Zhejiang University: ZJU scientists develop one-step enzymatic labeling approach

On September 30, the research team led by YI Wen at the Zhejiang University College of Life Sciences published a research article entitled “One-step enzymatic labeling reveals a critical role of O-GlcNAcylation in cell-cycle progression and DNA damage response” in the journal Angewandte Chemie International Edition.

O-linked N-acetylglucosamine (O-GlcNAcylation) is a prevalent and dynamic post-translational modification on serine or threonine residues of numerous proteins and it plays a vitally important role in various cellular processes. O-GlcNAc can be recycled by O-GlcNAc transferase (OGT) and O-GlcNAc hydrolase (OGA). In recent years, some chemical strategies have been developed to capture and profile O-GlcNAcylated proteins both in vitro and in cells. For example, by synthesizing GlcNAc bearing bio-orthogonal functional groups, researchers incorporate them into native OGlcNAcylated proteins, thereby permitting the detection and imaging of O-GlcNAcylated proteins within cells. In another strategy, researchers employ a two-step chemoenzymatic labeling method. In the first step, they apply a recombinant galactosyltransferase (GalT) to covalently transfer an azido-containing N-acetylgalactosamine (GalNAz) from the corresponding uridine diphosphate-linked sugar donor to OGlcNAc. In the second step, the azido functional group is derivatized with an alkynyl-containing small molecule reporter via Cu(I)-mediated azido-alkyl cycloaddition (CuAAC) reaction. The second step is vulnerable to incomplete reactions due to slow ligation or side reactions in the complicated cellular environment. Accumulating evidence indicates that glycosyltransferases could be engineered to expand the substrate scope. This has prompted YI Wen et al. to develop a remarkably sensitive one-step labeling strategy with an engineered GalT for capturing and profiling O-GlcNAcylated proteins.

In their study, YI Wen et al. analyzed the crystal structure of GaIT and surmised that K279A and K280A mutants could create a broader space for the C-2 position. Thanks to its better activity with Analogue 2, the K279A mutant was selected for further cellular studies. Researchers then applied the K279A mutant and Analogue 2 in the one-step labeling of O-GlcNAcylated proteins from HEK293T cells. A total of 740 proteins were identified at a less than 1% false discovery rate using the K279A mutant in the one-step procedure whereas 570 proteins were identified using the two-step procedure, confirming that the one-step procedure was more efficient in labeling.

FEN1, which was not discovered as an O-GlcNAcylated protein in any previous study, was identified as a potential glycoprotein. It is a multi-functional enzyme essential for both Okazaki fragment synthesis during DNA replication and DNA long-patch base excision repair. Serine 352 proved to be the major site of glycosylation in FEN1, but FEN1 was dynamically O-GlcNAcylated. In the S phase of the cell cycle, FEN1 was localized to the DNA replication foci through its interaction with PCNA. By deciphering the PFEN1-PCNA complex structure, researchers verified that S352 O-GlcNAcylation disrupts the FEN1/ PCNA interaction and contributes to an accumulation of DNA replication defects and DNA damage. In addition, O-GlcNAcylation of FEN1 can also enhance cells’ sensitivity to DNA damage agents.

“We provide an efficient method for profiling OGlcNAcylated proteins, and reveal a once-obscure mechanism of OGlcNAcylation in regulating cell cycle progression and DNA damage response,” said Prof. Yi. “This one-step labeling approach shows great promise to become a versatile tool to explore the function of O-GlcNAcylation in cells.”

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