From the Department of Biology, Massachusetts
Institute of Technology, Cambridge, Massachusetts 02139, the
¶ Department of Medicine, Harvard Medical School, Beth Israel
Hospital, Boston, Massachusetts 02215, and the
Département de Chimie, Ecole Normale Superieure,
75231 Paris Cédex 05, France
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ABSTRACT |
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The 3-O-sulfation of glucosamine
residues is an important modification during the biosynthesis of
heparan sulfate (HS). Our previous studies have led us to purify and
molecularly clone the heparan sulfate D-glucosaminyl
3-O-sulfotransferase (3-OST-1), which is the key enzyme
converting nonanticoagulant heparan sulfate (HSinact) to
anticoagulant heparan sulfate (HSact). In this study, we
expressed and characterized the full-length cDNAs of 3-OST-1
homologous genes, designated as 3-OST-2, 3-OST-3A, and
3-OST-3B as described in the accompanying paper (Shworak, N. W., Liu, J., Petros, L. M., Zhang, L., Kobayashi, M.,
Copeland, N. G., Jenkins, N. A., and Rosenberg, R. D. (1999)
J. Biol. Chem. 274, 5170-5184). All these cDNAs
were successfully expressed in COS-7 cells, and heparan sulfate
sulfotransferase activities were found in the cell extracts. We
demonstrated that 3-OST-2, 3-OST-3A, and
3-OST-3B are heparan sulfate D-glucosaminyl
3-O-sulfotransferases because the enzymes transfer sulfate
from adenosine 3'-phosphophate 5'-phospho-[35S]sulfate
([35S]PAPS) to the 3-OH position of glucosamine.
3-OST-3A and 3-OST-3B sulfate an identical
disaccharide. HSact conversion activity in the cell extract
transfected by 3-OST-1 was shown to be 300-fold greater than that in
the cell extracts transfected by 3-OST-2 and 3-OST-3A,
suggesting that 3-OST-2 and 3-OST-3A do not make
HSact. The results of the disaccharide analysis of the
nitrous acid-degraded [35S]HS suggested that 3-OST-2
transfers sulfate to GlcA2S-GlcNS and IdoA2S-GlcNS;
3-OST-3A transfers sulfate to IdoA2S-GlcNS. Our results
demonstrate that the 3-O-sulfation of glucosamine is
generated by different isoforms depending on the saccharide structures
around the modified glucosamine residue. This discovery has provided
evidence for a new cellular mechanism for generating a defined
saccharide sequence in structurally complex HS polysaccharide.
The cell surface and extracellular matrix contain heparan sulfate
proteoglycans which consist of a core protein and single or multiple
heparan sulfate (HS)1 side
chains. The highly charged HS side chains are the copolymers of
glucuronic acid (GlcA)/iduronic acid (IdoA) and
N-acetylated glucosamine (GlcNAc) with various
sulfations. HS binds to specific proteins such as antithrombin and
several growth factors, thereby regulates various biological processes
including anticoagulation and angiogenesis (1). The unique sulfation
pattern of a monosaccharide sequence within HS is believed to be
critical for binding to the target protein. The biosynthesis of HS
involves the formation of a polysaccharide backbone followed by serial
sulfation and epimerization reactions. The HS backbone is elongated by
HS copolymerase-dependent (2) transfer of GlcA and GlcNAc
to the tetrasaccharide linkage region present on core proteins. This
process creates a polysaccharide backbone of approximately 100 repeating disaccharide units (1). This polysaccharide is structurally
altered at GlcNAc to form N-sulfated glucosamine (GlcNS) by
heparan sulfate N-deacetylase/N-sulfotransferase (3-5). The heparan sulfate C5 epimerase subsequently
converts occasional GlcA to IdoA within the polysaccharide (6). Heparan sulfate (uronosyl) 2-O-sulfotransferase transfers sulfate
from PAPS to IdoA to form 2-O-sulfated iduronic acid
(IdoA2S) (7), and heparan sulfate (D-glucosaminyl)
6-O-sulfotransferase transfers sulfate to the 6-OH position
of glucosamine to form 6-O-sulfated glucosamine (8).
Although this mechanism provides a scheme for generating the average
structure of HS, it is unable to explain how HS with defined
monosaccharide sequences are produced.
We have delineated a mechanism for generating HS with a specific
monosaccharide sequence based on our investigation of the biosynthesis
of anticoagulant active HS (HSact). Our results show that
the synthesis of HSact requires a limiting factor, as well
as a precursor with a defined polysaccharide structure which can be
modified by the limiting factor. HSact, normally present as
1-10% of total HS, contains a structurally defined
antithrombin-binding pentasaccharide with a sequence of -GlcNSorAc6S-GlcA-GlcNS3S±6S-IdoA2S-GlcNS6S- (9, 10). Our previous work demonstrated the presence of a specific pathway for the
biosynthesis of HSact in L cells. Overexpression of
syndecan-4 core protein or chemical mutagenesis in wild-type L cells
suggest that HSact generation requires a limiting factor,
and the limiting factor was identified as heparan sulfate
3-O-sulfotransferase (3-OST-1, EC 2.8.2.23) (11-15).
We recently proposed that HS biosynthetic enzymes are present in
multiple isoforms, and each isoform has slightly different substrate
specificities and recognizes the monosaccharide sequences around the
modification sites (1). Indeed, the available data support this
hypothesis (16-18). As documented by several researchers including
ourselves, 3-O-sulfation of glucosamine is a rare
modification that constitutes the last step in the biosynthesis of HS
(14, 15, 19, 20). We have shown that 3-OST-1 requires an appropriate HS
precursor with defined monosaccharide sequence for sulfation to occur
and make HSact.2
It is likely that 3-O-sulfated HS are specifically involved
in other HS-related biological processes. If this is the case, studies of 3-O-sulfation should facilitate the identification of new
biologically active HS-binding proteins. The 3-OST-1, which we have
previously purified and molecularly cloned, only sulfates the
glucosamine in GlcA-GlcNS±6S. However, there are at least two
other types of 3-O-sulfated glucosamine that were previously
characterized. For example, the HS isolated from glomerular basement
membrane contains substantial amounts of an IdoA2S-GlcNS3S disaccharide unit (21, 22). Pejler et al. (23) also reported that a
heparin isolated from the clams Anomalocardia brasiliana and
Tivela mactroides contained IdoA-GlcNS3S6S disaccharide
(23). It is apparent that neither the IdoA2S-GlcNS3S nor the
IdoA-GlcNS3S6S is the product of 3-OST-1. These observations suggest
the possible existence of different heparan sulfate
3-O-sulfotransferases, which specifically sulfate the 3-OH
positions of glucosamine residues that are linked to various
sulfated/nonsulfated uronic acid residues at the nonreducing termini.
In this article, we report the expression and characterization of new
heparan sulfate 3-O-sulfotransferase isoforms, designated as
3-OST-2, 3-OST-3A, and 3-OST-3B that were
isolated as described in the accompanying article (24). We have found
that 3-OST-2 and 3-OST-3A catalyze the
3-O-sulfation of glucosamine in GlcA2S-GlcNS and
IdoA2S-GlcNS, respectively, and 3-OST-3A and
3-OST-3B sulfate an identical disaccharide. The HS modified
by 3-OST-2 and 3-OST-3A do not contain anticoagulant
activity. Our studies reveal the presence of different isoforms, each
of which sulfates the sugar residue in unique disaccharides or possibly
in oligosaccharide sequences. Thus, we provide a new mechanism for the
biosynthesis of HS with specific monosaccharide sequences and different
potential biological functions.
Materials
Plasmids pJL2.7, pJL3.3, and pJL3.6 containing 3-OST-2,
3-OST-3A and 3-OST-3B were isolated from human
brain and liver cDNA libraries (24). The [35S]PAPS
( Expression of 3-OST Isoforms
The expression plasmids for 3-OST-2 and 3-OST-3A
were constructed by inserting EcoRI/XbaI cleaved
fragments from pJL 2.7 and pJL 3.3 into pcDNA3 (Invitrogen) which
was digested with EcoRI/XbaI followed by
phosphatase treatment. The expression plasmid for 3-OST-3B
was similarly constructed by inserting the
HindIII/NotI cleaved fragment from pJL 3.6 into
pcDNA3 which was digested with HindIII/NotI.
The 3-OST-1 expression construct has been previously described (15).
The expression plasmids were transfected into exponentially growing
COS-7 cells by using DEAE dextran and dimethyl sulfoxide (15).
Measurement of 3-OST-2 and 3-OST-3A/-3B
Activities
The crude enzymes were extracted from transiently transfected
COS-7 cell pellets by vortexing 2.5 × 105 cells in
200 µl of ice-cold 0.25 M sucrose with 1% Triton X-100 (v/v), and the insoluble residue was removed by spinning at 10,000 × g for 10 min. To determine HS sulfotransferase activity,
the reaction contained 10-40 µg of cell extract protein, 200 nM unlabeled HSinact isolated from 33 cells
(14), 60 × 106 cpm of [35S]PAPS, 50 mM MES (Sigma), pH 7.0, 10 mM
MnCl2, 5 mM MgCl2, 75 µg/ml
protamine chloride (Sigma), 120 µg/ml bovine serum albumin, and 1%
Triton X-100 (v/v) in a final volume of 50 µl. The reaction mixture
was incubated at 37 °C for 2 h, quenched by heating to 100 °C for 2 min and spun at 14,000 × g for 2 min
to remove insoluble material. The sample was then subjected to a
200-µl DEAE-Sepharose column to isolate [35S]HS (14,
25). One unit of HS sulfotransferase activity was defined as the
transfer of 1 fmol (×10 Degradation of 35S-Labeled HS
Low pH Nitrous Acid Degradation of
[35S]HS--
Purified [35S]HS modified by
3-OST-2, 3-OST-3A, and 3-OST-3B (2 × 104 to 4 × 106 cpm) was mixed with 20 µg of porcine kidney HS (ICN), treated on ice with HNO2,
at pH 1.5 for 30 min, and then reduced under alkaline conditions with
0.5 M NaBH4 (26). The resultant
35S-disaccharides were mixed with
IdoA2S-[3H]AnMan6S (2 × 104 to 2 × 106 cpm) and desalted on a Bio-Gel P-2 gel column
(0.75 × 200 cm) which was equilibrated with 0.1 M
ammonium bicarbonate at a flow rate of 4 ml/h.
High Performance Liquid Chromatography--
Separation and
characterization of 35S-labeled disaccharides were carried
out by HPLC using either a C18-reversed phase column (0.46 × 25 cm) (RPIP-HPLC) (Vydac) or a Partisil-5 strong anion exchange column (0.46 × 25 cm) (SAX-HPLC) (Whatman). The
RPIP-HPLC was eluted with 4.2% acetonitrile for 45 min followed by 9%
acetonitrile for 15 min and then by 10.2% acetonitrile for 120 min in
10 mM ammonium dihydrophosphate and 1 mM
tetrabutylammonium dihydrophosphate (Sigma) at a flow rate of 0.5 ml/min (11, 12, 27); the SAX-HPLC was eluted with 30 mM
KH2PO4 for 80 min followed by a linear gradient from 30 to 400 mM KH2PO4 over 120 min at a flow rate of 0.8 ml/min (11,12).
Determination of the Structures of Peak-X and Peak-Y
Preparation of 3H-Labeled Di- and Monosaccharide
Standards--
Disaccharide IdoA3S-GlcNS3SOMe
(methyl-(3-O-sodium sulfonato-sodium
Epimerization and Chemical Desulfation of 35S- and
3H-Labeled Disaccharides--
35S-Labeled
disaccharides (5-10 × 104 cpm) were mixed with
appropriate 3H-labeled disaccharide standards (1-5 × 105 cpm) in 50 µl of anhydrous hydrazine (Aldrich)
containing 10 mg/ml hydrazine sulfate and incubated at 100 °C in a
sealed 1.0-ml vacuum hydrolysis tube (Pierce) for 16 h followed by
HIO3 oxidation as described by Shaklee and Conrad (29). The
partial acid hydrolysis of 35S-labeled disaccharide (2 × 104 cpm) was performed in 0.5 M
H2SO4 and incubated at 100 °C for 15 min as
described elsewhere (30). The partially desulfated [35S]disaccharide became susceptible to Iduronate 2-Sulfatase Digestion--
Because iduronate
2-sulfatase was extremely sensitive to phosphate (IC50 = 25-100 µM) (32), the disaccharide samples were desalted
extensively by using a combination of DEAE chromatography, P-2 gel, and
descending paper chromatography. Briefly, the RPIP-HPLC purified
[35S]disaccharides were diluted 5-10-fold with water to
bring the concentration of phosphate to less than 5 mM,
neutralized with 10 M NaOH and charged to a 1-ml
DEAE-Sephacel column (Sigma), which was equilibrated with 10 mM triethylammonium bicarbonate buffer (Sigma), pH 8.0. The
column was washed with 10 × 1 ml of 100 mM
triethylammonium bicarbonate, pH 8.0, and eluted with 400 mM triethylammonium bicarbonate, pH 8.0. The resultant
disaccharides were loaded on a P-2 gel column (0.75 × 200 cm)
equilibrated and eluted with 0.1 M ammonium bicarbonate at
a flow rate of 4 ml/h. The disaccharides were then loaded on 3MM paper
(Whatman) which was developed in ethyl acetate/formic acid/acetic
acid/water = 36:2:6:1 for 2 h (12, 13), and the disaccharides
were recovered by eluting from the 3MM paper with 3 × 0.2 ml of
water. The condition for iduronate 2-sulfatase digestion followed the
procedures reported by Bielicki et al. (32).
Determination of [35S]Sulfation Sites within
HS--
In the accompanying paper (24), we reported the isolation of
3-OST-2, 3-OST-3A, and 3-OST-3B cDNAs with
about 60% similarity to human 3-OST-1 and about 45% similarity to two
forms of human heparan sulfate
N-deacetylase/N-sulfotransferase in the proposed sulfotransferase domain (24). Because of such high homology, it seemed
likely that these cDNAs encode heparan sulfate sulfotransferase activities. To test this hypothesis, we incubated 40 µg of cell extracts protein from COS-7 cells transfected with 3-OST-2,
3-OST-3A, 3-OST-3B, or control vector with
known amounts of [35S]PAPS and 200 nM of
various unlabeled glycosaminoglycans including HS, heparin, chondroitin
sulfate A, B, and C, and keratan sulfate. As expected, the amount of
[35S]HS and was increased by 2.5-3-fold compared with
the control, whereas the remaining glycosaminoglycans failed to show a
detectable increase in [35S]sulfate incorporation,
suggesting that these cDNAs encode enzymes that specifically
transfer [35S]sulfate from [35S]PAPS to
heparan sulfate. Furthermore, the [35S]HS was
depolymerized by a mixture of heparitinase-I to IV, and the product
migrated as a tetrasaccharide on Bio-Gel P-2 and anion exchange-HPLC
(data not shown). It is worthwhile to note that 3-OST-2 and
3-OST-3A activities were not found in the medium unless the
transmembrane domains were removed.
The specific sites at which HS is sulfated by 3-OST-2,
3-OST-3A, or 3-OST-3B were determined by
incubating [35S]PAPS and unlabeled HS with cell extracts
from COS-7 cells transfected with expression constructs containing the
appropriate cDNAs. Because 3-OST-3A- and
3-OST-3B-modified [35S]HS yielded an
identical 35S-labeled disaccharide after the nitrous acid
degradation, we only present the data of 3-OST-3A. The
[35S]HS prepared with transfected cell extracts were
depolymerized with low pH (pH 1.5) nitrous acid followed by sodium
borohydride reduction and analyzed by Bio-Gel P-2 column (Fig.
1). The resultant gel filtration
chromatograms demonstrated that the majority of the depolymerized
[35S]HS (90 ± 2%) eluted at positions ranging from
disulfated disaccharide to free sulfate (Fig. 1). Compared with control
cell extracts (Fig. 1A), 3-OST-2 (Fig. 1B) and
3-OST-3A cell extracts (Fig. 1C) generated
2.4-fold greater amounts of 35S-disulfated
disaccharides.
The chromatographic fractions containing 35S-labeled counts
were pooled and then analyzed on RPIP-HPLC to determine the nature of
the labeled disaccharides (Fig. 2). The
control cell extracts exhibited [35S]sulfate and small
amounts of 35S-labeled IdoA2S-AnMan6S, which is a common
disaccharide of heparan sulfate (Fig. 2A). The 3-OST-2 cell
extracts possessed 35S-labeled Peak-X and Peak-Y (Fig.
2B), which accounted for 60-80% of
[35S]SO4 incorporated into HS by the
expressed enzyme. The separation between Peak-Y and
GlcA-[3H]AnMan3S6S was improved on SAX-HPLC (data not
shown). The 3-OST-3A cell extracts contained Peak-X (Fig.
2C), which accounted for about 70% of
[35S]SO4 incorporated into HS by the
expressed enzyme. Neither Peak-X nor Peak-Y coeluted with known
3H-labeled heparin disaccharides. It is of interest to note
that nearly 80% of the degraded 35S-labeled material was
eluted as tetrasaccharides and hexasaccharides on Bio-Gel P-2 column
when 3-OST-2- and 3-OST-3A-modified [35S]HS
were treated by hydrazinolysis followed by high pH (pH 5.5), low pH (pH
1.5) nitrous degradation and sodium borohydride reduction. We suspected
that this unexpected result was caused by a high degree of ring
contraction during the degradation of the [35S]HS.
We obtained secreted forms of 3-OST-2 and 3-OST-3A by
truncating the putative transmembrane domains and expressing in
Sf9 insect cells. These secreted enzymes sulfate the same
disaccharides as those of the cell extracts from the COS-7 cells
transfected with 3-OST-2 and 3-OST-3A. Furthermore,
3-OST-3A was successfully purified from the insect
cell serum-free medium by using Heparin-Toyopearl 650M and
3',5'-ADP-agarose chromatography and exhibited the expected molecular
weight on SDS gel.5 Taken
together, these results suggest that 3-OST-2 and 3-OST-3A are directly responsible for the HS sulfotransferase activities.
Determination of the Structures of Peak-X and Peak-Y--
To
determine the structure of Peak-X, it was mixed with
IdoA2S-[3H]AnMan6S (Fig.
3A) and exhaustively digested
with iduronate 2-sulfatase. A mono-[35S]sulfated
disaccharide and IdoA-[3H]AnMan6S (Fig. 3B)
were generated. The shift in retention time of the
35S-labeled peak on SAX-HPLC suggested that Peak-X
contained a 2-O-sulfated iduronic acid residue. The
mono-[35S]sulfated disaccharide was then digested with
The structure of Peak-Y was determined by a combination of partial
chemical desulfation followed by
Our data suggested that the structural difference between Peak-X and
Peak-Y is at the position of the proton on C5 of the uronic
acid residue. To confirm this hypothesis, Peak-Y and Peak-X were
epimerized by incubating samples with hydrazine containing 1%
hydrazine sulfate at 100 °C for 16 h followed by
HIO3 oxidation. The product from Peak-X coeluted with
Peak-Y, and the product from Peak-Y coeluted with Peak-X on RPIP-HPLC.
Furthermore, we have also found that the epimerized Peak-Y became
susceptible to iduronate 2-sulfatase. In contrast, Peak-Y is totally
resistant to the digestion of iduronate 2-sulfatase (data not shown).
To eliminate the possibility that Peak-X or Peak-Y contains a
3-O-sulfated uronic acid residue, we compared the elution
positions of Peak-X and IdoA3S-[3H]AnMan3S and Peak-Y and
GlcA3S-[3H]AnMan3S, respectively. As expected, Peak-X
(retention time = 95.0 min) did not coelute with
IdoA3S-[3H]AnMan3S (retention time = 97.4 min),
suggesting that Peak-X does not contain 3-O-sulfated
iduronic acid residue. Likewise, Peak-Y contains a
2-O-sulfated glucuronic acid residue, because Peak-Y
(retention time = 108.0 min) did not coelute with
GlcA3S-[3H]AnMan3S (retention time = 115.0 min).
Comparison of HSact Conversion Activities among 3-OST
Isoforms--
We previously reported that 3-OST-1 was a key enzyme for
generating HSact, and the activity was determined by the
HSact conversion assay (13, 14). We, therefore, compared
the HS sulfotransferase and HSact conversion activities
with fixed amount of COS-7 cell extracts (10 µg of protein)
containing the 3-OST isoforms. First, we determined the HS
sulfotransferase activities by mixing unlabeled HS and [35S]PAPS with the COS-7 cell extract transfected with
empty vector (control), 3-OST-1, 3-OST-2, and 3-OST-3A
respectively, as described under "Experimental Procedures."
3-OST-1, 3-OST-2, and 3-OST-3A showed a similar increase of
the HS sulfotransferase activity compared with the control. This
suggested that the three isoforms were expressed at similar level, as
well as that the substrate in the reaction has sufficient amount of
[35S]sulfate acceptor sites for each isoform (Fig.
5A). Second, we assayed for
the HSact conversion activity on these cell extracts by
mixing with 35S-labeled HSinact and unlabeled
PAPS as described under "Experimental Procedures." 3-OST-1 elevated
the level of HSact conversion activity by more than
300-fold above the detection limit (Fig. 5B). On the
contrary, the cell extracts transfected with control, 3-OST-2, and
3-OST-3A did not show any detectable HSact
conversion activity (detection limit = 0.5 units) (Fig.
5B).
The cDNAs, which have approximately 60% similarity to the
earlier cloned 3-OST-1 in the sulfotransferase domain, were
demonstrated to encode heparan sulfate 3-O-sulfotransferase
isoforms. The strategy used to characterize these cDNAs is the
following: 1) expression of the cDNAs in COS-7 cells by transient
transfection; 2) preparation of 35S-labeled HS by mixing
the [35S]PAPS and unlabeled HS with the COS-7 cell
extracts transfected with 3-OST-2, 3-OST-3A, and
3-OST-3B; 3) determination of the 35S labeling
site within the [35S]HS by degrading the HS with nitrous
acid at low pH (pH 1.5). A similar approach has been previously used to
characterize 3-OST-1, heparan sulfate (D-glucosaminyl)
6-O-sulfotransferase, and heparan sulfate (uronosyl)
2-O-sulfotransferase (7, 8, 14). Our data suggest that
3-OST-2, 3-OST-3A, and 3-OST-3B are heparan sulfate 3-O-sulfotransferases because they transfer
[35S]sulfate from [35S]PAPS to the 3-OH
position of the glucosamine residue, and 3-OST-3A and
3-OST-3B sulfates an identical disaccharide.
To our knowledge, the activities of 3-OST-2 and 3-OST-3A
have not been previously reported. The substrate specificities of 3-OST-2 and 3-OST-3A at the disaccharide level were
identified by determining the structures and [35S]sulfate
labeling sites of two 35S-labeled disaccharides designated
as Peak-X and Peak-Y. These two disaccharides are the products of low
pH nitrous acid-treated [35S]HS prepared by 3-OST-2 and
3-OST-3A. Peak-X was identified to be
IdoA2S-[35S]AnMan3S, and Peak-Y was identified to be
GlcA2S-[35S]AnMan3S. The structure GlcA2S-AnMan3S has not
been identified previously in naturally occurring heparin and heparan
sulfate. However, because of the potential side reactions during
chemical degradation of [35S]HS, the substrate
specificities of 3-OST-2 and 3-OST-3A have not been
conclusively determined. Such side reactions can only be controlled by
using chemically defined oligosaccharides with expected modifications.
The differences between the isoforms and 3-OST-1 are as follows:
3-OST-1 generates HSact, while 3-OST-2 and
3-OST-3A do not. In addition, the isoforms transfer sulfate
to uronyl-glucosamine disaccharides with different sulfated uronic acid
residues. For example, 3-OST-1 transfers sulfate to the 3-OH position
of the glucosamine within GlcA-GlcNS ± 6S; 3-OST-2 transfers
sulfate to the 3-OH position of the glucosamine within GlcA2S-GlcNS and
IdoA2S-GlcNS; 3-OST-3A transfers sulfate to the 3-OH
position of the glucosamine within IdoA2S-GlcNS. However, we cannot
rule out the possibility that 3-OST-2 and 3-OST-3A sulfate the 3-OH position of the glucosamine within GlcA2S-GlcNH2
and IdoA2S-GlcNH2,
respectively.6 Furthermore,
3-OST-1 is a secretary protein, because substantial amounts of the
activity were detected in the cell medium and no putative membrane
spanning region occurs within the primary sequence of this enzyme (14,
15). We found that 3-OST-2 and 3-OST-3A are membrane-bound
proteins, because their activities were detected only in cell extracts
and putative membrane-spanning regions occur within their primary
sequences (24).
The unique substrate specificities of 3-OST-2 and 3-OST-3A
suggest that their products possess distinct biological functions. Since Peak-X could be released from the 3-OST-3A-modified
[35S]HS by direct low pH nitrous treatment, it became
evident that this disaccharide is derived from
-GlcNS6S(?)-IdoA2S-GlcNS3S- sequence. It is very interesting to note
that -GlcNS6S(?)-IdoA2S-GlcNS3S- has been identified to be present in
substantial amounts in the glomerular basement membrane HS (21). This
substance is believed to regulate the permeability of glomeruli (33,
34). Indeed both 3-OST-3A and 3-OST-3B are
expressed in human kidney (24).
It is also very interesting to note that 3-OST-2 mRNA co-localizes
with HS that contains a high level of GlcA2S in human brain. Although
3-OST-2 modifies the substrate HS containing both IdoA2S- and
GlcA2S-linked glucosamine, the enzyme likely prefers to sulfate the
disaccharide containing GlcA2S because the product contains higher
level of Peak-Y when a low concentration of substrate was used in the
enzymatic reaction. For example, we have found that the ratio between
Peak-Y (GlcA2S-[35S]AnMan3S) and Peak-X
(IdoA2S-[35S]AnMan3S) in the 3-OST-2-modified HS was
increased from 0.5 to 1.5 as the concentration of substrate was
decreased from 200 to 20 nM.7 Furthermore,
3-OST-2 should sulfate the sequence containing GlcA2S with much higher
efficiency because the population of a sulfated glucuronic acid is
approximately 5-50-fold less frequent than that of a sulfated iduronic
acid in any given heparan sulfate (35-38). Our Northern analysis has
demonstrated that 3-OST-2 is predominantly expressed in human brain as
described in the accompanying article (24). Interestingly, the level of
GlcA2S in HS isolated from various sections of human brain was reported
to be 5-8-fold higher than that in the HS isolated from kidney and
aorta, respectively (39). This suggests a special biological function
of the HS modified by 3-OST-2 in human brain.
We have previously demonstrated that the antithrombin-binding site on
heparan sulfate is synthesized in a two-step process in which six
copies of a precursor structure are generated per polysaccharide chain
that possesses the correct positioning of all critical groups except
for the absence of the 3-O-sulfate group. In the final step
of the reaction, the amount of 3-OST-1 is up-regulated at a
transcriptional level which completes the formation of the
antithrombin-binding site and controls the supply of this site present
on the cell surface (40). It appears likely that parallel reaction
pathways might exist in which other precursor structures, of different
monosaccharide sequences, are generated. These precursors may be
recognized by sulfating enzymes distinct from 3-OST-1 but homologous to
it. This would result in multiple, distinct HS end products with
different biologic functions. In this situation the regulation of the
levels of enzymes homologous to 3-OST-1 might be expected to control
the production of non-anticoagulant HS.
In summary, our investigation has revealed a new cellular mechanism for
generating defined monosaccharide sequences within the structurally
complicated HS polysaccharide. The classical model of the HS
biosynthetic machinery involves serial sulfation and epimerization of
HS. However, this model is unable to explain the formation of specific
saccharide structures or the regulation of their levels. Our results
demonstrate that specific disaccharides containing
3-O-sulfated glucosamine can be generated by specialized isoforms of heparan sulfate 3-O-sulfotransferase, although
whether these isoforms recognize disaccharide or oligosaccharide
sequences remains to be determined. These isoforms are also expressed
at different levels in different human tissues. If this is the case for
all classes of HS biosynthetic enzymes, the individual isoforms of a
particular enzyme could play a key role in generating tissue-specific HS having defined saccharide sequence with unique biological functions. Therefore, the isolation and characterization of the cDNAs encoding specialized isoforms should allow us to understand how a specific monosaccharide sequence is generated. Furthermore, manipulation of the
level of the special isoform within a cell or animal tissue may provide
an excellent approach for studying the specific biological effects of HS.
INTRODUCTION
Top
Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
150 Ci/mmol) was prepared by incubating 0.4 mCi/ml
[35S]Na2SO4 (ICN) and 16 mM ATP with 5 mg/ml dialyzed yeast extract (Sigma) as
described previously (14). Iduronate 2-sulfatase,
-glucuronidase,
and
-iduronidase were purified from bovine liver (10). The following
[3H]disaccharide standards were prepared from
antithrombin-binding heparin octasaccharide (a gift from Dr. Lun H. Lam, Glycomed) (12):
GlcA-[3H]AnMan6S,3
IdoA-[3H]AnMan6S, IdoA2S-[3H]AnMan,
GlcA-[3H]AnMan3S, IdoA2S-[3H]AnMan6S, and
GlcA-[3H]AnMan3S6S.
15 mol) of sulfate from PAPS to
heparan sulfate under standard assay conditions. The procedure for
determining HSact conversion activity was based upon our
previous report (14). One unit of the HSact conversion
activity was defined as 0.5% increase of HSact per 20 min
under standard conditions.
-L-idopyranosyluronate)-(1
4)-2-deoxy-2-sodium
sulfonatamido-3-O-sodium-sulfonato-
-D-glucopyranoside) and GlcA3S-GlcNS3SOMe (methyl-(3-O-sodium
sulfonato-sodium
-D-glucopyranosyluronate)-(1
4)-2-deoxy-2-sodium sulfonatamido-3-O-sodium
sulfonato-
-D-glucopyranoside) were chemically synthesized (28). The structures of these two compounds were confirmed
by 1H NMR (2H2O, 250 Mhz).4
IdoA3S-[3H]AnMan3S and GlcA3S-[3H]AnMan3S
were prepared by low pH nitrous acid treatment of the corresponding
chemically synthesized disaccharide followed by a reduction with sodium
[3H]borohydride. GlcA2S-[3H]AnMan6S was
prepared by epimerization of IdoA2S-[3H]AnMan6S in the
presence of hydrazine and hydrazine sulfate. The products were
confirmed by chemical desulfation in the presence of 0.5 M
sulfuric acid followed by
-glucuronidase digestion. [3H]AnMan3S was prepared from
-D-glucosamine 3-O-sulfate (Sigma) by nitrous
acid treatment at pH 1.5 followed by NaB3H4
reduction. [3H]AnMan6S was prepared from
-glucuronidase-treated GlcA-[3H]AnMan6S.
-iduronidase
or
-glucuronidase which permitted the establishment of the structure
of the disaccharide as well as the position of
[35S]sulfate (14,31).
-Glucuronidase and
-Iduronidase
Digestion--
35S-Labeled disaccharide (1 × 104 cpm) was mixed with GlcA-[3H]AnMan6S or
GlcA-[3H]AnMan3S6S in 50 µl of 50 mM NaAcO,
pH 4.5, and 100 µl of purified
-glucuronidase (5,000 units) and
incubated at 37 °C for 36 h. For
-iduronidase digestion, the
35S-labeled disaccharide was mixed with
IdoA-[3H]AnMan6S (1-10 × 104 cpm) in
50 µl of buffer containing 250 mM sodium formate and 400 mM NaCl, pH 3.55, and 100 µl of
-iduronidase (8 units)
and incubated at 37 °C for 36 h.
RESULTS
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Fig. 1.
Bio-Gel P-2 fractionation of depolymerized
[35S]HS. [35S]HS generated by
incubating [35S]PAPS with unlabeled HSinact
and cell extracts prepared from COS-7 cells transfected with control
vector, 3-OST-2, or 3-OST-3A were depolymerized with low pH
nitrous acid (pH 1.5). The depolymerized [35S]HS was
mixed with IdoA2S- [3H]AnMan6S as an internal standard
and chromatographed on a column (0.75 × 200 cm) of Bio-Gel P-2
equilibrated with 0.1 M ammonium bicarbonate. The
three panels represent the profiles of the products of
[35S]HS prepared with 40 µg of cell extract of COS-7
cells transfected with vector alone (A), 3-OST-2
(B), and 3-OST-3A (C). Fractions of
one-half ml were collected, analyzed for 3H and
35S, and then were pooled as indicated by the solid
bar. The arrows indicate the elution positions of
hexa-, tetra-, and disaccharide.
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Fig. 2.
RPIP-HPLC of 35S-labeled HS
disaccharides. The disaccharides obtained by gel filtration
(pooled fractions in Fig. 1) were mixed with various
3H-labeled disaccharide standards as internal standards and
separated on RPIP-HPLC as described under "Experimental
Procedures." The three panels represent the chromatograms
of 35S-labeled disaccharides derived from
[35S]HS prepared with the cell extracts of COS-7 cells
transfected with vector alone (A), 3-OST-2 (B),
and 3-OST-3A (C). The arrows indicate
the elution positions of 3H-labeled disaccharide standards,
where 2 represents IdoA2S-[3H]AnMan,
3 represents GlcA-[3H]AnMan6S, 4 represents IdoA-[3H]AnMan6S, 5 represents
GlcA-[3H]AnMan3S6S, and 6 represents
IdoA2S-[3H]AnMan6S.
-iduronidase which generated a mono-[35S]sulfated
monosaccharide (Fig. 3C), coeluting with
[3H]AnMan3S but not with [3H]AnMan6S on
RPIP-HPLC (Fig. 3C, inset). This latter result
shows that [35S]sulfate was located at the 3-OH position
of the 2,5-anhydro-D-mannitol residue. Therefore, the data
suggest that Peak-X has the structure of
IdoA2S-[35S]AnMan3S.
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Fig. 3.
SAX-HPLC analysis of the iduronate
2-sulfatase and -iduronidase digested
Peak-X. Panel A shows the elution positions of Peak-X
and IdoA2S- [3H]AnMan6S on SAX-HPLC; Panel B
shows the elution positions of IdoA-[3H]AnMan6S and mono-
[35S]sulfated disaccharide yieled by the iduronate
2-sulfatase digested Peak-X; Panel C shows the elution
positions of [3H] AnMan6S and
mono-[35S]sulfated disaccharide digested with
-iduronidase (80 units/ml). The inset represents the
chromatography of the 35S-labeled monosaccharide on
RPIP-HPLC, which was eluted at a flow rate of 0.5 ml/min with 1.2%
acetonitrile in 1 mM tetrabutylammonium dihydrophosphate
and 10 mM ammonium dihydrophosphate. Under these
conditions, maximum resolution was obtained for
[3H]AnMan6S and [3H]AnMan3S.
-glucuronidase digestion (Fig.
4). The sulfate was removed from the
glucuronic acid residue by incubating Peak-Y and
GlcA-[3H]AnMan3S6S with 0.5 M sulfuric acid,
and analyzing the desulfated products on RPIP-HPLC. The
GlcA-[3H]AnMan3S6S, which serves as an internal standard,
allowed us to monitor the extent of the chemical desulfation and
determine the eluting position of monosulfated disaccharides on
RPIP-HPLC. The Peak-Y yielded a partially desulfated
mono-[35S]sulfated disaccharide, which coeluted with
GlcA-[3H]AnMan3S on RPIP-HPLC (Fig. 4). The structure of
the partially desulfated Peak-Y was confirmed to be
GlcA-[35S]AnMan3S by digesting with
-glucuronidase and
showing that the elution position of the 35S-labeled peak
was shifted from 55 to 9 min on SAX-HPLC (Fig. 4, inset).
Thus, the data suggest that the structure of Peak-Y is
GlcA2S-[35S]AnMan3S. It should be noted that Peak-Y prior
to partial acid desulfation was resistant to the action of
-glucuronidase (data not shown).
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Fig. 4.
HPLC analysis of chemically desulfated
Peak-Y. Peak-Y and GlcA-[3H]AnMan3S6S standard was
subjected to partial chemical desulfation and analyzed on RPIP-HPLC.
The inset shows the elution position of the partially
desulfated Peak-Y digested with -glucuronidase (50,000 units/ml) on
SAX-HPLC. The arrow in the inset represents the
elution position of the partially desulfated Peak-Y.
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Fig. 5.
Comparison of the capability of generating
HSact among 3-OST isoforms. Panel A shows
the HS sulfotransferase activity in the extract of COS-7 cell
transfected with empty vector (control), 3-OST-1, 3-OST-2,
and 3-OST-3A. The sulfotransferase activity was determined
by mixing unlabeled HS (200 nM) with
[35S]PAPS (60 × 106 cpm, 150 Ci/mmol)
and cell extracts (10 µg) in 50 µl of reaction buffer as described
under "Experimental Procedures." The 35S-labeled HS was
extracted by using DEAE chromatography and eluted with 1 M
sodium chloride. One unit of HS sulfotransferase activity was defined
as the transfer of 1 fmol (×10 15 mol) sulfate from PAPS
to heparan sulfate under standard assay conditions. Panel B
shows the HSact conversion activity in the extract of COS-7
cells transfected with empty vector (control), 3-OST-1,
3-OST-2, and 3-OST-3A. The HSact conversion
activity was determined by mixing
[35S]HSinact (1 × 105 cpm),
cell extracts (10 µg), and unlabeled PAPS (500 µM) in
50 µl of reaction buffer containing protamine. The amount of
converted [35S]HSact was determined by
measuring the 35S-labeled counts bound to ConA/AT affinity
chromatography. One unit of the HSact conversion activity
was defined as 0.5% increase of HSact per 20 min under
standard conditions. The presented data are the average of duplicate
determinations, and the error bar indicates the range.
DISCUSSION
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ACKNOWLEDGEMENT |
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We thank Peter Blaiklock for excellent effort in expressing the secreted form of 3-OST-2 in insect cells.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants 5-P01-HL41484.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.
§ Recipient of an American Heart Association, Massachusetts Affiliate, Postdoctoral Fellowship.
** Recipient of National Research Service Award Postdoctoral Fellowship.
To whom correspondence and reprint requests should be
addressed: Massachusetts Institute of Technology, 68-480, 77 Massachusetts Ave., Cambridge, MA 02139. Tel.: 617-253-8803; Fax:
617- 258-6553.
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ABBREVIATIONS |
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The abbreviations used are: HS, heparan sulfate; PAPS, adenosine 3'-phosphosphate 5'-phosphosulfate; GlcA, D-glucuronic acid; GlcNAc, N-acetyl-D-glucosamine; 3-OST, heparan sulfate D-glucosaminyl 3-O-sulfotransferase; GlcNS, N-sulfo-D-glucosamine; IdoA, L-iduronic acid; HSact, anticoagulant heparan sulfate; HSinact, nonanticoagulant heparan sulfate; IdoA2S, L-iduronic acid 2-O-sulfate; GlcA2S, D-glucuronic acid 2-O-sulfate; GlcNR'6S, N-acetyl-D-glucosamine 6-O-sulfate or N-sulfo-D-glucosamine 6-O-sulfate; GlcNS3S±6S, N-sulfo-D-glucosamine 3-O-sulfate or N-sulfo-D-glucosamine 3,6-O-bisulfate; HPLC, high performance liquid chromatography; SAX-HPLC, strong anion exchange-HPLC; RPIP-HPLC, reversed phase ion pairing HPLC; AnMan, 2,5-anhydro-D-mannitol; AnMan3S, AnMan6S, and AnMan3S6S, 2,5-anhydro-D-mannitol 3-O-sulfate, 6-O-sulfate and 3,6-O-bisulfate, respectively; AT, antithrombin; Mes, 4-morpholineethanesulfonic acid.
2 Zhang, L., Yoshida, K., Liu, J., and Rosenberg, R. D. (1999) J. Biol. Chem. 274, in press.
3
All 3H-labeled disaccharide and
monosaccharide standards used in the experiments were labeled at the
C1 position of 2,5-anhydromannitol. The structures of di-
and oligosaccharides were presented in an abbreviated format omitting
D-, L- 1
4, and
1
4 for each sugar residue in order to conserve space and improve the clarity. -GlcA-, -GlcNR'-, and -IdoA- represents the linkage of
4)-
-D-GlcA(1
,
4)-
-D-GlcNR'(1
, and
4)-
-L-IdoA-(1
, respectively.
4
S. Tripathy, J.-M. Mallet, M. Petitou, and P. Sina, manuscript in preparation.
5 J. Liu, Z. Shriver, P. Blaiklock, K. Yoshida, R. Sasisekharan, and R. D. Rosenberg, manuscript in preparation.
6 We have recently found that purified 3-OST-3A sulfates the glucosamine residue with free amino group. The conclusion was based on the results of the structural analysis of two 35S-labeled tetrasaccharides, which were isolated from heparin lyases degraded the [35S]HS [35S]sulfated by purified 3-OST-3A.
7 J. Liu, unpublished data.
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REFERENCES |
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