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O‐GlcNAc transferase (OGT) is the only enzyme that catalyzes the post‐translational modification of proteins at Ser/Thr with a single β‐N‐acetylglucosamine (O‐GlcNAcylation). Its activity has been associated with chronic diseases such as cancer, diabetes and neurodegenerative disease. Although numerous OGT substrates have been identified, its accepted substrate scope can still be refined. We report here an attempt to better define the peptide‐recognition requirements of the OGT active site by using mRNA display, taking advantage of its extremely high throughput to assess the substrate potential of a library of all possible nonamer peptides. An antibody‐based selection process is described here that is able to enrich an OGT substrate peptide from such a library, but with poor absolute recovery. Following four rounds of selection for O‐GlcNAcylated peptides, sequencing revealed 14 peptides containing Ser/Thr, but these were shown by luminescence‐coupled assays and peptide microarray not to be OGT substrates. By contrast, subsequent testing of an N‐terminal tag approach showed exemplary recovery. Our approach demonstrates the power of genetically encoded libraries for selection of peptide substrates, even from a very low initial starting abundance and under suboptimal conditions, and emphasizes the need to consider the binding biases of antibodies and both C‐ and N‐terminal tags in profiling peptide substrates by high‐throughput display., Improving the odds: In vitro mRNA display‐based ultra‐high‐throughput selection is shown here to be a feasible approach to selectively enrich peptide substrates of O‐GlcNAc transferase from the background of a random library. We identify and solve two crucial challenges to high‐throughput profiling of peptide substrates of glycosyltransferases, working towards a universally applicable system.

O-GlcNAc transferase (OGT) is the only enzyme that catalyzes the post-translational modification of proteins at Ser/Thr with a single β-N-acetylglucosamine (O-GlcNAcylation). Its activity has been associated with chronic diseases such as cancer, diabetes and neurodegenerative disease. Although numerous OGT substrates have been identified, its accepted substrate scope can still be refined. We report here an attempt to better define the peptide-recognition requirements of the OGT active site by using mRNA display, taking advantage of its extremely high throughput to assess the substrate potential of a library of all possible nonamer peptides. An antibody-based selection process is described here that is able to enrich an OGT substrate peptide from such a library, but with poor absolute recovery. Following four rounds of selection for O-GlcNAcylated peptides, sequencing revealed 14 peptides containing Ser/Thr, but these were shown by luminescence-coupled assays and peptide microarray not to be OGT substrates. By contrast, subsequent testing of an N-terminal tag approach showed exemplary recovery. Our approach demonstrates the power of genetically encoded libraries for selection of peptide substrates, even from a very low initial starting abundance and under suboptimal conditions, and emphasizes the need to consider the binding biases of antibodies and both C- and N-terminal tags in profiling peptide substrates by high-throughput display.

O-GlcNAcylation is a reversible and dynamic protein post-translational modification in mammalian cells. The O-GlcNAc cycle is catalyzed by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). O-GlcNAcylation plays important role in many vital cellular events including transcription, cell cycle regulation, stress response and protein degradation, and altered O-GlcNAcylation has long been implicated in cancer, diabetes and neurodegenerative diseases. Recently, numerous approaches have been developed to identify OGT substrates and study their function, but there is still a strong demand for highly efficient techniques. Here we demonstrated the utility of the peptide microarray approach to discover novel OGT substrates and study its specificity. Interestingly, the protein RBL-2, which is a key regulator of entry into cell division and may function as a tumor suppressor, was identified as a substrate for three isoforms of OGT. Using peptide Ala scanning, we found Ser 420 is one possible O-GlcNAc site in RBL-2. Moreover, substitution of Ser 420, on its own, inhibited OGT activity, raising the possibility of mechanism-based development for selective OGT inhibitors. This approach will prove useful for both discovery of novel OGT substrates and studying OGT specificity.