Metabolic Pathways for Natural and Unnatural Sialic Acids

Carbohydrates, along with lipids, nucleic acids, and amino acids constitute the four main building blocks of life. This thesis will focus on sialic acid, a unique, nine-carbon sugar that is critically important in humans. In Chapter 1, I will introduce sialic acid biology and the many diseases that result when sialic acid metabolism is disrupted. Furthermore, I will discuss metabolic carbohydrate engineering as a valuable tool for studying sialic acid biology. Lastly, I will introduce an ancient and conserved family of proteins, the glycosyltransferases, which are responsible for transferring sugars, such as sialic acid, onto glycoproteins and glycolipids. Disruptions in the sialic acid biosynthetic pathway are implicated in hereditary inclusion body myopathy (hIBM), a disease of aging. In Chapter 2, I determined that sialic acid biosynthesis alters the levels of UDP-GlcNAc, a product of the hexosamine biosynthetic pathway. This results in changes in the branch structure of N-glycans and impairs galectin-1 binding to cells. Furthermore, I found that sialylation of N-glycans can further influence N-glycan branching. Taken together, my work suggests unexplored mechanisms for hIBM pathogenesis. The use of unnatural sialic acids, such as SiaDAz, and metabolic carbohydrate engineering is enabling deeper understanding of the biological roles of sialic acid. In Chapter 3, I determined that different cell lines have impairments at different metabolic steps during SiaDAz synthesis. The results of my work reveals the importance of cell-specific enzymes during the metabolism of Ac4ManNDAz to ManDAz to SiaDAz. Furthermore, I found that cells poorly deprotect Ac5 1-OMe sialic acids, both natural and unnatural. The results of this chapter inspire strategies to improve the efficiency and generality of this technology. Mature glycans on the cell surface or on therapeutically active small molecules are formed by the action of glycosyltransferases. In Chapter 4, I apply statistical coupling analysis to reveal new insights into glycosyltransferases. Specifically, I was able to identify a network of key residues, termed a sector, which contained non-obvious key residues that appear to be important for catalytic function. Furthermore, sector residues correlated with surface sites that exert allosteric control over enzyme activity.