Mammalian sialyltransferase ST3Gal-II: its exchange sialylation catalytic properties allow labeling of sialyl residues in mucin-type sialylated glycoproteins and specific gangliosides.

Glycoproteins modulate biological function such as signaling, immune response and tissue development by interaction through terminal sugars (1). These sugars or glycans are commonly either N- linked to asparagine, or O- linked to Ser/Thr residues on protein scaffolds. Among these, mucins are O-glycan rich glycoproteins that often contain tandem repeats of peptide sequences characterized by a high content of Ser, Thr and Pro residues (2–4). Each tissue exhibits a unique pattern of mucinous proteins that can be modified under pathological conditions. Such regulation of mucin expression in cancer epithelial cells influences cell adhesion and tumor invasiveness. Moreover, cancer associated mucins that contain incomplete O-glycan chains are highly immunogenic and these are potential targets for immunotherapy. Whereas the study of N-glycans has progressed well, in part, due to the availability of highly-specific endoglycosidases that can release intact N-glycans, similar universal glycosidases for mucin-type O-glycans are not available. New tools for the study of O-glycosylation are thus desirable. The current study addresses this need by presenting a novel strategy to enzymatically radiolabel O-glycans, and also glycolipids. O-glycosylation is initiated by the attachment of GalNAc via α-linkages to hydroxyl groups of Ser/Thr that are exposed on the protein surface such as at coils, turns or linker regions. This step is catalyzed by a family of specific UDP GalNAc: polypeptide N-acetylgalacto-saminyltransferases (5,6). Further extension of these glycans is regulated, in large measure, by the distribution of glycosyltransferases and sulfotransferases that are primarily localized in the cellular Golgi (7,8). Sialic acid residues are typically found at the terminal non-reducing ends of O-glycans expressed both on cell-surface and secreted glycoproteins. The attachment of these residues is mediated by enzymes belonging to the sialyltransferase family. While these enzymes are typically thought to uni-directionally catalyze the transfer of sialic acid from a sugar nucleotide donor (CMP-NeuAc) to an acceptor substrate, we recently showed that this reaction can be reversible at least for the case of one mammalian/rat sialyltransferase ST3Gal-II (9). Here, using this process called `reverse sialylation', we demonstrated that ST3Gal-II can synthesize CMP-NeuAc from 5′-CMP and NeuAcα2,3Galβ1,3GalNAc- units of O-glycans, and glycolipids. In addition to this, we now report that this enzyme can also catalyze the direct exchange of NeuAc between CMP-NeuAc and the NeuAcα2,3Galβ1,3GalNAc- units of O-glycans and glycolipids, in the absence of exogenous 5'-CMP. While the precise mechanism for this exchange process is yet to be established, this may be partially attributed to the formation of 5'-CMP in the reaction mixture due to the break-down of CMP-NeuAc. These unique catalytic properties of ST3Gal-II could be utilized for the facile radiolabeling in vitro of sialyl residues in mucin-type structures.