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

• Published in: Glycobiology (Oxford) 2023-12, Vol.33 (12), p.1172-1181
• Main Authors: Mitchell, Conor W, Galan Bartual, Sergio, Ferenbach, Andrew T, Scavenius, Carsten, van Aalten, Daan M F
• Format: Article
• Language: English
• Subjects: Acetylglucosamine - metabolism; Acetylglucosaminidase - metabolism; Glycosylation; N-Acetylglucosaminyltransferases - genetics; N-Acetylglucosaminyltransferases - metabolism; Protein Processing, Post-Translational; Proteins - metabolism
• ISSN: 1460-2423; 1460-2423
• DOI: 10.1093/glycob/cwad086

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 CK2α, we demonstrate that near-homogeneous mono-S-GlcNAcylation of these proteins promotes DDX3X and CK2α 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.