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bioRxiv · 10.1101/2023.03.18.533286

Enzymatic assay for UDP-GlcNAc and its application in the parallel assessment of substrate availability and protein O-GlcNAcylation

Sunden, M.·Upadhyay, D.·Banerjee, R.·Sipari, N.·Fellman, V.·Kallijärvi, J.·Purhonen, J.
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Abstract

O-linked N-acetylglucosaminylation (O-GlcNAcylation) is a ubiquitous and dynamic yet still relatively poorly understood non-canonical glycosylation of intracellular proteins. Several vital branches of metabolism converge at the hexosamine biosynthetic pathway (HBP) to produce the substrate for protein O-GlcNAcylation the uridine diphosphate N-acetylglucosamine (UDP-GlcNAc). Availability of this metabolite is considered a key regulator of O-GlcNAcylation. Yet UDP-GlcNAc concentrations are rarely reported in studies exploring the HBP and O-GlcNAcylation, most likely because the methods to measure it restrict to specialized chromatographic procedures. To overcome this limitation, we introduce here an enzymatic method to quantify cellular and tissue UDP-GlcNAc. The method is based on O-GlcNAcylation of a substrate peptide by recombinant O-linked N-acetylglucosamine transferase (OGT) and detection of the modification with a specific antibody. The assay can be performed in dot blot or microplate formats. The key to successful assay was the removal of strong inhibition of OGT by the reaction side product, uridine diphosphate (UDP). We applied the assay to provide the first systematic report of UDP-GlcNAc concentrations in mouse tissues and cultured cells. Furthermore, we show how changes in UDP-GlcNAc levels correlate with O-GlcNAcylation and the expression of OGT and O-GlcNAcase (OGA).

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Sunden, M., Upadhyay, D., Banerjee, R., Sipari, N., Fellman, V., Kallijärvi, J., Purhonen, J.. 2023-03-19. Enzymatic assay for UDP-GlcNAc and its application in the parallel assessment of substrate availability and protein O-GlcNAcylation. https://doi.org/10.1101/2023.03.18.533286

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