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• W2029352605 abstract "4-Methylumbelliferyl-β-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-3GlcAβ1-4Xylβ1-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. 4-Methylumbelliferyl-β-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-3GlcAβ1-4Xylβ1-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. INTRODUCTIONIt has been reported that addition of a β-xyloside, such as p-nitrophenyl-β-D-xyloside, 4-methylumbelliferyl-β-D-xyloside (Xyl-MU),1( 1The abbreviations used are: Xyl-MU4-methylumbelliferyl-β-D-xylosideMU4-methylumbelliferoneXylxyloseSiasialic acidHPLChigh performance liquid chromatographyMEMminimum essential mediumPA2-aminopyridinePBSphosphate-buffered saline.) or benzyl-β-D-xyloside to cell culture medium induces elongation of glycosaminoglycan chains, which is initiated by the β-xyloside acting as a primer (1Okayama M. Kimata K. Suzuki S. J. Biochem.(Tokyo). 1973; 74: 1069-1073PubMed Google Scholar, 2Schwartz N.B. Galligani L. Ho P.-L. Dorfman A. Proc. Natl. Acad. Sci. U. S. A. 1974; 71: 4047-4051Crossref PubMed Scopus (143) Google Scholar, 3Fukunaga Y. Sobue M. Suzuki N. Kushida H. Suzuki S. Suzuki S. Biochim. Biophys. Acta. 1975; 381: 443-447Crossref PubMed Scopus (33) Google Scholar, 4Robinson H.C. Brett M.J. Tralaggan P.J. Lowther D.A. Okayama M. Biochem. J. 1975; 148: 25-34Crossref PubMed Scopus (111) Google Scholar, 5Kato Y. Kimata K. Ito K. Karasawa K. Suzuki S. J. Biol. Chem. 1978; 253: 2784-2789Abstract Full Text PDF PubMed Google Scholar, 6Kolset S.O. Ehlorsson J. Kjellén L. Lindahl U. Biochem. J. 1986; 238: 209-216Crossref PubMed Scopus (27) Google Scholar, 7Sobue M. Habuchi H. Ito K. Yonekura H. Oguri K. Sakurai K. Kamohara S. Ueno Y. Noyori R. Suzuki S. Biochem. J. 1987; 241: 591-601Crossref PubMed Scopus (80) Google Scholar, 8Lugemwa F.N. Esko J.D. J. Biol. Chem. 1991; 266: 6674-6677Abstract Full Text PDF PubMed Google Scholar, 9Fransson L.-Å. Havsmark B. Sakurai K. Suzuki S. Glycoconj. J. 1992; 9: 45-55Crossref PubMed Scopus (17) Google Scholar). 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 Galβ1-3Galβ1-4Xyl-MU, Galβ1-4Xyl-MU (10Takagaki K. Nakamura T. Kon A. Tamura S. Endo M. J. Biochem.(Tokyo). 1991; 109: 514-519Crossref PubMed Scopus (39) Google Scholar), in addition to glycosaminoglycan-MU, were synthesized. Freeze et al.(11Freeze H.H. Sampath D. Varki A. J. Biol. Chem. 1993; 268: 1618-1627Abstract Full Text PDF PubMed Google Scholar) reported that Siaα2-3Galβ1-4Xylβ1-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 β-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.(12Nakamura T. Izumi J. Takagaki K. Shibata S. Kojima K. Kato I. Endo M. Biochem. J. 1994; 304: 731-736Crossref PubMed Scopus (21) Google Scholar) and Izumi et al.(13Izumi J. Takagaki K. Nakamura T. Shibata S. Kojima K. Kato I. Endo M. J. Biochem.(Tokyo). 1994; 116: 524-529Crossref PubMed Scopus (18) Google Scholar) reported that GlcAβ1-4Xylβ1-MU and Xylβ1-4Xylβ1-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-3GlcAβ1-4Xylβ1-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 (14Chou D.K.H. Ilyas A.A. Evans J.E. Costello C. Quarles R.H. Jungalwala F.B. J. Biol. Chem. 1986; 261: 11717-11725Abstract Full Text PDF PubMed Google Scholar, 15Ariga T. Kohriyama T. Freddo L. Latov N. Saito M. Kon K. Ando S. Suzuki M. Hemling M.E. Rinehart Jr., K.L. Kusunoki S. Yu R.K. J. Biol. Chem. 1987; 262: 848-853Abstract Full Text PDF PubMed Google Scholar), and in fact this novel Xyl-MU-induced oligosaccharide was found also to be reactive with HNK-1.EXPERIMENTAL PROCEDURESMaterialsEagle'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 (16Takagaki K. Nakamura T. Kawasaki H. Kon A. Ohishi S. Endo M. J. Biochem. Biophys. Methods. 1990; 21: 209-215Crossref PubMed Scopus (21) Google Scholar). β-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). β-Glucuronidase was purified from rabbit liver using p-nitrophenyl-β-D-glucuronide as a substrate (17Nakamura T. Takagaki K. Majima M. Kimura S. Kubo K. Endo M. J. Biol. Chem. 1990; 265: 5390-5397Abstract Full Text PDF PubMed Google Scholar). The GlcA-Xyl-MU standard was the same as that reported previously (12Nakamura T. Izumi J. Takagaki K. Shibata S. Kojima K. Kato I. Endo M. Biochem. J. 1994; 304: 731-736Crossref PubMed Scopus (21) Google Scholar). 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. (18Takemoto H. Hase S. Ikenaka T. Anal. Biochem. 1985; 145: 245-250Crossref PubMed Scopus (157) Google Scholar).Cell CultureHuman 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% CO2 as described previously (10Takagaki K. Nakamura T. Kon A. Tamura S. Endo M. J. Biochem.(Tokyo). 1991; 109: 514-519Crossref PubMed Scopus (39) Google Scholar). The cells were plated at a density of 2 × 105/100-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 (16Takagaki K. Nakamura T. Kawasaki H. Kon A. Ohishi S. Endo M. J. Biochem. Biophys. Methods. 1990; 21: 209-215Crossref PubMed Scopus (21) Google Scholar).Enzymic Digestionβ-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 (17Nakamura T. Takagaki K. Majima M. Kimura S. Kubo K. Endo M. J. Biol. Chem. 1990; 265: 5390-5397Abstract Full Text PDF PubMed Google Scholar); 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 DegradationSmith degradation of the Xyl-MU-induced oligosaccharide was performed according to the method of Noble and Sturgeon (19Noble D.W. Sturgeon R.J. Carbohydr. Res. 1970; 12: 448-452Crossref Scopus (5) Google Scholar). An aliquot of purified sample was dissolved in 200 μl of 0.1 M sodium acetate buffer (pH 4.5) containing 0.015 M NaIO4 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 NaBH4 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 MethodsIn 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 (16Takagaki K. Nakamura T. Kawasaki H. Kon A. Ohishi S. Endo M. J. Biochem. Biophys. Methods. 1990; 21: 209-215Crossref PubMed Scopus (21) Google Scholar). The resulting PA-monosaccharides were identified and quantified by HPLC analysis on an Ultrasphere ODS column (16Takagaki K. Nakamura T. Kawasaki H. Kon A. Ohishi S. Endo M. J. Biochem. Biophys. Methods. 1990; 21: 209-215Crossref PubMed Scopus (21) Google Scholar).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 (20Takagaki K. Kojima K. Majima M. Nakamura T. Kato I. Endo M. Glycoconj. J. 1992; 9: 174-179Crossref PubMed Scopus (68) Google Scholar). 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 AnalysisDot-blot analysis of the Xyl-MU-induced oligosaccharide was performed using a modification of the method described by Sorrell et al.(21Sorrell J.M. Mahmoodian F. Caterson B. Cell Tissue Res. 1988; 252: 523-531Crossref PubMed Scopus (15) Google Scholar). The Xyl-MU-induced oligosaccharide, GlcAβ1-4Xylβ1-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, H2O2 and 2,2′-azino-di(3-ethyl-benzthiazoline sulfonate) were added.Solid Phase Binding AssayBinding of the Xyl-MU-induced oligosaccharide to HNK-1 monoclonal antibody was determined using a modification of a solid-phase binding assay procedure (22Cantarero L.A. Butler J.E. Osborne J.W. Anal. Biochem. 1980; 105: 375-382Crossref PubMed Scopus (251) Google Scholar). 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 GlcAβ1-4Xylβ1-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.RESULTSProduction of the Xyl-MU-induced Oligosaccharide by Cultured Human Skin FibroblastsHuman 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.Isolation of the Xyl-MU-induced OligosaccharideThe 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. V0, void volume; V, total bed volume. The fractions containing peak2 were recovered.View Large Image Figure ViewerDownload Hi-res image Download (PPT)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 (%).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Carbohydrate Composition of the Xyl-MU-induced OligosaccharideAn 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 (Table I).Table ICarbohydrate composition of 4-methylumbelliferyl β-D-xyloside-induced oligosaccharideTable ICarbohydrate composition of 4-methylumbelliferyl β-D-xyloside-induced oligosaccharideEnzymic Digestion of the Xyl-MU-induced OligosaccharideEnzymic digestion of the purified Xyl-MU-induced oligosaccharide was performed. An aliquot of the oligosaccharide was incubated with β-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 (12Nakamura T. Izumi J. Takagaki K. Shibata S. Kojima K. Kato I. Endo M. Biochem. J. 1994; 304: 731-736Crossref PubMed Scopus (21) Google Scholar). The sulfatase digestion product was incubated with β-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 β-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 β-glucuronidase; c, after incubation with sulfatase; d, after incubation with β-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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Mass Spectrometry of the Xyl-MU-induced OligosaccharideAn 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).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Smith Degradation of the Xyl-MU-induced OligosaccharideFrom 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-2GlcAβ1-4Xylβ1-MU, sulfate-O-3GlcAβ1-4Xylβ1-MU, or sulfate-O-4GlcAβ1-4Xylβ1-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-3GlcAβ1-4Xylβ1-MU (Fig. 6b).Figure 6:Possible structures of the Xyl-MU-induced oligosaccharide. a, sulfate-O-2GlcAβ1-4Xylβ1-MU; b, sulfate-O-3GlcAβ1-4Xylβ1-MU; c, sulfate-O-4GlcAβ1-4Xylβ1-MU.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Binding of the Xyl-MU-induced Oligosaccharide for HNK-1Dot-blot analysis of the Xyl-MU-induced oligosaccharide was performed. HNK-1 monoclonal antibody bound to sulfate-O-3GlcAβ1-4Xylβ1-MU but not to GlcAβ1-4Xylβ1-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-3GlcAβ1-4Xylβ1-MU for HNK-1 was found to be dose-dependent, but that of GlcAβ1-4Xylβ1-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, GlcAβ1-4Xylβ1-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-3GlcAβ1-4Xylβ1-MU; b, GlcAβ1-4Xylβ1-MU; c, Xyl-MU.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 8:Solid-phase binding assay of the Xyl-MU-induced oligosaccharide against HNK-1. The Xyl-MU-induced oligosaccharide and GlcAβ1-4Xylβ1-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 GlcAβ1-4Xylβ1-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-3GlcAβ1-4Xylβ1-MU; b, GlcAβ1-4Xylβ1-MUView Large Image Figure ViewerDownload Hi-res image Download (PPT)DISCUSSIONIt has been reported that the addition of a β-xyloside to cell culture medium induces elongation of glycosaminoglycan chains, which is initiated by the β-xyloside acting as a primer (1Okayama M. Kimata K. Suzuki S. J. Biochem.(Tokyo). 1973; 74: 1069-1073PubMed Google Scholar, 2Schwartz N.B. Galligani L. Ho P.-L. Dorfman A. Proc. Natl. Acad. Sci. U. S. A. 1974; 71: 4047-4051Crossref PubMed Scopus (143) Google Scholar, 3Fukunaga Y. Sobue M. Suzuki N. Kushida H. Suzuki S. Suzuki S. Biochim. Biophys. Acta. 1975; 381: 443-447Crossref PubMed Scopus (33) Google Scholar, 4Robinson H.C. Brett M.J. Tralaggan P.J. Lowther D.A. Okayama M. Biochem. J. 1975; 148: 25-34Crossref PubMed Scopus (111) Google Scholar, 5Kato Y. Kimata K. Ito K. Karasawa K. Suzuki S. J. Biol. Chem. 1978; 253: 2784-2789Abstract Full Text PDF PubMed Google Scholar, 6Kolset S.O. Ehlorsson J. Kjellén L. Lindahl U. Biochem. J. 1986; 238: 209-216Crossref PubMed Scopus (27) Google Scholar, 7Sobue M. Habuchi H. Ito K. Yonekura H. Oguri K. Sakurai K. Kamohara S. Ueno Y. Noyori R. Suzuki S. Biochem. J. 1987; 241: 591-601Crossref PubMed Scopus (80) Google Scholar, 8Lugemwa F.N. Esko J.D. J. Biol. Chem. 1991; 266: 6674-6677Abstract Full Text PDF PubMed Google Scholar, 9Fransson L.-Å. Havsmark B. Sakurai K. Suzuki S. Glycoconj. J. 1992; 9: 45-55Crossref PubMed Scopus (17) Google Scholar). Several Xyl-MU derivatives, as well as glycosaminoglycan-MU, have been obtained by incubating human skin fibroblasts in the presence of Xyl-MU (10Takagaki K. Nakamura T. Kon A. Tamura S. Endo M. J. Biochem.(Tokyo). 1991; 109: 514-519Crossref PubMed Scopus (39) Google Scholar). 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-3GlcAβ1-4Xylβ1-MU.3-O-Sulfated glucuronic acid has been reported to be present in chondroitin sulfate from cartilage of the king crab (23Seno N. Murakami K. Carbohydr. Res. 1982; 103: 190-194Crossref Scopus (29) Google Scholar) and a glycolipid from human peripheral nerve (14Chou D.K.H. Ilyas A.A. Evans J.E. Costello C. Quarles R.H. Jungalwala F.B. J. Biol. Chem. 1986; 261: 11717-11725Abstract Full Text PDF PubMed Google Scholar, 15Ariga T. Kohriyama T. Freddo L. Latov N. Saito M. Kon K. Ando S. Suzuki M. Hemling M.E. Rinehart Jr., K.L. Kusunoki S. Yu R.K. J. Biol. Chem. 1987; 262: 848-853Abstract Full Text PDF PubMed Google Scholar). It has been reported that glycosidases have transglycosylation activity as a result of the reverse reaction of hydrolysis (24Saitoh H. Takagaki K. Majima M. Nakamura T. Matsuki A. Kasai M. Narita H. Endo M. J. Biol. Chem. 1995; 270: 3741-3747Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Therefore, it is possible that the sulfated glucuronic acid residue was transferred from glycolipid, glycoprotein, or glycosaminoglycan through the activity of exo-β-glucuronidase. However, GlcAβ1-4Xylβ1-MU, considered to be a mediator of sulfate-O-3GlcAβ1-4Xylβ1-MU, was also detected in the culture medium, and its production was not inhibited by the addition of a β-glucuronidase inhibitor (12Nakamura T. Izumi J. Takagaki K. Shibata S. Kojima K. Kato I. Endo M. Biochem. J. 1994; 304: 731-736Crossref PubMed Scopus (21) Google Scholar). Accordingly, sulfate-O-3GlcAβ1-4Xyl-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 cultu" @default.
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