α2,3-Sialylation of Terminal GalNAcβ1–3Gal Determinants by ST3Gal II Reveals the Multifunctionality of the Enzyme

Enzymatic α2,3-sialylation of GalNAc has not been described previously, although some glycoconjugates containing α2,3-sialylated GalNAc residues have been reported. In the present experiments, recombinant soluble α2,3-sialyltransferase ST3Gal II efficiently sialylated the X2 pentasaccharide GalNAcβ1–3Galβ1–4GlcNAcβ1–3Galβ1–4Glc, globo-N-tetraose GalNAcβ1–3Galα1–4Galβ1–4Glc, and the disaccharide GalNAcβ1–3Gal in vitro. The purified products were identified as Neu5Acα2–3GalNAcβ1–3Galβ1–4GlcNAcβ1–3Galβ1–4Glc, Neu5Acα2–3GalNAcβ1–3Galα1–4Galβ1–4Glc, and Neu5Acα2–3GalNAcβ1–3Gal, 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 Neu5Acα2–3GalNAc 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 Galβ1–3GalNAc 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 Neu5Acα2–3GalNAc linkage of sialyl X2 was cleaved by sialidases from Arthrobacter ureafaciens and Clostridium perfringens, but resisted the action of sialidases from Newcastle disease virus andStreptococcus pneumoniae. Therefore, the latter two enzymes cannot be used to differentiate between Neu5Acα2–3GalNAc and Neu5Acα2–6GalNAc linkages, as has been assumed previously.

ST3Gal III Reactions-49 nmol of acceptor oligosaccharide and 100 nmol of CMP-Neu5Ac were incubated with 3.2 milliunits of rat recom-

Chromatographic Methods
The sialyltransferase reaction products were purified by gel filtration HPLC in a column of Superdex Peptide HR 10/30 with 50 mM NH 4 HCO 3 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 BIFLEX mass spectrometer (Bruker-Franzen Analytik, Bremen, Germany). The neutral oligosaccharides were analyzed essentially as in Ref. 25

NMR Spectroscopy
Prior to NMR experiments the saccharides (400 -600 nmol) were lyophilized twice from D 2 O and then dissolved in 40 l of D 2 O (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/t 1 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 1 H-13 C coupling constant was estimated to be 140 Hz, and ⌬ 2 was 63.5 ms. The spectral widths F 1 and F 2 were 11250 and 2400 Hz, respectively.
In order to resolve overlap in sialylated X 2 , an additional gradient HSQC spectrum (31) was measured on a Varian Unity 600 MHz instrument with a spectral width F 1 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 l of D 2 O and a conventional 5-mm tube was used. A matrix of 2k*256 points with 48 scans/t 1 value was recorded.
The 1 H and 13 C chemical shifts were referenced to internal acetone, 2.225 and 31.55 ppm, respectively.

RESULTS
The present report describes sialylation reactions catalyzed by the ␣2,3-sialyltransferase known as ST3Gal II (33) with oligosaccharide acceptors containing the terminal GalNAc␤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.
N-Acetylhexosaminidase Did Not Cleave Sialyl X 2 -Sialyl X 2 was incubated with jack bean ␤-N-acetylhexosaminidase under conditions that completely released the terminal N-acetylgalactosamine from non-sialylated X 2 . No cleavage product was seen  ␣2,3-Sialylation of GalNAc by ST3Gal II in a MALDI-TOF mass spectrum of the desalted reaction mixture of sialyl X 2 (Fig. 3a). The data suggest that the sialic acid is linked to the terminal N-acetylgalactosamine. Endo-␤-galactosidases from B. fragilis and E. freundii Cleaved Sialyl X 2 at Two Sites-The X 2 lipid-linked pentasaccharide is known to be cleaved at both internal galactosidic linkages by endo-␤-galactosidase from E. freundii, yielding GalNAc␤1-3Gal, GlcNAc␤1-3Gal, and Glc (2,34). In the present experiments, both B. fragilis and E. freundii endo-␤galactosidases also cleaved unconjugated sialyl X 2 hexasaccharide completely at two sites. MALDI-TOF mass spectrometry of the desalted digest revealed a sialic acid-containing trisaccharide of the composition Neu5Ac 1 HexNAc 1 Hex 1 and a neutral disaccharide of the composition HexNAc 1 Hex 1 (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 Neu5Ac␣2-3GalNAc␤1-3Gal and GlcNAc␤1-3Gal (Fig. 4).
NMR Spectroscopy of Sialyl X 2 Hexasaccharide-The 1 H and 13 C signals of sialyl X 2 were assigned from the one-dimensional experiment and from DQFCOSY, TOCSY, HSQC, HMQC, and HMBC spectra (Tables I-III).
Some features of the structural reporter group area in the  one-dimensional proton spectrum are significant for the structural analysis of sialyl X 2 (Table I and Fig. 5a). The presence of an ␣-linked Neu5Ac group was demonstrated by the presence of typical Neu5Ac␣ 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 X 2 , was assigned to GalNAc H3. The TOCSY spectrum (Fig. 6a) showed that this signal belongs to the Gal-NAc spin system, and the DQFCOSY spectrum identified it as the resonance of GalNAc H3 (not shown). Hence, ␣-sialylation of the X 2 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 ␣2,3-sialylation of Gal␤1-OR type saccharides (35,36). The chemical shift of the pivotal quaternary FC2 of the Neu5Ac unit was obtained from the HMBC spectrum of sialyl X 2 (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 Neu5Ac␣2-3Gal␤1-OR type saccharides (35,36) resemble closely the chemical shifts of this quaternary carbon in sialyl X 2 . With this background, the interglycosidic correlation FC2-EH3 of Fig. 7a shows unambiguously that sialyl X 2 contains a Neu5Ac2-3GalNAc linkage. No other interglycosidic correlations involving FC2 were detected in the HMBC spectrum of sialyl X 2 ; of particular significance is the absence of correlations of the type FC2-EH6.
The structure of the X 2 pentasaccharide core, indirectly established by Takeya et al. (18), was confirmed by the interresidual correlations present in the HMBC spectrum of sialyl X 2 (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 Neu5Ac␣2-3GalNAc Linkage of Sialyl X 2 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 X 2 hexasaccharide was incubated with several bacterial and viral sialidases in conditions that completely desialylated Neu5Ac␣2-3Gal␤1-4GlcNAc␤1-3Gal␤1-4GlcNAc. Sialyl X 2 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 ␣-glycosidic bonds of sialic acids, including the Neu5Ac␣2-6GalNAc bond (37)(38)(39). By contrast, less than 10% of sialyl X 2 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 Neu5Ac␣2-3Gal linkage, hydrolyzed poorly the Neu5Ac␣2-3GalNAc linkage.
NMR Spectroscopy of Sialylated Globo-N-tetraose-The onedimensional proton NMR spectrum (Fig. 5b), the TOCSY spectrum (Fig. 6b), and 1 H and 13 C resonances (Tables I-III) show that the Neu5Ac and the GalNAc residues of sialyl globo-Ntetraose are virtually identical to their counterparts in sialyl X 2 . This suggests that unconjugated globo-N-tetraose was sialylated by ST3Gal II in the same way as the X 2 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 Neu5Ac␣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- ␣2,3-Sialylation of GalNAc by ST3Gal II N-tetraose core of the sialylated product.
In the HMBC spectrum (Fig. 7c), interglycosidic correlations between the GalNAc E H1␣ and Gal D␣ C3, as well as between GalNAc E H1␤ and Gal D␤ C3 are visible.

DISCUSSION
The present report describes ␣2,3-sialylation of the distal GalNAc residue of the free X 2 pentasaccharide GalNAc␤1-3Gal␤1-4GlcNAc␤1-3Gal␤1-4Glc, globo-N-tetraose GalNAc␤1-3Gal␣1-4Gal␤1-4Glc, and the disaccharide GalNAc␤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 Neu5Ac␣2-3GalNAc linkage. The structure of the purified sialyl X 2 hexasaccharide was characterized by its molecular mass as obtained from MALDI-TOF mass spectrometry experiments, by enzymatic degradations, and by one-and twodimensional NMR spectroscopy. The presence of a Neu5Ac␣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 GalNAc␤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 Gal␤1-3GalNAc determinant (14), ST3Gal II also transfers to the isomeric GalNAc␤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 ␤1,4-galactosyltransferase, which is induced by ␣-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 ␤1,3-galactosyltransferase ␤3GalT-V, which transfers to both the terminal GalNAc of GalNAc␤1-3Gal␣1-4Gal␤1-4Glc and the terminal GlcNAc of GlcNAc␤1-3Gal␤1-4Glc (42), and the Core2GlcNAcTs, which transfer to the GalNAc of Gal␤1-3GalNAc␣-R and GlcNAc␤1-3GalNAc␣1-R, as well as to the Gal of GlcNAc␤1-3Gal␤1-R (43)(44)(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 Gal␤1-4(Fuc␣1-3)Glc and Gal␤1-4(Fuc␣1-3)GlcNAc, respectively (46 -48). Finally, the bovine colos-trum ␣2,6-sialyltransferase has also been shown to tolerate N-acetylation of C2 of the acceptor monosaccharide; it sialylates both the Gal of Gal␤1-4GlcNAc-R and the GalNAc of GalNAc␤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 Neu5Ac␣2-3GalNAc linkage in the sialylated X 2 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 Neu5Ac␣2-3Gal linkage under conditions that leave Neu5Ac␣2-6Gal and Neu5Ac␣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 Neu5Ac␣2-6GalNAc bonds (51)(52)(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.
␣2,3-Sialylation of the X 2 structure may play a role in bacteria-host interactions. The X 2 structure occurs in the lipooligosaccharide 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 ␣2,3-sialyltransferase, Lst, has relaxed acceptor specificity; it is able to use Nacetyllactosamine, lactose, and globotriose (Gal␣1-4Gal␤1-4Glc) as acceptors (56), and Lst from the strain 126E(L1) can even make both Neu5Ac␣2-3Gal and Neu5Ac␣2-6Gal linkages (57). To our knowledge it has not been tested whether the N. gonorrhoeae sialyltransferase uses X 2 -like structures as acceptors, but considering its relaxed acceptor specificity, it seems possible.
It has been suggested that the X 2 epitope on intestinal epithelium is the human receptor for C. difficile toxin A. X 2 glycosphingolipid has been shown to bind toxin A, but ␣2,3-sialylation of X 2 abolishes the binding (9). Therefore, sialylation of X 2 -like structures might be a protective measure against adhesion, and thus internalization and cytotoxic effects of C. difficile toxin A.