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

It is of great interest to probe glycosylation in primary neuron cultures. However, per-O-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-O-acetylated unnatural sugars was related to their reactions with protein cysteines via non-enzymatic S-glyco-modification. The modified proteins were enriched in biological functions related to microtubule cytoskeleton organization, positive regulation of axon extension, neuron projection development, and axonogenesis. We thus established MGL in cultured primary neurons without cytotoxicity using S-glyco-modification-free unnatural sugars including ManNAz, 1,3-Pr₂ManNAz, and 1,6-Pr₂ManNAz, which allowed for visualization of cell-surface sialylated glycans, probing the dynamics of sialylation, and large-scale identification of sialylated N-linked glycoproteins and the modification sites in primary neurons. Particularly, a total of 505 sialylated N-glycosylation sites distributed on 345 glycoproteins were identified by 1,6-Pr₂ManNAz.