Glycobiology, 2001, Vol. 11, No. 12 1043-1049
© 2001 Oxford University Press
Structural analysis of sulfated N-linked oligosaccharides in erythropoietin
2Division of Biological Chemistry and Biologicals, National Institute of Health Sciences, 1-18-1, Kamiyoga, Setagaya-ku, Tokyo 158-8501 Japan, and 3Division of Medical Devices, National Institute of Health Sciences, 1-18-1, Kamiyoga, Setagaya-ku, Tokyo 158-8501 Japan
Received on July 12, 2001; revised on September 5, 2001; accepted on September 7, 2001.
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Abstract |
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We previously demonstrated that high-performance liquid chromatography with electrospray ionization mass spectrometry (LC/MS) equipped with a graphitized carbon column (GCC) is useful for the structural analysis of carbohydrates in glycoproteins. Using LC/MS with GCC, sulfated N-linked oligosaccharides were found in erythropoietin (EPO) expressed in baby hamster kidney cells. Sulfation occurs in a part of the N-linked oligosaccharides in the EPO. Sulfated monosaccharide residue in the sulfated N-linked oligosaccharide was determined by exoglycosidase digestion followed by sugar mapping by LC/MS. The linkage position and branch-location of the sulfate group in the tetraantennary oligosaccharide were analyzed by 1H-nuclear magnetic resonance. It was suggested that sulfation occurs on the C-6 position of GlcNAc located in the GlcNAcß1-4Man
1-3 branch. Key words: erythropoietin/ESI-LC/MS/1H-NMR/sulfated oligosaccharide
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Introduction |
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The carbohydrates of glycoconjugates are highly diverse because of their variety of monosaccharide compositions, sequences, branching sites, linkages, and modifications involving phosphate and sulfate groups. A sulfate substituent has been shown to occur on C-3 of Gal (Spiro and Bhoyroo, 1988
; Hard et al., 1992; Karaivanova and Spiro, 1998; van Rooijen et al., 1998), C-4 of GalNAc (Green et al., 1985; Hard et al., 1992; Bergwerff et al., 1995; van Rooijen et al., 1998), and C-6 of GlcNAc (Roux et al., 1988; de Waard et al., 1991; Noguchi and Nakano, 1992; Shilatifard et al., 1993; Taguchi et al., 1996; Karaivanova and Spiro, 1998) in N-linked and O-linked oligosaccharides. Although the biological function of the sulfate group on glycoconjugates remains to be clarified, a number of studies suggest that sulfation is implicated in biological recognition, such as lymphocyte homing (Hemmerich and Rosen, 2000) and removal of pituitary glycoprotein hormones from the circulation (Fiete et al., 1991).
The presence of a sulfate group in N-linked oligosaccharides has been reported in glycoproteins, including lysosomal enzyme (Freeze and Wolgast, 1986), hen egg albumin (Yamashita et al., 1983), urokinase (Bergwerff et al., 1995), thyroglobulins (de Waard et al., 1991; Spiro and Bhoyroo, 1988), and pituitary hormones (Green et al., 1985) and in glycoproteins from zona pellucida (Noguchi and Nakano, 1992), thyroid plasma membrane (Edge and Spiro, 1984), human Tamm-Horsfall (Hard et al., 1992; van Rooijen et al., 1998), virus (Bernstein and Compans, 1992; Shilatifard et al., 1993), unfertilized eggs of Tribolodon hakonensis (Taguchi et al., 1996), and mammalian cell lines (Pierce and Arango, 1986; Roux et al., 1988; Sundblad et al., 1988). However, these reports are mostly based on the results of radioisotope labeling; there are only a few reports on the detailed structure of sulfated N-linked oligosaccharides in glycoproteins (de Waard et al., 1991; Hard et al., 1992; Noguchi and Nakano, 1992; Bergwerff et al., 1995; Taguchi et al., 1996; van Rooijen et al., 1998). This gap in the research is due to a lack of sensitive, specific analytical techniques for investigating sulfate substitution and destruction during the isolation and purification of the oligosaccharides.
We previously reported that high-performance liquid chromatography (LC) with electrospray ionization (ESI) mass spectrometry (MS) equipped with a graphitized carbon column (GCC) is useful for the structural analysis of carbohydrates in a glycoprotein (Kawasaki et al., 1999, 2000). Because many oligosaccharides can be analyzed rapidly without any derivertization or fractionation, our method is suitable for the elucidation of the detailed structure and distribution of oligosaccharides, including the minor components of glycoproteins. We demonstrated that LC/MS with GCC enabled more than 50 different sialylated fucosyl-complex-type oligosaccharides to be characterized in recombinant human erythropoietin (EPO) expressed in Chinese hamster ovary (CHO) cells (Kawasaki et al., 2000). In the present study, using LC/MS with GCC, we found that N-linked oligosaccharides are partly sulfated in EPO produced in baby hamster kidney (BHK) cells. We also determined the detailed structure of the most abundant sulfated oligosaccharides by exoglycosidase digestion followed by LC/MS and 1H-nuclear magnetic resonance (NMR) spectroscopy.
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Results |
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LC/MS of N-linked oligosaccharide alditols from EPO expressed in BHK cells
N-linked oligosaccharides alditols prepared from EPO expressed in BHK cells were analyzed by LC/MS with GCC using ammonium acetate/acetonitrile as an eluent in the negative ion mode. Figure 1A shows the total ion current (TIC) chromatogram of N-linked oligosaccharide alditols together with the carbohydrate structure of the major component in each peak. Figures 1B–G indicate the distributions of the major oligosaccharides. Monitored ions in Figures 1B–G are at m/z 11852– (disialylated biantennary, BiNA2), m/z 10083– (trisialylated triantennary, TriNA3), m/z 11303– (trisialylated tetraantennary or trisialylated triantennary containing N-acetyllactosamine, TertaNA3, or TriLac1NA3), m/z 12273– (TetraNA4), m/z 13493– (TetraLac1NA4), and m/z 14703– (TetraLac2NA4). The distributions of these sialylated oligosaccharides in EPO expressed in BHK cells are similar to those in CHO cells, however, two additional peaks, X1 and X2, appear in the TIC chromatogram drawn from EPO expressed in BHK cells.
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Figures 2A and B show the mass spectra of peaks X1 and X2, respectively. The observed mass of peak X1 is 3765 Da, which is not consistent with the theoretical mass of the typical complex-type oligosaccharide. Peak X1 is assumed to be TetraNA4 (3685.4 Da), the most abundant component, substituted by either a sulfate or phosphate group (80 Da). Likewise, the observed mass of peak X2 (3846 Da) corresponds with TetraNA4 substituted by two molecules of sulfate or phosphate. To determine the substituent group of peak X1, an alkaline phosphatase treatment followed by LC/MS was performed. The modified oligosaccharides were still detected at the same retention times by LC/MS after the treatment with alkaline phosphatase. The 31P-NMR measurement was carried out to confirm the presence of a phosphate group in peak X1 that had been fractionated by GCC after the removal of heterogeneity by a neuraminidase and ß-galactosidase treatment. No signal of a phosphate group was observed by 31P-NMR. These results suggest that peaks X1 and X2 do not contain a phosphate group. From these results, it is clear that peaks X1 and X2 can be assigned to monosulfated TetraNA4 and disulfated TetraNA4, respectively.
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Sulfation in other oligosaccharides in EPO was searched by LC/MS. Figure 3 shows the chromatograms of sulfated oligosaccharides at m/z 10353– (SO3-TriNA3), m/z 12543– (SO3-TetraNA4), m/z 12813– ([SO3]2-TetraNA4), m/z 13763– (SO3-TetraNA4Lac1), m/z 14033– ([SO3]2-TetraNA4Lac1), and m/z 14983– (SO3-TetraNA4Lac2). Sulfation was seen to occur in major oligosaccharides, and disulfation was found in the tetraantennay group. These results suggest that most of the N-linked oligosaccharides are partly sulfated in EPO.
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Exoglycosidase digestion followed by LC/MS of sulfated N-linked oligosaccharide alditols
To determine the sulfated monosaccharide residue, the N-linked oligosaccharide alditols from EPO were treated with neuraminidase, ß-galactosidase, and N-acetylhexosaminidase. The exoglycosidase-digested oligosaccharide alditols were then analyzed by LC/MS in the positive ion mode.
Figure 4A shows the TIC chromatogram of neuraminidase-treated oligosaccharides. Distributions of asialo-Bi, -Tri, -Tetra, -TetraLac1, and -TetraLac2 are indicated in Figures 4B–F, respectively, and those modified with a sulfate group are indicated in Figures 4G–K, respectively. Sulfated oligosaccharides were detected after the neuraminidase treatment, which suggests that the sulfated group is located on monosaccharide residues other than NeuAc.
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Figure 5A shows the TIC chromatogram of agalacto-oligosaccharides that had been prepared by a treatment with both neuraminidase and ß-galactosidase. Sulfated agalacto-oligosaccharides, such as SO3-Tetra (m/z 9762+), can still be observed in Figure 5A. This result suggests that the sulfate group substitutes to either GlcNAc on the nonreducing side or the fucosyltrimannosyl core. Next, LC/MS analysis was performed after the oligosaccharide alditols were treated with neuraminidase, ß-galactosidase plus N-acetylhexosaminidase. As shown in Figure 5B, the fucosyltrimannosyl core was detected as the most intense peak (m/z 1059+), and oligosaccharides in which one or two GlcNAc remain were also detected as peaks at m/z 1262+ and 1465+. Sulfation was found in the oligosaccharides that contain fucosyltrimannosyl core and one or two GlcNAc residues. In contrast, the sulfated fucosyltrimannosyl core (m/z 1139+) was not detected in the TIC chromatogram (Figure 5B). These results suggest that the sulfate group substitutes to GlcNAc at the nonreducing end.
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NMR study of sulfated oligosaccharide alditols
The branch location and linkage position of the sulfate group in the most abundant sulfated oligosaccharide was determined by 1H-NMR. As shown in our previous paper (Kawasaki et al., 2000
), the carbohydrates in EPO are highly heterogeneous and are separated into a number of fractions by GCC. Separating SO3-TetraNA4 from other components is quite complicated. To facilitate the isolation and assignment of sulfated oligosaccharides, GCC was used to fractionate sulfated agalacto-tetraantennary, which is generated from SO3-TetraNA4 by a neuraminidase and ß-galactosidase treatment. The 1H-NMR spectra of sulfated agalacto-tetraantennary and nonsulfated agalacto-tetraantennary, which was used as a reference oligosaccharide, are shown in Figures 6A and B, respectively. The chemical shifts of the anomeric protons are compiled in Table TI. The resonances of the anomeric protons of GlcNAc at the nonreducing end of nonsulfated agalacto-tetraantennary were assigned on the basis of the chemical shift values reported by Tomiya et al. (1988). The resonance of other protons were assigned from 2D 1H-1H chemical-shift-correlated spectroscopy (COSY) and homonuclear Hartmann-Hahn (HOHAHA) spectroscopy. Relevant regions of the 2D HOHAHA spectra of sulfated and nonsulfated agalacto-tetraantennary are presented in Figures 7A and B, respectively. The NMR spectrum of sulfated agalacto-tetraantennary is in good agreement with that of nonsulfated agalaco-tetraantennary, with the exceptions of a downfield shift of GlcNAc-7 H-1 and additional signals at 4.32 ppm and 4.42 ppm. From the 2D COSY spectrum, the signals at 4.32 ppm and 4.42 ppm can be assigned to GlcNAc H-6 and H-6', which have downfield-shifted from the bulk region at 3.73–3.86 ppm and 3.96–4.04 ppm, respectively. It has been reported that sulfate groups cause a downfield shift of the proton attached to the substituted carbon of about 0.5 ppm (Harris and Turvey, 1970). The downfield shift of GlcNAc H-6 and H-6' in Figure 6A can be explained by the sulfation of C-6 GlcNAc. Two-dimensional HOHAHA spectroscopy demonstrates the interconnection of the GlcNAc-7 H-1 signal with the SO3-GlcNAc H-6' signal at 4.46 ppm (Figure 7). These results suggest that the sulfated agalacto-tetraantennary bears 6-O-sulfated GlcNAc-7 residue in the GlcNAcß1-4Man1-3 branch.
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Discussion |
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Despite a number of studies on the carbohydrate structure of EPO expressed in CHO cells and BHK cells (Sasaki et al., 1987
, 1988; Nimtz et al., 1993; Hokke et al., 1995; Rush et al., 1995; Takeuchi et al., 1988; Tsuda et al., 1988), no report exists on sulfated N-linked oligosaccharides in EPO. In this article, using sugar mapping by LC/MS equipped with a GCC, we found that some major N-linked oligosaccharides are partly sulfated in EPO expressed in BHK cells. Some of these sulfated oligosaccharides are also found in EPO expressed in CHO cells (details to be published elsewhere). We previously reported that the response factors of neutral oligosaccharides were nearly the same in the positive ion mode (Kawasaki et al., 1999). Although the effect of sulfation on the response factors of oligosacccharides is unknown, one might be able to estimate that the 10–15% oligosaccharides are sulfated from the peak area ratio of SO3-Tetra/Tetra in the mass chromatogram (Figure 4A) and ultraviolet chromatogram (data not shown).
It has been reported that a sulfate group is located on C-3 Gal (Spiro and Bhoyroo, 1988; Hard et al., 1992; Karaivanova and Spiro, 1998; van Rooijen et al., 1998), C-4 GalNAc (Green et al., 1985; Hard et al., 1992; Bergwerff et al., 1995; van Rooijen et al., 1998), and C-6 GlcNAc (Roux et al., 1988; de Waard et al., 1991; Noguchi and Nakano, 1992; Shilatifard et al., 1993; Taguchi et al., 1996; Karaivanova and Spiro, 1998) in N-linked oligosaccharides. From the sugar map of exoglycosidase-digested oligosaccharides by LC/MS, it was indicated that the sulfation occurs on a GlcNAc on the nonreducing side in EPO. Sulfation of GlcNAc is facilitated on the C-6 position by Gal/GalNAc/GlcNAc 6-O-sulfotransferases (GSTs) (Hemmerich and Rosen, 2000; Hemmerich et al., 2001). The GST family has been recently discovered in humans and mice by several groups of researchers (Fukuta et al., 1995, 1998; Uchimura et al., 1998a,b,c; Bistrup et al., 1999; Lee et al., 1999; Hiraoka et al., 1999; Li and Tedder, 1999; Bhakta et al., 2000; Kitagawa et al., 2000; Hemmerich et al., 2001). Although no report has been made on GST in BHK cells, the sulfation of C-6 GlcNAc was suggested by the presence of keratan sulfate and the sulfation on the virus envelope glycoprotein by BHK cells (Karaivanova and Spiro, 1998; Pierce and Arango, 1986). In this study we fractionated the most abundant sulfated oligosaccharide, SO3-Tetra, as an agalacto form and subjected it to NMR study. From the downfield shift of about 0.5 ppm of the H-6 and H-6' protons of GlcNAc, it is suggested that sulfation occurs on the C-6 position of GlcNAc.
NMR analysis is also effective for determining the branch location of the sulfate group. The sulfate group on C-6 GlcNAc of the biantennary group was found on the Man1-3 branch (GlcNAc-5) in human Tamm-Horsfall glycoprotein (de Waard et al., 1991), and on both the Man1-3 and Man1-6 branches (GlcNAc-5 and -5') in glycotroteins from zona pellucida (Noguchi and Nakano, 1992). In our study, NMR spectroscopy suggested that the sulfate group in the sulfated tetraantennary is located on GlcNAc-7 in the GlcNAcß1-4Man1-3 branch.
In this study we demonstrated that the LC/MS with GCC is useful for the structural analysis of sulfated oligosaccharides. The modified residue can be determined by exoglycosidase digestion followed by sugar mapping with LC/MS. Furthermore, GCC is suitable for the isolation of oligosaccharides for the NMR study because of the use of a volatile mobile phase. Our method is applicable to the structural analysis of carbohydrates in various glycoproteins.
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Materials and methods |
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Recombinant human EPO was produced in BHK cells and purified according to the method described previously (Tsuda et al., 1988
). N-Glycosidase F (PNGase F) and neuraminidase (Arthrobacter ureafaciens) were obtained from Roche Diagnostics GmbH (Mannheim, Germany) and Nacalai Tesque (Kyoto, Japan), respectively. ß-Galactosidase (jackbean) and ß-N-acetylhexosaminidase (jackbean) were purchased from Seikagaku-Kogyo (Tokyo). All other chemicals used were of the highest purity available.
Preparation of reduced oligosaccharides
Erythropoietin (400 µg) was dissolved in 500 µl of sodium phosphate buffer, pH 6.2, and incubated with 10 U PNGase F at 37°C for 18 h. Protein was precipitated with 1.7 ml of cold ethanol. The supernatant was dried, and the oligosaccharides were dissolved in 100 µl of H2O. To the oligosaccharide solution, 0.5 M NaBH4 (100 µl) was added, and the mixture was incubated at 25°C for 2 h. Diluted acetic acid (20 µl) was added to the mixture to decompose excess NaBH4. A 20-µl volume of the sample was injected into the LC/MS.
Exoglycosidase digestion of N-linked oligosaccharide alditols from EPO
Oligosaccharide alditols from 200 µg of EPO were dissolved in 100 mM ammonium acetate buffer, pH 4.5, and incubated with neuraminidase (40 mU), ß-galactosidase (400 mU), or N-acetylhexosaminidase (400 mU) at 37°C for 18 h. The reaction mixture was applied to Supelclean Envi-Carb (Supelco, Bellefonte, PA), and the tube was washed with H2O to remove salts. Oligosaccharide alditols were eluted with 30% acetonitrile containing 5 mM ammonium acetate.
LC of oligosaccharide alditols from EPO
High-performance LC was carried out using a Finnigan spectra system consisting of a p4000 pump and UV2000. The GCC used was Hypercarb 5 µ (100 x 2.1 mm, Hypersil, UK). The eluents were 5 mM ammonium acetate (A pump), and 50% CH3CN containing 5 mM ammonium acetate (B pump). The flow rate was 0.2 ml/min, and the effluent was monitored at 206 nm.
• Gradient condition 1 for sialylated oligosaccharide alditols: 30–55% of B in 80 min.
• Gradient condition 2 for asialo-oligosaccharide alditols: 20–45% of B in 60 min.
• Gradient condition 3 for agalacto-oligosaccharide alditols and fucosyltrimannosylcore alditol: 18–30% of B in 50 min.
ESI MS of oligosaccharide alditols
Mass spectra were recorded on a Finnigan TSQ 7000 triple-stage quadruple mass spectrometer equipped with an electrospray ion source (Finnigan Instruments, San Jose, CA). The ESI voltage was set at 4500 V, and the capillary temperature was 225°C. The electron multiplier was set at 1000–1200 V. The pressure of the sheath gas was 70 psi, and that of the auxiliary gas was 10 U.
• Analytical condition 1 for sialylated oligosaccharide alditols: polarity, negative; mass range, m/z 1000–1600; scan time, 2 min.
• Analytical condition 2 for asialo- and agalacto-oligosaccharide and fucosyl trimannosylcore alditols: polarity, positive; mass range, m/z 800–1800; scan time, 2 min.
NMR
Sulfated and nonsulfated agalacto-tetraantennary oligosaccharide alditols from EPO (5 mg) was prepared by the method described above and purified by GCC/LC. The elution was performed with linear acetonitrile gradient from 9.5 to 11.5% in 100 min in 5 mM ammonium acetate at a flow rate of 0.2 ml/min. The fraction was exchanged twice in 99.95% D2O and lyophilized. Sulfated and nonsulfated agalacto-tetraantennary oligosaccharide alditols were dissolved in D2O with 3-mm tubes. 1H-NMR experiments were performed on a JEOL A-600 (Tokyo) at 35°C. In 2D COSY and HOHAHA analyses, the 1000 x 500 data points were processed with square sine bell functions for resolution enhancement in the f2 and f1 dimensions, respectively, and were zero-filled to give 2000 x 2000 real data points. All measurements were performed using JEOL software.
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Abbreviations |
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BHK, baby hamster kidney; Bi, biantennary; CHO, Chinese hamster ovary; COSY, 1H-1H chemical shift-correlated spectroscopy; EPO, erythropoietin; ESI, electrospray ionization; GCC, graphitized carbon column; GST, Gal/GalNAc/GlcNAc 6-O-sulfotransferase; HOHAHA, homonuclear Hartmann-Hahn; Lac, N-acetyllactosamine; LC/MS, liquid chromatography with mass spectrometry; NA, N-acetylneuramic acid; NMR, nuclear magnetic resonance; PNGase F, N-glycosidase F; Tetra, tetraantennary; TIC, total ion current; Tri, triantennary.
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Footnotes |
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1 To whom correspondence should be addressed
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References |
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