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Abstract
The protein alpha-dystroglycan (α-DG) is highly glycosylated with O-Man and O-GalNAc glycans through serine or threonine within its mucin-like domain. Defects in O-mannosylation of α-DG lead to dystroglycanopathies, which involve congenital muscular dystrophies and neurological defects. Furthermore, hypoglycosylation of α-DG leads to defective axonal guidance and neuronal migration as well as mosaic spacing of neurons during retinal development. Interactions between α-DG and its extracellular matrix (ECM) ligands require α-DG to be extended by O-mannose structures based on the M3 core structure by the glycosyltransferase POMGNT2. The core M3 can be extended with matriglycan, which is a repeating disaccharide that binds LG-domain containing proteins in the ECM. There is currently no method to identify the M3 core without the presence of matriglycan. Therefore, it remains a possibility that unextended M3 structures exist, but there is currently no tool to identify core M3 glycans without the presence of the repeating disaccharide. 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. In efforts to overcome stability issues of O-linked glycoside immunogens, a novel C-linked glycoside mimic (C-Man-Thr) of O-mannose-Threonine was synthesized. This biomimetic has the potential to be used as an antigen to generate antibodies to identify these core O-mannose glycans. Extension of the C-linked glycoside into C-linked glycopeptides to generate antibodies has the potential to provide improved reagents to generate a more comprehensive profile of sites that are modified with O-mannose glycans. Another route of enhanced detection includes bioorthogonal tagging with an azido-modified form of UDP-GlcNAc and subsequentially clicking with a bioorthoganol biotin tag to enrich for modified protein sites. Potentially novel O-mannosylated proteins were enriched using this methodology.