Metabolic oligosaccharide engineering (MOE) has fundamentally contributed to our understanding of protein glycosylation. Efficient MOE reagents are activated into nucleotide-sugars by cellular biosynthetic machineries, introduced into glycoproteins and traceable by bioorthogonal chemistry. Despite their widespread use, the metabolic fate of many MOE reagents is only beginning to be mapped. While metabolic interconnectivity can affect probe specificity, poor uptake by biosynthetic salvage pathways may impact probe sensitivity and trigger side reactions. Here, we use metabolic engineering to turn the weak alkyne-tagged MOE reagents Ac₄GalNAlk and Ac₄GlcNAlk into efficient chemical tools to probe protein glycosylation. We find that bypassing a metabolic bottleneck with an engineered version of the pyrophosphorylase AGX1 boosts nucleotide-sugar biosynthesis and increases bioorthogonal cell surface labeling by up to two orders of magnitude. A comparison with known azide-tagged MOE reagents reveals major differences in glycoprotein labeling, substantially expanding the toolbox of chemical glycobiology.

中文翻译：

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通过工程焦磷酸化酶优化代谢寡糖工程与 Ac4GalNAlk 和 Ac4GlcNAlk

代谢寡糖工程 (MOE) 从根本上促进了我们对蛋白质糖基化的理解。高效的 MOE 试剂通过细胞生物合成机器活化成核苷酸糖，引入糖蛋白并通过生物正交化学进行追踪。尽管它们被广泛使用，但许多 MOE 试剂的代谢命运才刚刚开始绘制。虽然代谢相互联系会影响探针特异性，但生物合成补救途径的吸收不良可能会影响探针敏感性并引发副反应。在这里，我们使用代谢工程将弱炔标记的 MOE 试剂 Ac ₄ GalNAlk 和 Ac ₄GlcNAlk 成为探测蛋白质糖基化的有效化学工具。我们发现，用焦磷酸化酶 AGX1 的工程版本绕过代谢瓶颈可促进核苷酸 - 糖的生物合成，并将生物正交细胞表面标记提高两个数量级。与已知的叠氮标记 MOE 试剂的比较揭示了糖蛋白标记的主要差异，大大扩展了化学糖生物学的工具箱。