Research Article: WGA-based lectin affinity gel electrophoresis: A novel method for the detection of O-GlcNAc-modified proteins

Date Published: July 7, 2017

Publisher: Public Library of Science

Author(s): Yuji Kubota, Ko Fujioka, Mutsuhiro Takekawa, David D. Roberts.

http://doi.org/10.1371/journal.pone.0180714

Abstract

Post-translational modification with O-linked β-N-acetylglucosamine (O-GlcNAc) occurs selectively on serine and/or threonine residues of cytoplasmic and nuclear proteins, and dynamically regulates their molecular functions. Since conventional strategies to evaluate the O-GlcNAcylation level of a specific protein require time-consuming steps, the development of a rapid and easy method for the detection and quantification of an O-GlcNAcylated protein has been a challenging issue. Here, we describe a novel method in which O-GlcNAcylated and non-O-GlcNAcylated forms of proteins are separated by lectin affinity gel electrophoresis using wheat germ agglutinin (WGA), which primarily binds to N-acetylglucosamine residues. Electrophoresis of cell lysates through a gel containing copolymerized WGA selectively induced retardation of the mobility of O-GlcNAcylated proteins, thereby allowing the simultaneous visualization of both the O-GlcNAcylated and the unmodified forms of proteins. This method is therefore useful for the quantitative detection of O-GlcNAcylated proteins.

Partial Text

Protein post-translational modifications (PTMs) regulate various properties of proteins such as stability, subcellular localization, and catalytic activity. Of these PTMs, O-linked β-N-acetylglucosamine modification (O-GlcNAcylation) is a type of protein glycosylation in which a single-sugar, N-acetylglucosamine, is added to the hydroxyl moiety of serine and threonine residues of cytoplasmic and nuclear proteins. Previous studies have shown that thousands of proteins in cells are modified with O-GlcNAc [1]. Protein O-GlcNAcylation is dynamically and reversibly regulated by the paired enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) [2, 3]. OGT catalyzes the addition of a GlcNAc moiety from a uridine diphosphate (UDP)-GlcNAc to target proteins, while OGA removes the O-GlcNAc from the modified proteins. In contrast to protein phosphorylation that is regulated by many kinases and phosphatases, OGT and OGA are the only enzymes responsible for the protein O-GlcNAcylation cycle in a cell. Since both O-GlcNAcylation and phosphorylation occur at serine and threonine residues, these two types of PTMs are mutually exclusive on the same target residue. In addition, these modifications can suppress each other within a protein even when they do not take place on the same residues [4]. This dynamic crosstalk between phosphorylation and O-GlcNAcylation has been frequently found in various cellular proteins, and the molecular function of the modified proteins dramatically changes according to the modification state [4].

All reagents and solvents in this study were analytical grade or better.

 

Source:

http://doi.org/10.1371/journal.pone.0180714

 

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