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Review
. 2020 Jun 12;1(3):98-109.
doi: 10.1039/d0cb00052c. eCollection 2020 Aug 1.

Tools for functional dissection of site-specific O-GlcNAcylation

Andrii Gorelik  1 , Daan M F van Aalten  1   2
Affiliations
Review

Tools for functional dissection of site-specific O-GlcNAcylation

Andrii Gorelik et al. RSC Chem Biol. .

Abstract

Protein O-GlcNAcylation is an abundant post-translational modification of intracellular proteins with the monosaccharide N-acetylglucosamine covalently tethered to serines and threonines. Modification of proteins with O-GlcNAc is required for metazoan embryo development and maintains cellular homeostasis through effects on transcription, signalling and stress response. While disruption of O-GlcNAc homeostasis can have detrimental impact on cell physiology and cause various diseases, little is known about the functions of individual O-GlcNAc sites. Most of the sites are modified sub-stoichiometrically which is a major challenge to the dissection of O-GlcNAc function. Here, we discuss the application, advantages and limitations of the currently available tools and technologies utilised to dissect the function of O-GlcNAc on individual proteins and sites in vitro and in vivo. Additionally, we provide a perspective on future developments required to decipher the protein- and site-specific roles of this essential sugar modification.

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Conflict of interest statement

There are no conflicts of interest to declare.

Figures

Fig. 1
Fig. 1. O-GlcNAc cycling on nucleocytoplasmic proteins. (a) OGT and OGA control the addition and removal of GlcNAc on serine and threonine residues. (b) Domain architecture and structures of OGT (composite of PDB: 1W3B and PDB: 5C1D) and OGA (PDB: 5M7R, lacks the disordered region). Catalytic site residues K842 (OGT) and D175 (OGA) are coloured in red. GT domain – glycosyltransferase domain. NLS – nuclear localization sequence, GH domain – glycoside hydrolase domain.
Fig. 2
Fig. 2. Methods for generating protein-specific O-GlcNAcylation. (a) In vitro OGT reaction. (b) Co-expression of OGT with its substrate in E. coli, followed by purification. KanR – kanamycin resistance, AmpR – ampicillin resistance. (c) Proximity OGT approach for protein-selective O-GlcNAcylation in cells. nGFP – anti-GFP nanobody. Blue squares denote O-GlcNAc.
Fig. 3
Fig. 3. Site-specific chemical biology approaches to study O-GlcNAcylation in vitro. (a) Expressed protein ligation. (b) “Tag-and-modify” dehydroalanine approach. POI – protein of interest.
Fig. 4
Fig. 4. Approaches to study site-specific O-GlcNAcylation in vivo. (a) Principle of a genetic code expansion approach with a metabolically stable Cys-S-GlcNAc amino acid: (1). UAA uptake, (2). tRNA aminoacylation, (3). UAA translation in response to a stop codon, (4). Production of a modified protein. (b) Genetic recoding approach to introduce site-specific S-GlcNAc mimicry of O-GlcNAcylation. Blue squares denote GlcNAc. POI – protein of interest.
None
Andrii Gorelik and Daan van Aalten
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References

    1. Aebersold R. et al., How many human proteoforms are there? Nat. Chem. Biol. 2018;14:206–214. doi: 10.1038/nchembio.2576. - DOI - PMC - PubMed
    1. Klein T. Eckhard U. Dufour A. Solis N. Overall C. M. Proteolytic Cleavage - Mechanisms, Function, and ‘omic’ Approaches for a Near-Ubiquitous Posttranslational Modification. Chem. Rev. 2018;118:1137–1168. doi: 10.1021/acs.chemrev.7b00120. - DOI - PubMed
    1. Yau R. Rape M. The increasing complexity of the ubiquitin code. Nat. Cell Biol. 2016;18:579–586. doi: 10.1038/ncb3358. - DOI - PubMed
    1. Moremen K. W. Tiemeyer M. Nairn A. V. Vertebrate protein glycosylation: Diversity, synthesis and function. Nat. Rev. Mol. Cell Biol. 2012;13:448–462. doi: 10.1038/nrm3383. - DOI - PMC - PubMed
    1. Ardito F. Giuliani M. Perrone D. Troiano G. Muzio L. L. The crucial role of protein phosphorylation in cell signaling and its use as targeted therapy (Review) Int. J. Mol. Med. 2017;40:271–280. doi: 10.3892/ijmm.2017.3036. - DOI - PMC - PubMed