Glycobiology Advance Access originally published online on June 15, 2005
Glycobiology 2005 15(10):1051-1060; doi:10.1093/glycob/cwi092
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Development of structural analysis of sulfated N-glycans by multidimensional high performance liquid chromatography mapping methods
2 Graduate School of Pharmaceutical Sciences, Nagoya City University, 3–1 Tanabe-dori, Mizuho-ku, Nagoya 467–8603, Japan; 3 CREST, JST, 4-1-8 Honcho, Kawaguchi 332–1102, Japan; 4 GLYENCE Co., Ltd. 1-4-6 Masaki, Naka-ku, Nagoya 460–8690, Japan; 5 Program of Molecular Pathology, Aichi Cancer Center, Research Institute, 1–1 Kanokoden, Chikusa-ku, Nagoya 464–8681, Japan; and 6 Department of Anatomy and Program in Immunology, University of California, San Francisco, CA 94143–0452, USA
1 To whom correspondence should be addressed; e-mail: kkato{at}phar.nagoya-cu.ac.jp
Received on May 10, 2005; revised on June 6, 2005; accepted on June 8, 2005
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Abstract |
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Although the biological importance of sulfated oligosaccharides has been widely recognized, there are only a few reports that describe detailed structures of sulfated N-glycans. This is largely due to the lack of a convenient method to identify structures of sulfated glycans found in low incidence. Here we develop multidimensional high performance liquid chromatography (HPLC) mapping methods for rapid and convenient identification of sulfated N-glycans. By using adequate quantities of sulfated N-glycans derived from LS12 cells, which are transfected with sulfotransferase cDNA, 40 different sulfated glycans have been successfully mapped. Furthermore, we have applied the HPLC data to identification of isomeric products resulting from an enzymatic reaction of N-acetylglucosamine 6-O-sulfotransferase-1 in vitro and revealed that this enzyme preferentially catalyzes sulfation of the GlcNAcß1
2Man
13Man branch in a biantennary acceptor. Key words: branch specificity / GlcNAc6ST-1 / HPLC mapping / N-glycans / sulfated oligosaccharides
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Introduction |
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TopAbstractIntroductionResults and discussionMaterials and methodsReferences |
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Sulfated glycans expressed on glycoproteins play important roles in the biological functions, such as lymphocyte homing through L-selectin (Rosen, 1999
), degradation of pituitary gland hormones (Fiete et al., 1991), and adhesion of neural cells (Martini et al., 1992). It is important to identify the structures of individual sulfated glycans to understand the mechanisms of their biological functions. However, there are only a few reports that describe detailed structures of sulfated N-glycans in glycoproteins. Although nuclear magnetic resonance (NMR) spectroscopy could be a powerful method to determine structures of sulfated oligosaccharides (van Rooijen et al., 1998; Gallego et al., 2001), it requires large quantities of samples. Mass spectrometric (MS) method is rapid and sensitive but has difficulty in discriminating isomeric structures (Liedtke et al., 2001; Wheeler and Harvey, 2001).
To identify the structures of N-glycans, we have been developing a three-dimensional (3-D) sugar mapping technique using pyridyl-2-aminated (PA) derivatives of oligosaccharides (Tomiya et al., 1988; Takahashi et al., 1995). This method uses three different high performance liquid chromatography (HPLC) columns (diethylaminoethyl [DEAE], octadecyl silica [ODS], and amide) to separate PA-oligosaccharides and requires only picomole quantities of samples. The structures are identified on the basis of their elution positions on these HPLC columns by direct comparison with the HPLC data of available reference compounds. The HPLC data of 
500 different N-glycans accumulated so far are now available in a web application, GALAXY (Takahashi and Kato, 2003). However, it is still difficult to identify the structures of sulfated oligosaccharides by HPLC map, because little HPLC data of these oligosaccharides are obtained as yet (Wakabayashi et al., 1999; Wakabayashi et al., 2001).
In this study, we collected sulfated N-glycans derived from mammalian cells cotransfected with both N-acetylglucosamine 6-O-sulfotransferase-1 (GlcNAc6ST-1) and 1–3 fucosyltransferase VII (Fuc-T VII) cDNAs (Kimura et al., 1999). The structures of these sulfated oligosaccharides were successfully determined by multidimensional HPLC mapping technique, that is the 3-D sugar mapping technique combined with exoglycosidase digestion and MS data. In addition, we applied the HPLC data of the sulfated oligosaccharides to identification of isomeric products resulting from an enzymatic reaction of GlcNAc6ST-1 in vitro and revealed the branch specificity of this enzyme.
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Results and discussion |
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Isolation of sulfated N-glycans derived from LS12 cells
The glycoproteins were extracted by 3-(1-pyridinio)-1-propanesulphonate (3-(I-pyridinio)-1-propanesulphonate [NDSB]-201) from LS12 cells, which are derived from a human endothelial cell line (ECV304) (Takahashi et al., 1990
) by cotransfection with both GlcNAc6ST-1 and Fuc-T VII cDNAs (Kimura et al., 1999). By glycoamidase A digestion, the glycans were released from a glycoprotein fraction of LS12 cells. Each reducing end of the released glycans was labeled with 2-aminopyridine. Figure 1A shows the elution profile on a DEAE-5PW column of the PA-glycans derived from the glycoproteins from LS12 cells. A number of anionic fractions were detected possibly because the PA-glycans were sialylated as well as sulfated. These fractions were subjected to a sialidase treatment, because we focused on the sulfated asialooligosaccharides as a first step of collection of the HPLC data of sulfated oligosaccharides. The desialylated products were applied onto a DEAE-5PW column to fractionate anionic asialooligosaccharides (fractions I and II) (Figure 1B). Fractions I and II were applied to a TSK gel Amide-80 column (amide) individually and gave eleven (a–k) and three (a–c) major peaks on their elution profiles, respectively (Figure 1C and D). These individual fractions were further applied onto an ODS column. The fraction a in Figure 1C (derived from fraction I) was further fractionated into two peaks on the ODS column (data not sown). These fractions are designated as I-a-1 and I-a-2. Similar notation will be used for other fractions. We could isolate 21 different anionic asialooligosaccharides derived from LS12 cells and record the elution values of these glycans on the amide and ODS columns (Table I). Matrix-assisted laser desorpsion/ionization time-of-flight mass spectrometric (MALDI-TOF-MS) data indicated that all of these 21 asialooligosaccharides were sulfated.
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Structural analysis of sulfated N-glycans derived from LS12 cells
The procedure of structural analyses of these sulfated glycans would be exemplified by the determination of structure of glycan corresponding to fraction I-g-1, which was eluted at 11.3 glucose unit value on the ODS column (GU[ODS]) and 7.5 glucose unit value on the amide column (GU[amide]). The molecular mass of this glycan determined by MALDI-TOF-MS analysis was 2238 Da, which corresponds to (Hex)5(HexNAc)4(DeoxyHex)3(HSO3)1PA. Upon HCl treatment, this glycan released a sulfate group, giving rise to a biantennary oligosaccharide, Galß14 (Fuc13)GlcNAcß12Man16(Galß14(Fuc13)GlcNAcß12Man13)Manß14GlcNAcß14(Fuc1 6)GlcNAc-PA.
We postulate that the original oligosaccharide, I-g-1 is monosulfated at a GlcNAc residue in one of two branches of this structure. To identify the sulfated GlcNAc residue, fraction I-g-1 was subjected to various glycosidase treatments and desulfation by HCl treatment and the resultant PA-oligosaccharides were mapped based on their GU(amide) and molecular mass values (Figure 2). The glycan I-g-1 was resistant to ß-galactosidase but changed to the product A upon removal of three fucose residues by -fucosidase digestion. Then, the product A was converted into the product B releasing two galactose residues by the ß-galactosidase treatment. A ß-N-acetylglucosaminidase treatment of product B gave rise to product C releasing one N-acetylglucosamine residue. Finally, desulfation of product C gave rise to product D whose GU(amide), GU(ODS) and molecular mass values are in good agreement with those of known reference PA glycan, Man16(GlcNAcß12 Man13)Manß14GlcNAcß1 4GlcNAc-PA (Code No. 100.2 in GALAXY). By co-chromatography on the ODS column, we confirmed the identity between product D and this PA-oligosaccharide.
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Taking account of the reaction specificities of the enzymes expressed by the transfected cells, we estimated precursor structures at each reaction step as follows: (A) Galß14GlcNAcß12Man16(Galß14(SO3H6)GlcNAcß12Man13)Manß14GlcNAcß14GlcNAc-PA, (B) GlcNAcß12Man16(SO3H6GlcNAcß1 2Man13)Manß14GlcNAcß14GlcNAc-PA, (C) Man16(SO3H6GlcNAcß12Man13)Manß14GlcNAcß14GlcNAc-PA (Table II). These structures were confirmed individually by co-chromatography of the desulfated products of A and B with the corresponding candidates. On the basis of all these data, we concluded that the starting material I-g-1 is the biantennary complex type glycan with sulfated and unsulfated Lewis X structures at the Man1–3 and Man1–6 branches, respectively, Galß14(Fuc13)GlcNAcß12Man16(Galß14 (Fuc13)(SO3H6)GlcNAcß12Man13)Manß14GlcNAcß14(Fuc16)GlcNAc-PA.
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In a similar way, we identified 21 sulfated oligosaccharides derived from LS12 cells, which are bi-, tri-, and tetraantennary complex-type glycans sulfated at the GlcNAc residues in the GlcNAcß12Man13Man and/or GlcNAcß12Man16Man branches (Table I). These results suggest a tendency of GlcNAc6ST-1 to sulfate preferentially at the GlcNAcß12Man13Man branch. Furthermore, we identified 19 sulfated oligosaccharides derived from the original sulfated oligosaccharides by various glycosidase digestions and HCl treatments and recorded their HPLC data (Table II). Four monosulfated oligosaccharides with a sulfate group at the GlcNAcß12Man16Man branch (1S2 series) are derived from a disulfated oligosaccharide (code No. 2S1-210.43) by partial desulfation and exoglycosidase treatments.
Inspection of the present HPLC data indicates that monosulfation of either GlcNAcß12Man13Man or GlcNAcß12Man16Man causes 1 decrease in GU(amide) but has little or no effect on GU(ODS). For example, although GU(amide) and GU(ODS) values of the non-sulfated biantennary oligosaccharide, GlcNAcß1 2Man16(GlcNAcß12Man13)Manß14GlcNAcß14GlcNAc-PA (code No. 200.1), have been reported to be 8.9 and 5.1, respectively (Tomiya et al., 1988), those of both monosulfated analogs derived from it, that is GlcNAcß12Man16(SO3H6GlcNAcß12Man13) Manß14GlcNAcß14GlcNAc-PA (code No. 1S1-200.1) and SO3H6GlcNAcß12Man16(GlcNAcß12 Man13)Manß14GlcNAcß14GlcNAc-PA (code No. 1S2-200.1) are 9.2 and 4.1, respectively (Table II).
In the previous study (Lee et al., 1990), it has been shown that the elution position of a given PA-glycan could be represented by the sum of the contribution of individual monosaccharide unit at a specific position (unit contribution). However this is obviously not the case for the sulfate groups. Namely, GU(amide) and GU(ODS) values of the disulfated biantennary oligosaccharide, SO3H6GlcNAcß1 2Man16(SO3H6GlcNAcß12Man13)Manß14GlcNAcß14GlcNAc-PA (code No. 2S1-200.1), are 8.6 and 3.3, respectively (Table II). Thus, additivity of contribution of the two sulfate units to elution time holds for GU(amide) but not for GU(ODS), that is 0.3 decrease upon disulfation. One possible explanation is that disulfation induces significant conformational alteration of oligosaccharides by electrostatic repulsion between the sulfate groups.
Branch specificity of GlcNAc6ST-1
The HPLC map thus established will facilitate identification of sulfated oligosaccharides. In this study, we applied the HPLC data to quantification of products of a reaction catalyzed by recombinant GlcNAc6ST-1 expressed by COS7 cells as a fusion protein with protein A (protein A-fused GlcNAc6ST-1) to characterize branch specificity of this enzyme. In the previous study (Uchimura et al., 2002), GlcNAc6ST-1 catalyzes the transfer of sulfate from radioactive adenosine 3'-phosphate 5'-phosphosulfate (PAPS) to the C-6 position of non-reducing GlcNAc residue. Here we use the biantennary complex type glycan with GlcNAc residues at both non-reducing ends, GlcNAcß12Man1 6(GlcNAcß12Man13)Manß14GlcNAcß14(Fuc16)GlcNAc-PA was utilized as a substrate of GlcNAc6ST-1. This PA oligosaccharide was treated with protein A-fused GlcNAc6ST-1 in the presence of adenosine 3’-phosphate 5’-phosphosulfate (PAPS), adenosine 5'-monophosphate sodium salt, and NaF for 2 days. Figure 3A and 3B compares the elution profiles on amide column of the accepter glycan and the reaction mixtures, respectively. On the elution profile of the reaction mixture, beside the acceptor (peak c), two peaks a and b appeared, which were assigned to mono- and disulfated oligosaccharides based on their elution time. It was not possible to distinguish two possible monosulfated oligosaccharides, that is GlcNAcß1 2Man16(SO3H6GlcNAcß12Man13)Manß14GlcNAcß14(Fuc16)GlcNAc-PA and SO3H 6Glc NAcß12Man16(GlcNAcß12Man13)Manß14GlcNAcß14(Fuc16)GlcNAc-PA, solely on the basis of the elution time data, because these two isomers have identical GU(amide) and GU(ODS) values (Tables I and II). To discriminate these isomeric structures, the fraction b was treated with N-acetylhexosaminidase. The digestion mixture gave two peaks (d and e) with a intensity ratio of 2:1 on the elution profile (Fig. 3C). These two fractions were further applied on an amide column individually and show identical elution time, that is 4.2 GU(amide). Inspection of these data allowed us to assign peaks d and e to Man16(SO3H6GlcNAcß12Man13)Manß14GlcNAcß14(Fuc16)GlcNAc-PA (code No. 1S1-110.2) and SO3H6GlcNAcß12Man16(Man13)Manß1 4GlcNAcß14(Fuc16)GlcNAc-PA (code No. 1S2-110.1), respectively. Because virtually no peaks corresponding to the starting materials nor any possible byproducts were detected in the elution profiles of the reaction mixture, we conclude that the intensity ratio of peaks d to e represents the relative amount of GlcNAcß12Man16(SO3H 6GlcNAcß12Man13)Manß14GlcNAcß14(Fuc16)GlcNAc-PA and SO3H6GlcNAcß12Man1 6(GlcNAcß12Man13)Manß14GlcNAcß14(Fuc1 6)GlcNAc-PA, which in turn reflects incidence of monosulfation at each branch. Based on the fluorescence intensity of each fraction, the ratio of these reaction products was calculated as follows: SO3HGlcNAcß1 2Man16(SO3H6GlcNAcß12Man13)Manß14GlcNAcß14(Fuc16)GlcNAc-PA, 24 %; 6GlcNAcß1 2Man16(SO3H6GlcNAcß12Man13)Manß14GlcNAcß14(Fuc16)GlcNAc-PA,51%;SO3H 6GlcNAcß12Man16(GlcNAcß12Man13)Man ß14GlcNAcß14(Fuc16)GlcNAc-PA, 25%. These data indicate that GlcNAc6ST-1 preferentially sulfates the GlcNAcß12Man13Man branch, which is consistent with the preferential sulfation at this branch in LS12 cells (Table I).
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As far as we know, this is the first report of characterization of branch specificity of sulfotransferase against N-glycans.
In this study, we developed analytical methods of sulfated N-glycan structures by the HPLC map including 40 different sulfated glycans. This method enables us to identify and profile the sulfated glycans using subpicomole quantities of PA-oligosaccharides and is therefore much more conventional than NMR spectroscopy and gas chromatography-electron ionization mass spectrometry (GC-EI-MS) linkage analysis, which are time-consuming and require nanomole quantities of oligosaccharides. Furthermore, this method is promising for discriminating isomeric structures, for example 1S1-110.2 and 1S2-110.1, which cannot be achieved solely by MS analysis.
We could successfully apply the HPLC data to identify the reaction products of GlcNAc6ST-1 and characterization of this enzyme. Many of sulfotransferases in glycan biosynthesis have so far been identified (Hemmerich et al., 2001; Kusche-Gullberg and Kjellen, 2003). This method is now available to facilitate profiling of sulfated N-glycans and investigation of precise substrate specificity of the sulfotransferases.
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Enzymes
ß-Galactosidase and ß-N-acetylhexosaminidase from jack beans and glycoamidase A from sweet almonds were purchased from Seikagaku Kogyo (Tokyo, Japan).
-Sialidase from Arthrobacter ureafaciens was from Nacalai Tesque (Kyoto, Japan). -Fucosidase from bovine kidney was from Boehringer-Mannheim (Mannheim, Germany). Trypsin and chymotrypsin were from Sigma Chemical (St. Louis, MO).
Reference oligosaccharides
The pyridylamino derivatives of isomalto-oligosaccharides (degree of polymerization of glucose residues = 4–20) and the PA derivatives of neutral N-linked oligosaccharides of code Nos. 100.1, 100.2, 110.1, 110.2, 200.1, 200.2, 200.3, 200.4, 210.1, 210.2, 210.3, 210.4, 300.8, and 310.8 were purchased from Seikagaku Kogyo.
Other chemicals
The following materials were purchased from the sources indicated: dithiothreitol (DTT), Nacalai Tesque; iodoacetic acid (IAA) and NaF, Wako (Osaka, Japan); 3-(1-pyridinio)-1-propanesulphonate (NDSB-201), (Calbiochem, La Jolla, CA); 2,5-dihydroxybenzoic acid (DHB), adenosine 5¢-monophosphate sodium salt (AMP) and adenosine 3¢-phosphate 5¢-phosphosulfate, Sigma Chemical.
Preparation of sulfated N-glycans
To collect small quantity of sulfated oligosaccharides, we utilized LS12 cells, which had been cotransfected with both GlcNAc6ST-1 and Fuc-T VII cDNAs (Kimura et al., 1999). LS12 cells were grown to confluence in Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA) containing 10 % fetal calf serum. 4 x 108 cells were harvested and washed three times with 20 mM Tris–HCl, pH 8.0 containing 150 mM NaCl. Finally, the cells were suspended in 20 mL of 20 mM Tris–HCl, pH 8.0 containing 150 mM NaCl and 1 M NDSB-201. The cell suspension was sonicated, incubated at 4°C for 6 h and then centrifuged at 10,000 x g to provide protein fraction (supernatant). The supernatant was incubated in the presence of 10 mM DTT for 1 h at 37°C and then in the presence of 10 mM IAA for 1 h at 37°C in dark. The reaction mixture was dialyzed against water and dried by lyophilizer. The sample was incubated in the presence of 20 nM trypsin and 20 nM chymotripsin in 50 mM ammonium bicarbonate buffer, pH 8.5 at 37°C for 10 h. Then the N-glycans were released from proteolytic fractions with 5 mU glycoamidase A in 50 mM ammonium acetate buffer, pH 4.0 at 37°C for 10 h.
After removal of the peptide materials by Sep Pack (Waters, Milford, MS) according to manufacturer instructions, the reducing ends of the N-glycans were derivatized with 2-aminopyridine under conditions described previously (Yamamoto et al., 1989).
Isolation and characterization of sulfated N-glycans by multidimensional mapping
All procedures including chromatographic conditions used in this work have been reported previously (Nakagawa et al., 1995; Takahashi et al., 1995). After digestion of sialidase under the condition as described below, the desialylated PA-oligosaccharide mixture derived from LS12 cells was separated on a TSK gel DEAE-5PW column (Tosoh, Tokyo, Japan) according to their sulfate group content. Then, mono-, and di-sulfated PA-glycans were individually separated and isolated sequentially on a TSK-gel Amide-80 column and a Shim-pack HRC-octadecyl silica (ODS) column (Shimadzu, Kyoto, Japan). The elution volumes of each sulfated N-glycan both on the amide and the ODS columns were recorded and expressed as the glucose units. In this study, we determined the structures of six neutral N-glycans, which had not been recorded in the GALAXY database, that is Galß14GlcNAcß12M
16(Galß14 (Fuc13)GlcNAcß12Man13)Manß14GlcNAcß4 GlcNAc-PA, Galß14(Fuc13)GlcNAcß12Man16 (Galß14GlcNAcß12Man13)Manß14GlcNAcß1 4GlcNAc-PA, Galß14GlcNAcß12Man16(Galß1 4(Fuc13)GlcNAcß12Man13)Manß14GlcNAcß14(Fuc16)GlcNAc-PA, Galß14(Fuc13)GlcNAc ß12Man16(Galß14(Fuc13)GlcNAcß1 2Man 1 3)Manß14GlcNAcß14(Fuc16)GlcNAc-PA, Galß1 4(Fuc13)GlcNAcß12Man16(Galß1 4GlcNAcß12Man13)(GlcNAcß14)Manß14GlcNAcß14(Fuc16)GlcNAc-PA and Galß14GlcNAcß1 2Man16(Galß14(Fuc13)GlcNAcß12Man13) (GlcNAcß14)Manß14GlcNAcß14(Fuc16)GlcNAc-PA, by the HPLC mapping combined with MALDI-TOF-MS analyses and exoglycosidase digestion, before structural analyses of sulfated oligosaccharides.
MALDI-TOF-MS analysis
The isolated oligosaccharides derived from LS12 cells were subjected to MALDI-TOF-MS spectrometric analysis. MALDI-TOF-MS data were acquired in the positive and negative modes using Axima-CFR (Shimadzu) operated in the liner mode. The matrix solution was prepared as follows: DHB (10 mg) was dissolved in 1:1 (v/v) of acetonitrile/water (1 mL). Stock solutions of PA-glycans were prepared by dissolving them in pure water. 1 µL of matrix solution was applied on the target spot of plate, and 1 µL of sample solution was added, and then dried by warm air.
Desulfation
After the desalting, sulfated glycans was incubated in 1 M HCl at 37°C for 4 h. The resultings were subjected to MALDI-TOF-MS analysis. Approximately 50–65% sulfate groups were liberated by the HCl treatment.
Exoglycosidase digestion
Exoglycosidase digestion was used to characterize structures of sulfate oligosaccharides. Each PA-glycan isolated from LS12 cells was digested with exoglycosidases (-fucosidase, ß-galactosidase, ß-N-acetylglucosaminidase, and -sialidase) under conditions described previously (Nakagawa et al., 1995).
Purification of GlcNAc6ST-1 fused with protein A
COS7 cells on 75 cm2 of cultural flask (Corning, NY) were transfected with 4 µg of relevant plasmid, pcDNA-GlcNAc6ST-1 (Uchimura et al., 2002) using Lipofectamine plus (Invitrogen) according to manufacturer instructions. After 24 h of culture in Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum, the medium was replaced with Dulbecco’s modified Eagle’s medium containing 2% IgG-free fetal calf serum. The cells were cultured for an additional 96 h. Subsequently, the culture medium was collected and concentrated to 1 mL by Amicon Ultra-15 (Millipore, Billerica, MS). The recombinant protein A-fused GlcNAc6ST-1 expressed in the medium was adsorbed to IgG-Sepharose (20 µl resin/1 mL of culture medium) at 4°C for 3 h. The resin was collected by centrifugation and washed three times with 50 mM Tris–HCl, pH 7.5. Finally, the resin was suspended in 20 µL of 50 mM Tris–HCl, pH 7.5, and used as the enzyme.
Assay of GlcNAc6ST-1 activity toward PA oligosaccharide
GlcNAcß12Man16(GlcNAcß12Man13)Manß 4GlcNAcß14(Fuc16)GlcNAc-PA was utilized as a accepter of protein A-fused GlcNAc6ST-1. The standard reaction mixture contained 1 µmol of Tris–HCl, pH 7.5, 0.4 µmol of MnCl2, 0.08 µmol of AMP, 24 µmol of NaF, 50 pmol of PA-oligosaccharide (Code No.210.1 in GALAXY), 300 pmol of PAPS, 0.1% Triton X-100, and 20 µL of the fusion protein suspension in final volume of 40 µL. After incubation at 30°C for 2 days, aliquots of 5 µL of the reaction mixture were applied onto an amide column. Then, the separated glycans were individually injected onto an ODS column and identified based on the HPLC map including sulfated oligosaccharides.
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We thank Mamiko Nishimura for her contribution at an early stage of this work. This work was supported in part by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan and by a grant for Research on Health Sciences focusing on Drug Innovation from the Japan Health Sciences Foundation.
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Abbreviations |
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DEAE, diethylaminoethyl; DeoxyHex, deoxyhexose; Fuc, fucose; Gal, galactose; GlcNAc, N-acetylglucosamine; GU(amide), glucose unit value on the amide column; GU(ODS), glucose unit value on the ODS column; Hex, hexose; HexNAc, N-acetylhexosamine; HPLC, high performance liquid chromatography; MALDI-TOF-MS, matrix-assisted laser desorpsion/ionization time-of-flight mass spectrometry; Man, mannose; NDSB, 3-(I-pyridinio)-1-propanesulphonate; ODS, octadecyl silica; PAPS, adenosine 3’-phosphate 5’-phosphosulfate
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References |
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