Protein glycosylation events that happen early in the secretory pathway are often dysregulated during tumorigenesis. These events can be probed, in principle, by monosaccharides with bioorthogonal tags that would ideally be specific for distinct glycan subtypes. However, metabolic interconversion into other monosaccharides drastically reduces such specificity in the living cell. Here, we use a structure-based design process to develop the monosaccharide probe GalNAzMe that is specific for cancer-relevant Ser/Thr-N-acetylgalactosamine (O-GalNAc) glycosylation. By virtue of a branched N-acylamide side chain, GalNAzMe is not interconverted by epimerization to the corresponding N-acetylglucosamine analog like conventional GalNAc-based probes. GalNAzMe enters O-GalNAc glycosylation but does not enter other major cell surface glycan types including Asn (N)-linked glycans. We equip cells with the capacity to biosynthesize the nucleotide-sugar donor UDP-GalNAzMe from a caged precursor. Tagged with a bioorthogonal azide group, GalNAzMe serves as an O-glycan specific reporter in superresolution microscopy, chemical glycoproteomics, a genome-wide CRISPR knock-out (KO) screen, and imaging of intestinal organoids. GalNAzMe is a precision tool that allows a detailed view into the biology of a major type of cancer-relevant protein glycosylation.

中文翻译：

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代谢物精确标记可选择性探测O-连接的N-乙酰半乳糖胺糖基化

分泌途径早期发生的蛋白质糖基化事件通常在肿瘤发生过程中失调。这些事件原则上可以通过具有生物正交标签的单糖进行探测，理想情况下，这些标签应对不同的聚糖亚型具有特异性。但是，代谢相互转化为其他单糖会大大降低这种在活细胞中的特异性。在这里，我们使用基于结构的设计过程来开发针对与癌症相关的Ser / Thr- N的单糖探针GalNAzMe-乙酰半乳糖胺（O-GalNAc）糖基化。借助于支链的N-酰胺基侧链，GalNAzMe不会通过差向异构化转变为相应的N-乙酰氨基葡萄糖类似物，就像传统的基于GalNAc的探针一样。GalNAzMe进入O-GalNAc糖基化，但不进入其他主要细胞表面聚糖类型，包括与Asn（N）连接的聚糖。我们使细胞具有从笼状前体中生物合成核苷酸糖供体UDP-GalNAzMe的能力。带有生物正交叠氮化物基团的GalNAzMe在超分辨率显微镜，化学糖蛋白组学，全基因组CRISPR敲除（KO）筛查和肠道类器官成像中充当O-聚糖特异性报告分子。GalNAzMe是一种精密工具，可让您详细了解与癌症相关的蛋白质糖基化的主要类型的生物学特性。