Metabolic Glycan Labeling in Primary Neurons Enabled by Unnatural Sugars with No -Glyco-Modification.

It is of great interest to probe glycosylation in primary neuron cultures. However, per--acetylated clickable unnatural sugars, which have been routinely utilized in metabolic glycan labeling (MGL) for analyzing glycans, showed cytotoxicity to cultured primary neurons and thus led to the speculation that MGL was not compatible with primary neuron cell cultures. Here, we uncovered that neuron cytotoxicity of per--acetylated unnatural sugars was related to their reactions with protein cysteines via non-enzymatic -glyco-modification.

Emerging roles of O-glycosylation in regulating protein aggregation, phase separation, and functions.

Protein O-glycosylation is widely identified in various proteins involved in diverse biological processes. Recent studies have demonstrated that O-glycosylation plays crucial and multifaceted roles in modulating protein amyloid aggregation and liquid-liquid phase separation (LLPS) under physiological conditions. Dysregulation of these processes is closely associated with human diseases such as neurodegenerative diseases (NDs) and cancers.

O-GlcNAcylation modulates liquid-liquid phase separation of SynGAP/PSD-95.

Liquid-liquid phase separation (LLPS) of SynGAP and PSD-95, two abundant proteins that interact in the postsynaptic density (PSD) of neurons, has been implicated in modulating SynGAP PSD enrichment in excitatory synapses. However, the underlying regulatory mechanisms remain enigmatic. Here we report that O-GlcNAcylation of SynGAP acts as a suppressor of LLPS of the SynGAP/PSD-95 complex.

Artificial Cysteine S-Glycosylation Induced by Per-O-Acetylated Unnatural Monosaccharides during Metabolic Glycan Labeling.

The unexpected, non-enzymatic S-glycosylation of cysteine residues in various proteins by per-O-acetylated monosaccharides is described. This artificial S-glycosylation greatly compromises the specificity and validity of metabolic glycan labeling in living cells by per-O-acetylated azido and alkynyl sugars, which has been overlooked in the field for decades. It is demonstrated that the use of unacetylated unnatural sugars can avoid the artifact formation and a corrected list of O-GlcNAcylated proteins and O-GlcNAc sites in HeLa cells has been assembled by using N-azidoacetylgalactosamine (GalNAz).

Quantitative time-resolved chemoproteomics reveals that stable -GlcNAc regulates box C/D snoRNP biogenesis.

-linked GlcNAcylation (-GlcNAcylation), a ubiquitous posttranslational modification on intracellular proteins, is dynamically regulated in cells. To analyze the turnover dynamics of -GlcNAcylated proteins, we developed a quantitative time-resolved -linked GlcNAc proteomics (qTOP) strategy based on metabolic pulse-chase labeling with an -GlcNAc chemical reporter and stable isotope labeling with amino acids in cell culture (SILAC). Applying qTOP, we quantified the turnover rates of 533 -GlcNAcylated proteins in NIH 3T3 cells and discovered that about 14% exhibited minimal removal of -GlcNAc or degradation of protein backbones.