(Received for publication, March 26, 1997, and in revised form, May 15, 1997)
From the Department of Biochemistry, Kobe Pharmaceutical
University, Higashinada-ku, Kobe 658, Japan and the
Department of Biochemistry, Imperial College,
London SW7 2AZ, United Kingdom
We previously isolated novel tetrasaccharides
containing 3-O-sulfated glucuronic acid from king crab
cartilage chondroitin sulfate K and demonstrated that the disaccharide
units containing 3-O-sulfated glucuronic acid were
decomposed by chondroitinase ABC digestion (Sugahara, K., Tanaka, Y.,
Yamada, S., Seno, N., Kitagawa, H., Haslam, S. M., Morris, H. R., and
Dell, A. (1996) J. Biol. Chem. 271, 26745-26754). The
findings indicated the necessity to re-evaluate the disaccharide
compositions of chondroitin sulfate preparations purified from other
biological sources and analyzed using the above enzyme. In this study,
to evaluate squid cartilage chondroitin sulfate E a series of
even-numbered oligosaccharides were isolated after exhaustive digestion
with sheep testicular hyaluronidase and subsequent fractionation by gel
chromatography. The tetrasaccharide fraction was subfractionated by
high performance liquid chromatography on an amine-bound silica column.
Systematic structural analysis of five major fractions, h,
l, m, n, and q, by fast
atom bombardment mass spectrometry, enzymatic digestions in conjunction
with capillary electrophoresis, and 500-MHz 1H NMR
spectroscopy revealed one disulfated, three trisulfated, and one
tetrasulfated tetrasaccharide structure: fraction h,
GlcA1-3GalNAc(4S)
1-4GlcA
1-3GalNAc(4S); fraction
l,
GlcA(3S)
1-3GalNAc(6S)
1-4GlcA
1-3GalNAc(4S); fraction m,
GlcA(3S)
1-3GalNAc(4S)
1-4GlcA
1-3GalNAc(4S); fraction
n,
GlcA
1-3GalNAc(4S,6S)
1-4GlcA
1-3GalNAc(4S); and
fraction q,
GlcA(3S)
1-3GalNAc(4S,6S)
1-4GlcA
1-3GalNAc(4S), where 3S, 4S,
and 6S represent 3-O-, 4-O- and
6-O-sulfate, respectively. The structures found in
fractions h and m as well as the unsaturated counterpart of that found in fraction n have been reported,
whereas those in fractions l and q are novel in
that they contained unusual disulfated and trisulfated disaccharide
units where GlcA(3S) is directly linked to GalNAc(6S) and
GalNAc(4S,6S), respectively. These novel tetrasaccharide sequences are
distinct from those found in other chondroitin sulfate isoforms and may
play key roles in the biological functions and activities of
chondroitin sulfate E not only from squid cartilage but also from
mammalian cells and tissues.
Chondroitin sulfate (CS)1 proteoglycans are ubiquitous components of the extracellular matrix of connective tissues and are also found at the surface of many cell types and in intracellular secretory granules. They play several key roles in the normal physiology of animal tissues, regulating cell migration, cell recognition, and tissue morphogenesis (for reviews, see Refs. 1-3). Immunological studies using CS-specific antibodies have revealed the developmentally regulated expression of the epitopes in the rodent fetus and in the rat central nervous system (for reviews, see Refs. 4 and 5). Some CS epitopes are distributed differentially in distinct tissues and in functionally distinct domains within these tissues (6). These observations suggest that CS chains differing in degree and profile of sulfation perform distinct functions in development.
Oversulfated CS isoforms contain rare structural building units; thus,
they may form domain structures that interact specifically with other
molecules. Oversulfated CSs were originally discovered in cartilages of
shark (7) and invertebrates such as squid and horseshoe crab (king
crab) (Ref. 8; for a review, see Ref. 9). Oversulfated CS isoforms from
the shark, squid, and horseshoe crab cartilage are characterized by the
distinct disulfated disaccharide units,
GlcA(2S)1-3GalNAc(6S), GlcA
1-3GalNAc(4S,6S), and
GlcA(3S)
1-3GalNAc(4S), where 2S, 3S, 4S, and 6S represent
2-O-, 3-O-, 4-O-, and
6-O-sulfate and are designated as CS-D, CS-E, and CS-K,
respectively (10-12). Oversulfated CSs are not limited to
invertebrates or marine vertebrates and have been identified in various
tissues and cells from higher land-dwelling vertebrates. CS-D and CS-E
have been found in human rib cartilage in small proportions (13).
Another disaccharide unit, IdceA-GalNAc(4S,6S), has also been
identified in dermatan sulfate (DS) from hagfish notochord (14),
mammalian liver (15), and bovine kidney (16). This type of DS is
designated as DS-E in this article.
Although the specific physiological functions of oversulfated CS isoforms have not been clarified, they show differentiation-associated expression. Elevated CS-E synthesis has been observed in the terminal differentiation of embryonic chick chondrocytes (17). Oversulfated CS isoforms are differentiation markers for different mast cell (MC) subsets (Refs. 18 and 19; for a review, see Ref. 20). While heparin is found in the secretory granules of "connective tissue" type MC, as typified by the "serosal" MC (21), CS-E or DS-E is usually found in those of "mucosal" MC, "bone marrow-derived" MC, and related cells (22-26). A proposed role for secretory granule proteoglycans involves their concentration and the stabilization of secretory granule enzymes (27). Close association between histamine and CS-E proteoglycan release has also been observed in human colonic mucosa, which is assumed to contain mast cells (24). DS-E has been demonstrated in guinea pig peritoneal macrophages (28). Kolset et al. (29, 30) showed CS-E and DS-E in human monocyte-derived macrophages as well as CS-E or CS-D in mature peritoneal macrophages and suggested that CS-E could be a marker for differentiation of monocytes into macrophages. Recently, Edwards et al. (31) demonstrated that human macrophages exhibited the differentiation-associated expression of CS-E or DS-E, which bound to lipoprotein lipase, and hypothesized that the marked increase in the synthesis of the surface proteoglycans containing CS-E/DS-E increased the macrophage uptake of plasma low density lipoprotein, i.e. the atherogenic potential of these cells.
CS-E exhibits biological activities in vitro. An in
vitro anticoagulant property has been reported for squid cartilage
CS-E (32). This property is mediated primarily by acceleration of heparin cofactor II interaction with thrombin (33). Thrombomodulin, which is an integral membrane protein with anticoagulant activity, bears a single CS or DS chain with a unique
GalNAc(4S,6S)1-4GlcA
1-3GalNAc(4S,6S) sequence of a CS-E type at
the nonreducing terminus (34). The CS or DS chain is involved in its
anticoagulant activity through direct binding to thrombin and through
activating protein C (for a review, see Ref. 35). More recently,
monocyte procoagulant activities of purified coagulation factors, VIIIa
and IXa, were reported to be specifically inhibited on monocyte
surfaces by size-defined CS-E oligosaccharides, indicating that the
CS-E expressed on monocyte membranes inhibits factor X-activating
reactions in the intrinsic pathway (36). Squid cartilage CS-E and
murine bone marrow-derived MC CS-E (or DS-E) were noted to specifically inhibit the function of activated properdin in the alternative complement pathway (37). The former activates the contact (Hageman factor) system of plasma in vitro as does heparin (38).
In view of the importance of CS chains in various biological systems, we have been conducting systematic structural studies of various CS isoforms including CS-D, CS-E, and CS-K. Recent studies have revealed that the structural complexity and diversity of CS chains resulted from an extensive series of modifications by various sulfation reactions (39-43). Novel oligosaccharides that contained an unusual GlcA(3S) residue were isolated from CS-K of king crab cartilage (44, 45). To our surprise, when these oligosaccharides were digested with chondroitinase ABC, the resultant disaccharide units containing a GlcA(3S) residue were undetectable when monitored by UV-absorbance and were assumed to have been decomposed during the enzyme treatment. Thus, we reinvestigated squid cartilage CS-E that had been characterized by chondroitinase ABC digestion (11, 46) to see if it contained GlcA(3S).
In this study, five tetrasaccharides were isolated after digestion of a commercial squid cartilage CS-E preparation with hyaluronidase instead of chondroitinase ABC to prevent degradation of possible structures that may contain GlcA(3S) residues. The analysis by 500-MHz 1H NMR spectroscopy in conjunction with fast atom bombardment-mass spectrometry (FAB-MS) unambiguously demonstrated GlcA(3S) residues in some of the major tetrasaccharide components. Preliminary findings were reported in abstract form (47).
CS isoform preparations (super special grade),
including squid cartilage CS-E, whale cartilage CS-A, shark cartilage
CS-C, and shark cartilage CS-D, were purchased from Seikagaku Corp., Tokyo, Japan. A king crab cartilage CS-K peptide preparation (48) was a
gift from the late Dr. N. Seno (Ochanomizu University, Tokyo). Six
unsaturated standard CS disaccharides, chondroitinase ABC (EC 4.2.2.4),
and AC-II (EC 4.2.2.5) were purchased from Seikagaku Corp.. Sheep
testicular hyaluronidase (EC 3.2.1.35) was obtained from Sigma. Bio-Gel
P-10 and Sephadex G-25 (fine) were obtained from Bio-Rad and Pharmacia
Biotech Inc., respectively. 2-Aminoacridone (AMAC) and
NaCNBH3 were obtained from LAMBDA Corp. (Graz, Austria) and
Aldrich, respectively. The following tetrasaccharides were prepared
from shark cartilage CS-D as described (41):
GlcA1-3GalNAc(6S)
1-4GlcA
1-3GalNAc(6S), GlcA
1-3GalNAc(4S)
1-4GlcA
1-3GalNAc(4S),
GlcA
1-3GalNAc(6S)
1-4GlcA
1-3GalNAc(4S), GlcA(2S)
1-3GalNAc(6S)
1-4GlcA
1-3GalNAc(6S),
GlcA
1-3Gal NAc(4S)
1-4GlcA(2S)
1-3GalNAc(6S), and
GlcA(2S)
1-3GalNAc(6S)
1-4GlcA
1-3GalNAc(4S).
GlcA(3S)
1-3GalNAc(4S)
1-4GlcA
1-3GalNAc(4S) and
GlcA(3S)
1-3GalNAc(4S)
1-4GlcA(3S)
1-3Gal NAc(4S) were
prepared from king crab cartilage as reported (44).
Various
CS isoform preparations (CS-A, CS-C, CS-D, CS-E, and CS-K) of 0.4 mg
each were digested with 50 mIU of chondroitinase ABC in a total volume
of 400 µl of Tris-HCl, pH 8.0, containing 0.06 M sodium
acetate at 37 °C. At appropriate time intervals, a 10-µl aliquot
of the incubation mixture was withdrawn and mixed with 0.01 M HCl to terminate the reaction. Unsaturated
oligosaccharide products were determined to monitor the reaction by
absorbance at 232 nm using the average millimolar absorption
coefficient of 5.5. The digestion rate was calculated based on the
amount of the resultant unsaturated oligosaccharides relative to the uronic acid value determined for each CS isoform by the carbazole reaction (49) (Fig. 1). Analysis of the chondroitinase ABC digest of
CS-E was carried out by HPLC on an amine-bound silica PA03 column to
determine the disaccharide composition as reported (50).
Preparation of Oligosaccharide Fractions
A commercial squid
cartilage CS-E (100 mg) was digested with 10 mg (approximately 15,000 National formulary units) of sheep testicular hyaluronidase in a total
volume of 2.0 ml of 50 mM sodium phosphate buffer, pH 6.0, containing 150 mM NaCl (1 National formulary unit
corresponds to the amount of the enzyme which hydrolyzes 74 µg of
hyaluronate/min) (51, 52). Digestion proceeded at 37 °C for 18 h, and then an additional 8 mg (6000 National formulary units) of the
enzyme was added and the incubation was continued for 7 h to
complete the digestion. Thereafter, the digest was mixed with 0.44 ml
of 30% trichloroacetic acid and centrifuged at 2500 rpm for 10 min.
The precipitate was washed with 0.5 ml of 5% trichloroacetic acid. The
combined supernatant obtained from the trichloroacetic acid
precipitation was extracted with ether, and the aqueous phase was
neutralized with 1 M Na2CO3. Then
the sample was applied to a Bio-Gel P-10 column (1.6 × 95 cm)
using 1 M NaCl, 10% ethanol as the eluent. Eluates were
monitored by absorbance at 210 nm, which is attributable mainly to
carbonyl groups. Fractions I-IX were separately pooled as indicated in Fig. 2, concentrated, desalted through a column (1.5 × 46 cm) of
Sephadex G-25 (fine) using distilled water as the eluent, and lyophilized. In this study, the smallest size fraction, fraction IX,
was subfractionated into fractions a-v as indicated in Fig. 3 by HPLC. HPLC was performed in an LC-10AS system (Shimadzu Co., Kyoto, Japan) using a linear NaH2PO4 gradient
from 16 to 798 mM over a 60-min period at a flow rate of
1.0 ml/min at room temperature (50). The eluates were monitored by
absorbance at 210 nm. Each peak was purified by rechromatography under
the same conditions as the first step and desalted as described above.
Each peak was quantified by the carbazole method, using GlcA as a
standard (49).
Digestion of the Isolated Oligosaccharides with Chondroitinase ABC or AC-II
Each isolated oligosaccharide (1 nmol as GlcA) was digested using 5 mIU of chondroitinase AC-II or 10 mIU of chondroitinase ABC as described (39). Reactions were terminated by boiling for 1 min, and the reaction mixture was analyzed by capillary electrophoresis after derivatization with AMAC as described below.
CE Analysis of Underivatized Oligosaccharides and the AMAC DerivativesThe homogeneity of each purified oligosaccharide fraction was judged by CE (53) as well as by HPLC. For derivatization of oligosaccharide fractions, samples (1 nmol each) were first digested with chondroitinase AC-II as described above and concentrated to dryness in a vacuum concentrator. Then the digests were derivatized with AMAC according to Jackson (54). The labeled disaccharides were analyzed by CE according to the method of Kitagawa et al. (55). The electrophoresed fractions of the underivatized oligosaccharides and the AMAC derivatives were detected by absorption at 185 or 254 nm, respectively.
FAB-MS and 500-MHz 1H NMR SpectroscopyThe
sugar and sulfate compositions of oligosaccharides were determined by
FAB-MS. FAB mass spectra of the oligosaccharide samples were obtained
using a VG Analytical ZAB-2SE 2FPD mass spectrometer fitted with a
cesium ion gun operated at 20-25 kV. Data were acquired and processed
using the VG Analytical Opus software. Monothioglycerol was used as the
matrix. 1H NMR spectra of the oligosaccharides were
measured on a Varian-500 at a probe temperature of 26 or 60 °C as
reported (39, 41). 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 (56). Tetrasaccharides for NMR analysis
were repeatedly exchanged in 2H2O with
intermediate lyophilization.
The previous study on the GlcA(3S)-containing oligosaccharides isolated from king crab cartilage CS-K indicated that the disaccharide units containing GlcA(3S) were decomposed by the action of chondroitinase ABC (44) and that re-evaluation of disaccharide compositions was required for CS preparations that were purified from other biological sources and analyzed using the above enzyme. Therefore, in this study, various CS isoforms were first digested using chondroitinase ABC to compare the degree of unsaturated oligosaccharide formation from various CS isoforms.
Differential Susceptibility of Various CS Isoforms to Chondroitinase ABCVarious individual CS isoforms were digested
with chondroitinase ABC, and the reactions were monitored by absorbance
at 232 nm caused by HexA produced by the eliminase action of the
enzyme. The digestion degree was calculated based on the comparison of the amounts of the resultant unsaturated oligosaccharides estimated by
the absorption at 232 nm and the uronic acid values determined for each
parent CS isoform by the carbazole reaction. CS-A, -C, and -D yielded
unsaturated products corresponding to 121, 118, and 121% of each
parent isoform, respectively (Fig. 1). The apparent overproduction of the products was most likely due to the use of the
average millimolar absorption coefficient (5.5) to calculate the unsaturated products. In contrast, the digestion of king crab cartilage CS-K produced unsaturated oligosaccharides corresponding to
only 46% of the uronic acid contained in the parent polymer, consistent with the recent finding that GlcA(3S)-containing
disaccharide units produced were destroyed by the enzymatic action and
undetectable when monitored by absorbance at 232 nm (44). The
production of UV-absorbing materials from squid cartilage CS-E reached
90% of the level calculated based on the uronic acid content but was significantly lower when compared with that of CS-A, CS-C, or CS-D. The
disaccharide composition analysis by HPLC of the digest showed
HexA
1-3GalNAc,
HexA
1-3GalNAc(4S),
HexA
1-3GalNAc(6S), and
HexA
1-3GalNAc(4S,6S) in a molar
ratio of 8.8:21.1:8.6:61.5. No other components were detected by
absorbance at 232 nm. The findings may indicate that CS-E also
contained the GlcA(3S) structure that was destroyed by the
chondroitinase ABC treatment as in the case of CS-K. Therefore, squid
cartilage CS-E was digested by hyaluronidase to obtain oligosaccharide
fragments for structural analysis as below.
A commercial
preparation of squid cartilage CS-E was exhaustively digested with
sheep testicular hyaluronidase. Although the previous study
demonstrated that CS-E as well as CS-D were less susceptible to
testicular hyaluronidase when compared with CS-K and CS-A, it was
digested to a significant degree (44). The digest was size-fractionated
by gel filtration on a Bio-Gel P-10 column. A number of peaks were
observed when monitored by absorbance at 210 nm that were caused
primarily by carbonyl groups, and they were divided into nine
fractions, I-IX, as indicated in Fig. 2. The large peak
around fraction number 85 was attributable to the buffer salts. The
elution profile was markedly different from that reported for king crab
CS-K (44) and that for a commercial CS-D preparation (41), reflecting
the differential susceptibility of the CS isoforms to the enzyme
probably due to the linkage specificity of the enzyme. There were no
fragments larger than octasaccharides among the CS-K degradation
products, and there were small proportions of fragments larger than
hexasaccharides among the CS-D degradation products. In contrast,
larger oligosaccharides were predominant among the CS-E degradation
products as shown in Fig. 2, being consistent with the relative
unsusceptibility of CS-E to the hyaluronidase action (see Fig. 1 in
Ref. 44). In this study, the smallest size fraction, fraction IX, was
investigated. It represented approximately 6.4% of the resultant
oligosaccharides and was judged to contain tetrasaccharides based on
the well defined mechanism of action of hyaluronidase (57, 58). It was
subfractionated into fractions a-v as indicated in Fig.
3 by HPLC on an amine-bound silica column. Five major
fractions h, l, m, n, and
q, marked by asterisks, were further purified by
rechromatography to apparent homogeneity as judged by HPLC and CE (data
not shown). Then their structures were analyzed as described below. As
shown in Fig. 3, fraction h was eluted at the position of an
authentic disulfated saturated tetrasaccharide
GlcA1-3GalNAc(4S)
1-4GlcA
1-3GalNAc (4S). Fractions l, m, and n were eluted at the
positions of authentic saturated tetrasaccharides with three sulfate
groups, whereas fraction q was eluted at the position of
those with three or four sulfate groups. The elution position of
fraction l was shortly after that of authentic
GlcA
1-3GalNAc(4S)
1-4GlcA(2S)
1-3GalNAc(6S), and that
of fraction m was the same as that of authentic
GlcA(3S)
1-3GalNAc(4S)
1-4GlcA
1-3GalNAc(4S) or
GlcA(2S)
1-3GalNAc(6S)
1-4GlcA
1-3GalNAc(4S).
The amounts of the purified oligosaccharides in fractions
h, l, m, n, and
q from 100 mg of CS-E are summarized in Table
I together with the FAB-MS data.
|
FAB-MS analyses of the underivatized
oligosaccharide samples in the negative ion mode defined their
molecular weights, from which the composition and the maximum number of
O-sulfate groups present in each fraction were inferred, as
in the case of heparin, heparan sulfate, and CS-K oligosaccharides (44,
53, 59). In the negative ion mode FAB spectrum, alkali-metal-attached
molecular ions of the type [M + xNa (x + 1)H]
(M represents the fully
protonated acid forms of oligosaccharides) were preferentially
observed. Representative FAB spectra are shown in Fig.
4, and assignments of the molecular ion signals afforded by each of the analyzed fractions are summarized in Table I.
The molecular ion signal clusters at m/z 1037, 1059, and
1081 afforded by fraction n corresponded, respectively, to
[M + Na 2H]
, [M + 2Na
3H]
and [M + 3Na
4H]
of a trisulfated saturated tetrasaccharide
HexA2HexNAc2 (OSO3H)3 (Fig. 4A). The molecular ion signal clusters at
m/z 1161, 1183, and 1205 afforded by fraction q
corresponded, respectively, to [M + 3Na
4H]
, [M + 4Na
5H]
,
and [M + 5Na
6H]
of a tetrasulfated
saturated tetrasaccharide,
HexA2HexNAc2(OSO3H)4 (Fig. 4B). The molecular ion signal clusters at
m/z 957, 979, and 1001 afforded by fraction h
corresponded, respectively, to [M + Na
2H]
,
[M +2Na
3H]
, and [M + 3Na
4H]
of a disulfated saturated
tetrasaccharide,
HexA2HexNAc2(OSO3H)2 (Table I). The molecular ion signal clusters at m/z 1059, 1081, and 1103 afforded by fraction l corresponded,
respectively, to [M + 2Na
3H]
,
[M + 3Na
4H]
, and [M + 4Na
5H]
of a trisulfated saturated tetrasaccharide
HexA2HexNAc2(OSO3H)3 (Table I). The molecular ion signal clusters at m/z 1037, 1059, 1081, and 1103 afforded by fraction m corresponded,
respectively, to [M + Na
2H]
,
[M + 2Na
3H]
, [M + 3Na
4H]
, and [M + 4Na
5H]
of a trisulfated saturated tetrasaccharide,
HexA2 HexNAc2(OSO3H)3 (Table I).
The disaccharide composition of the isolated tetrasaccharides was determined by our microanalytical procedure (55). The chondroitinase AC-II digest of each tetrasaccharide was labeled with the fluorophore, AMAC. Then disaccharides were analyzed by CE. Chondroitinase AC-II, which is a bacterial eliminase, should degrade a CS-tetrasaccharide to yield 1 mol each of an unsaturated disaccharide unit and a saturated disaccharide unit derived from the reducing and the nonreducing terminus, respectively. The high resolution CE quantitatively resolves each authentic unsaturated disaccharide and the corresponding saturated disaccharide with a sulfation profile identical to that of the former. Therefore, this procedure not only gives the disaccharide composition but also gives information about the sequential arrangement of disaccharide units in a given oligosaccharide sequence (43, 55).
CE analysis of the chondroitinase AC-II digest of fraction h
after the AMAC derivatization demonstrated AMAC-derivatives of GlcA1-3GalNAc(4S) (Di-4S) and
HexA
1-3GalNAc (4S)
(
Di-4S) in a molar ratio of 0.90:1.00 (Fig.
5A). The molar ratio was determined using the
integrated peak areas reported for each standard CS-disaccharide (55).
The disaccharides, Di-4S and
Di-4S, which share the same sulfation
profile and differ only in the nonreducing terminal uronic acid
residues in terms of saturation or unsaturation, were distinctly
separated as described. The saturated disaccharide unit Di-4S was
derived from the nonreducing terminus, whereas the unsaturated
disaccharide unit
Di-4S was derived from the reducing terminus. The
internal uronic acid is GlcA but not IdceA, since the tetrasaccharide
was digested by chondroitinase AC-II. Hence, the compound in fraction
h has the disulfated tetrasaccharide structure
GlcA
1-3GalNAc(4S)
1-4GlcA
1-3Gal NAc(4S).
CE analysis of the chondroitinase AC-II digest of fraction n
after the AMAC derivatization showed AMAC derivatives of
GlcA1-3GalNAc(4S,6S) (Di-diSE) and
Di-4S in a molar
ratio of 1.20:1.00 (Fig. 5D), the former being derived from
the nonreducing terminus and the latter from the reducing terminus.
Hence, the major compound in fraction n has the
hexasaccharide structure
GlcA
1-3GalNAc(4S,6S)
1-4GlcA
1-3GalNAc (4S).
CE analysis of the chondroitinase AC-II digest of fraction l
after AMAC derivatization showed AMAC derivatives of Di-4S and an
unidentified component X in a peak area ratio of 1.00:0.63, respectively (Fig. 5B). Likewise, fraction m
resulted in AMAC derivatives of
Di-4S and an unidentified component
Y in a peak area ratio of 1.00:0.64, and fraction q yielded
AMAC derivatives of
Di-4S and an unidentified component Z in a peak
area ratio of 1.00:0.68. The unidentified components X and Y migrated
to the positions of AMAC derivatives of disulfated disaccharide units, whereas the component Z migrated to the position of an AMAC derivative of a trisulfated disaccharide unit. The results indicated that the
compounds in fractions l, m, and q
share the disaccharide unit GlcA
1-3GalNAc(4S) on the reducing sides
but differ in the disaccharide structures on the nonreducing sides. The
smaller peak areas of the unidentified peaks compared with that of
Di-4S are probably due to the structural differences between
Di-4S and the unidentified compounds. However, the structure
determination of the compound was not possible due to the lack of
authentic compounds and had to await 1H NMR analysis as
described below.
Each isolated
tetrasaccharide fraction was characterized by 500-MHz 1H
NMR spectroscopy. The sites of sulfation and the types of the internal
uronic acid residues were determined by 1H NMR analysis.
The one-dimensional 1H NMR spectra of fractions
l, n, and q and two-dimensional HOHAHA spectra of fractions l and q are depicted as
representatives in Figs. 6 and 7,
respectively. The chemical shifts were assigned by two-dimensional
HOHAHA and correlation spectroscopy analyses (41, 60), and the NMR data
are summarized in Table II. The resonances were well
resolved in the structural-reporter group regions between 4.4 and 5.3 ppm and at around 2.0 ppm, being separated from other signals in the
bulk region (3.6-4.3 ppm). The resonances between 4.4 and 5.3 ppm are
characteristic of anomeric protons, whereas those at around 2.0 ppm are
characteristic of the NAc protons of GalNAc. When recorded at 60 °C
to suppress the disturbance by the HOD line, the weak resonances at
around 4.8 ppm were recognizable in the one-dimensional spectra (Fig.
6), which are shown in the insets. The types of the two
uronic acid residues in each isolated tetrasaccharide were identified
as GlcA based on the chemical shifts ( 4.46-4.59) of the anomeric
proton signals. Anomeric proton signals of an
IdceA and a
GlcA in
oligosaccharides derived from DS and CS are observed at around
5.0-5.2 and 4.5-4.8, respectively (39, 40, 61, 62). Two NAc proton
signals were observed for each tetrasaccharide component. They were
assigned by comparison with those of CS oligosaccharides reported
previously (39, 41, 60).
|
The one-dimensional spectrum (not shown) of fraction h was
recorded at 26 °C and was identical with those reported previously for the disulfated tetrasaccharide
GlcA1-3GalNAc (4S)
1-4GlcA
1-3GalNAc(4S) isolated from
shark cartilage CS-D (fraction 2 in Ref. 41). The NMR data are
summarized in Table II together with those of the other fractions.
Thus, the structure of the compound in fraction h is as
follows, consistent with the results from the enzymatic analysis.
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In this study, CS-E tetrasaccharides were prepared by testicular
hyaluronidase digestion, which exhibits transglycosylation activity
through the reverse reaction of hydrolysis (64, 65). However, since
disaccharides do not serve as acceptors, the isolated tetrasaccharides
most likely originated from the natural sequences as has been noted
(43). The possibility that they were released from higher oligo- or
polysaccharide chains newly formed by transglycosylation reactions is
unlikely, due to the low concentrations of such chains. Indeed, the
structures of the tetra- and hexasaccharides prepared by hyaluronidase
digestion of CS-D (41, 43) were in agreement with those of the
oligosaccharides prepared by chondroitinase digestion (39, 42). The
structures of the isolated tetrasaccharides strongly indicate that the
enzyme preferentially cleaves the N-acetylgalactosaminidic linkage in sequences containing GalNAc(4S)1-4GlcA
1-3GalNAc and GalNAc(4S)
1-4GlcA(3S)
1-3GalNAc but not that in the
GalNAc(4S,6S)
1-4GlcA sequence. The susceptibility of the
N-acetylgalactosaminidic linkage to GlcA(3S) has been
observed for king crab CS-K that contains a high proportion of GlcA(3S)
and is extensively digested into small oligosaccharides (44). This
markedly contrasts the insusceptibility of this linkage to
chondroitinase AC-II and to the decomposition of GlcA(3S) residues upon
chondroitinase ABC treatment (44, 45). The elution profile observed in
Fig. 2 probably reflects the CS-E structure that contains a high
content (61%) of GalNAc(4S,6S)
1-4GlcA units (46) resistant to the
hyaluronidase action.
The isolated tetrasaccharides in fractions h and
m as well as an unsaturated counterpart of that in fraction
n have been reported (39, 41, 44), whereas those in
fractions l and q are novel in that they contain
GlcA(3S) directly linked to GalNAc(6S) and GalNAc(4S,6S) forming
unusual disaccharide units GlcA(3S)1-3GalNAc(6S) and
GlcA(3S)
1-3GalNAc (4S,6S), respectively. These disaccharide structures have been suggested for oversulfated CS isolated from squid
skin (66-68) as will be discussed below. The present study demonstrated their structures in the novel tetrasaccharide sequences. Thus, the GlcA(3S)-containing structure in squid cartilage CS-E seems
to have been missed in previous studies where a disaccharide composition analysis was performed by chondroitinase ABC digestion (11,
39, 46). The lower recovery of unsaturated oligosaccharide products
from the chondroitinase ABC digest (Fig. 1) seems to indicate the
probable decomposition of the GlcA(3S)-containing disaccharide units.
The GlcA(3S) content in the CS-E preparation may be roughly estimated
to be up to 10% based on the recovery of unsaturated oligosaccharides.
The structures of oversulfated CSs isolated from other biological
sources, especially those containing GalNAc(4S,6S), should be
reinvestigated to see if GlcA(3S) is contained.
Unique fucose-branched CS isolated from sea cucumber also contains GlcA(3S) (69) as well as a disaccharide unit GlcA-GalNAc(4S,6S) (70), although the sequential arrangement of these structural elements in the polysaccharide chain has not been reported. A proteoglycan bearing oversulfated CS chains has also been isolated from squid skin, and unsaturated counterparts of the disaccharide units (GlcA(2S)-GalNAc(6S), GlcA(3S)-GalNAc(4S) and GlcA-GalNAc(4S,6S)) as well as the GlcA(3S)-containing trisulfated disaccharide unit have been noted (66, 67). The proposal of the GlcA(3S)-containing structure for the disulfated and the trisulfated units was based on the elution positions on HPLC, the reactivity to the HNK-1 antibody, and the resistance to periodate oxidation. However, there is an apparent discrepancy between our findings and the isolation procedures used in the above studies. Karamanos et al. (67, 68) have reported that the GlcA(3S)-containing disaccharide units were obtained by digestion of squid skin CS with chondroitinase AC (presumably AC-II as in Karamanos et al. (71) although not specified), which strikingly contrasts to the findings by us and others that such structures were resistant to this particular enzyme (44, 45, 69). We could obtain GlcA(3S)-containing oligosaccharides only by hyaluronidase digestion (12, 44, 45). This discrepancy remains to be clarified.
The structures found in fractions l and q are
hybrids. The compound in fraction l contained structural
elements for CS-A (GalNAc(4S)), CS-C (GalNAc(6S)), and CS-K (GlcA(3S))
in a single sequence, whereas that in fraction q contained
elements for CS-A, CS-K, and CS-E (GalNAc(4S,6S)). A tetrasaccharide,
HexA-GalNAc(4S,6S)-GlcA(2S)-GalNAc(6S), with a hybrid structure of
the CS-D and CS-E structural elements had been isolated from shark fin
cartilage CS after chondroitinase AC-I digestion, taking advantage of
the resistant nature of the GalNAc-GlcA(2S) to the enzyme (46). This
finding and ours may indicate that various combinations of the
structural units representative of different CS isoforms result in more
domain structures than anticipated, which would be embedded in
oversulfated CS chains. It should be emphasized that immunological
studies using CS-specific monoclonal antibodies including MO-225 that
interact specifically with this hybrid structure (46) revealed
developmentally regulated expression of distinct CS epitopes during the
odontogenesis in the mouse fetus (4). Thus, rare hybrid structural
domains would be extremely unique so that they would specifically be
recognized by other molecules and be involved in various biological
processes. The structural bases for the in vitro activities
of squid cartilage CS-E and for the physiological functions of
mammalian CS-E/DS-E have to be reinvestigated in regard to the new
structural feature demonstrated in this study. It remains to be
clarified whether or not the novel sequences exist in chondroitin
sulfate chains of mammalian origin and whether or not they have
specific functions.
It is well known that GlcA(3S) is detected in a glycolipid isolated
from human peripheral nerves using the mouse monoclonal antibody HNK-1,
raised against human natural killer cells (72, 73). The terminal
GlcA(3S) residue is essential for the immunoreactivity, and the
carbohydrate epitope is expressed also on glycoproteins (74). The
trisaccharide sequence GlcA(3S)1-3Gal
1-4GlcNAc has been
demonstrated for the epitope on bovine peripheral myelin glycoprotein
P0 (75). The HNK-1-reactive carbohydrates are temporally and spatially
regulated during the development of the nervous system (76) and
implicated in cell-cell adhesion as well as in the recognition of
neurons and astrocytes (77). In addition, the HNK-1-reactive
carbohydrate is a ligand for selectins, which are leukocyte-endothelial
cell adhesion molecules (78). It has been reported that the HNK-1
antibody reacted with an intact CS polymer from squid skin (67). It
will be interesting to investigate whether GlcA(3S)-containing CS
chains exist in the nervous system.
Biosynthetic mechanisms for the production of GlcA(3S) in oversulfated
CSs or HNK-1 antigens are unknown. There may exist multiple
3-O-sulfotransferases, which synthesize CS-K, CS-E, and the
GlcA(3S)-containing HNK-1 epitopes in glycolipids and glycoproteins, although none of them has been demonstrated. In contrast, two 6-O-sulfotransferases involved in the synthesis of
GalNAc(4S,6S) have been reported. The one that synthesizes internal
GalNAc(4S,6S) units was demonstrated in squid cartilage (79). The other
6-O-sulfotransferase that specifically transfers a sulfate
group to the GalNAc(4S) residue at the nonreducing terminus to form
GalNAc(4S,6S) was discovered in human serum (79, 80). GalNAc(4S,6S) has
been found at the nonreducing terminus of the newly synthesized CS in
the in vitro culture of chick and rat embryo cartilage (81, 82) and in the cell culture of chick chondrocytes and rat
chondrosarcoma (17, 83) and is suggested as the possible chain
termination signal in the CS biosynthesis. In the recent enzymological
studies, sulfated CS tetra- or hexasaccharides with
GlcA1-3GalNAc(4S,6S) or GlcA(3S)
1-3GalNAc(4S) at the
nonreducing ends did not serve as acceptors for chondroitin GalNAc
transferase (84), supporting the notion of the termination signal.
Interestingly, CS-E but not CS-C synthesis in fetal calf articular
cartilage in culture was stimulated by dibutyryl cyclic AMP, indicating
specific control mechanisms for the CS-E biosynthesis (85). Regulatory
mechanisms for the CS-E synthesizing system including the putative
3-O-sulfotransferase will be interesting to investigate.
We thank Tomoko Hazeki for technical assistance and Dr. Makiko Sugiura (Kobe Pharmaceutical University) for recording the NMR spectra.