The active site of O-GlcNAc transferase imposes constraints on substrate sequence

Abstract

O-GlcNAc transferase (OGT) glycosylates a diverse range of intracellular proteins with O-linked N-acetylglucosamine (O-GlcNAc), an essential and dynamic post-translational modification in metazoans. Although this enzyme modifies hundreds of proteins with O-GlcNAc, it is not understood how OGT achieves substrate specificity. In this study, we describe the application of a high-throughput OGT assay to a library of peptides. We mapped sites of O-GlcNAc modification by electron transfer dissociation MS and found that they correlate with previously detected O-GlcNAc sites. Crystal structures of four acceptor peptides in complex with Homo sapiens OGT suggest that a combination of size and conformational restriction defines sequence specificity in the -3 to +2 subsites. This work reveals that although the N-terminal TPR repeats of OGT may have roles in substrate recognition, the sequence restriction imposed by the peptide-binding site makes a substantial contribution to O-GlcNAc site specificity.

Mapping and Quantification of Over 2000 O-linked Glycopeptides in Activated Human T Cells with Isotope-Targeted Glycoproteomics (Isotag)

“…Glycosites from human T cells show only a weak homology sequence, in agreement with previous reports (50,51). The absence of a strict consensus motif as well as the possible transfer of UDP-GlcNAc to a Ser/Thr near the originally targeted Ser/Thr on a protein has stymied the study of O-GlcNAc function.…”

Elucidating crosstalk mechanisms between phosphorylation and O-GlcNAcylation

“…In sharp contrast, OGT has a much more restricted substrate sequence specificity (22); thus O-GlcNAcylation typically occurs at only one or a few Ser/Thr residues within these Ser/Thr-rich sequences. It has been suggested that there is little OGT substrate bias toward a specific amino acid at P+1 (22), in contrast to our finding that Pro at P+1 blocks O-GlcNAcylation. We investigated, therefore, whether Pro could act to guide OGT to specific Ser/Thr sites.…”

“…3A). This motif is based on several sets of reported data, namely the O-GlcNAcylation motif extracted from the 26 best-known OGT substrates (22), the PX(S/T) O-GlcNAc motif described for HLA class I-bound peptides (23), the protein sequences O-GlcNAcylated in this study (SI Appendix, Table S2), and the finding reported in this paper that phosphorylation at P−3 hampers O-GlcNAcylation. Using bioinformatics analysis, we found that these sequences of four amino acids not only are very common in the human proteome (Fig.…”

“…Based on prediction software (34) and the best known O-GlcNAcylation motif (22), O-GlcNAcylation is predicted to occur on Ser202, Thr205, and Ser208 in Tau ND, no O-GlcNAcylation detected; x, two heptad repeats from RNA polymerase II; thus this 14mer peptide represents any/all of these repeats. *Proteins with (S/T)PX(S/T) motif for which evidence exists that the first S/T can be phosphorylated.…”

O-GlcNAcylation Antagonizes Phosphorylation of CDH1 (CDC20 Homologue 1)

“…1 and 2). Consistent with our results, Cdh1 was recently identified in a screen using in vitro OGT assays on substrate peptides derived from annotated protein kinase substrates (37).…”