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(Received for publication, December 11,
1995; and in revised form, February 6, 1996) From the
Porcine intestinal heparin was extensively digested with Flavobacterium heparinase and size-fractionated by gel
chromatography. Subfractionation of the hexasaccharide fraction by
anion exchange high pressure liquid chromatography yielded 10
fractions. Six contained oligosaccharides derived from the repeating
disaccharide region, whereas four contained glycoserines from the
glycosaminoglycan-protein linkage region. The latter structures were
reported recently (Sugahara, K., Tsuda, H., Yoshida, K., Yamada, S., de
Beer, T., and Vliegenthart, J. F. G.(1995) J. Biol. Chem. 270,
22914-22923). In this study, the structures of one tetra- and
five hexasaccharides from the repeat region were determined by chemical
and enzymatic analyses as well as 500-MHz Heparin is a highly sulfated, linear polysaccharide that has
various biological activities such as inhibition of blood coagulation
(Marcum and Rosenberg, 1989), modulation of cellular proliferation
(Clowes and Karnovsky, 1977; Thornton et al., 1983),
potentiation of angiogenesis (Folkman and Ingber, 1989), and
interactions with various growth factors (Maciag et al., 1984;
Shing et al., 1984; Klagsbrun and Shing, 1985; Nakamura et
al., 1986). The basic polymeric structure of heparin is an
alternating repeat sequence of the disaccharide units
Recent structural studies
of the binding domains to ATIII (For review see Lindahl(1989)) and
basic fibroblast growth factor (Maccarana et al., 1993) are
the best known examples showing the relationships between the
complicated fine structures and biological functions. The ATIII-binding
site requires a minimal pentasaccharide sequence uniquely
3-O-sulfated on the central GlcN residue. This specific
pentasaccharide has been shown to be primarily responsible for the
anticoagulant activity of heparin (Lindahl et al., 1983). It
has also been demonstrated that the binding domain to basic fibroblast
growth factor requires a 2-O-sulfated IdoA residue and N-sulfated GlcN residue(s) for its specific interaction with
the growth factor. Some structural variability has been observed within
both binding sequences (Lindahl et al., 1984; Yamada et
al., 1993; Maccarana et al., 1993). We have been
studying the basic primary structure of heparin to clarify the
structural basis of its various biological activities. Previously, we
demonstrated its structural variability by isolating six glycoserines
from the carbohydrate-protein linkage region (Sugahara et al.,
1992, 1995) and a number of tetrasaccharides from the repeating
disaccharide region of porcine intestinal heparin after extensive
digestion with bacterial heparin lyases (Yamada et al., 1993,
1994, 1995). In this study, we isolated and characterized five
hexasaccharide structures from the repeating disaccharide region of the
same heparin preparation after extensive enzymatic digestion to
investigate the structure beyond the above tetrasaccharide sequences.
These included three hitherto unreported hexasaccharide structures and
structural variants with an apparent biosynthetic precursor-product
relationship for the ATIII-binding site. Materials-Stage 14 heparin was purchased from
American Diagnostica (New York, NY) and purified by DEAE-cellulose
chromatography as reported previously (Sugahara et al., 1992).
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, Japan).
Figure 1:
Capillary
electrophoresis of the isolated hexasaccharide fractions. The isolated
hexasaccharide fractions (1.0 nmol each) were subjected to
electrophoresis as described under ``Experimental
Procedures.'' A, Fr. b-15; B, Fr. b-19; C, Fr. b-20; D, Fr. b-22; E, Fr.
b-24.
Figure 2:
HPLC
analysis of the enzyme digests of Fr. b-15. Fr. b-15 (0.5 nmol) was
digested with heparitinase I (A) and successively with
2-sulfatase and then heparitinase I (B) or with heparitinase V (C) as described under ``Experimental Procedures.''
The digest was subjected to HPLC on an amine-bound silica column using
a linear gradient of NaH
Figure 3:
HPLC analysis of the enzyme digests of Fr.
b-24. Fr. b-24 (0.25 nmol) was digested with a mixture of heparitinase
I and heparinase (A), heparinase (B), heparitinase I (C), or 2-sulfatase and then heparitinase I successively (D) as described under ``Experimental Procedures.''
For the HPLC conditions, see the legend to Fig. 2.
The major component in Fr. b-24 accounted for 75% of the
UV-absorbing materials in this fraction as judged by capillary
electrophoresis (Fig. 1E). Exhaustive digestion of this
fraction with a mixture of heparinase and heparitinase I yielded
Fr. b-19 was resolved into several subcomponents by capillary
electrophoresis, the major component accounting for only 54% of the
UV-absorbing materials in this fraction (Fig. 1B), but
it was not possible to fractionate it preparatively into its
subcomponents. Therefore, it was first digested with 2-sulfatase and
then the digest was analyzed by HPLC. The 2-sulfatase treatment
resulted in a peak shift of 8 min of 61% of the parent compound on
HPLC, indicating that the major product lost one sulfate group (data
not shown). The major product, designated as Fr. b-19S, was isolated
and subjected to structural analysis. The yield was 167 nmol/100 mg of
the starting heparin. It was degraded by heparitinase I, yielding
almost exclusively Heparitinase I digestion of both Fr. b-20 and -22 resulted in two
unsaturated components, the trisulfated disaccharide Sensitivities of the compounds in Fr. b-20 and -22 to 2-sulfatase
were examined to characterize the sequential arrangement of the
constituent di- and tetrasaccharide units. After 2-sulfatase digestion,
Fr. b-20 and -22 gave a single peak on HPLC, which eluted approximately
10 min earlier than the corresponding parent compound, indicating that
the major compound in each fraction had a sulfate group on the C-2
position of the
The
resultant di- and/or tetrasaccharide(s) from each hexasaccharide
fraction were isolated by gel filtration on Bio-Gel P-2 and were
analyzed by HPLC. These fractions obtained from Fr. b-15, -20, or -22
were confirmed to contain a disaccharide and a tetrasaccharide
component as judged from their elution positions on HPLC (data not
shown) (Bienkowski and Conrad, 1985). The tetrasaccharide component
presumably derived from the reducing side of each of these original
hexasaccharides was subjected to digestion with human liver
Figure 4:
Deaminative cleavage of Fr. b-19S yielded no appreciable
tetrasaccharides but two kinds of disaccharide components, which eluted
at around the positions of Fr. b-24 was also degraded into a disaccharide and a tetrasaccharide
component (data not shown) as judged from the gel filtration profiles
of the deaminative cleavage products and the elution positions on the
subsequent HPLC of the isolated di- and tetrasaccharides. The
disaccharide eluted at around the position of
The internal uronic acid residue of
each isolated hexasaccharide was unambiguously identified by 500-MHz
Figure 5:
One-dimensional 500-MHz
The two internal uronic acid residues of the hexasaccharide in Fr.
b-15 were identified as IdoA and GlcA, based on the chemical shifts
( Likewise, the structures of the major compounds in three other
fractions were determined and summarized in Table 3. Some of the
chemical shifts of Fr. b-20 were not assigned due to the low quality of
the spectra.
The five sulfated hexasaccharide structures isolated in this
study share the pentasulfated hexasaccharide backbone
Heparin and heparan sulfate have been demonstrated to exhibit
various biological activities (Kjellén and
Lindahl, 1991). Especially their specific interactions with various
growth factors have recently attracted much attention. However, the
functional domain structures elucidated to date are limited to only a
few examples, including the minimum pentasaccharide sequences for ATIII
binding and basic fibroblast growth factor binding, which have been
demonstrated as
GlcN(6S) The hexasaccharides isolated in this
study appear to be large enough to potentially exhibit binding
activities toward growth factors or other functional proteins. None of
them, however, showed ATIII-mediated inhibition of factor Xa as
examined according to Morita et al.(1977). Although the
hexasaccharides in Fr. b-20 and -22 contain a 3-sulfated
GlcN(NS) residue at their reducing termini, they lack a part
of the minimum pentasaccharide sequence on their reducing sides, i.e. IdoA(2S) The most interesting structural feature of
the isolated hexasaccharides is that they include three overlapping
pairs of structural variants with an apparent biosynthetic
precursor-product relationship for the ATIII-binding site.
Structurally, the hexasaccharide in Fr. b-15 is a pro-form of that in
Fr. b-22. The former lacks the 3-sulfate group on GlcN-1 of the latter.
Likewise, the hexasaccharide in Fr. b-20 is a pro-form of that in Fr.
b-22, the former lacking the 6-sulfate of GlcN-1 of the latter. The
hexasaccharide in Fr. b-15 can be considered as a pro-form of that in
Fr. b-24, where the former lacks the 2-sulfate on IdoA-4 of the latter.
Among the above hexasaccharides, the previously isolated structures
found in Fr. b-15 and -22 (Linhardt et al., 1992) have been
subjects of much discussion as biosynthetic precursors and product,
respectively, of the ATIII-binding site as described below. Only
about one-third of chains of commercial porcine intestinal heparin
contain an ATIII-binding site and have a high affinity for ATIII. The
critical 3-O-sulfation of GlcN required for the ATIII-high
affinity concludes the biosynthesis of the ATIII-binding site (Kusche et al., 1988). Based upon the isolation of a precursor
tetrasaccharide sequence, which lacked the 3-O-sulfate group
from heparin chains with ATIII-low affinity, Kusche et al. (1990) proposed that essentially each low affinity chain would
contain a potential but not utilized 3-O-sulfation site.
Linhardt et al.(1992) challenged this hypothesis, proposing
that the existence of a low affinity heparin may not simply be the
result of the incomplete action of 3-O-sulfotransferase in the
final step, but rather some earlier step involved in the formation of
the precursor sites may be primarily responsible for high and low ATIII
affinity heparins. Recently, Razi and Lindahl(1995) proposed an
intriguing hypothesis that the 3-O-sulfotransferase may be
inhibited by a sulfated saccharide sequence outside the
3-O-sulfate acceptor region based upon the observation that an
octasaccharide fraction isolated from ATIII-low affinity heparin,
unlike low affinity heparin polysaccharide (Kusche et al.,
1990), yielded high affinity components following incubation with a
GlcN 3-O-sulfotransferase preparation. The multiple
pro-form structures demonstrated in this study probably do not
represent precursors and products but rather reflect structural
variants that have diverged during the modification reactions in the as
yet unresolved but precisely programmed biosynthetic scheme.
Practically, they will be valuable acceptor substrates for
sulfotransferases to investigate such biosynthetic mechanisms by which
the production of specific functional carbohydrate sequences is
regulated. For example, it would be of interest to examine whether the
hexasaccharide structures in Fr. b-15 and -20 serve as acceptor
substrates for IdoA 2-O-sulfotransferase and GlcN
6-O-sulfotransferase (Habuchi et al., 1995) to
produce the hexasaccharide structures found in Fr. b-24 and -22,
respectively. It will be intriguing to evaluate these hexasaccharides
as regulatory elements as well because they may control biosynthetic
modifying enzymes. They will also be useful for characterizing
heparin/heparan sulfate-degrading enzymes including both the bacterial
heparinase/heparitinases and mammalian heparanases. The former enzymes
are essential tools for structural studies (Yoshida et al.,
1989), whereas the latter have been implicated in tumor metastasis (for
review see Nakajima et al.(1988)), T cell adhesion (Gilat et al., 1995), and chemoattractant functions (Hoogewerf et
al., 1995).
Volume 271,
Number 18,
Issue of May 3, 1996 pp. 10495-10502
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
STRUCTURAL VARIANTS WITH APPARENT BIOSYNTHETIC PRECURSOR-PRODUCT
RELATIONSHIPS FOR THE ANTITHROMBIN III-BINDING SITE (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
H NMR
spectroscopy. The tetrasaccharide has the hexasulfated structure
typical of heparin. The five hexa- or heptasulfated hexasaccharides
share the common core pentasulfated structure
HexA(2S)
1-4GlcN(NS,6S)1-4IdoA/GlcA
1-4GlcN(6S)1-4GlcA1-
4GlcN(NS) with one or two additional sulfate groups
(HexA, GlcN, IdoA, and GlcA represent
4-deoxy--L-threo-hex-4-enepyranosyluronic acid, D-glucosamine, L-iduronic acid, and D-glucuronic acid, whereas 2S, 6S, and NS stand for
2-O-, 6-O-, and 2-N-sulfate, respectively).
Three components have the following hitherto unreported structures:
HexA(2S)1-4GlcN(NS,6S)1-4GlcA1-4GlcN(NS,6S)1-4GlcA1-4GlcN(NS,6S),
HexA(2S)1-4GlcN(NS,6S)1-4IdoA1-4GlcNAc(6S)1-4GlcA1-4GlcN(NS,3S),
and
HexA(2S)1-4GlcN(NS,6S)1-4IdoA(2S)1-4GlcNAc(6S)1-4GlcA1-
4GlcN(NS,6S). Two of the five hexasaccharides are structural
variants derived from the antithrombin III-binding sites containing
3-O-sulfated GlcN at the reducing termini with or without a
6-O-sulfate group on the reducing N,3-disulfated GlcN
residue. Another contains the structure identical to that of the above
heptasulfated antithrombin III-binding site fragment but lacks the
3-O-sulfate group and therefore is a pro-form for the binding
site. Another has an extra sulfate group on the internal IdoA residue
of this pro-form and therefore can be considered to have diverged from
the binding site in the biosynthetic pathway. Thus, the isolated
hexasaccharides in this study include the three overlapping pairs of
structural variants with an apparent biosynthetic precursor-product
relationship, which may reflect biosynthetic regulatory mechanisms of
the binding site.
4IdoA/GlcA14GlcN1, which can be
variably sulfated (for reviews see Rodén(1980),
Gallagher and Lyon(1989), and Lindahl(1989)). It is synthesized on a
serine residue of a protein core named ``serglycin'' through
a specific structure, the so-called carbohydrate-protein linkage
region:
GlcA1-3Gal1-3Gal1-4Xyl1-O-Ser
(Lindahl and Rodén, 1965). Although the principal
structure of the repeating disaccharide region, known as the regular
region (Casu, 1985), is composed of the major trisulfated disaccharide
unit,
4IdoA(2-sulfate)14GlcN(N,6-disulfate)1,
undersulfation and substantial structural variability are observed in
the irregular region, which is distributed along the chain flanked by
the regular region and accounts for approximately one quarter of the
heparin polysaccharide chain. The structural variability is often the
basis of a wide variety of domain structures with a number of
biological activities ascribed to heparin.
-Glycuronate-2-sulfatase (EC 3.1.6.-), abbreviated
as 2-sulfatase, and heparitinase V (no EC number) were purified from Flavobacterium heparinum (McLean et al., 1984) and Flavobacterium sp. Hp206 (Yoshida et al.,
1989), respectively. Sephadex gels were from Pharmacia Biotech Inc.,
and Bio-Gel resins were from Bio-Rad. NaB
H
(15
Ci/mmol) was supplied by American Radiolabeled Chemicals, Inc. (St.
Louis, MO). 4-Methylumbelliferyl--L-iduronide was from
Sigma, and p-nitrophenyl--D-glucuronide was from
Nacalai Tesque (Kyoto, Japan). Seven standard unsaturated disaccharides
were prepared from heparin as reported previously (Yamada et
al., 1992). Standard heparin disaccharides prepared by deaminative
cleavage (Shively and Conrad, 1976a, 1976b) were gifts from Dr. H. E.
Conrad, University of Illinois. -Glucuronidase (EC 3.2.1.31)
purified to homogeneity from Ampullaria (freshwater apple
shell) hepatopancreas (Tsukada and Yoshino, 1987) was obtained from
Tokyo Zouki Chemical Co., Tokyo. Human liver -iduronidase (EC
3.2.1.76) was purified as reported previously (Freeman and Hopwood,
1992).Preparation and Purification of Hexasaccharides
Stage 14
heparin was purified by anion exchange chromatography and digested with
heparinase, and the digest was fractionated into fractions a-d by
gel filtration as described previously (Sugahara et al.,
1995). Fractions c and d contained tetra- and disaccharides,
respectively, which were derived from the repeating disaccharide region
as characterized by HPLC ()(data not shown). Fraction a
contained larger oligosaccharides and glycoserines/glycopeptides that
were derived from the glycosaminoglycan-protein linkage region.
Fraction b containing mainly hexasaccharides was subfractionated by
HPLC on an amine-bound silica column (Sugahara et al., 1995)
and structurally characterized in this study. Each peak was purified by
rechromatography under the same conditions as the first step and
desalted by gel filtration through a Sephadex G-25 column.Digestion of Fraction b-19 with 2-Sulfatase and
Subfractionation of the Digest
Fraction b-19 (170 nmol) was
incubated with 60 mIU of 2-sulfatase in a total volume of 120 µl of
6.7 mM CHCOONa, pH 6.5, containing 0.05% bovine
serum albumin at 37 °C for 140 min. The reaction was terminated by
boiling for 1 min, and the digest was fractionated by HPLC on an
amine-bound silica column as described below.Digestion of the Isolated Hexasaccharides with
Heparinase, Heparitinases, or 2-Sulfatase
Each isolated
hexasaccharide (0.5-1.0 nmol) was digested using 1-5 mIU of
heparinase, heparitinase I, II, or V, or 2-sulfatase as described
previously (Sugahara et al., 1992; Yamada et al.,
1994). Successive enzymatic digestion of a given hexasaccharide with
2-sulfatase and then heparitinase I was also carried out as reported
(Yamada et al., 1995). Reactions were terminated by boiling
for 1 min, and the reaction mixture was analyzed by HPLC as described
below.HPLC and Capillary Electrophoresis
Fractionation
and analysis of unsaturated oligosaccharides were carried out by HPLC
on an amine-bound silica PA03 column using a linear gradient of
NaH
PO basically as described previously, except
that a linear gradient of NaHPO was made from
16-800 mM over 90 min (Sugahara et al., 1992).
Eluates were monitored by absorption at 232 nm. Capillary
electrophoresis was carried out to examine the purity of each isolated
fraction in a Waters capillary ion analyzer as reported previously
(Sugahara et al., 1994). The electrophoretic fractions were
examined by absorption at 185 nm due to carbonyl groups because its
sensitivity was higher than that of detection at 232 nm (Sugahara et al., 1995).Nitrous Acid Degradation
Each heparin
hexasaccharide (5.0 nmol) was treated at room temperature with
HNO at pH 1.5 for 30 min (Shively and Conrad, 1976a), and
the resultant di- and/or tetrasaccharides were reduced under alkaline
conditions with [H]sodium borohydride (0.50 mCi)
as reported previously (Yamada et al., 1995). Labeled
oligosaccharides were separated by gel filtration chromatography on a
column (1.0 
115 cm) of Bio-Gel P-2 using 0.25 M NHHCO/7% propanol as an eluent. Di- and
tetrasaccharide fractions were separately pooled, lyophilized at least
three consecutive times to ensure complete removal of the ammonium
bicarbonate, and then reconstituted in water. These fractions were
separated, respectively, by HPLC on an amine-bound silica column at a
flow rate of 1 ml/min using a stepwise gradient of
NaHPO. Samples were collected at 1.0-min
intervals for radioactivity measurement in an Aloka LSC-700 liquid
scintillation counter. Individual disaccharide peaks were identified by
comparison with authentic heparin disaccharides as reported previously
(Bienkowski and Conrad, 1985).
Tetrasaccharides obtained by deamination of
hexasaccharides in Fr. b-15, -20, and -22 were tested for their
sensitivities to -Iduronidase and -Glucuronidase Digestion of
Tetrasaccharides-iduronidase and -glucuronidase to determine
the isomer type of the uronic acid residue exposed at the nonreducing
termini. Each [H]tetrasaccharide (8 pmol)
corresponding to approximately 4 10 cpm was
digested using 17.7 mIU of -iduronidase or 81.5 mIU of
-glucuronidase in a total volume of 30 µl of 20 mM NaOH-formic acid buffer, pH 3.0 (Freeman and Hopwood, 1992) or 50
mM acetic acid-NaOH buffer, pH 4.5 (Tsukada and Yoshino, 1987)
at 37 °C overnight. One IU of -iduronidase or
-glucuronidase is defined as the amount of enzyme that produces 1
µmol of uronic acid/min from
4-methylumbelliferyl--L-iduronide or p-nitrophenyl--D-glucuronide, respectively.500-MHz
Hexasaccharides for NMR analysis were fully
sodiated using a Dowex 50-X8 (NaH NMR
Spectroscopy
form) column (7
18 mm) and then repeatedly exchanged in HO with intermediate lyophilization. 500-MHz H NMR spectra of hexasaccharides were measured on a Varian
VXR-500 at a probe temperature of 26 °C as reported previously
(Yamada et al., 1993). 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 HO (Vliegenthart et al., 1983).Other Analytical Methods
Uronic acid was
determined by the carbazole method (Bitter and Muir, 1962). Unsaturated
uronic acid was spectrophotometrically quantified based upon an average
millimolar absorption coefficient of 5.5 at 232 nm (Yamagata et
al., 1968). Amino sugars were quantified after acid hydrolysis in
3 M HCl at 100 °C for 16 h using a Beckman 6300E amino
acid analyzer (Sugahara et al., 1987).
Isolation of the Oligosaccharides
Purified stage
14 heparin from porcine intestine was exhaustively digested with
heparinase and fractionated into fractions a-d by gel filtration
on Cellulofine GCL-90 m (Sugahara et al., 1995). Amino sugar
and uronic acid analyses showed that fraction b contained approximately
3 mol each of GlcN and HexA/mol of HexA (data not shown), i.e. hexasaccharides. Fraction b was subfractionated by HPLC on an
amine-bound silica column into Fr. b-1 to b-26 (Sugahara et
al., 1995). Nine major fractions, Fr. b-5, -6, -10, -15, -17, -19,
-20, -22, and -24, were further purified by rechromatography. They
altogether accounted for 77 mol% (as HexA) of the oligosaccharides
obtained from fraction b. Fr. b-5, -6, and -10 contained glycoserines
derived from the glycosaminoglycan-protein linkage region as reported
previously (Sugahara et al., 1995). The other fractions, Fr.
b-15, -17, -19, -20, -22, and -24, were subjected to structural
analysis below in this study. These individual fractions gave a single
peak on HPLC but were approximately 95, 99, 54, 66, 96, and 75% pure,
respectively, when examined by capillary electrophoresis (Fig. 1). Fr. b-17 contained the previously reported
hexasulfated tetrasaccharide
HexA(2S)1-4GlcN(NS,6S)1-4IdoA(2S)1-4GlcN(NS,6S)
(Linker and Hovingh, 1984) as confirmed by H NMR
spectroscopy, where 2S, 6S, and NS represent 2-O-,
6-O-, and 2-N-sulfation, respectively (data not
shown). Apparently, this tetrasaccharide was partitioned into the
hexasaccharide fraction due to its heavily sulfated structure. The
amounts of the analyzed fractions isolated from 100 mg of the starting
purified heparin are summarized in Table 1.
Enzymatic Analysis
The disaccharide compositions
of the isolated oligosaccharide fractions were determined by digestion
with heparitinase(s) and/or heparinase, followed by HPLC on an
amine-bound silica column. All the above oligosaccharide fractions
except for Fr. b-17 were enzymatically degraded into approximately 3
mol of disaccharide units or 1 mol each of a di- and a tetrasaccharide
unit. As representative chromatograms, those obtained with Fr. b-15 and
b-24 are shown in Fig. 2and Fig. 3, respectively. Fr.
b-15 yielded DiHS-6S, DiHS-diS, and
DiHS-triS upon heparitinase I digestion as shown in Fig. 2A; their recoveries were 113, 105, and 124%,
respectively, when taking the UV absorbance of the parent
oligosaccharide(s) in Fr. b-15 as 100% (Table 1). Thus, the major
component in this fraction was a hexasulfated hexasaccharide composed
of a monosulfated, a disulfated, and a trisulfated disaccharide unit.
Its sensitivity to 2-sulfatase was examined to localize the
HexA(2S)-containing disaccharide unit DiHS-triS in the
hexasaccharide sequence. The enzyme acts only on the HexA(2S)
structure at the nonreducing end (McLean et al., 1984). When
Fr. b-15 was successively digested with 2-sulfatase and then
heparitinase I, it yielded DiHS-6S and DiHS-diS with recoveries of 92 and 204%, respectively, indicating that
DiHS-triS had been located at the nonreducing end and converted to
DiHS-diS by successive digestion (Fig. 2B). When digested with heparitinase V (Yoshida, et al., 1989), Fr. b-15 gave rise to equimolar amounts of
DiHS-diS and a presumable tetrasulfated
tetrasaccharide (Fig. 2C), indicating that
DiHS-diS was derived from the other terminus, i.e. the reducing end. Therefore, the structure of the compound in Fr.
b-15 is
HexA(2S)-GlcN(NS,6S)-HexA-GlcNAc(6S)-HexA-GlcN(NS,6S).
PO from 16 to 800
mM over 90 min. Elution positions of the standard
disaccharides isolated from heparin/heparan sulfate are indicated in A: 1, DiHS-0S; 2, DiHS-6S; 3, DiHS-NS; 4,
DiHS-diS; 5, DiHS-diS; 6, DiHS-diS; 7, DiHS-triS. The
peaks marked by asterisks are often observed between 35 and 40
min upon high sensitivity analysis and are due to an unknown substance
eluted from the column resin. The broad peaks observed at around 5 and
10 min were derived from the incubation buffer or the enzyme
preparation.
DiHS-triS, DiHS-diS, and DiHS-diS with recoveries of 158, 96, and 125%, respectively (Fig. 3A). Upon incubation with heparinase only, Fr.
b-24 was degraded into two unsaturated components, DiHS-triS and a
component that eluted near the elution position of a tetrasulfated
tetrasaccharide, with recoveries of 171 and 110%, respectively (Fig. 3B), whereas it was degraded by heparitinase I
into two unsaturated components, DiHS-diS and a
component that eluted near the elution position of a pentasulfated
tetrasaccharide, with recoveries of 100 and 106%, respectively (Fig. 3C). These results together indicate that the
major component in Fr. b-24 is a pentasulfated hexasaccharide composed
of equimolar amounts of three disaccharide units corresponding to
DiHS-triS, DiHS-diS, and
DiHS-diS, and that the excess recovery of
DiHS-triS upon heparinase or heparinase/heparitinase I digestion
was probably due to degradation of contaminating oligosaccharide(s).
The sequential arrangement of the three disaccharide units in the major
hexasaccharide was determined based upon the sensitivity to
2-sulfatase. When digested successively with 2-sulfatase and then
heparitinase I, the presumably pentasulfated tetrasaccharide peak
shifted to a position corresponding to the loss of one sulfate group on
HPLC, indicating that DiHS-triS had been located on the
nonreducing terminal side of the major compound in Fr. b-24. Therefore,
the structure of the major compound in this fraction is proposed as
HexA(2S)-GlcN(NS,6S)-HexA(2S)-GlcNAc(6S)-HexA-GlcN(NS,6S). DiHS-diS (Table 1).
Therefore, the major component in Fr. b-19S was deduced to be a
hexasulfated hexasaccharide composed of 3 mol of the disulfated
disaccharide unit corresponding to DiHS-diS, i.e. HexA-GlcN(NS,6S)-HexA-GlcN(NS,6S)-HexA-GlcN(NS,6S).
Consequently, the structure of the major component in the parent
fraction b-19 was
HexA(2S)-GlcN(NS,6S)-HexA-GlcN(NS,6S)-HexA-GlcN(NS,6S). DiHS-triS and
a component that eluted near the elution position of the tri- or
tetrasulfated tetrasaccharide (data not shown). Recoveries of the di-
and tetrasaccharide components from Fr. b-20 or -22 were 109 and 62% or
122 and 106%, respectively (Table 1). The lower recoveries of the
presumable tetrasaccharide components of these fractions compared with
those of their counterpart disaccharides suggested that the excess
disaccharides were derived from minor components in these fractions
consistent with the results of capillary electrophoresis. The
presumable tetrasaccharide components from both Fr. b-20 and -22 were
resistant to heparinase and heparitinases I and II (data not shown),
probably due to the 3-O-sulfation of the reducing GlcN as
reported previously for the ATIII-binding site-derived tetrasaccharides
(Yamada et al., 1993). The presumable tetrasaccharides were
co-chromatographed on HPLC with the authentic tetrasaccharides
containing 3-O-sulfated GlcN residue (Yamada et al.,
1993), demonstrating that the tri- and tetrasulfated tetrasaccharides
derived from Fr. b-20 and -22 were identical to
HexA-GlcNAc(6S)-GlcA-GlcN(NS, 3S) and
HexA-GlcNAc(6S)-GlcA-GlcN(NS, 3S,6S), respectively. HexA residue at the nonreducing terminus.
Therefore, the structures of the major compounds in Fr. b-20 and -22
were
HexA(2S)-GlcN(NS,6S)-HexA-GlcNAc(6S)-GlcA-GlcN(NS,3S)
and
HexA(2S)-GlcN(NS,6S)-HexA-GlcNAc(6S)-GlcA-GlcN(NS,3S,6S),
respectively.HPLC Analysis of the Di- and Tetrasaccharides Formed by
HNO
To
identify the internal uronic acid residues in the hexasaccharides of
the isolated fractions, nitrous acid degradation products of each
fraction were analyzed by HPLC. Bacterial lyase treatment converts the
original structures of internal uronic acid, GlcA and IdoA in the
oligosaccharides into the common 4,5-unsaturated,
4-deoxy-/NaBH Treatment-L-threo-hex-4-enepyranosyluronic acid.
In contrast, nitrous acid treatment preserves the original uronic acid
structures despite loss of an N-sulfate group and production
of an artificial structure, anhydromannitol, at the reducing end of the
resultant oligosaccharides (Shively and Conrad, 1976a, 1976b).-iduronidase (Freeman and Hopwood, 1992) and Ampullaria -glucuronidase (Tsukada and Yoshino, 1987) to identify the
uronic acid residues at the nonreducing termini. After
-iduronidase digestion, a part (40, 57, or 60%) of the
tetrasaccharide peak derived from Fr. b-15, -20, or -22 eluted
7-14 min earlier than the corresponding parent compound on HPLC.
As representative chromatograms, those obtained with the
tetrasaccharide from Fr. b-22 are shown in Fig. 4. Upon
-glucuronidase digestion, however, the tetrasaccharide fraction
obtained from each of the three fractions was totally resistant to the
action of the enzyme (data not shown). These results suggest that the
nonreducing terminal uronic acid of the major tetrasaccharide obtained
from Fr. b-15, -20, or -22 by nitrous acid treatment is not GlcA but
rather IdoA. The reason for the partial insensitivity to the action of
-iduronidase is unclear at present but may have been due to side
product(s) of the deamination reactions. It has been reported that the
so-called ring contraction tetrasaccharides can be formed during
deamination reactions (Bienkowski and Conrad, 1985). Based upon the
above results, the structures of the major components in these
fractions were deduced as follows: Fr. b-15,
HexA(2S)-GlcN(NS,6S)-IdoA-GlcNAc(6S)-HexA-GlcN(NS,6S);
Fr. b-20,
HexA(2S)-GlcN(NS,6S)-IdoA-GlcNAc(6S)-GlcA-GlcN(NS,
3S); Fr. b-22,
HexA(2S)-GlcN(NS,6S)-IdoA-GlcNAc(6S)-GlcA-GlcN(NS,
3S,6S).
-Iduronidase digestion of the
tetrasaccharide prepared from Fr. b-22 by
HNO/NaBH treatment. Fr. b-22 was
subjected to nitrous acid depolymerization at pH 1.5, and the resultant H-labeled tetrasaccharides were isolated by gel filtration
chromatography on a Bio-Gel P-2 column and analyzed by HPLC on an
amine-bound silica column using a stepwise salt gradient as indicated
by the dashed line. Fractions were collected at 1-min
intervals at a flow rate of 1 ml/min, and their radioactivity was
determined by liquid scintillation counting. A, the presumable
tetrasaccharide (7500 cpm) derived from Fr. b-22; B, the
-iduronidase digest of the tetrasaccharide (3900
cpm).
HexA-anMan
(6S) and
GlcA-anMan(6S), respectively, in a molar ratio of 1.0:1.7.
These results indicate that both internal uronic acid residues of the
major hexasaccharide component in Fr. b-19S and its parent fraction
b-19 are GlcA. Thus, the structure of the major hexasaccharide in Fr.
b-19S and -19 was deduced respectively as follows: Fr. b-19S,
HexA-GlcN(NS,6S)-GlcA-GlcN(NS,6S)-GlcA-GlcN(NS,6S);
Fr. b-19,
HexA(2S)-GlcN(NS,6S)-GlcA-GlcN(NS,6S)-GlcA-GlcN(NS,6S). HexA(2S)-anMan(6S) on HPLC. Deaminative cleavage
products of this fraction were not further analyzed because the
internal uronic acid residues were easily identified as IdoA(2S) and
nonsulfated GlcA by 500-MHz H NMR analysis as described
below.500-MHz
All the
individual hexasaccharides were analyzed by 500-MHz H NMR AnalysisH NMR
to confirm the structures proposed above. 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 (Yamada et
al., 1993) and heparan sulfate (Sugahara et al., 1994).
The NMR data obtained in this study for the hexasaccharides are
summarized in Table 2.
H NMR spectroscopy based upon the chemical shifts of the
anomeric proton signals and the coupling constants J
. Anomeric proton signals of an IdoA and a
GlcA residue in heparin/heparan sulfate oligosaccharides are
observed at around 5.2-5.0 and 4.7-4.5, respectively
(Merchant et al., 1985; Yamada et al., 1995). The
coupling constants J of IdoA and GlcA
in heparin/heparan sulfate oligosaccharides are approximately 3.0 and
8.0 Hz, respectively (Horne and Gettins, 1992; Yamada et al.,
1995). In the spectrum of Fr. b-24, two internal uronic acid residues
were identified as IdoA and GlcA based on the chemical shifts of the
anomeric proton signals, at 5.184 and 4.576 (Fig. 5), and
the coupling constants J, 3.0 and 7.5 Hz,
respectively. The chemical shifts of H-1 and H-2 of the IdoA residue
were shifted downfield by approximately 0.2 and 0.6 ppm, respectively,
when compared with those of the nonsulfated IdoA residue of the
tetrasaccharides isolated from bovine kidney heparan sulfate (Sugahara et al., 1994), supporting the 2-sulfation of IdoA-4 of the
compound in Fr. b-24 (Yamada et al., 1994). Based upon these
NMR data and the sequential arrangement of the disaccharide units
determined by enzymatic analysis, the following structure is proposed
for the major compound in this fraction: Fr. b-24,
HexA(2S)-GlcN(NS,6S)-IdoA(2S)-GlcNAc(6S)-GlcA-GlcN(NS,6S).
H NMR
spectrum of the structure in Fr. b-24 recorded in HO. The numbers and letters in the spectrum refer to the corresponding residues in the
structure.
5.024 and 4.566) of the anomeric proton signals, respectively.
Analysis of the deamination products showed that the IdoA residue was
located at position 4, suggesting in turn that the GlcA residue is
located at position 2. Thus, the following structure is proposed for
the major compound in this fraction: Fr. b-15,
HexA(2S)-GlcN(NS,6S)-IdoA-GlcNAc(6S)-GlcA-GlcN(NS,6S).
HexA(2S)1-4GlcN(NS,6S)1-4IdoA/GlcA1-4GlcN(6S)1-4GlcA1-4GlcN(NS)
with one or two additional sulfate groups on GlcN-1, GlcN-3, and/or
IdoA-4. Two of these, Fr. b-15 and -22, have been isolated previously
(Linhardt et al., 1992), whereas the other three (Fr. b-19,
-20, and -24) were isolated for the first time as discrete structures
in this study. All the hexasaccharides contain the common trisulfated
disaccharide unit on their nonreducing sides and the GlcN(NS)
residue at their reducing termini, reflecting the substrate specificity
of heparinase used for digestion of the starting heparin. These
structural features are in good agreement with the established
specificity of heparinase that cleaves the glucosaminidic linkage in
the GlcN(NS)1-4IdoA(2S) sequence in a polymer
(Linker and Hovingh, 1984; Merchant et al., 1985) and that in
the
GlcN(NS)1-4IdoA(2S)1-4GlcN(NS,6S)
sequence in small oligosaccharides (Yamada et al., 1994,
1995).1-4GlcA1-4GlcN(NS,3S)1-4IdoA(2S)1-4GlcN(NS,6S)
(Lindahl et al., 1983) and
GlcA1-4GlcN(NS)1-4HexA1-4GlcN(NS)1-4IdoA(2S)
(Maccarana et al., 1993), respectively. The oligosaccharide
sequences for high affinity binding to acidic fibroblast growth factor,
fibroblast growth factor 4, and hepatocyte growth factor have been
partially characterized (Bârzu et al.,
1989; Ishihara, 1994; Guimond et al., 1993; Lyon et
al., 1994), but the essential sulfate groups in these sequences
have not yet been identified.1-4GlcN(NS,6S). The isolated
hexasaccharides are not expected to contain the functional domains for
binding to basic fibroblast growth factor. The hexasaccharides, except
for that in Fr. b-24, do not have an IdoA(2S) residue essential for
binding to basic fibroblast growth factor. Although the hexasaccharide
in Fr. b-24 contains an IdoA(2S) residue in its sequence, it lacks the
disaccharide extension GlcA1-4GlcN(NS) of the basic
fibroblast growth factor binding sequence on the nonreducing side. It
remains to be determined whether the binding domains to the other
growth factors or biologically active proteins are embedded in the
isolated hexasaccharides.
)HexA or
HexA,
4-deoxy--L-threo-hex-4-enepyranosyluronic acid;
anMan, 2,5-anhydromannitol; DiHS-0S,
HexA(1-4)GlcNAc; DiHS-6S,
HexA(1-4)GlcNAc(6-sulfate); DiHS-NS,
HexA(1-4)GlcN(N-sulfate);
DiHS-diS,
HexA(1-4)GlcN(N,6-disulfate);
DiHS-diS,
HexA(2-sulfate)(1-4)GlcN(N-sulfate);
DiHS-diS,
HexA(2-sulfate)(1-4)GlcNAc(6-sulfate);
DiHS-triS,
HexA(2-sulfate)(1-4)GlcN(N,6-disulfate); NS, 2-N-sulfate; 2S, 2-O-sulfate; 3S,
3-O-sulfate; 6S, 6-O-sulfate; ATIII, antithrombin
III, Fr., fraction(s).
We thank Dr. Makiko Sugiura (Kobe Pharmaceutical
University) for recording the NMR spectra and Nariko Fujikawa for
excellent technical assistance.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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