binders derived from heavy-chain only antibodies that have been developed to recognize endogenous proteins or common protein tags. To develop a targeted O-GlcNAc writer and eraser, we fused a nanobody to an engineered O-GlcNAc editor that has reduced activity on endogenous O-GlcNAc substrates. The O-GlcNAc editor is then recruited to a desired target protein through the nanobody, where O-GlcNAc levels are selectively enhanced or reduced.To generate a selective O-GlcNAc writer, we converted full-length OGT with 13.5 tetratricopeptide repeats (TPRs) to a shorter form with 4.5 TPRs [OGT(4)] and fused this truncated OGT(4) to a nanobody for target protein O-GlcNAcylation. The reduction of the TPR domain of OGT reduces its activity on endogenous substrates. Fusion of the nanobody to the TPR domain regains enhanced selectivity for desired target proteins through either tagged targets or endogenous targets. We additionally performed a systematic investigation of the impact of truncations to the TPR domain on substrate and glycosite selection using chemical glycoproteomics. We found that the first four TPRs of OGT enhance substrate scope, while the last four TPRs of OGT contribute to glycosite selection. These data provide a foundation to analyze how perturbations to the TPR domain and expression of OGT isoforms affects the glycosylation of substrates, which will be critical for future efforts in protein engineering of OGT, the biology of OGT isoforms, and diseases associated with the TPR domain of OGT.To develop a selective O-GlcNAc eraser, we developed a nanobody-fused split OGA system for target protein deglycosylation in cells. OGA is naturally cleaved by caspase-3 during apoptosis, which served as a site for systematic evaluation of a split OGA with reduced activity for endogenous substrates. Systematic evaluation of the split OGA fragments afforded a nanobody-split OGA that has selective activity against the target protein recognized by the nanobody and limited deglycosidase activity against the broader O-GlcNAc proteome. The nanobody-split OGA system is also compatible with several nanobodies, which increases versatility for the protein tags and targets used during implementation.With a protein-selective O-GlcNAc writer and eraser now in hand, we have begun to evaluate the effect of O-GlcNAc on target proteins in cells and in vivo. The application of the system to some of the thousands of proteins modified by O-GlcNAc, including kinases, transcription factors, E3 ligases, and structural proteins, have begun to reveal functions in protein stabilization, regulation of protein–protein interactions, and downstream signaling outcomes and phenotypes. The use of these methods to reveal new insights to O-GlcNAc functions will enable novel insights to the physiological role of O-GlcNAc and approaches to engineer these biological signals in the future.19Special LectureChristina M. Woo
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