<i>O</i>-linked β-<i>N</i>-acetylglucosamine (O-GlcNAc) is an essential post-translational modification found on serine and threonine residues of cytoplasmic, nuclear, and mitochondrial proteins. O-GlcNAc cycling is controlled in a nutrient-responsive manner by a single pair of enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). Studies have revealed that O-GlcNAcylation is involved in many cellular processes such as cell cycle control, signaling pathways, transcriptional regulation, and protein stability. Aberrant O-GlcNAcylation is implicated in various pathologies including cancer, diabetes, and neurodegeneration. Recently, we developed a protein engineering approach to write and erase O-GlcNAc from desired target proteins. Our approach utilizes nanobody-tagged versions of OGT and OGA (termed nanobody-OGT and nanobody-splitOGA respectively) to selectively install or remove O-GlcNAc from a target protein tagged with the antigen recognized by the nanobody. To further optimize the nanobody fusions for selective O-GlcNAcylation of proteins, we engineered a conditionally stable nanobody-OGT and nanobody-splitOGA system. Destabilizing mutations in the nanobody sequence lead to conditional stability only in the presence of the antigen. Application of the conditionally stable nanobody-OGT and nanobody-splitOGA system to evaluate the contribution of O-GlcNAc to target proteins in the cell will be described.

Utilizing nanobodies as proximity-directing groups for protein-selective glycosylation in cells

Fall 2022 abstracts, posters, and presentations will launch in mid-September.

ACS Fall 2022

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.