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

It is of great interest to probe glycosylation in primaryneuroncultures. However, per-O-acetylated clickable unnaturalsugars, which have been routinely utilized in metabolic glycan labeling(MGL) for analyzing glycans, showed cytotoxicity to cultured primaryneurons and thus led to the speculation that MGL was not compatiblewith primary neuron cell cultures. Here, we uncovered that neuroncytotoxicity of per-O-acetylated unnatural sugarswas related to their reactions with protein cysteines via non-enzymatic S-glyco-modification. The modified proteins were enrichedin biological functions related to microtubule cytoskeleton organization,positive regulation of axon extension, neuron projection development,and axonogenesis. We thus established MGL in cultured primary neuronswithout cytotoxicity using S-glyco-modification-freeunnatural sugars including ManNAz, 1,3-Pr(2)ManNAz, and 1,6-Pr(2)ManNAz, which allowed for visualization of cell-surface sialylatedglycans, probing the dynamics of sialylation, and large-scale identificationof sialylated N-linked glycoproteins and the modification sites inprimary neurons. Particularly, a total of 505 sialylated N-glycosylationsites distributed on 345 glycoproteins were identified by 1,6-Pr(2)ManNAz.

Automated summary

It was found that per-O-acetylated clickable unnatural sugars used in metabolic glycan labeling (MGL) for analyzing glycans were cytotoxic to cultured primary neurons. This cytotoxicity was due to the non-enzymatic S-glyco-modification reactions of the sugars with protein cysteines, resulting in modified proteins associated with biological functions related to microtubule cytoskeleton organization and neuron projection development. By using S-glyco-modification-free unnatural sugars, MGL without cytotoxicity was established in cultured primary neurons, allowing for visualization of cell-surface sialylated glycans and large-scale identification of sialylated N-linked glycoproteins and modification sites.