Novel strategies for studying O-GlcNAc glycosylation Glycosylation is an essential regulatory process for posttranslational modification of cellular proteins. European scientists developed innovative methodology for studying an unusual type of glycosylation with emerging clinical interest. Fundamental Research © molekuul_be, Shutterstock O-GlcNAc glycosylation is an unusual form of protein glycosylation that entails the modification of serine and threonine residues of nuclear and cytoplasmic proteins by the covalent attachment of N-acetylglucosamine (GlcNAc). The process involves the single-sugar modification of a protein, and in contrast to conventional protein glycosylation, O-GlcNAcylation is not further elaborated into complex glycans. This process is highly dynamic through enzymatic addition and removal by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), respectively. O-GlcNAc is emerging as an essential process for cell survival and is implicated in key biological processes including nutrient sensing as well as human diseases such as diabetes and cancer. Despite its biological importance, the functional roles and molecular details of O-GlcNAc modification remain to be elucidated alongside the consensus sequence for the site of glycosylation. This has been hampered by the lack of methods and tools to identify and study O-GlcNAc proteins’ modification. To address this, the EU-funded GLCNAC-PROBE project developed novel chemical and enzymatic strategies to recapitulate and probe O-GlcNAcylation in vitro and in vivo. They followed a ‘tag-and-modify’ approach to enable the identification of O-GlcNAc-modified proteins and their specific glycosylation sites. In particular, the researchers employed an enzymatic approach to convert the natural GlcNAc-nucleotide into an unnatural analogue bearing a chemical moiety. Following extensive optimisation, the resultant GlcNAc-nucleotide was successfully recognised and utilised by OGT to modify model peptides. They then used the chemical moiety as the basis for bio-orthogonal labelling of the modified protein with appropriate probes such as the biotin affinity tag. The GLCNAC-PROBE chemoenzymatic methodology is expected to aid in the prospective identification of O-GlcNAc glycosylated proteins and allow their study. Given the clinical importance of O-GlcNAc glycosylation, it will shed light on their functional significance and the molecular mechanisms underlying this dynamic posttranslational modification. Long-term, this will translate into an improved understanding of the role of O-GlcNAc glycosylation in various diseases and their potential for therapeutic targeting. Keywords O-GlcNAc glycosylation, posttranslational modification, O-GlcNAc transferase, GLCNAC-PROBE, biotin
