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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Key words: erythropoietin/ESI-LC/MS/1H-NMR/sulfated oligosaccharide
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Introduction |
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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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Figure 4A shows the TIC chromatogram of neuraminidase-treated oligosaccharides. Distributions of asialo-Bi, -Tri, -Tetra, -TetraLac1, and -TetraLac2 are indicated in Figures 4BF, respectively, and those modified with a sulfate group are indicated in Figures 4GK, 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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Discussion |
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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 Man
1-3 and Man
1-6 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 GlcNAcß1-4Man
1-3 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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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: 3055% of B in 80 min.
Gradient condition 2 for asialo-oligosaccharide alditols: 2045% of B in 60 min.
Gradient condition 3 for agalacto-oligosaccharide alditols and fucosyltrimannosylcore alditol: 1830% 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 10001200 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 10001600; scan time, 2 min.
Analytical condition 2 for asialo- and agalacto-oligosaccharide and fucosyl trimannosylcore alditols: polarity, positive; mass range, m/z 8001800; 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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Footnotes |
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References |
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