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The O-GlcNAc Modification of Recombinant Tau Protein and Characterization of the O-GlcNAc Pattern for Functional Study

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Tau Protein

Abstract

The neuronal microtubule-associated tau protein is characterized in vivo by a large number of post-translational modifications along the entire primary sequence that modulates its function. The primary modification of tau is phosphorylation of serine/threonine or tyrosine residues that is involved in the regulation of microtubule binding and polymerization. In neurodegenerative disorders referred to as tauopathies including Alzheimer’s disease, tau is abnormally hyperphosphorylated and forms fibrillar inclusions in neurons progressing throughout different brain area during the course of the disease. The O-β-linked N-acetylglucosamine (O-GlcNAc) is another reversible post-translational modification of serine/threonine residues that is installed and removed by the unique O-GlcNAc transferase (OGT) and O-GlcNAc hydrolase (OGA), respectively. This modification was described as a potential modulator of tau phosphorylation and functions in the physiopathology. Moreover, reducing protein O-GlcNAc levels in the brain upon treatment of tauopathy mouse models with an OGA inhibitor reveals a beneficial effect on tau pathology and neurodegeneration. However, whether the role of tau O-GlcNAcylation is responsible of the protective effect against tau toxicity remains to be determined. The production of O-GlcNAc modified recombinant tau protein is a valuable tool for the investigations of the impact of O-GlcNAcylation on tau functions, modulation of interactions with partners and crosstalk with other post-translational modifications, including but not restricted to phosphorylation. We describe here the in vitro O-GlcNAcylation of tau with recombinant OGT for which we provide an expression and purification protocol. The use of the O-GlcNAc tau protein in functional studies requires the analytical characterization of the O-GlcNAc pattern. Here, we describe a method for the O-GlcNAc modification of tau protein with recombinant OGT and the analytical characterization of the resulting O-GlcNAc pattern by a combination of methods for the overall characterization of tau O-GlcNAcylation by chemoenzymatic labeling and mass spectrometry, as well as the quantitative, site-specific pattern by NMR spectroscopy.

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Acknowledgments

The NMR facilities were funded by the Région Nord, CNRS, Pasteur Institute of Lille, European Community (FEDER), French Research Ministry, and the University of Sciences and Technologies of Lille I. We acknowledge support from the TGE RMN THC (FR-3050, France) and the Research Federation FRABio (Univ. Lille, CNRS, FR 3688, FRABio, “Structural & Functional Biochemistry of Biomolecular Assemblies”) for providing the scientific and technical environment. This study was supported by a grant from the LabEx (Laboratory of Excellence) DISTALZ (Development of Innovative Strategies for a Transdisciplinary approach to Alzheimer’s disease), by the Mizutani Foundation for Glycoscience (grant number 180122) and in part by the French government funding agency (Agence Nationale de la Recherche).

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El Hajjar, L., Bridot, C., Nguyen, M., Cantrelle, FX., Landrieu, I., Smet-Nocca, C. (2024). The O-GlcNAc Modification of Recombinant Tau Protein and Characterization of the O-GlcNAc Pattern for Functional Study. In: Smet-Nocca, C. (eds) Tau Protein. Methods in Molecular Biology, vol 2754. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3629-9_14

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