An overview of tools to decipher O-GlcNAcylation from historical approaches to new insights

Dupas, Thomas; Betus, Charlotte; Blangy-Letheule, Angélique; Pelé, Thomas; Persello, Antoine; Denis, Manon; Lauzier, Benjamin

doi:10.1016/j.biocel.2022.106289

Cited by 6 publications

(9 citation statements)

References 171 publications

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“…5b). Currently, detection of O- GlcNAcylation is performed in fixed cells or bulk extracts using O- GlcNAc sensitive antibodies 44 .…”

Section: Resultsmentioning

confidence: 99%

Subcellular imaging of lipids and sugars using genetically encoded proximity sensors

Moore,

Brea,

Knittel

et al. 2024

Preprint

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Live cell imaging of lipids and other metabolites is a long-standing challenge in cell biology. Bioorthogonal labeling tools allow for the conjugation of fluorophores to several phospholipid classes, but cannot discern their trafficking between adjacent organelles or asymmetry across individual membrane leaflets. Here we present fluorogen-activating coincidence sensing (FACES), a chemogenetic tool capable of quantitatively imaging subcellular lipid pools and reporting their transbilayer orientation in living cells. FACES combines bioorthogonal chemistry with genetically encoded fluorogen-activating proteins (FAPs) for reversible proximity sensing of conjugated molecules. We first validate this approach for quantifying discrete phosphatidylcholine pools in the ER and mitochondria that are trafficked by lipid transfer proteins. We then show that transmembrane domain-containing FAPs can be used to reveal the membrane asymmetry of multiple lipid classes that are generated in the trans-Golgi network. Lastly, we show that FACES is a generalizable tool for subcellular bioorthogonal imaging by measuring changes in mitochondrial N-acetylhexosamine levels. These results demonstrate the use of fluorogenic tags for spatially-defined molecular imaging.

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“…5b). Currently, detection of O- GlcNAcylation is performed in fixed cells or bulk extracts using O- GlcNAc sensitive antibodies 44 .…”

Section: Resultsmentioning

confidence: 99%

Subcellular imaging of lipids and sugars using genetically encoded proximity sensors

Moore,

Brea,

Knittel

et al. 2024

Preprint

View full text Add to dashboard Cite

show abstract

“…The enrichment strategies roughly fall into two categories. One category involves direct capture of O- GlcNAcylated proteins by antibodies or lectins that recognize the GlcNAc moiety ( Yin et al, 2021 ; Hu et al, 2022 ; Dupas et al, 2022 ; Saha et al, 2021 ; Maynard and Chalkley, 2021 ; Ma et al, 2021a ; Ma et al, 2021b ). O- GlcNAc antibodies including RL2 and CTD110.6, as well as O- GlcNAc-binding lectins such as wheat germ agglutinin (WGA), are commonly used for enrichment.…”

Section: Introductionmentioning

confidence: 99%

“…In addition, the catalytic-dead mutant of Cp OGA that retains the ability to recognize O- GlcNAcylated substrates was successfully repurposed to concentrate many developmental regulators from Drosophila embryo lysates ( Selvan et al, 2017 ). Another category of enrichment strategies relies on chemoenzymatic or metabolic labeling ( Yin et al, 2021 ; Hu et al, 2022 ; Dupas et al, 2022 ; Saha et al, 2021 ; Maynard and Chalkley, 2021 ; Ma et al, 2021a ; Ma et al, 2021b ). Azido-modified intermediates, such as N -azidoacetylglucosamine (GlcNAz) and N -azidoacetylgalactosamine (GalNAz), are used to introduce specific tags (e.g.…”

Section: Introductionmentioning

confidence: 99%

Tissue-specific O-GlcNAcylation profiling identifies substrates in translational machinery in Drosophila mushroom body contributing to olfactory learning

Yu,

Liu,

Zhang

et al. 2024

eLife

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O-GlcNAcylation is a dynamic post-translational modification that diversifies the proteome. Its dysregulation is associated with neurological disorders that impair cognitive function, and yet identification of phenotype-relevant candidate substrates in a brain-region specific manner remains unfeasible. By combining an O-GlcNAc binding activity derived from Clostridium perfringens OGA (CpOGA) with TurboID proximity labeling in Drosophila, we developed an O-GlcNAcylation profiling tool that translates O-GlcNAc modification into biotin conjugation for tissue-specific candidate substrates enrichment. We mapped the O-GlcNAc interactome in major brain regions of Drosophila and found that components of the translational machinery, particularly ribosomal subunits, were abundantly O-GlcNAcylated in the mushroom body of Drosophila brain. Hypo-O-GlcNAcylation induced by ectopic expression of active CpOGA in the mushroom body decreased local translational activity, leading to olfactory learning deficits that could be rescued by dMyc overexpression-induced increase of protein synthesis. Our study provides a useful tool for future dissection of tissue-specific functions of O-GlcNAcylation in Drosophila, and suggests a possibility that O-GlcNAcylation impacts cognitive function via regulating regional translational activity in the brain.

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“…One category involves direct capture of O-GlcNAcylated proteins by antibodies or lectins that recognize the GlcNAc moiety [27][28][29][30][31][32][33] . O-GlcNAc antibodies including RL2 and CTD110.6, as well as O-GlcNAc-binding lectins such as wheat germ agglutinin (WGA), are commonly used for enrichment.…”

Section: Introductionmentioning

confidence: 99%

“…In addition, the catalytic-dead mutant of CpOGA that retains the ability to recognize O-GlcNAcylated substrates was successfully repurposed to concentrate many developmental regulators from Drosophila embryo lysates 34 . Another category of enrichment strategies relies on chemoenzymatic or metabolic labeling [27][28][29][30][31][32][33] . Azido-modified intermediates, such as N-azidoacetylglucosamine (GlcNAz) and N-azidoacetylgalactosamine (GalNAz), are used to introduce specific tags (e.g.…”

Section: Introductionmentioning

confidence: 99%

Olfactory learning inDrosophilarequires O-GlcNAcylation of mushroom body ribosomal subunits

Yu,

Liu,

Zhang

et al. 2023

Preprint

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O-GlcNAcylation is a dynamic post-translational modification that diversifies the proteome. Its dysregulation is associated with neurological disorders that impair cognitive function, and yet identification of phenotype-relevant candidate substrates in a brain-region specific manner remains unfeasible. By combining an O-GlcNAc binding activity derived fromClostridium perfringensOGA (Cp OGA) with TurboID proximity labeling inDrosophila, we developed an O-GlcNAcylation profiling tool that translates O-GlcNAc modification into biotin conjugation for tissue-specific candidate substrates enrichment. We mapped the O-GlcNAc interactome in major brain regions ofDrosophilaand found that components of the translational machinery, including many ribosomal subunits, were abundantly O-GlcNAcylated in the mushroom body, the computational center of theDrosophilabrain. Hypo-O-GlcNAcylation induced by ectopic expression of active Cp OGA in the mushroom body decreased local ribosomal activity, leading to olfactory learning deficits that could be rescued by increasing ribosome biogenesis. Our study reveals that O-GlcNAcylation contributes to the links between protein synthesis and cognitive function in the brain learning center, and provides a useful tool for future dissection of tissue-specific functions of O-GlcNAcylation inDrosophila.

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An overview of tools to decipher O-GlcNAcylation from historical approaches to new insights

Cited by 6 publications

References 171 publications

Subcellular imaging of lipids and sugars using genetically encoded proximity sensors

Subcellular imaging of lipids and sugars using genetically encoded proximity sensors

Tissue-specific O-GlcNAcylation profiling identifies substrates in translational machinery in Drosophila mushroom body contributing to olfactory learning

Olfactory learning inDrosophilarequires O-GlcNAcylation of mushroom body ribosomal subunits

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