From the
4-Methylumbelliferyl-
It has been reported that addition of a
In the present study, a
novel Xyl-MU derivative produced by cultured human skin fibroblasts in
the presence of Xyl-MU was isolated and analyzed. Its structure was
sulfate-O-3GlcA
Mass spectra of
the Xyl-MU-induced oligosaccharide were obtained using an ion spray
mass spectrometer (Sciex API-III, Thornhill, Ontario, Canada) equipped
with an atmospheric pressure ionization source, as described
previously
(20) . Each sample (0.1 nmol/ml) was dissolved in 0.5
mM ammonium acetate/acetonitrile (50:50) and injected at 2
µl/min using a micro-HPLC syringe pump (pump 22, Harvard Apparatus
Inc., MA).
It has been reported that the addition of a
3-O-Sulfated glucuronic acid has been reported to be
present in chondroitin sulfate from cartilage of the king crab
(23) and a glycolipid from human peripheral
nerve
(14, 15) . It has been reported that glycosidases
have transglycosylation activity as a result of the reverse reaction of
hydrolysis
(24) . Therefore, it is possible that the sulfated
glucuronic acid residue was transferred from glycolipid, glycoprotein,
or glycosaminoglycan through the activity of exo-
A glycolipid with 3-O-sulfated glucuronic acid
was reported to react with the mouse monoclonal antibody HNK-1, raised
against human natural killer cells
(14, 15) , and
sulfate-3GlcA
Previously, we reported that some
oligosaccharides were present in the culture medium of human
fibroblasts, which were unrelated to glycosaminoglycan, as
GlcA
Data are
expressed as molar ratios relative to 4-methylumbelliferone. ND, not
detected.
-D-xyloside (Xyl-MU) was
added to the medium of cultured human skin fibroblasts. After
incubation, the culture medium was pooled, and the Xyl-MU-induced
oligosaccharides in the medium were purified by gel filtration
chromatography. A novel Xyl-MU derivative was obtained, in addition to
the previously reported Xyl-MU derivatives such as Gal-Gal-Xyl-MU,
Gal-Xyl-MU, Sia-Gal-Xyl-MU, GlcA-Xyl-MU, and Xyl-Xyl-MU. The novel
Xyl-MU derivative was purified using gel-filtration chromatography and
high performance liquid chromatography and then subjected to
carbohydrate composition analysis, enzymic digestion, Smith
degradation, and ion spray mass spectrometric analysis. The results
indicated that it was
sulfate-O-3GlcA
1-4Xyl
1-MU. The structure of
the nonreducing terminal of this Xyl-MU-induced oligosaccharide was the
same as that of the oligosaccharide chain of a human peripheral
nerve-derived glycolipid, reactive with the mouse monoclonal antibody
HNK-1, and this Xyl-MU-induced oligosaccharide also reacted with HNK-1.
These results suggest that the oligosaccharide, which is structurally
identical to that of human peripheral nerve-derived glycolipid
synthesized by nervous tissue and related to cell adhesion, is
synthesized also by mesenchymal cells.
-xyloside, such as
p-nitrophenyl-
-D-xyloside,
4-methylumbelliferyl-
-D-xyloside (Xyl-MU),
(
)
or benzyl-
-D-xyloside to cell culture medium
induces elongation of glycosaminoglycan chains, which is initiated by
the
-xyloside acting as a
primer
(1, 2, 3, 4, 5, 6, 7, 8, 9) .
In a previous study, we observed synthesis of 4-methylumbelliferone
(MU) derivatives by human skin fibroblasts cultured in medium
containing Xyl-MU. As a result, it was clarified that synthetic
intermediates of Xyl-MU-induced glycosaminoglycan
(glycosaminoglycan-MU), such as
Gal
1-3Gal
1-4Xyl-MU,
Gal
1-4Xyl-MU
(10) , in addition to
glycosaminoglycan-MU, were synthesized. Freeze et al.(11) reported that
Sia
2-3Gal
1-4Xyl
1-MU, which is related to
glycolipid sugar chains, was synthesized in cultures of Chinese hamster
ovary and human melanoma cells using Xyl-MU as a primer. This suggested
that
-xyloside could act as a primer for the synthesis of
glycolipid sugar chains as well as glycosaminoglycan chains in cultured
cells. Furthermore, Nakamura et al.(12) and Izumi
et al.(13) reported that
GlcA
1-4Xyl
1-MU and Xyl
1-4Xyl
1-MU, which
are unrelated to glycosaminoglycans or glycolipids, were elongated from
Xyl-MU. The presence of synthetic mechanisms for Xyl-MU-initiated
bioactive oligosaccharides, unrelated to glycosaminoglycan or
glycolipid, is of considerable interest.
1-4Xyl
1-MU. The structure of
the nonreducing terminal site of the oligosaccharide was the same as
that of the oligosaccharide chain of a human peripheral nerve-derived
glycolipid, which reacts with a mouse monoclonal antibody,
HNK-1
(14, 15) , and in fact this novel Xyl-MU-induced
oligosaccharide was found also to be reactive with HNK-1.
Materials
Eagle's minimum essential medium
(MEM), fetal bovine serum, and penicillin-streptomycin solution
(penicillin 100 milliunits/ml and streptomycin 100 µg/ml) were
purchased from Life Technologies Inc. Xyl-MU was purchased from Nacalai
Tesque Inc. (Kyoto, Japan). The 2-aminopyridine (PA) used was the same
as that reported previously
(16) . -Xylosidase (from
Aspergillus niger), sulfatase (from Helix pomatia),
alkaline phosphatase (from calf intestine), and saccharo-1,4-lactone
were purchased from Sigma. Sephadex G-15 was purchased from Pharmacia
Biotech Inc. The mouse monoclonal antibody HNK-1 was purchased from
Cosmo Bio Co. (Tokyo, Japan).
-Glucuronidase was purified from
rabbit liver using p-nitrophenyl-
-D-glucuronide
as a substrate
(17) . The GlcA-Xyl-MU standard was the same as
that reported previously
(12) . Standard PA-Xyl was purchased
from Takara Shuzo Co. (Kyoto, Japan). PA-GlcA was synthesized and
purified according to the method of Takemoto et
al.
(18) .
Cell Culture
Human skin fibroblasts were cultured
in Eagle's MEM containing 10% fetal bovine serum and 1%
penicillin-streptomycin solution at 37 °C in a humidified air
atmosphere containing 5% CO as described
previously
(10) . The cells were plated at a density of 2
10
/100-mm plastic dish (Corning Glass Works, Corning, NY)
and then subcultured after being grown to confluence. Fibroblasts at
passage 4-7 were used for the study. Confluent cultured
fibroblasts were incubated for 72 h in Eagle's MEM containing 0.5
mM Xyl-MU at 37 °C, and the culture medium was recovered.
High Performance Liquid Chromatography (HPLC)
A
high performance liquid chromatograph (Hitachi L-6200, Hitachi Co.,
Tokyo, Japan) connected to a fluorescence spectrometer (Hitachi F-1050,
Hitachi Co.) was used. Xyl-MU derivatives were detected by their
fluorescence at an excitation wavelength of 325 nm and an emission
wavelength of 380 nm. PA-monosaccharides were detected by their
fluorescence at an excitation wavelength of 320 nm and an emission
wavelength of 400 nm. Gel-filtration HPLC of the MU-derivatives was
performed using a Shodex OHpak KB-803 column (8 300 mm, Shoko
Co., Tokyo, Japan) with 0.2 M NaCl as the solvent at a column
temperature of 30 °C and a flow rate of 1 ml/min. Reverse-phase
HPLC was performed using a Shodex C18-5B column (4.6
250
mm, Shoko) with a linear gradient of distilled water-acetonitrile. The
PA-sugars were identified by analysis using an Ultrasphere ODS column
(4.6
250 mm, Beckman Instruments, Inc., Palo Alto, CA) with 1%
acetonitrile in 0.25 M sodium citrate buffer (pH 4.0) as the
solvent
(16) .
Enzymic Digestion
-Glucuronidase digestion of
the Xyl-MU-induced oligosaccharide was performed in 0.1 M
sodium acetate buffer (pH 4.5) at 37 °C for 12 h, as described
previously
(17) ; sulfatase digestion was performed in 0.1
M sodium acetate buffer (pH 4.5) at 37 °C for 4 h;
alkaline phosphatase digestion was performed in 0.1 M Tris-HCl
buffer (pH 8.0) at 37 °C for 4 h; and xylosidase digestion was
performed in 0.1 M sodium acetate buffer (pH 4.0) at 37 °C
for 6 h.
Smith Degradation
Smith degradation of the
Xyl-MU-induced oligosaccharide was performed according to the method of
Noble and Sturgeon
(19) . An aliquot of purified sample was
dissolved in 200 µl of 0.1 M sodium acetate buffer (pH
4.5) containing 0.015 M NaIO and incubated at 4
°C for 120 h in the dark. Forty microliters of ethylene glycol was
added and allowed to react for 1 h at 20 °C. This was followed by
the addition of 60 µl of 0.25 M NaBH
in 0.1
M sodium borate buffer (pH 8.0), and the mixture was allowed
to react for 18 h. Then, the pH was adjusted to 4.0 by the addition of
acetic acid, and the solution was evaporated to dryness repeatedly in
the presence of methanol under reduced pressure to remove the borate.
The resulting reduced samples were mixed with 0.1 N HCl and
allowed to stand at 20 °C for 30 min, after which each reaction was
stopped by the addition of an equal volume of 0.1 N NaOH.
Analytical Methods
In order to determine their
sugar composition, samples were hydrolyzed in 2 N HCl at 100
°C for 4 h and then pyridylaminated, as described previously (16).
The resulting PA-monosaccharides were identified and quantified by HPLC
analysis on an Ultrasphere ODS column
(16) .
Dot-blot Analysis
Dot-blot analysis of the
Xyl-MU-induced oligosaccharide was performed using a modification of
the method described by Sorrell et al.(21) . The
Xyl-MU-induced oligosaccharide, GlcA1-4Xyl
1-MU and
Xyl-MU (1 nmol each) were applied to the same sheet of nitrocellulose.
The sheet was incubated with phosphate-buffered saline (PBS) containing
5% bovine serum albumin at 20 °C for 1 h and then washed with PBS
and incubated in PBS containing HNK-1 monoclonal antibody (10
µg/ml) at 4 °C for 24 h. The sheet was then washed with PBS
containing 0.05% Tween 20, and incubated with peroxidase-conjugated
goat anti-mouse antibody at 20 °C for 2 h. After washing with PBS
containing 0.05% Tween 20, H
O
and
2,2`-azino-di(3-ethyl-benzthiazoline sulfonate) were added.
Solid Phase Binding Assay
Binding of the
Xyl-MU-induced oligosaccharide to HNK-1 monoclonal antibody was
determined using a modification of a solid-phase binding assay
procedure
(22) . A Corning® enzyme-linked immunosorbent assay
plate was coated with 0, 5, 10, or 20 µg of HNK-1 monoclonal
antibody (dissolved in 100 µl of 0.1 M carbonate buffer,
pH 9.6) and incubated at 37 °C for 3 h. Then, the plate was washed
with PBS and blocked with 100 µl of PBS containing 2% bovine serum
albumin at 37 °C for 2 h. The plate was washed with PBS 3 times.
The Xyl-MU-induced oligosaccharide and GlcA1-4Xyl
1-MU
(40 pmol each), dissolved in 100 µl of PBS, were then added, and
the plate was incubated at 4 °C for 24 h, followed by washing with
PBS containing 0.05% Tween 20 3 times. In order to solubilize the
Xyl-MU-induced oligosaccharides binding to HNK-1, 50 µl of 0.1
M glycine-HCl buffer (pH 2.5) was added, and the plate was
incubated at 4 °C for 48 h. To estimate the HNK-1-binding
Xyl-MU-induced oligosaccharides, the recovered Xyl-MU-induced
oligosaccharides were subjected to HPLC.
Production of the Xyl-MU-induced Oligosaccharide by
Cultured Human Skin Fibroblasts
Human skin fibroblasts were
incubated for 72 h in Eagle's MEM containing 0.5 mM
Xyl-MU at 37 °C. The pooled medium (20 l) was dialyzed, and the
dialyzable fraction was concentrated with a lyophilizer. In order to
purify the MU derivatives, the concentrated sample was subjected to gel
filtration on a Sephadex G-15 column (4.1 150 cm), which was
equilibrated and eluted with distilled water at a flow rate of 50 ml/h
(Fig. 1). The fluorescence intensity of the eluate was monitored
at excitation and emission wavelengths of 325 and 380 nm, respectively.
The presence of Sia-Gal-Xyl-MU (Fig. 1, arrow1), GlcA-Xyl-MU (Fig. 1, arrow3), Gal-Gal-Xyl-MU (Fig. 1, arrow4), Gal-Xyl-MU (Fig. 1, arrow5),
and Xyl-MU (Fig. 1, arrow6) was confirmed
using gel-filtration HPLC (Shodex OHpak KB-803). Moreover, a fraction
containing an unknown fluoro-labeled oligosaccharide was detected
(Fig. 1, arrow2). Then, the fractions
containing the novel Xyl-MU-induced oligosaccharide were collected and
purified, and the structure of the Xyl-MU-induced oligosaccharide was
analyzed.
Figure 1:
Gel filtration chromatography on
Sephadex G-15 of the dialyzable fraction of the culture medium. Twenty
liters of pooled culture medium concentrated by lyophilization was
dialyzed against distilled water; the dialyzable fraction was
concentrated and applied to a Sephadex G-15 column (4.1 150
cm), which was equilibrated and eluted with distilled water at a flow
rate of 50 ml/h, and 25-ml fractions were collected. The eluate was
monitored with a fluorescence detector at excitation and emission
wavelengths of 325 and 380 nm, respectively. The arrows denote
the Xyl-MU-induced oligosaccharides: 1, Sia-Gal-Xyl-MU;
2, novel Xyl-MU-induced oligosaccharide; 3,
GlcA-Xyl-MU; 4, Gal-Gal-Xyl-MU; 5, Gal-Xyl-MU;
6, Xyl-MU. V
, void volume; V,
total bed volume. The fractions containing the novel Xyl-MU-induced
oligosaccharide were recovered.
Isolation of the Xyl-MU-induced
Oligosaccharide
The fractions containing the Xyl-MU-induced
oligosaccharide were recovered, concentrated, and applied to a Sephadex
G-15 column (2.1 27 cm) equilibrated with 0.1 M acetic
acid (Fig. 2). In addition to the peak of Sia-Gal-Xyl-MU, another
fluoro-labeled peak (Fig. 2, peak2) was
obtained. This peak was detected as a single one on reverse-phase HPLC
using Shodex C18-5B, and was recovered, rechromatographed with
the same column, and used for analyses as a purified sample
(Fig. 3). The yield of the purified Xyl-MU-induced
oligosaccharide was 300 nmol from 20 liters of the pooled medium.
Figure 2:
Gel-filtration rechromatography on
Sephadex G-15. The recovered fractions from Sephadex G-15 (Fig. 1) of
the dialyzable fraction of the culture medium were applied to a
Sephadex G-15 column (2.1 27 cm) equilibrated and eluted with
0.1 M acetic acid at a flow rate of 36 ml/h, and 3-ml
fractions were collected. The eluate was monitored with a fluorescence
detector. The arrows denote the Xyl-MU-induced
oligosaccharides: 1, Sia-Gal-Xyl-MU; 2, novel
Xyl-MU-induced oligosaccharide. V
, void volume;
V, total bed volume. The fractions containing peak2 were recovered.
Figure 3:
Reverse-phase HPLC of the Xyl-MU-induced
oligosaccharide. HPLC was performed using a Shodex C18-5B column
(4.6 250 mm) with a linear gradient of distilled
water-acetonitrile, and the eluate was monitored with a fluorescence
detector. The Xyl-MU-induced oligosaccharide was included in the
fractions indicated by the bar. The fractions were collected
and used for analyses as a purified sample. Solidline is fluorescence intensity, and dashedline is
concentration of acetonitrile (%).
Carbohydrate Composition of the Xyl-MU-induced
Oligosaccharide
An aliquot of the purified Xyl-MU-induced
oligosaccharide was subjected to acid hydrolysis in 2 N HCl at
100 °C for 4 h and then pyridylaminated. The resulting PA-sugars
were identified and quantified using an Ultrasphere ODS column. The
Xyl-MU-induced oligosaccharide was composed of MU, xylose, and
glucuronic acid in molar ratios of 1.0:0.73:1.1 but contained no
galactose, glucosamine, and galactosamine ().
Enzymic Digestion of the Xyl-MU-induced
Oligosaccharide
Enzymic digestion of the purified Xyl-MU-induced
oligosaccharide was performed. An aliquot of the oligosaccharide was
incubated with -glucuronidase and then subjected to gel-filtration
HPLC on a Shodex OHpak KB-803 column. The oligosaccharide did not shift
from the control position after digestion (Fig. 4b), nor
did it shift after digestion with alkaline phosphatase (data not
shown). The peak of the oligosaccharide was shifted to a position
corresponding to GlcA-Xyl-MU after sulfatase digestion
(Fig. 4c). The results of analysis with Smith
degradation and mass spectrometry of the sulfatase-digestion product
indicated that the glucuronic acid residue was linked at the C-4
position of xylose on this oligosaccharide, as already reported for
GlcA-Xyl-MU
(12) . The sulfatase digestion product was incubated
with
-glucuronidase, and the elution time of the oligosaccharide
after digestion was shifted to that of Xyl-MU (Fig. 4d).
The digestion product was incubated with
-xylosidase, and the peak
was shifted to that of MU (data not shown). These results indicated
that the sequence of the carbohydrate components of this Xyl-MU-induced
oligosaccharide was sulfate-GlcA-Xyl-MU.
Figure 4:
Analysis by HPLC of the Xyl-MU-induced
oligosaccharide after incubation with various enzymes. The column used
was a Shodex OHpak KB-803 (8 300 mm), which was eluted with 0.2
M NaCl at flow rate of 1 ml/min. The eluate was monitored with
a fluorescence detector. a, before enzymic digestion;
b, after incubation with
-glucuronidase; c,
after incubation with sulfatase; d, after incubation with
-glucuronidase following sulfatase digestion. The arrows denote the positions of Xyl-MU derivatives: 1, novel
Xyl-MU-induced oligosaccharide; 2, GlcA-Xyl-MU; 3,
Xyl-MU.
Mass Spectrometry of the Xyl-MU-induced
Oligosaccharide
An aliquot of the purified Xyl-MU-induced
oligosaccharide was subjected to ion spray mass spectrometry. The
spectrum showed a major peak at m/z 563
(Fig. 5a), and therefore this peak was analyzed as the
precursor ion for fragmentation by tandem mass spectrometric analysis.
Four product ion peaks with mass numbers of 97, 175, 307, and 483 were
obtained and identified as (sulfuric acid-H),
(MU-H)
, ((Xyl-MU)-H)
, and
((GlcA-Xyl-MU)-H)
, respectively
(Fig. 5b). Thus, the structure of this Xyl-MU-induced
oligosaccharide was identified as sulfate-GlcA-Xyl-MU.
Figure 5:
Mass
spectra of the Xyl-MU-induced oligosaccharide. a, the
Xyl-MU-induced oligosaccharide; b, product ions on tandem mass
spectrometric analysis spectrum of Xyl-MU-induced oligosaccharide using
m/z 563 as the precursor ion
(a).
Smith Degradation of the Xyl-MU-induced
Oligosaccharide
From the analytical results described above, the
Xyl-MU-induced oligosaccharide appeared to be GlcA-Xyl-MU with a
sulfated glucuronic acid residue. Therefore, its structure was most
likely to be sulfate-O-2GlcA1-4Xyl
1-MU,
sulfate-O-3GlcA
1-4Xyl
1-MU, or
sulfate-O-4GlcA
1-4Xyl
1-MU (Fig. 6,
a-c). In order to examine the sulfate to glucuronic acid
linkage position, an aliquot of the purified Xyl-MU-induced
oligosaccharide was subjected to Smith degradation. The degradation
product was hydrolyzed in 2 N HCl at 100 °C for 4 h,
pyridylaminated, and analyzed by HPLC on an Ultrasphere ODS column.
PA-glucuronic acid was detected. If the sulfate had been linked at any
position other than the C-3 position of glucuronic acid, the glucuronic
acid would have been cleaved and thus not detected as PA-glucuronic
acid. Therefore, this result indicated that the structure of the
oligosaccharide was sulfate-O-3GlcA
1-4Xyl
1-MU
(Fig. 6b).
Figure 6:
Possible structures of the Xyl-MU-induced
oligosaccharide. a,
sulfate-O-2GlcA1-4Xyl
1-MU; b,
sulfate-O-3GlcA
1-4Xyl
1-MU; c,
sulfate-O-4GlcA
1-4Xyl
1-MU.
Binding of the Xyl-MU-induced Oligosaccharide for
HNK-1
Dot-blot analysis of the Xyl-MU-induced oligosaccharide
was performed. HNK-1 monoclonal antibody bound to
sulfate-O-3GlcA1-4Xyl
1-MU but not to
GlcA
1-4Xyl
1-MU and Xyl-MU (Fig. 7). Binding of
the Xyl-MU-induced oligosaccharide to HNK-1 was also analyzed using
solid-phase binding assay. As indicated on Fig. 8, the affinity
of sulfate-O-3GlcA
1-4Xyl
1-MU for HNK-1 was
found to be dose-dependent, but that of GlcA
1-4Xyl
1-MU
was not detected.
Figure 7:
Dot-blot analysis of the Xyl-MU-induced
oligosaccharide against HNK-1 on a nitrocellulose membrane. The
Xyl-MU-induced oligosaccharide, GlcA1-4Xyl
1-MU, and
Xyl-MU (1 nmol each) were applied to the same sheet of nitrocellulose.
The nitrocellulose sheet was blocked and incubated with HNK-1
monoclonal antibody at 4 °C for 24 h, and then incubated with
peroxidase-conjugated goat anti-mouse antibody at 20 °C for 2 h.
After washing, peroxidase substrate was added. a,
sulfate-O-3GlcA
1-4Xyl
1-MU; b,
GlcA
1-4Xyl
1-MU; c,
Xyl-MU.
Figure 8:
Solid-phase binding assay of the
Xyl-MU-induced oligosaccharide against HNK-1. The Xyl-MU-induced
oligosaccharide and GlcA1-4Xyl
1-MU (40 pmol each),
dissolved in 100 µl of PBS, were added to a Corning®
enzyme-linked immunosorbent assay plate coated with 0, 5, 10, or 20
µg of HNK-1 monoclonal antibody, and then the plate was incubated
at 4 °C for 24 h. The plate was then washed 3 times with PBS
containing 0.05% Tween 20, and Xyl-MU-induced oligosaccharides were
solubilized by incubation with 0.1 M glycine-HCl buffer (pH
2.5) at 4 °C for 48 h. The recovered Xyl-MU-induced
oligosaccharides were applied to a Shodex OHpak KB-803 column, and the
Xyl-MU-induced oligosaccharide (
) and
GlcA
1-4Xyl
1-MU (
) were detected on the basis of
their fluorescence intensity. The insets show HPLC
chromatograms of the Xyl-MU-induced oligosaccharides recovered from the
well coated with 20 µg of HNK-1. a,
sulfate-O-3GlcA
1-4Xyl
1-MU; b,
GlcA
1-4Xyl
1-MU
-xyloside to
cell culture medium induces elongation of glycosaminoglycan chains,
which is initiated by the
-xyloside acting as a
primer
(1, 2, 3, 4, 5, 6, 7, 8, 9) .
Several Xyl-MU derivatives, as well as glycosaminoglycan-MU, have been
obtained by incubating human skin fibroblasts in the presence of
Xyl-MU
(10) . In this study, human skin fibroblasts were cultured
in the presence of Xyl-MU, a large quantity of medium was recovered and
concentrated, and a minor unknown Xyl-MU derivative was detected by
HPLC. This Xyl-MU derivative was purified using gel filtration
chromatography and HPLC and then subjected to carbohydrate composition
analysis, enzyme digestion, Smith degradation, and ion spray mass
spectrometric analysis. The results indicated that its structure was
sulfate-O-3GlcA
1-4Xyl
1-MU.
-glucuronidase.
However, GlcA
1-4Xyl
1-MU, considered to be a mediator of
sulfate-O-3GlcA
1-4Xyl
1-MU, was also detected
in the culture medium, and its production was not inhibited by the
addition of a
-glucuronidase inhibitor
(12) . Accordingly,
sulfate-O-3GlcA
1-4Xyl-MU was considered to be a
product of human skin fibroblasts utilizing Xyl-MU as a primer. This is
the first report of an oligosaccharide having sulfated glucuronic acid
at the nonreducing terminal derived from cultured human skin
fibroblasts.
1-4Xyl
1-MU also reacted with HNK-1.
Karamanos et al.
(25) reported that HNK-1 reacted with
chondroitin sulfate from squid skin and that oversulfated
-disaccharides containing 3-sulfated glucuronic acid
inhibited the reaction. The glycolipid reactive with HNK-1 and its
sugar chain have been reported to inhibit the outgrowth of neurites and
astrocytic processes and to decrease the adhesion of neurons and
astrocytes
(26) . The HNK-1-reactive glycolipid has been reported
to be a ligand for selectins, cell adhesion molecules implicated in
leukocyte-endothelial cell adhesion, and platelet adhesion
(27) .
Although the structures of the HNK-1-reactive sugar chains have not
been precisely determined, some glycoproteins have been reported to
react with HNK-1 and to act as adhesion molecules in the nervous
system
(28, 29, 30, 31) . The
HNK-1-reactive oligosaccharide has been considered to be related to the
nervous and immune systems, but it was revealed in this study that an
HNK-1-reactive oligosaccharide was also produced by fibroblasts, which
are mesenchymal cells. This finding indicates that the HNK-1-reactive
oligosaccharide may be involved in the differentiation, growth, and
adhesion of mesenchymal cells.
1-4Xyl
1-MU
(12) and
Xyl
1-4Xyl
1-MU
(13) . Freeze et al.(11) reported that
Sia
2-3Gal
1-4Xyl
1-MU was synthesized in
cultures of Chinese hamster ovary and human melanoma cells with Xyl-MU
as a primer, and they concluded that
Sia
2-3Gal
1-4Xyl
1-MU was related to the
synthesis of glycolipid and not to that of
glycosaminoglycan
(11) . This Xyl-MU-induced oligosaccharide was
considered to be derived from GlcA-Xyl-MU
(12) . The structure,
3-O-sulfate-GlcA, is present in glycosaminoglycans
(23) and glycolipids
(14, 15) . Thus it seems that
there are various pathways of oligosaccharide synthesis initiated by
Xyl-MU, such as glycosaminoglycan-MU synthesis, glycolipid
oligosaccharide synthesis, and the unknown oligosaccharide synthetic
pathway (GlcA-Xyl-MU and Xyl-Xyl-MU). At present, it is unclear why so
many kinds of oligosaccharides are derived from Xyl-MU.
Table:
Carbohydrate composition of 4-methylumbelliferyl
-D-xyloside-induced oligosaccharide
-D-xyloside; MU,
4-methylumbelliferone; Xyl, xylose; Sia, sialic acid; HPLC, high
performance liquid chromatography; MEM, minimum essential medium; PA,
2-aminopyridine; PBS, phosphate-buffered saline.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.