Exploiting O-GlcNAc Transferase promiscuity to dissect site-specific O-GlcNAcylation

Mitchell, C. W.; Bartual, S. G.; Ferenbach, A. T.; van Aalten, D. M. F.

doi:10.1101/2023.07.31.551258

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bioRxiv · 10.1101/2023.07.31.551258

Exploiting O-GlcNAc Transferase promiscuity to dissect site-specific O-GlcNAcylation

Mitchell, C. W.·Bartual, S. G.·Ferenbach, A. T.·van Aalten, D. M. F.

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Abstract

Protein O-GlcNAcylation is an evolutionary conserved post-translational modification catalysed by the nucleocytoplasmic O-GlcNAc transferase (OGT) and reversed by O-GlcNAcase (OGA). How site-specific O-GlcNAcylation modulates a diverse range of cellular processes is largely unknown. A limiting factor in studying this is the lack of accessible techniques capable of producing homogeneously O-GlcNAcylated proteins, in high yield, for in vitro studies. Here, we exploit the tolerance of OGT for cysteine instead of serine, combined with a co-expressed OGA to achieve site-specific, highly homogeneous mono-glycosylation. Applying this to DDX3X, TAB1, and CK2, we demonstrate that near-homogeneous mono-S-GlcNAcylation of these proteins promotes DDX3X and CK2 solubility and enables production of mono-S-GlcNAcylated TAB1 crystals, albeit with limited diffraction. Taken together, this work provides a new approach for functional dissection of protein O-GlcNAcylation.

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Mitchell, C. W., Bartual, S. G., Ferenbach, A. T., van Aalten, D. M. F.. 2023-07-31. Exploiting O-GlcNAc Transferase promiscuity to dissect site-specific O-GlcNAcylation. https://doi.org/10.1101/2023.07.31.551258

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