Browsing by Subject "Polysaccharides"
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Item Development of Chemical Tools to Discover and Characterize Sialic Acid Mediated Interactions(2014-02-04) Parker, Randy B.; Lehrman, Mark A.; Kohler, Jennifer J.; Phillips, Margaret A.; Brown, Kathlynn C.Glycosylation refers to the addition of carbohydrates onto proteins, lipids, and other biomolecules. Proteins and lipids on the cell surface are frequently found to be glycosylated. This glycosylation can have a number of functions, one of which is to mediate specific binding interactions between the glycans of glycoconjugates and glycan binding proteins. Such glycan-mediated interactions are implicated in numerous cell-signaling processes. Many of these glycan-mediated interactions involve a class of carbohydrates known as sialic acids. The presence of sialic acid on a glycan can recruit specific sialic acid binding partners. Alternatively, sialic acids can mask underlying glycan epitopes, thereby preventing protein binding. There are several challenges in studying sialic acid mediated interactions. Chapter 1 describes the importance of sialic acids as well as the challenges in studying their interactions. Sialidases are enzymes that hydrolytically cleave sialic acid from its underlying glycan. In this regard, sialidases have the potential to impact numerous cellular processes in a dynamic fashion. Relatively little is known about the substrates of mammalian sialidases. Chapter 2 describes a novel assay that takes advantage of chemical labeling of sialic acids to characterize sialidase substrate specificity towards various complex glycans. Chapter 3 focuses on efforts to develop assays to identify the protein components of sialidase substrates in a cellular context. Finally, Chapter 4 describes the development of a series of probes to discover novel sialic acid mediated interactions. Taken together, this work describes several new techniques that will allow for better understanding of sialic acid mediated interactions.Item Metabolic Pathways for Natural and Unnatural Sialic Acids(2014-07-25) Pham, Nam Dinh; Seemann, Joachim; Mangelsdorf, David J.; Chen, Zhijian J.; Kohler, Jennifer J.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.