The Metabolic Chemical Reporter Ac46AzGal Could Incorporate Intracellular Protein Modification in the Form of UDP-6AzGlc Mediated by OGT and Enzymes in the Leloir Pathway.

Jiajia Wang,Jingying Chen,Peiyu Dong,Yinhang Wen,Lu Zheng,Yingyi Chen,Wenxuan Pan,Jing Ma,Wei Cao,Xia Li,Xueke Zeng,Biao Dou

doi:10.3389/fchem.2021.708306

Jiajia Wang, Jingying Chen + Show 10 more

Open Access

https://doi.org/10.3389/fchem.2021.708306

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Abstract

Galactose is a naturally occurring monosaccharide used to build complex glycans that has not been targeted for labeling as a metabolic reporter. Here, we characterize the cellular modification of proteins by using Ac46AzGal in a dose- and time-dependent manner. It is noted that a vast majority of this labeling of Ac46AzGal occurs intracellularly in a range of mammalian cells. We also provided evidence that this labeling is dependent on not only the enzymes of OGT responsible for O-GlcNAcylation but also the enzymes of GALT and GALE in the Leloir pathway. Notably, we discover that Ac46AzGal is not the direct substrate of OGT, and the labeling results may attribute to UDP-6AzGlc after epimerization of UDP-6AzGal via GALE. Together, these discoveries support the conclusion that Ac46AzGal as an analogue of galactose could metabolically label intracellular O-glycosylation modification, raising the possibility of characterization with impaired functions of the galactose metabolism in the Leloir pathway under certain conditions, such as galactosemias.

Highlights

O-GlcNAc modification of serine and threonine residues of intracellular proteins is ubiquitous in eukaryotic cells (Schwein and Woo, 2020; Zol-Hanlon and Schumann, 2020; Ma et al, 2021)
The highly conserved Leloir pathway is composed of three enzymes: galactokinase (GALK), galactose-1phosphate uridylyltransferase (GALT), and UDP galactose 4′-epimerase (GALE)
We challenge the Metabolic chemical reporters (MCRs) per-O-acetylated-6-Azido6-deoxy-Galactose (Ac46AzGal, Figure 1) for metabolic labeling of intracellular O-glycosylation proteins since the success labeling of Ac46AzGlc mediated with substrate promiscuity of O-GlcNAc transferase (OGT)

Summary

Introduction

O-GlcNAc modification of serine and threonine residues of intracellular proteins is ubiquitous in eukaryotic cells (Schwein and Woo, 2020; Zol-Hanlon and Schumann, 2020; Ma et al, 2021). We challenge the MCR per-O-acetylated-6-Azido6-deoxy-Galactose (Ac46AzGal, Figure 1) for metabolic labeling of intracellular O-glycosylation proteins since the success labeling of Ac46AzGlc mediated with substrate promiscuity of OGT.

Results

Conclusion