Unique enzymatic properties of human ST3Gal sialyltransferases: Reversible sialylation and synthesis of CMP-sialic acid in human blood
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Glycosylation is a ubiquitous posttranslational modification found on most secretory proteins that are either soluble or that are cell surface bound. Structures formed as a result of glycosylation regulate biological processes, including, but not limited to inflammation and immunity [1-3]. A family of 200 enzymes called glycosyltransferases (GlycoTs) mediates Glycosylation. Among these, the focus of the current project is on enzymes called sialyltransferases (SialylTs). Commonly, these enzymes transfer sialic acid, its most common form in mammals being N-acetylneuraminic acid (Neu5Ac or NANA), to terminal glycan structures or acceptors . In this regard, SialylTs mediate the formation of α2,3, α2,6 and α2,8 linkages by the attachment of sialic acid to the 3-, 6- and 8- positions of acceptors. Among these linkages, α2,3 linked sialic acid terminated structures, catalyzed by the α2,3sialylTs are ubiquitously found in nature. As illustrated in Table I, α2,3 sialylation commonly occurs on the terminal galactose residues of three types of glycans that are termed Type I, II and III structures. Enzymes that mediate the formation of such Neu5Acα2,3Galactose structures belong to the 'ST3Gal family'. Since these terminally sialylated glycans are involved in various inflammatory processes, studies of the ST3Gals are significant. In this context, sialylated Type II structures are precursors to sLe X type carbohydrate ligands present on leukocyte surfaces . Such ligands recognize adhesion molecules belonging to the selectin family in the vasculature. Recognition of sLe X by selectins, mediates leukocyte homing to sites of the inflammatory insult . sLe a type structures that are formed on tumor cells can also bind selectins. Traditionally, sialylation reactions are thought to proceed unidirectionally--a process termed 'forward sialylation' (Figure 1A). Recent reports including my studies with human ST3Gals (isoforms ST3Gal I, II, III, IV and VI) support the proposition that this reaction can be reversible under some circumstances -- a process termed 'reverse sialylation' ( Figure 1B) . Additionally, while sialylation is typically considered to be a Golgi resident process, the presence of these ST3Gal enzymes in blood suggests the capacity of organisms to catalyze previously unrecognized post-Golgi glycosylation related reactions . Based on these propositions, this research focuses on the characterization of human ST3Gals using synthetic, natural and cell-based glycoprobes. Further, comprehensive characterization is conducted by considering the catalytic nature of 'reverse' and trans-sialidase like 'exchange' activity. These studies are performed using radioactivity assay and confirmed by the development of method using LC-MS. Overall, the dissertation presents experimental results indicating an active role of soluble ST3Gals in extracellular sialylation. These reactions may prominently occur using a range of cell types including neutrophils, platelets, and erythrocytes.