Revealing and Exploiting the Biochemistry of O-GlcNAc through Protein Semisynthesis

O-GlcNAcylation is a dynamic posttranslational modification regulated by the enzymes O-GlcNAc transferase (OGT) and O-GlcNAc hydrolase (OGA). It involves the attachment of N-acetylglucosamine to serine or threonine residues of proteins in the cytosol, nucleus, and mitochondria. As a dynamic and abundant modification, O-GlcNAcylation functions as a sensor of the cell's metabolic state. Fluctuations in O-GlcNAc levels of the adenosine (O-GlcNAc) signal cellular stress or metabolic changes and have been implicated in various human diseases. The overall impact of this modification is protein-dependent, underscoring the importance of studying its biochemical consequences in a protein- and site-specific manner. To achieve this, enzymatic and chemical strategies have been developed to incorporate O-GlcNAc into peptides and proteins. These synthetic glycopeptides and glycoproteins have been instrumental in elucidating how O-GlcNAcylation influences protein structure, function, and diverse biochemical pathways. Recently, the O-GlcNAcylation has also emerged as a tool for glycosylation-assisted folding of proteins and as a solubility tag for the chemical synthesis of glycopeptides and proteins. Here, we overview the current methods enabling the preparation of specific O-GlcNAc-modified proteins and highlight recent developments.