Studying the O-GlcNAcome of human placentas using banked tissue samples

Abstract

O-GlcNAcylation is a dynamic modulator of signaling pathways, equal in magnitude to the widely studied phosphorylation. With the rapid development of tools for its detection at the single protein level, the O-GlcNAc modification rapidly emerged as a novel diagnostic and therapeutic target in human diseases. Yet, mapping the human O-GlcNAcome in various tissues is essential for generating relevant biomarkers. In this study, we used human banked tissue as a sample source to identify O-GlcNAcylated protein targets relevant to human diseases. Using human term placentas, we propose (1) a method to clean frozen banked tissue of blood proteins; (2) an optimized protocol for the enrichment of O-GlcNAcylated proteins using immunoaffinity purification; and (3) a bioinformatic workflow to identify the most promising O-GlcNAc targets. As a proof-of-concept, we used 45 mg of banked placental samples from two pregnancies to generate intracellular protein extracts depleted of blood protein. Then, antibody-based O-GlcNAc enrichment on denatured samples yielded over 2000 unique HexNAc PSMs and 900 unique sites using 300 μg of protein lysate. Due to efficient sample cleanup, we also captured 82 HexNAc proteins with high placental expression. Finally, we provide a bioinformatic tool (CytOVS) to sort the HexNAc proteins based on their cellular localization and extract the most promising O-GlcNAc targets to explore further. To conclude, we provide a simple 3-step workflow to generate a manageable list of O-GlcNAc proteins from human tissue and improve our understanding of O-GlcNAcylation’s role in health and diseases.

Regulation of Glycogen Synthase Kinase-3β by Phosphorylation and O-β-Linked N-Acetylglucosaminylation: Implications on Tau Protein Phosphorylation

“…Among the known O-GlcNAc sites (https://www.oglcnac.mcw.edu/), Thr38, Thr39 and Thr43, which we have also identified, were previously found from human embryonic stem cells by multiple reaction monitoring MS (MRM-MS) . Thr309, not found in this study, was recently captured from tissues of human placentas . None of the O-GlcNAc residues within the disordered N- and C-terminal domains identified in this study have been previously described.…”

“…79 Thr309, not found in this study, was recently captured from tissues of human placentas. 76 None of the O-GlcNAc residues within the disordered N-and C-terminal domains identified in this study have been previously described. However, the role of the Cterminal domain of GSK3β, which shares only 36% identity with the α isoform, and of its O-GlcNAc modifications remain to be defined.…”

“…The structure of a GSK3β complex with the autoinhibitory pSer9-GSK3 peptide highlights a competitive occupation of the phosphate-binding pocket involved in the binding of primed substrates in which substrate priming may be a selective mechanism of regulation of GSK3 activity toward the large number of potential substrates (Figure A). , Besides the phosphorylation-dependent regulation of GSK3β kinase activity, the potential role of O-GlcNAcylation on modulation of GSK3β functions is still intriguing. The interplay between phosphorylation and O-GlcNAcylation has been evoked in cellular regulatory processes, and O-GlcNAcylation of GSK3β has been described without deciphering its function among the plethora of GSK3β activities. ,,− …”