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.探究原代神经元培养（primary neuron cultures）中的糖基化具有重要研究价值。然而，常规用于代谢糖标记（metabolic glycan labeling, MGL）以分析糖链的过O-乙酰化可点击非天然糖（per-O-acetylated clickable unnatural sugars），会对培养的原代神经元产生细胞毒性，由此引发了“代谢糖标记与原代神经元培养不兼容”的推测。本研究发现，过O-乙酰化非天然糖的神经元细胞毒性与其通过非酶促S-糖基修饰（non-enzymatic S-glyco-modification）与蛋白质半胱氨酸残基发生反应密切相关。经修饰的蛋白质富集于微管细胞骨架组织（microtubule cytoskeleton organization）、轴突生长正向调控（positive regulation of axon extension）、神经元突起发育（neuron projection development）及轴突发生（axonogenesis）等生物学功能通路中。基于此，我们使用不含S-糖基修饰的非天然糖（包括ManNAz、1,3-Pr2ManNAz及1,6-Pr2ManNAz），建立了无细胞毒性的原代神经元代谢糖标记体系，该体系可实现细胞表面唾液酸化糖链的可视化、唾液酸化动力学的探究，以及大规模鉴定原代神经元中的唾液酸化N-连接糖蛋白（sialylated N-linked glycoproteins）及其修饰位点。尤为值得一提的是，借助1,6-Pr2ManNAz共鉴定到分布于345个糖蛋白上的505个唾液酸化N-糖基化位点。