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

TY - JOUR

T1 - Exploiting O-GlcNAc transferase promiscuity to dissect site-specific O-GlcNAcylation

AU - Mitchell, Conor

AU - Galan Bartual, Sergio

AU - Ferenbach, Andrew T.

AU - Scavenius, Carsten

AU - van Aalten, Daan M. F.

N1 - Copyright: © The Author(s) 2023. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: [email protected].

PY - 2023/12

Y1 - 2023/12

N2 - Protein O-GlcNAcylation is an evolutionary conserved post-translational modification catalysed by the nucleocytoplasmic O-GlcNAc transferase (OGT) and reversed by OGlcNAcase (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.

AB - Protein O-GlcNAcylation is an evolutionary conserved post-translational modification catalysed by the nucleocytoplasmic O-GlcNAc transferase (OGT) and reversed by OGlcNAcase (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.

KW - Methodology

KW - O-GlcNAc

KW - O-GlcNAc Transferase

KW - S-GlcNAc

U2 - 10.1093/glycob/cwad086

DO - 10.1093/glycob/cwad086

M3 - Article

C2 - 37856504

SN - 0959-6658

VL - 33

SP - 1172

EP - 1181

JO - Glycobiology

JF - Glycobiology

IS - 12

ER -