Influence of diversity in sialic acid presentation on human neuraminidase enzyme activity

Field: Biochemistry, Genetics and Molecular Biology (Life Sciences)

Sialic acids are an incredibly diverse family of carbohydrates that serve many roles in biological systems. Their presentation is influenced not only by structural modification of the sialic acid, but also by attachment through glycosidic linkages and their presence on different glycoconjugates. Many unusual sialic acids are unstable and difficult to study. Our group has an ongoing interest in sialic acid metabolism by the human neuraminidase enzymes (hNEU). The metabolism of unstable sialic acids such as 9-O-acetylated sialic acids (Neu5,9Ac2) and polymers of sialic acid (polysialic acid, polysia) by hNEU has not been systematically defined. In this thesis we present studies towards understanding hNEU metabolism of sialic acids which vary in monosaccharide structure and glycosidic linkage. Chapter 2 describes our initial study of hNEU activity towards Neu5,9Ac2 by monitoring enzyme activity on simple fluorescent substrates based on 4MU-NANA (4-methylumbelliferyl α-D-N-acetylneuraminic acid). The substrate preference observed for NEU4 towards these simple substrates disagreed with those observed for octyl sialyllactoside mimics of the ganglioside GM3. We also observed an increase in the preference for Neu5Ac over Neu5,9Ac2 for the α(2→6)-linked octyl sialyllactosides compared to the α(2→3)-linked substrates. These results confirmed the importance of considering the context of sialic acid presentation when studying its metabolism and inspired us to continue studying hNEU activity towards Neu5,9Ac2 on different sialoside substrates. We expanded our study of hNEU activity towards Neu5,9Ac2 substrates in Chapter 3 to encompass more of the diversity of sialic acid presentation. We began by studying hNEU activity towards a Neu5,9Ac2-enriched glycoprotein, which required the optimization of an assay used to detect bacterial neuraminidase activity on Neu5,9Ac2 glycoproteins. For all four hNEU isoenzymes, we observed a large preference for Neu5Ac over Neu5,9Ac2 such that our assay could only detect the release of Neu5Ac. We then expanded the scope of our study to include α(2→8)-linked sialosides. We designed a panel of disialo substrates based on the ganglisoside GD3 with Neu5,9Ac2 sialic acids and developed an HPLC assay to monitor hNEU kinetics on α(2→8)-linked sialic acids. The assay will be valuable for the study of hNEU activity on labeled gangliosides. The challenge of studying metabolism of α(2→8)-linked sialic acids is apparent when considering polymers of α(2→8)-linked sialic acids, known as polysialic acid. The unique chemical and physical properties of polysia include enhanced susceptibility to acid-catalyzed hydrolysis. In Chapter 4 we detail the first systematic investigation of the activity of isoforms of hNEU towards polysialic acid. We found that NEU1, NEU3, and NEU4 hydrolyzed short polymers with degree of polymerization (DP) of 3-8. We did not detect hNEU activity on longer polymers (DP > 10), suggesting alternative mechanisms for polysialic acid regulation in vivo. The work described in this thesis fills in longstanding questions in our understanding of sialic acid metabolism by hNEU enzymes. In doing this work, we have expanded chemical biology methods available for the investigation of sialic acid metabolism and the important roles of monosaccharide and glycosidic linkage diversity in substrate presentation. Overall, this work highlights the importance of considering the influence of sialic acid presentation on the role of sialosides in biological systems.