Native detection of protein <i>O</i>-GlcNAcylation by gel electrophoresis

Fu, Chuan; Aalten, Daan M. F. van

doi:10.1039/c9an02506e

Cited by 6 publications

(4 citation statements)

References 37 publications

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“…The utility of sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS‐PAGE)‐based assay with a specific lectin‐embedded acrylamide gel layer was demonstrated for the separation of the N‐glycosylated form from the unmodified forms of the protein [87] . Similar electrophoretic strategies have been applied for the detection of endogenously O‐GlcNAcylated proteins [88,89] . In these approaches, a standard acrylamide gel is engineered to contain a layer of copolymerized O‐GlcNAc‐binding receptor between the stacking and the separating gel (Figure 6B).…”

Section: Detection and Identification Of O‐glcnacylation And Its Binding Partnersmentioning

confidence: 99%

“…To address this issue the van Aalten group replaced WGA with a catalytically impaired bacterial OGA mutant, Cp OGA(D298N) with better qualities of affinity and specificity [89] . In this approach, Cp OGA(D298N) is first functionalized to contain acrylamide moieties in several lysine residues of the enzyme.…”

Section: Detection and Identification Of O‐glcnacylation And Its Binding Partnersmentioning

confidence: 99%

“…Then the acrylamide‐tagged inactive OGA mutant is subjected to polymerization to form a gel layer of the copolymerized inactive OGA mutant. Using this method, the authors determined kinetic parameters of OGT by quantifying O‐GlcNAc stoichiometry on the model samples [89] . However, the O‐GlcNA‐receptor‐based electrophoretic detection methods largely depend on the availability of antibodies that are to be used.…”

Section: Detection and Identification Of O‐glcnacylation And Its Binding Partnersmentioning

confidence: 99%

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Advances in Strategies and Tools Available for Interrogation of Protein O‐GlcNAcylation

Kim

2021

ChemBioChem

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The attachment of a single O‐linked β‐N‐acetylglucosamine (O‐GlcNAc) to serine and threonine residues of numerous proteins in the nucleus, cytoplasm, and mitochondria is a reversible post‐translational modification (PTM) and plays an important role as a regulator of various cellular processes in both healthy and disease states. Advances in strategies and tools that allow for the detection of dynamic O‐GlcNAcylation on cellular proteins have helped to enhance our initial and ongoing understanding of its dynamic effects on cellular stimuli and given insights into its link to the pathogenesis of several chronic diseases. Furthermore, chemical genetic strategies and related tools have been successfully applied to a myriad of biological systems with a new level of spatiotemporal and molecular precision. These strategies have started to be used in studying and controlling O‐GlcNAcylation both in vivo and in vitro. In this minireview, overviews of recent advances in molecular tools being applied to the detection and identification of O‐GlcNAcylation on cellular proteins as well as on individual proteins are provided. In addition, chemical genetic strategies that have already been applied or are potentially usable in O‐GlcNAc functional are also discussed.

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Section: Detection and Identification Of O‐glcnacylation And Its Binding Partnersmentioning

confidence: 99%

Section: Detection and Identification Of O‐glcnacylation And Its Binding Partnersmentioning

confidence: 99%

Section: Detection and Identification Of O‐glcnacylation And Its Binding Partnersmentioning

confidence: 99%

See 1 more Smart Citation

Advances in Strategies and Tools Available for Interrogation of Protein O‐GlcNAcylation

Kim

2021

ChemBioChem

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“…Mutation of the catalytic residue Asp298 to Asn (D298N) inactivates the enzyme but retains its ability to bind O-GlcNAcylated peptides 28 . Taking advantage of this property, far western and gel electrophoresis methods have been developed 29,30 , and the mutant protein has been successfully used to enrich and profile O-GlcNAcylated proteins in vitro 26 .…”

Section: Introductionmentioning

confidence: 99%

A mutant bacterial O-GlcNAcase visualizes a progressive decline of protein O-GlcNAcylation in early Drosophila embryos critical for neurodevelopment

Zhang

Wang

et al. 2022

Preprint

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Protein O-GlcNAcylation, a monosaccharide posttranslational modification maintained by two evolutionarily conserved enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), is a major nutrient sensor integrating key metabolic pathways. While mutations in OGT have recently been associated with neurodevelopmental disorders, the dynamics and function of protein O-GlcNAcylation during early embryogenesis remain elusive. Here we develop a new fluorescent probe to visualize O-GlcNAcylation levels in live Drosophila early embryos. Our study shows that protein O-GlcNAcylation declines as the embryos develop to the mid-blastula transition when the facultative heterochromatin makes its first appearance. Lowering O-GlcNAcylation levels by exogenous OGA activity promotes the polycomb group O-GlcNAc protein Polyhomeotic (Ph) to form nuclear foci and K27 trimethylation of histone H3. This enhanced facultative heterochromatin formation fine-tunes the expression of several neurodevelopmental genes including short of gastrulation (sog). We provide evidence that perturbation of O-GlcNAcylation during early embryogenesis affects learning ability in adulthood, highlighting the importance of O-GlcNAcylation homeostasis for the development of the nervous system.

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Chemical Tools for Decoding the Functions of O-GlcNAcylation

Hao,

Liu,

Deng

et al. 2024

Chemistry and Biology of O-GlcNAcylation

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Native detection of protein O-GlcNAcylation by gel electrophoresis

Abstract: O-GlcNAcylated proteins are spatially separated by a novel gel (SOPAGE), enabling the measurement of O-GlcNAc stoichiometry and dynamics.

Cited by 6 publications

References 37 publications

Advances in Strategies and Tools Available for Interrogation of Protein O‐GlcNAcylation

Advances in Strategies and Tools Available for Interrogation of Protein O‐GlcNAcylation

A mutant bacterial O-GlcNAcase visualizes a progressive decline of protein O-GlcNAcylation in early Drosophila embryos critical for neurodevelopment

Chemical Tools for Decoding the Functions of O-GlcNAcylation

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