A Major Common Trisulfated Hexasaccharide Core Sequence,
Hexuronic
Acid(2-Sulfate)-Glucosamine(N-Sulfate)-Iduronic
Acid-N-Acetylglucosamine-Glucuronic
Acid-Glucosamine(N-Sulfate), Isolated from the Low Sulfated
Irregular Region of Porcine Intestinal Heparin*
Shuhei
Yamada,
Yukari
Yamane,
Hiromi
Tsuda,
Keiichi
Yoshida
, and
Kazuyuki
Sugahara§
From the Department of Biochemistry, Kobe Pharmaceutical
University, Higashinada-ku, Kobe 658 and
Tokyo
Research Institute of Seikagaku Corp.,
Higashiyamato-shi, Tokyo 207, Japan
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ABSTRACT |
The major structure of the low sulfated irregular
region of porcine intestinal heparin was investigated by characterizing the hexasaccharide fraction prepared by extensive digestion of the
highly sulfated region with Flavobacterium heparinase and subsequent size fractionation by gel chromatography. Structures of a
tetrasaccharide, a pentasaccharide, and eight hexasaccharide components
in this fraction, which accounted for approximately 19% (w/w) of the
starting heparin representing the major oligosaccharide fraction
derived from the irregular region, were determined by chemical and
enzymatic analyses as well as 1H NMR spectroscopy. Five
compounds including one penta- and four hexasaccharides had hitherto
unreported structures. The structure of the pentasaccharide with a
glucuronic acid at the reducing terminus was assumed to be derived from
the reducing terminus of a heparin glycosaminoglycan chain and may
represent the reducing terminus exposed by a tissue
endo-
-glucuronidase involved in the intracellular post-synthetic
fragmentation of macromolecular heparin. Eight out of the 10 isolated
oligosaccharides shared the trisaccharide sequence,
-4IdceA
1-4GlcNAc
1-4GlcA
1-, and its reverse sequence,
-4GlcA
1-4GlcNAc
1-4IdceA
1-, was not found. The latter has not
been reported to date for heparin/heparan sulfate, indicating the
substrate specificity of the D-glucuronyl C-5 epimerase. Furthermore, seven hexasaccharides shared the common trisulfated hexasaccharide core sequence
HexA(2-sulfate)
1-4GlcN(N-sulfate)
1-4IdceA
1-4GlcNAc
1-4GlcA
1-4GlcN(N-sulfate) which contained the above trisaccharide sequence (
HexA, IdceA, GlcN,
and GlcA represent
4-deoxy-
-L-threo-hex-4-enepyranosyluronic acid, L-iduronic acid, D-glucosamine, and
D-glucuronic acid, respectively) and additional sulfate
groups. The specificity of the heparinase used for preparation of
the oligosaccharides indicates the occurrence of the common
pentasulfated octasaccharide core sequence,
-4GlcN(N-sulfate)
1-4HexA(2-sulfate)1-4GlcN(N-sulfate)
1-4IdceA
1-4GlcNAc
1-4GlcA
1-4GlcN(N-sulfate)
1-4HexA(2-sulfate)1-, where the central hexasaccharide is flanked by
GlcN(N-sulfate) and HexA(2-sulfate) on the nonreducing and
reducing sides, respectively. The revealed common sequence constituted
a low sulfated trisaccharide representing the irregular region
sandwiched by highly sulfated regions and should reflect the control
mechanism of heparin biosynthesis.
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INTRODUCTION |
Heparin is a highly sulfated co-polymer of glucosamine and uronic
acid residues that are alternatively 1
4-linked. Most of the heparin
molecule is accounted for by the major trisulfated disaccharide
repeating unit,
4IdceA(2-sulfate)
1
4GlcN(N,6-disulfate)
1
. This repeating sequence forms highly sulfated regions and represents at
least 75% heparin from porcine intestine (1). Undersulfation and
substantial structural variability are observed in the rest of the
region which is called the irregular region and distributed along the
chain flanked by the fully sulfated region composed of the trisulfated
disaccharide units, accounting for approximately one-quarter of the
heparin polysaccharide chain (for reviews see Refs. 2-4).
Heparin exhibits a wide range of biological activities such as
inhibition of blood coagulation (5), modulation of cellular proliferation (6, 7), potentiation of angiogenesis (8), and
interactions with various growth factors (9-12). These activities result from the ability of heparin to interact with various proteins causing their activation, deactivation, or stabilization. Interactions between heparin and proteins generally depend on the presence of
sulfate groups. Some proteins such as lipoprotein lipase (13), thrombin
(14), and platelet factor 4 (15) bind to the highly sulfated region
consisting exclusively of the trisulfated disaccharide unit in a
seemingly nonspecific fashion. Still, many other proteins are thought
to require specific sequences for binding, and the precise requirement
for individual sulfate groups may vary from one protein to another.
However, it should be noted that heparin is oversulfated and contains
not only the essential sulfate groups but also additional nonessential
ones for protein binding. The contribution of the irregular region to
the biological activities of heparin is not well understood
mainly because of the difficulty in analyzing the variably sulfated
structure. However, it is well known that the
antithrombin-binding minimum pentasaccharide sequence GlcN(6S)1
1-4GlcA
1-4GlcN(NS,3S)
1-4IdceA(2S)
1-4GlcN(NS,6S)
(16, 17) is embedded over the connecting border of the high
and low sulfated regions and contains both low and high sulfated
disaccharide units. Hence, it is conceivable that the low sulfated
irregular region is involved in some other active domains as well.
Biosynthetic reactions required to generate heparin sequences include
the formation of an initial, simple polysaccharide structure,
4GlcA
1
4GlcNAc
1
, that is subsequently modified through
N-deacetylation/N-sulfation of GlcNAc units, C-5
epimerization of GlcA to IdceA units, and O-sulfation at C-2
of IdceA and C-6 of GlcN units (for review see Ref. 4). The
undersulfated region of the final product heparin is thought to have
escaped such modifications. However, the mechanism by which certain
residues are selected for modification is not understood, partly
because of a lack of sequence information on relatively large fragments
although certain rules about the sequential arrangement of various
disaccharide units have been noted (4).
In this study we isolated and systematically characterized 10 oligosaccharide structures from the undersulfated region of porcine
intestinal heparin after extensive digestion with heparinase, which
acts only on the highly sulfated repeating region, to investigate the
variability and/or regularity, if any, of the low sulfated region. A
majority of these oligosaccharides were revealed to share a common
trisulfated hexasaccharide core sequence.
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EXPERIMENTAL PROCEDURES |
Enzymes and Oligosaccharides--
Stage 14 heparin was purchased
from American Diagnostica, New York, and purified by DEAE-cellulose
chromatography as reported (18). Heparinase (EC 4.2.2.7) and purified
heparitinases I (EC 4.2.2.8) and II (no EC number) were obtained from
Seikagaku Corp., Tokyo.
4,5-Glycuronate-2-sulfatase (EC
3.1.6.-), abbreviated as
hexuronate-2-sulfatase, was purified from
Flavobacterium heparinum (19).
-Glucuronidase (EC
3.2.1.31) purified to homogeneity from Ampullaria
(freshwater apple shell) hepatopancreas (20) was obtained from Tokyo
Zouki Chemical Co., Tokyo. Human liver
-iduronidase (EC 3.2.1.76) was a gift from Dr. J. J. Hopwood, Adelaide Children's Hospital (21). The hexasaccharide fraction was prepared from stage 14 heparin
after heparinase digestion as described (22). A peptide (Arg-Gly)15 was custom-synthesized by Peptide Institute,
Inc., Osaka.
Digestion of the Isolated Oligosaccharides with Heparinase,
Heparitinases, or
Hexuronate-2-Sulfatase--
Each isolated
oligosaccharide (0.5-1.0 nmol) was digested using 1-5 mIU of
heparinase, heparitinase I and/or II, or
hexuronate-2-sulfatase as
described previously (18, 23, 24). Successive enzymatic digestion of a
given oligosaccharide with
hexuronate-2-sulfatase and then
heparitinase I or II was also carried out as reported (22). Reactions
were terminated by boiling for 1 min, and the reaction mixture was
analyzed by HPLC as reported (18).
-Iduronidase and
-Glucuronidase Digestion of
Tetrasaccharides Obtained by Deamination--
Tetrasaccharides
obtained by deamination of the hexasaccharides in fractions 6-26 and
6-27 were tested for their sensitivities to
-iduronidase and
-glucuronidase to determine the isomer type of the uronic acid
residue exposed at the nonreducing termini. Each heparin hexasaccharide
(2.0 nmol) was treated at room temperature with HNO2 at pH
1.5 for 30 min (25), and the resultant di- and/or tetrasaccharides were
reduced under alkaline conditions using [3H]sodium
borohydride (0.50 mCi) as reported (22). Each resultant [3H]tetrasaccharide (2.6 pmol) corresponding to
approximately 2.9 × 103 dpm was digested using 17.7 mIU
-iduronidase or 48.9 mIU
-glucuronidase as reported (22).
500-MHz 1H NMR Spectroscopy--
Oligosaccharides
for NMR analysis were fully sodiated using a Dowex 50-X8
(Na+ form) column (7 × 18 mm) and then repeatedly
exchanged in 2H2O with intermediate
lyophilization. 500-MHz 1H NMR spectra of hexasaccharides
were measured on a Varian VXR-500 at a probe temperature of 26 °C as
reported (26, 27). Chemical shifts are given relative to sodium
4,4-dimethyl-4-silapentane-1-sulfonate but were actually measured
indirectly relative to acetone (
2.225) in
2H2O (28).
Matrix-assisted Laser Desorption Ionization (MALDI)
Time-of-flight (TOF) Mass Spectrometry--
MALDI TOF mass spectra of
a sulfated heparin oligosaccharide were recorded on a Konpact MALDI 1 (Shimadzu/Kratos) linear instrument in the positive ion mode at Toray
Research Center, Kanagawa, Japan. Ultraviolet MALDI experiments were
carried out using an N2 laser (Laser Science, Newton, MA;
337-nm wavelength, 3-ns pulse width). The ions were accelerated to 20 keV energy. Caffeic acid was used as a matrix at a concentration of 10 mg/ml in 1:1 water/MeCN mixture. A synthetic peptide
(Arg-Gly)15 was used as a complexing agent to shield the
negatively charged groups of a sulfated oligosaccharide according to
Juhasz and Biemann (29). Aqueous solutions of a heparin oligosaccharide
(10 pmol/µl) and the peptide (10 pmol/µl) were mixed in advance and
then diluted with an equimolar proportion of the matrix solution. Of
this sample/matrix solution, 0.5-1.0 µl was placed on the probe
surface and dried under a stream of air.
Other Analytical Methods--
Uronic acid was determined by the
carbazole method (30). Unsaturated uronic acid was
spectrophotometrically quantified based upon an average millimolar
absorption coefficient of 5.5 at 232 nm (31). Amino acids and amino
sugars were quantified after acid hydrolysis in 6 M HCl at
110 °C for 20 h or 3 M HCl at 100 °C for 16 h, respectively, using a Beckman 6300E amino acid analyzer (32).
Capillary electrophoresis was carried out to examine the purity of each
isolated fraction in a Waters capillary ion analyzer as reported
(33).
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RESULTS |
Isolation of the Oligosaccharides--
Purified stage 14 heparin
from porcine intestine was exhaustively digested with heparinase and
fractionated into fractions 1-8 by gel filtration on a column of
Bio-Gel P-10 (22). Fraction 8 contained disaccha-rides, and the
major fraction 7, which contained tetrasaccha-rides, was
characterized previously (22). In this study fraction 6 was
characterized. Fractions 6, 7, and 8 represented approximately 19, 24, and 32% (w/w) of the starting heparin, respectively. Amino sugar and
uronic acid analyses showed that fraction 6 contained hexasaccharides.
Fraction 6 was subfractionated by HPLC on an amine-bound silica column
into fractions 6-1 to -37, as indicated in Fig.
1. Twelve fractions, 6-1, -6, -17, -22, -23, -25, -26, -27, -30, -31, -32, and -34, were further purified by
rechromatography. They altogether accounted for 61 mol% (as
HexA)
of the oligosaccharides obtained from fraction 6. These individual
fractions gave a single peak on HPLC but were 98, 91, 81, 57, 75, 95, 66, 89, 82, 80, 93, and 99% pure, respectively, when examined by
capillary electrophoresis (results not shown). Characterization of
fractions 6-1 and 6-6, which turned out to contain dermatan
sulfate-derived oligosaccharides, will be described elsewhere. In this
article, analyses of the other fractions are described.

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Fig. 1.
HPLC fractionation of the hexasaccharide
fraction. The hexasaccharide fraction obtained from gel filtration
on Bio-Gel P-10 was separated into subfractions 6-1 to 6-37, on an
amine-bound silica column using an NaH2PO4
gradient (indicated by the dashed line). Fraction 6-28 was
the right shoulder of fraction 6-27 and was removed by
rechromatography. For experimental details, see "Experimental
Procedures."
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Enzymatic Characterization of the Isolated
Oligosaccharides--
Disaccharide compositions of the isolated
oligosaccharides were determined by digestion with heparitinases I
and/or II, followed by HPLC analysis on an amine-bound silica column.
Substrate specificities of the three heparin lyases are shown in Fig.
2. Most of the fractions except for
fractions 6-32 and 6-34 were degraded into disaccharides by the
enzymes, although fractions 6-32 and 6-34 were degraded into
approximately 1 mol each of a di- and a tetrasaccharide unit. The
results are summarized in Table I.
Recoveries of the oligosaccharides in Table I were calculated taking
the absorbance of the parent oligosaccharide in each fraction as 100%.
Excess or insufficient recoveries of the products were observed in some
cases partly due to the products derived from possible impurities and
partly due to the use of the average millimolar absorption coefficient of 5.5 at 232 nm obtained from unsaturated disaccharides (31) for
quantification of oligosaccharides. Millimolar absorption coefficients
of individual oligosaccharides deviate from the average value to
various degrees. As representative chromatograms, those obtained with
fractions 6-23 and -27 are shown in Fig.
3. Fractions 6-23 and -27 yielded
HexA-GlcNAc(6S) and
HexA(2S)-GlcN(NS,6S) in a molar
ratio of 1.0:1.4 and
HexA-GlcNAc(6S),
HexA-GlcN(NS,6S), and
HexA(2S)-GlcN(NS,6S) in a molar
ratio of 0.9:1.0:1.1, respectively, upon heparitinase I digestion. A
sequential arrangement of the resultant disaccharide units in each
parent oligosaccharide was determined based upon the results of
enzymatic digestions using heparitinases I, II, and
hexuronate-2-sulfatase (Table I) and 500-MHz 1H NMR
analysis.

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Fig. 2.
Substrate specificities of the heparin
lyases. The arrows indicate the glycosidic linkages
cleaved by heparin lyases. A, heparinase (heparinase I);
B, heparitinase I (heparinase III); C,
heparitinase II (heparinase II). These enzymes can cleave both glucosaminidic linkages (marked by double dagger) to GlcA
and IdceA in an eliminative fashion (22, 23, 34). The C-3 OH of the
glucosamine residue (marked by an asterisk) adjacent to the
disaccharide cleavage site has to be free for sensitivity to
heparitinases I and II (26).
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Table I
Enzymatic analysis of the isolated oligosaccharides
After each oligosaccharide fraction was incubated with heparitinase I,
with 2-sulfatase and then heparitinase II, or with heparitinase II, the
reaction products were characterized by HPLC. Recoveries of the
disaccharides calculated based on absorption at 232 nm taking the
absorbance of the parent oligosaccharide(s) in each fraction as 100%
are shown in parentheses.
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Fig. 3.
HPLC analysis of the heparitinase I and/or II
digests of the isolated oligosaccharides. The oligosaccharides
were digested with heparitinases I and/or II and fractionated by HPLC
on an amine-bound silica column. A, the heparitinase I
digest of fraction 6-23 (0.5 nmol); B, the heparitinase I
digest of fraction 6-27 (0.5 nmol). Elution positions of the standard
disaccharides isolated from heparin/heparan sulfate are indicated in
A as follows: 1, HexA-GlcNAc; 2,
HexA-GlcNAc(6S); 3,
HexA(2S)-GlcNAc; 4,
HexA-GlcN(NS); 5,
HexA-GlcN(NS,6S); 6,
HexA(2S)-GlcN(NS); 7,
HexA(2S)-GlcNAc(6S); 8,
HexA(2S)-GlcN(NS,6S). The peak
marked by an asterisk around 35 min is often observed upon
high sensitivity analysis and is due to an unknown substance eluted
from the column resin.
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500-MHz 1H NMR Analysis--
All the individual
oligosaccharides were also analyzed by 500-MHz 1H NMR, and
individual monosaccharide units were identified based on the chemical
shifts of the proton signals and the coupling constants
J1,2. Chemical shifts were assigned by
two-dimensional homonuclear Hartmann-Hahn and correlation spectroscopy
analyses (data not shown) as reported for the sulfated oligosaccharides isolated previously from heparin (26) and heparan sulfate (33). Although some fractions were still mixtures as revealed by capillary electrophoresis, it was possible to extract sequence information about
the major compound in these fractions by taking advantage of
1H NMR spectroscopy. Since peak heights of resonances
reflect molar ratios of the components, signals for the major compound
in the mixtures could be easily distinguished from those of the minor impurities. The internal uronic acid residues of each isolated oligosaccharide were unambiguously identified based upon the chemical shifts of the anomeric proton signals and the coupling constants J1,2. Anomeric proton signals of an
IdceA and
a
GlcA residue in heparin/heparan sulfate oligosaccharides are
observed around
5.2-5.0 and 4.7-4.5, respectively (22, 35). The
coupling constants J1,2 of
IdceA and
GlcA
in heparin/heparan sulfate oligosaccharides are approximately 3.0 and
8.0 Hz, respectively (22, 36). The NMR data obtained in this study for
the oligosaccharides are summarized in Table
II.
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Table II
1H chemical shifts of the constituent monosaccharides of the
isolated oligosaccharides derived from heparin
Chemical shifts are given in ppm downfield from internal sodium
4,4-dimethyl-4-silapentane-1-sulfonate but were actually measured indirectly relative to acetone ( 2.225 ppm) in 2H2O
at 26 °C. The estimated error for the values to two decimal places
was only ±0.01 ppm because of partial overlap of signals. That for the
values to three decimal places was ±0.002 ppm. Coupling constants
J1,2 (in Hz) are given in
parentheses.
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Fraction 6-25--
When fraction 6-25 was digested with
heparitinase II, it yielded equimolar amounts of
HexA(2S)-GlcNAc,
HexA-GlcN(NS,6S), and
HexA(2S)-GlcN(NS) (Table I). Upon successive
digestion with
hexuronate-2-sulfatase and then heparitinase II, it
yielded equimolar amounts of
HexA(2S)-GlcNAc,
HexA-GlcN(NS,6S), and
HexA-GlcN(NS) (Table I). These results indicated that the
disaccharide unit on the nonreducing terminus was
HexA(2S)-GlcN(NS). When digested with
heparitinase I, fraction 6-25 gave rise to equimolar amounts of
HexA-GlcN(NS,6S) and a component that eluted
near the elution position of a trisulfated tetrasaccharide (Table I),
indicating that
HexA-GlcN(NS,6S) was derived
from the reducing end. Therefore, the compound in fraction 6-25 is a
pentasulfated hexasaccharide with a sequence of
HexA(2S)-GlcN(NS)-HexA(2S)-GlcNAc-HexA-GlcN(NS,6S).
In the spectrum of fraction 6-25, six individual saccharide
residues were readily identified. Two internal uronic acid residues were determined as IdceA-4 and GlcA-2 based on
the chemical shifts of the anomeric proton signals, at
5.192 and 4.574, respectively, as well as the coupling constants
J1,2 of 2.0 and 8.0Hz, respectively. The
chemical shifts of H-1 and H-2 of the IdceA residue of the compound in
fraction 6-25 were shifted downfield by approximately 0.2 and 0.6 ppm,
respectively, when compared with those of the nonsulfated IdceA residue
of the oligosaccharides isolated from bovine kidney heparan sulfate or
porcine intestinal heparin (22, 33). In contrast, the proton chemical
shifts of the GlcA residue of the compound in this fraction were very similar to those of the nonsulfated GlcA residue. These results indicated the 2-sulfation of IdceA-4 and the nonsulfation of
GlcA-2 of this compound. Based upon these NMR data and the
sequential arrangement of the disaccharide units determined by
enzymatic analysis, the structure of the major compound in fraction
6-25 has been determined as the following.
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Fraction 6-30--
When fraction 6-30 was digested with
heparitinase II, it yielded approximately equimolar amounts of
HexA(2S)-GlcNAc,
HexA-GlcN(NS,6S), and
HexA(2S)-GlcN(NS,6S) (Table
I). Taking the UV absorbance of the parent hexasaccharide in fraction
6-30 as 100%, the total recovery of these disaccharide products was
greater than 300%, suggesting that the millimolar absorption
coefficient of the parent hexasaccharide may be smaller than those of
the produced disaccharides. Upon successive digestion with
hexuronate-2-sulfatase and then heparitinase II, it yielded
HexA-GlcN(NS,6S) and
HexA(2S)-GlcNAc with the recoveries of 183 and 112%,
respectively (Table I). These results indicated that
HexA(2S)-GlcN(NS,6S) was located on
the nonreducing terminus. When digested with heparitinase I, fraction
6-30 gave rise to equimolar amounts of
HexA-GlcN(NS,6S) and a component that eluted
near the elution position of a tetrasulfated tetrasaccharide (Table I),
indicating that
HexA-GlcN(NS,6S) was derived
from the reducing end. Therefore, the compound in fraction 6-30 is a
hexasulfated hexasaccharide with a sequence of
HexA(2S)-GlcN(NS,6S)-HexA(2S)-GlcNAc-HexA-GlcN(NS,6S).
In the spectrum of fraction 6-30, two internal uronic acid
residues were identified as IdceA-4 and GlcA-2
based on the chemical shifts of the anomeric proton signals, at
5.203 and 4.572, respectively, as well as the coupling constants
J1,2 of 1.5 and 7.5 Hz, respectively. The
chemical shifts of H-1 and H-2 of the IdceA residue of the compound in
fraction 6-30 were shifted downfield by approximately 0.2 and 0.6 ppm,
respectively, when compared with those of the nonsulfated IdceA residue
of the oligosaccharides isolated from bovine kidney heparan sulfate or porcine intestinal heparin (22, 33). In contrast, the proton chemical
shifts of the GlcA residue of the compound in this fraction were very
similar to those of the nonsulfated GlcA residue. These results
indicated the 2-sulfation of IdceA-4 and the nonsulfation of
GlcA-2 of this compound. Based upon these NMR data and the
sequential arrangement of the disaccharide units determined by
enzymatic analysis, the structure of the major compound in fraction
6-30 has been determined as the following.
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Fraction 6-22--
Fraction 6-22 was resolved into several
subcomponents by capillary electrophoresis (data not shown), the major
component accounting for only 57% of the UV absorbing materials in
this fraction. However, it was not possible to fractionate it
preparatively into its subcomponents. Therefore, it was first digested
with
hexuronate-2-sulfatase and then the digest was analyzed by
HPLC. The
hexuronate-2-sulfatase digest gave four peaks in a molar
ratio of 7:24:54:15, all of which eluted 11-16 min earlier than the
parent fraction (data not shown), indicating that fraction 6-22 was a
mixture of at least four different compounds. The major product,
designated as fraction 6-22S-1, was isolated and subjected to
structural analysis. The yield of fraction 6-22S-1 was 42 nmol/100 mg
starting heparin. When examined by capillary electrophoresis, fraction 6-22S-1 was 69% pure (results not shown). Upon heparitinase II digestion, fraction 6-22S-1 yielded
HexA-GlcN(NS) and
HexA(2S)-GlcNAc with the recoveries of 220 and 66%,
respectively, taking the UV absorbance of the total parent
oligosaccharides in fraction 6-22S-1 as 100% (Table I). Amino sugar
and uronic acid analyses showed that fraction 6-22S-1 contained a
hexasaccharide as a major compound. These results altogether indicate
that the major compound in this fraction was composed of 2 mol of
HexA-GlcN(NS) and 1 mol of
HexA(2S)-GlcNAc.
The excess recovery of
HexA-GlcN(NS) upon heparitinase II
digestion over the other product was probably due to the disaccharide produced by degradation of contaminating oligosaccharide(s). Since this
fraction was isolated after
hexuronate-2-sulfatase digestion, the
disaccharide unit on the nonreducing terminus of the parent hexasaccharide was not
HexA(2S)-GlcNAc but
HexA-GlcN(NS). Heparitinase I digestion of both fractions
6-22 and 6-22S-1 resulted in mainly two unsaturated components, the
monosulfated disaccharide
HexA-GlcN(NS) and a presumable
tetrasaccharide component that eluted near the elution position of the
tri- or disulfated tetrasaccharide. Recoveries of the di- and
tetrasaccharide components from fraction 6-22 or 6-22S-1 were 56 and
49% or 91 and 83%, respectively (Table I). The low recoveries of the
components from fraction 6-22 were due to the corresponding low content
of the major component in the fraction. Since the presumable
tetrasaccharide peak from fraction 6-22 was shifted by the prior
hexuronate-2-sulfatase digestion to the position corresponding to
the loss of one sulfate group on HPLC, the tetrasaccharide component
was derived from the nonreducing terminus. Therefore, the structure of
the compound in fraction 6-22S-1 is
HexA-GlcN(NS)-HexA(2S)-GlcNAc-HexA-GlcN(NS).
Consequently, the major compound in the parent fraction 6-22 is a
tetrasulfated hexasaccharide with a sequence of
HexA(2S)-GlcN(NS)-HexA(2S)-GlcNAc-HexA-GlcN(NS).
In the spectrum of fraction 6-22S-1, two internal uronic acid
residues were identified as IdceA-4 and GlcA-2 based on the chemical shifts of the anomeric proton signals, at
5.217 and 4.542, respectively, as well as the coupling constants J1,2 of 2.0 and 8.0 Hz, respectively. The
chemical shifts of H-1 and H-2 of the IdceA residue of the compound in
fractions 6-22S-1 were shifted downfield by approximately 0.2 and 0.6 ppm, respectively, when compared with those of the nonsulfated IdceA
residue of the oligosaccharides isolated from bovine kidney heparan
sulfate or porcine intestinal heparin (22, 33). In contrast, the proton chemical shifts of the GlcA residue of the compound in this fraction were very similar to those of the nonsulfated GlcA residue. These results indicated the 2-sulfation of IdceA-4 and the
nonsulfation of GlcA-2 of this compound. Based upon these
NMR data and the sequential arrangement of the disaccharide units
determined by enzymatic analysis, the structure of the major compound
in fraction 6-22S-1 has been determined as the following: fraction 6-22S-1,
HexA
1-4GlcN(NS)
1-4IdceA(2S)
1-4GlcNAc
1-4GlcA
1-4GlcN(NS). Since fraction 6-22S-1 was isolated after
hexuronate-2-sulfatase digestion as described above, the major
compound in the parent fraction 6-22 contained the following structure.
Fractions 6-17, -27, -32, and -34--
The spectral data of
fraction 6-27 (Table II) were indistinguishable from those of fraction
b-15 obtained from porcine intestinal heparin reported
previously (24). Hence, the major compound in fraction 6-27 was
identical with
HexA(2S)
1-4GlcN(NS,6S)
1-4IdceA
1-4GlcNAc(6S)
1-4GlcA
1-4GlcN(NS,6S). Likewise, 1H NMR data of fractions 6-17, -32, and -34 (Table II) were also indistinguishable from those of fractions VII,
b-20, and b-22, respectively, that were obtained
from porcine intestinal heparin as reported previously (22, 24),
indicating that the major compounds in fractions 6-17, -32, and -34 were identical with those in the compounds in the above fractions,
respectively. These structures are in good agreement with the results
obtained from enzymatic characterization (Table I). Therefore, the
structures of the compounds in those fractions are as follows.
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Fraction 6-31--
The spectral data of fraction 6-31 were
compared with those of fraction b-19S, which were obtained
from porcine intestinal heparin after
hexuronate-2-sulfatase
digestion as reported previously (24). No significant differences were
observed except for the downfield shifts (
0.05-0.84 ppm) of the
proton signals belonging to the nonreducing terminal
HexA. It should
be noted that in our previous report (24), the resonances of H-2 and -3 of the compound in fraction b-19S had been interchanged by
mistake. The results indicated that the major compound in fraction 6-31 has an additional sulfate group on C-2 of the nonreducing terminal
HexA in the structure for the compound in fraction b-19S. Therefore, the structure of the compound in fraction 6-31 is the following, and this structure is in good agreement with the results obtained from the enzymatic analysis (Table I).
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Fraction 6-26--
Upon exhaustive heparitinase I digestion,
fraction 6-26 yielded
HexA-GlcNAc(6S),
HexA-GlcN(NS), and
HexA(2S)-GlcN(NS,6S) with the
recoveries of 53, 67, and 67%, respectively, taking the UV absorbance
of the total parent oligosaccharides in fraction 6-26 as 100% (Table
I). The low recoveries of the major unsaturated disaccharides were
probably due to the corresponding low content of the major component in
fraction 6-26. The major compound in this fraction was a pentasulfated
hexasaccharide composed of equimolar amounts of the above three
components and accounted for only 53%, as judged by the recovery of
one of the three major disaccharides. The sensitivity of this fraction
to
hexuronate-2-sulfatase indicated that the disaccharide unit
on the nonreducing terminus was
HexA(2S)-GlcN(NS,6S) (Table I).
Thus, the major compound in this fraction was a pentasulfated hexasaccharide with a sequence of either
HexA(2S)-GlcN(NS,6S)-HexA-GlcNAc(6S)-HexA-GlcN(NS) or
HexA(2S)GlcN(NS,6S)-HexA-GlcN(NS)-HexA-GlcNAc(6S).
The two internal uronic acid residues of the hexasaccharide in fraction
6-26 were identified as IdceA and GlcA, based on the chemical shifts
(
4.997 and 4.538) of the anomeric proton signals and the coupling
constants J1,2 (1.5 and 8.0 Hz), respectively. Analysis of the deamination products identified the location of internal uronic acid residues. A tetrasaccharide component derived from
the reducing side of the parent hexasaccharide was obtained by nitrous
acid degradation. It was sensitive to
-iduronidase but resistant to
-glucuronidase (results not shown), indicating that the uronic acid
HexA-4 at the nonreducing end was IdceA, and the GlcA
residue was in turn localized at position 2 of the
hexasaccharide. Thus, the sequence of the major compound in fraction
6-26 is
HexA(2S)-GlcN(NS,6S)-IdceA-GlcNAc(6S)-GlcA-GlcN(NS), which was confirmed by 500-MHz 1H NMR spectroscopy (Table
II). The proton chemical shifts of
HexA-6, GlcN-5, IdceA, GlcNAc-3, and GlcA of the compound in fraction 6-26 were very similar to those of
HexA-6, GlcN-5, IdceA-4, GlcNAc-3, and
GlcA-2 of the compound in fraction 6-27. Those of
GlcN-1 were also analogous to those of GlcN-1 of
the compound in fraction 6-27 except for the upfield shifts of H-5,
H-6, and H-6
. These results indicated that the compound in fraction
6-26 lacks a sulfate group on C-6 of the GlcN-1 in the
structure of the compound in fraction 6-27. Therefore, the structure of
the major compound in fraction 6-26 is the following.
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Fraction 6-23--
Amino sugar and uronic acid analyses showed
that fraction 6-23 contained 1.8 mol of HexA and 1.9 mol of GlcN/mol of
HexA, where the GlcN value has been corrected for the degradation
(16%) during acid hydrolysis, indicating that the major compound in this fraction is a pentasaccharide. Upon exhaustive heparitinase I
digestion of fraction 6-23, the absorption at 232 nm doubled, and
HexA-GlcNAc(6S) and
HexA(2S)-GlcN(NS,6S) were observed
with recoveries of 88 and 121%, respectively, taking the UV absorbance of the total parent oligosaccharides in fraction 6-23 as 100% (Fig.
3A and Table I). The expected unsaturated uronic acid
residue to be derived from the reducing end was not detected probably since it was labile and decomposed into an
-keto acid as reported previously for the enzymatic digestions of unsaturated di- and trisaccharide from chondroitin sulfate (37, 38). The excess recovery of
HexA(2S)-GlcN(NS,6S) over the other
products was probably due to the disaccharide derived from
contaminating oligosaccharide(s). When fraction 6-23 was digested
successively with
hexuronate-2-sulfatase and then heparitinase II,
it yielded
HexA-GlcN(6S) and
HexA-GlcN(NS,6S) (Table I). These results
indicated the presence of the nonreducing end tetrasaccharide sequence
of
HexA(2S)-GlcN(NS,6S)-HexA-GlcNAc(6S)-. However, it remained to be determined whether the reducing terminal uronic acid was sulfated or not.
To define the pentasaccharide structure, fraction 6-23 was analyzed by
mass spectrometry. However, it was not possible to obtain spectra of
good quality by fast atom bombardment-ionization mass spectrometry
unlike for heparin tetrasaccharides (23) probably due to the high
negative charge. The fraction was successfully analyzed by MALDI TOF
mass spectrometry, where the negatively charged groups of the
oligosaccharide were shielded with a synthetic peptide as a complexing
agent (Arg-Gly)15 (29). Internal calibration by the peptide
yielded a molecular ion signal of the protonated 1:1 complex at
m/z 4434 (data not shown). After subtracting the contribution of the protonated peptide (m/z
3217), the molecular mass of the oligosaccharide was calculated to be
1217 Da, in reasonable agreement with the theoretical value (1213 Da)
for an unsaturated tetrasulfated pentasaccharide
HexA1HexA2HexNAc1HexN1(OSO3H)3. The mass accuracy of the present method could be lower (±0.2-0.3%) (29) than that obtained using a spectrometer equipped with the recently
developed delayed ion extraction device (39) but still sufficed to
determine the number of saccharide units and sulfate groups present.
Hence, the major compound in this fraction is a pentasaccharide with a
sequence of
HexA(2S)-GlcN(NS,6S)-HexA-GlcNAc(6S)-HexA.
The spectrum of fraction 6-23 (Fig. 4)
contained additional H-1 signals in the anomeric region when compared
with fraction 6-17 containing tetrasaccharides. Characteristic H-1
resonances at
5.2 and 4.6 led to identification of
GlcA and
GlcA residues at the reducing end, respectively (38), confirming
that the reducing terminal sugar residue was GlcA. The signals of the
nonreducing terminal trisaccharide region were very similar to those of
fraction 6-27, showing the presence of the structural element,
HexA(2S)-GlcN(NS,6S)-IdceA-. Compared with the chemical shifts of the protons belonging to the
reducing terminal GlcA residue of the reference compound,
HexA
1-3GalNAc(4-sulfate)
1-4GlcA, isolated from commercial
pig skin dermatan sulfate (38), no significant differences were observed, confirming the presence of a GlcA residue at the reducing terminus of the compound in fraction 6-23. The NMR data indicated that
the compound in fraction 6-23 has the pentasaccharide structure which
contains a GlcA residue extension on the reducing side. Based upon the
NMR data, the MALDI TOF mass data, and the sequential arrangement of
the disaccharide units determined by the enzymatic analysis, the
structure of the compound in fraction 6-23 was deduced as the following
tetrasulfated pentasaccharide structure.

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Fig. 4.
One-dimensional 500-MHz 1H NMR
spectrum of the structure in fraction 6-23 recorded in
2H2O. The numbers and
letters in the spectrum refer to the corresponding residues
in the structure.
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DISCUSSION |
In this study, we determined the structures of a tetra-, a penta-,
and eight hexasaccharides isolated from the heparin hexasaccharide fraction, which was prepared by the extensive digestion of porcine intestinal heparin with Flavobacterium heparinase. Five
subfractions, 6-22, -23, -25, -26, and -30, were isolated for the first
time as discrete structures. Since heparinase cleaves most
glucosaminidic linkages in the highly sulfated region (22, 40), which
accounts for three-quarters of a heparin polysaccharide chain, but does not cleave those in the less sulfated irregular region scattered along
the polysaccharide chain being flanked by the highly sulfated region,
the isolated oligosaccharides are derived from the irregular region.
Isolation of the heparin hexa- and larger oligosaccharides has been
limited. Two hexasaccharides,
HexA(2S)-GlcN(NS,6S)-IdceA(2S)-GlcN(NS,6S)-IdceA(2S)-GlcN(NS,6S) and
HexA(2S)-GlcN(NS,6S)-IdceA(2S)-GlcN(NS,6S)-GlcA-GlcN(NS,6S), have been isolated from the heparinase digests of both bovine lung heparin (41, 42) and porcine intestinal heparin (43). Two others
obtained by heparinase digestion of porcine intestinal heparin have
been determined as
HexA(2S)-GlcN(NS,6S)-IdceA(2S)-GlcN(NS,6S)-IdceA(2S)-GlcN(NS) and
HexA(2S)-GlcN(NS,6S)-IdceA(2S)-GlcN(NS,6S)-IdceA-GlcNAc(6S) (43). Another,
IdceA(2S)-GlcN(NS,6S)-IdceA(2S)-GlcN(NS,6S)-IdceA(2S)-2,5-anhydromannitol(6S), which is an HNO2 degradation product of porcine
mucosal heparin, has also been isolated (44). However, all these are
considered to be derived from the highly sulfated region. A few larger
oligosaccharides have also been isolated from the highly sulfated
region (41, 45-47). In contrast, the oligosaccharides isolated in this
study are derived from the irregular region of heparin. The isolation of hexa- and larger oligosaccharides from the irregular region has been
limited to those derived from the antithrombin III-binding site, and
the structural variability of several such oligosaccharides has been
summarized and discussed (24, 26, 48).
The isolated pentasaccharide appears to be derived from the reducing
end of a parent heparin glycosaminoglycan chain. During biosynthetic
processing, heparin glycosaminoglycan chains are synthesized on the
core protein as part of a proteoglycan, released from macromolecular
proteoglycans by an endo-
-glucuronidase, and stored in mast cell
granules that may be discharged from cells suitably stimulated
(49-51). The pentasaccharide in fraction 6-23 could be derived from
the newly formed reducing end exposed by the specific
endo-
-glucuronidase. Endoglycosidase activities toward
heparin/heparan sulfate have also been found in various other cells and
tissues (for a review, see Ref. 52). Some heparanases are secreted from
cells to play a role in remodeling basement membranes after injury or
at inflammation sites. Other heparanases are intracellular and
important for degrading cell surface heparan sulfate proteoglycans once
they have been internalized. One glucuronidic linkage cleavable by the
endoglucuronidase of human platelets is in the sequence
-GlcNAc-GlcA-GlcN(NS)- (53). However, the substrate
specificities of most of the endoglycosidases for heparin/heparan sulfate have been only partially characterized. One approach to investigate substrate specificity of endo-
-glucuronidases is to
analyze the reducing and nonreducing terminal structures of heparin
glycosaminoglycan chains, which would reflect the specific sites
cleaved by endoglycosidases. The nonreducing terminus of a heparin
glycosaminoglycan chain will be isolated as a saturated oligosaccharide
after digestions with heparin lyases (23, 54). The oligosaccharides in
fractions 6-26, -27, -32, and -34, which contain the pentasaccharide
sequence of the compound in fraction 6-23, may be useful for
elucidating the structural requirement for recognition by an
endo-
-glucuronidase. Bame and Robson (55) have recently
characterized the reducing terminal structures of heparanase-derived
short heparan sulfate chains isolated from Chinese hamster ovary cells
and proposed a model for the heparanase action.
As many as 8 of the 10 isolated oligosaccharide components shared the
trisaccharide sequence -4IdceA
1-4GlcNAc
1-4GlcA
1- (Table
III). In striking contrast, its reverse
sequence -4GlcA
1-4GlcNAc
1-4IdceA
1- was not found and has not
been reported to date for heparin/heparan sulfate to our knowledge.
Although Lindahl (4) previously referred in a review to the lack of
identification of the disaccharide sequence -4GlcNAc
1-4IdceA
1-
in heparin/heparan sulfate, this is the first demonstration of the
common trisaccharide sequence in the oligosaccharides isolated from the
irregular region of heparin. These findings indicate that the
-4GlcNAc
1-4GlcA
1- sequence does not serve as a substrate for the
epimerase involved in heparin/heparan sulfate biosynthesis.
Alternatively or in addition, GlcNAc
N-deacetylase/N-sulfotransferase may not attack
the -4IdceA
1-4GlcNAc
1-4GlcA
1- sequence. It should be noted
that the trisaccharide sequence was found to be sulfated either at C-2
of the IdceA residue (fractions 6-22, -25, and -30) or at C-6 of the
GlcNAc residue (fractions 6-23, -26, -27, -32, and -34), suggesting
that the -4GlcA
1-4GlcNAc
1-4IdceA
1- sequence serves as a
substrate for IdceA 2-O-sulfotransferase or GlcNAc
6-O-sulfotransferase. One of the five heparin
hexasaccharides reported previously (24) contained the disulfated
trisaccharide sequence
-4IdceA(2S)
1-4GlcNAc(6S)
1-4GlcA
1-,
indicating a possible conversion of a monosulfated to the disulfated
trisaccharide sequence. Seven hexasaccharides isolated in this
study shared the common trisulfated hexasaccharide core sequence
HexA(2S)1-4GlcN(NS)
1-4IdceA
1-4GlcNAc
1-4GlcA
1-4GlcN(NS) which contained the above trisaccharide sequence. The specificity of
the heparinase used for preparation of the oligosaccharides strongly
indicates the occurrence of the common pentasulfated octasaccharide
core sequence,
-4GlcN(NS)
1-4HexA(2S)1-4GlcN(NS)
1-4IdceA
1-4GlcNAc
1-4GlcA
1-4GlcN(NS)
1-4HexA(2S)1-, where the hexasaccharide is flanked by GlcN(NS) and
HexA(2S) on the nonreducing and reducing sides,
respectively. The common sequence revealed for the first time by the
present systematic analysis turned out to be a low sulfated
trisaccharide representing the irregular region sandwiched by highly
sulfated regions and should reflect the control mechanism of heparin
biosynthesis.
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Table III
Structures of the unsaturated oligosaccharides isolated from the low
sulfated region of porcine intestinal heparin
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ACKNOWLEDGEMENTS |
We thank Dr. Makiko Sugiura (Kobe
Pharmaceutical University) for recording the NMR spectra and
Tomomi Okumura for excellent technical assistance.
 |
FOOTNOTES |
*
This work was supported in part by the Science Research
Promotion Fund from the Japan Private School Promotion Foundation (to
K. S.), a grant from Ciba-Geigy Foundation (Japan) for the Promotion
of Science (to S. Y.), a grant from Japan Health Sciences Foundation
(to K. S.), Grants-in-Aid for Encouragement of Young Scientists
09772034 (to S. Y.), Exploratory Research Grant 08877338 (to K. S.),
and Scientific Research (B) Grant 09558082 (to K. S.) from the
Ministry of Education, Science, Culture, and Sports of Japan.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed. Tel.: 81-78-441-7570;
Fax: 81-78-441-7569; E-mail: k-sugar{at}kobepharma-u.ac.jp.
1
The abbreviations used are: 6S,
6-O-sulfate; NS, 2-N-sulfate;
2S, 2-O-sulfate; 3S,
3-O-sulfate; HPLC, high performance liquid chromatography;
HexA, hexuronic acid;
HexA,
4-deoxy-
-L-threo-hex-4-enepyranosyluronic acid;MALDI TOF, matrix-assisted laser desorption ionization
time-of-flight.
 |
REFERENCES |
-
Perlin, A. S.,
Mackie, D. M.,
and Dietrich, C. P.
(1971)
Carbohydr. Res.
18,
185-192[CrossRef][Medline]
[Order article via Infotrieve]
-
Rodén, L.
(1980)
in
The Biochemistry of Glycoproteins and Proteoglycans (Lennarz, W. J., ed), pp. 267-371, Plenum Publishing Corp., New York
-
Gallagher, J. T.,
and Lyon, M.
(1989)
in
Heparin (Lane, D. A., and Lindahl, U., eds), pp. 135-158, Edward Arnold, London
-
Lindahl, U.
(1989)
in
Heparin (Lane, D. A., and Lindahl, U., eds), pp. 159-189, Edward Arnold, London
-
Marcum, J. A.,
and Rosenberg, R. D.
(1989)
in
Heparin (Lane, D. A., and Lindahl, U., eds), pp. 275-294, Edward Arnold, London
-
Clowes, A.,
and Karnovsky, M.
(1977)
Nature
265,
625-626[Medline]
[Order article via Infotrieve]
-
Thornton, S. C.,
Mueller, S. H.,
and Levine, E. M.
(1983)
Science
222,
623-625[Medline]
[Order article via Infotrieve]
-
Folkman, J.,
and Ingber, D. E.
(1989)
in
Heparin (Lane, D. A., and Lindahl, U., eds), pp. 317-333, Edward Arnold, London
-
Maciag, T.,
Mehlman, T.,
Friesel, R.,
and Schreiber, A. B.
(1984)
Science
225,
932-935[Medline]
[Order article via Infotrieve]
-
Shing, Y.,
Folkman, J.,
Sullivan, R.,
Butterfield, C.,
Murray, J.,
and Klagsbrun, M.
(1984)
Science
223,
1296-1299[Medline]
[Order article via Infotrieve]
-
Klagsbrun, M.,
and Shing, Y.
(1985)
Proc. Natl. Acad. Sci. U. S. A.
82,
805-809[Abstract]
-
Nakamura, T.,
Teramoto, H.,
and Ichihara, A.
(1986)
Proc. Natl. Acad. Sci. U. S. A.
83,
6489-6493[Abstract]
-
Parthasarathy, N.,
Goldberg, I. J.,
Sivaram, P.,
Mulloy, B.,
Flory, D. M.,
Wagner, W. D.
(1994)
J. Biol. Chem.
269,
22391-22396[Abstract/Free Full Text]
-
Bourin, M.-C.,
and Lindahl, U.
(1993)
Biochem. J.
289,
313-330[Medline]
[Order article via Infotrieve]
-
Maccarana, M.,
and Lindahl, U.
(1993)
Glycobiology
3,
271-277[Abstract]
-
Lindahl, U.,
Bäckström, G.,
and Thunberg, L.
(1983)
J. Biol. Chem.
258,
9826-9830[Abstract/Free Full Text]
-
Atha, D. H.,
Stephens, A. W.,
and Rosenberg, R. D.
(1984)
Proc. Natl. Acad. Sci. U. S. A.
81,
1030-1034[Abstract]
-
Sugahara, K.,
Yamada, S.,
Yoshida, K.,
de Waard, P.,
Vliegenthart, J. F. G.
(1992)
J. Biol. Chem.
267,
1528-1533[Abstract/Free Full Text]
-
McLean, M. W.,
Bruce, J. S.,
Long, W. F.,
Williamson, F. B.
(1984)
Eur. J. Biochem.
145,
607-615[Abstract]
-
Tsukada, T.,
and Yoshino, M.
(1987)
Comp. Biochem. Physiol.
86,
565-569
-
Freeman, C.,
and Hopwood, J. J.
(1992)
Biochem. J.
282,
899-908[Medline]
[Order article via Infotrieve]
-
Yamada, S.,
Murakami, T.,
Tsuda, H.,
Yoshida, K.,
and Sugahara, K.
(1995)
J. Biol. Chem.
270,
8696-8705[Medline]
[Order article via Infotrieve]
-
Yamada, S.,
Sakamoto, K.,
Tsuda, H.,
Yoshida, K.,
Sugahara, K.,
Khoo, K.-H.,
Morris, H. R.,
Dell, A.
(1994)
Glycobiology
4,
69-78[Abstract]
-
Tsuda, H.,
Yamada, S.,
Yamane, Y.,
Yoshida, K.,
Hopwood, J. J.,
Sugahara, K.
(1996)
J. Biol. Chem.
271,
10495-10502[Abstract/Free Full Text]
-
Shively, J. E.,
and Conrad, H. E.
(1976)
Biochemistry
15,
3932-3942[Medline]
[Order article via Infotrieve]
-
Yamada, S.,
Yoshida, K.,
Sugiura, M.,
Sugahara, K.,
Khoo, K.-H.,
Morris, H. R.,
Dell, A.
(1993)
J. Biol. Chem.
268,
4780-4787[Abstract/Free Full Text]
-
Sugahara, K.,
Tsuda, H.,
Yoshida, K.,
Yamada, S.,
de Beer, T.,
Vliegenthart, J. F. G.
(1995)
J. Biol. Chem.
270,
22914-22923[Abstract/Free Full Text]
-
Vliegenthart, J. F. G.,
Dorland, L.,
and Van Halbeek, H.
(1983)
Adv. Carbohydr. Chem. Biochem.
41,
209-374
-
Juhasz, P.,
and Biemann, K.
(1995)
Carbohydr. Res.
270,
131-147[CrossRef][Medline]
[Order article via Infotrieve]
-
Bitter, M.,
and Muir, H.
(1962)
Anal. Biochem.
4,
330-334
-
Yamagata, T.,
Saito, H.,
Habuchi, O.,
and Suzuki, S.
(1968)
J. Biol. Chem.
243,
1523-1535[Abstract/Free Full Text]
-
Sugahara, K.,
Okamoto, H.,
Nakamura, M.,
Shibamoto, S.,
and Yamashina, I.
(1987)
Arch. Biochem. Biophys.
258,
391-403[Medline]
[Order article via Infotrieve]
-
Sugahara, K.,
Tohno-oka, R.,
Yamada, S.,
Khoo, K.-H.,
Morris, H. R.,
Dell, A.
(1994)
Glycobiology
4,
535-544[Abstract]
-
Yoshida, K.,
Miyazono, H.,
Tawada, A.,
Kikuchi, H.,
and Morikawa, K.
(1989)
in
Proceedings 10th International Symposium Glycoconjugates (Sharon, N., Lis, H., Duskin, D., and Kahane, I., eds), pp. 330-331, Jerusalem, Israel
-
Merchant, Z. M.,
Kim, Y. S.,
Rice, K. G.,
Linhardt, R. J.
(1985)
Biochem. J.
229,
369-377[Medline]
[Order article via Infotrieve]
-
Horne, A.,
and Gettins, P.
(1992)
Carbohydr. Res.
225,
43-57[CrossRef][Medline]
[Order article via Infotrieve]
-
Linker, A.,
Hoffman, P.,
Meyer, K.,
Sampson, P.,
and Korn, E. D.
(1960)
J. Biol. Chem.
235,
3061-3065[Medline]
[Order article via Infotrieve]
-
Sugahara, K.,
Takemura, Y.,
Sugiura, M.,
Kohno, Y.,
Yoshida, K.,
Takeda, K.,
Khoo, K.-H.,
Morris, H. R.,
Dell, A.
(1994)
Carbohydr. Res.
255,
165-182[CrossRef][Medline]
[Order article via Infotrieve]
-
Sugiyama, E.,
Hara, A.,
Uemura, K.,
and Taketomi, T.
(1997)
Glycobiology
7,
719-724[Abstract]
-
Linker, A.,
and Hovingh, P.
(1984)
Carbohydr. Res.
127,
75-94[CrossRef][Medline]
[Order article via Infotrieve]
-
Al-Hakim, A.,
and Linhardt, R. J.
(1990)
Electrophoresis
11,
23-28[Medline]
[Order article via Infotrieve]
-
Mallis, L. M.,
Wang, H. M.,
Loganathan, D.,
and Linhardt, R. J.
(1989)
Anal. Chem.
61,
1453-1458[Medline]
[Order article via Infotrieve]
-
Larnkjaer, A.,
Hansen, S. H.,
and Østergaard, P. B.
(1995)
Carbohydr. Res.
266,
37-52[CrossRef][Medline]
[Order article via Infotrieve]
-
Bârzu, T.,
Lormeau, J.-C.,
Petitou, M.,
Michelson, S.,
and Choay, J.
(1989)
J. Cell. Physiol.
140,
538-548[Medline]
[Order article via Infotrieve]
-
Chai, W.,
Hounsell, E. F.,
Bauer, C. J.,
Lawson, A. M.
(1995)
Carbohydr. Res.
269,
139-156[CrossRef][Medline]
[Order article via Infotrieve]
-
Larnkjaer, A.,
Nykjaer, A.,
Olivecrona, G.,
Thøgersen, H.,
and Østergaard, P. B.
(1995)
Biochem. J.
307,
205-214[Medline]
[Order article via Infotrieve]
-
Pervin, A.,
Gallo, C.,
Jandik, K. A.,
Han, X.-J.,
Linhardt, R. J.
(1995)
Glycobiology
5,
83-95[Abstract]
-
Toida, T.,
Hileman, R. E.,
Smith, A. E.,
Vlahova, P. I.,
Linhardt, R. J.
(1996)
J. Biol. Chem.
271,
32040-32047[Abstract/Free Full Text]
-
Ögren, S.,
and Lindahl, U.
(1975)
J. Biol. Chem.
250,
2690-2697[Abstract]
-
Robinson, H. C.,
Horner, A. A.,
Höök, M.,
Ögren, S.,
and Lindahl, U.
(1978)
J. Biol. Chem.
253,
6687-6693[Medline]
[Order article via Infotrieve]
-
Thunberg, L.,
Bäckström, G.,
Wasteson, Å.,
Robinson, H. C.,
Ögren, S.,
Lindahl, U.
(1982)
J. Biol. Chem.
257,
10278-10282[Abstract/Free Full Text]
-
Hopwood, J. J.
(1989)
in
Heparin (Lane, D. A., and Lindahl, U., eds), pp. 191-227, Edward Arnold, London
-
Oldberg, Å.,
Heldin, C.-H.,
Wasteson, Å.,
Busch, C.,
and Höök, M.
(1980)
Biochemistry
19,
5755-5762[Medline]
[Order article via Infotrieve]
-
Nader, H. B.,
Porcionatto, M. A.,
Tersariol, I. L. S.,
Pinhal, M. A. S.,
Oliveira, F. W.,
Moraes, C. T.,
Dietrich, C. P.
(1990)
J. Biol. Chem.
265,
16807-16813[Abstract/Free Full Text]
-
Bame, K. J.,
and Robson, K.
(1997)
J. Biol. Chem.
272,
2245-2251[Abstract/Free Full Text]
-
Linhardt, R. J.,
Wang, H. M.,
Loganathan, D.,
and Bae, J. H.
(1992)
J. Biol. Chem.
267,
2380-2387[Abstract/Free Full Text]
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