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

Abstract

Enzymatic ␣2,3-sialylation of GalNAc has not been described previously, although some glycoconjugates containing ␣2,3-sialylated GalNAc residues have been reported. In the present experiments, recombinant soluble ␣2,3-sialyltransferase ST3Gal II efficiently sialylated the 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 in vitro. The purified products were identified as Neu5Ac␣2-3GalNAc␤1-3Gal␤1-4GlcNAc␤1-3Gal␤1-4Glc, Neu5Ac␣2-3GalNAc␤1-3Gal␣1-4Gal␤1-4Glc, and Neu5Ac␣2-3GalNAc␤1-3Gal, respectively, by matrixassisted laser desorption/ionization time-of-flight mass spectrometry, enzymatic degradations, and oneand two-dimensional NMR-spectroscopy. In particular, the presence of the Neu5Ac␣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 Gal␤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 X 2 pentasaccharide. The Neu5Ac␣2-3GalNAc linkage of sialyl X 2 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 Neu5Ac␣2-3GalNAc and Neu5Ac␣2-6GalNAc linkages, as has been assumed previously.Enzymatic ␣2,3-sialylation of GalNAc 1 has not been described before, although structures containing ␣2,3-sialylated GalNAc residues, like sialyl X 2 , have been reported. The X 2 epitope, GalNAc␤1-3Gal␤1-4GlcNAc␤1-3Gal␤1-4Glc, and its distally ␣2,3-sialylated form have been characterized from glycosphingolipids on human red blood cells (1-3). Immunohistochemical experiments with the anti-X 2 monoclonal antibody TH2 suggest the presence of X 2 and sialylated X 2 also in human white blood cells, liver, spleen, lung, kidney, colon, pancreas, brain, and placenta (3). High concentration of X 2 glycolipid has been reported in gastric tumor tissue (4). Sialyl globoside (Neu5Ac␣2-3GalNAc␤1-3Gal␣1-4Gal␤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 (Neu5Ac␣2-3GalNAc␤1-4GlcNAc) from snake venom serine proteases contains ␣2,3-sialylated GalNAc (7, 8). It has been suggested that the X 2 epitope is the human receptor for Clostridium difficile toxin A (9). The X 2 structure also occurs in the lipooligosaccharide of Neisseria gonorrhoeae strain F62 (10).The known mammalian ␣2,3-sialyltransferases, ST3Gal I-VI, 2 transfer sialic acid...

Human α2,3-Sialyltransferase (ST3Gal II) Is a Stage-specific Embryonic Antigen-4 Synthase

“…globo-and ganglio-series, could be sialylated by the same enzyme. Recently ␣2,3-sialylation of GalNAc␤1,3Gal determinant by ST3Gal II was demonstrated (28), indicating that ST3Gal II is a multifunctional enzyme. The fact that levels of ST3Gal II mRNA are not necessarily consistent with those of MSGb5 in RCC cell lines may also suggest that the enzyme plays the other roles.…”

Enterovirus 70 Binds to Different Glycoconjugates Containing α2,3-Linked Sialic Acid on Different Cell Lines

“…O-linked glycans containing terminal sialic acid ␣2,3 linked to galactose, although the possibility that EV70 has the capacity to interact with glycans containing ␣2,6-linked sialic acid cannot be completely ruled out. V. cholerae sialidase cleaves terminal sialic acid but not sialic acid from branched internal positions, and S. pneumoniae sialidase, which efficiently stripped cells of EV70 binding sites, exhibits specificity for sialic acid that is ␣2,3 linked to galactose but is unable to release sialic acid linked to N-acetylgalactosamine in vitro (66). The ␣2,3-sialyltransferases used in this study sialylate galactose residues (53) ( Table 1) and restored EV70 attachment sites to sialidase-treated cells to a much greater degree than did an ␣2,6-specific sialyltransferase.…”

Glycosphingolipids of human embryonic stem cells

“…Sialyl-globotetraosylceramide has not been reported in normal human tissues, but has been characterized in human teratocarcinoma cells [ 32 ], and in muscles affected by amyothropic lateral sclerosis [ 33 ]. Terminal NeuAcα3GalNAc is a rare sequence, but is found in the sialyl- x 2 glycosphingolipid (NeuAcα3GalNAcβ3Galβ4GlcNAcβ3Galβ4Glcβ1Cer; [ 34 ]), and α2,3-sialylation of the terminal GalNAc of x 2 and globotetraosylceramide by the α2,3-sialyltransferase ST3Gal II has been demonstrated [ 35 ].…”