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Originally published In Press as doi:10.1074/jbc.M105715200 on July 30, 2001

J. Biol. Chem., Vol. 276, Issue 40, 37141-37148, October 5, 2001
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alpha 2,3-Sialylation of Terminal GalNAcbeta 1-3Gal Determinants by ST3Gal II Reveals the Multifunctionality of the Enzyme

THE RESULTING Neu5Acalpha 2-3GalNAc LINKAGE IS RESISTANT TO SIALIDASES FROM NEWCASTLE DISEASE VIRUS AND STREPTOCOCCUS PNEUMONIAE*

Suvi ToivonenDagger , Olli AitioDagger §, and Ossi RenkonenDagger

From the Dagger  Institute of Biotechnology and Department of Biosciences and the § Viikki Drug Discovery Technology Center, Department of Pharmacy, University of Helsinki, 00014 Helsinki, Finland

Received for publication, June 20, 2001, and in revised form, July 19, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Enzymatic alpha 2,3-sialylation of GalNAc has not been described previously, although some glycoconjugates containing alpha 2,3-sialylated GalNAc residues have been reported. In the present experiments, recombinant soluble alpha 2,3-sialyltransferase ST3Gal II efficiently sialylated the X2 pentasaccharide GalNAcbeta 1-3Galbeta 1-4GlcNAcbeta 1-3Galbeta 1-4Glc, globo-N-tetraose GalNAcbeta 1-3Galalpha 1-4Galbeta 1-4Glc, and the disaccharide GalNAcbeta 1-3Gal in vitro. The purified products were identified as Neu5Acalpha 2-3GalNAcbeta 1-3Galbeta 1-4GlcNAcbeta 1-3Galbeta 1-4Glc, Neu5Acalpha 2-3GalNAcbeta 1-3Galalpha 1-4Galbeta 1-4Glc, and Neu5Acalpha 2-3GalNAcbeta 1-3Gal, respectively, by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, enzymatic degradations, and one- and two-dimensional NMR-spectroscopy. In particular, the presence of the Neu5Acalpha 2-3GalNAc linkage was firmly established in all three products by a long range correlation between Neu5Ac C2 and GalNAc H3 in heteronuclear multiple bond correlation spectra. Collectively, the data describe the first successful sialyltransfer reactions to the 3-position of GalNAc in any acceptor. Previously, ST3Gal II has been shown to transfer to the Galbeta 1-3GalNAc determinant. Consequently, the present data show that the enzyme is multifunctional, and could be renamed ST3Gal(NAc) II. In contrast to ST3Gal II, ST3Gal III did not transfer to the X2 pentasaccharide. The Neu5Acalpha 2-3GalNAc linkage of sialyl X2 was cleaved by sialidases from Arthrobacter ureafaciens and Clostridium perfringens, but resisted the action of sialidases from Newcastle disease virus and Streptococcus pneumoniae. Therefore, the latter two enzymes cannot be used to differentiate between Neu5Acalpha 2-3GalNAc and Neu5Acalpha 2-6GalNAc linkages, as has been assumed previously.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Enzymatic alpha 2,3-sialylation of GalNAc1 has not been described before, although structures containing alpha 2,3-sialylated GalNAc residues, like sialyl X2, have been reported. The X2 epitope, GalNAcbeta 1-3Galbeta 1-4GlcNAcbeta 1-3Galbeta 1-4Glc, and its distally alpha 2,3-sialylated form have been characterized from glycosphingolipids on human red blood cells (1-3). Immunohistochemical experiments with the anti-X2 monoclonal antibody TH2 suggest the presence of X2 and sialylated X2 also in human white blood cells, liver, spleen, lung, kidney, colon, pancreas, brain, and placenta (3). High concentration of X2 glycolipid has been reported in gastric tumor tissue (4). Sialyl globoside (Neu5Acalpha 2-3GalNAcbeta 1-3Galalpha 1-4Galbeta 1-4Glc) has been characterized from human teratocarcinoma cells (5) and from muscles affected by amyotrophic lateral sclerosis (6) by chromatographic behavior, exoglycosidase treatments, and antibody reactivity. Additionally, sialyl LacdiNAc (Neu5Acalpha 2-3GalNAcbeta 1-4GlcNAc) from snake venom serine proteases contains alpha 2,3-sialylated GalNAc (7, 8). It has been suggested that the X2 epitope is the human receptor for Clostridium difficile toxin A (9). The X2 structure also occurs in the lipo-oligosaccharide of Neisseria gonorrhoeae strain F62 (10).

The known mammalian alpha 2,3-sialyltransferases, ST3Gal I-VI,2 transfer sialic acid to the galactose in Galbeta 1-4GlcNAc, Galbeta 1-3GlcNAc, or Galbeta 1-3GalNAc, and show some promiscuity among the three acceptor types, as well as overlapping acceptor specificities with each other (11-13). To elucidate the biosynthetic route to the Neu5Acalpha 2-3GalNAc linkage, we tested commercial recombinant alpha 2,3-sialyltransferases for their ability to sialylate enzymatically synthesized, unconjugated X2 pentasaccharide. We found that ST3Gal II, but not ST3Gal III, efficiently catalyzed the transfer of Neu5Ac to this acceptor, generating Neu5Acalpha 2-3GalNAcbeta 1-3Galbeta 1-4GlcNAcbeta 1-3Galbeta 1-4Glc. ST3Gal II transferred Neu5Ac in alpha 2,3-linkage also to the GalNAc termini of free globo-N-tetraose, GalNAcbeta 1-3Galalpha 1-4Galbeta 1-4Glc, and the disaccharide GalNAcbeta 1-3Gal. Previously, ST3Gal II has been shown to act on the core 1 disaccharide Galbeta 1-3GalNAc, and on glycolipids containing a terminal Galbeta 1-3GalNAcbeta 1-OR sequence (14). The emerging dual acceptor specificity of ST3Gal II makes it one of the few glycosyltransferases reported to date that are capable of transferring to different monosaccharide residues.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials

UDP-Gal was a gift from Prof. B. Ernst (University of Basel, Basel, Switzerland). Lacto-N-neotetraose was a gift from Prof. R. D. Cummings (University of Oklahoma, Oklahoma City, OK). Globo-N-tetraose was from Accurate Chemical and Scientific Corporation (Westbury, NY), GalNAcbeta 1-3Gal was from Dextra (Reading, United Kingdom). Neu5Acalpha 2-3Galbeta 1-4GlcNAcbeta 1-3Galbeta 1-4GlcNAc was enzymatically synthesized as described in Ref. 17. UDP-GalNAc, CMP-Neu5Ac, bovine milk beta 1,4-galactosyltransferase, and jack bean beta -N-acetylhexosaminidase were from Sigma. Rat recombinant ST3Gal II (alpha 2,3-(O)-sialyltransferase), rat recombinant ST3Gal III (alpha 2,3-(N)-sialyltransferase), and Streptococcus pneumoniae sialidase were from Calbiochem (La Jolla, CA). Jack bean beta -galactosidase and Escherichia freundii endo-beta -galactosidase were from Seikagaku Corp. (Tokyo, Japan). Bacteroides fragilis endo-beta -galactosidase and Newcastle disease virus sialidase were from Roche Molecular Biochemicals (Basel, Switzerland). Arthrobacter ureafaciens sialidase was from Glyko (Novato, CA). Clostridium perfringens sialidase was from New England Biolabs (Beverly, MA). Dowex AG-50, Dowex AG-1, and Bio Gel P-4 were from Bio-Rad. Superdex Peptide HR 10/30 and Mono-Q columns were from Amersham Pharmacia Biotech (Uppsala, Sweden). D2O was from Cambridge Isotope Laboratories (Woburn, MA).

Synthesis of the X2 Pentasaccharide

GalNAcbeta 1-3Galbeta 1-4GlcNAcbeta 1-3Galbeta 1-4Glc (X2) was synthesized essentially as described in Ref. 18, using a distinct ammonium sulfate precipitate of human serum as the enzyme source (19), UDP-GalNAc as the donor, and the tetrasaccharide Galbeta 1-4GlcNAcbeta 1-3Galbeta 1-4Glc as the acceptor. The product, representing the putative X2 pentasaccharide, GalNAcbeta 1-3Galbeta 1-4GlcNAcbeta 1-3Galbeta 1-4Glc, was isolated from the reaction mixture by gel filtration chromatography. Takeya et al. (18) have shown by methylation analysis that the product formed from lactose under these reaction conditions is GalNAcbeta 1-3Galbeta 1-4Glc. We established the structure of the putative X2 pentasaccharide by the following set of experiments. First, its MALDI-TOF mass spectrum revealed two peaks at m/z 933.33 and 949.33, which were assigned to [M + Na]+ and [M + K]+ of HexNAc1(Galbeta 1-4GlcNAcbeta 1-3Galbeta 1-4Glc), respectively (calculated m/z 933.32 and 949.29, respectively). Second, the terminal HexNAc was sensitive to jack bean beta -N-acetylhexosaminidase (data not shown), but it was not GlcNAc as it could not be galactosylated by beta 1,4-galactosyltransferase from bovine milk (data not shown). Finally, the HMBC spectrum of the sialyl X2 revealed that the distal beta -GalNAc and the peridistal Gal of the X2 core were joined by a 1,3-bond (Fig. 7a).

Enzymatic Methods

ST3Gal II Reactions-- 600 nmol of acceptor oligosaccharide and 2.4 µmol of CMP-Neu5Ac were incubated with 40 milliunits of rat recombinant ST3Gal II (alpha 2,3-(O)-sialyltransferase, EC 2.4.99.4) in 50 mM sodium cacodylate, pH 6.0, 0.02% NaN3, 0.05% bovine serum albumin, and 8 mM MnCl2 in a reaction volume of 600 µl for 6 days at room temperature. 20 milliunits of fresh enzyme was added on day 3. The reaction was terminated by boiling for 3 min.

ST3Gal III Reactions-- 49 nmol of acceptor oligosaccharide and 100 nmol of CMP-Neu5Ac were incubated with 3.2 milliunits of rat recombinant ST3Gal III (alpha 2,3-(N)-sialyltransferase, EC 2.4.99.5) in 100 mM MOPS-NaOH, pH 7.5, 0.02% NaN3, and 8 mM MnCl2 in a reaction volume of 12.5 µl for 6 days at room temperature. The reaction was terminated by boiling for 3 min.

Other Enzymatic Methods-- beta 1,4-Galactosyltransferase reactions were performed with bovine milk beta 1,4-galactosyltransferase (EC 2.4.1.90) essentially as described in Ref. 20. N-Acetylhexosaminidase reactions were performed by using jack bean beta -N-acetylhexosaminidase (EC 3.2.1.52) essentially as described in Refs. 21 and 22. Reactions with B. fragilis and E. freundii endo-beta -galactosidases (EC 3.2.1.103) were performed essentially as described in Ref. 23. Sialidase reactions were performed in a 40-µl reaction volume and 75 µM substrate concentration. The reactions were incubated 20 h in 37 °C and terminated by boiling for 3 min. A. ureafaciens sialidase reactions were done in 100 mM sodium phosphate buffer, pH 5, with 40 milliunits of enzyme; C. perfringens sialidase reactions in 50 mM sodium phosphate buffer, pH 4.5, with 167 milliunits of enzyme; Newcastle disease virus sialidase reactions in 50 mM sodium phosphate buffer, pH 5.5, with 8 milliunits of enzyme; and S. pneumoniae sialidase reactions in 50 mM sodium phosphate buffer, pH 4.5, with 20 milliunits of enzyme.

Chromatographic Methods

The sialyltransferase reaction products were purified by gel filtration HPLC in a column of Superdex Peptide HR 10/30 with 50 mM NH4HCO3 as eluant, followed by anion exchange HPLC in a column of MonoQ (5/5) essentially as described in Ref. 24. The oligosaccharides were quantitated by comparing their UV 214 absorbance to external GlcNAc and Neu5Ac.

Mass Spectrometry

Matrix-assisted laser desorption/ionization mass spectrometry of reaction products was performed with a BIFLEXTM mass spectrometer (Bruker-Franzen Analytik, Bremen, Germany). The neutral oligosaccharides were analyzed essentially as in Ref. 25 and the sialylated oligosaccharides as in Refs. 26 and 27.

NMR Spectroscopy

Prior to NMR experiments the saccharides (400-600 nmol) were lyophilized twice from D2O and then dissolved in 40 µl of D2O (99.996 atom %). The NMR experiments were carried out on a Varian Unity 500 spectrometer at 23 °C using a gHX nano-NMR probe (Varian). A spinning rate of 2000 Hz was used. In recording one-dimensional proton spectra, a modification of the water-eliminated Fourier transformation sequence (28) was used. The DQFCOSY and TOCSY experiments were carried out essentially as in Ref. 29.

For the gradient HMQC (30) and gradient HMBC experiments (31, 32) (32 and 128 scans/t1 value, respectively), matrices of 2k*256 and 2k*128 points were recorded and zero-filled to 2k*512 and 2k*256 points, respectively and a shifted sine-bell function was used. The average 1H-13C coupling constant was estimated to be 140 Hz, and Delta 2 was 63.5 ms. The spectral widths F1 and F2 were 11250 and 2400 Hz, respectively.

In order to resolve overlap in sialylated X2, an additional gradient HSQC spectrum (31) was measured on a Varian Unity 600 MHz instrument with a spectral width F1 of 6000 Hz (The C1:s, CH3 of NAc:s, and C3 of Neu5Ac were folded). In this experiment the glycan (590 nmol) was dissolved in 600 µl of D2O and a conventional 5-mm tube was used. A matrix of 2k*256 points with 48 scans/t1 value was recorded.

The 1H and 13C chemical shifts were referenced to internal acetone, 2.225 and 31.55 ppm, respectively.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The present report describes sialylation reactions catalyzed by the alpha 2,3-sialyltransferase known as ST3Gal II (33) with oligosaccharide acceptors containing the terminal GalNAcbeta 1-3Gal determinant. The acceptors and products are depicted in Fig. 1, which also shows the one-letter symbols of the constituent monosaccharides, used for describing the NMR data.


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  		Fig. 1.   Present alpha 2,3-sialylation reactions catalyzed by ST3Gal II.

Sialylation of the X2 Pentasaccharide, GalNAcbeta 1-3Galbeta 1-4GlcNAcbeta 1-3Galbeta 1-4Glc-- The X2 pentasaccharide GalNAcbeta 1 -3Galbeta 1-4GlcNAcbeta 1-3Galbeta 1-4Glc (600 nmol) was incubated with CMP-Neu5Ac (2.4 µmol) and rat recombinant sialyltransferase ST3Gal II (alpha 2,3-(O)-sialyltransferase) as described under "Experimental Procedures." The sialylated product (590 nmol) was isolated by gel filtration and anion exchange HPLC in pure form. In negative ion mode MALDI-TOF mass spectrometry, the purified product gave a peak at m/z 1200.44, which was assigned to [M - H]- of Neu5Ac1[GalNAcbeta 1-3Galbeta 1-4GlcNAcbeta 1-3Galbeta 1-4Glc] (calculated monoisotopic m/z 1200.42) (Fig. 2a).


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  		Fig. 2.   MALDI-TOF mass spectra of the ST3Gal II reaction products of X2 (a), globo-N-tetraose (b), and GalNAcbeta 1-3Gal (c).

In contrast to ST3Gal II, rat recombinant ST3Gal III (alpha 2,3-(N)-sialyltransferase) sialylated less than 0.5% of the X2 pentasaccharide when incubated under conditions that completely sialylated Galbeta 1-4GlcNAc (data not shown).

N-Acetylhexosaminidase Did Not Cleave Sialyl X2-- Sialyl X2 was incubated with jack bean beta -N-acetylhexosaminidase under conditions that completely released the terminal N-acetylgalactosamine from non-sialylated X2. No cleavage product was seen in a MALDI-TOF mass spectrum of the desalted reaction mixture of sialyl X2 (Fig. 3a). The data suggest that the sialic acid is linked to the terminal N-acetylgalactosamine.


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  		Fig. 3.   a, negative mode MALDI-TOF mass spectrum of the desalted reaction mixture of N-acetylhexosaminidase digestion of sialyl X2. N-Acetylhexosaminidase did not cleave sialyl X2, as the only peak in the spectrum is at [M - H]- of intact sialyl X2, m/z 1200.65. b, positive mode MALDI-TOF mass spectrum of the desalted reaction mixture of endo-beta -galactosidase digestion of sialyl X2. Endo-beta -galactosidase cleaved sialylated X2 into product di- and trisaccharides. The labeled peaks can be assigned to sodium and potassium adducts of Neu5Ac1HexNAc1Hex1 (697.20, 713.17, 719.24, and 735.17) and HexNAc1Hex1 (406.39 and 422.23).

Endo-beta -galactosidases from B. fragilis and E. freundii Cleaved Sialyl X2 at Two Sites-- The X2 lipid-linked pentasaccharide is known to be cleaved at both internal galactosidic linkages by endo-beta -galactosidase from E. freundii, yielding GalNAcbeta 1-3Gal, GlcNAcbeta 1-3Gal, and Glc (2, 34). In the present experiments, both B. fragilis and E. freundii endo-beta -galactosidases also cleaved unconjugated sialyl X2 hexasaccharide completely at two sites. MALDI-TOF mass spectrometry of the desalted digest revealed a sialic acid-containing trisaccharide of the composition Neu5Ac1HexNAc1Hex1 and a neutral disaccharide of the composition HexNAc1Hex1 (Fig. 3b). Additionally, Glc was probably formed in the reaction, but it could not be identified in the spectrum among the matrix peaks. In view of the NMR data described below, the oligosaccharide products were identified as Neu5Acalpha 2-3GalNAcbeta 1-3Gal and GlcNAcbeta 1-3Gal (Fig. 4).


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  		Fig. 4.   Scheme of endo-beta -galactosidase cleavage of sialyl X2.

NMR Spectroscopy of Sialyl X2 Hexasaccharide-- The 1H and 13C signals of sialyl X2 were assigned from the one-dimensional experiment and from DQFCOSY, TOCSY, HSQC, HMQC, and HMBC spectra (Tables I-III).

                              
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Table I
1H NMR chemical shifts (ppm) of structural reporter groups of the present acceptors and reaction products at 23 °C
ND, not determined; ---, not appropriate.

                              
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Table II
1H chemical shifts (ppm) of the sialylated reaction products
ND, not determined; ---, not appropriate.

                              
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Table III
13C chemical shifts (ppm) of the sialylated reaction products
ND, not determined; ---, not appropriate.

Some features of the structural reporter group area in the one-dimensional proton spectrum are significant for the structural analysis of sialyl X2 (Table I and Fig. 5a). The presence of an alpha -linked Neu5Ac group was demonstrated by the presence of typical Neu5Acalpha H3 resonances at 1.628 and 2.696 ppm (35-36). A resonance at 4.189 ppm, not seen in the spectrum of non-sialylated X2, was assigned to GalNAc H3. The TOCSY spectrum (Fig. 6a) showed that this signal belongs to the GalNAc spin system, and the DQFCOSY spectrum identified it as the resonance of GalNAc H3 (not shown). Hence, alpha -sialylation of the X2 pentasaccharide was associated with a sizable downfield shift of the GalNAc H3 resonance from the bulk of the ring proton signals. Quite similar changes have been reported for the Gal H3 upon alpha 2,3-sialylation of Galbeta 1-OR type saccharides (35, 36).


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  		Fig. 5.   Expansions of 1H NMR spectra of X2 sialylated by ST3Gal II (a), globo-N-tetraose sialylated by ST3Gal II (b), and the disaccharide GalNAcbeta 1-3Gal sialylated by ST3Gal II (c). The one-letter symbols of the monosaccharide residues are shown in Fig. 1.


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  		Fig. 6.   Expansions of TOCSY spectra of X2 sialylated by ST3Gal II (a), globo-N-tetraose sialylated by ST3Gal II (b), and the disaccharide GalNAcbeta 1-3Gal sialylated by ST3Gal II (c).The one-letter symbols of the monosaccharide residues are shown in Fig. 1.

The chemical shift of the pivotal quaternary FC2 of the Neu5Ac unit was obtained from the HMBC spectrum of sialyl X2 (Fig. 7a), which reveals clear correlations at 100.80 ppm with the two Neu5Ac FH3:s, and with the GalNAc EH3. These correlations arise from a quaternary carbon because, at 100.80 ppm, no correlations were detected in the HMQC spectrum (data not shown). The reported Neu5Ac C2 chemical shifts (100.1-100.3 ppm), but not the C1 chemical shifts (174-175 ppm), in Neu5Acalpha 2-3Galbeta 1-OR type saccharides (35, 36) resemble closely the chemical shifts of this quaternary carbon in sialyl X2. With this background, the interglycosidic correlation FC2-EH3 of Fig. 7a shows unambiguously that sialyl X2 contains a Neu5Ac2-3GalNAc linkage. No other interglycosidic correlations involving FC2 were detected in the HMBC spectrum of sialyl X2; of particular significance is the absence of correlations of the type FC2-EH6.


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  		Fig. 7.   HMBC spectra of X2 sialylated by ST3Gal II (a), globo-N-tetraose sialylated by ST3Gal II (b), and the disaccharide GalNAcbeta 1-3Gal sialylated by ST3Gal II (c). The one-letter symbols of the monosaccharide residues are shown in Fig. 1.

The structure of the X2 pentasaccharide core, indirectly established by Takeya et al. (18), was confirmed by the interresidual correlations present in the HMBC spectrum of sialyl X2 (Fig. 7a); the positions of the four glycosidic linkages of the pentasaccharide core were confirmed by the four correlations BH1-AC4, CH1-BC3, DH1-CC4, and EH1-DC3 (for the monosaccharide denotations, see Fig. 1). These data validate many of the assignments in Tables II and III.

The Neu5Acalpha 2-3GalNAc Linkage of Sialyl X2 Was Cleaved by Sialidases from C. perfringens and A. ureafaciens, but Was Resistant to Sialidases from Newcastle Disease Virus and S. pneumoniae-- The enzymatically synthesized sialyl X2 hexasaccharide was incubated with several bacterial and viral sialidases in conditions that completely desialylated Neu5Acalpha 2-3Galbeta 1-4GlcNAcbeta 1-3Galbeta 1-4GlcNAc. Sialyl X2 was completely desialylated by treatments with the sialidases from C. perfringens and A. ureafaciens, as shown by gel filtration chromatography and MALDI-TOF mass spectrometry of the reaction mixtures (Table IV). These enzymes are known to cleave alpha -glycosidic bonds of sialic acids, including the Neu5Acalpha 2-6GalNAc bond (37-39). By contrast, less than 10% of sialyl X2 was desialylated by the sialidases from Newcastle disease virus (Fig. 8) and S. pneumoniae (data not shown) (Table IV). The data imply that the sialidases from Newcastle disease virus and S. pneumoniae, which are able to cleave the Neu5Acalpha 2-3Gal linkage, hydrolyzed poorly the Neu5Acalpha 2-3GalNAc linkage.

                              
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Table IV
Desialylation of sialyl X2 and a control oligosaccharide by different sialidases
The amounts of desialylated products were assayed by gel filtration chromatography and MALDI-TOF mass spectrometry.


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  		Fig. 8.   Positive mode MALDI-TOF mass spectrum of the digest obtained by incubating sialyl X2 with Newcastle disease virus sialidase under conditions that completely desialylated Neu5Acalpha 2-3Galbeta 1-4GlcNAcbeta 1-3Galbeta 1-4GlcNAc. The peaks at m/z 1224.66, 1240.69, 1246.65, and 1262.69 can be assigned to intact sialyl X2, whereas the small peak at m/z 933.37 is the desialylated product.

Sialylation of Globo-N-tetraose, GalNAcbeta 1-3Galalpha 1-4Galbeta 1-4Glc-- Unconjugated globo-N-tetraose GalNAcbeta 1-3Galalpha 1-4Galbeta 1-4Glc (600 nmol) was incubated with CMP-Neu5Ac (2.4 µmol) and rat recombinant ST3Gal II as described under "Experimental Procedures." The sialylated product (450 nmol) was isolated by gel filtration and anion exchange HPLC in pure form. In negative ion mode MALDI-TOF mass spectrometry, the purified product gave a peak at m/z 997.28, which was assigned to [M - H]- of Neu5Ac1[GalNAcbeta 1-3Galalpha 1-4Galbeta 1-4Glc] (calculated monoisotopic m/z 997.34) (Fig. 2b).

NMR Spectroscopy of Sialylated Globo-N-tetraose-- The one-dimensional proton NMR spectrum (Fig. 5b), the TOCSY spectrum (Fig. 6b), and 1H and 13C resonances (Tables I-III) show that the Neu5Ac and the GalNAc residues of sialyl globo-N-tetraose are virtually identical to their counterparts in sialyl X2. This suggests that unconjugated globo-N-tetraose was sialylated by ST3Gal II in the same way as the X2 pentasaccharide, at position 3 of the terminal GalNAc residue. This notion was confirmed by the downfield shift of GalNAc H3 of globo-N-tetraose (40, 22) that was caused by sialylation (Table I). The best proof of the presence of Neu5Acalpha 2-3GalNAc linkage in the sialyl globo-N-tetraose was obtained from the HMBC spectrum (Fig. 7b). This spectrum shows a correlation between Neu5Ac FC2 and GalNAc EH3. The correlations EH1-DC3, DH1-BC4, and BH1-AC4 in the HMBC spectrum of sialyl globo-N-tetraose identified correctly the glycosidic linkages of the globo-N-tetraose core of the sialylated product.

Sialylation of the Free Disaccharide GalNAcbeta 1-3Gal-- The disaccharide GalNAcbeta 1-3Gal (600 nmol) was incubated with CMP-Neu5Ac (2.4 µmol) and rat recombinant ST3Gal II as described under "Experimental Procedures." The sialylated product (400 nmol) was isolated by gel filtration and anion exchange HPLC in pure form. In negative ion mode MALDI-TOF mass spectrometry, the purified product gave a peak at m/z 673.06, which was assigned to [M - H]- of Neu5Ac1[GalNAcbeta 1-3Gal] (calculated monoisotopic m/z 673.23) (Fig. 2c).

NMR Spectroscopy of Sialylated GalNAcbeta 1-3Gal-- The 1H and 13C signals of the sialylated GalNAcbeta 1-3Gal were fully assigned (Tables I-III). The structural reporter group resonances of the 1D proton spectrum of the sialylated GalNAcbeta 1-3Gal (Fig. 5c and Table I) resemble closely their counterparts in sialyl X2 and sialyl globo-N-tetraose (Table I). The TOCSY spectrum (Fig. 6c) reveals that the GalNAc spin system, too, is virtually identical with those of sialyl X2 and sialyl globo-N-tetraose. Finally, the HMBC spectrum (Fig. 7c) confirms the presence of Neu5Acalpha 2-3GalNAc linkage by showing a clear correlation between GalNAc EH3 and Neu5Ac FC2. Due to vicinity of the reducing end, several GalNAc protons resonate at two different fields (Table I). The two signals belong to the alpha  (Gal Dalpha ) and beta  (Gal Dbeta ) anomeric forms of the oligosaccharide. In the HMBC spectrum (Fig. 7c), interglycosidic correlations between the GalNAc E H1alpha and Gal Dalpha C3, as well as between GalNAc E H1beta and Gal Dbeta C3 are visible.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The present report describes alpha 2,3-sialylation of the distal GalNAc residue of the free X2 pentasaccharide GalNAcbeta 1-3Galbeta 1-4GlcNAcbeta 1-3Galbeta 1-4Glc, globo-N-tetraose GalNAcbeta 1-3Galalpha 1-4Galbeta 1-4Glc, and the disaccharide GalNAcbeta 1-3Gal by the recombinant sialyltransferase ST3Gal II, as shown in Fig. 1. These reactions represent the first successful enzymatic in vitro syntheses of the Neu5Acalpha 2-3GalNAc linkage. The structure of the purified sialyl X2 hexasaccharide was characterized by its molecular mass as obtained from MALDI-TOF mass spectrometry experiments, by enzymatic degradations, and by one- and two-dimensional NMR spectroscopy. The presence of a Neu5Acalpha 2-3GalNAc bond was firmly established by the HMBC spectrum, which revealed a long range correlation between Neu5Ac C2 and GalNAc H3. The sialylated products obtained from globo-N-tetraose and the disaccharide GalNAcbeta 1-3Gal were characterized in the same way, but enzymatic degradations were not performed. Compared with previous reports on structural analysis of Neu5Ac2-3GalNAc determinants of naturally expressed glycans, the experiments of the present report are more NMR-oriented, and are not based on methylation analysis.

Our present data, showing that, in addition to the Galbeta 1-3GalNAc determinant (14), ST3Gal II also transfers to the isomeric GalNAcbeta 1-3Gal determinant, establish that the enzyme represents one of the few glycosyltransferases that are capable of transferring to different monosaccharide residues in the acceptor, challenging the dogma "one glycosyltransferase, one glycosidic linkage" (41). The best known of these is beta 1,4-galactosyltransferase, which is induced by alpha -lactalbumin to transfer to glucose instead of N-acetylglucosamine (20). Glycosyltransferases that transfer to different monosaccharide residues without requiring an additional modifier molecule include the beta 1,3-galactosyltransferase beta 3GalT-V, which transfers to both the terminal GalNAc of GalNAcbeta 1-3Galalpha 1-4Galbeta 1-4Glc and the terminal GlcNAc of GlcNAcbeta 1-3Galbeta 1-4Glc (42), and the Core2GlcNAcTs, which transfer to the GalNAc of Galbeta 1-3GalNAcalpha -R and GlcNAcbeta 1-3GalNAcalpha 1-R, as well as to the Gal of GlcNAcbeta 1-3Galbeta 1-R (43-45). A fourth example is the human fucosyltransferases III, V, and VI, which transfer to the Glc of lactose as well as to the GlcNAc of N-acetyllactosamine, generating Galbeta 1-4(Fucalpha 1-3)Glc and Galbeta 1-4(Fucalpha 1-3)GlcNAc, respectively (46-48). Finally, the bovine colostrum alpha 2,6-sialyltransferase has also been shown to tolerate N-acetylation of C2 of the acceptor monosaccharide; it sialylates both the Gal of Galbeta 1-4GlcNAc-R and the GalNAc of GalNAcbeta 1-4GlcNAc-R (49), suggesting an acceptor recognition mechanism similar to that of ST3Gal II discussed here. We suggest that ST3Gal II, and the other multifunctional glycosyltransferases, may bind their multiple acceptors by recognizing identical sets of saccharide atoms that belong to several monosaccharide residues, and form identical patterns.

The Neu5Acalpha 2-3GalNAc linkage in the sialylated X2 hexasaccharide resisted cleavage by sialidases from Newcastle disease virus and S. pneumoniae. The Newcastle disease virus sialidase is known to exhibit strict specificity for hydrolysis of the Neu5Acalpha 2-3Gal linkage under conditions that leave Neu5Acalpha 2-6Gal and Neu5Acalpha 2-6GalNAc bonds intact (50). Similar data have been reported for the sialidase from S. pneumoniae (manufacturer's specifications). Neu5Ac-GalNAc linkages that resist the action of Newcastle disease virus sialidase have been regarded as Neu5Acalpha 2-6GalNAc bonds (51-53). Our cleavage data show that this conclusion is not necessarily valid.

Globoside is expressed abundantly in human tissues. Therefore, it is remarkable that, although globo-N-tetraose is readily sialylated by ST3Gal II, as shown by the present experiments, sialyl globoside appears to be rare; its presence has been reported only in human embryonal carcinoma cells (5) and in muscles affected by amyotrophic lateral sclerosis (6). The reasons for the low expression levels of sialyl globoside are unknown, but association of globoside with other biomolecules than ST3Gal II, or low expression levels of ST3Gal II in cells expressing globoside may be involved.

alpha 2,3-Sialylation of the X2 structure may play a role in bacteria-host interactions. The X2 structure occurs in the lipo-oligosaccharide of N. gonorrhoeae strain F62 (10). Sialylation of lipo-oligosaccharide converts gonococci into serum resistance (reviewed in Ref. 54), possibly by camouflaging bacterial surface from the host by molecular mimicry of human cell surface glycoconjugates (55). The N. gonorrhoeae alpha 2,3-sialyltransferase, Lst, has relaxed acceptor specificity; it is able to use N-acetyllactosamine, lactose, and globotriose (Galalpha 1-4Galbeta 1-4Glc) as acceptors (56), and Lst from the strain 126E(L1) can even make both Neu5Acalpha 2-3Gal and Neu5Acalpha 2-6Gal linkages (57). To our knowledge it has not been tested whether the N. gonorrhoeae sialyltransferase uses X2-like structures as acceptors, but considering its relaxed acceptor specificity, it seems possible.

It has been suggested that the X2 epitope on intestinal epithelium is the human receptor for C. difficile toxin A. X2 glycosphingolipid has been shown to bind toxin A, but alpha 2,3-sialylation of X2 abolishes the binding (9). Therefore, sialylation of X2-like structures might be a protective measure against adhesion, and thus internalization and cytotoxic effects of C. difficile toxin A.

    ACKNOWLEDGEMENTS

We thank Jari Natunen and Leena Penttilä for helpful discussions and Hannu Maaheimo for critically reading the manuscript. We thank Prof. B. Ernst for UDP-Gal and Prof. R. D. Cummings for lacto-N-neotetraose.

    FOOTNOTES

* This work was supported by Grants 38042, 40901, and 44318 from the Academy of Finland; Grant 40896 from the Technology Development Center, Helsinki; a grant from the Emil Aaltonen Foundation; and the Graduate School of Bioorganic Chemistry, University of Turku. Portions of the data in this report were presented in poster form at the 20th International Carbohydrate Symposium, August 27-September 1, 2000, Hamburg, Germany (15) and at Glycobiology 2000, November 8-11, 2000, Boston, MA (16).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom all correspondence should be addressed: Biomedicum Helsinki, P. O. Box 63, University of Helsinki, 00014 Helsinki, Finland. Tel.: 358-9-191-25117; Fax: 358-9-19125155; E-mail: ossi.renkonen@helsinki.fi.

Published, JBC Papers in Press, July 30, 2001, DOI 10.1074/jbc.M105715200

2 The abbreviated nomenclature of sialyltransferases is based on Ref. 33.

    ABBREVIATIONS

The abbreviations used are: GalNAc, N-acetyl-D-galactosamine; DQFCOSY, double-quantum-filtered correlated spectroscopy; Gal, D-galactose; Glc, D-glucose; GlcNAc, N-acetyl-D-glucosamine; HexNAc, N-acetylhexosamine; HMBC, heteronuclear multiple bond correlation; HMQC, heteronuclear multiple quantum coherence; HSQC, heteronuclear single quantum coherence; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; m/z, mass to charge ratio; Neu5Ac, N-acetylneuraminic acid; TOCSY, total correlation spectroscopy; X2, GalNAcbeta 1-3Galbeta 1-4GlcNAcbeta 1-3Galbeta 1-4Glc; HPLC, high performance liquid chromatography; MOPS, 4-morpholinepropanesulfonic acid.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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