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 ¶ Tokyo Research
Institute of Seikagaku Corp.,
Higashiyamato-shi, Tokyo 207, Japan
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ABSTRACT |
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To understand the mechanisms that control
anticoagulant heparan sulfate (HSact) biosynthesis,
we previously showed that HSact production in the F9 system
is determined by the abundance of 3-O-sulfotransferase-1 as
well as the size of the HSact precursor pool. In this
study, HSact precursor structures have been studied by
characterizing [6-3H]GlcN metabolically labeled F9 HS
tagged with 3-O-sulfates in vitro by
3'-phosphoadenosine 5'-phospho-35S and purified
3-O-sulfotransferase-1. This later in vitro
labeling allows the regions of HS destined to become the antithrombin
(AT)-binding sites to be tagged for subsequent structural studies. It
was shown that six 3-O-sulfation sites exist per
HSact precursor chain. At least five out of six
3-O-sulfate-tagged oligosaccharides in HSact
precursors bind AT, whereas none of 3-O-sulfate-tagged
oligosaccharides from HSinact precursors bind AT. When
treated with low pH nitrous or heparitinase, 3-O-sulfate-tagged HSact and
HSinact precursors exhibit clearly different structural
features. 3-O-Sulfate-tagged HSact
hexasaccharides were AT affinity purified and sequenced by chemical and enzymatic degradations. The 3-O-sulfate-tagged
HSact hexasaccharides exhibited the following
structures,
Different heparin/heparan sulfate
(HS)1 sequences bind to a
large number of growth factors and cytokines (1-6), enzymes (7), protease inhibitors (8-12), virus proteins (13), and selectins (14).
Such sequences are usually synthesized in the right place (15) and at
the right time (16, 17). These HS species are involved in development,
angiogenesis, lipid metabolism, coagulation, virus infection, and
inflammation (18-22). To synthesize HS oligosaccharide sequences that
bind to these specific protein ligands probably requires the synthesis
of specific HS precursor structures. HS precursor structures are then
acted upon by a unique sulfotransferase, which is positioned at the end
of the biosynthetic pathways and whose levels control the concentration
of the specific HS component that is the product of the biosynthetic pathway.
The pentasaccharide sequence,
GlcNAc/NS6S-GlcUA-GlcNS3S6S-IdceA2S-GlcNS6S,
represents the minimum sequence for antithrombin (AT) binding, where
the boldface 3S and 6S constitute the most critical elements involved
in the interaction (8, 9). The first sugar residue is either
N-acetylated or N-sulfated. The third sugar
residue is either with or without a 6-O-sulfate, depending
on the source of heparin from which these sequences were originally
characterized (23). The AT-binding sequence also exists in HS, but such
sequences have never been fully characterized due to limited materials.
HSact produced by endothelial cells (1% total HS) is
responsible in part for the nonthrombogenic properties of blood vessels
(24). Even though it constitutes a small portion of
HStotal, the relative abundance of the AT-binding sequence
is at least 10-fold greater than would be predicted by completely
random assembly of disaccharide constituents (25). These observations
suggest that production of the AT-binding sequence requires the
coordinated action of several biosynthetic enzymes, i.e. the
enzymes that catalyze chain polymerization, GlcNAc
N-deacetylation and N-sulfation, GlcUA
epimerization, 2-O-sulfation of uronic acid residues, and 3-O- and 6-O-sulfation of glucosaminyl residues.
Multiple forms of N-sulfotransferases (26-30) and
3-O-sulfotransferases
(31)2 have been reported
recently. Due to the substrate specificity of the enzymes involved, the
initial distribution of N-sulfate groups strongly influences
the subsequent epimerization and O-sulfations (32, 33). The
downstream O-sulfation may also be able to influence N-sulfation as well (34). However, the mechanisms that
control the coordinated action of these enzymes to generate
AT-binding sequences or other sequences in HS and heparin are unknown.
To delineate the biosynthetic pathway that regulates HSact
synthesis, our laboratory has purified as well as molecularly cloned 3-O-ST-1 (EC 2.8.2.23). It was demonstrated that
3-O-ST-1, existing in limited amount, acts upon
HSact precursor to produce HSact and
HSinact precursor to produce 3-O-sulfated
HSinact (31, 35). When 3-O-ST-1 is no longer
limiting in the F9 cell system, the capacity for HSact
generation is determined by the abundance of HSact
precursors (36). We have reported that overall HSact and
HSinact structures are different at the disaccharide level
in the F9 cell system. In vitro 3-O-sulfation
with purified 3-O-ST-1 can tag the regions of the
HSact precursors destined to become AT-binding sites and
allow the HSact precursors to be captured. The tagged
regions can then be structurally examined (36). Based on the above
observations, we have studied the HSact and
HSinact precursor structures by characterizing
[6-3H]GlcN metabolically labeled F9 HS tagged with
3-O-sulfates in vitro by PAP35S and
purified 3-O-ST-1. We have found that HSact and
HSinact precursors in F9 cells have different epimerization
and O-sulfation patterns around the 3-O-sulfate
acceptor sites in the intact glycosaminoglycan (GAG) chain. We have
sequenced the 3-O-sulfate-tagged HSact precursor
hexasaccharides that represent the 3-O-sulfate acceptor sites in the HSact precursors, which are destined to become
AT-binding sites. This region contains five hexasaccharides,
i.e.
( Cell Culture--
F9 cells were grown on gelatin (Sigma) coated
(0.1%) tissue culture dishes in Dulbecco's modified Eagle's medium
(DMEM) containing 10% heat-inactivated calf serum (Irvine Scientific),
penicillin G (100 units/ml), and streptomycin sulfate (100 µg/ml)
under an atmosphere of 5% CO2, 95% air and 100% relative
humidity. All tissue culture media and reagents were purchased from
Life Technologies, Inc., unless otherwise indicated.
HS Preparation--
The methods for HS preparation have been
described previously (36). In brief, cell monolayers were labeled with
100-1000 µCi/ml sodium [35S]sulfate (carrier-free,
ICN) overnight by incubation in sulfate-deficient DMEM, penicillin G
(100 units/ml), and 10% (v/v) fetal bovine serum that had been
exhaustively dialyzed against phosphate-buffered saline and
supplemented with 200 µM Na2SO4.
[6-3H]Glucosamine labeling were conducted in 100 µCi/ml
D-[6-3H]glucosamine (40 Ci/mmol, ICN)
overnight in 1 mM glucose DMEM with 10% (v/v) dialyzed
fetal bovine serum. GAGs were isolated from both monolayer and media.
Purified GAGs were resuspended in 0.5 M NaBH4
in 0.4 M NaOH and incubated at 4 °C for 24 h to release the chains from the core protein.
The quality of HS chains prepared was analyzed by anion exchange HPLC
(TSK DEAESW, 8 cm × 7.5 mm inner diameter, Tosohaas Inc.).
Samples were eluted with a linear gradient of 0.2 to 1 M
NaCl in 10 mM KH2PO4, pH 6.0, containing 0.2% CHAPS at a flow rate of 1 ml/min, and radioactivity in
the effluent was determined by on-line liquid scintillation
spectrometry (Packard).
The absolute amounts of the unlabeled HS were determined by hydrolyzing
HS for 3 h with 6 N HCl and 0.1% phenol (v/v) at
100 °C and determining the levels of glucosamine on an Applied
Biosystems model 420 Amino Acid Derivatizer with on-line model 130A PTC
amino acid analyzer.
HSact and HSinact Precursors Were Tagged
by 3-O-Sulfation with Purified 3-O-ST-1 and PAPS--
The standard
50-µl reaction contains 400 units of purified
3-O-sulfotransferase-1 (3-O-ST-1) (EC 2.8.2.23),
60 µg of bovine serum albumin, metabolically labeled
[3H]HS, [35S]HS, or cold HS chains prepared
from F9 cells suspended in 1 mM CaCl2, 5 mM MgCl2, 10 mM MnCl2,
0.375 µg/ml protamine, 0.4 mg/ml chondroitin sulfate, 1% Triton
X-100, 50 mM MES (pH 7.0), and 1 mM PAPS or 25 µM (1 × 108 cpm) PAP35S.
The reactions were incubated at 37 °C overnight and then terminated by boiling for 1 min. After adding 100 µg of chondroitin sulfate as
cold carrier, the radiolabeled HS was purified by phenol/chloroform extraction, DEAE-Sepharose chromatography, and ethanol precipitation. After washing the pellets with 0.5 ml of 75% ethanol,
3-O-sulfate-tagged HSact and HSinact
precursors were separated by AT affinity assay as described below.
Separation of 3-O-Sulfate-tagged HSact and
HSinact Precursors by AT Affinity Assay--
AT affinity
assay was used as described previously (36). In brief, AT complexes
were created by mixing 3-O-sulfate-tagged HStotal in 500 µl of HB (150 mM NaCl, 10 mM Tris-Cl (pH 7.4)) with 2.5 mM AT, 100 µg
of chondroitin sulfate, 0.002% Triton X-100, 1 mM each of
CaCl2, MgCl2, and MnCl2. 60 µl of
HB containing ~50% concanavalin A-Sepharose 4B was then added. AT
complexes were bound to concanavalin A by mixing the reaction mixtures
for 1 h at room temperature. Beads were pelleted by a brief
centrifugation at 10,000 × g. The supernatant was
collected, and the beads were washed three times with 1.25 ml of HB
containing 0.0004% Triton X-100. The supernatant and washing solutions
were combined as 3-O-sulfate-tagged HSinact
precursors. 3-O-Sulfate-tagged HSact precursors
were eluted with three successive 200-µl washes of HB containing 1.0 M NaCl, 0.0004% Triton X-100 and pooled. After adding 100 µg of chondroitin sulfate as cold carrier to
3-O-sulfate-tagged HSact precursors, the pooled
3-O-sulfate-tagged HSact and HSinact
precursors were cleaned by phenol/chloroform extraction followed by
DEAE-Sepharose chromatography and ethanol precipitation. After washing
the pellets with 0.5 ml of 75% ethanol and dried briefly by Speed-Vac,
3-O-sulfate-tagged HSact and HSinact
precursors were resuspended in H2O and used for chemical
and enzymatic structural studies.
Low pH Nitrous Acid
Degradation--
3-O-Sulfate-tagged HSact and
HSinact precursors or 3-O-sulfate-tagged tetra-
or hexasaccharide were mixed with 10 µg of bovine kidney HS (ICN),
treated at room temperature with low pH nitrous acid (pH 1.5) for 10 min, and then reduced under alkaline conditions with 0.5 M
NaBH4 for another 10 min. Bio-Gel P6 or P2 chromatography as described below was used to analyze further the resultant products.
Bio-Gel P2 and P6 Gel Filtration Chromatography of Chemically or
Enzymatically Degraded 3-O-Sulfate-tagged HSact and
HSinact Precursors--
Bio-Gel P2 (0.75 × 200 cm)
and P6 (0.75 × 200 cm) columns were equilibrated with 100 mM ammonium bicarbonate at a flow rate of 4 ml/h. 200 µl
of radiolabeled sample mixed with dextran blue (5 µg) and phenol red
(5 µg) was loaded on the column. 0.4 ml per fraction was collected. A
10-µl sample from each fraction was counted for 3H and/or
35S radioactivity unless otherwise indicated. The desired
fractions were pooled and dried by Speed-Vac to remove ammonium
bicarbonate and used for further analysis. A small amount of free
sulfate generated during the experimental procedures was quantitated by re-running the collected disaccharide fractions on a polyamine HPLC.
The free sulfate is subtracted from disaccharides to get the accurate
di-, tetra-, and oligosaccharide quantitations indicated under
"Results."
Digestion of 3-O-Sulfate-tagged HSact and
HSinact Precursors with Heparitinase I, Heparitinase II,
Heparinase, and Heparitinase IV--
Heparitinase I (EC 4.2.2.8),
heparitinase II (no EC number), heparinase (EC 4.2.2.7) were obtained
from Seikagagu; heparitinase IV was from Dr. Yoshida, Seikagagu Corp.,
Tokyo, Japan. Heparitinase I recognizes the following sequences:
GlcNAc/NS±6S(3S?)-
The digestion of 3-O-sulfate-tagged HSact and
HSinact precursors was carried out in 100 µl of 40 mM ammonium acetate (pH 7.0) containing 1 mM
CaCl2 with 2 milliunits of enzyme or 2 milliunits of each heparitinase I, heparitinase II, and heparinase. The digestion was
incubated at 37 °C overnight unless otherwise indicated.
PAMN High Performance Liquid Chromatography--
Separation and
characterization of 3-O-sulfate-tagged tetra- and
hexasaccharides were carried out by HPLC using an amine-bound silica
PA01 column (0.46 × 25 cm) (PAMN-HPLC) (YMC). 100-µl aliquots containing 2 nmol of each six known heparan sulfate disaccharide standards (Seikagagu) were included with all analytic runs. The PAMN-HPLC was eluted with H2O for 5 min followed by
0-100% KH2PO4 linear gradient for 100 min at
a flow rate of 1 ml/min. The elution of cold disaccharide standards was
detected by on-line UV monitoring, and radiolabeled disaccharides were
monitored by on-line liquid scintillation spectrometry (Packard
Instrument Co.). Each fraction represents a 0.5-ml elution volume.
Sequential Enzymatic Degradation of
3-O-35S-Sulfate-tagged tetrasaccharides were
digested with 10 milliunits of The HSact Precursors Contain Six Potential AT-binding
Sites Whose Structure Is Completed by in Vitro 3-O-Sulfation--
We
have previously reported that F9 cells make 1% HSact and
29% HSact precursors, and the metabolically labeled
GlcUA-anManR335S (6%) and
GlcUA-anManR335S635S (12%)
accounted for about 18% of O-sulfated disaccharides in 1%
F9 HSact (36). The high percentage of
3-O-sulfated disaccharides in the biosynthesized
HSact chain suggests that there are multiple potential
3-O-sulfation sites in the F9 HSact precursor
chain. To estimate this parameter, [6-3H]GlcN
metabolically labeled HSact precursor was isolated by
exhaustive conversion to HSact by exposure in
vitro to pure 3-O-ST-1 as well as PAPS and subsequent captured by AT affinity chromatography. The
3-O-sulfate-tagged HSact precursors were
subjected to a mixture of heparitinase I, heparitinase II, and
heparinase treatment. According to a previous publication (38), the
combined heparitinase I, heparitinase II, and heparinase digestion
should depolymerize all the 3-O-sulfate containing sites into tetrasaccharides and the remaining sugar residues into
disaccharides. The depolymerized materials were sized by Bio-Gel P2
chromatography (Fig. 1). 15% of
3H counts are resistant to digestion and remained as
tetrasaccharides. In contrast, 0.5% of 3H counts are
resistant to digestion and remained as tetrasaccharides in
[6-3H]GlcN metabolically labeled F9 HS control (data not
shown).
The di- and tetrasaccharides indicated by solid bar in Fig.
1 were collected and subjected to polyamine HPLC chromatography (Fig.
2). Each disaccharide peak on HPLC was
assigned by comparing to standards ("Experimental Procedures"). The
two tetrasaccharide peaks that are resistant to the combined
treatment of heparitinase I, heparitinase II, and heparinase digestion
are likely to represent the 3-O-sulfate-containing
tetrasaccharides. We will subsequently show that the first
tetrasaccharide peak has the structure
The di- and tetrasaccharides from Fig. 2 were further analyzed to
estimate the number of 3-O-sulfation sites per
HSact precursor chain. We first assessed the molecular
weight for F9 HS by SDS-polyacrylamide gel electrophoresis as described
previously (39), where Mr = 2.112 × protein M-21027. HS exhibits an average molecular weight
(Mr) of 42,000 (data not shown). Based on this and the average disaccharide molecular mass of 500 Da, there are about
84 disaccharides per chain, i.e. 82 disaccharides in the repetitive -(UA-GlcNAc/S)n- region and one tetrasaccharide GlcUA-Gal-Gal-Xyl- in the linkage region. Assuming the average HS
consists of 82 disaccharides in the repetitive -(UA-GlcNAc/S)n- region, the number of di- and tetrasaccharides per
3-O-sulfated HSact precursor chain are
summarized in Table I. There are 4 ×
The sum of N-sulfates, 2-O-sulfate,
6-O-sulfates, and 3-O-sulfates for each sugar
residues is provided in Table I and equals 100% of the total sulfate
groups per 3-O-sulfate-tagged chain, which corresponds to a
total of 71 sulfate groups. Of these, 37 sulfate groups or 52% are
O-sulfates. Furthermore, since 3-O-sulfates comprise 14% of all metabolically labeled O-sulfates (36).
We can calculate that each endogenous HSact chain contains
5.2 3-O-sulfation sites (37 × 14%).
By treating cold F9 HS with PAP35S and 3-O-ST-1,
we found that 6 pmol of [35S]sulfates from
PAP35S are transferred to 3-O-sulfate positions
in 1 pmol of F9 HSact precursors (data not shown). Thus,
from the evidence presented above, we concluded that there are six
3-O-sulfate acceptor sites per HSact precursor
chain, which can be captured as 3-O-sulfated
tetrasaccharides as indicated above.
We then estimated the number of 3-O-sulfate acceptor sites
per HSinact precursor chain. We previously reported that
metabolically labeled GlcUA-anManR335S (5%)
and GlcUA-anManR335S635S (5%)
accounted for 10% of O-sulfated disaccharides in retinoic acid and dibutyryl cAMP plus theophilline F9 HSinact.
However, endogenous retinoic acid and dibutyryl cAMP plus theophilline F9 HSinact are composed of 3-O-sulfated
HSinact as well as HSact and
HSinact precursors that have never been
3-O-sulfated (36). To obtain a more accurate estimate, cold
F9 HS was exhaustively converted with PAP35S and
3-O-ST-1. After AT affinity fractionation to remove all 3-O-sulfate-tagged HSact precursors, we found
that 6 pmol of [35S]sulfates from PAP35S are
transferred to 3-O-sulfate positions in 1 pmol of F9
HSinact precursors (data not shown). Therefore, we conclude
that there are six 3-O-sulfate acceptor sites in
HSinact precursors as well.
3-O-Sulfate-tagged Tetrasaccharide Structures--
To characterize
the two tetrasaccharide structures in Fig. 2B, we repeated
the experiment to obtain the tetrasaccharides using different F9 HS
preparations. To this end, either cold F9 HS was tagged with
radiolabeled 3-O-sulfates or 35SO4
metabolically labeled HS was tagged with cold 3-O-sulfates in vitro by PAPS/PAP35S and purified
3-O-ST-1. Affinity purified 3-O-sulfate-tagged HSact precursors were subjected to heparitinase I,
heparitinase II, and heparinase digestion. The radiolabeled
tetrasaccharides were collected from Bio-Gel P2 chromatography. The
identical two tetrasaccharide peaks with the same 2 to 1 ratio of
tetrasaccharide I to tetrasaccharide II were found after polyamine HPLC
chromatography among the three 3-O-sulfate-tagged
tetrasaccharide sources (data not shown). This result suggests that we
can characterize the tetrasaccharides from any
3-O-sulfate-tagged F9 HSact precursors.
The 3-O-35S-tagged tetrasaccharide I and
tetrasaccharide II from cold F9 HSact were separated by
polyamine HPLC chromatography and collected for the tetrasaccharide
sequencing analysis. A fraction of each tetrasaccharide collected was
re-run on polyamine HPLC (Fig. 3, A and B). Tetrasaccharide I and tetrasaccharide
II were first treated with low pH nitrous acid, which cleaves the
GlcNS±6S-GlcUA/IdceA±2S linkage but not the
GlcNAc±6S-GlcUA/IdceA±2S linkage in the tetrasaccharides. However,
after low pH nitrous acid treatment and NaBH4 reduction, both tetrasaccharide peaks remained as tetrasaccharides as judged by
the Bio-Gel P2 profile (data not shown), but the elution time on
polyamine HPLC was altered (Fig. 3, C and D). The
alteration in this parameter corresponds to the lost of one
N-sulfate and ring contraction from the reducing end GlcN
residue of both tetrasaccharides.
The tetrasaccharides collected were then treated with
Further GlcNAc 6-O-sulfatase treatment results in the loss
of one sulfate as judged by polyamine HPLC (Fig. 3, G and
H). After
We carried out a similar experiment to determine the
3-O-35S-tagged HSinact
tetrasaccharide structures by using
3-O-35S-tagged F9 HSinact
precursors. The combined treatment with heparitinase I, heparitinase II, and heparinase treatment generates both
Peak II (56%) and peak III (29%) have the same retention time as the
tetrasaccharides in Fig. 3, C and D. The
identical polyamine HPLC profiles as in Fig. 3, E
The elution position of peak I on polyamine HPLC suggests that this
tetrasaccharide may have only one sulfate group. After Five of Six 3-O-Sulfate-tagged Oligosaccharides in
HSact Precursors Bind to AT--
Both HSact
and HSinact precursors contain about six
3-O-sulfation sites per chain. 3-O-Sulfate-tagged
HSact precursors binds to AT, and
3-O-sulfate-tagged HSinact precursors do not.
The next question we asked is how many of the 3-O-sulfate
acceptor sites (potential AT-binding sites) in 3-O-sulfate-tagged HSact precursors actually
bind to AT. We reasoned that heparitinase I digestion should leave
AT-binding sites intact since all the 3-O-sulfate-tagged
HSact precursors remained as 3-O-sulfate-tagged
tetrasaccharide after heparitinase I, heparitinase II, and heparinase
digestion (Fig. 1). If HS has the same AT-binding sequence as in
heparin, i.e. GlcNAc6S-GlcUA-GlcNS3S±6S-IdceA2S-GlcNS6S,
the reducing end IdceA2S-GlcNS6S cannot be cleaved by
heparitinase I ("Experimental Procedures" (38)). To this end,
3-O-ST-1 and PAP35S-labeled, AT affinity
purified HSact and HSinact were digested with 2 milliunits of heparitinase I overnight. A fraction of the digested
materials from the 3-O-sulfate-tagged HSact and
HSinact precursors were used in the AT affinity assay. We
found that, for the digested HSact, the extent of AT
binding was approximately equal to the undigested HSact
control. To check the heparitinase I digestion reaction, the rest of
the digested 3-O-sulfate-tagged HSact and
HSinact materials were analyzed by Bio-Gel P-6
chromatography. The digestion patterns were reproducibly obtained, and
sample profiles are provided (Fig. 5).
For 3-O-sulfate-tagged HSact precursors, the
data show that 85% remained larger than hexasaccharides, 14% were
tetrasaccharides, and 0.5% were disaccharides (Fig. 5A). For 3-O-sulfate-tagged HSinact precursors, only
14% remained larger than hexasaccharides, 52% were tetrasaccharides,
33% were disaccharides (Fig. 5B).
Each fraction of oligosaccharides equal to or larger than
hexasaccharides in 3-O-sulfate-tagged HSact and
HSinact precursors were collected and lyophilized for the
AT affinity assay. All the oligosaccharides from
3-O-sulfate-tagged HSact precursors bound to AT,
but the oligosaccharides from 3-O-sulfate-tagged HSinact precursors did not. Since 85% of the
3-O-sulfate-tagged HSact oligosaccharides bind
to AT and there are six 3-O-sulfation sites per chain,
6 × 85% = 5.1. It implies that at least five out of six
3-O-sulfate acceptor sites possess the correct positioning of all critical groups except for the 3-O-sulfate for AT
binding in all HSact precursor chains. This experiment also
suggests that HSact and HSinact precursors have
distinct sulfation patterns around the potential 3-O-sulfate
acceptor sites.
The majority of 3-O-sulfate-tagged HSinact
precursors should have GlcUA at their reducing end, i.e.
-GlcUA-GlcNS3S±6S-
It is interesting to note that 33% of
3-O-sulfate-tagged HSinact precursors can
be degraded into disaccharides and 0.5% of
3-O-sulfate-tagged HSact precursors can be
degraded into disaccharides. This result implies that
3-O-sulfation is not the only factor that determines whether 3-O-sulfated sequences are preserved as tetrasaccharides
following the digestion with heparitinase.
Different Sulfation Distribution around 3-O-Sulfate Acceptor Sites
in HSact and HSinact Precursors--
To
compare further the sulfation patterns in HSact and
HSinact precursors, [6-3H]GlcN metabolically
labeled HS was treated with 3-O-ST-1 and PAP35S.
The 3H and 35S double-labeled HS was AT
affinity fractionated into 3-O-sulfate-tagged HSact and HSinact precursors. The low pH
nitrous-treated and NaBH4 reduced products were analyzed by
Bio-Gel P-6 chromatography (Fig. 6).
These conditions depolymerize HS between GlcNS and GlcUA/IdceA
residues, liberating oligosaccharides whose length reflects the spacing
between N-sulfated GlcN units. The 3H
radioactivity profiles are similar; therefore, the distribution of
GlcNS residues are similar in HSact and HSinact
precursors outside 3-O-sulfate acceptor sites. However,
3-O-sulfate acceptor sites in HSact and
HSinact precursors are different because their
35S radioactivity profiles are distinct. The
3-O-sulfate acceptor sites in HSact precursors
should be located in
GlcUA/IdceA-GlcNAc6S-GlcUA-GlcNS335S±6S
sequences since 88% of 3-O-sulfated materials remained as
tetrasaccharides (Fig. 6A). In contrast,
3-O-sulfate acceptor sites in HSinact precursors
should be located mainly in
GlcUA/IdceA-GlcNS±6S-GlcUA-GlcNS335S±6S
sequences since 60% of 3-O-sulfated materials was cleaved into disaccharides (Fig. 6B).
3-O-Sulfate-tagged HSact Hexasaccharide
Structures--
The tetrasaccharide analysis, heparitinase I, and low
pH nitrous acid treatment data imply that the dominant
3-O-sulfate acceptor sites in HSact precursor
are
GlcUA/IdceA-GlcNAc6S-GlcUA-GlcNS335S±6S-IdceA±2S,
whereas the dominant 3-O-sulfate acceptor sites in
HSinact precursor are
GlcUA/IdceA-GlcNS/Ac±6S-GlcUA-GlcNS335S±6S-GlcUA.
These results further clarify why 3-O-sulfate-tagged HSact precursors bind AT and 3-O-sulfate-tagged
HSinact precursors do not. To obtain the minimum AT-binding
sequence in 3-O-sulfate-tagged HSact precursors,
the 3H and 35S double-labeled HSact
and HSinact precursors were subjected to limited digestion
with heparitinase I and heparitinase II. According to recently
published work on the exolytic and processive mechanism of
depolymerization of HS by heparitinase II (41), this digestion
condition should preserve some of the AT-binding hexasaccharide
structures. The digested products were analyzed by Bio-Gel P-6
chromatography (Fig. 7). The major
hexasaccharide peak from 3-O-sulfate-tagged
HSact precursors was collected and lyophilized. AT affinity
purified, 3H and 35S double-labeled
hexasaccharides were loaded on a polyamine HPLC column (Fig.
8). Two peaks (hexasaccharide I and
hexasaccharide II) were found and collected for further analysis.
Hexasaccharide I and hexasaccharide II were digested with
heparitinase IV. All the hexasaccharides were degraded into di- and
tetrasaccharides, which were separated and collected after Bio-Gel P2
chromatography (data not shown). The 3H-labeled
disaccharides from hexasaccharide I and hexasaccharide II both
co-eluted with
To determine the identity of the UA2S in both
hexasaccharides, hexasaccharide I and hexasaccharide II were treated
with low pH nitrous acid and NaBH4 reduced. The products
were fractionated into disaccharide and tetrasaccharide by Bio-Gel P2
chromatography and collected for further analysis. The
3H-labeled disaccharides from both hexasaccharide I and
hexasaccharide II coeluted with
IdceA235S-anManR635S standard on
ion pairing reverse phase HPLC (data not shown). Tetrasaccharide from
hexasaccharide I eluted at the same position as
Based on the data presented in this paper, the differences between
3-O-sulfate acceptor sites in HSact and
HSinact precursors are summarized in Fig.
10. It is important to note that the
heterogeneity in the HSinact precursors results in a
generation of a large number of potential 3-O-sulfate
acceptor sites. Those structures for which we have evidence are
provided in the figure. In this regard, the second sugar residue in
HSinact precursors is either a GlcNS (60%) or GlcNAc
(40%), and the fifth sugar residue in HSinact precursors
is either a GlcUA (85%) or IdceA (15%). Furthermore, the
6-O-sulfate groups on residue 2 and 6 of the
3-O-sulfate acceptor site on the HSinact
precursors can be present or absent. These alterations distinguish the
potential 3-O-sulfate acceptor sites of the
HSinact precursors from that of HSact
precursors whose sequence is uniquely defined. The many possible combinations of alterations in the HSinact precursor
acceptor sites reflect the underlying heterogeneity of the
HSinact precursors. For this reason, we are unable to
preserve the major 3-O-sulfate acceptor hexasaccharide
sequence in HSinact precursors and cannot quantitatively
provide the actual sequence of the 3-O-sulfate acceptor
sites in HSinact precursors. However, it should be
emphasized that the critical 6-O-sulfate is always present
in HSact precursors, whereas it can be missing in
HSinact precursors.
This article presents an analysis of AT-binding HSact
precursor sequences in F9 embryonal carcinoma cells. At least five out of six 3-O-sulfate acceptor oligosaccharides possess the
correct positioning of all critical AT-binding groups except
3-O-sulfate in all HSact precursor chains. It is
interesting to note that four AT-binding oligosaccharides in each LTA
cell HSact chain are present as shown by an AT-protected
heparin lyase assay in our
laboratory.3 To possess
multiple AT-binding sites in each HSact chain suggests that
HSact biosynthesis is not a random process but a highly
repetitive, highly organized operation. Based on the data presented in
this paper, the schematic model for the structure of HSact
precursors in F9 cells is advanced in Fig.
11. In this model, there are six
potential AT-binding hexasaccharides that exhibited all groups
necessary for AT binding except for the 3-O-sulfate moieties. Six potential AT-binding hexasaccharides are equal to 22% of
the chain length of HSact precursors (18 of 82 disaccharides per chain) and contain 26 of 37 O-sulfates in
3-O-sulfate-tagged HSact precursors. Beyond the
six potential AT-binding hexasaccharides, there are 5 × UA-[6-3H]GlcNAc6S-GlcUA-[6-3H]GlcNS335S±6S-IdceA2S-[6-3H]GlcNS6S.
The underlined 6- and 3-O-sulfates constitute the most critical groups for AT binding in view of the fact that the precursor hexasaccharides possess all the elements for AT binding except for the
3-O-sulfate moiety. The presence of five potential
AT-binding precursor hexasaccharides in all HSact precursor
chains demonstrates for the first time the processive assembly of
specific sequence in HS. The difference in structures around potential
3-O-sulfate acceptor sites in HSact and
HSinact precursors suggests that these precursors might be
generated by different concerted assembly mechanisms in the same cell.
This study permits us to understand better the nature of the HS
biosynthetic pathway that leads to the generation of specific
saccharide sequences.
INTRODUCTION
Top
Abstract
Introduction
References
UA-[6-3H]GlcNAc6S-GlcUA-[6-3H]GlcNS335S±6S-IdceA2S-[6-3H]GlcNS6S)5,
which possess the correct positioning of all critical groups except for
the absence of the 3-O-sulfate residue required for AT
binding in all HSact precursor chains. This finding
demonstrates for the first time the overall structure of the
HSact precursors. This information permits us to speculate
about the nature of the HS biosynthetic pathway that leads to the
generation of specific oligosaccharide sequences.
EXPERIMENTAL PROCEDURES
-Elimination was stopped by adding 5-µl aliquots of 5 M acetic acid until bubble
formation ceased. The released GAG chains were purified by
DEAE-Sepharose (Sigma) chromatography followed by ethanol precipitation
and then resuspended in water. GAGs were digested with 20 milliunits of chondroitinase ABC (Seikagaku) in 50 mM Tris-HCl and 50 mM sodium acetate buffer (pH 8.0). Complete digestion of
chondroitin sulfate by chondroitinase ABC was ensured by monitoring the
extent of conversion of the carrier to disaccharides (100 µg = 1.14 absorbance units at 232 nm). HS was purified from
chondroitinase-degraded products by phenol/chloroform extraction and
ethanol precipitation. After washing the pellets with 0.5 ml of 75%
ethanol, HS was suspended in H2O for further analysis.
GlcUA-GlcNAc/NS±6S. The arrow indicates the
cleavage site. Heparitinase II has broad sequence recognition,
GlcNAc/NS±6S(3S?)-
GlcUA/IdceA±2S-GlcNAc/NS±6S. Heparinase and
heparitinase IV recognizes the sequences:
GlcNAc/NS±3S±6S-
IdceA2S-GlcNAc/NS±6S. The reaction products and
references can be found in the Seikagagu's catalog.
4,5-Tetrasaccharides--
4,5Glycuronidase
(no EC number) and
4,5glycuronate
2-O-sulfatase (no EC number) were from Dr. Yoshida,
Seikagagu Corp., Tokyo, Japan.
-N-Acetylglucosamine
6-O-sulfate sulfatase (6-O-sulfatase) (no EC
number) and
-N-acetylglucosaminidase (no EC number) were purified from bovine kidney in our laboratory. No other enzymatic activity was detected in these enzyme preparations.
GlcUA-[3H]anManR3S and
GlcUA-[3H]anManR3S6S standards were
prepared as described previously (31). GlcUA-anManR335S and
GlcUA-anManR335S6S standards were prepared from
3-O-35S-sulfate-tagged cold F9 HS. In brief,
after hydrazinolysis, 3-O-35S-sulfate-tagged F9
HS was treated with high pH nitrous acid (pH 4.0) and low pH nitrous
acid (pH 1.5). The resultant disaccharides were reduced with
NaBH4 and desalted by Bio-Gel P2 chromatography. 3-O-35S-Sulfate-tagged disaccharides were
collected by ion pairing reverse phase HPLC as previously reported
(37). The identity of GlcUA-anManR335S and
GlcUA-anManR335S6S was further confirmed by
co-chromatography on ion pairing reverse phase HPLC with
GlcUA-[3H]anManR3S and
GlcUA-[3H]anManR3S6S standards.
GlcUA-anManR335S and
GlcUA-anManR335S6S are the only disaccharides
generated by 3-O-ST-1 and PAP35S tagging of cold
F9 HS.
4,5glycuronate
2-O-sulfatase or
4,5glycuronidase in a total
volume of 100 µl of 50 mM imidazole HCl buffer (pH 6.5)
at 37 °C overnight. 5% (5 µl) of the reaction mixture was
combined with six HS disaccharide UV standards and GlcUA-[3H]anManR3S6S standard (2000 cpm) in a
total volume of 100 µl and analyzed by polyamine HPLC. Completion of
the digestion was confirmed by polyamine HPLC. 40 µl of 0.5 M sodium acetate (pH 5.5), 55 µl of H2O, and
10 µl 6-O-sulfatase (8 µg) were added to 95 µl of
reaction mixture. The 6-O-sulfatase digestion was completed in 2 days at 37 °C as monitored by polyamine HPLC as described above. 100 µl of 6-O-sulfatase-treated materials was
diluted with 300 µl of H2O. 2 µl of 16 M
acetic acid and then 5 µl of
-N-acetylglucosaminidase (3.5 µg) were added (pH 4.2).
-N-Acetylglucosaminidase
digestion was completed overnight at 37 °C as monitored by polyamine
HPLC.
RESULTS
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Fig. 1.
Bio-Gel P2 fractionation of heparitinase
I-, heparitinase II-, and heparinase-digested
3-O-sulfate-tagged HSact
precursors. [6-3H]GlcN metabolic HS were treated
with 3-O-ST-1 and PAPS. 3-O-Sulfate-tagged
[3H]HS was AT affinity fractionated into
HSact and HSinact.
3-O-Sulfate-tagged [3H]HSact
precursors were digested with 2 milliunits each of heparitinase I,
heparitinase II, and heparinase overnight. The products were analyzed
by Bio-Gel P2 chromatography ("Experimental Procedures"). The
fractions indicated by solid bars were pooled as
tetrasaccharides and disaccharides.
UA-GlcNAc6S-GlcUA-GlcNS3S and the second peak
has the structure
UA-GlcNAc6S-GlcUA-GlcNS3S6S.
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Fig. 2.
Polyamine HPLC profile of di- and
tetrasaccharides. [3H]Di- and
[3H]tetrasaccharides from
3-O-sulfatetagged HSact precursors
collected after a Bio-Gel P2 column (Fig. 1) were further resolved by
polyamine HPLC. The elution times of the disaccharide peaks were
compared with those of authentic standards and numerically labeled
accordingly. The tetrasaccharide fractions indicated by solid
bars were pooled as tetrasaccharide I (1) and
tetrasaccharide II (2). The numbers correspond
with numbers and names in Table I, which summarizes the relative
abundance of each of the di- and tetrasaccharides.
UA-GlcNAc6S-GlcUA-GlcNS3S and 2 ×
UA-GlcNAc6S-GlcUA-GlcNS3S6S per chain, therefore a total of six 3-O-sulfate sites per
HSact precursor chain.
Di- and tetrasaccharide compositions of 3-O-sulfate-tagged
HSact precursors
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Fig. 3.
Polyamine HPLC analysis of sequential treated
3-O-35S-tagged tetrasaccharides.
3-O-35S-Tagged HSact precursor
tetrasaccharides collected from polyamine HPLC were sequentially
treated with low pH nitrous acid,
4,5glycuronidase, 6-O-sulfatase, and
N-acetylglucosaminidase and were resolved by polyamine
HPLC chromatography (see "Experimental Procedures"). A,
tetrasaccharide I; B, tetrasaccharide II; C and
D, after low pH nitrous acid and NaBH4
treatment; E and F, followed by
4,5glycuronidase; G and H,
6-O-sulfatase; I and J,
N-acetylglucosaminidase treatments. The arrow
indicates the elution position of the internal
GlcUA-[3H]anManR3S6S standard.
GlcA indicates GlcUA.
4,5glycuronate 2-O-sulfatase. The retention
time of tetrasaccharide I and tetrasaccharide II remained the same,
whereas the 2-O-sulfate in the internal
4,5UA2S-GlcNS UV standard was completely removed as
judged by the shift of UV absorbance on polyamine HPLC. This result
indicates that there are no 2-O-sulfates on
4,5UA in either tetrasaccharide (data not shown).
Indeed,
4,5glycuronidase removed a sugar residue from
the non-reducing end in both tetrasaccharides. The polyamine HPLC
profiles of
4,5glycuronidase-treated samples are shown
in Fig. 3, E and F.
-N-acetylglucosaminidase treatment,
the resulting disaccharides coeluted with
GlcUA-[3H]anManR3S and
GlcUA-[3H]anManR3S6S standards, respectively
(Fig. 3, I and J). From the above results, we
concluded that the first tetrasaccharide peak has the structure
4,5UA-GlcNAc6S-GlcUA-GlcNS3S (Fig.
3A) and the second peak has the structure
4,5UA-GlcNAc6S-GlcUA-GlcNS3S6S
(Fig. 3B).
4,5di-
(35%) and
4,5tetrasaccharides (65%) on Bio-Gel P2
chromatography (data not shown). 40% of total
3-O-35S-sulfate-tagged F9 HSinact
precursors remained as tetrasaccharides after low pH nitrous acid and
NaBH4 treatment of the combined heparitinase I,
heparitinase II, and heparinase-digested materials. The
tetrasaccharides were resolved by polyamine HPLC. Three tetrasaccharide
peaks were observed (Fig. 4).
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Fig. 4.
Polyamine HPLC profile of
3-O-35S-tagged HSinact
precursor tetrasaccharides. 3-O-35S-Tagged
HSinact precursors were digested with a mixture of
heparitinase I, heparitinase II, and heparinase. Tetrasaccharides
obtained after Bio-Gel P2 chromatography were treated with low pH
nitrous acid and NaBH4 and subjected to Bio-Gel P2
chromatography again. Tetrasaccharides recovered after low pH nitrous
acid and NaBH4 treatment were resolved by polyamine HPLC.
Peak I, peak II, and peak III were collected for
further structural analysis.
J, were
observed upon sequential
4,5glycuronidase,
6-O-sulfatase, and
-N-acetylglucosaminidase
treatments of the collected peak II and peak III (data not shown).
Therefore, peak II has the structure
4,5UA-GlcNAc6S-GlcUA-anManR3S
and the peak III has the structure
4,5UA-GlcNAc6S-GlcUA-anManR3S6S.
This result indicates that 34% (85 × 40%) of the
3-O-sulfate acceptor sites in HSinact precursors
are the same at the
4,5tetrasaccharide level as in
HSact precursors. The 3-O-sulfate-tagged
HSact and HSinact precursor
4,5tetrasaccharides do not bind to AT (data not shown).
In view of the fact that
GlcNAc6S-GlcUA-GlcNS3S6S-IdceA2S has 150-fold less affinity for AT (Kd = 4.6 × 10
6 M) compared with that of the
pentasaccharide,
GlcNAc6S-GlcUA-GlcNS3S6S-IdceA2S-GlcNS6S (Kd = 3.0 × 10
8 M)
(40), 3-O-sulfate-tagged HSinact precursors with
the critical 3-O and 6-O failed to
bind to AT because they do not have proper sugar sequences toward the
reducing end of the 3-O-sulfate-tagged GlcN residues.
4,5glycuronidase treatment, the resulting products
eluted at the same position as GlcNAc-GlcUA-
anManR3S in Fig. 3G. After
-N-acetylglucosaminidase treatment, it coeluted with
GlcUA-[3H]anManR3S standard. From the above
evidence, we concluded that peak I has the structure of
4,5UA-GlcNAc-GlcUA-anManR3S.
The critical 6-O-sulfate group at residue 2 is
missing in this tetrasaccharide. This observation explains the fact
that a population of 3-O-sulfate-tagged
HSinact precursors within the
GlcNAc-GlcUA-GlcNS3S context failed to bind to AT. However,
we cannot quantitate how many 6-O-sulfates are missing in
the 3-O-sulfate-tagged HSinact precursors within
the GlcNS-GlcUA-GlcNS335S context
because heparitinase treatment does not preserve all the
3-O-sulfate acceptor sites as tetrasaccharides as compared with the 3-O-sulfate-tagged HSact precursors.
Our inability to capture all of the 3-O-sulfate-tagged tetrasaccharides from the HSinact precursors is probably
due to the heterogeneity in the structure of these precursors. This
heterogeneity results from the fact that we have not, as yet,
identified the ligands for 3-O-sulfated HSinact
materials and hence cannot purify the 3-O-sulfated
HSinact precursors.
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Fig. 5.
Bio-Gel P6 fractionation of heparitinase
I-digested 3-O-35S-tagged
HSact and HSinact precursors. Cold F9 HS
was treated with 3-O-ST-1 and PAP35S. The
3-O-35S-tagged HS were AT affinity fractionated
into HSact and HSinact.
3-O-35S-Tagged HSact and
HSinact precursors were digested with 2 milliunits of
heparitinase I overnight. The products were analyzed by Bio-Gel P6
chromatography ("Experimental Procedures"). A,
3-O-35S-tagged HSact precursors;
B, 3-O-35S-tagged HSinact
precursors. n = the number of monosaccharide units in
each peak.
GlcUA, since GlcUA residues at
this position are apparently cleaved by heparitinase I in order to
generate 85% of 3-O-sulfated di- (33%) and
tetrasaccharides (52%). In contrast,
GlcUA-GlcNS3S±6S-IdceA±2S should
be the main 3-O-sulfate-tagged HSact sequence
since it is uncleavable by heparitinase I.
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Fig. 6.
Bio-Gel P6 fractionation of low pH
nitrous-treated and NaBH4-reduced
3-O-35S-tagged
[3H]HSact and
[3H]HSinact precursors.
[6-3H]GlcN metabolically labeled HS was treated with
3-O-ST-1 and PAP35S. The 3H- and
35S-doubled-labeled HS were AT affinity fractionated into
HSact and HSinact. The low pH nitrous-treated
and NaBH4-reduced 3-O-35S-tagged
[3H]HSact and
[3H]HSinact precursors were analyzed by
Bio-Gel P6 chromatography (see "Experimental Procedures").
A, 3-O-35S-tagged
[3H]HSact precursors; B,
3-O-35S-tagged
[3H]HSinact precursors. , 3H
cpm;
, 35S cpm. n = the number of
monosaccharide units in each peak.
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Fig. 7.
Bio-Gel P6 fractionation of limit digested
3-O-35S-tagged
[3H]HSact and
[3H]HSinact precursors by heparitinase I and
heparitinase II. 3-O-35S-Tagged
[3H]HSact and
[3H]HSinact precursors were digested with 1 milliunit each of heparitinase I and heparitinase II for 30 min. The
products were analyzed by Bio-Gel P6 chromatography (see
"Experimental Procedures"). The fractions indicated by the
solid bar were pooled for further analysis. A,
3-O-35S-tagged
[3H]HSact precursors; B,
3-O-35S-tagged
[3H]HSinact precursors. , 3H
cpm;
, 35S cpm. n = the number of
monosaccharide units in each peak.
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Fig. 8.
Polyamine HPLC profile of AT-binding
3-O-sulfate-tagged hexasaccharides.
Hexasaccharides produced by heparitinase I and heparitinase II limited
depolymerization of 3-O-35S-tagged
[3H]HSact precursors as in Fig. 7 were AT
affinity purified and separated by polyamine HPLC. The
hexasaccharide I and the hexasaccharide II
resolved by polyamine HPLC were collected for further analysis.
UA2S-GlcNS6S UV standard on polyamine HPLC
(Fig. 9, A and B).
The 3H and 35S double-labeled tetrasaccharide
from hexasaccharide I has the structure
UA-[3H]GlcNAc6S-GlcUA-[3H]GlcNS335S.
The tetrasaccharide from hexasaccharide II has the structure
UA-[3H]GlcNAc6S-GlcUA-[3H]GlcNS3S6S
because they have the same retention time on polyamine HPLC as
tetrasaccharide I in Fig. 3A and tetrasaccharide II in Fig.
3B. This experiment suggests that the AT-binding
hexasaccharide I has the structure
[6-3H]GlcNAc6S-GlcUA-[6-3H]GlcNS335S-UA2S-[6-3H]GlcNS6S
and the hexasaccharide II has the structure
[6-3H]GlcNAc6S
-GlcUA-[6-3H]GlcNS335S6S-UA2S-
[6-3H]GlcNS6S.
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Fig. 9.
Polyamine HPLC profiles of the disaccharides
generated from the reducing ends of AT-binding
3-O-sulfate-tagged hexasaccharides.
Hexasaccharide I and hexasaccharide II (Fig. 8) were digested with 2 milliunits of heparitinase IV overnight. The di- and tetrasaccharides
were separated by Bio-Gel P2 chromatography. The disaccharides
collected were resolved by polyamine HPLC. The arrow
indicates the elution position of the internal
4,5UA2S-GlcNS6S UV standard.
UA-GlcNAc6S-GlcUA-anManR3S on
polyamine HPLC (Fig. 3C), and tetrasaccharide from
hexasaccharide II eluted at the same position as
UA-GlcNAc6S-GlcUA-anManR3S6S on polyamine HPLC (Fig. 3D). Combining both hexasaccharide
enzymatic and low pH nitrous acid analysis data, we concluded that
hexasaccharide I has the structure
UA-GlcNAc6S-GlcUA-GlcNS3S-IdceA2S-GlcNS6S and hexasaccharide II has the structure
UA-GlcNAc6S-GlcUA-GlcNS3S6S-IdceA2S-GlcNS6S.
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Fig. 10.
3-O-Sulfate-tagged
hexasaccharide structures in F9 HSact and
HSinact precursors. The structures are based on
the following data: 1) Six 3-O-sulfate acceptor sites per
HSact and HSinact precursors; 2) Low
pH nitrous acid, heparitinase I, and heparitinase II degradations of
3-O-sulfate-tagged HSact and
HSinact precursors; 3) tetrasaccharide structures in
3-O-sulfate-tagged HSact and
HSinact precursors; 4) hexasaccharide structures in
3-O-sulfate-tagged [3H]HSact
precursors. The boxes in 3-O-sulfate-tagged
HSinact precursors correspond to the sulfate residues
existing in 3-O-sulfate-tagged HSact precursors.
The 3-O-sulfate-tagged HSact and
HSinact precursor sequences are different at all levels.
IdA indicates IdceA; GlcA indicates GlcUA.
DISCUSSION
UA-GlcNAc6S, 3 ×
UA2S-GlcNS, 3 ×
UA-GlcNS6S, 16 ×
UA-GlcNS, and 37 ×
UA-GlcNAc
disaccharides to complete the structure of the HSact
precursor chain (Table I). 5 ×
UA-GlcNAc6S, 3 ×
UA2S-GlcNS, and 3 ×
UA-GlcNS6S are the 11 O-sulfated residues outside the AT-binding domain that need
to be placed to complete the primary structure of HSact
precursor (Fig. 11).
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Fig. 11.
Schematic model of
3-O-sulfate-tagged HSact precursor
structures derived from the analysis HS from F9 cells. The model
is based on the data presented in this paper, i.e. there are
six 3-O-sulfation sites per chain and all
3-O-sulfate acceptor oligosaccharides bind to AT in
3-O-sulfate-tagged HSact precursors. It implies
that HSact precursor formation involves correct
N-sulfation, epimerization, 6-O-, and
2-O-sulfation at all six potential 3-O-acceptor
sites per chain during the biosynthesis. IdA indicates
IdceA; GlcA indicates GlcUA.
The reason we proposed that there are six instead of five potential
AT-binding hexasaccharides in F9 HSact precursors is that
we could place five out of six UA2S-GlcNS6S residues in
potential AT-binding domains in F9 HSact precursors. We
suspect that heparitinase I may contain trace heparitinase II
contaminants that generate 15% of the tetrasaccharides in
3-O-sulfate-tagged HSact precursors (Fig. 5).
More likely, endo-D-glucuronidases may cleave mature HS
chains limitedly in cultured F9 cells before HS chains were prepared
for our structural studies (see "Experimental Procedures"). The
general presence of endo-D-glucuronidases in variety of
tissue and cells has been reported (41-48).
Endo-D-glucuronidases recognize the sequences
GlcUA/IdceA-GlcNAc/NS±6S-GlcUA-
GlcNAc/NS±3S±6S-IdceA2S-GlcNS±6S. The arrow indicates the cleavage site (49, 50). The cleavage eliminates
both AT- and potential AT-binding sites (50). The endo-D-glucuronidases extensively cleave the newly
synthesized heparin (Mr 60,000-100,000) to
generate fragments that are stored in cytoplasmic granules of mast
cells (Mr 5,000-25,000). This may explain why
commercial heparin (Mr 5,000-25,000) contains limited numbers of AT-binding sites per chain (38). In contrast, endo-D-glucuronidases may cleave in a limited fashion the
endogenous HS chains (48, 49). This limited cleavage may explain why we
can place five out of six
UA2S-GlcNS6S residues in
potential AT-binding domains in F9 HSact precursors.
It is apparent from our model that there is a template for HSact precursor formation that requires correct N-sulfation, epimerization, 2-O-, and 6-O-sulfation at all six sites along the chain during the biosynthesis. What remains to be explained is how this is accomplished. F9 HSact chain contains almost all the detectable 3-O-sulfated disaccharides (GlcUA-anManR3S and GlcUA-anManR3S6S) in HStotal. The 3-O-sulfated disaccharides accounted for about 18% of O-sulfated disaccharides in the HSact chain (36). Our current study showed that there are six 3-O-sulfate acceptor sites per HSact chain. Therefore, 3-O-sulfates are added in an all or none manner to the six 3-O-sulfate acceptor sites in HSact precursor in a concerted fashion to generate the AT-binding sites during biosynthesis. Based on this observation, our current model is that after chain polymerization, all other chain modification reactions, i.e. N-sulfation, epimerization, 2-O-, 6-O-sulfation, occur at all six regions in the same fashion as 3-O-sulfation of HSact precursors. To explain this concerted mechanism, we propose that N-STs recognize and sulfate a three-dimensional feature of the nascent polymerized HS chain (51) and therefore produce a definite spacing of N-sulfations. Epimerase may lay out different epimerization patterns due to different N-sulfation patterns, or 2-O-/6-O-sulfation occurring before epimerization. These patterns may be further amplified depending on whether the 6-O-sulfation or 2-O-sulfation occurs first (the initial action of 6-O-ST excludes subsequent function of 2-O-ST, whereas the initial action of the 2-O-ST allows subsequent function of 6-O-ST (52)). The structure of each subpopulation HS is determined by the sequential action of modification enzymes, i.e. pathway, in the Golgi apparatus. In other words, different pathways during HS modification generate different HS structures in the same cell.
This study shows that F9 cells produce different HS structures. The previous HSact-deficient mutants and 3-O-ST-1 studies in our laboratory suggested that the same cells make both HSact and HSinact precursors (35, 53), and the same HS proteoglycan core proteins carry both HSact and HSinact chains (54). A Chinese hamster ovary cell mutant defective in N-ST makes both fully sulfated and undersulfated HS chains (55). A human colon carcinoma cell makes both fully sulfated and undersulfated HS chains (56). All these observations suggest that different biosynthesis schemes that generate different HS structures occur in the same cells. In addition to the increasing numbers of specific HS domain structural studies in different tissues and cells (57-61), we suggest that different HS biosynthetic pathways exist, which generate HS with different structures and biologic functions. Evaluation of the different pathways will eventually delineate how the HS biosynthesis is regulated.
Currently we do not know where HSact and HSinact precursor biosynthesis pathways diverge. The constant presence of the critical 6-O-sulfate groups only in HSact precursors indicates that the pathway is either set up by or diverges before the critical 6-O-sulfation. It is possible that specific isoforms of 6-O-ST are involved in HSact precursor pathways, and the existing structures around the 3-O-sulfate acceptor site favor the action of a 6-O-ST isoform. Furthermore, the existence of auxiliary proteins that modulate the action of N-ST, epimerase, 2-O-ST, and 6-O-ST for HSact precursor formation have not been excluded.
In conclusion, the decision to synthesize HSact or
HSinact depends on the presence of specific HS precursor
intermediates, specific modification enzymes, and perhaps auxiliary
factors in the Golgi biosynthesis machinery. Currently, we have
obtained a series of Chinese hamster ovary mutants defective in
HSact precursor formation. Complementation of these
mutations will provide us with molecular details about the required
elements for HSact precursor biosynthesis. This information
should allow us to formulate a more definitive model of the
HSact biosynthetic pathway. This model will be evaluated by
reconstituting the sequential biosynthetic apparatus using different
recombinant modification enzymes/proteins.
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ACKNOWLEDGEMENTS |
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We thank Peter Blaiklock for careful reading of this manuscript and members of the Rosenberg laboratory for their insightful comments.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants 5-P01-HL41484 and HL66385.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 National Research Service Award Postdoctoral Fellowship.
** To whom correspondence and reprint requests should be addressed: Massachusetts Institute of Technology, Bldg. 68-480, 77 Massachusetts Ave., Cambridge, MA 02139. Tel.: 617-253-8804; Fax: 617-258-6553; E-mail: rdrrosen{at}MIT.EDU.
2
Liu, J., Shworak, N. W., Sina, P.,
Schwartz, J. J., Zhang, L., Fritze, L. M. S., and Rosenberg, R. D. (1999) J. Biol. Chem. 274, in press.
3 J. Liu and R. D. Rosenberg, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: HS, heparan sulfate; HSact, anticoagulantly active heparan sulfate; HSinact, anticoagulantly inactive heparan sulfate; CHAPS, 3-[(3-cholamidopropy)dimethylammonio]-1-propanesulfonic acid; DMEM, Dulbecco's modified Eagle's medium; GAG, glycosaminoglycan; PAPS, 3'-phosphoadenosine 5'-phosphosulfate; AT, antithrombin; 3-O-ST-1, glucosaminyl 3-O-sulfotransferase-1; N-ST, N-deacetylase/N-sulfotransferase; 2-O-ST, iduronic/glucuronic acid 2-O-sulfotransferase; 6-O-ST, glucosaminyl 6-O-sulfotransferase; GlcUA, glucuronic acid; IdceA, iduronic acid; HPLC, high pressure liquid chromatography; MES, 4-morpholineethanesulfonic acid.
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