IP-LC-MSMS Enables Identification of Three Tau O‑GlcNAcylation Sites as O‑GlcNAcase Inhibition Pharmacodynamic Readout in Transgenic Mice Overexpressing Human Tau

• Published in: Journal of proteome research Vol. 22; no. 4; pp. 1309 - 1321
• Main Authors: Bijttebier, Sebastiaan, Rodrigues Martins, Dina, Mertens, Liesbeth, Grauwen, Karolien, Bruinzeel, Wouter, Willems, Roland, Bartolomé-Nebreda, José Manuel, Theunis, Clara, Bretteville, Alexis, Ebneth, Andreas, Dillen, Lieve
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 07.04.2023
• Subjects: Acetylglucosamine - pharmacology; Animals; beta-N-Acetylhexosaminidases - genetics; biomarkers; brain; genetically modified organisms; Humans; hydrolases; mass spectrometry; Mice; Mice, Transgenic; N-Acetylglucosaminyltransferases - genetics; N-Acetylglucosaminyltransferases - metabolism; peptides; pharmacodynamics; Phosphorylation; proteome; Tandem Mass Spectrometry; tau Proteins - chemistry; IP-LC-MSMS; O-GlcNAc hydrolase inhibition; Alzheimer’s disease; P301S transgenic mouse brain homogenates; tau O-GlcNAcylation
• ISSN: 1535-3893; 1535-3907; 1535-3907
• DOI: 10.1021/acs.jproteome.2c00822

O-β-linked N-acetylglucosaminylation (O-GlcNAcylation) modulates tau phosphorylation and aggregation: the pharmacological increase of tau O-GlcNAcylation upon treatment with inhibitors of O-GlcNAc hydrolase (OGA) constitutes a potential strategy to tackle neurodegenerative diseases. Analysis of tau O-GlcNAcylation could potentially be used as a pharmacodynamic biomarker both in preclinical and clinical studies. The goal of the current study was to confirm tau O-GlcNAcylation at S400 as a pharmacodynamic readout of OGA inhibition in P301S transgenic mice overexpressing human tau and treated with the OGA inhibitor Thiamet G and to explore if additional O-GlcNAcylation sites on tau could be identified. As a first step, an immunoprecipitation-liquid chromatography-mass spectrometry (IP-LC-MS) methodology was developed to monitor changes in O-GlcNAcylation around S400 of tau in mouse brain homogenate (BH) extracts. Second, additional O-GlcNAc sites were identified in in-house produced recombinant O-GlcNAcylated human tau at relatively high concentrations, thereby facilitating collection of informative LC-MS data for identification of low-concentration O-GlcNAc-tryptic tau peptides in human transgenic mouse BH extracts. This strategy enabled, for the first time, identification of three low abundant N-terminal and mid-domain O-GlcNAc sites of tau (at S208, S191, and S184 or S185) in human transgenic mouse BH. Data are openly available at data.mendeley.com (doi: 10.17632/jp57yk9469.1; doi: 10.17632/8n5j45dnd8.1; doi: 10.17632/h5vdrx4n3d.1).