Identification and Characterization of GRASP55 O-GlcNAcylation.

Xiaoyan Zhang

Methods in molecular biology (Clifton, N.J.) 2023

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O-GlcNAcylation is a posttranslational modification of proteins that adds a single sugar, β-N-acetylglucosamine (GlcNAc), to Ser/Thr residues. Extensive studies have been conducted to identify and characterize substrates of this modification but mostly focused on cytosolic and nuclear proteins. This chapter describes a detailed protocol used to determine the O-GlcNAcylation of GRASP55, the first O-GlcNAcylated Golgi protein identified, including how its O-GlcNAcylation level responds to glucose deprivation. In addition, this chapter also provides a detailed method to express and purify O-GlcNAcylated GRASP55 in bacteria for further in vitro functional assays. This protocol could be applied to other Golgi proteins that are potentially O-GlcNAcylated. © 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.