One‐Step Enzymatic Labeling Reveals a Critical Role of O‐GlcNAcylation in Cell‐Cycle Progression and DNA Damage Response

Abstract

O‐linked N‐acetylglucosamine (O‐GlcNAcylation) is a ubiquitous post‐translational modification of proteins that is essential for cell function. Perturbation of O‐GlcNAcylation leads to altered cell‐cycle progression and DNA damage response. However, the underlying mechanisms are poorly understood. Here, we develop a highly sensitive one‐step enzymatic strategy for capture and profiling O‐GlcNAcylated proteins in cells. Using this strategy, we discover that flap endonuclease 1 (FEN1), an essential enzyme in DNA synthesis, is a novel substrate for O‐GlcNAcylation. FEN1 O‐GlcNAcylation is dynamically regulated during the cell cycle. O‐GlcNAcylation at the serine 352 of FEN1 disrupts its interaction with Proliferating Cell Nuclear Antigen (PCNA) at the replication foci, and leads to altered cell cycle, defects in DNA replication, accumulation of DNA damage, and enhanced sensitivity to DNA damage agents. Thus, our study provides a sensitive method for profiling O‐GlcNAcylated proteins, and reveals an unknown mechanism of O‐GlcNAcylation in regulating cell cycle progression and DNA damage response.

Endo‐M Mediated Chemoenzymatic Approach Enables Reversible Glycopeptide Labeling for O‐GlcNAcylation Analysis

“…Usually, a two‐step labeling is needed for the enrichment of glycoproteins, in which GalT1(Y289L) is utilized as the enzyme to label O ‐GlcNAc moieties by GalNAc with alkynyl or nitrine groups, followed by click chemistry to couple biotin tags for further enrichment (Scheme 1a) [7] . Recently, a one‐step chemoenzymatic strategy using the double‐mutant of GalT1 (K279A/Y289L) was developed for O ‐GlcNAcylation analysis, which directly labeled glycoproteins with GalNAc‐biotin to enhance the efficiency of the following glycoprotein enrichment [8] . To further determine the location of O ‐GlcNAcylation, the glycopeptides need to be released by cleavable linkers under acidic or UV conditions as reported in previous chemoenzymatic labeling studies [9] .…”

“…[7] Recently, a one-step chemoenzymatic strategy using the double-mutant of GalT1 (K279A/Y289L) was developed for O-GlcNAcylation analysis, which directly labeled glycoproteins with GalNAc-biotin to enhance the efficiency of the following glycoprotein enrichment. [8] To further determine the location of O-GlcNAcylation, the glycopeptides need to be released by cleavable linkers under acidic or UV conditions as reported in previous chemoenzymatic labeling studies. [9] In our recent work, O-GlcNAcylated peptides were first chemoenzymatically labeled with a Gal moiety, followed by chemical oxidation to efficiently introduce aldehyde groups.…”

O-GlcNAcylation mapping of single living cells by in situ quantitative SERS imaging

“…O-GlcNAcylation plays a crucial role in regulating DNA replication, cell-cycle progression and mitosis, and is related to cancerization. [31][32][33] It also protects genome integrity by modifying histones, kinases and scaffold proteins in the face of the altered cell cycle. 34 Therefore, the quantification of O-GlcNAcylation during cell cycle is of great importance.…”

Nascent Glycoproteome Reveals That N-Linked Glycosylation Inhibitor-1 Suppresses Expression of Glycosylated Lysosome-Associated Membrane Protein-2

“…O-linked glycosylation and N-linked glycosylation are the two most important forms of protein glycosylation ( Sun et al, 2021 ). Unlike the O-linked glycosylation, N-linked glycosylation is one type of biological processes that proteins undergo during de novo synthesis ( Xu et al, 2018 ; Karki et al, 2021 ; Tian et al, 2021 ). N-linked protein glycosylation mainly begins from endoplasmic reticulum (ER).…”