Sialylated cell surface glycoproteins and glycolipids are involved in host-pathogen interactions, molecular recognition and cell adhesion, immune cell regulation and antibody function, and accordingly, dysregulated sialylation is associated with pathology and is a hallmark of some diseases. In particular, ST6GalNAc1 adds Neu5Ac sialic acid in an α2,6 linkage to GalNAc-Ser/Thr residues resulting in the STn antigen, which is implicated in cancer progression and survival. To understand the enzymatic basis of STn antigen synthesis, we have characterized the recombinant form of the human enzyme purified from a mammalian expression system. ST6GalNAc1 preferentially acts on O-glycans as substrates in a glycoprotein microarray experiment and not N-glycans and does not require divalent cations for function. We characterize the Km value of the enzyme for the sugar donor, CMP-Neu5Ac, using asialo-bovine submaxillary mucin as a substrate, and the Km for acceptors GalNAc-Ser and GalNAc-Thr. ST6GalNAc1 contains a transmembrane domain, a stem region with multiple O-glycosylation sites, and a catalytic domain with three N-glycosylation sites. We characterized the N- and O-glycomes of the released glycans by MS, and additionally the site-specific glycoform profiles by glycopeptide analysis with LC-MS/MS. Like other mammalian sialyltransferases ST6GalNAc1 is a monomer by analytical size exclusion chromatography with multi-angle light scattering, and the stem domain contributes to a significantly increased hydrodynamic radius by dynamic light scattering measurements. Taken together, these biochemical data provide a framework for understanding the function of the mammalian sialyltransferase ST6GalNAc1.

Characterization of the human α2,6 sialyltransferase ST6GalNAc1

256th National Meeting (2018)

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