New Method to Detect Protein O-GlcNAcylation without S-Glyco Modification
On April 19, 2024, the team of Prof. Dr. Valentin Wittmann from the University of Konstanz published a research paper entitled "Next-Generation Metabolic Glycosylation Reporters Enable Detection of Protein O-GlcNAcylation in Living Cells without S-Glyco Modification" in Angewandte Chemie International Edition.
Current detection of O-GlcNAcylated proteins in living cells mainly faces the following two major problems.
Carbohydrate derivatives used in traditional Metabolic Glycoengineering (MGE) are usually fully acetylated to increase membrane permeability and thus promote cellular uptake. It has recently been found that the use of fully acetylated carbohydrate derivatives can lead to non-specific attachment of proteins to cysteine side chains in non-enzymatic reactions, resulting in S-glycosylation, a large number of false positive results, and affect the glycoproteomic identification of O-GlcNAcylated proteins.
Commonly used reporter groups include azides and alkynes. They can react in a Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) or a strain-promoted azide-alkyne cycloaddition (SPAAC). However, due to the toxicity of Cu(I) or the nonspecific reaction of cyclooctynes with thiols present in cells, the application of these two reactions in living cells is severely limited, resulting in low labeling efficiency or inaccurate labeling.
Therefore, it is necessary to find new detection methods to avoid S-glycosylation and improve the detection accuracy of O-GlcNAcylated proteins in living cells. This study developed a protected GlcNAc-1-phosphate that can be labeled through a cell-friendly inverse-electron-demand Diels-Alder (IEDDA) reaction, thereby bypassing the traditional S-glycosylation pathway and successfully realizing the detection of O-GlcNAcylated proteins in living cells, providing new tools and methods for the study and visualization of O-GlcNAcylated proteins.
Fig. 1 Investigation of protein O-GlcNAcylation by MGE using conventional peracetylated sugars and protected GlcNAc-1-phosphates. (Kufleitner, et al., 2024)
It is a chemical modification method in which sugar molecules are connected to sulfur groups (such as the sulfur atom of cysteine) in proteins or nucleic acid molecules through chemical bonds. S-glycosylation mainly occurs on cysteine residues in proteins, but it may also occur on other sulfur-containing amino acids or nucleic acid molecules, which has an important impact on biological processes such as protein function and cell signaling.
This method is used to detect glycosylation in various cell lines and even in vivo, including protein O-GlcNAcylation. Synthetic carbohydrate derivative A (equipped with a chemical reporter group X) is added to the cell culture medium, absorbed and metabolized by the cells, and finally incorporated into the cell Glycans through an enzymatic mechanism. In this process, Glycosyltransferase acts as a catalyst to transfer sugar molecules to specific positions of the target molecule (protein or lipid), forming glycosidic bonds, thereby changing the structure, stability and function of the target molecule.
The IEDDA reaction is a [4+2] cycloaddition reaction between an electron-deficient diene and an electron-rich dienophile. In contrast to the normal Diels-Alder (DA) reaction, the electron transfer direction of the IEDDA reaction is from the highest occupied molecular orbital (HOMO) of the dienophile to the lowest unoccupied molecular orbital (LUMO) of the diene. It has the advantages of good biocompatibility, high reaction specificity, and fast reaction rate, and has a wide range of applications in the field of Bioorthogonal Chemistry.
The research team has developed a new method that bypasses the pathway that leads to S-glyco modification, thereby detecting protein O-GlcNAcylation in living cells without S-glyco modification.
Norbornene-modified GlcNAc derivatives (such as Ac3GlcNNorboc-1-P(SATE)2) can be accepted by cells in an OGT-dependent manner and can be used for visualization of protein O-GlcNAcylation in living cells by Förster resonance energy transfer (FRET) technology.
The experimental results showed that the intermolecular FRET between eGFP and TAMRA-labeled GlcNNorboc residues was negligible, indicating that Ac3GlcNNorboc-1-P(SATE)2 is an effective reporter gene for studying protein O-GlcNAcylation in living cells and can be used in combination with confocal fluorescence lifetime imaging microscopy (FLIM)-FRET imaging technology.
A new method has been developed to detect protein O-GlcNAcylation in living cells without S-glyco modification. This innovation enables the researchers to observe and analyze the dynamic changes of protein O-GlcNAcylation more directly and accurately.
A new GlcNAc derivative was designed and synthesized, which contains a protected phosphate group at the anomeric position and can be labeled by a cell-friendly IEDDA reaction. This derivative not only improves the sensitivity and specificity of detection, but also provides more possibilities for subsequent research.
Combining FRET technology, spatial resolution of protein O-GlcNAcylation sites is achieved. By constructing POI-eGFP fusion protein and using FLIM-FRET imaging technology, researchers can intuitively observe the distribution of protein O-GlcNAcylation in cells, providing a powerful tool for in-depth study of its function and regulatory mechanism.
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