Study of Glycan-Mediated Interactions by Photocrosslinking GlcNAc and GalNAc Analogs

Glycans, along with nucleic acids, proteins and lipids, are essential elements of life. Glycans are involved in multiple biological processes, such as cell signaling, cell adhesion and host pathogen interactions, many of which are achieved through interactions with other molecules, especially glycan binding proteins. However, there are multiple challenges in the study of glycan-mediated interactions. One of the main challenges in glycobiology is the low affinity of glycan binding events, making them difficult to characterize. To address this challenge, diazirine-functionalized sugar analogs that can be metabolically incorporated into cellular glycans were designed. Upon UV irradiation, a covalent bond forms between the diazirine group and nearby molecules. The formation of a covalent bond makes it easier to characterize glycan-mediated interactions using various methods, including Western blot, immunoprecipitation and proteomics. In this thesis, I will focus on diazirine-functionalized N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc) analogs, GlcNDAz and GalNDAz. I will demonstrate the incorporation of GlcNDAz into N-linked glycans using complementary methods and the applications of GlcNDAz in identification of binding partners of two glycan binding proteins, galectin-1 and cholera toxin B subunit. In addition, I will demonstrate the preliminary results I have on GalNDAz incorporation and photocrosslinking, which indicate that GalNDAz may be incorporated into cellular glycans. Chapter 1 introduces the background of this thesis, including basics of glycans and lectins, metabolic glycan engineering, photocrosslinking groups and photocrosslinking sugar analogs. In chapter 2, the incorporation of GlcNDAz into cellular N-linked glycans is demonstrated. Chapter 3 focuses on applications of GlcNDAz in crosslinking of galectin-1 and cholera toxin B subunit, and in identification of their binding partners by crosslinking and proteomics. Chapter 4 includes the preliminary data on applications of GlcNDAz in identification of galectin-1 binding partners during lysosomal damage and the function of galectin-1 in cellular response to lysosomal damage. In chapter 5, the preliminary results on GalNDAz incorporation and photocrosslinking are examined.

Author dc:creator

• Wu, Han

Contributors dc:contributor

• Lehrman, Mark A.
• Shiloh, Michael
• Tu, Benjamin
• Kohler, Jennifer J.

• Acetylglucosamine
• Cell Membrane
• Cross-Linking Reagents
• Glycoproteins
• Acetylgalactosamine

Language dc:language

en

Identifier

1482732371

OAI identifier oai:identifier

oai:utswmed-ir.tdl.org:2152.5/10446