Characterization of a Heparan Sulfate 3-O-Sulfotransferase-5, an Enzyme Synthesizing a Tetrasulfated Disaccharide*
Hideo Mochizuki
,
Keiichi Yoshida ¶,
Masanori Gotoh
||,
Shigemi Sugioka
,
Norihiro Kikuchi
**,
Yeon-Dae Kwon
**,
Akira Tawada
,
Kennichi Maeyama
,
Niro Inaba

,
Toru Hiruma

,
Koji Kimata ¶¶ and
Hisashi Narimatsu
||||
From the
Glycogene Function Team, Research Center
for Glycoscience, National Institute of Advanced Industrial Science and
Technology (AIST), Central-2, 1-1-1 Umezono, Tsukuba, Ibaraki 305-8586, Japan,
the
Central Research Laboratories, Seikagaku
Corporation, 3-1253 Tateno, Higashi-yamato, Tokyo 207-0021, Japan, the
¶Mizutani Foundation for Glycoscience, Sen-i
Kaikan, 3-1-11 Nihonbashi-honcho, Chuo-ku, Tokyo 103-0023, Japan,
||Amersham Biosciences KK, 3-25-1 Hyakunincho,
Shinjuku-ku, Tokyo 169-0073, Japan, **Mitsui Knowledge
Industry Co., Ltd., 1 Honcho, Nakano-ku, Tokyo 164-8721, Japan,

JGS Japan Genome Solutions, Inc., 51
Komiya-cho, Hachioji, Tokyo 192-0031, Japan, the

Frontier Research Division, Fundamental
Research Department, Fujirebio, Inc., 51 Komiya-cho, Hachioji, Tokyo 192-0031,
Japan, and the ¶¶Institute for Molecular
Science of Medicine, Aichi Medical University, Nagakute, Aichi 480-1195,
Japan
Received for publication, February 21, 2003
, and in revised form, April 22, 2003.
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ABSTRACT
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Heparan sulfate D-glucosaminyl 3-O-sulfotransferases
(3-OSTs) catalyze the transfer of sulfate from 3'-phosphoadenosine
5'-phosphosulfate (PAPS) to position 3 of the glucosamine residue of
heparan sulfate and heparin. A sixth member of the human 3-OST family, named
3-OST-5, was recently reported (Xia, G., Chen, J., Tiwari, V., Ju, W., Li,
J.-P., Malmstrom, A., Shukla, D., and Liu, J. (2002) J. Biol. Chem.
277, 3791237919). In the present study, we cloned putative catalytic
domain of the human 3-OST-5 and expressed it in insect cells as a soluble
enzyme. Recombinant 3-OST-5 only exhibited sulfotransferase activity toward
heparan sulfate and heparin. When incubated heparan sulfate with
[35S]PAPS, the highest incorporation of35S was observed,
and digestion of the product with a mixture of heparin lyases yielded two
major35S-labeled disaccharides, which were determined as
HexA-GlcN(NS,3S,6S) and
HexA(2S)-GlcN(NS,3S) by further
digestion with 2-sulfatase and degradation with mercuric acetate. However,
when used heparin as acceptor, we identified a highly sulfated disaccharide
unit as a major product. This had a structure of
HexA(2S)-GlcN(NS,3S,6S). Quantitative real-time PCR analysis revealed
that 3-OST-5 was highly expressed in fetal brain, followed by adult brain and
spinal cord, and at very low or undetectable levels in the other tissues.
Finally, we detected a tetrasulfated disaccharide unit in bovine intestinal
heparan sulfate. To our knowledge, this is the first report to describe not
only the natural occurrence of tetrasulfated disaccharide unit but also the
enzymatic formation of this novel structure.
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INTRODUCTION
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Heparan sulfate proteoglycans
(HSPGs)1 are
ubiquitously present on the cell surface and in the extracellular matrix and
have divergent structures and functions
(13).
Many biological functions of HSPGs are mediated by interactions between the
heparan sulfate chain and a variety of proteins, including protease
inhibitors, heparin-binding growth factors, extracellular matrix components,
protease, and lipoprotein lipase
(48).
Moreover, some pathogens exploit heparan sulfate of the host cell surface,
which binds to coat proteins or cell surface proteins of pathogens, at the
invasion (9,
10). Most of the interactions
between heparan sulfate and various functional proteins occur in certain
regions of the heparan sulfate chain with specific sulfated monosaccharide
sequences such as binding sites for antithrombin III (AT III)
(11), acidic fibroblast growth
factor (12), basic fibroblast
growth factor
(1316),
and hepatocyte growth factor
(17,
18). The ability of cells to
produce heparan sulfate with such sequences depends on the specific mechanisms
of heparan sulfate biosynthesis
(19,
20). Heparan sulfate is
initially synthesized as a polymer of a disaccharide repeat sequence,
-glucuronic acid-
1,4-N-acetylglucosamine-
1,4-. This
polymer is then N-deacetylated/N-sulfated and subsequently
undergoes epimerization of glucuronic acid (GlcA) to iduronic acid (IdoA),
2-O-sulfation of uronic acid, and 6-O-sulfation of
glucosamine residues. Additionally, a rare but functionally important
modification, 3-O-sulfation of the glucosamine residue, also occurs
(21). Most of the enzymes
involved in the biosynthesis of heparan sulfate have been purified and cloned
(22,
23). Some of them have been
shown to be present as isoforms. These isoforms play important roles in
generating the specific and diverse structures of heparan sulfate. Four
isoforms of N-deacetylase/N-sulfotransferase have been
reported. These isoforms are distinguishable from each other with respect to
the extent of N-sulfation of heparan sulfate, the ratio of
N-deacetylase to N-sulfotransferase activities and the
expression pattern in various tissues
(2426).
Three isoforms of heparan sulfate 6-O-sulfotransferase were found to
have different specificities and different expression patterns
(27). Six isoforms of
3-O-sulfotransferase (3-OST) with different spectra of acceptor
substrate specificity have also been reported. 3-OST-1 transfers sulfate to
GlcA-GlcN(NS,±6S) and produces the GlcA-GlcN(NS,3S,±6S) unit
essential to the AT III binding domain
(28,
29). 3-OST-2 transfers sulfate
to GlcA/IdoA(2S)-GlcN(NS) units
(30). 3-OST-3A transfers
sulfate to N-unsubstituted glucosamine residues and forms
IdoA(2S)-GlcN(3S,±6S) units
(31). 3-OST-3B has a
sulfotransferase domain 99.2% identical to that of 3-OST-3A and also forms
identical reaction products
(21). 3-OST-3-modified heparan
sulfate specifically binds to glycoprotein D (gD) of herpes simplex virus
type-1 (HSV-1) and also makes cells susceptible to HSV-1 entry
(32,
33). The substrate specificity
of 3-OST-4 has not been reported
(34). 3-OST-5 was recently
cloned and its reaction products were identified as GlcA-aMan(3S,6S),
IdoA(2S)-aMan(3S) and IdoA(2S)-aMan(3S,6S) by nitrous acid degradation.
Interestingly, 3-OST-5-modified heparan sulfate binds to AT III and gD
(35).
In this study, we cloned human 3-OST-5, and focused on the characterization
of reaction products. Our results indicate novel substrate specificities and
tissue distribution of 3-OST-5. Finally, we demonstrated the novel
tetrasulfated disaccharide structure in natural heparan sulfate.
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EXPERIMENTAL PROCEDURES
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Materials[35S]PAPS was purchased from
PerkinElmer Life Sciences (Boston, MA). Sodium [3H]borohydride was
obtained from ICN Biomedicals (Irvine, CA). Heparinase, heparitinase I,
heparitinase II, an unsaturated glucosaminoglycan disaccharide kit,
chondroitin sulfate A, chondroitin sulfate C, dermatan sulfate, keratan
sulfate, hyaluronic acid, and Cellulofine GCL-25-sf were from Seikagaku
Corporation (Tokyo, Japan). Heparin from porcine intestinal mucosa,
D-glucosamine N-3-disulfate (GlcN(NS,3S)), and
D-glucosamine N-3,6-trisulfate (GlcN(NS,3S,6S)) were from
Sigma. The TSKgel G2500PW column was from Tosoh (Tokyo, Japan). The
CarboPacTM PA1 column was from Dionex (Sunnyvale, CA). The
SuperdexTM Peptide HR10/30 column was from Amersham Biosciences (Amersham
Place, UK). BioGel P-4 was from Bio-Rad Laboratories (Hercules, CA). Heparan
sulfate from bovine kidney and from bovine intestine and 2-sulfatase,
4,5-glycuronate-2-sulfatase from
Flavobacterium heparinum, were provided by Seikagaku Corporation.
Construction and Purification of 3-OST-5 Protein Fused with FLAG
PeptideThe putative catalytic domain of 3-OST-5 (amino acids
63346) was cloned and expressed as a secreted protein fused with a FLAG
peptide in insect cells according to the instruction manual of GATEWAYTM
Cloning Technology (Invitrogen, Groningen, Netherlands). Because the catalytic
domain is encoded by a single exon
(35), an
1-kb DNA
fragment containing the entire exon II was amplified by PCR using the human
genomic DNA (Clontech), as a template, and two primers,
5'-ACTGGGGAACCAGAAAAATGAAAAG-3' and
5'-GTGTCTCCAGGCACAACACATAGTG-3'. The PCR product was used as a
template in a nested PCR to amplify the DNA fragment containing the putative
catalytic domain. The forward primer was
5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCTTTAAGCGTGGCCTGCTGCACGAG-3' and
the reverse primer was
5'-GGGGACCACTTTGTACAAGAAAGCTGGGTTTAGGGCCAGTTCAATGTCCTCCC-3'. The
amplified fragment was cloned into a pDONRTM 201 vector (Invitrogen) and
subsequently cloned into a vector pFBIF linearized by NcoI to yield
pFBIF containing the catalytic domain of 3-OST-5 (pFBIF-3OST). pFBIF is an
expression vector derived from pFastBac1 (Invitrogen) and contains a fragment
encoding a signal peptide of human immunoglobulin (MHFQVQIFSFLLISASVIMSRG),
the FLAG peptide (DYKD-DDDK) and the conversion site for the GATEWAYTM
system. DH10BAC competent cells (Invitrogen) were transformed with
the pFBIF-3OST and then bacmid DNA was prepared from the cells. Recombinant
virus was prepared according to the instruction manual of BAC-TO-BAC
Baculovirus Expression Systems (Invitrogen). Sf21 insect cells (BD Pharmingen,
San Diego, CA) were infected with the recombinant virus and incubated at 27
°C until the survival rate was less than 50% to yield secreted recombinant
3-OST-5 proteins fused with the FLAG peptide. The secreted enzyme was purified
using anti-FLAG M1 monoclonal antibody agarose affinity gel (Sigma). The
culture media and affinity gel mixed overnight at 4 °C were centrifuged
for 5 min, and the supernatant was aspirated. The affinity gel was washed
twice with 50 mM TBS(50 mM Tris-HCl, pH 7.4, and 150
mM NaCl) containing 1 mM CaCl2, and
resuspended in 50 mM TBS to obtain a 50% slurry. The immobilized
enzyme was stable at 80 °C for at least six months.
Assay for Sulfotransferase ActivityThe standard reaction
mixture contained 50 mM imidazole-HCl, pH 6.8, 75 µg/ml of
protamine chloride, 0.5 mM (as hexosamine) acceptor substrates, 1.5
µM [35S]PAPS (about 5 x 105 dpm),
and 1 µl of immobilized enzyme in a final volume of 50 µl. Acceptor
substrates used to examine the substrate specificity were heparan sulfate,
heparin, chondroitin sulfate A, chondroitin sulfate C, dermatan sulfate,
keratan sulfate, and hyaluronic acid. After incubation at 37 °C for 20
min, the reaction was stopped by heating at 100 °C for 1 min. The reaction
mixture was filtrated with an Ultrafree-MC (MILLIPORE, Bedford, MA) and 60
µg of chondroitin sulfate C was added as a carrier. 35S-Labeled
substrates were precipitated with 3 volumes of ethanol containing 1.3%
potassium acetate and separated completely from [35S]PAPS and its
degradation products by gel filtration HPLC on a TSKgel G2500PW column (0.75
x 30 cm) equilibrated with 0.2 M NaCl at a flow rate of 0.6
ml/min and the column temperature of 35 °C. Fractions of 0.3 ml were
collected and the radioactivity was determined by liquid scintillation
counting.
Preparation of Oligosaccharides from HeparinIt was reported
that the heparinase digestion of heparin produces about 45% each disaccharide
and tetrasaccharide, and a few percent hexasaccharide
(36,
37). Two milligrams of heparin
was digested with 0.2 units of heparinase and applied to a BioGel P-4 column
(1.5 x 140 cm) equilibrated with 0.1 M ammonium bicarbonate.
Elution positions of each oligosaccharide were monitored at 232 nm.
Disaccharide, tetrasaccharide and hexasaccharide fractions were pooled and
desalted by lyophilization.
HPLC Analysis on an Anion-exchange Column and a Gel Filtration
ColumnAnion-exchange (SAX) separation was performed on a
CarboPacTM PA1 column (4 x 250 mm) as described
(38). A combination of five
linear LiCl gradients was used, from 30 to 180 mM (05 min),
from 180 to 570 mM (58 min), from 0.57 to 1.14 M
(815 min), from 1.14 to 2.1 M (1520 min), and from
2.1 to 2.28 M (2028 min) followed by 2.28 M (from
28 min). The flow rate was 0.8 ml/min, and the column temperature was 40
°C. Fractions were collected and the radioactivity was determined by
liquid scintillation counting. To determine the molecular size of
oligosaccharides, gel filtration HPLC was performed on a SuperdexTM
Peptide HR10/30 column (1 x 30 cm, x 2 columns in series) as
described (39). The column was
equilibrated with 0.2 M NaCl at a flow rate of 0.8 ml/min, at room
temperature.
Preparation of 35S-Labeled Heparan Sulfate and Digestion
with Heparin LyasesOne milligram of heparan sulfate from bovine
kidney and [35S]PAPS (6.6 x 107 dpm) was incubated
with 0.5 ml of standard reaction mixture containing 67 µl of immobilized
enzyme at 37 °C for 3 h. After the incubation, the reaction mixture was
filtrated, and 35S-labeled heparan sulfate was precipitated with
ethanol as described above. The precipitate was dried at room temperature. The
35S-labeled heparan sulfate was digested with a mixture of 0.5
units of heparinase, 0.3 units of heparitinase I, and 0.2 units of
heparitinase II in 0.3 ml of 20 mM sodium acetate buffer (pH 7.0)
containing 2 mM calcium acetate at 37 °C for 2 h. The reaction
was stopped by heating at 100 °C for 1 min, and the mixture was filtrated.
The digested products were subjected to chromatography on a BioGel P-4 column
(1.5 x 140 cm) equilibrated with 0.1 M ammonium bicarbonate
at a flow rate of 5 ml/h. Fractions of 2 ml were collected. The fractions
indicated by horizontal bars in Fig.
2, named P4-1 and P4-2, were pooled, lyophilized and completely
desalted with a gel filtration column.

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FIG. 2. Gel filtration chromatography on a BioGel P-4 column of
35S-labeled heparan sulfate digested with a mixture of heparin
lyases. 35S-Labeled heparan sulfate digested with a mixture of
heparin lyases was applied to a BioGel P-4 column as described under
"Experimental Procedures." Fractions of 2 ml were collected, and
radioactivity was determined. The fractions indicated by horizontal
bars, named P4-1 and P4-2, were pooled.
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Digestion with 2-SulfataseThe 35S-labeled
disaccharides were digested with 4 milliunits of 2-sulfatase in 20
mM sodium acetate buffer (pH 6.5) containing 1.5 mg/ml of bovine
serum albumin. After incubation at 37 °C for 2 h, the reaction was stopped
by heating at 100 °C for 1 min. The digested products were analyzed by
HPLC on a SAX column as described above.
Determination of Glucosamine ResidueThe
35S-labeled disaccharides were treated with mercuric acetate to
remove unsaturated uronic acid residues as described
(40). The disaccharide
fraction was mixed with an equal volume of 70 mM mercuric acetate
(pH 5.0) and incubated at room temperature for 10 min. The reaction products,
[35S]sulfated glucosamines, were reduced with sodium borohydride as
described (41) and analyzed by
HPLC on a SAX column as described above. GlcN(NS,3S) and GlcN(NS,3S,6S) were
reduced with sodium [3H]borohydride and used as a standard.
Preparation of 35S-Labeled Heparin and Digestion with
Heparin Lyases35S-Labeled heparin was prepared by
incubating 0.3 mg of heparin with [35S]PAPS (2.3 x
107 dpm) and 20 µl of immobilized enzyme in a standard reaction
mixture. The 35S-labeled heparin was precipitated with ethanol and
digested with a mixture of heparin lyases as described above. The digested
products were subjected to HPLC on a SAX column as described above. Fractions
of 0.24 ml were collected and the radioactivity was determined. A peak
fraction at 30.1 min (indicated by a white arrow in
Fig. 6) was applied to a
Cellulofine G-25-sf column (1 x 28 cm) equilibrated with distilled water
to remove LiCl. Radioactive fractions were pooled and concentrated under
vacuum.
Quantitative Analysis of the 3-OST-5 Transcripts in Human Tissues by
Real-time PCRFor quantification of 3-OST-5 transcripts, we
employed the real-time PCR method, as described in detail previously
(42). Total RNAs derived from
various human tissues were purchased from Clontech and cDNAs were synthesized
with the SuperScriptTM First-strand Synthesis System for RT-PCR
(Invitrogen). To obtain the control DNA of 3-OST-5, a DNA fragment containing
exon I and the 5'-terminal region of exon II was amplified by PCR using
the Marathon ReadyTM cDNA, as a template, and two primers,
5'-TCTATCGCAGGCTGCAGCAGTCCT-3' and
5'-GGAACTCGTGCAGCAGGCCACGC-3'. Standard curves for the 3-OST-5 and
the endogenous control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
cDNAs, were generated by serial dilution of the control DNA of 3-OST-5, and a
pCR2.1 vector (Invitrogen) containing the GAPDH gene. The primer sets
and the probes for 3-OST-5 were as follows: the forward primer was
5'-GCCAGAGTTGGGAGCTTGG-3', the reverse primer was
5'-ACCCAGTCGACCTTCAATGG-3', and the probe for 3-OST-5 was
5'-TAGGCTACAACCCATTTG-3' with a minor groove binder
(43). PCR products were
continuously measured with an ABI PRISM 7700 Sequence Detection System
(Applied Biosystems, Foster City, CA). The relative amounts of 3-OST-5
transcripts were normalized to the amount of GAPDH transcript in the same
cDNA.
Analysis of Bovine Intestinal Heparan SulfateBovine
intestinal heparan sulfate (0.2 mg) was digested with a mixture of heparin
lyases as described above. The digested products were reduced with sodium
[3H]borohydride and subjected to HPLC on a SAX column as outlined
above. 35S-Labeled tetrasulfated disaccharide obtained from
3-OST-5-modified heparin in this study was reduced with sodium borohydride and
used as a standard.
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RESULTS
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Specificity for Acceptor SubstrateThe putative catalytic
domain of 3-OST-5 was cloned and expressed, and the sulfotransferase activity
was examined as described in "Experimental Procedures." As
expected, 3-OST-5 exhibited sulfotransferase activity toward heparan sulfate
and heparin. On the other hand, it exhibited no activity toward chondroitin
sulfate A, chondroitin sulfate C, dermatan sulfate, keratan sulfate, and
hyaluronic acid. The activity of 3-OST-5 for heparan sulfate is presented as
100%, and the other activities are given as relative values in
Table I.
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TABLE I Acceptor substrate specificity of 3-OST-5
Sulfotransferase activities were assayed using various glycosaminoglycans
as acceptor substrates as described under "Experimental
Procedures."
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Digestion of 35S-Labeled Heparan Sulfate with Heparin
Lyases35S-Labeled heparan sulfate, which was prepared
by incubating heparan sulfate with [35S]PAPS and the recombinant
3-OST-5, was digested with a mixture of heparin lyases and analyzed by HPLC on
a SAX column as described under "Experimental Procedures." When
monitored at 232 nm, six known disaccharide units of heparan sulfate,
HexA-GlcNAc,
HexA-GlcN(NS),
HexA-GlcNAc(6S),
HexA-GlcN(NS,6S),
HexA(2S)-GlcN(NS), and
HexA(2S)-GlcN(NS,6S), were identified
(Fig. 1A). Several
radioactive peaks were observed, but no peak was identified at the position of
those six units (Fig.
1B). Therefore, the 35S-labeled products were
different from these six disaccharides. The digestion products were applied to
a gel filtration column of BioGel P-4, and two major 35S
radioactive peaks were obtained (Fig.
2). The fractions indicated by a horizontal bar in
Fig. 2, named P4-1 and P4-2,
were pooled and desalted for further analysis. To confirm the molecular size,
these two fractions were analyzed by HPLC on a gel filtration column. The
elution profile of standard oligosaccharides prepared from heparin is shown in
Fig. 3A.
35S-Labeled substances, P4-1 and P4-2 in
Fig. 2, were eluted at the
position of the tetrasaccharide and disaccharide, respectively
(Fig. 3, B and
C). These two fractions were analyzed by HPLC on a SAX
column, and each fraction gave two components. Judging from the elution
position, P4-1 and P4-2 corresponded to peaks 1 and 3, and peaks 2 and 4,
respectively, which were four major peaks observed in
Fig. 1B. To confirm
the resistance to enzyme digestion, P4-1 was treated with the heparin lyase
mixture again. No change was observed in the appearance of peak 1 and peak 3
during HPLC analysis on a SAX column. Moreover, this fraction is also
resistant to nitrous acid degradation. The P4-1 was not characterized further,
since these tetrasaccharides encountered some difficulty in further
identification using enzymes (see "Discussion").

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FIG. 3. HPLC analysis on a gel filtration column of oligosaccharides derived
from 35S-labeled heparan sulfate. Standard oligosaccharides
were prepared from heparin as described in "Experimental
Procedures." Then, 5 µg of each oligosaccharide, hexasaccharide
(Hexa), tetrasaccharide (Tetra), and disaccharide
(Di), was applied to a SuperdexTM Peptide HR10/30 column
(panel A). The conditions for HPLC were as described under
"Experimental Procedures." Panels B and C shows
analysis of radioactive fractions obtained from the BioGel P-4 column,
P4-1 and P4-2 in Fig.
2, respectively.
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Structural Analysis of 35S-Labeled Disaccharides from
Heparan SulfateThe 35S-labeled disaccharide fraction
(P4-2) was digested with 2-sufatase as described under "Experimental
Procedures," and analyzed by HPLC on a SAX column.
Fig. 4A shows the
analysis of P4-2. With the enzyme digestion
(Fig. 4B), only one
component, peak 2 in panel A, shifted its retention time to
an earlier position. The result indicates that peak 2 has a
HexA(2S) residue, but not peak 1. To confirm the location of
sulfate group of glucosamine residues, P4-2 was treated with mercuric acetate
to remove unsaturated uronic acids. The reaction products were reduced with
sodium borohydride and analyzed by HPLC on a SAX column
(Fig. 5A). By
comparison with 3H-labeled standards
(Fig. 5B), peak
a and peak b were identified as reduced forms of GlcN(NS,3S) and
GlcN-(NS,3S,6S). Judging from peak area, peak a and peak b
originated from peak 2 and peak 1 in
Fig. 4A, respectively.
From these data, peak 1 and peak 2 in
Fig. 4A were
determined as
HexA-GlcN(NS,3S,6S) and
HexA(2S)-GlcN(NS,3S),
respectively. Fig. 9, A and
B show schematic drawings of the analysis described
above.

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FIG. 4. HPLC analysis of 35S-labeled disaccharides digested with
2-sulfatase. 35S-Labeled disaccharides derived from
3-OST-5-modified heparan sulfate (P4-2 in
Fig. 2) were digested with
2-sulfatase as described under "Experimental Procedures." The
digested products were analyzed by HPLC on a SAX column (panel B).
Panel A shows analysis of untreated P4-2. A dotted line
indicates the shifted peak.
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FIG. 5. HPLC analysis of 35S-labeled disaccharides treated with
mercuric acetate. 35S-Labeled disaccharides derived from
3-OST-5-modified heparan sulfate (P4-2 in
Fig. 2) were treated with
mercuric acetate to remove unsaturated uronic acid residues as described under
"Experimental Procedures." The reaction products were reduced with
sodium borohydride and analyzed by HPLC on a SAX column (panel A).
Panel B shows analysis of sulfated glucosamine standards, GlcN(NS,3S)
and GlcN(NS,3S,6S), reduced with sodium [3H]borohydride. A letter
R attached to the name means reduced form.
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Highly Sulfated Structure of 35S-Labeled
Heparin35S-Labeled heparin was prepared by incubating
heparin with [35S]PAPS and the recombinant 3-OST-5 and digested
with a mixture of heparin lyases as described under "Experimental
Procedures." When the digested products were separated by HPLC on a SAX
column, major radioactivity was detected at the retention time of 30.1 min,
later than the positions of trisulfated disaccharides
(Fig. 6). The fraction
indicated by a white arrow in Fig.
6 was pooled and desalted for further analysis. The
35S-labeled product was confirmed to be a disaccharide by HPLC on a
gel filtration column (data not shown). On 2-sulfatase digestion, the
35S radioactive peak exhibited a shift in retention time to an
earlier position (Fig. 7),
indicative of a
HexA(2S) residue. The retention time of the
2-sulfatase-treated product corresponded to that of a disaccharide isolated
from 3-OST-5-modified heparan sulfate (peak 1 in
Fig. 4A), its
structure being determined as
HexA-GlcN(NS,3S,6S) as described above.
Therefore,
HexA(2S)-GlcN(NS,3S,6S) was expected. To confirm the
location of sulfate group of glucosamine residue, 35S-labeled
disaccharide was treated with mercuric acetate, reduced with sodium
borohydride, and analyzed by HPLC on a SAX column
(Fig. 8A). The
position of the 35S radioactive peak corresponded to that of
3H-labeled GlcN (NS,3S,6S) standard
(Fig. 8B). From these
data, the 35S-labeled disaccharide from 3-OST-5-modified heparin
was determined as
HexA(2S)-GlcN(NS,3S,6S).
Fig. 9C shows a
schematic drawing of the analysis described above.

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FIG. 7. HPLC analysis of 35S-labeled disaccharide digested with
2-sulfatase. 35S-Labeled disaccharide derived from
3-OST-5-modified heparin (white arrow in
Fig. 6) was digested with
2-sulfatase as described under "Experimental Procedures." The
digested product was analyzed by HPLC on a SAX column (panel B).
Panel A shows analysis of untreated 35S-labeled
disaccharide. A dashed line indicates the shifted peak.
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FIG. 8. HPLC analysis of 35S-labeled disaccharide treated with
mercuric acetate. 35S-Labeled disaccharide derived from
3-OST-5-modified heparin (white arrow in
Fig. 6) was treated with
mercuric acetate to remove unsaturated uronic acid residue as described under
"Experimental Procedures." The reaction product was reduced with
sodium borohydride and analyzed by HPLC on a SAX column (panel A).
Panel B shows analysis of sulfated glucosamine standards, GlcN(NS,3S)
and GlcN(NS,3S,6S), reduced with sodium [3H]borohydride. A letter
R attached to the name means reduced form.
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Quantitative Analysis of the 3-OST-5 Transcripts in Human
TissuesWe determined the tissue distribution and expression levels
of the 3-OST-5 transcripts by a real-time PCR method. The expression levels of
3-OST-5 in various tissues were shown as a relative amount compared with the
GAPDH transcripts (Fig. 10).
The transcripts were highly expressed in fetal brain, followed by adult brain
and spinal cord. Cerebellum, colon and skeletal muscle expressed the 3-OST-5
transcripts at a relatively low level. The expression levels in the remaining
tissues were very low or undetectable.

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FIG. 10. Quantitative analysis of 3-OST-5 transcripts in human tissues by
real-time PCR. Standard curves for 3-OST-5 and GAPDH were generated by
serial dilution of control DNA or plasmid DNA as described under
"Experimental Procedures." The expression level of 3-OST-5 was
normalized to that of the GAPDH transcript, which was measured in the same
cDNAs. Data were obtained from triplicate experiments and are given as the
mean ± S.D. PBMC, peripheral blood mononuclear cell.
|
|
Identification of Tetrasulfated Disaccharide in Bovine Intestinal
Heparan SulfateTo confirm the presence of the natural
tetrasulfated disaccharide unit in heparan sulfate, we chose bovine intestinal
heparan sulfate as a natural source because of the availability of highly
purified material. The bovine intestinal heparan sulfate was digested with a
mixture of heparin lyases as described under "Experimental
Procedures." The digested products were reduced with sodium
[3H]borohydride and subjected to HPLC on a SAX column
(Fig. 11). Six major
3H radioactive peaks were identified as known disaccharides by
comparing with disaccharide standards reduced with sodium borohydride.
35S-Labeled tetrasulfated disaccharide obtained from
3-OST-5-modified heparin as described above was reduced with sodium
borohydride and used as a standard. Its elution position is shown in
Fig. 11 as
Di-tetraSR. Weak but apparent
radioactivity was detected at this position (inset of
Fig. 11). To confirm the
molecular size, the peak fraction (indicated by a white arrow) was
analyzed by the HPLC on a gel filtration column. The 3H-labeled
substance was eluted at the position of the tetrasulfated disaccharide
standard (data not shown).
 |
DISCUSSION
|
---|
The cloning and characterization of a sixth member of the human 3-OST
family, named 3-OST-5, was recently reported. Xia et al.
(35) employed low pH nitrous
acid degradation to analyze the reaction products and identified three
3-O-sulfated disaccharides, GlcA-aMan(3S,6S), IdoA(2S)-aMan(3S), and
IdoA(2S)-aMan(3S,6S). In the case of 3-O-sulfated glucosamine, both
N-sulfated and N-unsubstituted residues are susceptible to
low pH nitrous acid degradation, therefore the N-substituents of the
glucosamine residue was unidentified
(31). They also confirmed that
the 3-OST-5-modified heparan sulfate binds to AT III and gD. In addition,
transfection of the plasmid expressing 3-OST-5 makes CHO cells susceptible to
HSV-1. From these results, they concluded that 3-OST-5 has the activities of
both 3-OST-1 and 3-OST-3. The reaction specificity of 3-OST-1 and 3-OST-3 is
the formation of GlcA-GlcN-(NS,3S,±6S) and Ido(2S)-GlcN(3S,±6S),
respectively (31,
44).
In the present study, we employed enzyme digestion to analyze the reaction
products. 35S-Labeled heparan sulfate, which was prepared by
incubating heparan sulfate with [35S]PAPS and recombinant 3-OST-5,
was digested with a mixture of heparin lyases. Two major
35S-labeled disaccharides obtained from the digests were determined
as
HexA-GlcN(NS,3S,6S) and
HexA(2S)-GlcN(NS,3S). These two
disaccharides appear to resemble the reaction products of 3-OST-1 and 3-OST-2,
respectively, although the types of uronic acids are unidentified.
It has been believed that the glucosaminidic linkage adjacent to a
disaccharide unit containing a 3-O-sulfated glucosamine residue is
resistant to heparin lyases
(45,
46). To date, unsaturated
disaccharides containing a 3-O-sulfated glucosamine residue have not
been isolated. On the other hand, Rhomberg et al.
(47) reported that a
decasaccharide containing a 3-O-sulfated glucosamine residue was
depolymerized by heparinase II, and confirmed that the reaction product
contained an unsaturated 3-O-sulfated disaccharide unit in the
non-reducing terminal. They also examined a shorter oligosaccharide resistant
to heparinase II and concluded that the length of the oligosaccharide
determines the susceptibility to the enzyme. Accordingly, it is considered
that the resistance of tetrasaccharides isolated in this study to the enzyme
digestion may be due to the short length of the chain. Interestingly, the
enzyme digestion of 3-OST-3-modified heparan sulfate produces no disaccharide
containing a 3-O-sulfated glucosamine residue
(31). At this time, the
reaction specificity of heparin lyases especially for the
3-O-sulfated structure is still obscure. A more detailed analysis of
the sulfated monosaccharide sequence around the modification site of 3-OST-5
will be helpful for solving this problem.
In addition to heparan sulfate, we analyzed 3-OST-5-modified heparin. HPLC
analysis of 35S-labeled heparin digested with a mixture of heparin
lyases showed a very different elution profile compared with heparan sulfate.
A prominent peak was observed at the later position of trisulfated
disaccharides, and its structure was determined as
HexA(2S)-GlcN
(NS,3S,6S). As far as we know, this is the first report to show the enzyme
catalyzes the reaction to form the tetrasulfated disaccharide unit. A small
peak detected at the elution position of tetrasulfated disaccharide in
Fig. 1B (peak
5) indicates enzymatic formation of this structure with heparan sulfate
as an acceptor substrate.
3-OST-5 was highly expressed in fetal brain, followed by adult brain and
spinal cord. Some other tissues, including skeletal muscle, also expressed
3-OST-5 at a relatively low level. Xia et al.
(35) did not examine fetal
brain in their Northern blot analysis. They also did not detect the expression
of 3-OST-5 in the brain, and concluded that 3-OST-5 is predominantly expressed
in skeletal muscle. This discrepancy is unexplained, although real time-PCR is
a more quantitative method than Northern blot analysis. 3-OST-2 and 3-OST-4
were also predominantly expressed in human brain. 3-OST-1 and 3-OST-3 were
widely expressed in human tissues
(34). Heparan sulfate in the
nervous system is predominantly expressed on proteoglycans of either the
syndecan family (48) or the
glypican family (49). A number
of investigations have revealed that the development of the central nervous
system is controlled by the spatiotemporal expression of these proteoglycans
(50). Therefore, it can be
expected that the 3-OSTs specifically expressed in the central nervous system
play an important role in forming specific domain structures of heparan
sulfate to bind some functional proteins necessary to control the central
nervous system.
In the present study, we demonstrated the enzymatic formation of a
tetrasulfated disaccharide unit in vitro. No evidence of this novel
structure in natural heparan sulfate or heparin has been reported as far as we
know. Therefore, we focused on the detection of the tetrasulfated disaccharide
unit in natural heparan sulfate. We chose bovine intestinal heparan sulfate as
a natural source because of the availability of highly purified material, even
though the expression level of 3-OST-5 is relatively low in human intestine.
As the result of analysis, bovine intestinal heparan sulfate was found to
contain about 0.15% tetrasulfated disaccharide (mol/mol of total
disaccharides). The retention time of tetrasulfated disaccharide on a
SAX-column shifted markedly to an early position on reduction with sodium
borohydride, although the retention times of six major disaccharides were not
changed significantly by the reduction. Interestingly, the same phenomenon was
observed with
HexA-GlcN(NS,3S,6S) and
HexA(2S)-GlcN(NS,3S).
Therefore this is a characteristic of disaccharides containing a
3-O-sulfated glucosamine residue.
Although 3-OST-5 is the only enzyme known to catalyze the formation of the
tetrasulfated disaccharide unit at this time, there is no evidence that the
novel structure detected in this study was synthesized by the 3-OST-5 in
bovine intestine. The generation of transgenic and gene knockout animal models
could provide not only the answer to this question but also important
information about the biological role of 3-OST-5 or its reaction products
including tetrasulfated disaccharide.
 |
FOOTNOTES
|
---|
* This work was supported by the R&D Project of the Industrial Science
and Technology Frontier Program (R&D for Establishment and Utilization of
a Technical Infrastructure for Japanese Industry) supported by the New Energy
and Industrial Technology development Organization (NEDO). The costs of
publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section 1734
solely to indicate this fact. 
||||
To whom correspondence should be addressed: Glycogene Function Team, Research
Center for Glycoscience, National Institute of Advanced Industrial Science and
Technology (AIST), Central-2, 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Japan.
Tel.: 81-298-61-3200; Fax: 81-298-61-3201; E-mail:
h.narimatsu{at}aist.go.jp.
1 The abbreviations used are: HSPG, heparan sulfate proteoglycan; AT III,
antithrombin III; gD, glycoprotein D; 3-OST, heparan sulfate
D-glucosaminyl 3-O-sulfotransferase; HSV-1, herpes simplex
virus type-1; GlcA, D-glucuronic acid; IdoA, L-iduronic
acid; GlcN, D-glucosamine; GlcNAc,
N-acetyl-D-glucosamine; aMan,
2,5-anhydro-D-mannitol;
HexA, 4,5-unsaturated uronic acid;
2S, 2-O-sulfate; NS, 2-N-sulfate; 3S, 3-O-sulfate;
6S, 6-O-sulfate;
Di-0S,
HexA-GlcNAc;
Di-NS,
HexA-GlcN(NS);
Di-6S,
HexA-GlcNAc(6S);
Di-diS1,
HexA-GlcN(NS,6S);
Di-diS2,
HexA(2S)-GlcN(NS);
Di-triS1,
HexA(2S)-GlcN(NS,6S);
Di-triS2,
HexA-GlcN(NS,3S,6S);
Di-triS3,
HexA(2S)-GlcN(NS,3S);
Di-tetraS,
HexA(2S)-GlcN (NS,3S,6S); PAPS,
3'-phosphoadenosine 5'-phosphosulfate; GAPDH,
glyceraldehyde-3-phosphate dehydrogenase; SAX, strong anion-exchange; HPLC,
high performance liquid chromatography. 
 |
ACKNOWLEDGMENTS
|
---|
We thank Hiroshi Kikuchi for excellent technical assistance, Dr. Mamoru
Kyogashima for helpful support, and Dr. Hiroko Habuchi for critical reading of
the manuscript.
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