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

Author:

Mitchell Conor W123,Galan Bartual Sergio1,Ferenbach Andrew T1,Scavenius Carsten1,van Aalten Daan M F123

Affiliation:

1. Department of Molecular Biology and Genetics, Aarhus University , Universitetsbyen 81, 8000 Aarhus , Denmark

2. Division of Molecular , Cell, and Developmental Biology, , Dow St., Dundee, DD1 5EH , United Kingdom

3. School of Life Sciences, University of Dundee , Cell, and Developmental Biology, , Dow St., Dundee, DD1 5EH , United Kingdom

Abstract

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.

Funder

Wellcome Trust Investigator Award

Novo Nordisk Foundation Laureate

BBSRC EASTBIO

Publisher

Oxford University Press (OUP)

Subject

Biochemistry

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