From the Department of Biochemistry, Osaka University
Medical School, Suita, Osaka 565-0871, the ¶ Department of
Molecular Medicine, Research Institute, Osaka Medical Center for
Maternal and Child Health, Izumi, Osaka 594-1101, the
Department
of Biochemistry, Teikyo University School of Medicine, Tokyo 173-8605, and the ** Institute of Organ Transplants, Reconstructive Medicine and
Tissue Engineering, Shinshu University Graduate School of Medicine,
Matsumoto 390-8621, Japan
Received for publication, June 28, 2000, and in revised form, October 10, 2000
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ABSTRACT |
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A novel sulfotransferase gene
(designated GP3ST) was identified on human
chromosome 2q37.3 based on its similarity to the cerebroside
3'-sulfotransferase (CST) cDNA (Honke, K., Tsuda, M., Hirahara, Y.,
Ishii, A., Makita, A., and Wada, Y. (1997) J. Biol.
Chem. 272, 4864-4868). A full-length cDNA was obtained by reverse transcription-polymerase chain reaction and 5'- and 3'-rapid amplification of cDNA ends analyses of human colon mRNA. The
isolated cDNA clone predicts that the protein is a type II
transmembrane protein composed of 398 amino acid residues. The amino
acid sequence indicates 33% identity to the human CST sequence. A
recombinant protein that is expressed in COS-1 cells showed no CST
activity, but did show sulfotransferase activities toward
oligosaccharides containing nonreducing Sulfated glycoconjugates, whose sulfate groups are biologically
relevant, occur in a wide range of biological compounds (reviewed in
Ref. 1), including glycoproteins, proteoglycans, glycolipids, and
polysaccharides. The enzymes responsible for the sulfation of these
compounds, sulfotransferases, utilize in common the sulfate donor
5'-phosphoadenosine 3'-phosphosulfate
(PAPS).1
We recently reported on the purification of the glycolipid
3-sulfotransferase (cerebroside sulfotransferase (CST);
galactosylceramide sulfotransferase, EC 2.8.2.11) to homogeneity
from human renal cancer cells (2) and the cloning of the human CST
cDNA on the basis of the amino acid sequence of the purified enzyme
(3). The deduced amino acid sequence shows no overall homology to other sulfotransferases, except for the PAPS-binding motifs, suggesting that
CST has a different evolutionary origin (3).
Carbohydrate structures with 3'-sulfo- Materials--
[35S]PAPS (72.5 GBq/mmol) was
purchased from PerkinElmer Life Sciences. Unlabeled PAPS,
lacto-N-fucopentaose I
(Fuc Cloning of Human GP3ST cDNA--
One microgram of
total RNA isolated from various human tissues (OriGene Technologies,
Rockville, MD) was reverse-transcribed with random hexamers and then
subjected to PCR. PCR was carried out using Taq DNA
polymerase and two pairs of primers: CF2 (5'-TCACCAACATCATGTTC-3', corresponding to human GP3ST cDNA nucleotides
284-300 in Fig. 1A) and CR2 (5'-TGAGGCAGGTTGAACCT-3',
corresponding to nucleotides 490-506); and CF1
(5'-CCAACGACACCTTCTAC-3', corresponding to nucleotides 524-540) and
CR1 (5'-GGAACGGGATGTTCTTG-3', corresponding to nucleotides 1302-1318),
which are located in the putative exons of the CST-like candidate gene
in the CEB1 cosmid (GenBankTM/EBI accession number
AF048727). The PCR products were electrophoresed on 1.5% agarose gel.
DNA fragments with the expected sizes of 223 and 795 base pairs,
respectively, were excised and subcloned into pT7 Blue(R) (Novagen,
Madison, WI) and sequenced as described below. The determined sequences
were identical to those in the CEB1 cosmid, and the cloned DNA
fragments were designated LIKE-C1 and LIKE-C2.
The 5'-end of human GP3ST cDNA was cloned by the rapid
amplification of cDNA ends (RACE) (21) using a 5'-RACE system kit (Life Technologies, Inc.). According to the manufacturer's recommended protocol, 1 µg of total RNA from human colon (OriGene Technologies) was reverse-transcribed with the GP3ST gene-specific
antisense primer CR2. The first strand cDNA was tailed at the
3'-end by terminal deoxynucleotidyltransferase with dATP and then
subjected to PCR. PCR was accomplished using Taq DNA
polymerase, a 3'-RACE adapter primer
(5'-GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTTTT-3'), and the GP3ST
gene-specific antisense primer CR3 (5'-GACCCCACGCCTTCCACGTA-3', corresponding to nucleotides 436-455). The PCR product was
electrophoresed on 1.5% agarose gel, and the DNA was transferred to a
nylon membrane (Roche Molecular Biochemicals, Tokyo) and hybridized
with a digoxigenin-labeled DNA probe synthesized using LIKE-C1 as a
template. Southern hybridization was carried out at 42 °C overnight
in hybridization buffer containing 50% formamide, 5× SSC, 2%
blocking reagent (Roche Molecular Biochemicals), 0.1%
N-lauroylsarcosine, and 0.02% SDS. Positive bands
were cut out, subcloned into pT7 Blue(R), and sequenced.
The 3'-end of human GP3ST cDNA was cloned using a
3'-RACE system kit (Life Technologies, Inc.) in a similar manner.
According to the manufacturer's protocol, 1 µg of human colon total
RNA was reverse-transcribed with the 3'-RACE adapter primer and
subsequently subjected to PCR. PCR was performed using Taq
DNA polymerase, the abridged universal amplification primer
(5'-GGCCACGCGTCGACTAGTAC-3'), and the GP3ST
gene-specific sense primer CF5 (5'-TCAAGAACCACACGCAGATC-3', corresponding to nucleotides 1115-1134). The PCR product was
electrophoresed on 1.5% agarose gel and subjected to Southern blotting
as described above, except that the hybridization probe was synthesized
using LIKE-C2 as a template.
DNA Sequencing--
The subcloned DNAs were sequenced by the
dideoxy chain termination method using a Dye Terminator cycle
sequencing kit (PerkinElmer Life Sciences) with a DNA sequencer
(Applied Biosystems Model 377).
Overexpression of Human GP3ST in COS-1 Cells and Sulfotransferase
Assay--
To obtain a GP3ST cDNA clone that contained
the entire open reading frame, RT-PCR was accomplished using total RNA
from human colon as a template. PCR was performed using Taq
DNA polymerase and a set of primers (CF3, 5'-CCAGAGGCCAAGATGATG-3',
corresponding to nucleotides 121-138 in Fig. 1A); and CR6,
5'-GGAGAGAGGAGCTGGTGT-3', corresponding to nucleotides 1362-1379). The
PCR product with the expected size of 1259 base pairs was subcloned
into pT7 Blue(R) and sequenced as described above. The obtained clone
was digested with EcoRI and SalI, and the GP3ST
open reading frame fragment was inserted into the EcoRI and
SalI sites of pSVK3 (Amersham Pharmacia Biotech), yielding
pSV-GP3ST. COS-1 cells (1 × 106) that had been
pre-cultured for 1 day in a 10-cm diameter dish were transfected with
10 µg of pSV-GP3ST and 30 µl of LipofectAMINE (Life Technologies,
Inc.). After 72 h, the cells were washed twice with 10 ml of cold
phosphate-buffered saline, harvested with 1 ml of phosphate-buffered
saline using a silicon scraper, centrifuged at 1500 rpm for 2 min,
sonicated in 0.3 ml of ice-cold Tris-buffered saline containing 0.1%
Triton X-100, and assayed for sulfotransferase activity according to
the method for CST activity (2). When oligosaccharides were used as
acceptor substrates, water was employed as the eluant in DEAE A25
column chromatography as a substitute for the organic solvent.
GP3ST Activity Assay Using PA-Lc4 and PA-nLc4--
The standard
incubation mixture contained the following components in a total volume
of 25 µl: 100 mM MES (pH 6.2), 10 mM MnCl2, 1% Lubrol PX, 1 mM PAPS, 0.2 mM PA-Lc4 or PA-nLc4, and 7.5 µl of enzyme source. After
incubation at 37 °C for 2 h, the reaction was terminated by
boiling for 2 min. The sample was centrifuged at 15,000 rpm for 5 min,
and 20 µl of the supernatant was transferred to a new tube, followed
by dilution with 180 µl of water; and 50 µl of the diluted solution
was injected onto a TSKgel SuperQ-5PW column (7.5 × 75 mm; Tosoh)
equipped with a Shimadzu LC-VP HPLC system. Elution was
performed with a linear gradient of 0-0.2 M ammonium
acetate (pH 9.0) and monitored with a fluorescence spectrophotometer
(excitation, 320 nm; and emission, 400 nm).
1H NMR Analysis of the Reaction Product--
For
preparation of the NMR sample, a large-scale GP3ST reaction was carried
out using PA-Lc4 as a substrate, and the reaction product was isolated
by anion-exchange chromatography as described above. The eluate of the
anion-exchange chromatography was then directly injected onto a TSKgel
ODS-80TM column (4.6 × 75 mm; Tosoh). Elution was carried out at
55 °C with 20 mM ammonium acetate and 0.01% 1-butanol
(pH 4.0) at a flow rate of 1.0 ml/min and monitored as described above.
A single peak was detected. The effluent corresponding to the peak was
collected and dried. Approximately 17 nmol of the reaction product, as
evaluated by fluorescence intensity, was deuterium-exchanged; dissolved
in 0.1 ml of D2O; and subjected to 1H NMR
analysis as described previously (22, 23), except for the use of
microtubes (Shigemi, Tokyo). The 1H signals of the
pyridylaminated oligosaccharides were assigned based on the
1H-1H DQF-COSY, two-dimensional HOHAHA,
and two-dimensional ROESY experiments in a Joel GX-400 spectrometer at
30 °C. In the case of the DQF-COSY and two-dimensional HOHAHA
experiments, the data matrices of 256 (t1) × 4096 (t2) points, acquired with a spectral width of 2000 Hz, were
zero-filled to 1048 × 4096 data points. The mixing time of the
two-dimensional HOHAHA was set at 100 ms, and the two-dimensional ROESY
spectrum was acquired with a 200-ms spin-lock period with a spectral
width of 5 kHz.
Northern Blot Analysis--
Samples containing 20 µg of total
RNAs from human brain, heart, skeletal muscle, kidney, stomach, small
intestine, colon, lung, liver, and testis (OriGene Technologies) were
denatured in 50% (v/v) formamide, 6% (v/v) formaldehyde, and 20 mM MOPS (pH 7.0) at 65 °C; electrophoresed on 1%
agarose gel containing 6% formaldehyde; and then transferred to a
nylon membrane. The membrane was hybridized with a digoxigenin-labeled
( In Situ Hybridization of GP3ST Transcripts--
To detect
GP3ST transcripts in normal human tissues, in
situ hybridization was carried out using an RNA probes with
nonradioactive labeling as described previously (24).
GP3ST gene-specific nucleotide sequence (nucleotides
720-861 in Fig. 1A) was amplified by PCR using a 5'-primer
(5'-CCCAAGCTTCGGCTTCGACCCCAA-3') and a 3'-primer (5'-CGGAATTCCAGCGCAGCCGGCGC-3') (HindIII
and EcoRI sites are underlined). This amplified DNA fragment
was cloned into the HindIII and EcoRI sites of
pGEM-3Zf (+) (Promega), and the resultant vector was used for
construction of the RNA probe. A digoxigenin-labeled antisense RNA
probe was obtained using a HindIII-cut template and T7 RNA
polymerase with a DIG RNA labeling mixture (Roche Molecular Biochemicals). Similarly, a sense probe was prepared for negative control experiments using an EcoRI-cut template and SP6 RNA
polymerase with the DIG RNA labeling mixture.
Paraffin blocks of normal human tissues, including colon, liver,
placenta, kidney, and brain, fixed for 24 h in 20% buffered Formalin (pH 7.4) were selected from the pathology file of the Central
Clinical Laboratories of the Shinshu University Hospital (Matsumoto,
Japan). The deparaffinized tissue sections were immersed in 0.2 M HCl for 20 min and then digested with 100 µg/ml
proteinase K at 37 °C for 20 min, followed by post-fixation with 4%
paraformaldehyde. These slides were rinsed with 2 mg/ml glycine,
acetylated for 10 min in 0.25% acetic anhydride in 0.1 M
triethanolamine (pH 8.0), and then defatted with chloroform and
air-dried. After prehybridization with 50% deionized formamide and 2×
SSC for 1 h at 45 °C, these slides were hybridized with 0.5 mg/ml antisense or sense probe in 50% deionized formamide, 2.5 mM EDTA (pH 8.0), 300 mM NaCl, 1× Denhardt's
solution, 10% dextran sulfate, and 1 mg/ml brewers' yeast tRNA at
45 °C for 48 h.
After hybridization, the slides were washed with 50% formamide and 2×
SSC for 1 h at 45 °C and digested with 10 mg/ml RNase A at
37 °C for 30 min. After sequential washing with 2× SSC and 50%
formamide at 45 °C for 1 h and 1× SSC and 50% formamide at 45 °C for 1 h, the sections were subjected to
immunohistochemistry for detection of the hybridized probes using an
alkaline phosphatase-conjugated anti-digoxigenin antibody (Roche
Molecular Biochemicals). The alkaline phosphatase reaction was
visualized with 5-bromo-4-chloro-3-indolyl phosphate and nitro blue
tetrazolium in the presence of levamisole and 10% polyvinyl alcohol. A
control study using the sense probe showed no specific reactivity.
cDNA Cloning and the Predicted Protein Sequences of a Novel
Sulfotransferase--
When the GenBankTM/EMBL/DDBJ DNA
Data Bank was searched for a sequence homologous to the coding region
of the human CST gene (3), a cosmid clone containing the minisatellite
CEB1 (GenBankTM/EBI accession number AF048727),
located on human chromosome 2q37.3, included a significantly similar
sequence (25). The CEB1 cosmid includes two exons with open
reading frames that together cover 1076 base pairs of the human CST
mRNA (25). To investigate the issue of whether the
CEB1 candidate gene actually encodes a functional
sulfotransferase, we elected to clone its cDNA from human tissues.
First, the cDNA fragments LIKE-C1 (nucleotides 284-506 in Fig.
1A) and LIKE-C2 (nucleotides
524-540), which were found in the putative exons of the
CEB1 candidate gene, were obtained by RT-PCR using total
RNAs from various human tissues. Since the RT-PCR product was strongly
detected in the colon (Fig. 5), we employed human colon as a source of
mRNA for subsequent cDNA cloning. The 5'- and 3'-ends of the
cDNA of the sulfotransferase candidate were obtained by RACE
analyses as described under "Experimental Procedures."
Fig. 1A shows the DNA and deduced amino acid sequences of
the cloned cDNA (termed GP3ST). The deduced protein is a
type II membrane protein composed of 398 amino acids with a molecular mass of 46,092 Da and has six potential N-glycosylation
sites. Its sequence exhibits 33% identity to the human CST sequence
(Fig. 1B) and contains PAPS-binding motifs (26), suggesting
that the cDNA encodes a sulfotransferase.
Substrate Specificity of GP3ST--
To investigate the issue of
whether the isolated cDNA encodes a functional sulfotransferase
and, if so, on what substrates the sulfotransferase acts, the cloned
cDNA was inserted into a mammalian expression vector, pSVK3
(pSV-GP3ST) and overexpressed in COS-1 cells. We performed a substrate
specificity experiment using [35S]PAPS as the sulfate
donor, various carbohydrate compounds as acceptors, and cellular
lysates of pSV-GP3ST-introduced COS-1 cells as an enzyme source (Table
I). A recombinant protein expressed in
COS-1 cells showed no CST activity, but did show significant sulfotransferase activities toward oligosaccharides containing nonreducing Sulfotransferase Assay Using PA-Lc4 or PA-nLc4 as an
Acceptor--
To further characterize the cloned sulfotransferase, we
developed a novel sulfotransferase assay system using pyridylaminated oligosaccharides as acceptors. This system was originally developed for use in the structural analysis of oligosaccharides (27) and was
later applied to glycosyltransferase assays (28). To separate the
acidic products from the neutral substrates, anion-exchange chromatography was employed. A typical elution pattern of the reaction
products when PA-Lc4 or PA-nLc4 was used as an acceptor is shown in
Fig. 2. The arrows indicate
the elution positions of the products. When COS-1 cell lysates
transfected with pSV-GP3ST were used as an enzyme source, a robust peak
was detected at the positions indicated by the arrows (Fig.
2, b and d), whereas COS-1 cells transfected with
the pSVK3 vector alone showed no significant peak at the indicated
positions (Fig. 2, a and c). The yield of GP3ST
products was increased relative to incubation time (over a period of
2 h) in a linear fashion, as well as at enzyme concentrations up
to 1 mg/ml (data not shown).
Identification of the Reaction Product--
To investigate the
position where the sulfate group was transferred, a 1H NMR
analysis of the enzyme product was performed. For this, a large-scale
reaction was performed using PA-Lc4 as a donor, and the product was
isolated by sequential HPLC with anion-exchange chromatography and
C18 reverse-phase chromatography as described under
"Experimental Procedures."
H1-H4 of the Gal residues in PA-Lc4 and the sulfation product
(PA-Lc4-S) were assigned by DQF-COSY and two-dimensional HOHAHA. As
shown in Fig. 3, the H2-H4 signals of
the terminal Gal (IV) residue in PA-Lc4-S were shifted to the
lower field by 0.14, 0.67, and 0.38 ppm, respectively, indicating
unequivocally that the hydroxyl group at C-3 was esterified by a
sulfate. On the other hand, the signals of the internal Gal (II)
residue in PA-Lc4-S remained unchanged relative to those in PA-Lc4. To
investigate whether or not C-6 was sulfated, the H5 protons were
assigned from the two-dimensional ROESY spectra in which the cross-peak between H1 and H5 was observed. The difference in chemical shift of the
H5 proton in Gal (IV) was 0.06 ppm (Table
II), which is typical for the sulfation
at C-3 of the terminal residue (22, 29). The additional downfield shift
by ~0.2 ppm, which should result from the sulfation at C-6 (22, 30),
was not observed. These results clearly show that GP3ST is a
Some Properties of GP3ST--
The optimal pH was found to be
between 6.0 and 6.5 (Fig. 4) when a MES
buffer was used. The effect of divalent cations on GP3ST activity is
summarized in Table III. GP3ST
essentially has no requirement for divalent cations; and in fact,
Cu2+ and Zn2+ strongly inhibited the
sulfotransferase activity. The Km values for PA-Lc4,
PA-nLc4, and PAPS were found to be 220, 480, and 240 µM,
respectively (Table IV). Although the
Km value for PA-nLc4 was higher than that for
PA-Lc4, the Vmax for PA-nLc4 was higher than
that for PA-Lc4, and Vmax/Km values were nearly equal for PA-Lc4 and PA-nLc4, suggesting that GP3ST
is able to act on both type 1 and type 2 chains with a similar efficiency.
Tissue-specific Expression of the GP3ST Gene--
Northern blot
and RT-PCR analyses using total RNAs from various human tissues
consistently showed that GP3ST is ubiquitously transcribed
(Fig. 5). Among the examined organs,
GP3ST mRNA was found to be highly expressed in heart,
stomach, colon, liver, and spleen.
Expression of GP3ST Transcripts in Normal Human Tissues--
Since
the Northern blot analysis revealed that GP3ST is
ubiquitously transcribed, we performed in situ hybridization
using an RNA probe specific for GP3ST to determine the cell
types that actually express GP3ST transcripts (Fig.
6). Among the various human tissues
examined, a specific signal for GP3ST was detected in
epithelial cells lining the lower to middle layer of the crypts in
colonic mucosa, hepatocytes surrounding the central vein of the liver,
extravillous cytotrophoblasts in the basal plate and septum of the
placenta, renal tubules of the kidney, and neuronal cells of the
cerebral cortex.
We report herein the identification of a novel functional
sulfotransferase gene (named GP3ST) based on the similarity
of its DNA sequence to that of the CST gene (3). The primary structure of GP3ST showed an overall similarity to that of CST with 33% identity. GP3ST catalyzes the transfer of a sulfate group to C-3 of the
nonreducing CST acts on various nonreducing terminal The reaction product of GP3ST,
3'-sulfo-Gal The expression of the GP3ST gene was found to be ubiquitous,
with a relatively high level in the colon, where a considerable amount
of mucin is generated and secreted. GP3ST may be involved in the
biosynthesis of sulfated mucin in the human colon. A decreased level of
mucin sulfation has been observed in colon carcinomas and ulcerative
colitis (43-46). This decrease in mucin sulfation is associated with
reduced Gal-3'-sulfotransferase activity (14, 47). In some human
breast cancer cell lines, the similar sulfotransferase activity is much
lower than in normal mammary cells (16). It would be interesting to
study the expression of the GP3ST gene in tumor and normal
tissues of these secreting organs. The high level of GP3ST
expression in heart might be involved in the production of the tumor
necrosis factor- To measure the enzyme activity of a large number of samples, it is
essential to establish a simple and sensitive assay method. The method
developed in this study does not require radioisotopes and is a
convenient and very sensitive assay method for GP3ST using a unique
fluorescent-labeled pyridylaminated oligosaccharide as an acceptor
substrate. The method is sufficiently sensitive for the detection of
0.5 pmol of reaction product and can be applied to other
sulfotransferase assays. In conclusion, we report the cloning of
the cDNA of -galactosides such as
N-acetyllactosamine, lactose, lacto-N-tetraose
(Lc4), lacto-N-neotetraose (nLc4), and Gal
1-3GalNAc
-benzyl (O-glycan core 1 oligosaccharide). To characterize the cloned sulfotransferase, a
sulfotransferase assay method was developed that uses pyridylaminated
(PA) Lc4 and nLc4 as enzyme substrates. The enzyme product using PA-Lc4
as an acceptor was identified as
HSO3-3Gal
1-3GlcNAc
1-3Gal
1-4Glc-PA by
two-dimensional 1H NMR. Kinetics studies suggested that
GP3ST is able to act on both type 1 (Gal
1-3GlcNAc-R) and type 2 (Gal
1-4GlcNAc-R) chains with a similar efficiency. In
situ hybridization demonstrated that the GP3ST gene
is expressed in epithelial cells lining the lower to middle layer of
the crypts in colonic mucosa, hepatocytes surrounding the central vein
of the liver, extravillous cytotrophoblasts in the basal plate and
septum of the placenta, renal tubules of the kidney, and neuronal cells
of the cerebral cortex. The results of this study indicate the
existence of a novel
-Gal-3'-sulfotransferase gene family.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Gal linkages have been found
in both N-glycans (4, 5) and O-glycans (6-13) of glycoproteins, and the
-Gal-3'-sulfotransferase activities
responsible for the synthesis of these glycoproteins have been
demonstrated (4, 13-16). The
-Gal-3'-sulfotransferase synthesizing
O-glycans has been demonstrated to be different from CST
(13). Thus far, of the sulfotransferases that act on glycoproteins, the
molecular cloning of Gal-6'-sulfotransferase and
GlcNAc-6-sulfotransferase has been accomplished (17-20). However, no
-Gal-3'-sulfotransferase genes responsible for the biosynthesis of
3'-sulfo-
-Gal linkages in glycoproteins have been cloned. The fact
that CST transfers a sulfate group to C-3 of the nonreducing terminal
-galactoside in glycolipids prompted us to undertake a search for a
novel
-Gal-3'-sulfotransferase gene using CST cDNA sequence as a probe.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-2Gal
1-3GlcNAc
1-3Gal
1-4Glc), lacto-N-fucopentaose II
(Gal
1-3(Fuc
1-4)GlcNAc
1-3Gal
1-4Glc), lacto-N-fucopentaose III
(Gal
1-4(Fuc
1-3)GlcNAc
1-3Gal
1-4Glc), N-acetyllactosamine, GalNAc
-benzyl,
Gal
1-3GalNAc
-benzyl, Gal
1-4Gal, Gal
1-4Gal, and
methyl-
-Gal were from Sigma. Lacto-N-tetraose (Gal
1-3GlcNAc
1-3Gal
1-4Glc; Lc4) and
lacto-N-neotetraose (Gal
1-4GlcNAc
1-3Gal
1-4Glc; nLc4) were purchased from Seikagaku Kogyo (Tokyo, Japan).
Methyl-
-Gal and lactose were from Nacalai Tesque (Kyoto, Japan).
Galactosylceramide and lactosylceramide were prepared in our laboratory
as described previously (2). PA-Lc4 and PA-nLc4 were synthesized by
pyridylamination of lacto-N-tetraose and
lacto-N-neotetraose, respectively, using a GlycoTAG reagent
kit (Takara, Shiga, Japan) with an automated pyridylamination apparatus
(GlycoTAG, Takara).
)-strand RNA probe made from human GP3ST cDNA.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
cDNA and deduced amino acid sequences of
human GP3ST. A, the predicted amino
acid sequence is indicated by the single-letter amino acid code below
the nucleotide sequence. The putative transmembrane portion is
double-underlined. The potential N-glycosylation
site is indicated by an asterisk. The presumed PAPS-binding
sites are boxed. B, shown is a comparison of the amino acid
sequences of human GP3ST and human CST (hCST) (3).
Asterisks below the sequences show identical residues.
-galactosides such as N-acetyllactosamine,
lactose, Lc4, nLc4, and Gal
1-3GalNAc
-benzyl
(O-glycoside core 1 oligosaccharide). On the other hand, the
COS-1 cells transfected with pSV-CST (3) or with pSVK3 alone did not
show sulfotransferase activity toward any of the oligosaccharides
examined (data not shown).
Substrate specificity of GP3ST
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Fig. 2.
Typical elution profile of the reaction
mixture upon anion-exchange chromatography. A reaction mixture,
using PA-Lc4 (a and b) or PA-nLc4 (c
and d) as a substrate and COS-1 cell lysates transfected
with the pSVK3 vector (a and c) or pSV-GP3ST
(b and d) as an enzyme source, was applied to a
TSKgel SuperQ-5PW column (7.5 × 75 mm) and eluted with a linear
gradient of 0-0.2 M ammonium acetate (pH 9.0).
Fluorescence was detected at excitation and emission wavelengths of 320 and 400 nm, respectively. The arrows indicate the elution
positions of the products.
-Gal-3'-sulfotransferase that transfers a sulfate group to C-3 of
the nonreducing
-galactosyl residue.
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Fig. 3.
Proton NMR spectra of the substrate and the
sulfation product. Shown are cross-sections from the
two-dimensional HOHAHA spectra through the H1 protons in the terminal
Gal (IV) residue in PA-Lc4 (A) and PA-Lc4-S
(B).
Chemical shift of the galactosyl protons in PA-Lc4 and the enzyme
product (PA-Lc4-S)
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Fig. 4.
Effect of pH on GP3ST activity.
Incubations were carried out at the indicated pH values under standard
assay conditions using PA-Lc4 as a substrate in the following buffers:
MES ( ), triethanolamine (
), and Tris (
).
Metal requirements of GP3ST
Kinetics studies of GP3ST
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Fig. 5.
Expression of the GP3ST gene
in various human tissues. a, Northern blotting. First,
20 µg of total RNA from the indicated organs was electrophoresed,
blotted, and hybridized with a digoxigenin-labeled ( )-strand RNA
probe made from the full-length human GP3ST cDNA.
Lane 1, brain; lane 2, heart; lane 3,
skeletal muscle; lane 4, kidney; lane 5, stomach;
lane 6, small intestine; lane 7, colon;
lane 8, liver; lane 9, testis; lane
10, lung; lane 11, spleen; lane 12,
placenta. The sizes of the molecular markers are indicated on the
right. b, RT-PCR analysis. Next, 1 µg of total RNA from
the indicated organs was reverse-transcribed with a random primer. The
resulting cDNA was amplified by PCR using a set of GP3ST primers:
CF2 (nucleotides 284-300 in Fig. 1A) and CR2 (nucleotides
490-506). The PCR product was electrophoresed, blotted, and hybridized
with a digoxigenin-labeled DNA probe synthesized using the
GP3ST cDNA as a template. The observed PCR products
showed the predicted size of 223 bases. Lane 1, brain;
lane 2, heart; lane 3, skeletal muscle;
lane 4, kidney; lane 5, stomach; lane
6, small intestine; lane 7, colon; lane 8,
lung; lane 9, liver; lane 10, spleen; lane
11, testis; lane 12, placenta.
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Fig. 6.
Expression of human
GP3ST transcripts in various human
tissues. GP3ST mRNA was detected in epithelial cells located
in the lower to middle layer of colonic mucosa (a and
f), hepatocytes surrounding the central vein of the liver
(b and g), extravillous cytotrophoblasts in the
placental septum (c and h), renal tubules of the
kidney (d and i), and neuronal cells of the
cerebral cortex (e and j). In situ
hybridization for GP3ST was carried out using the antisense
probe (a-e) and the sense probe as a control
(f-j). Bars = 100 µ m.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactoside in oligosaccharides such as CST. The
similarities of structure and function between CST and GP3ST argue that
they are members of the
-Gal-3'-sulfotransferase family. Similarly,
several isoforms of this carbohydrate sulfotransferase have been found,
e.g. heparan-sulfate
N-deacetylase/N-sulfotransferase (31-33),
heparan-sulfate glucosaminyl-3-O-sulfotransferase (34, 35),
heparan-sulfate 6-O-sulfotransferase (36),
chondroitin-sulfate/keratan-sulfate 6-O-sulfotransferase
(17, 37), and GlcNAc-6-sulfotransferase (18-20). The distinct
regulation of gene expression and the different substrate specificities
of these isoforms may give rise to the specificity and diversity of
sulfated glycans that are often found in a particular cell type.
-galactosides in
glycolipids such as galactosylceramide, lactosylceramide,
galactosyl-1-alkyl-2-acyl-sn-glycerol, and
galactosyldiacylglycerol, but it does not act on oligosaccharides that
are not associated with lipid (Ref. 2 and this study), suggesting that
CST recognizes the nonreducing
-galactoside of an oligosaccharide
chain attached to a lipid moiety. On the other hand, GP3ST acts only on
the nonreducing terminal
-galactoside of an oligosaccharide without
lipid, suggesting that this enzyme is involved in the biosynthesis of
sulfated glycoproteins. The substrate specificity of GP3ST is similar
to that of the mucin 3'-sulfotransferase activity in rat colonic mucosa
(15), although GP3ST prefers N-acetyllactosamine to the
O-glycan core 1 structure. Since the sulfation of core 1 prevents the branching reaction to form a core 2 structure (15), GP3ST
may regulate the biosynthesis of core 2. GP3ST also acts on
methyl-
-galactoside to some extent, like the mucin
3'-sulfotransferase in human respiratory mucosa (13). Since
N-acetyllactosamine is a good substrate for GP3ST, it may
catalyze the sulfation of the N-acetyllactosamine structures of N-glycans like the calf thyroid 3'-sulfotransferase (4). The issue of whether GP3ST actually acts on N-glycans,
O-glycans, or both remains to be solved.
1-3GlcNAc
1-3Gal
1-4Glc, can be an
oligosaccharide ligand for L-selectin in vitro (38).
Furthermore, the 3'-sulfo-Lea
(Gal
1-3(Fuc
1-4)GlcNAc-R) and -LeX
(Gal
1-4(Fuc
1-3)GlcNAc-R) structures were revealed to be more potent ligands for L-selectin than 3'-sialyl-Lea and
-LeX (38, 39), although they have been observed only in
epithelia (9-11). Alternatively, the 3'-sulfated oligosaccharide
chains on epithelia might be involved in the adhesion of microorganisms such as Helicobacter pylori (40). GP3ST might be involved in the biosynthesis of 3'-sulfo-Lea and -LeX
epitopes. In the substrate specificity experiment (Table I), lacto-N-tetraose and lacto-N-neotetraose served
as good substrates for the cloned 3'-sulfotransferase, whereas neither
lacto-N-fucopentaose II nor lacto-N-fucopentaose
III served as a substrate. This result suggests that 3'-sulfation of
the nonreducing terminal Gal residue occurs prior to the
3/4-fucosylation of the penultimate GlcNAc residue in the biosynthetic
pathway of 3'-sulfo-Lea and -LeX structures, as
3'-sialylation occurs before the 3/4-fucosylation in the synthetic
pathway of 3'-sialyl-Lea and -LeX (41, 42).
-inducible sulfated ligand for L-selectin in human
cardiac microvascular endothelial cells (48).
-Gal-3'-sulfotransferase that is involved in the
biosynthesis of glycoprotein and propose a novel
-Gal-3'-sulfotransferase gene family.
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ACKNOWLEDGEMENT |
---|
We thank Maiko Hashizume for technical assistance.
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FOOTNOTES |
---|
* This work was supported by Grant-in-aid 10178104 for Scientific Research on Priority Areas from the Ministry of Education, Science, and Culture of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence reported in this paper has been submitted to the DDBJ/GenBankTM/EBI Data Bank with accession number AB040610.
§ To whom correspondence should be addressed: Dept. of Biochemistry, Osaka University Medical School, Rm. B1, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan. Tel.: 81-6-6879-3421; Fax: 81-6-6879-3429; E-mail: khonke@biochem.med.osaka-u.ac.jp.
Published, JBC Papers in Press, October 11, 2000, DOI 10.1074/jbc.M005666200
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ABBREVIATIONS |
---|
The abbreviations used are: PAPS, 5'-phosphoadenosine 3'-phosphosulfate; CST, cerebroside sulfotransferase; Lc4, lacto-N-neotetraose; nLc4, lacto-N-neotetraose; PA, pyridylaminated; RT-PCR, reverse transcription-polymerase chain reaction; RACE, rapid amplification of cDNA ends; MES, 2-(N-morpholino)ethanesulfonic acid; HPLC, high performance liquid chromatography; DQF, double quantum filter; HOHAHA, homonuclear Hartmann-Hahn spectroscopy; ROESY, rotational nuclear overhauser effect spectroscopy; MOPS, 3-(N-morpholino)propanesulfonic acid; PA-Lc4-S, sulfated pyridylaminated lacto-N-neotetraose.
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