©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
HNK-1-reactive Novel Oligosaccharide, Sulfate-O-3GlcA1 4Xyl1-(4-methylumbelliferone), Synthesized by Cultured Human Skin Fibroblasts (*)

Shigeru Shibata (1), Keiichi Takagaki (1), Toshiya Nakamura (1), Jun Izumi (1), Kaoru Kojima (2), Ikunoshin Kato (2), Masahiko Endo (1)(§)

From the (1) Department of Biochemistry, Hirosaki University School of Medicine, 5 Zaifu-cho, Hirosaki 036, Japan and the (2) Research Institute for Glycotechnology, 82-4 Zaifu-cho, Hirosaki 036, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

4-Methylumbelliferyl--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-3GlcA1-4Xyl1-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.


INTRODUCTION

It has been reported that addition of a -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 Gal1-3Gal1-4Xyl-MU, Gal1-4Xyl-MU (10) , in addition to glycosaminoglycan-MU, were synthesized. Freeze et al.(11) reported that Sia2-3Gal1-4Xyl1-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 GlcA1-4Xyl1-MU and Xyl1-4Xyl1-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.

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-3GlcA1-4Xyl1-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.


EXPERIMENTAL PROCEDURES

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) .

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).

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, GlcA1-4Xyl1-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, HO 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 GlcA1-4Xyl1-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.


RESULTS

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-2GlcA1-4Xyl1-MU, sulfate-O-3GlcA1-4Xyl1-MU, or sulfate-O-4GlcA1-4Xyl1-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-3GlcA1-4Xyl1-MU (Fig. 6b).


Figure 6: Possible structures of the Xyl-MU-induced oligosaccharide. a, sulfate-O-2GlcA1-4Xyl1-MU; b, sulfate-O-3GlcA1-4Xyl1-MU; c, sulfate-O-4GlcA1-4Xyl1-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-3GlcA1-4Xyl1-MU but not to GlcA1-4Xyl1-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-3GlcA1-4Xyl1-MU for HNK-1 was found to be dose-dependent, but that of GlcA1-4Xyl1-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, GlcA1-4Xyl1-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-3GlcA1-4Xyl1-MU; b, GlcA1-4Xyl1-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 GlcA1-4Xyl1-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 GlcA1-4Xyl1-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-3GlcA1-4Xyl1-MU; b, GlcA1-4Xyl1-MU




DISCUSSION

It has been reported that the addition of a -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-3GlcA1-4Xyl1-MU.

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--glucuronidase. However, GlcA1-4Xyl1-MU, considered to be a mediator of sulfate-O-3GlcA1-4Xyl1-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-3GlcA1-4Xyl-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.

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-3GlcA1-4Xyl1-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.

Previously, we reported that some oligosaccharides were present in the culture medium of human fibroblasts, which were unrelated to glycosaminoglycan, as GlcA1-4Xyl1-MU (12) and Xyl1-4Xyl1-MU (13) . Freeze et al.(11) reported that Sia2-3Gal1-4Xyl1-MU was synthesized in cultures of Chinese hamster ovary and human melanoma cells with Xyl-MU as a primer, and they concluded that Sia2-3Gal1-4Xyl1-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

Data are expressed as molar ratios relative to 4-methylumbelliferone. ND, not detected.



FOOTNOTES

*
This work was supported by Grants-in-aid 04454153, 05274107, and 05680518 for Scientific Research from the Ministry of Education, Science and Culture of Japan and from the Mizutani Foundation for Glycoscience. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Fax: 8000-0172-32-3796.

The abbreviations used are: Xyl-MU, 4-methylumbelliferyl--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.


REFERENCES
  1. Okayama, M., Kimata, K., and Suzuki, S.(1973) J. Biochem.(Tokyo) 74, 1069-1073 [Medline] [Order article via Infotrieve]
  2. Schwartz, N. B., Galligani, L., Ho, P.-L., and Dorfman, A.(1974) Proc. Natl. Acad. Sci. U. S. A. 71, 4047-4051 [Abstract]
  3. Fukunaga, Y., Sobue, M., Suzuki, N., Kushida, H., Suzuki, S., and Suzuki, S.(1975) Biochim. Biophys. Acta 381, 443-447 [Medline] [Order article via Infotrieve]
  4. Robinson, H. C., Brett, M. J., Tralaggan, P. J., Lowther, D. A., and Okayama, M.(1975) Biochem. J. 148, 25-34 [Medline] [Order article via Infotrieve]
  5. Kato, Y., Kimata, K., Ito, K., Karasawa, K., and Suzuki, S.(1978) J. Biol. Chem. 253, 2784-2789 [Medline] [Order article via Infotrieve]
  6. Kolset, S. O., Ehlorsson, J., Kjellén, L., and Lindahl, U.(1986) Biochem. J. 238, 209-216 [Medline] [Order article via Infotrieve]
  7. Sobue, M., Habuchi, H., Ito, K., Yonekura, H., Oguri, K., Sakurai, K., Kamohara, S., Ueno, Y., Noyori, R., and Suzuki, S.(1987) Biochem. J. 241, 591-601 [Medline] [Order article via Infotrieve]
  8. Lugemwa, F. N., and Esko, J. D.(1991) J. Biol. Chem. 266, 6674-6677 [Abstract/Free Full Text]
  9. Fransson, L.-Å., Havsmark, B., Sakurai, K., and Suzuki, S.(1992) Glycoconj. J. 9, 45-55 [Medline] [Order article via Infotrieve]
  10. Takagaki, K., Nakamura, T., Kon, A., Tamura, S., and Endo, M. (1991) J. Biochem.(Tokyo) 109, 514-519 [Abstract]
  11. Freeze, H. H., Sampath, D., and Varki, A.(1993) J. Biol. Chem. 268, 1618-1627 [Abstract/Free Full Text]
  12. Nakamura, T., Izumi, J., Takagaki, K., Shibata, S., Kojima, K., Kato, I., and Endo, M.(1994) Biochem. J. 304, 731-736 [Medline] [Order article via Infotrieve]
  13. Izumi, J., Takagaki, K., Nakamura, T., Shibata, S., Kojima, K., Kato, I., and Endo, M.(1994) J. Biochem.(Tokyo) 116, 524-529 [Abstract]
  14. Chou, D. K. H., Ilyas, A. A., Evans, J. E., Costello, C., Quarles, R. H., and Jungalwala, F. B.(1986) J. Biol. Chem. 261, 11717-11725 [Abstract/Free Full Text]
  15. Ariga, T., Kohriyama, T., Freddo, L., Latov, N., Saito, M., Kon, K., Ando, S., Suzuki, M., Hemling, M. E., Rinehart, K. L., Jr., Kusunoki, S., and Yu, R. K.(1987) J. Biol. Chem. 262, 848-853 [Abstract/Free Full Text]
  16. Takagaki, K., Nakamura, T., Kawasaki, H., Kon, A., Ohishi, S., and Endo, M.(1990) J. Biochem. Biophys. Methods 21, 209-215 [Medline] [Order article via Infotrieve]
  17. Nakamura, T., Takagaki, K., Majima, M., Kimura, S., Kubo, K., and Endo, M.(1990) J. Biol. Chem. 265, 5390-5397 [Abstract/Free Full Text]
  18. Takemoto, H., Hase, S., and Ikenaka, T.(1985) Anal. Biochem. 145, 245-250 [Medline] [Order article via Infotrieve]
  19. Noble, D. W., and Sturgeon, R. J.(1970) Carbohydr. Res. 12, 448-452
  20. Takagaki, K., Kojima, K., Majima, M., Nakamura, T., Kato, I., and Endo, M.(1992) Glycoconj. J. 9, 174-179 [Medline] [Order article via Infotrieve]
  21. Sorrell, J. M., Mahmoodian, F., and Caterson, B.(1988) Cell Tissue Res. 252, 523-531 [Medline] [Order article via Infotrieve]
  22. Cantarero, L. A., Butler, J. E., and Osborne, J. W.(1980) Anal. Biochem. 105, 375-382 [Medline] [Order article via Infotrieve]
  23. Seno, N., and Murakami, K.(1982) Carbohydr. Res. 103, 190-194
  24. Saitoh, H., Takagaki, K., Majima, M., Nakamura, T., Matsuki, A., Kasai, M., Narita, H., and Endo, M.(1995) J. Biol. Chem. 270, 3741-3747 [Abstract/Free Full Text]
  25. Karamanos, N. K., Aletras, A. J., Antonopoulos, C. A., and Hjerpe, A. (1994) Biochimie(Paris) 76, 79-82 [Medline] [Order article via Infotrieve]
  26. Künemund, V., Jungalwala, F. B., Fischer, G., Chou, D. K. H., Keilhauer, G., and Schachner, M.(1988) J. Cell Biol. 106, 213-223 [Abstract]
  27. Needham, L. K., and Schnaar, R. L.(1993) Proc. Natl. Acad. Sci. U. S. A. 90, 1359-1363 [Abstract]
  28. McGarry, R. C., Helfand, S. L., Quarles, R. H., and Roder, J. C.(1983) Nature 306, 376-378 [Medline] [Order article via Infotrieve]
  29. Kruse, J., Mailhammer, R., Wernecke, H., Faissner, A., Sommer, I., Goridis, C., and Schachner, M.(1984) Nature 311, 153-155 [Medline] [Order article via Infotrieve]
  30. Kruse, J., Keilhauer, G., Faissner, A., Timpl, R., and Schachner, M. (1985) Nature 316, 146-148 [Medline] [Order article via Infotrieve]
  31. Bollensen, E., and Schachner, M.(1987) Neurosci. Lett. 82, 77-82 [CrossRef][Medline] [Order article via Infotrieve]

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