A mouse brain chondroitin sulfate (CS)
proteoglycan, DSD-1-PG, bears the DSD-1 epitope and has neurite
outgrowth promoting properties. Shark cartilage CS-C inhibits the
interactions between the DSD-1-specific monoclonal antibody 473HD and
the CS chains of the DSD-1-PG, which is expressed on the mouse glial
cells (Faissner, A., Clement, A., Lochter, A., Streit, A., Mandl, C.,
and Schachner, M. (1994) J. Cell Biol. 126, 783-799). On
the other hand, several hexasaccharides isolated from commercial shark
cartilage CS-D, which contains a higher proportion of characteristic D
units (GlcUA(2-sulfate)
1-3GalNAc(6-sulfate)) as compared with CS-C,
has the A-D tetrasaccharide sequence composed of an A disaccharide
unit (GlcUA
1-3GalNAc(4-sulfate)) and a D disaccharide unit
(Nadanaka, S. and Sugahara, K. (1997) Glycobiology 7, 253-263). In this study, the biological activities and the structure
of shark cartilage CS-D were investigated. CS-D inhibited the
interactions between monoclonal antibody 473HD and DSD-1-PG and also
promoted neurite outgrowth of embryonic day 18 hippocampal neurons.
Eight octasaccharide fractions were isolated from CS-D after partial
digestion with bacterial chondroitinase ABC by means of gel filtration
chromatography and anion-exchange high performance liquid
chromotography to investigate the frequency and the arrangement of the A-D tetrasaccharide unit in the polymer sequence. Structural analysis performed by a combination of enzymatic digestions with 500-MHz 1H NMR spectroscopy demonstrated that the isolated
octasaccharides shared the common core structure
HexA
1-3GalNAc
1-4(GlcUA
1-3GalNAc)3 with
four, five, and six sulfate esters at various hydroxyl groups in
different combinations. In the structure,
HexA and GlcUA
represent 4-deoxy-
-L-threo-hex-4-enepyranosyluronic
acid and glucuronic acid, respectively. No D-D tetrasaccharide sequence
was found, and discrete D disaccharide units were demonstrated
exclusively as A-D tetrasaccharide units in either an A-D-A or an A-D-C
hexasaccharide sequence in the five octasaccharides that represented
about 5.0% (w/w) of the starting polysaccharides (C denotes the
disaccharide GlcUA
1-3GalNAc(6-sulfate)). It remains to be
determined whether such characteristic hexasaccharide sequences present
in shark cartilage CS-D serve as functional domain structures
recognized by some protein ligands.
 |
INTRODUCTION |
Chondroitin sulfate
(CS)1 chains occur in animal
tissues usually as proteoglycans (PGs), in which these polysaccharide
chains are covalently attached to a core protein. CS-PGs are present on
cell surfaces and in the extracellular matrix of most tissues and are
involved in many biological activities. However, the structure of CS
chains is only poorly understood. The backbone structure of CS chains
consists of repeating disaccharide units, each containing a glucuronic
acid (GlcUA) residue and an N-acetylgalactosamine (GalNAc)
residue. Diversity in ester-sulfation of both saccharide residues
results in structural variability of CS chains, which is assumed to be
the basis for its functional diversity. Immunological studies using
monoclonal antibodies (mAbs) have revealed that the sulfation profile
of CS chains changes with concomitant specific spatio-temporal patterns
in various tissues, suggesting that CS isoforms differing in sulfation
position and degree perform distinct functions in development (1).
Disaccharide units with various sulfation profiles are non-randomly
distributed in the CS chains as detected by mAbs (2).
It had been shown previously that the CS-PG named DSD-1-PG from
developing mouse brain is located on the surface of immature glial
cells in the central nervous system and promotes neurite outgrowth (3).
The neurite outgrowth stimulating capacity of DSD-1-PG was strongly
reduced by mAb 473HD, which recognizes a glycosaminoglycan epitope
present in DSD-1-PG. Surprisingly, shark cartilage CS-C inhibited the
interactions between the mAb 473HD and the DSD-1 epitope (3). In
agreement with this observation, neurite outgrowth promotion was also
abolished by treatment with chondroitinase ABC, which removes the CS
chains of the PG (3). It can be concluded from these data that the
particular CS structure recognized by mAb 473HD is of physiological
relevance in the context of neurite outgrowth. In addition, the rat
brain 6B4 CS-PG/phosphacan, which corresponds to the extracellular
region of a receptor-like protein-tyrosine phosphatase, PTP
/RPTP
,
was demonstrated to interact with pleiotrophin through the CS chains
and the core protein, and to regulate pleiotrophin-induced neurite
outgrowth (4). The binding was specifically inhibited by shark
cartilage CS-C. Structurally, shark cartilage CS chains possess a
unique D disaccharide unit GlcUA(2S)
1-3GalNAc(6S) in addition to
the common A and C disaccharide units (GlcUA
1-3GalNAc(4S) and
GlcUA
1-3GalNAc(6S)), where 2S, 4S, and 6S denote 2-O-,
4-O- and 6-O-sulfate, respectively (5). D units
have been found not only in shark cartilage CS but also in the CS of
the mouse mast cells derived from immune lymph nodes. The CS-D composed
of A and D disaccharide units occurs transiently, functioning as an
important phenotypic marker that distinguishes different mast cell
subsets (6). CS in the basement membrane of embryonic mouse tooth germ
also possesses D disaccharide units and appears transiently. A, C, and
D units are produced in this order in a time-dependent
manner (1), as demonstrated using mAbs including MO-225, which
specifically recognizes the epitope containing a D disaccharide unit
(7). Although there is accumulating evidence for the biological
importance of CS-D, structural information regarding the sequential
arrangement of its disaccharide units and its biosynthetic mechanisms
remain unclear.
To investigate the structural features and the biosynthetic mechanism
of shark cartilage CS, we have been analyzing the structures of
oligosaccharides prepared by bacterial chondroitinase or testicular hyaluronidase digestion of a shark cartilage CS-D isoform, which contains a larger proportion of D disaccharide units than CS-C (21.2 versus 9.62%). Commercial CS-D and CS-C isoforms are
purified from cranial cartilages of a shark strain Rhizoprionodon
acutus (Rüppell) and a mixture of cartilages from various
body parts of multiple shark strains, mainly Prionace glauca
(Linnaeus), respectively, according to the manufacturer. Previous
findings revealed a unique A-D tetrasaccharide sequence,
GlcUA
1-3GalNAc(4S)
1-4GlcUA(2S)
1-3GalNAc(6S), in one
tetrasaccharide and in a few hexasaccharides (8-10). The A-D
tetrasaccharide sequence is recognized by the mAb MO-225 (7), and its
epitope is transiently expressed and disappears during developmental
maturation of mouse odontoblasts, suggesting the epitope to be
biologically functional (1). The aim of this study was to investigate
the biological activity of CS-D especially in the context of neurite
outgrowth, to clarify the distribution of D disaccharide units and A-D
sequences along CS-D chains, and to characterize the sulfation profiles
surrounding them by analyzing structures of larger oligosaccharides
(i.e. octasaccharides). Preliminary data have been reported
in abstract form (11).
 |
EXPERIMENTAL PROCEDURES |
Materials--
The following materials and enzymes were
purchased from Seikagaku Corp., Tokyo, Japan: a sodium salt preparation
(super special grade) of shark cartilage CS-D, six unsaturated
CS-disaccharides, chondroitinase AC-II (EC 4.2.2.5), conventional
chondroitinase ABC, and highly purified chondroitinase ABC
(commercialized as a protease-free preparation) (EC 4.2.2.4), and
chondro-4-O-sulfatase and chondro-6-O-sulfatase
abbreviated as CS-4-sulfatase (EC 3.1.6.9) and CS-6-sulfatase (EC
3.1.6.10), respectively.
4,5Hexuronate-2-O-sulfatase (abbreviated as
hexuronate-2-sulfatase) purified from Flavobacterium
heparinum (12) was provided by Dr. K. Yoshida, Seikagaku Corp. The
CS-D octasaccharide fraction was obtained as reported (8) after
digestion with highly purified chondroitinase ABC followed by gel
filtration chromatography on a column of Bio-Gel P-10. The following
authentic sulfated tetrasaccharides were isolated previously from shark
cartilage CS-D (13):
HexA
1-3GalNAc(4S)
1-4GlcUA
1-3GalNAc(4S),
HexA
1-3GalNAc(6S)
1-4GlcUA
1-3GalNAc(4S),
HexA
1-3GalNAc(6S)
1-4GlcUA
1-3GalNAc(6S),
HexA(2S)
1-3GalNAc(6S)
1-4GlcUA
1-3GalNAc(4S), and
HexA(2S)
1-3GalNAc(6S)
1-4GlcUA
1-3GalNAc(6S).
HexA
1-3GalNAc(6S)
1-4GlcUA
1-3GalNAc was prepared by
the digestion of
HexA
1-3GalNAc(6S)
1-4GlcUA
1-3GalNAc(6S)
with CS-6-sulfatase.
Cell Culture and Bioassays--
Hippocampal neuron cultures were
established from embryonic day 18 rat brain according to the standard
procedures (14) with some modifications: hippocampi were dissected in a
37 °C Ca-Mg-free Hank's balanced salt solution supplemented with
0.6% (w/v) glucose and 7 mM HEPES-NaOH, pH 7.4, treated
with 0.25% (w/v) trypsin at 37 °C, washed three times with a
Ca-Mg-free Hank's balanced salt solution, and dissociated by repeated
passage through two fire-polished Pasteur pipettes of different sizes.
Cells were plated at a density of 8000 cells/cm2 on glass
coverslips precoated with 15 µg/ml poly-DL-ornithine (catalog no. P-0671, Sigma, Deisenhofen, Germany) in 0.1 M
borate buffer, pH 8.1, and subsequently with 5 µg/ml uronic acid
equivalents of different CS isoforms in phosphate-buffered saline
(PBS). The cultures were kept in a humidified atmosphere with 5%
CO2 in minimum Eagle's medium containing the N2
supplements of Bottenstein and Sato (15), 0.1% (w/v) ovalbumin, and
0.1 mM pyruvate. After 24 h in culture the cells were
fixed with 4% (w/v) paraformaldehyde for 20 min at room temperature,
washed three times with PBS, permeabilized with 0.1% (w/v) Triton
X-100 in PBS for 15 min, and stained with mAb to
-tubulin (clone DM
1A; Sigma) and peroxidase-derivatized secondary antibodies to mouse IgG
(Dianova), as described (16). For quantitative analysis, 100 cells were
analyzed per coverslip; the neurons growing one fiber exceeding a cell
diameter in length were counted as process-bearing, and the
corresponding fraction was given as percentage. At least six
independent experiments per parameter and condition were carried
out.
Enzyme-linked Immunosorbent Assay (ELISA)--
Competition
ELISAs were performed as described (3). In brief, DSD-1-PG preparations
were absorbed overnight at 4 °C to polyvinylpyrrolidone 96-well
plates (Falcon Plastics, Cockeysville, PA) at 0.5 µg/ml uronic acid
equivalents, diluted in 0.1 M NaHCO3, pH 8.0 to
100 µl/well. The wells were blocked with 1% (w/v) bovine serum
albumin in PBS plus 0.05% (v/v) Tween 20 (PBS-Tween) at least for
1 h at room temperature. The purified mAb 473HD (0.5-1 µg/ml)
was preincubated with glycosaminoglycans (GAGs) (10 µg/ml) in the
blocking buffer for 2 h at room temperature and used for ELISA on
DSD-1-PG in the presence of the competitors for 1 h at room
temperature. After three washes with PBS-Tween, a secondary goat
anti-rat IgM antibody derivatized with peroxidase (Dianova, Hamburg,
Germany) was incubated in the blocking buffer (1:5000) for 1 h at
room temperature. Thereafter, the wells were washed three times with
PBS-Tween and the ELISA was developed with
2,2
-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (17). The colored
reaction product was quantified in an ELISA reader at 405 nm (Titertec
Multiscan, Flow Laboratories, Inc.).
Enzymatic Analysis--
Enzymatic digestion of the isolated
oligosaccharides with chondroitinase AC-II or highly purified
chondroitinase ABC was carried out using 0.5 nmol of each
oligosaccharide substrate and 10 mIU of the enzyme in a total volume of
30 µl of the appropriate buffer at 37 °C for 30 min as described
(13). After incubation, the reaction mixtures were boiled at 100 °C
for 1 min, cooled to room temperature, mixed with 370 µl of 16 mM NaH2PO4, and analyzed by HPLC.
For successive enzymatic digestion of fraction VIb, the fraction (1 nmol as
HexA) was first incubated with 10 mIU of highly purified
chondroitinase ABC in a total volume of 20 µl of 50 mM
Tris-HCl buffer, pH 8.0, containing 60 mM sodium acetate and 100 µg/ml bovine serum albumin at 37 °C for 60 min and boiled at 100 °C for 1 min to terminate the reaction. Half of the sample was analyzed by HPLC, whereas the other half was mixed with 10 µl
each of water and a
hexuronate-2-sulfatase (2.5 mIU) solution in 20 mM sodium acetate buffer, pH 6.5, containing 0.15% bovine serum albumin and incubated at 37 °C for 120 min. The reaction was
terminated and analyzed by HPLC as above. For time-course experiments
of chondroitinase AC-II digestion of the oligosaccharides, each
fraction containing 0.5 nmol (as
HexA) of an oligosaccharide was
incubated with 1 mIU of chondroitinase AC-II in a total volume of 30 µl of the appropriate buffer at 37 °C for 1.5, 3, 15, 30, or 150 min. The reaction was terminated and analyzed by HPLC as above.
HPLC and Capillary Electrophoresis (CE)--
Fractionation and
analysis of unsaturated oligosaccharides were carried out by HPLC on an
amine-bound silica PA03 column using a linear gradient of
NaH2PO4 at a flow rate of 1 ml/min at room temperature as described for the separation of CS-di- and
-tetrasaccharides (13, 18, 19). Eluates were monitored by absorption at
232 nm. The separated fractions were concentrated and desalted through a column of Sephadex G-25. CE was carried out to examine the purity of
each isolated oligosaccharide fraction using a fused silica capillary
and 25 mM sodium phosphate buffer, pH 3.0, in a Waters capillary ion analyzer with a negative polarity power supply (20). The
electrophoresed fractions were detected by absorption at 185 nm.
Other Analytical Methods--
Oligosaccharides produced by
enzymatic digestions were quantified based on the absorbance
(E232 = 5500 M
1
cm
1) caused by the
4,5 sites of the uronic
acid at the non-reducing ends (21).
500-MHz 1H NMR Spectroscopy--
Octasaccharide
fractions were repeatedly exchanged in D2O with
intermediate lyophilization. Their 1H NMR spectra were
measured on a Varian VXR-500 at a probe temperature of 15, 26, or
60 °C (22-24). 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 D2O (25).
 |
RESULTS |
Interactions with the mAb 473HD and the Neurite Outgrowth Promoting
Activity of Shark Cartilage CS-D--
Previous studies suggested that
the mAb 473HD reacted with the DSD-1 epitope, which was contained in
CS-polymers covalently linked to the core of DSD-1-PG. The epitope was
proved to be necessary for the neurite outgrowth promoting properties
of DSD-1-PG on embryonic day 18 hippocampal neurons, as shown by
digestion of the culture substrate with chondroitinase ABC (3). The
stimulatory effect of DSD-1-PG on neurite elongation was strongly
reduced by mAb 473HD (3), supporting the functional importance of the epitope. In this study, to circumscribe the molecular nature of the
epitope in more detail, various GAGs were tested for their ability to
interfere with mAb 473HD binding to its antigen. Of these, CS-C and
CS-D but not the other GAGs competed the binding of mAb 473HD to
DSD-1-PG. According to these competition assays, CS-C and -D contained
the DSD-1 structure, in contrast to the other GAGs and dextran sulfate
(Fig. 1). In view of the asserted functional properties of the DSD-1 epitope, these GAGs were probed for
neurite outgrowth promoting properties. In this functional assay, CS-D
and CS-E most efficiently enhanced the fraction of neurite-bearing
cells, as compared with other CS isoforms (Fig. 2). Only the entire DSD-1-PG was able to
promote neurite outgrowth more strongly than CS-D and CS-E. CS-C, which
also contains the DSD-1 epitope according to the competition ELISA, was
not efficient in this assay. This probably reflects a lower
concentration of the DSD-1 structure in CS-C preparations, as shown by
competition ELISAs with serially diluted
GAGs.2 Therefore, CS-D was
chosen for further structural analysis. The observed neurite outgrowth
promoting activity of CS-E will be discussed below (see
"Discussion").

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Fig. 1.
Competition of mAb 473HD binding by soluble
GAGs. mAb 473HD was preincubated with individual various GAGs and
used for ELISA on DSD-1-PG in the presence of the GAGs. The percent of
control was calculated using the equation: percent of control = OD405 nm test/OD405 nm control. The
experiments were performed at least in triplicate. The results of one
representative experiment are shown. Dex.S, dextran sulfate;
HA, hyaluronic acid; KS, keratan sulfate;
HS, heparan sulfate; Hep, heparin.
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Fig. 2.
Promotion of neurite outgrowth of hippocampal
neurons by different chondroitin sulfate isoforms. Embryonic day
18 hippocampal neurons were seeded and cultured on glass coverslips,
which were precoated with different CS isoforms and DSD-1-PG as
described under "Experimental Procedures." The graph shows the
percent increase of the fraction of neurite-bearing cells on the
different substrates compared with the polyornithine control. The
number of experiments is given in parentheses. The
experiments were statistically evaluated with Student's t
test. ns, not significant; x, 0.01 < p < 0.05; xx, 0.001 < p < 0.01; xxx, 0.001 > p.
Abbreviations in parentheses (ns and
xx) indicate statistics between the data obtained with the
indicated sample and the polyornithine control.
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Isolation of the Octasaccharide Fraction--
To isolate
octasaccharide fragments from shark cartilage CS-D, chondroitinase ABC
rather than testicular hyaluronidase was used because the former does
not have transglycosylation activity unlike the latter, which may
generate unnatural compounds by such an activity. Two different
commercial preparations of chondroitinase ABC are available: a
conventional one and a highly purified one. Conventional chondroitinase
ABC contains a minor extra protein (Mr 100,000)
in addition to the major one corresponding to the highly purified
enzyme (Mr 98,000) as examined by SDS-PAGE (see "Discussion" of Ref. 13; see also Ref. 26). Enzymatically, a highly
purified preparation does not digest tetrasaccharides (13). It shows an
exolytic action on CS-hexasaccharides removing only the disaccharide
units on the non-reducing termini and leaving the tetrasaccharides on
the reducing side undigested (8), contrary to the previously proposed
concept of its non-random endolytic activity (27). However,
conventional chondroitinase ABC digests tetrasaccharides
efficiently. Hence, the highly purified preparation was used under
limited incubation conditions to obtain relatively large fragments
(i.e. octasaccharides) from shark cartilage CS-D.
A commercial preparation of shark cartilage CS-D was partially digested
with highly purified chondroitinase ABC, and the digest was
size-fractionated by gel filtration into di-, tetra-, hexa-, octa-,
deca-, dodeca-, and tetradecasaccharides using a column of Bio-Gel P-10
as reported (8). The octasaccharide fraction was subfractionated into
fractions I-VIII by HPLC on an amine-bound silica column as indicated
in Fig. 3. Fraction II contained at least
two compounds: the CS-4-sulfatase-sensitive major (77%) and
CS-4-sulfatase-resistant minor compound (23%). Under mild incubation
conditions, CS-4-sulfatase acts preferentially on compounds bearing a
GalNAc(4S) residue at the reducing end (13, 28). Therefore, fraction II
was digested with CS-4-sulfatase, and the digest was subjected to HPLC
fractionation to separate the digested and undigested compounds. The
desulfated compound resulting from the major component eluted
approximately 10 min earlier than the corresponding parent compound on
HPLC (not shown). It was isolated separately from the undigested minor
compound and designated as fraction II-S. Fractions V and VI were
further purified by rechromatography into subfractions Va and Vb in a
molar ratio of 44:56, and into VIa and VIb in a molar ratio of 38:62,
respectively. Since fraction Va contained at least two components, the
major component Va-I that represented 92% of fraction Va was obtained
after CS-4-sulfatase digestion followed by HPLC. Among the isolated
fractions, the major fractions I, II-S, IV, Va-I, Vb, VIa, VIb, and
VIII were seemingly pure, giving a symmetrical single peak on HPLC and
CE (data not shown), and subjected to structural analysis as described below.

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Fig. 3.
HPLC fractionation of the octasaccharide
fraction. The octasaccharide fraction obtained from gel filtration
on Bio-Gel P-10 (8) was separated into fractions I-VIII on an
amine-bound silica column using a linear
NaH2PO4 gradient from 0.31 to 0.71 M over a 75-min period. For the experimental details, see
"Experimental Procedures."
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Enzymatic Characterization of the Isolated Octasaccharide Fractions
I, II, IV, VIa, VIb, and VIII Using Chondroitinase AC-II and
Sulfatases--
Oligosaccharides in the isolated fractions were first
characterized by enzymatic digestions in conjunction with HPLC before 1H NMR analysis. The disaccharide composition of each
isolated octasaccharide fraction was determined by chondroitinase AC-II digestion in conjunction with HPLC, and the findings are summarized in
Table I. All octasaccharides in the
isolated fractions were digested completely into disaccharides by
chondroitinase AC-II, indicating that all the internal uronic acid
residues in each component are GlcUA and not L-iduronic
acid (IdceA).
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Table I
Disaccharide composition of the isolated octasaccharide fractions
Each octasaccharide fraction was digested with chondroitinase
AC-II, and the products were identified and quantified by HPLC as
described under "Experimental Procedures." Similar results were obtained using a conventional commercial chondroitinase ABC instead of chondroitinase AC-II.
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The results obtained from enzymatic studies with fraction VIb are
described below as representatives. Digestion of fraction VIb with
chondroitinase AC-II gave three major products,
Di-4S,
Di-6S, and
Di-diSD, in a molar ratio of 2:1:1 as quantified by HPLC
(Table I and Fig. 4D). The
peaks around 10 min did not contain
Di-0S and were derived from the
enzyme preparation. The sequential arrangement of these disaccharide
units was first characterized using three types of sulfatases:
hexuronate-2-sulfatase, CS-4-sulfatase, and CS-6-sulfatase. The
enzyme
hexuronate-2-sulfatase removes a sulfate group only from the
C-2 position of a
HexA residue located at the non-reducing terminus
(12), whereas the enzyme CS-6-sulfatase removes only a sulfate group on
C-6 of the GalNAc residue at the reducing terminus of oligosaccharides
(28). Although CS-4-sulfatase can remove sulfates from internal GalNAc
residues under harsh incubation conditions as well as from the GalNAc
residue at the reducing terminus, it acts predominantly on the latter GalNAc under limited incubation conditions (28). The major component in
fraction VIb was completely digested by CS-4-sulfatase but resistant to
CS-6-sulfatase (Table II), indicating
that one of the 4-sulfated GalNAc residues is located at the reducing
terminus. The major component representing 95% of this fraction was
resistant to
hexuronate-2-sulfatase (Table II), indicating that it
has a non-sulfated
HexA residue at the non-reducing end. The
sequential arrangement of the disaccharide units was finally determined
by time-course digestion experiments using chondroitinase AC-II, which
has an exolytic action (27). At the indicated time intervals, the
disaccharide units released sequentially from the non-reducing terminus
of the octasaccharide were determined by HPLC. It should be noted that
relatively mild incubation conditions were used to monitor the early
reaction products from partial digestions. The predominant early
digestion product was
Di-6S followed by
Di-4S (Fig. 4,
A and B), suggesting that the major compound
contained the sequence
HexA
1-3GalNAc(6S)
1-4GlcUA
1-3GalNAc(4S) on the non-reducing side. Hence, the major compound in fraction VIb contained the following structure with a C-A-D-A sequence, which was also supported by the data from chondroitinase ABC digestion experiments (see below).

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Fig. 4.
Time-course experiments of chondroitinase
AC-II digestion of the isolated fraction VIb. Fraction VIb (0.5 nmol) was digested with chondroitinase AC-II (1 mIU) for 1.5 (A), 30 (B), or 150 min (C). Each
digest was analyzed by HPLC on an amine-bound silica column using a
linear NaH2PO4 gradient from 16 to 800 mM over a 90-min period. Fraction VIb (0.5 nmol) was also
digested with 10 mIU of the enzyme for 20 min for complete digestion
(D). The elution positions of the following authentic hexa-
and tetrasaccharides are indicated by open arrows: Hexa-,
HexA 1-3GalNAc(4S) 1-4GlcUA(2S) 1-3GalNAc(6S) 1-4GlcUA 1-3GalNAc(4S); Tetra-,
HexA(2S) 1-3GalNAc(6S) 1-4GlcUA 1-3GalNAc(4S). Peaks eluted before 8 min are attributable to the buffer salts and those around 10 min are derived from the enzyme preparation. The peak eluted
at 25 min in B-D corresponded to Di-diSD.
The elution positions of authentic unsaturated disaccharides are
indicated in the top panel by arrows.
a, Di-0S; b, Di-6S; c, Di-4S;
d, Di-diSD; e,
Di-diSE; f, Di-triS.
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Table II
Action of sulfatases on the isolated octasaccharides
Each octasaccharide was digested with hexuronate-2-, CS-4-, or
CS-6-sulfatase, and the digest was analyzed by HPLC.
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Fraction VIb--
Fraction VIb structure is
HexA
1-3GalNAc(6S)
1-4GlcUA
1-3GalNAc(4S)
1-4GlcUA(2S)
1-3GalNAc(6S)
1-4GlcUA
1-3GalNAc(4S).
Likewise, fractions I, II-S, IVa, VIa, and VIII were successfully
analyzed by enzymatic digestions in conjunction with HPLC. The results
obtained from sulfatase digestion experiments are summarized in Table
II. The enzymes CS-4- and CS-6-sulfatase removed a sulfate group from
70% and 12% of the compounds in fraction IV, respectively, as judged
by HPLC (data not shown), indicating that the compounds in the major
and the minor subfractions designated as IVa and IVb have a 4-sulfated
and a 6-sulfated GalNAc residue at the reducing terminus, respectively.
To deduce the structures of the major components in fractions IV, VIa,
and VIII, time-course digestion experiments using chondroitinase AC-II
were also carried out, and the results are shown in Fig.
5. Notably, in these experiments, the D-A
linkage seems to have been cleaved by chondroitinase AC-II relatively
slowly when compared with the D-C linkage as shown in Figs. 4 and 5.
Fraction IV containing a D-C sequence on the non-reducing side was
completely digested into disaccharides under the relatively mild
incubation conditions used, whereas fractions VIa, VIb, and VIII
containing a D-A sequence was digested relatively slowly showing a
certain degree of resistance of the linkage against the enzyme. Based
on the results from enzymatic studies (Tables I and II, Fig. 5), the
following structures were deduced. The structure of the major compound
in fraction II was deduced from that in fraction II-S. The
Di-6S
unit detected upon chondroitinase AC-II digestion of fractions VIa and
VIII and the
Di-4S unit generated from fraction I (Table I) were
considered to be derived from minor components.

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Fig. 5.
Time-course experiments of chondroitinase
AC-II digestion of fractions IV, VIa, and VIII. Individual
fractions IV, VIa, and VIII (0.5 nmol each) were incubated with
chondroitinase AC-II (1 mIU) for the indicated time intervals, and each
digest was analyzed by HPLC on an amine-bound silica column. Each
digestion was incomplete since it was carried out under limited
conditions. A, fraction IV; B, fraction VIa;
C, fraction VIII. Percent recoveries were calculated based
on the peak areas in the HPLC chromatograms and are expressed as
percentages relative to the amount of each parent octasaccharide
fraction used for digestion. , Di-4S; , Di-6S; ,
Di-diSD; , tetrasaccharide; , hexasaccharide; ,
octasaccharide.
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Fractions I, II, IVa, VIa, and VIII--
Fraction I structure is
HexA
1-3GalNAc(6S)
1-4GlcUA
1-3GalNAc(6S)
1-4GlcA
1-3GalNAc(6S)
1-4GlcUA
1-3GalNAc(6S).
Fraction II structure is
HexA
1-3GalNAc(6S)
1-4GlcUA
1-3GalNAc(6S)
1-4GlcUA
1-3GalNAc(6S)
1-4GlcUA
1-3GalNAc(4S). Fraction IVa structure is
HexA(2S)
1-3GalNAc(6S)
1- 4GlcUA
1-3GalNAc(6S)
1-4GlcUA
1-3GalNAc(6S)
1-4GlcA
1-3GalNAc(4S). Fraction VIa structure is
HexA
1- 3GalNAc(4S)
1-4GlcUA
1-3GalNAc(4S)
1-4GlcUA(2S)
1-3GalNAc(6S)
1-4GlcUA
1-3GalNAc(4S). Fraction VIII structure is
HexA(2S)
1-3GalNAc(6S)
1-4GlcUA
1-3GalNAc(4S)
1-4GlcUA(2S)
1-3GalNAc(6S)
1-4GlcUA
1-3GalNAc(4S).
Characterization of Highly Purified Chondroitinase ABC Using the
Structurally Defined Octasaccharides--
For the structural analysis
of fractions Va and Vb, highly purified chondroitinase ABC was an
indispensable tool along with chondroitinase AC-II and the sulfatases.
This enzyme acts on unsaturated hexasaccharides in an exolytic fashion,
removing an unsaturated disaccharide unit from the non-reducing
terminus of hexasaccharides irrespective of the sulfation profiles (8).
To further investigate the mechanism of action of this enzyme, the
structurally defined octasaccharides described above were digested
exhaustively by the enzyme and the products were analyzed by HPLC.
The results obtained with fraction VIb are described below as
representatives. Digestion of fraction VIb with highly purified chondroitinase ABC yielded two disaccharide units (
Di-6S and
Di-4S) and two unsaturated tetrasaccharides with recoveries of 86%,
98%, 90%, and 18%, respectively (Table
III, Fig.
6A). The major and the minor
tetrasaccharides were detected at the elution positions of the
authentic tetrasaccharides
HexA(2S)
1-3GalNAc(6S)
1-4GlcUA
1-3GalNAc(4S) and
HexA
1-3GalNAc(6S)
1-4GlcUA
1-3GalNAc(4S), respectively. The major tetrasaccharide was shifted to the position of
HexA
1-3GalNAc(6S)
1-4GlcUA
1-3GalNAc(4S) upon
subsequent
hexuronate-2-sulfatase digestion (Fig.
6B), confirming the structure of the above major
tetrasaccharide derived from the tetrasaccharide portion on the
reducing side of the octasaccharide. In contrast, the minor C-A
tetrasaccharide was assumed to be derived from the non-reducing side of
the major octasaccharide because a D-A, but no C-A, tetrasaccharide was
produced in a time-course digestion experiment using chondroitinase
AC-II that has an exolytic activity to release disaccharide units, but
not tetrasaccharide units (Fig. 5, B and C).
Thus, highly purified chondroitinase ABC must have split the major
octasaccharide in the case of fraction VIb into two tetrasaccharides by
18%.
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Table III
Analysis of the chondroitinase ABC digests of the isolated
octasaccharide fractions
Each octasaccharide fraction was digested with highly purified
chondroitinase ABC, and the reaction products were identified and
quantified by HPLC as described under "Experimental Procedures."
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Fig. 6.
Enzymatic analysis of the isolated fraction
VIb. The isolated fraction VIb was digested with highly purified
chondroitinase ABC (A) or successively with highly purified
chondroitinase ABC and then hexuronate-2-sulfatase (B).
Each digest was analyzed by HPLC on an amine-bound silica column using
a linear NaH2PO4 gradient from 16 to 800 mM over a 90-min period. The elution positions of the
authentic unsaturated disaccharides are indicated at the top
of A. The peaks marked by asterisks were often observed upon high sensitivity analysis and were due to an unknown substance that
eluted from the column resin. For the peaks eluted before 15 min, see
the legend to Fig. 4. The elution positions of the following authentic
tetrasaccharides are indicated in A and B: 1,
HexA(2S) 1-3GalNAc(6S) 1-4GlcUA 1-3GalNAc(4S);
2, HexA 1-3GalNAc(6S) 1-4GlcUA 1-3GalNAc(4S). The elution positions of authentic unsaturated disaccharides are indicated in the top panel by arrows.
a, Di-0S; b, Di-6S; c, Di-4S;
d, Di-diSD; e,
Di-diSE; f, Di-triS.
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Likewise, the specificity of highly purified chondroitinase ABC was
investigated by digesting fractions I-S, II-S, IV, and VIa, and the
results are summarized in Table III. The data altogether indicated that
the enzyme digested preferentially the tetrasaccharide portion on the
non-reducing side of the octasaccharides into disaccharides in an
exolytic fashion, although it split octasaccharides into two
tetrasaccharides to a small extent in some cases. The exolytic action
seems to be dominant over the endolytic action, if not exclusive.
Although the results obtained from digestion of fraction IV
demonstrated a minor tetrasaccharide fragment having the same sequence
as that of the tetrasaccharide on the non-reducing side, the results
may indicate that fraction IV contained at least two octasaccharide
components and that the major and one of the minor tetrasaccharides
were derived from the above tetrasaccharide sequences on the reducing
side of each octasaccharide, respectively. The production of the major
tetrasaccharide sequence was in agreement with the notion that the
enzyme exolytically digested the tetrasaccharide portion on the
non-reducing side of the major octasaccharide component IVa in fraction
IV. The production of the minor tetrasaccharide sequence was
consistent with the findings from the CS-6-sulfatase digestion
experiment where the minor component in fraction IV was susceptible to
the enzyme. However, there is the possibility that the enzyme split the
major octasaccharide into two tetrasaccharides to a small extent (6%).
Structural analyses of fractions Va and Vb were then performed, taking
advantage of this mechanism of action of highly purified chondroitinase
ABC.
Enzymatic Characterization of Fractions Va and Vb Using
Chondroitinase AC-II, Sulfatase, and Highly Purified Chondroitinase
ABC--
Fraction Vb gave rise to three major disaccharides,
Di-6S,
Di-4S, and
Di-diSD, in a molar ratio of 2:1:1 upon
chondroitinase AC-II digestion as quantified by HPLC (Table I). The
sequential arrangement of these disaccharide units was determined by
digestions using chondroitinase AC-II and highly purified
chondroitinase ABC. The predominant early product generated upon
digestion with the former enzyme was
Di-6S, followed by
Di-4S
(data not shown), suggesting that the tetrasaccharide structure on the
non-reducing side of the major compound was
HexA
1-3GalNAc(6S)
1-4GlcUA
1-3GalNAc(4S). Digestion of
fraction Vb with the latter enzyme yielded three disaccharide units
(
Di-6S,
Di-4S, and
Di-diSD) and two unsaturated tetrasaccharides in molar proportions of 74:80:22:81:24, taking the
parent octasaccharide as 100 (Table III). The major and the minor
tetrasaccharides were detected at the elution positions of the
authentic tetrasaccharides
HexA(2S)
1-3GalNAc(6S)
1-4GlcUA
1-3GalNAc(6S) and
HexA
1-3GalNAc(6S)
1-4GlcUA
1-3GalNAc(4S), respectively. Fraction Vb was sensitive to CS-6-sulfatase (Table II),
indicating that the major compound in fraction Vb contained a
GalNAc(6S) residue at the reducing terminus. Product analysis in the
time-course digestion experiments for fraction Vb using chondroitinase
AC-II demonstrated a single tetrasaccharide (D-C) species (data not shown), which was most likely derived from the reducing side. Thus, the
major compound in fraction Vb was judged to contain the octasaccharide
structure with a C-A-D-C sequence shown below. The minor
tetrasaccharide structure
HexA
1-3GalNAc(6S)
1-4GlcUA
1-3GalNAc(4S) produced by
highly purified chondroitinase ABC was most likely derived from the
non-reducing side of the major octasaccharide due to the cleavage of
the central hexosaminidic linkage by the enzyme.
Fraction Vb--
Fraction Vb structure is
HexA
1-3GalNAc(6S)
1-4GlcUA
1-3GalNAc(4S)
1-4GlcUA(2S)
1-3GalNAc(6S)
1-4GlcUA
1-3GalNAc(6S).
Likewise, fraction Va-I was structurally analyzed. Digestion of
fraction Va-I with highly purified chondroitinase ABC yielded three
disaccharide units (
Di-6S,
Di-4S, and
Di-diSD) and
two unsaturated tetrasaccharides in molar proportions of
22:192:16:93:26, taking the parent octasaccharide as 100 (Table III).
The major and the minor tetrasaccharides were detected at the elution
positions of the authentic tetrasaccharides
HexA(2S)
1-3GalNAc(6S)
1-4GlcUA
1-3GalNAc(6S) and
HexA
1-3GalNAc(6S)
1-4GlcUA
1-3GalNAc(4S), respectively
(data not shown). The major octasaccharide component in fraction
Va-I contained the structure with an A-A-D-C sequence shown below. The
minor tetrasaccharide was probably derived from a minor octasaccharide whether it is from the reducing or non-reducing side.
Fraction Va-I--
Fraction Va-I structure is
HexA
1-3GalNAc(4S)
1-4GlcUA
1-3GalNAc(4S)
1-4GlcUA(2S)
1-3GalNAc(6S)
1-4GlcUA
1-3GalNAc(6S).
500-MHz 1H NMR Spectroscopy--
Fractions Vb, VIa,
VIb, and VIII were obtained in amounts large enough to allow solid
structural determination by 500-MHz 1H NMR spectroscopy in
addition to the enzymatic characterization described above. The
one-dimensional spectrum of fraction Vb recorded at 26 °C is shown
in Fig. 7. Signals at 4.4-5.5 ppm were
identified as H-1 resonances of the constituent saccharide residues by
comparison with the NMR spectra of unsaturated CS-tetrasaccharides (13) and CS-hexasaccharides (8). Since the H-1 resonances of
GalNAc residues and 2-O-sulfated GlcUA residues as well as the H-4
resonances of the 4-O-sulfated GalNAc residues of the
octasaccharides in all the fractions were obscured by the HOD signal
when spectra were recorded at 26 °C, they were also recorded at
15 °C for fraction VIb and at 60 °C for fractions Vb, VIa, and
VIII. The spectrum of fraction Vb recorded at 60 °C is shown in the
inset of Fig. 7. Proton signals in the one-dimensional
spectra were assigned using two-dimensional HOHAHA and COSY spectra as
described (8, 13).

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Fig. 7.
Structural reporter group regions of the
500-MHz 1H NMR spectrum of fraction Vb recorded in
2H2O at 26 °C. The inset
shows the spectrum recorded at 60 °C. The letters and
numbers refer to the corresponding residues in the
structures. U, GlcUA; U, HexA;
G, GalNAc.
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The two-dimensional HOHAHA and COSY spectra of fraction Vb are shown in
Fig. 8. The assignment pathway for
GalNAc-1 from H-1 to H-4 signals is shown in the COSY
spectrum measured at 26 °C (Fig. 8A). Beginning with the
GalNAc-1 H-1 proton signal at
5.213, a cross-peak
showing the connectivity between the H-1 and the H-2 (
4.280)
protons was found. The connectivities of the H-2 to the H-3 (
4.030)
and then to the H-4 resonance (
4.19) were sequentially established.
Although the connectivity of the H-4 to the H-5 resonances was not
observed, the H-5 signal (
4.340) was assigned in the
one-dimensional spectrum based on comparison with the corresponding
signals belonging to the tetra- and hexasaccharides of the same
series, such as
HexA
1-3GalNAc(6S)
1-4GlcUA
1-3GalNAc(4S) and
HexA
1-3GalNAc(6S)
1-4GlcUA
1-3GalNAc(6S)
1-4GlcUA
1-3GalNAc(4S) (8, 13). The cross-peaks of the H-5 with the H-6 and H-6
atoms
were observed at
4.19 and 4.14, respectively, as indicated in Fig.
8A. In a similar fashion, other proton signals were assigned starting with the H-1 resonance of each of the other sugar residues, except for
HexA-8 and GalNAc-5. The
HexA-8 H-1 signal (
5.181) was identified by the
sequential assignment of the cross-peaks starting with the
characteristic H-4 signal (
5.874) in the COSY spectrum. The
GalNAc-5 H-1 signal was assigned beginning with the H-4
resonance characteristic for a 4-O-sulfated GalNAc residue (see below). The anomeric resonances of the GalNAc and GlcUA residues were easily discriminated by the difference in chemical shifts. Anomeric resonances of the GalNAc-3, -5, and
-7 residues or the GlcUA-2, -4, and
-6 residues were distinguished from one another, respectively, as described below.

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Fig. 8.
Two-dimensional HOHAHA and COSY spectra of
fraction Vb. A, COSY spectrum recorded at 26 °C;
B, COSY spectrum recorded at 60 °C; C,
two-dimensional HOHAHA spectrum recorded at 60 °C. The
letters and numbers refer to the corresponding
residues in the structures. For abbreviations, see the legend to Fig.
7. In A, the assignment pathways of proton signals for
G-1, U-2, U-6, and U-8 are indicated, where cross-peaks belonging
to G-1 are boxed and those belonging to U-8 are
connected by bold lines. Cross-peaks between H-1 and H-2 for
G-1, G-3, G-5, and G-7 are also indicated. In B, the
assignment pathways of protons for U-4 and G-5 are indicated, where
cross-peaks belonging to the former are circled. In
C, protons belonging to G-3, G-5, and G-7 are indicated on
the H-3, H-2, and H-2 tracks, respectively, although the G-3 H-3 and
G-7 H-2 tracks overlapped each other.
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The anomeric resonances of GalNAc-3, 5 and
7 were clustered at
4.55-4.58 in the one-dimensional
spectrum (Fig. 7). Starting with the H-4 resonance (
4.74) of the
GalNAc-5 residue, other proton resonances of this residue
including the H-1 (
4.60) were assigned in the COSY spectrum
recorded at 60 °C (Fig. 8B). The interrelationship among
the GalNAc-5 H-1, H-2, H-3, and H-4 was confirmed in the
HOHAHA spectrum recorded at 60 °C (Fig. 8C). In our
previous study, the H-1 signal of GalNAc-5 of the four unsaturated CS-hexasaccharides was localized in a lower magnetic field
than that of GalNAc-3 in the same sequence, reflecting the substitution of the GalNAc-5 residue by the
HexA residue
at the non-reducing terminus (8). Therefore, the strong
cross-peak that was observed at
4.58 in the COSY spectrum and
assigned above to GalNAc-5 H-1 was judged to be overlapped
with that of GalNAc-7 of the octasaccharide in fraction Vb
(Fig. 8A). The remaining H-1 resonance at
4.55 in the
COSY spectrum (Fig. 8A) was assigned to GalNAc-3
and was found to have a similar chemical shift to those (
4.55) of
the 6-O-sulfated GalNAc-3 residues in the
hexasaccharides
HexA
1-3GalNAc(6S)
1-4GlcUA
1-3GalNAc(6S)
1-4GlcUA
1-3GalNAc(4S) and
HexA(2S)
1-3GalNAc(6S)
1-4GlcUA
1-3GalNAc(6S)
1-4GlcUA
1-3GalNAc(4S) (8). Since no cross-peak was detected between the
GalNAc-3 H-4 and H-5 or between the GalNAc-7
H-4 and H-5, the H-6 signal was found at
4.22 in the
one-dimensional spectrum by comparison with the corresponding signals
belonging to the tetrasaccharide
HexA
1-3GalNAc(6S)
1-4GlcUA
1-3GalNAc(4S) and the
hexasaccharide
HexA
1-3GalNAc(6S)
1-4GlcUA
1-3GalNAc(6S)
1-4GlcUA
1-3GalNAc(4S) (8, 13). The GalNAc-3 H-1 and H-6 were observed on
the H-3 track, and the GalNAc-7 H-1 and H-6 were
found on the H-2 track in the HOHAHA spectrum, although the two tracks
overlapped (Fig. 8C), supporting the connectivities between
the H-1 and the H-6 protons of each GalNAc residue.
GlcUA-2 was readily identified by the anomerization effects
caused by GalNAc-1, which resulted in doubling of the
anomeric proton signal of this GlcUA residue. The anomeric resonances
of GlcUA-2 were observed at
4.56 and 4.510 (Fig. 7).
The GlcUA-2 H-1 resonance of the
-anomer was assigned to
the latter based on the closer resemblance to that of the
GlcUA-4 H-1 (
4.458) that is
-linked, whereas the
remaining proton resonance at
4.56 was assigned to that of
GlcUA-2 H-1 of the
-anomer. The H-1 resonance at
4.458 was assigned to GlcUA-6 based on the similarity in
chemical shift to that of the corresponding GlcUA-4 H-1
(
4.464) of the hexasaccharide
HexA
1-3GalNAc(6S)
1-4GlcUA
1-3GalNAc(4S)
1-4GlcUA
1-3GalNAc(4S) that shares the same tetrasaccharide sequence on the non-reducing side (8). The anomeric signal observed at
4.76 in the
one-dimensional spectrum measured at 60 °C (Fig. 7,
inset) was assigned to the 2-O-sulfated
GlcUA-4 residue, based on the two-dimensional spectra and the similar chemical shift to that of the H-1 signals (
4.718 and 4.734) of the 2-O-sulfated GlcUA-4 residues
belonging to the authentic tetrasaccharides
GlcUA(2S)
1-3GalNAc(6S)
1-4GlcUA
1-3GalNAc(6S) and
GlcUA(2S)
1-3GalNAc(6S)
1-4GlcUA
1-3GalNAc(4S),
respectively (9). The assignment pathways of the proton signals
belonging to GlcUA-2, -6 and
HexA-8 are indicated in Fig. 8A, whereas that for GlcUA-4 is shown in Fig. 8B.
The resonances observed around
2.0 are characteristic of the
N-acetyl methyl protons of GalNAc. Three NAc signals
belonging to the four GalNAc residues were found for each
octasaccharide component. Generally, the NAc signal of a penultimate
GalNAc residue substituted by
HexA or GlcUA is located in a lower
magnetic field than that of the other GalNAc residues in the same
sequence. In contrast, that of the reducing GalNAc is observed in a
higher magnetic field than those of the other GalNAc residues (8-10, 13). Thus, the NAc signals belonging to the GalNAc-7 and
GalNAc-1 residues in each octasaccharide were readily
assigned, and the remaining signal with stronger intensity observed
between the NAc signals of GalNAc-1 and -7 was
assigned to both GalNAc-3 and -5 residues.
Modification by O-sulfation causes downfield shifts of
protons bound to the O-sulfated carbon atoms by
approximately 0.4-0.7 ppm (29). A downfield shift of the H-6, 6
(
0.4 ppm) of GalNAc-1, -3, and
-7, the H-4 (
0.5 ppm) of GalNAc-5, and
the H-2 (
0.7 ppm) of GlcUA-4 of the octasaccharide
in fraction Vb as compared with those of the non-sulfated residues of
authentic disaccharides (30) indicated 6-O-sulfation of
GalNAc-1, 3 and 7,
4-O-sulfation of GalNAc-5 and
2-O-sulfation of GlcUA-4, respectively. Likewise, the sulfation profiles of the major compounds in the other
octasaccharide fractions were firmly established (Table IV).
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Table IV
1H chemical shifts of structural reporter groups of the
constituent monosaccharides of the isolated octasaccharides
Chemical shifts are given in ppm downfield from internal sodium
4,4-dimethyl-4-silapentane-1-sulfonate but were actually measured indirectly to acetone in 2H2O ( 2.225) at 26 °C.
ND, not determined.
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DISCUSSION |
Previous studies indicated that mAb 473HD strongly reduced the
stimulatory effect of DSD-1-PG on neurite outgrowth and that shark
cartilage CS-C, which contains D disaccharide units, specifically inhibited the interactions between mAb 473HD and the CS chains of the
DSD-1-PG isolated from mouse brains (3). In this study, biological
activities and the structure of shark cartilage CS-D, which contains a
higher proportion of D units as compared with CS-C, were investigated.
Consistent with the interpretation that the DSD-1-epitope is required
for the neurite outgrowth promoting properties of DSD-1-PG, CS-D but
not CS-C adsorbed to the culture substrate increased the fraction of
neurite-bearing hippocampal neurons. For this reason, CS-D was selected
for further structural studies. The significant neurite outgrowth
promoting activity observed with squid cartilage CS-E was unexpected
and seemingly contradictory to the observation that it did not exhibit
significant inhibition of the mAb binding to DSD-1-PG (Fig. 1).
However, it is noteworthy that a tetrasaccharide isolated from shark
fin cartilage contained a unique sequence
HexA-GalNAc(4S,6S)-GlcUA(2S)-GalNAc(6S) with both an E and a D
disaccharide unit, and was recognized by mAb MO-225 (7). The intriguing
but puzzling observations concerning the CS-E activity remain to be
pursued.
Commercial shark cartilage CS-D is composed of four kinds of
disaccharide units (A, GlcUA
1-3GalNAc(4S); C,
GlcUA
1-3GalNAc(6S); D, GlcUA(2S)
1-3GalNAc(6S); and
E, GlcUA
1-3GalNAc(4S,6S)) in a molar ratio of 26.9:44.2:21.2:2.0 as
described in the specification provided by the manufacturer. A D unit
has been demonstrated on the immediate reducing side of an A unit,
forming an A-D tetrasaccharide sequence characteristic of shark
cartilage CS-D. This A-D sequence has been found in three minor
unsaturated hexasaccharides, corresponding to A-D-C, A-A-D, A-D-A
sequences, prepared by partial chondroitinase ABC digestion as well as
in one tetra- (A-D) and two hexasaccharides (D-A-D and C-A-D) prepared
by exhaustive testicular hyaluronidase digestion as summarized in Table
V. The propriety of the use of testicular
hyaluronidase with a transglycosylation activity for preparing
oligosaccharides has been discussed (10). In this study, eight
octasaccharides were isolated from the same source after partial
digestion with chondroitinase ABC, which has no transglycosylation
activity. These octasaccharides, composed of A, C, or D units but
devoid of an E unit, included two tetrasulfated (fractions I and II),
five pentasulfated (fractions IV, Va, Vb, VIa, and VIb), and one
hexasulfated (fraction VIII) structure. The A-D sequence was found in
five major fractions in the following sequences as shown in Table V;
A-A-D-C (Va), C-A-D-C (Vb), A-A-D-A (VIa), C-A-D-A (VIb), and D-A-D-A
(VIII). Thus, the A-D sequence was not unusual as previously presumed
(10), but appeared rather frequently. Although the octasaccharide
fraction analyzed in this study accounted for only approximately 7.5%
(w/w) of the starting CS-D, the revealed sequences indicated that the
disaccharide unit located on the immediate non-reducing side of an A-D
sequence was an A, C or D unit, whereas that on the reducing side was
not a D unit but an A or C unit, forming an A-D-A or an A-D-C
hexasaccharide sequence (i.e.
GlcUA-GalNAc(4S)-GlcUA(2S)-GalNAc(6S)-GlcUA-GalNAc(4S or 6S)).
In other words, D units were not found consecutively as a D-D
sequence unlike A-A or C-C sequences, as has been suggested based on
the structural studies of the tetrasaccharides generated by digestion
with specific chondroitinases AC-I, AC-II, and AC-III (31). The
characteristic hexasaccharide sequences most likely reflect the
biosynthetic mechanisms: specificities of 2-O- and 6-O-sulfotransferases responsible for producing a D
disaccharide unit. It remains to be determined whether such
hexasaccharide sequences are associated indeed with biological
functions such as a neurite outgrowth promoting activity, and whether
the D unit is contained in the CS chains of DSD-1-PG. A preliminary
disaccharide composition analysis after chondroitinase AC-II digestion
of a limited amount of purified DSD-1-PG showed a peak representing approximately 5% of the total disaccharide units at the elution position of the authentic
Di-diSD unit on
HPLC.3 The D unit has been
identified not only in shark cartilage, but also in mouse mast cells
derived from immune lymph nodes (6) and in the basement membrane of
mouse tooth germ for a brief period (1). Characteristic sequences
including D units may serve as functional domain structures recognized
by some protein ligands. Thus, the structural arrangement of D units
along CS chains, its function, and biosynthetic mechanism are of
particular interest.
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Table V
Oligosaccharides isolated from shark cartilage CS-D
The oligosaccharides that have been so far prepared by digestion of
commercial shark cartilage CS-D either with chondroitinase ABC or with
testicular hyaluronidase and isolated by gel filtration and subsequent
ion-exchange HPLC are summarized. Fr., fraction.
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Chondroitinase ABC, which is an indispensable analytical tool, has
recently become a subject for discussion in terms of its purity and
mechanism of action. Until recently, it had been believed to be an
endoeliminase that splits
1,4-galactosaminidic bonds of CS and
dermatan sulfate chains. However, our recent findings raised questions
concerning its purity and mechanism of action. The conventional
commercial preparation contained a minor protein component in addition
to a major one corresponding to the highly purified enzyme as revealed
by SDS-PAGE, and the latter enzyme did not digest tetrasaccharides (see
the second paragraph of "Results"; see also Ref. 13). In addition,
highly purified chondroitinase ABC preferentially acted on the
unsaturated hexasaccharides in an exolytic fashion, releasing only the
disaccharide units at the non-reducing ends (8). On the other hand, the
conventional preparation of chondroitinase ABC has been claimed to have
a non-random endolytic action (27). In contrast, the same preparation
has also been reported to release di-, tetra-, and hexasaccharides from
the non-reducing ends of CS polysaccharides in an exolytic fashion
(32). Thus, it had become necessary to define its endolytic or exolytic
actions to apply it as an analytical tool. Very recently, after the
completion of this work, Hamai et al. (26) reported purifying two chondroitinase ABCs from Proteus vulgaris and
crystallizing them. One enzyme, designated exochondroitinase ABC, was
demonstrated to be an exoeliminase preferentially acting on CS tetra-
and hexasaccharides to yield respective disaccharides, and corresponded
to the minor protein bond observed in the conventional enzyme
preparation by SDS-PAGE in our previous study (13). The other,
designated endochondroitinase ABC, corresponded to the highly purified
enzyme and was shown to act on polymer CS and dermatan sulfate chains
endolytically, whereas it did not act on tetrasaccharides, which is in
good agreement with our findings (13).
In this study, however, exolytic actions of this latter enzyme on
octasaccharides were revealed. Structural analysis of highly purified
chondroitinase ABC digests of fractions Vb and VIb indicated that the
enzyme split the individual octasaccharides into two tetrasaccharide
fragments acting on the central galactosaminidic linkages by
approximately 20% (Table III), whereas the other octasaccharides were
digested exclusively, if not completely, in an exolytic action releasing disaccharide units sequentially from the non-reducing ends.
The activity that produced two tetrasaccharides from an octasaccharide
is probably attributable to an endolytic activity as claimed by Hamai
et al. (26). In conclusion, highly purified chondroitinase
ABC (endochondroitinase ABC) that shows primarily an endolytic action
on CS/dermatan sulfate polymers (26) has a strong tendency to exert an
exolytic action when acting on oligosaccharides such as hexa- and
octasaccharides. It cleaves disaccharide units from the non-reducing
ends always when acting on hexasaccharides and can cleave
tetrasaccharide fragments probably in an endolytic fashion when acting
on octasaccharides depending on the sequences although the
disaccharide-releasing exolytic activity still dominates the
tetrasaccharide-releasing endolytic activity. Thus, the enzyme is
useful for determining the tetrasaccharide sequence located on the
reducing side of at least hexa-, octa-, and maybe larger oligosaccharides as well. Investigation of the size and sequence dependence of its action will make it more useful. It is interesting to
note that chondroitinase ABC from F. heparinum has also been reported to degrade CS and dermatan sulfate chains in an exolytic fashion, but beginning from the reducing end of the molecules (33) in
contrast to chondroitinase ABC from P. vulgaris used in this
study.
We thank Dr. Makiko Sugiura (Kobe
Pharmaceutical University) for recording the NMR spectra and Dr. Tonny
De Beer (University of Colorado) for the critical review of the
manuscript.