Zafar Abdul, Sridhar Sandhya, Bineva-Todd Ganka, Cioce Anna, Abdulla Nadia, Chang Vincent, Malaker Stacy A, Hewings David S, Schumann Benjamin
Chemical Glycobiology Laboratory, The Francis Crick Institute NW1 1AT London UK.
Department of Chemistry, Imperial College London W12 0BZ London UK
RSC Chem Biol. 2024 Aug 22;5(10):1002-9. doi: 10.1039/d4cb00093e.
Glycosylation is a ubiquitous modification of proteins, necessitating approaches for its visualization and characterization. Bioorthogonally tagged monosaccharides have been instrumental to this end, offering a chemical view into the cell biology of glycans. Understanding the use of such monosaccharides by cellular biosynthetic pathways has expanded their applicability in cell biology, for instance through the strategy named Bio-Orthogonal Cell-specific TAgging of Glycoproteins (BOCTAG). Here, we show that the cellular use of two azide-tagged analogues of the monosaccharide -acetylgalactosamine (GalNAzMe and GalNPrAz) can be promoted through expression of two biosynthetic enzymes. More precisely, cellular expression of the bacterial kinase NahK and the engineered human pyrophosphorylase AGX1 led to biosynthesis of the corresponding activated nucleotide-sugars and subsequent bioorthogonal tagging of the cellular glycoproteome. We explore the use of both sugars for BOCTAG, demonstrating the visualization of cell surface glycosylation tagged with GalNPrAz in a specific cell line in a co-culture system. Our work adds to the toolbox of glycoprotein analysis in biomedicine.
糖基化是蛋白质普遍存在的一种修饰，因此需要对其进行可视化和表征的方法。生物正交标记的单糖在这方面发挥了重要作用，为聚糖的细胞生物学提供了化学视角。了解细胞生物合成途径对这类单糖的利用，扩大了它们在细胞生物学中的应用范围，例如通过名为糖蛋白生物正交细胞特异性标记（BOCTAG）的策略。在此，我们表明，通过表达两种生物合成酶，可以促进细胞对两种叠氮化物标记的单糖类似物——N-乙酰半乳糖胺（GalNAzMe和GalNPrAz）的利用。更确切地说，细菌激酶NahK和工程化的人类焦磷酸化酶AGX1在细胞中的表达导致了相应的活化核苷酸糖的生物合成，随后对细胞糖蛋白组进行了生物正交标记。我们探索了将这两种糖用于BOCTAG，证明了在共培养系统中特定细胞系中用GalNPrAz标记的细胞表面糖基化的可视化。我们的工作为生物医学中糖蛋白分析的工具箱增添了内容。
