Truncation of the TPR domain of OGT alters substrate and glycosite selection

Nucleocytoplasmic O-linked N-acetylglucosamine (O-GlcNAc) is an essential post-translational modification that is installed to thousands of protein substrates by O-GlcNAc transferase (OGT). Substrate selection by OGT and its isoforms is primarily mediated by the tetratricopeptide repeat (TPR) domain, yet the impact of truncations to the TPR domain on substrate and glycosite selection remains unresolved. Here, we report the effects of TPR truncations on the substrate and glycosite selection of OGT against the model protein GFP-JunB and the surrounding O-GlcNAc proteome in U2OS cells. Truncation of the TPR domain of OGT maintains glycosyltransferase activity but alters subcellular localization in cells. Examination of the glycoproteome across the TPR truncations revealed the broadest substrate activity from the canonical nucleocytoplasmic OGT, with the greatest changes in O-GlcNAc occurring on proteins associated with mRNA splicing processes. Glycosite analysis revealed alterations to the O-GlcNAc consensus sequence globally and differential glycosite selectivity on GFP-JunB as a function of the OGT TPR isoform. This dataset provides 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 protein engineering of OGT, the biology of OGT isoforms, and diseases associated with the TPR domain of OGT.