing 10 mM Tris-HCl
(pH 7.4), 250 mM sucrose, 10 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride. Aliquots of the homogenates
prepared were assayed for the Dopa/tyrosine sulfotransferase activities
or subjected to the Western blot analysis for the presence of the
immunoreactive enzyme using rabbit anti-rat liver Dopa/tyrosine
sulfotransferase antiserum. The conditions for the Western blot
analysis were as described previously(33) .
Miscellaneous
Methods
PAP[
S] (15 Ci/mmol) was
synthesized from ATP and [
S]sulfate using the
sulfate-activating enzymes ATP sulfurylase and adenosine
5`-phosphosulfate kinase from Bacillus stearothermophilus as
described previously(34) . SDS-PAGE was performed on 10%
polyacrylamide gels using the method of Laemmli(30) . The
native molecular weight (M
) of the purified
Dopa/tyrosine sulfotransferase was determined by gel filtration
chromatography using a Sephacryl S-200 column (2.6
90 cm).
Molecular weight standards including bovine serum albumin (M
67,000), ovalbumin (M
43,000), carbonic anhydrase (M
29,000),
chymotrypsinogen A (M
25,800), and cytochrome c (M
12,400) were used for calibration.
Protein determination was based on the method of Bradford (35) with bovine serum albumin as the standard.
RESULTS
Purification of the Rat Liver Dopa/Tyrosine
Sulfotransferase
Preliminary experiments showed that
similar to several mammalian cell lines previously
studied(19, 20) , the Dopa/tyrosine sulfotransferase
was present predominantly in the cytosolic fraction of the rat liver.
The specific activity of the enzyme purified from the rat liver
cytosol, with L-Dopa as the substrate, was determined to be
2,153 pmol/min/mg protein, indicating a 760-fold purification over its
specific activity in the rat liver cytosol (Table 1). It was
noted that, during the elution from the DEAE Bio-Gel A anion exchange
chromatography step, the Dopa/tyrosine sulfotransferase activity was
well separated from the major phenol sulfotransferase activity (data
not shown). As shown in Fig. 1, the purified Dopa/tyrosine
sulfotransferase migrated as a single protein band upon SDS-PAGE under
reducing conditions. A key step during the purification of the
Dopa/tyrosine sulfotransferase was the first ATP-agarose affinity
chromatography, which furnished a considerably greater efficiency of
purification than the other two types of column chromatography (cf.Fig. 1, lanes 2-4). A second ATP-agarose
affinity chromatography was used in the final step of purification to
remove small amounts of contaminating proteins and, at the same time,
improve the specific activity of the purified Dopa/tyrosine
sulfotransferase by removing denatured enzyme molecules that had lost
the PAPS (nucleotide) binding activity. In this last purification step,
the Dopa/tyrosine sulfotransferase was eluted not with an NaCl gradient
but with a PAPS gradient. Because PAPS is a co-substrate needed during
the sulfation reaction, its presence in the purified Dopa/tyrosine
sulfotransferase fraction may also help stabilize the Dopa/tyrosine
sulfotransferase. No significant loss of the enzyme activity was
observed during several weeks of storage at 4 °C. However, freezing
and thawing readily caused the purified Dopa/tyrosine sulfotransferase
to lose its enzymatic activity.
Figure 1:
SDS gel electrophoretic patterns of the
Dopa/tyrosine sulfotransferase-containing fractions collected at
different purification steps. Samples prepared were subjected to
SDS-PAGE followed by Coomassie Blue staining. Lane 1, rat
liver cytosol; lane 2, eluate from the first Bio-Gel HTP
hydroxylapatite column; lane 3, eluate from DEAE Bio-Gel A
column; lane 4, eluate from the first ATP-agarose column; lane 5, eluate from the second Bio-Gel HTP hydroxylapatite
column; lane 6, eluate from the second ATP-agarose
column.
Characterization of the Purified Rat Liver
Dopa/Tyrosine Sulfotransferase
The purified rat
liver Dopa/tyrosine sulfotransferase was subjected to characterization
with respect to its physicochemical and enzymatic properties as
described below.
Molecular Weight
Based on its
electrophoretic mobility relative to the molecular weight standards
co-electrophoresed during the SDS-PAGE under reducing conditions (Fig. 1), the minimum molecular weight of the purified
Dopa/tyrosine sulfotransferase was determined to be approximately
33,000. Gel filtration chromatography revealed a molecular weight of
approximately 34,000 for the native Dopa/tyrosine sulfotransferase
(figure not shown). These results combined indicate that the purified
Dopa/tyrosine sulfotransferase was present in monomeric form.
Partial Amino Acid Sequences
Repeated
attempts to determine the N-terminal amino acid sequence of the
purified Dopa/tyrosine sulfotransferase were unsuccessful, indicating
that the enzyme, similar to some other cytosolic
sulfotransferases(36, 37) , is N-blocked.
Upon digestion with endoproteinase Lys-C, three proteolytic fragments
were purified by HPLC and sequenced. Fig. 2shows the alignment
of the three partial amino acid sequences with the homologous sequences
of other sulfotransferases reported previously. Although the
Dopa/tyrosine sulfotransferase displayed higher degrees of sequence
homology to phenol (aryl) sulfotransferases from mammalian animals,
some degrees of homology to the sequences of even the two plant
flavonol sulfotransferases (fcFST3 and fcFST4`) were observed. Among
the 18 sulfotransferases listed for comparison, one unknown rat liver
sulfotransferase (cDNA clone ST1B1; previously identified by Yamazoe et al.(38) ) contains sequences identical to the three
partial amino acid sequences determined for the purified Dopa/tyrosine
sulfotransferase. However, the deduced amino acid sequence of the
full-length cDNA encoding the Dopa/tyrosine sulfotransferase (see
below) was found to differ from that of clone ST1B1 at position 68
(with a glycine residue instead of a glutamic acid residue).
Figure 2:
Comparison of the partial amino acid
sequences of the rat liver Dopa/tyrosine sulfotransferase to the
homologous sequences of other cytosolic sulfotransferases. Boxed
residues indicate the amino acid residues identical to those of
the rat liver Dopa/tyrosine sulfotransferase. The enzymes listed
include the rat liver phenol sulfotransferase (rPST)(55) , mouse liver phenol sulfotransferase (mPST)(56) , human liver thermostable phenol
sulfotransferase (hPST(TS))(57) , human liver
thermolabile phenol sulfotransferase (hPST(TL))(58) ,
rat liver ST1B1 gene product (r??ST)(38) , rat liver
hydroxyarylamine sulfotransferase (rHAST)(59) , bovine
placental estrogen sulfotransferase (bEST)(60) ,
guinea pig adrenocortical estrogen sulfotransferase (gpEST)(61) , rat liver estrogen sulfotransferase (rEST)(62) , human liver estrogen sulfotransferase (hEST)(63) , human liver hydroxysteroid
sulfotransferase (hHSST)(64) , mouse liver
hydroxysteroid sulfotransferase (mHSST)(65) , guinea
pig adrenal hydroxysteroid sulfotransferase (gpHSST)(66) , rat liver senescence marker protein (rSMP2)(67) , rat liver hydroxysteroid
sulfotransferase I (rHSST1)(68) , rat liver
hydroxysteroid sulfotransferase II (rHSST2)(69) , Flaveria chloraefolia flavonol 3-sulfotransferase (fcFST3), and flavonol 4`-sulfotransferase (fcFST4`)(70) .
Enzymatic Sulfation of Tyrosine and Dopa
Isomers
During the purification process, the activity of
the Dopa/tyrosine sulfotransferase was determined using DL-m-tyrosine or L-Dopa as substrates. The
purified enzyme was found to display enzymatic activities toward all
tyrosine and Dopa isomers tested except DL-o-tyrosine.
pH Optimum
As shown in Fig. 3,
with L-Dopa as the substrate, the pH optimum of the purified
Dopa/tyrosine sulfotransferase was determined to be 9.25. Although
considerably lower enzymatic activities were observed in Ches buffer
when compared with other buffers (Taps, Ampso, and Caps) at
corresponding pH values, the pH optimum remained unchanged at 9.25.
Figure 3:
pH dependence of the purified
Dopa/tyrosine sulfotransferase activity. The Dopa/tyrosine
sulfotransferase 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.
Substrate Specificity
To study further
the substrate specificity of the purified Dopa/tyrosine
sulfotransferase, tyrosine and Dopa isomers, thyroid hormones and their
analogs, and compounds previously shown to be substrates for
``simple phenol (P) form'' or ``monoamine (M)
form'' phenol sulfotransferases were tested in the standard assay
described under ``Experimental Procedures.'' Results obtained
are compiled in Table 2. Of the Dopa and tyrosine isomers tested,
both L-Dopa and D-Dopa gave much higher specific
activities than those obtained using the tyrosine isomers. Among the
tyrosine isomers, the specific activity obtained with DL-m-tyrosine as the substrate was 2 orders of
magnitude higher than those obtained with L-p-tyrosine or D-p-tyrosine as the
substrate. Both p-nitrophenol, a substrate for the P form
phenol sulfotransferase, and dopamine, a substrate for the M form
phenol sulfotransferase, yielded specific activities of the same order
of magnitude as those obtained using the Dopa isomers.
Kinetic Parameters
To determine the
apparent K
values of the Dopa/tyrosine
sulfotransferase for L-Dopa and D-Dopa, the enzymatic
assays were performed in the presence of varying concentrations of L- or D-Dopa with a constant 14 µM of
PAPS. Results obtained indicated that in the low concentration range, L-Dopa was sulfated at higher rates than was D-Dopa.
When the concentration was above 0.25 mM, however, higher
rates of sulfation were observed for D-Dopa. Based on the
Lineweaver-Burk plots plotted using the data obtained, the apparent K
values of the Dopa/tyrosine sulfotransferase for L-Dopa and D-Dopa were determined to be 0.76 and 3.44
mM, respectively (Table 3). Their corresponding V
values were calculated to be, respectively,
3,521 and 13,699 pmol/min/mg protein. The kinetic constants for p-nitrophenol and dopamine, two commonly used substrates for
phenol sulfotransferases, were also determined. As shown in Table 3, the apparent K
values of the
Dopa/tyrosine sulfotransferase for p-nitrophenol and dopamine,
calculated from the Lineweaver-Burk plots, were 30.9 and 0.24
mM, respectively. Their corresponding V
values were calculated to be, respectively, 125,000 and 5,435
pmol/min/mg protein (Table 3). It is worthwhile pointing out that
although the apparent K
values for L-Dopa, D-Dopa, and p-nitrophenol are
different, their calculated V
/K
values are similar. Because the same concentration of the
purified enzyme was used in these experiments, the K
/K
or the catalytic
efficiency of the Dopa/tyrosine sulfotransferase for these substrates
is almost the same. With triiodothyronine as the substrate, a strong
substrate inhibition effect was observed when the concentration of
triiodothyronine was greater than 0.5 mM.
Effects of Cationic Salts on the Enzymatic
Activity
The effects of cationic salts on the Dopa/tyrosine
sulfotransferase activity were measured. The addition of different
divalent cationic salts, such as MgCl
, MnCl
,
and CoCl
, exerted virtually no effects on the activities of
the purified enzyme. With L-Dopa as the substrate, increasing
concentrations of NaCl or KCl resulted in the inhibition of the
Dopa/tyrosine sulfotransferase activity. A 50% decrease in the
enzymatic activity was found when the concentration of NaCl or KCl in
the reaction mixture was increased to 200 mM.
Effect of DCNP on the Enzymatic
Activity
DCNP, a commonly used inhibitor for aryl (phenol)
sulfotransferases(1, 2, 21) , was tested for
its inhibitory effect on the Dopa/tyrosine sulfotransferase activity.
It was found that the purified Dopa/tyrosine sulfotransferase was
inhibited by submillimolar levels of DCNP, with an IC
of
approximately 7
10
M. A similar
IC
value for DCNP has previously been determined for the
thermolabile M form but not the thermostable P form of human phenol
sulfotransferase(1, 22) .
Molecular Cloning of the Rat Liver
Dopa/Tyrosine Sulfotransferase
To determine
unequivocally the identity of the rat liver Dopa/tyrosine
sulfotransferase as a novel enzyme, we have cloned and sequenced its
cDNA. Repeated screening of the rat liver Lambda ZAP II cDNA library
yielded 14 positive cDNA clones. The three largest cDNA inserts,
ranging from 1,500 to 2,400 base pairs, were subjected to preliminary
nucleotide sequencing. The analysis revealed that one of them,
designated clone D/TST-11, contained an initiation codon and a 3`
region encoding the poly(A) tail and thus appeared to contain the
full-length sequence. The nucleotide and deduced amino acid sequences
are presented in Fig. 4. Because the purified Dopa/tyrosine
sulfotransferase was found to be N-blocked, the ATG codon
encoding the N-terminal methionine residue was assigned based on i) the
predicted molecular weight that matches the data from SDS-PAGE and gel
filtration chromatography and ii) the sequence alignment in comparison
with known aryl sulfotransferases (see below). The open reading frame,
beginning at base residue 91, encompasses 897 nucleotides and encodes a
299-amino acid polypeptide. The predicted molecular weight, 34,762, is
in agreement with the results (33,000 and 34,000, respectively)
obtained through SDS-PAGE and gel filtration chromatography using the
purified Dopa/tyrosine sulfotransferase. The termination codon, located
at nucleotide residues 988-990, was followed by a 292-nucleotide
3`-untranslated sequence that includes a poly(A) tract. Two
polyadenylation signals (ATTAAA) (39) located 180 and 19
nucleotides, respectively, upstream from the poly(A) tract were found.
The authenticity of the cDNA was indicated by the inclusion of the
three partial amino acid sequences obtained through direct amino acid
sequencing of the purified Dopa/tyrosine sulfotransferase, and by the
expression of the functionally active recombinant enzyme that
cross-reacted with the antiserum against the purified Dopa/tyrosine
sulfotransferase (see below). As shown in Fig. 5, the deduced
amino acid sequence of the rat liver Dopa/tyrosine sulfotransferase
(rD/TST) cDNA displays 72.2/52.6, 72.6/52.4, 72.5/51.9, and 71.9/53.6%
similarity/identity to the amino acid sequences of rat liver phenol
sulfotransferase, human thermolabile phenol sulfotransferase, human
thermostable phenol sulfotransferase, and mouse phenol sulfotransferase
(based on analysis using the Program Manual for the Wisconsin package,
version 8). The deduced amino acid sequence of the Dopa/tyrosine
sulfotransferase differed from that of clone ST1B1 (38) by a
glycine residue instead of a glutamic acid residue at position 68.
Figure 4:
Nucleotide and deduced amino acid
sequences of the rat liver Dopa/tyrosine sulfotransferase cDNA.
Nucleotides are numbered in the 5` to 3` direction with the adenosine
of the translation initiation codon designated as +1. The
translation stop codon is indicated by an asterisk. The
polyadenylation signals and the two primer sequences used in the
reverse transcriptase-PCR are underlined. The three partial
amino acid sequences determined using the purified enzyme are double underlined.
Figure 5:
Amino acid sequence comparison of the rat
liver Dopa/tyrosine sulfotransferase with rat liver phenol
sulfotransferase (rPST), human liver thermolabile phenol
sulfotransferase (hPST(TL)), human liver thermostable phenol
sulfotransferase (hPST(TS)), mouse liver phenol
sulfotransferase (mPST), rat liver hydroxylamine
sulfotransferase (rHAST), rat liver estrogen sulfotransferase (rEST), human liver estrogen sulfotransferase (hEST),
guinea pig adrenocortical estrogen sulfotransferase (gpEST),
and bovine placental estrogen sulfotransferase (bEST). The
sequences are aligned with the N-terminal methionine residue of the rat
liver Dopa/tyrosine sulfotransferase designated as position 1. Residues
conserved among at least 5 out of the 10 sulfotransferases are shown in reverse print. See the legend to Fig. 2for
references.
Expression of the Cloned Rat Liver Dopa/Tyrosine
Sulfotransferase in COS-7 Cells
The recombinant protein was
expressed in COS-7 cells and subjected to functional characterization
and examination of the immunoreactivity toward the rabbit antiserum
against the purified rat liver Dopa/tyrosine sulfotransferase. As shown
in Fig. 6, a 33-kDa protein cross-reactive toward the antiserum
against the purified Dopa/tyrosine sulfotransferase was expressed
specifically when the COS-7 cells were transfected with an expression
vector (pSMG
CMV) that contained the full-length cDNA encoding the
Dopa/tyrosine sulfotransferase. When the cell homogenates were assayed
for the Dopa/tyrosine sulfotransferase activity, it was found that the
sample prepared from the cells transfected with the expression vector
inserted with the full-length cDNA indeed exhibited a highly elevated
Dopa/tyrosine sulfotransferase activity (Table 4).
Figure 6:
Expression of the recombinant rat liver
Dopa/tyrosine sulfotransferase in COS-7 cells. The figure shows the
autoradiograph taken from the Immobilon-P membrane used in the Western
blot analysis for the presence of the recombinant rat liver
Dopa/tyrosine sulfotransferase. Samples analyzed were homogenates
prepared from untransfected COS-7 cells (lanes 1 and 2), COS-7 cells transfected with pSMG
CMV only (lanes
3 and 4), and COS-7 cells transfected with pSMG
CMV
harboring the full-length cDNA encoding the Dopa/tyrosine
sulfotransferase (lanes
5-8).
DISCUSSION
Since the discovery of the excretion of free TyrS in human
urine (4) , the questions concerning the functional relevance
and the formation of TyrS by the enzymatic sulfation of tyrosine have
remained unresolved for nearly forty years. A consensus formed
(following Huttner's discovery of the widespread occurrence of
the post-translational tyrosine sulfation of eukaryotic
proteins(14) ) is that free TyrS is generated primarily through
the turnover of tyrosine sulfated proteins in vivo. We have
indeed demonstrated earlier (16) that exogenous tyrosine
S-sulfated proteins added to the medium could be
endocytosed by cultured cells and degraded intracellularly to generate
free Tyr[
S]. A metabolic labeling experiment
using the same cells, however, showed a considerable discrepancy
between the amount of the free Tyr[
S] generated
and the amount of tyrosine
S-sulfated proteins turned over
during a 48-h time course monitored. This finding had prompted our
interest in investigating further the possibility of the sulfation of
free tyrosine.
In our recent studies(19, 20) , we
have obtained conclusive evidence that sulfation of L-p-tyrosine does occur in several mammalian cell
lines. It is, however, unclear why mammalian cells should carry out the
sulfation of an amino acid needed for protein synthesis. To convert L-p-tyrosine to L-p-TyrS, a
compound destined for excretion(4) , would seem to be
counterproductive in terms of cellular economy. The question that
should be raised then is whether L-p-tyrosine truly
represents the physiological substrate of the enzyme, initially
designated the ``tyrosine sulfotransferase''(19) .
Using HepG2 human hepatoma cells as a model, we have demonstrated in a
more recent study
that other tyrosine derivatives, e.g. Dopa and m-tyrosine isomers, are in fact better
substrates for sulfation than is L-p-tyrosine. These
results are summarized in the schematic diagram shown in Fig. 7.
In view of the large number of aryl sulfotransferases that have been
identified, it is tempting to ask whether, in mammalian cells, there is
one single enzyme catalyzing the sulfation of all Dopa and tyrosine
isomers or instead that there are multiple sulfotransferases
responsible for the sulfation of individual Dopa and tyrosine isomers.
To find an answer to this question, we have decided to isolate the
enzyme(s) from rat liver for further characterization.
Figure 7:
Schematic diagram illustrating the
sulfation of Dopa and tyrosine isomers.
The rat liver
has been more exhaustively studied with regard to aryl
sulfotransferases (1, 2) than other mammalian tissues.
At least six different types of aryl sulfotransferases have been
identified and characterized(13, 23, 24) .
The rat liver is therefore the best model for investigating whether the
Dopa/tyrosine sulfotransferase activities are associated with a new
enzyme(s) or instead a known aryl sulfotransferase(s). In the present
study, a single Dopa/tyrosine sulfotransferase was purified from the
rat liver. It was noted that for all five chromatography steps during
the purification, single symmetric peaks of elution of the
Dopa/tyrosine sulfotransferase, as monitored by standard assays using
either L-Dopa or DL-m-tyrosine as substrate, were
observed. The existence of multiple enzymes catalyzing the sulfation of
individual Dopa and tyrosine isomers, therefore, seemed unlikely.
SDS-PAGE and gel filtration chromatography revealed the enzyme to be
present in the monomeric form. In contrast to these data obtained with
the purified Dopa/tyrosine sulfotransferase, most, if not all, of the
known aryl sulfotransferases were shown to be present in the dimeric
form(21, 24) . The purified rat liver Dopa/tyrosine
sulfotransferase was found to be capable of catalyzing the sulfation of
all Dopa and tyrosine isomers, except DL-O-tyrosine.
The specific activity of the purified enzyme with L-Dopa as
the substrate was 2,153.4 pmol/min/mg protein. This value is
considerably lower than those previously reported for phenol
sulfotransferases from rat liver with either simple phenols or
monoamines as
substrates(1, 2, 21, 22) . However,
some sulfotransferases that utilize endogenous compound as substrates, e.g. the tyrosylprotein sulfotransferase (5,700 pmol/min/mg) (40) and the dopamine-sulfating sulfotransferase (7,735
pmol/min/mg) (41) , also displayed specific activities in the
same order of magnitude as that determined for the Dopa/tyrosine
sulfotransferase. Furthermore, because tyrosine and Dopa are important
precursors for the synthesis of proteins and/or catecholamines, there
may be regulatory mechanisms in vivo for the Dopa/tyrosine
sulfotransferase activity. Thyroid hormones triiodothyronine and
thyroxine, as well as dopamine and p-nitrophenol, could also
be used as substrates by the purified Dopa/tyrosine sulfotransferase.
Although the broad substrate specificity seems to be a rule rather than
exception for aryl sulfotransferases that have been studied, it should
be pointed out that these latter compounds (thyroid hormones, dopamine,
and p-nitrophenol) are more effectively used by other aryl
(phenol) sulfotransferases (42, 43) with K
values 2-3 orders of magnitude lower than
those determined for the purified rat liver Dopa/tyrosine
sulfotransferase. Furthermore, the Dopa/tyrosine sulfotransferase
represents the only known enzyme that is capable of catalyzing the
sulfation of Dopa and tyrosine isomers. In contrast to the HepG2
Dopa/tyrosine sulfotransferase, which showed higher activities toward D-form Dopa and tyrosine isomers and a remarkable divalent
cation dependence,
the rat liver enzyme displayed higher
activities toward the L-form substrates and showed no
significant changes in activity in the presence of a variety of
divalent cations.
Initial attempts to determine the N-terminal amino
acid sequence of the purified Dopa/tyrosine sulfotransferase showed it
to be N-blocked. Three internal partial amino acid sequences were
obtained by sequencing the HPLC-purified fragments derived from the
digestion of the purified enzyme with endoproteinase Lys-C. The
alignment of the partial amino acid sequences of the rat liver
Dopa/tyrosine sulfotransferase with the homologous sequences from other
sulfotransferases provided the first clue that the Dopa/tyrosine
sulfotransferase is a novel enzyme. The three partial amino acid
sequences, however, completely matched those found in the deduced amino
acid sequence of an unidentified sulfotransferase cDNA clone (ST1B1)
reported by Yamazoe et al.(38) . Because the ST1B1
cDNA clone encodes the only rat liver sulfotransferase that remains
unidentified to date, we decided to clone and express it in COS-7 cells
for functional characterization with respect to its identity as the
Dopa/tyrosine sulfotransferase. Two regions, WDNKCKM and WKNYFTM, of
the deduced amino acid sequence of the ST1B1 clone were chosen for
designing degenerate oligonucleotide primers for reverse
transcriptase-PCR. Using the 452-nucleotide PCR product as the probe
for screening, a full-length cDNA clone was isolated and sequenced. The
deduced amino acid sequence of the isolated cDNA contained the three
partial amino acid sequences derived from the protein sequencing of the
purified Dopa/tyrosine sulfotransferase and was found to be identical
to that of clone ST1B1 except for a glycine residue instead of a
glutamic acid residue at position 68. Whether this difference reflects
the presence of isozymes in the rat liver remains to be clarified. The
identity of the cDNA isolated was further verified by the expression in
transfected COS-7 cells of a functional 33-kDa Dopa/tyrosine
sulfotransferase that displayed immunologic cross-reactivity toward the
antiserum against the purified rat liver Dopa/tyrosine
sulfotransferase. These results have thus unequivocally confirmed the
identity of the rat liver Dopa/tyrosine sulfotransferase as a novel
enzyme, being distinct from all known sulfotransferases previously
characterized.
The important question remains of whether the
sulfation of Dopa and tyrosine isomers is functionally relevant.
Although the precise physiological involvement of the Dopa/tyrosine
sulfotransferase still awaits further clarification, some possibilities
could be put forth by taking into account the metabolic roles of its
substrates, in particular L-Dopa and L-m-tyrosine. L-Dopa is generally known as
the biosynthetic precursor of catecholamines including dopamine,
norepinephrine, and epinephrine(44) . L-meta-Tyrosine has been shown to be present in
vivo(45, 46) and is capable of crossing the
blood-brain barrier(47) . Quantitative analysis showed that
although L-p-tyrosine represents the predominant
species, L-m-tyrosine constitutes a significant amount (2.8%)
of the total tyrosine circulating in blood (46) . Using bovine
adrenal medulla extract or rat brain homogenate, it has been
demonstrated that L-m-tyrosine was produced through
the meta-hydroxylation of L-phenylalanine (48, 49) . Furthermore, in vivo studies have
shown that L-m-tyrosine could be converted to L-Dopa (50, 51, 52) or m-tyramine(47, 53) , a decarboxylated product
of L-m-tyrosine with neurotransmitter activity.
Considering that L-Dopa and L-m-tyrosine are
both involved in the biosynthesis of neurotransmitters, it would be
important to regulate the concentrations of these compounds in
vivo. A hypothetical role for the sulfation of L-Dopa and L-m-tyrosine therefore is that under normal
circumstances, sulfation may be employed as a safeguard against the
overproduction of L-Dopa and L-meta-tyrosine
that if not prevented, might lead to the overproduction of
catecholamines and consequently some neurological problems. When L-Dopa or L-m-tyrosine exceeds the normal
concentration range, sulfation reaction may provide a mechanism by
which they can be readily excreted. In this regard, the Dopa/tyrosine
sulfotransferase may occupy a unique position related to the
neurotransmitter metabolism. For other Dopa and tyrosine isomers, as
well as thyroid hormones (triiodothyronine and thyroxine), a similar
role for sulfation in facilitating their excretion can also be
proposed. It is to be pointed out that the sulfation of dopamine and
other catecholamines has been reported(2, 54) . The
enzyme responsible for the sulfation of catecholamines, the M form
phenol sulfotransferase, has been shown to be predominantly present in
neuronal cells(41) . Whereas the M form phenol sulfotransferase
serves to catalyze the sulfation of catecholamines that may have
already exerted their neurotransmitter function, the Dopa/tyrosine
sulfotransferase discovered in our studies functions to catalyze the
sulfation of their biosynthetic precursors (L-Dopa, L-p-tyrosine, and L-m-tyrosine),
thereby preventing the overproduction of catecholamines.
Finally, it
is to be noted that the co-elution with synthetic L-p-TyrS standard upon ion-exchange column
chromatography was the major, if not exclusive, criteria used for the
identification of free TyrS excreted in mammalian
urine(4, 5, 6) . This procedure, however, is
unlikely to provide enough resolution needed to distinguish between
different sulfated tyrosine and/or Dopa isomers. It will be important
to investigate, using more precise methods, the true identity (or
identities) of the TyrS excreted in mammalian urine. Such information
will be valuable in delineating the real substrate(s) for the
Dopa/tyrosine sulfotransferase in vivo.