Department of Chemistry, Imperial College London, 80 Wood Lane, W12 0BZ London, United Kingdom.
The Chemical Glycobiology Laboratory, The Francis Crick Institute, 1 Midland Road, NW1 1AT London, United Kingdom.
ACS Chem Biol. 2021 Oct 15;16(10):1961-1967. doi: 10.1021/acschembio.1c00034. Epub 2021 Apr 9.
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 AcGalNAlk and AcGlcNAlk 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.
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。有效的 MOE 试剂通过细胞生物合成机制被激活为核苷酸糖，然后被引入糖蛋白中，并可通过生物正交化学进行追踪。尽管它们被广泛应用，但许多 MOE 试剂的代谢命运才刚刚开始被描绘出来。虽然代谢的相互关联性可能会影响探针的特异性，但生物合成补救途径的摄取不良可能会影响探针的灵敏度并引发副反应。在这里，我们使用代谢工程将弱炔基标记的 MOE 试剂 AcGalNAlk 和 AcGlcNAlk 转化为高效的化学工具，用于探测蛋白质糖基化。我们发现，通过工程化的焦磷酸化酶 AGX1 绕过代谢瓶颈可以促进核苷酸糖的生物合成，并将生物正交的细胞表面标记提高多达两个数量级。与已知的叠氮标记的 MOE 试剂的比较揭示了糖蛋白标记的主要差异，这大大扩展了化学糖生物学的工具包。
