| Glycobiology | Pages |
A novel 4-methylumbelliferyl-[beta]-d-xyloside derivative, sulfate-O-3-xylosyl[beta]1-(4-methylumbelliferone), isolated from culture medium of human skin fibroblasts, and its role in methylumbelliferone-initiated glycosaminoglycan biosynthesis
Introduction
Results
Discussion
Materials and methods
Acknowledgments
Abbreviations
References
A novel 4-methylumbelliferyl-[beta]-d-xyloside derivative, sulfate-O-3-xylosyl[beta]1-(4-methylumbelliferone), isolated from culture medium of human skin fibroblasts, and its role in methylumbelliferone-initiated glycosaminoglycan biosynthesis
Introduction
Glycosaminoglycan (GAG) chains are covalently attached to the core protein through a linkage region, GlcA[beta]1-3Gal[beta]1-3Gal[beta]1-4Xyl[beta]1-O-Ser. Although the mechanisms involved in GAG biosynthesis are not yet fully understood, it has been demonstrated that GAG biosynthesis is initiated by the transfer of a xylose residue from UDP-Xyl to a serine residue of the core protein, followed by stepwise addition of individual monosaccharides from UDP-sugars by a series of glycosyltransferase reactions (Rodén, 1980).
It has been reported that addition of a [beta]-xyloside, such as p-nitrophenyl-[beta]-d-xyloside, 4-methylumbelliferyl-[beta]-d-xyloside (Xyl-MU), or benzyl-[beta]-d-xyloside to cell culture medium induces elongation of GAG chains, which is initiated by the [beta]-xyloside acting as a primer (Okayama et al., 1973; Schwartz et al., 1974; Fukunaga et al., 1975; Robinson et al., 1975; Kato et al., 1978; Kolset et al., 1986; Sobue et al., 1987; Lugemwa and Esko, 1991; Fransson et al., 1992). We have also reported that culture of human skin fibroblasts in the presence of Xyl-MU results in the synthesis of synthetic intermediates of Xyl-MU-induced GAG (GAG-MU), such as Gal[beta]1-3Gal[beta]1-4Xyl[beta]1-MU, Gal[beta]1-4Xyl[beta]1-MU (Takagaki et al., 1991), in addition to GAG-MU. Freeze et al. have reported that SA[alpha]2-3Gal[beta]1-4Xyl[beta]1-MU was synthesized in cultures of Chinese hamster ovary cells and human melanoma cells using Xyl-MU as a primer (Freeze et al., 1993). Furthermore, we have reported that GlcA[beta]1-4Xyl[beta]1-MU (Nakamura et al., 1994), Xyl[beta]1-4Xyl[beta]1-MU (Izumi et al., 1994), and sulfate-O-3-GlcA[beta]1-4Xyl[beta]1-MU (Shibata et al., 1995), which are unrelated to GAG, were elongated from Xyl-MU.
In the present study, Xyl-MU derivatives produced by cultured human skin fibroblasts in the presence of Xyl-MU were isolated and analyzed. The results indicated that a novel Xyl-MU derivative, sulfate-O-3-Xyl-MU, in addition to the already known Xyl-MU derivatives, was present in the culture medium. No structure similar to the sulfated xylose of Xyl-MU has been reported previously. It was demonstrated that this Xyl-MU derivative was involved in the control of Xyl-MU-initiated GAG biosynthesis.
Results
Isolation of a novel Xyl-MU derivative
Human skin fibroblasts were cultured for 72 h in the presence of 0.5 mM Xyl-MU. The medium (20 l) was pooled, lyophilized, and dialyzed. The dialyzable fraction was recovered and concentrated with a lyophilizer, then loaded on a Sephadex G-15 column (Figure
Figure 1. Gel-filtration chromatography on Sephadex G-15. The dialyzable fraction of culture medium was concentrated and applied to a Sephadex G-15 column (4.1 × 150 cm). Elution was performed with distilled water at 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 fractions indicated by the horizontal bar were collected and used for further purification.
Figure 2. Ion-exchange chromatography on DEAE-Sephacel. The fractions recovered from Sephadex G-15 chromatography were applied to a DEAE-Sephacel column (2.3 × 40 cm). Elution was performed with a linear gradient of 0-1.0 M NaCl at a flow rate of 30 ml/h, and 10 ml fractions were collected. The eluate was monitored with a fluorescence detector. The fractions indicated by the horizontal bar were collected and used for further purification.
Figure 3. Reverse-phase HPLC on Shodex C18-10F. HPLC was performed using a Shodex C18-10F column (20 × 250 mm), with a linear gradient of distilled water-acetonitrile. The eluate was monitored with a fluorescence detector. The fractions indicated by the horizontal bar were collected and used for further purification.
Figure 4. Gel-filtration HPLC on Shodex OHpak KB-802. HPLC was performed using a Shodex OHpak KB-802 column (8 × 300 mm) with 20% acetonitrile. The eluate was monitored with a fluorescence detector. The fractions were collected and used for analysis as a purified sample.
Enzyme digestion of the Xyl-MU derivative
Substance [Igr] was composed of only MU and xylose in a molar ratio of about 1:1 (data not shown). Therefore, enzyme digestion of substance [Igr] was performed. An aliquot of substance [Igr] was incubated with alkaline phosphatase and then subjected to gel-filtration HPLC on a Shodex OHpak KB-802 column. The peak of substance [Igr] was not shifted from the control position after digestion (Figure
Figure 5. Analysis by HPLC of the Xyl-MU derivative after incubation with various enzymes. (A) before enzymic digestion; (B) after incubation with alkaline phosphatase; (C) after incubation with sulfatase. The arrows denote the positions of Xyl-MU derivatives: 1, novel Xyl-MU derivative; 2, Xyl-MU.
Mass spectrometry of the Xyl-MU derivative
An aliquot of substance [Igr] was subjected to ion-spray mass spectrometery. The spectrum showed a major peak at m/z 386.5 (Figure
Figure 6. Mass spectra of the Xyl-MU derivative. (A) The Xyl-MU derivative; (B) product ions on tandem mass spectrometric analysis spectrum of the Xyl-MU derivative using m/z 386.5 as the precursor ion.
Smith degradation of the Xyl-MU derivative
From the analytical results described above, substance [Igr] appeared to be Xyl-MU with a sulfated xylose residue. In order to examine the sulfate-to-xylose linkage position, aliquots of substance [Igr] and Xyl-MU as a control were subjected to Smith degradation, and the degradation product was analyzed by HPLC on a Shodex OHpak KB-802 column. As a result, Xyl-MU was found to be degraded but substance [Igr] was not (Figure
Figure 7. Analysis by HPLC of the Xyl-MU derivative after Smith degradation. (A) and (B) Xyl-MU; (C) and (D), the Xyl-MU derivative. (A) and (C), before Smith degradation; (B) and (D), after Smith degradation. Incorporation of sulfate into the Xyl-MU derivatives
Xyl-MU or Gal-Xyl-MU or Gal-Gal-Xyl-MU was incubated with [35S]PAPS using a homogenate prepared from cultured skin fibroblasts as an enzyme source. When Xyl-MU was incubated with [35S]PAPS, a new peak was detected and the product was identified as sulfate-O-3-Xyl-MU by HPLC (Figure
Figure 8. Analysis by HPLC of the Xyl-MU derivatives after incubation with [35S]PAPS. Incubation was performed as described in Materials and Methods, using the following acceptors: (A) none; (B) Xyl-MU; (C) Gal-Xyl-MU; (D) Gal-Gal-Xyl-MU. Transfer of galactose to Xyl-MU and sulfate-Xyl-MU
Xyl-MU or sulfate-O-3-Xyl-MU was incubated with UDP-[3H]Gal using the same homogenate as that described above. When Xyl-MU was incubated with UDP-[3H]Gal, a new peak was detected and the product was identified as Gal[beta]1-4Xyl-MU by HPLC (Figure
Figure 9. Analysis by HPLC of the Xyl-MU derivative after incubation with UDP-[3H]Gal. (A) and (C), before incubation; (B) and (D), after incubation. Incubation was performed as described in Materials and methods, using the following acceptors: (A) and (B), Xyl-MU; (C) and (D), sulfate-O-3-Xyl-MU.
We have reported that several Xyl-MU derivatives, as well as GAG-MU, were obtained by incubating human skin fibroblasts in the presence of Xyl-MU (Takagaki et al., 1991; Freeze et al., 1993; Nakamura et al., 1994; Izumi et al., 1994; Shibata et al., 1995). In the present study, human skin fibroblasts were cultured in the presence of Xyl-MU. A large amount of the medium was then recovered, concentrated and purified, and a novel Xyl-MU derivative was isolated, in addition to the previously reported Xyl-MU derivatives. This new Xyl-MU derivative was subjected to carbohydrate composition analysis, enzyme digestion, ion-spray mass spectrometric analysis, and Smith degradation, and the results indicated that the structure of the Xyl-MU derivative was sulfate-O-3-Xyl-MU. The amount of this derivative (0.0125 µM) was much less than that of the other Xyl-MU derivatives, GAG-MU (0.22 µM), Gal-Gal-Xyl-MU (0.67 µM), and Gal-Xyl-MU (0.17 µM) (Takagaki et al., 1991).
When Xyl-MU was incubated with [35S]PAPS, [35S]sulfate-Xyl-MU was produced (Figure
Recently, phosphorylation of xylose (Oegema et al., 1984; Fransson et al., 1985; Sugahara et al., 1992a,b) and/or sulfation of galactose (Sugahara et al., 1992b; de Waard et al., 1992; Yamada et al., 1995) in the linkage region was reported. However, in the present study, incorporation of sulfate into Gal-Xyl-MU and Gal-Gal-Xyl-MU was not observed (Figure
Previously, we reported that culture of human skin fibroblasts in the presence of Xyl-MU resulted in the synthesis of GAG-MU and its synthetic intermediates such as Gal-Gal-Xyl-MU, Gal-Xyl-MU (Takagaki et al., 1991). In the present study, a sulfated Xyl-MU, sulfate-O-3-Xyl-MU, was detected in the culture medium of human skin fibroblasts. When Xyl-MU was used as an acceptor, galactose was transferred to xylose, but when sulfate-O-3-Xyl-MU was used, galactose was not transferred. Moreover, sulfated residues were not found in the linkage region of the Xyl-MU-induced GAG (Takagaki et al., 1991). These results suggest that sulfate-O-3-Xyl-MU is involved in the control of Xyl-MU-initiated GAG elongation by inhibiting the transfer of galactose to xylose. Materials
Eagle's minimum essential medium (MEM), fetal bovine serum, penicillin-streptomycin solution (penicillin 100 mU/ml and streptomycin 100 µg/ml), and fungizone were purchased from Gibco (Grand Island, NY). Xyl-MU was purchased from Nacalai Tesque Inc. (Kyoto, Japan). Sulfatase (from Aspergillus niger) and alkaline phosphatase (from calf intestine) were purchased from Sigma Chemical Co. (St. Louis, MO). Sephadex G-15 and DEAE-Sephacel were purchased from Pharmacia LKB Biotech. (Uppsala, Sweden). [35S]PAPS (1.6 Ci/mmol) was purchased from Dupont/NEN (Wilmington, DE). UDP-[3H]galactose (15 Ci/mmol) was purchased from American Radiolabeled Chemicals Inc. (St. Louis, MO). Other reagents and chemicals were obtained from commercial sources. 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% CO2 as described previously (Takagaki et al., 1991). The cells were plated at a density of 2 × 105/100 mm plastic dish and then subcultured after being grown to confluency. Fibroblasts at passage 4-7 were used for this study. High-performance liquid chromatography (HPLC)
A high-performance liquid chromatograph (Hitachi L-6200, Hitachi Co., Tokyo, Japan) equipped with a fluorescence spectrometer (Hitachi F-1050, Hitachi Co.) was used. The Xyl-MU derivatives were detected by their fluorescence at excitation and emission wavelengths of 325 and 380 nm, respectively. Reverse-phase HPLC was performed using a Shodex C18-10F column (20 × 250 mm, Shoko Co., Tokyo, Japan) or C18-5B column (4.6 × 250 mm, Shoko Co.) with a linear gradient of distilled water-60% acetonitrile as the eluent at a column temperature of 30°C and flow rate of 3.0 or 0.5 ml/min, respectively.
Gel-filtration HPLC was performed using a Shodex OHpak KB-802 column (8 × 300 mm, Shoko Co.) or OHpak SB-803 column (8 × 300 mm, Shoko Co.) with 20% acetonitrile or 5% acetonitrile as the eluent at a column temperature of 30°C and flow rate of 0.5 ml/min. Carbohydrate composition analysis was performed using an Ultrasphere ODS column (4.6 × 250 mm, Beckman Instruments Inc., Palo Alto, CA) with 0.25 M sodium citrate buffer (pH 4.0), containing 1% acetonitrile (Takagaki et al., 1990). Purification of the Xyl-MU derivatives
Human skin fibroblasts were cultured for 72 h in Eagle's MEM containing 0.5 mM Xyl-MU. The culture medium from a total 2000 dishes was collected and pooled up to 20 l, concentrated with a lyophilizer to 500 ml, and then dialyzed against distilled water. The dialyzable fraction (5 l) was concentrated with a lyophilizer to 300 ml. Then, 100 ml of the dialysate 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. The fluorescence intensity of the eluate was monitored at excitation and emission wavelengths of 325 and 380 nm, respectively. The fractions exhibiting fluorescence were collected, concentrated, and subjected to ion-exchange on a DEAE-Sephacel column (2.3 × 40 cm), which was equilibrated with distilled water and eluted with a linear gradient of 0-1.0 M NaCl at a flow rate of 30 ml/h. The fractions exhibiting fluorescence were collected, concentrated, and subjected to reverse-phase HPLC on a Shodex C18-10F column. The fractions exhibiting fluorescence were collected and subjected to gel-filtration HPLC on a Shodex OHpak KB-802 column. Enzyme digestion
Sulfatase digestion was performed in 0.1 M sodium acetate buffer (pH 4.5) at 37°C for 4 h, and alkaline phosphatase digestion was performed in Tris-HCl buffer (pH 8.0) at 37°C for 4 h. Smith degradation
Smith degradation of the Xyl-MU derivative was done according to the method of Noble and Sturgeon (1970). 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 ethyleneglycol was added and allowed to react for 1 h at 20°C. This was followed by 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 addition of acetic acid, and the solution was evaporated to dryness repeatedly in the presence of methanol under reduced pressure to remove the borate. 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 (Takagaki et al., 1990). The resulting PA-monosaccharides were identified and quantified by HPLC analysis on an Ultrasphere ODS column (Takagaki et al., 1990).
The mass spectrum of the Xyl-MU derivative was obtained on a API- [Igr][Igr][Igr] triple-quadrupole mass spectrometer (Sciex, Thornhill, Ontario, Canada) equipped with an atmospheric-pressure ionization source, as described previously (Takagaki et al., 1992). The sample was dissolved in 0.5 mM ammonium acetate/acetonitrile (50:50) and injected at 2 µl/min with a micro-HPLC syringe pump (pump 22, Harvard Apparatus Inc., MA). Incubation with [35S]PAPS and MU-linkage oligosaccharides
Xyl-MU, Gal-Xyl-MU or Gal-Gal-Xyl-MU as an acceptor was incubated with [35S]PAPS. Human skin fibroblasts (2 × 107 cells) were homogenized in a Dounce homogenizer using 10 strokes and centrifuged at 700 × g for 20 min, and the resulting supernatant was used as the enzyme solution. The reaction mixture contained the following components in a final volume of 50 µl: 50 mM MES buffer (pH 6.5), 3 mM MnCl2, 12 mM MgCl2, 100 mM KCl, 0.12 mM [35S]PAPS as a donor, 25 mM d-galactal as an inhibitor of endogenous [beta]-galactosidase, 0.2 mM Xyl-MU, Gal-Xyl-MU or Gal-Gal-Xyl-MU as an acceptor, and 10 µl of enzyme solution. After incubation for 3 h at 37°C, the reaction was stopped by boiling at 100°C for 3 min. The reaction mixture was then centrifuged at 10,000 × g for 5 min. The supernatant fractions were subjected to reverse-phase HPLC on a Shodex C18-5B column and gel-filtration HPLC on a Shodex OHpak KB-802 column. Incubation with UDP-[3H]Gal and the Xyl-MU derivative
The Xyl-MU derivative as an acceptor was incubated with UDP-[3H]Gal according to a modification of the method of Higuchi et al. (1994). Human skin fibroblasts (4 × 106 cells) were homogenized by brief sonication and centrifuged at 110 × g for 5 min, and the resulting supernatant was used as the enzyme solution. The reaction mixture contained the following components in a final volume of 50 µl: 80 mM MES buffer (pH 5.5), 15 mM MnCl2, 100 mM KCl, 0.05 mM UDP-[3H]Gal as a donor, 25 mM D-galactal, 0.05 mM Xyl-MU derivative or Xyl-MU as an acceptor, and 10 µl of enzyme solution. After incubation for 3 h at 37°C, the reaction was stopped by boiling at 100°C for 3 min. The reaction mixture was then centrifuged at 10,000 × g for 5 min. The supernatant fractions were subjected to reverse-phase HPLC on a Shodex C18-5B column and gel-filtration HPLC on a Shodex OHpak SB-803 column.
This work was supported in part by Grants-in-Aid for Scientific Research (08457032, 09240202, and 09358013) from the Ministry of Education, Science, Sports and Culture of Japan.
Xyl-MU, 4-methylumbelliferyl-[beta]-d-xyloside; MU, 4-methylumbelliferone; Xyl, xylose; Gal, galactose; GlcA, glucuronic acid; SA, sialic acid; GAG, glycosaminoglycan; PAPS, 3[prime]-phosphoadenosine-5[prime]-phosphosulfate; HPLC, high-performance liquid chromatography.
Discussion
Materials and methods
Acknowledgments
Abbreviations
References
3To whom correspondence should be addressed at: Department of Biochemistry, Hirosaki University School of Medicine, 5 Zaifu-cho, Hirosaki 036-8562, Japan
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