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.