(Received for publication, April 18, 1995; and in revised form, July 18, 1995)
From the
Previously we isolated a tetrasaccharide-serine and a
hexasaccharide-serine from the carbohydrate-protein linkage region of
porcine intestinal heparin after digestion with a mixture of Flavobacterium heparinase and heparitinases I and II
(Sugahara, K., Yamada, S., Yoshida, K., de Waard, P., and Vliegenthart,
J.F.G.(1992) J. Biol. Chem. 267, 1528-1533). In this
study four longer carbohydrate sequences (I-IV) attached to Ser
or a dipeptide (Ser-Gly or Gly-Ser), which accounted for at least 18.2%
of the total linkage region, were isolated from the same heparin
preparation after digestion with heparinase only. IV was successfully
isolated only after subsequent digestion with glycuronate-2-sulfatase.
Their structures were determined by chemical and enzymatic analyses and H NMR spectroscopy and found to be the following octa- and
decasaccharide sequences attached to Ser in a molar ratio of
1.1:2.3:1.0:1.3:
HexA(2S)
1-4GlcN(NS,6S)
1-4GlcA
1-4GlcNAc
1-4GlcA
1-3Gal
1-3Gal
1-4Xyl
1-O-Ser
(I),
HexA(2S)
1-4GlcN(NS,6S)
1-4IdoA
1-4GlcNAc
1-4GlcA
1-3Gal
1-3Gal
1-4Xyl
1-O-Ser
(II),
HexA(2S)
1-4GlcN(NS,6S)
1-4IdoA
1-4GlcNAc
1-4GlcA
1-4GlcNAc
1-4GlcA
1-3Gal
1-3Gal
1-4Xyl
1-O-Ser
(III),
HexA
1-4GlcN(NS,6S)
1-4IdoA
1-4GlcNAc(6S)
1-4GlcA
1-3Gal
1-3Gal
1-4Xyl
1-O-Ser
(IV) (
HexA, GlcA, IdoA, and GlcN represent 4,5-unsaturated
hexuronic acid, D-glucuronic acid, L-iduronic acid,
and D-glucosamine, whereas 2S, 6S, and NS stand for
2-sulfate, 6-sulfate, and N-sulfate, respectively). I and II
contained 1 mol of Gly in addition to Ser. The four structures indicate
that sulfation in heparin chains takes place on the monosaccharide
residues located in closer vicinity to the core protein than found for
heparan sulfate chains and that there exist at least several heparin
subclass chains with different linkage region structures. The
significance of the isolated structures is discussed in relation to the
biological functions and the biosynthetic mechanisms of heparin.
Heparin exerts a variety of 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 acidic and basic fibroblast growth factors (Maciag et al., 1984; Shing et al., 1984; Klagsbrun and Shing, 1985). Some of these activities seem to reside within the complex fine structure of heparin. It is generally accepted that heparin expresses most of these activities by mimicking in vitro the physiological activities of heparan sulfate through its structure, similar to that of heparan sulfate. However, the structure-function relationships of heparin/heparan sulfate are not fully understood.
The basic polymeric common structure of heparin
and heparan sulfate is an alternating repeat sequence of
4GlcA
/IdoA
1
4GlcN
1
, (
)which
can be variably sulfated (for reviews see
Rodén(1980), Gallagher and Lyon(1989), and
Lindahl(1989)). Heparin contains more sulfate and IdoA but less N-acetyl groups and GlcA as compared with heparan sulfate.
Sulfate groups can be located at C-2 of hexuronic acid and C-2, C-3,
and/or C-6 of glucosamine residue and add the structural complexity to
the carbohydrate backbone to form various active domain structures
responsible for a number of biological activities. Recent structural
studies of the binding domains to antithrombin III (for review see
Lindahl(1989)) and basic fibroblast growth factor (Habuchi et
al., 1992; Turnbull et al., 1992; Tyrrell et
al., 1993; Maccarana et al., 1993) are the best known
examples showing the relationships between the fine structure and
biological functions.
Heparin and heparan sulfate are synthesized on
the specific serine residues of the core polypeptides through the
unique carbohydrate-protein linkage region,
-3Gal1-3Gal
1-4Xyl
1-O-Ser, which is
also shared by chondroitin sulfate and dermatan sulfate. It has not
been clarified yet how these glycosaminoglycans diverge in biosynthesis
into different structures from the same trisaccharide sequence. The
biosynthetic sorting mechanism of glucosaminoglycans (heparin/heparan
sulfate) and galactosaminoglycans (chondroitin sulfate and dermatan
sulfate) has been an enigma. Although unique modifications by
phosphorylation and sulfation of the linkage trisaccharide sequence
have been demonstrated (Oegema et al., 1988; Fransson et
al., 1985; Sugahara et al., 1988, 1991, 1992b; de Waard et al., 1992), no evidence has been presented for the
involvement of these modifications in the biosynthetic sorting
mechanism. The importance of the amino acids near the heparan sulfate
attachment site has also been pointed out (Zhang and Esko, 1994), but
whether peptide sequences are the primary determinants for heparan
sulfate synthesis remains to be determined. Differences between
biosynthesis of heparin and heparan sulfate are not well understood
either. Although they share a number of common structural features,
there are several structural differences. Heparan sulfate has a long
nonsulfated sequence consisting of at least eight repeating units
(-4GlcA
1-4GlcNAc
1-)
in the vicinity of the
linkage region (For review see Gallagher and Lyon(1989); Lyon et
al.(1994)), whereas heparin has a shorter nonsulfated sequence and
appears to be modified by sulfation near the linkage region (For review
see Lindahl(1989); Rosenfeld and Danishefsky(1988); Sugahara et
al. (1992a)). However, the exact modified structure of this region
of a heparin chain has not been investigated in detail yet.
We have
been analyzing the structure of the carbohydrate-protein linkage region
of various sulfated glycosaminoglycans to investigate the
structure-function relationships and the biosynthetic mechanisms of
these glycosaminoglycans (Sugahara et al., 1988, 1991, 1992a,
1992b, 1994, 1995; de Waard et al., 1992). Previously we
isolated two glycoserines,
HexA
1-3Gal
1-3Gal
1-4Xyl
1-O-Ser
(glycoserine I) and
HexA
1-4GlcNAc(6S)
1-4GlcA
1-3Gal
1-3Gal
1-4Xyl
1-O-Ser
(glycoserine II) from the linkage region of heparin after exhaustive
digestion with a mixture of heparinase and heparitinases I and II
(Sugahara et al., 1992a). In the present study we isolated and
characterized larger glycoserines and glycopeptides after digestion
with only heparinase in order to investigate the structure beyond the
above tetra- and hexasaccharide sequences.
Figure 1:
Fractionation
of the heparinase digest by gel filtration. Purified stage 14 heparin
was exhaustively digested by heparinase and gel-filtrated on a column
of Cellulofine GCL-90-m. Fractions were monitored by absorption at 232
nm () and the carbazole reaction (
) and pooled as
indicated.
NMR spectra of fractions b-5 and b-6 were recorded
on a Bruker AMX-500 or AMX-600 spectrometer (Department of NMR
spectroscopy, Utrecht University) operated at a probe temperature of
292, 295, or 300 K. One-dimensional spectra and a double
quantum-filtered correlation spectroscopy spectrum of fraction b-5 were recorded as described previously (Piantini et
al., 1982; Derome and Williamson, 1990; Hård et
al., 1992). Two-dimensional NOESY experiments were performed with
a mixing time of 150-200 ms (Jeener et al., 1979).
Two-dimensional TOCSY spectra were recorded using a clean-MLEV-17
spin-lock pulse sequence of 100 ms, preceded by a 2.5-ms trim-pulse
(Braunschweiler and Ernst, 1983; Bax and Davis, 1985; Griesinger et
al., 1988). In all two-dimensional experiments the HOH
resonance was presaturated during the relaxation delay and additionally
during the NOE-mixing time in NOESY-experiments. Phase-sensitive
detection was achieved by the time-proportional phase increment method
(Marion and Wüthrich, 1983). Two-dimensional
spectra were recorded with 300-512 t
experiments, and 80-160 free induction decays of 2048 or
4096 data points were collected per t
increment.
Data sets were processed using the Bruker UXNMR software package. In
short, time domain data were zero-filled, multiplied by a phase-shifted
sine bell function, and after Fourier transformation base
line-corrected with fifth order polynomal fits.
NMR spectra of fraction b-10S-II were recorded on a Varian VXR-500 spectrometer (Kobe Pharmaceutical University) at a probe temperature of 299 K as previously reported (Yamada et al., 1992).
Fraction b was subfractionated by HPLC
on an amine-bound silica column, and peaks were designated fractions b-1 to b-26 as shown in Fig. 2. Nine major fractions,
5, 6, 10, 15, 17, 19, 20, 22, and 24, were purified by
rechromatography, and the amino acid analysis was performed for each
fraction. Based upon the detected serine, approximately 41, 12, and 28%
of the linkage region in fraction b was recovered in fractions b-5, -6, and -10, which accounted for 3.6, 1.1, and 2.7% of
the applied HexA, respectively. Preparative HPLC yielded 239, 71,
and 166 nmol (as Ser) of fractions b-5, -6, and -10,
respectively, per 100 mg of the purified heparin. No appreciable amount
of serine was recovered in the other peaks, most of which contained
unsaturated hexasaccharides derived from the repeating disaccharide
region as judged from the ratio of uronic acid and GlcN to
HexA.
Structural studies of these hexasaccharides are in progress.
Figure 2:
Subfractionation of fraction b by HPLC on
an amine-bound silica column. Oligosaccharide fraction b
obtained by gel filtration was subfractionated into fractions b-1 to b-26 on an amine-bound silica column using a linear
gradient of NaHPO
from 0.2 to 1.0 M over 90 min. Fractions b-5, b-6, and b-10, which
contained Ser, are marked by asterisks. Fraction b-10
was digested by 2-sulfatase, and the digest, designated fraction b-10S, was separated into three subfractions, b-10S-I, -II, and -III, by HPLC as shown in the inset using the same conditions. The elution position of the parent
fraction b-10 is indicated by an arrow.
The
separated fractions b-5, -6, and -10 gave a single symmetrical
peak upon HPLC. In the preliminary experiments their sensitivity to
2-sulfatase was examined to evaluate their purity and structural
characteristics. 2-Sulfatase acts only on the 4,5-unsaturated hexuronic
acid 2-sulfate structure at the nonreducing end of a saccharide chain
(McLean et al., 1984). Fractions b-5, -6, and -10
were all sensitive to the enzyme, as expected from the linkage
specificity of heparinase (see above), indicating that the major
compound in each fraction has a sulfate group at the C-2 position of
the HexA (not shown). After 2-sulfatase digestion, fractions b-5 and -6 gave a single peak, which eluted approximately 10
min earlier than the corresponding parent compound on HPLC (not shown),
supporting the homogeneity of fractions b-5 and b-6. In
contrast, a 2-sulfatase digest of fraction b-10, designated
fraction b-10S, gave three peaks, fractions b-10S-I,
-II and -III, in a molar ratio of 21:58:21, which eluted approximately
8 min earlier than the parent fraction (Fig. 2, inset),
indicating that fraction b-10 was a mixture of at least three
components. Since it was not possible to resolve fraction b-10
into its subcomponents preparatively, it was first digested with
2-sulfatase, and then the digest was fractionated by HPLC. The major
peak b-10S-II, the yield of which was about 90 nmol from 100
mg of the starting heparin, was isolated and subjected to structural
analysis. Fractions 10S-I and -III were not analyzed due to their
limited amounts.
Figure 3: Capillary electrophoresis of the isolated linkage region fractions. Fraction b-5 (A), b-6 (B) or b-10S-II (C) was subjected to electrophoresis as described under ``Experimental Procedures.'' The peak at around 4 min is presumably due to a non-carbohydrate contaminant.
As shown in Table 1, chemical analysis showed that fraction b-5 contained HexA, HexA, GlcN, Ser, and Gly in a molar
ratio of 1.00:1.73:1.96:1.11:0.93. The disaccharide analysis of
fraction b-5, carried out by exhaustive heparitinase I
digestion followed by HPLC on an amine-bound silica column, gave rise
to equimolar amounts of
DiHS-0S,
DiHS-triS, and a component
that eluted shortly before
DiHS-0S, corresponding to glycoserine I
derived from the carbohydrate-protein linkage region of heparin
(Sugahara et al., 1992a) (Fig. 4B). These
results indicate that fraction b-5 most likely contained two
isomeric octasaccharide-peptides, each of which was composed of 1 mol
each of the nonsulfated and the trisulfated disaccharide units and
HexA1-3Gal
1-3Gal
1-4Xyl
1-O-Ser
(Gly).
Figure 4:
HPLC analysis of the enzyme digests of
fraction b-5. Fraction b-5 was digested by heparitinase I (panelB) or by heparitinase II alone (panelC) as described under ``Experimental
Procedures.'' The digest was subjected to HPLC on an amine-bound
silica column using a linear gradient of NaHPO
from 16 to 530 mM over 60 min. Elution positions of the
standard disaccharides isolated from heparin/heparan sulfate are
indicated in panelA. 1,
DiHS-0S; 2,
DiHS-6S; 3,
DiHS-NS; 4,
DiHS-diS
; 5,
DiHS-diS
; 6,
DiHS-triS. Glycoserine I is the tetrasaccharide-serine
HexA
1-3Gal
1-3Gal
1-4Xyl
1-O-Ser
reported previously (Sugahara et al., 1992a). The peak marked
by an asterisk at around 35 min is often observed upon high
sensitivity analysis and is due to an unknown substance eluted from the
column resin.
Heparitinase II digestion of fraction b-5 yielded
equimolar amounts of two components, namely DiHS-triS and a
component that eluted near the elution position of the nonsulfated
hexasaccharide,
HexA
1-4GlcNAc
1-4GlcA
1-3Gal
1-3Gal
1-4Xyl,
derived from the carbohydrate-protein linkage region of bovine kidney
heparan sulfate (Sugahara et al., 1994) (Fig. 4C). The results are summarized in Table 2and are consistent with the previous observation that the
hexasaccharide structure is resistant to heparitinase II (Fig. 5) (Sugahara et al., 1994). Together the above
results indicate that the two components in fraction b-5 share
the common structure,
HexA(2S)
1-4GlcN(NS,6S)
1-4HexA1-4GlcNAc
1-4HexA1-3Gal
1-3Gal
1-4Xyl
1-O-Ser(Gly)
with only a subtle difference between them.
Figure 5:
Specificities of heparitinases I and II.
Enzymatic action of heparitinases I and II on the isolated linkage
compounds are shown by arrows with the Roman numerals I and IIabove and below each structure,
respectively. A thickarrow shows a preference for
the indicated linkage over the other(s). a, the
octasaccharide-peptides in fractions b-5-I and b-5-II; b, the decasaccharide-serine in fraction b-6; c, the octasaccharide-serine in fraction b-10S-II. *,
it is noted that this linkage in the octasaccharide-serine in fraction b-10S-II was cleaved by heparitinase I, whereas the
corresponding linkage in glycoserine II
HexA-GlcNAc(6S)-GlcA-Gal-Gal-Xyl-Ser was not (Sugahara et al. (1992a), and see
``Discussion'').
Heparitinase II digestion of fraction b-6
produced mainly two components (Table 2), DiHS-triS and one
that eluted approximately 6 min later than
HexA
1-4GlcNAc
1-4GlcA
1-3Gal
1-3Gal
1-4Xyl
derived from heparan sulfate (Sugahara et al., 1994), and
which therefore was assumed to be a nonsulfated octasaccharide-serine
HexA
1-4GlcNAc
1-4HexA1-4GlcNAc
1-4HexA1-3Gal
1-3Gal
1-4Xyl
1-O-Ser.
When exhaustively digested using 6 times the amount of enzyme used
under the standard conditions, fraction b-6 gave rise to
equimolar amounts of
DiHS-0S,
DiHS-triS, and a presumably
nonsulfated hexasaccharide-serine (data not shown). These results
indicate that the major compound in fraction b-6 is a
trisulfated decasaccharide-serine
HexA(2S)
1-4GlcN(NS,6S)
1-4HexA1-4GlcNAc
1-4HexA1-4GlcNAc
1-4HexA1-3Gal
1-3Gal
1-4Xyl
1-O-Ser.
When digested with heparitinase II, this compound
yielded equimolar amounts of DiHS-diS
and the
component eluted at the position of glycoserine II (Sugahara et
al., 1992a) (Table 2), indicating that the disulfated
disaccharide unit,
DiHS-diS
, was located at the
nonreducing terminus. Thus, the structure of the compound in fraction b-10S-II is
HexA
1-4GlcN(NS,6S)
1-4HexA1-4GlcNAc(6S)
1-4HexA1-3Gal
1-3Gal
1-4Xyl
1-O-Ser.
Figure 6:
One-dimensional 600-MHz H NMR
spectra of the structures in fractions b-5 and b-6, recorded in
H
0 at 300 K. The Arabicnumerals in the spectra refer to the corresponding residues in the
structures. A, fraction b-5; B, fraction b-6.
Figure 7: Two-dimensional TOCSY (A) and NOESY (B) spectra of the structures in fraction b-5 recorded at 300 and 292 K, respectively. Anomeric protons are indicated by verticallines, and the number at the top corresponds to the monosaccharide number. Resonances of the core region overlap and are indicated in italics. Cross-peaks on the H-1 tracks are labeled by proton number in the TOCSY spectrum. In the NOESY spectrum only trans-glycosidic NOEs on the H-1 tracks are labeled. The doubledigitnumber represents the monosaccharide unit followed by the proton number.
The similar intensities of the resonances at 5.377,
5.335, and
4.948 suggest that they belong to the major
compound (b-5-I). The resonance at
5.377 stems from the anomeric
proton of an N,6-disulfated glucosamine residue (Fig. 6A). This assignment is based on the upfield
shift out of the bulk region of the H-2 resonance (
3.279)
reflecting N-sulfation (Horne and Gettins, 1991; Yamada et
al., 1994), and the downfield shifts out of the bulk region of the
H-5 (
3.985) and the hydroxymethyl-proton signals (
4.203,
4.353), indicating sulfation at the 6-position (Fig. 6A) (Horne
and Gettins, 1991; Sugahara et al., 1992a; Yamada et
al., 1994). The presence of an NAc resonance (
2.034), and
the characteristic TOCSY pattern observed on the H-1 track (Fig. 7A) show that the signal at
5.335 stems
from the anomeric proton of a nonsulfated glucosamine residue (Sugahara et al., 1994). The presence of an iduronic acid residue is
deduced from the small coupling constant (2 Hz) observed on the signal
at
4.948 and its characteristic TOCSY pattern observed on the H-1
track (Fig. 7A, Table 3) (Sugahara et
al., 1994). The sequence of the major compound b-5-I is
unequivocally established by NOEs between
HexA(2S)-8 H-1
and GlcN(NS,6S)-7 H-4 and H-6, between
GlcN(NS,6S)-7 H-1 and IdoA-6 H-3 and H-4,
between IdoA-6 H-1 and GlcNAc-5 H-4, and between
GlcNAc-5 H-1 and GlcA-4 H-4 (Fig. 7B).
The minor compound (b-5-II) also contains an N,6-disulfated
and a nonsulfated glucosamine residue as judged from the TOCSY patterns
on their H-1 tracks at 5.566 and
5.359, respectively (Fig. 7A) (Sugahara et al., 1994; Yamada et al., 1994). NOEs between GlcNAc H-1 and GlcA-4 H-4
and the one between
HexA(2S)-8 H-1 and GlcN(NS,6S)
H-4 and H-6, locate these glucosamine residues at positions 5 and 7 in the oligosaccharide sequence. The resonance at
4.510 ppm, displaying a coupling constant of 8 Hz, and its
characteristic TOCSY-pattern on the H-1 track show the presence of a
GlcA residue (Sugahara et al., 1994; Yamada et al.,
1994). The NOE between GlcN(NS,6S)-7 H-1 and GlcA H-4
and the one between GlcA H-1 and H-4 of GlcNAc-5 locate this
residue at position 6 in the sequence. The resonance of the
anomeric proton of GlcN(NS,6S)-7 (
5.566) has
shifted downfield by
0.19 with respect to the corresponding
signal of the major compound, in agreement with the previously reported
observation that the GlcN(NS,6S) H-1 reports on the identity
of the preceding hexuronic acid (Horne and Gettins, 1991). In
conclusion, the two compounds in fraction b-5 are as follows: b-5-I
(
HexA(2S)
1-4GlcN(NS,6S)
1-4IdoA
1-4GlcNAc
1-4GlcA
1-3Gal
1-3Gal
1-4Xyl
1-O-Ser)
and b-5-II
(
HexA(2S)
1-4GlcN(NS,6S)
1-4GlcA
1-4GlcNAc
1-4GlcA
1-3Gal
1-3Gal
1-4Xyl
1-O-Ser).
The one-dimensional H NMR spectrum of fraction b-6 is shown in Fig. 6B. The TOCSY spectrum of
fraction b-6 (not shown) is very similar to that of fraction b-5, especially with respect to the signals stemming from the
core region, GlcA-Gal-Gal-Xyl-O-Ser and those of the terminal
HexA(2S) residue (Table 3), showing the presence of these
structural elements (van Halbeek et al., 1982; Horne and
Gettins, 1991; Sugahara et al., 1992a; Yamada et al.,
1994). Characteristic TOCSY patterns on the H-1 tracks at
5.364,
5.375, and
5.347 led to identification of one N,6-disulfated and two nonsulfated glucosamine residues
(Sugahara et al., 1994; Yamada et al., 1994).
Furthermore, both an IdoA and a GlcA residue are present based on their
characteristic chemical shifts (Sugahara et al., 1994; Yamada et al., 1994) determined from the TOCSY pattern and
intraresidue NOEs. The sequence of this decasaccharide was elucidated
by a series of trans-glycosidic H-1 to H-4 NOEs, an NOE between
HexA H-1 and H-6 of GlcN(NS,6S), and one between
GlcN(NS,6S) H-1 and IdoA H-3, showing that fraction b-6 contains the following structure: b-6,
HexA(2S)
1-4GlcN(NS,6S)
1-4IdoA
1-4GlcNAc
1-4GlcA
1-4GlcNAc
1-4GlcA
1-3Gal
1-3Gal
1-4Xyl
1-O-Ser.
Fraction b-10S-II was analyzed by one-dimensional, TOCSY,
and correlation spectroscopy spectra (not shown), and the NMR data are
summarized in Table 3. Based on these NMR data the following
trisulfated octasaccharide-serine is proposed for the structure of the
compound in fraction b-10S-II: b-10S-II,
HexA
1-4GlcN(NS,6S)
1-4IdoA
1-4GlcNAc(6S)
1-4GlcA
1-3Gal
1-3Gal
1-4Xyl
1-O-Ser.
Since fraction b-10S-II was isolated after 2-sulfatase
digestion as described above, the major compound in the parent fraction b-10 contains the following structure: b-10,
HexA(2S)
1-4GlcN(NS,6S)
1-4IdoA
1-4GlcNAc(6S)
1-4GlcA
1-3Gal
1-3Gal
1-4Xyl
1-O-Ser.
In this study we identified one deca- and three octasaccharide structures in the linkage-derived fractions b-5-I, -5-II, -6, and -10, which accounted for at least 7.4, 3.5, 3.2, and 4.1 mol % of the total linkage region of porcine intestinal heparin, respectively. Since they were prepared using only heparinase, they represent most likely the molecules from which glycoserines I (a tetrasaccharide-serine) and II (a hexasaccharide-serine) were previously produced by exhaustive digestion with a mixture of heparinase and heparitinases I and II (Sugahara et al., 1992a). Presumably, glycoserine I had been produced partly from the compounds corresponding to fractions b-5-I, b-5-II, and b-6, whereas glycoserine II had been produced at least from the compound corresponding to fraction b-10 (see Fig. 5for the enzyme specificities).
Although the majority of the linkage structures with even longer sequences were recovered in fraction a and remain to be investigated, the molecules isolated in this study contain hitherto unreported structural features in the extended region beyond the sequences found in glycoserines I and II. They share the trisulfated disaccharide unit in common at the nonreducing termini, indicating that the trisulfated unit characteristic of heparin begins emerging even in the second and the third disaccharide units from the carbohydrate attachment site. The GlcNAc in the first disaccharide unit can be 6-sulfated as in the compound in fraction b-10, but it cannot be N-sulfated. The sugar residue at the first uronic acid position is always GlcA but never IdoA in these four molecules, which is in contrast to the recent finding of both GlcA and IdoA at this position in dermatan sulfate from bovine aorta (Sugahara et al., 1995). The second and the third uronic acid could be IdoA when located adjacent to the trisulfated disaccharide unit as in the compounds in fractions b-5-I, b-10, and b-6. However, the uronic acid next to the trisulfated disaccharide unit was not always IdoA but could be GlcA as in the compound in fraction b-5-II. The above structural characteristics of these four molecules may have some implications in the expression of biological functions and in the biosynthetic mechanisms of heparin.
The trisulfated disaccharide unit characteristic of heparin and heparan sulfate were demonstrated to begin appearing nearer the linkage region in heparin as compared with heparan sulfate. A heparin chain with an IdoA-containing segment closer to the linkage region would be more flexible and mobile around the core protein due to the specific conformational properties of sulfated or nonsulfated iduronic acid, which appears to be present in dynamic equilibrium of different conformations (Casu, 1989). In contrast, heparan sulfate has a long nonsulfated stretch of more than eight repeating disaccharide units, which are assumed to contain only GlcA (Gallagher and Lyon, 1989; Lindblom et al., 1991; Lyon et al., 1994) and therefore would be rather rigid in the proximal portion to the linkage region but plastic in the distal portion.
The present study indicates that there are at least four subclasses of heparin chains different in structure of the linkage region and/or in length of the nonsulfated sequence proximal to the protein core. It is likely that there exist other subclass chains in fraction a, and it is possible that different chains have different patterns of modification. It remains to be determined whether biologically active domain structures such as the binding domains to the antithrombin III and basic fibroblast growth factor are found on a specific subclass chain and where along a heparin chain they are embedded. Since the linkage region is first constructed in biosynthesis, differences in the structure of the linkage region may influence that of the repeating disaccharide region to be synthesized thereafter. It should be noted that the anticoagulant-conferring area appears to occur about 20 disaccharide units away from the linkage region (Rosenfeld and Danishefsky, 1988). The observed heterogeneity in the linkage region also raises questions of whether the different chains are derived from different core proteins and whether they come from identical or different sites of a single core protein. Answers to these questions require further investigation.
The present work provided some useful
information about the substrate specificities of heparitinases I and
II, which are essential tools for structural studies of heparin/heparan
sulfate. Previously, heparitinase I was shown to cleave glucosaminidic
linkages bound to nonsulfated GlcA and IdoA except for two
glucosaminidic linkages: the one linked to the GlcA residue
substituting the 3-sulfated glucosamine residue in the antithrombin
III-binding sequence (Yamada et al., 1993) and the one linked
to the GlcA residue located between the Gal and the 6-sulfated GlcNAc
found in glycoserine II (Sugahara et al., 1992a). In this
study, however, all the glucosaminidic linkages in the four molecules
including the octasaccharide in fraction b-10S-II were cleaved
by this enzyme (Fig. 5c). It may be that the enzyme
acts on the glucosaminidic linkage in the sequence
-GlcNAc(6S)-GlcA-Gal- when it is located in an octasaccharide (b-10S-II) but not in a hexasaccharide (glycoserine II).
Digestability of the glucosaminidic linkages in fraction b-6
suggests some linkage preference of this enzyme. Although all of the
three glucosaminidic linkages were cleaved by the enzyme under harsh
conditions, only the middle linkage was cleaved under milder conditions
for partial digestion (see ``Experimental Procedures''),
yielding a tetrasaccharide and a linkage hexasaccharide-serine but no
disaccharides, as illustrated in Fig. 5b. The enzyme
appears to prefer the linkage between the two nonsulfated disaccharide
units to the other two, consistent with the notion that the enzyme acts
on the relatively low sulfated region of a heparan sulfate chain
(Linhardt et al., 1990). Heparitinase I digestion of fraction b-5 under limited conditions resulted in both di- and
tetrasaccharides, suggesting the comparable sensitivity of the two
glucosaminidic linkages to this enzyme as illustrated in Fig. 5a. Heparitinase II has been demonstrated to have
a broad specificity (Linhardt et al., 1990; Nader et
al., 1990; Yamada et al., 1994, 1995) acting on every
-glucosaminidic bond in heparin except for the two unique
hexosaminidic bonds: the one in the structure
-4GlcNAc(6S)
1-4GlcA
1-4GlcN(NS,3S,6S)
found within the antithrombin III-binding domain (Yamada et
al., 1993) and the one in the structure
-4HexA1-4GlcNAc
1-4GlcA
1-3Gal
1- of the
carbohydrate-protein linkage region (Fig. 5) (Sugahara et
al., 1994). In this study it was demonstrated that this enzyme
cleaves the hexosaminidic linkage adjacent to a trisulfated
disaccharide unit more preferentially than the one next to a
nonsulfated disaccharide unit (Fig. 5b).