O-GlcNAc Engineering on a Target Protein in Cells with Nanobody-OGT and Nanobody-splitOGA

Curr Protoc. 2021 May;1(5):e117. doi: 10.1002/cpz1.117.


The monosaccharide O-linked N-acetyl glucosamine (O-GlcNAc) is an essential and dynamic post-translational modification (PTM) that decorates thousands of nucleocytoplasmic proteins. Interrogating the role of O-GlcNAc on a target protein is crucial yet challenging to perform in cells. We recently reported a pair of methods to selectively install or remove O-GlcNAc on a target protein in cells using an engineered O-GlcNAc transferase (OGT) or split O-GlcNAcase (OGA) fused to a nanobody. Target protein O-GlcNAcylation and de-O-GlcNAcylation complements methods to interrogate the role of O-GlcNAc on a global scale or at individual glycosites. Herein, we describe a protocol for utilizing the nanobody-OGT and nanobody-splitOGA systems to screen for O-GlcNAc functionality on a target protein. We additionally include associated protocols for the detection of O-GlcNAc and cloning procedures to adapt the method for the user's target protein of interest. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Target protein O-GlcNAcylation of JunB using nanobody-OGT Basic Protocol 2: Target protein deglycosylation of Nup62 using nanobody-splitOGA Alternate Protocol: Verification of the O-GlcNAc state of a tagged target protein through chemoenzymatic labeling Support Protocol: Cloning of new nanobody-OGT/nanobody-splitOGA and target protein pairs.

Keywords: O-GlcNAc; deglycosylation; glycosylation; nanobodies; post-translational modification; proximity-direction.

MeSH terms

  • Acetylglucosamine
  • Glucosamine*
  • N-Acetylglucosaminyltransferases* / genetics
  • Protein Processing, Post-Translational
  • Proteins


  • Proteins
  • N-Acetylglucosaminyltransferases
  • Glucosamine
  • Acetylglucosamine