Ashley Carter Graduate Student, Department of Chemistry University of Georgia Learn more about the speaker Thursday, March 4, 2021 - 11:10am ONLINE ONLY Organic Seminar Glycosylation of certain proteins by oxygen-linked mannose (O-mannose) is known as O-mannosylation, and this process is essential for growth and development in animals.1-4 Defective O-mannosylation of α-dystroglycan, the most well studied O-mannosylated mammalian protein, leads to congenital muscular dystrophies and neurological defects.4-6 Mannose is linked to proteins through the serine or threonine side chain oxygen. Glycosyl transferase enzymes then extend these O-mannose structures with other sugar residues to form polysaccharides, known as core O-mannose glycans. No current tools exist to directly detect these core O-mannose glycans. Therefore, it remains a possibility that other O-mannose modified structures exist, but there is no detection method available to identify these structures. In efforts to address this limitation, we are synthesizing chemical tools to directly identify these O-mannose core glycans. Development of antibodies to the core O-mannose glycans would help to identify these core glycans and ultimately better understand their function. Generating antibodies against this epitope presents a challenge since the oxygen in the glycosidic linkage is vulnerable to enzymatic degradation. However, carbon-linked (C-linked) glycosides, in which carbon replaces the normal oxygen in the glycosidic linkage, are resistant to enzymatic degradation and provide robust immunogens.7-9 In efforts to overcome stability issues of O-linked glycoside immunogens, we are synthesizing a C-linked glycoside mimic (C-Man-Thr) of O-mannose-Threonine that has the potential to be used as an antigen to generate antibodies to identify these core O-mannose glycans. Another method to identify these core O-mannose glycans is via click chemistry, using azido-carbohydrate mimics.10-11 Our synthetic efforts have been directed toward developing two azido-sugar derivatives, UDP-N-acetylazidoglucosamine (UDP-GlcNAz) and UDP-4-azido-glucouronic acid (UDP-4-azidoGlcA), which are azido derivatives of sugars which are added to the O-mannose core glycans by glycosyltransferase enzymes. The UDP-azido sugars will be used as donors for the glycosyl transferase enzymes. This will allow the enzyme-modified protein sites to display a unique azide functionality that can then be modified with the addition of an alkyne-functionalized biotin derivative, allowing isolation of products for analysis to identify protein sites that are modified by the glycosyl transferase enzymes. This click chemistry method coupled with the use of the C-linked glycoside to generate antibodies have the potential to provide powerful chemical tools for identifying novel O-mannose modified protein sites. References Halmo, S. M., Singh, D., Patel, S.P., Wang, S., Eldin, M., Boons, G., Moremen, K. W., Live, D., Wells, L. Protein O-Linked Mannose β-1,4-N-Acetylglucosaminyl-transferase 2 (POMGNT2) is a Gatekeeper Enzyme for Functional Glycosylation of α-Dystroglycan. Biol. Chem. 2017, 292: 2101-2109. doi:10.1074/jbc.M116.764712. Clements, R., Turk, R., Campbell, K. P., Campbell, K.W. Dystroglycan Maintains Inner Limiting Membrane Integrity to Coordinate Retinal Development. J. Neurosci. 2017, 37 (35): 8559-8574; DOI: 10.1523/JNEUROSCI.0946-17.2017. http://www.jneurosci.org/content/37/35/8559.long. Sheikh, M.S., Halmo, S., Wells, L. Recent advancements in understanding mammalian O-mannosylation. Glycobiology. 2017, 27 (9): 806-819. https://doi.org/10.1093/glycob/cwx062. Wright, K. 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