C-Terminal Tag Location Hampers in Vitro Profiling of OGT Peptide Substrates by mRNA Display

Jie Shi, Suhela Sharif, Cyril Balsollier, Rob Ruijtenbeek, Roland J. Pieters, Seino A.K. Jongkees*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

Abstract

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.

Original languageEnglish
Pages (from-to)666-671
Number of pages6
JournalChemBioChem
Volume22
Issue number4
Early online date6 Oct 2020
DOIs
Publication statusPublished - 15 Feb 2021

Keywords

  • mRNA display
  • O-GlcNAc transferase
  • O-GlcNAcylation
  • peptide selection
  • substrate specificity

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