Department of Chemistry, Stanford University, Stanford, CA 94305.
The Chemical Glycobiology Laboratory, The Francis Crick Institute, NW1 1AT London, United Kingdom.
Proc Natl Acad Sci U S A. 2020 Oct 13;117(41):25293-25301. doi: 10.1073/pnas.2007297117. Epub 2020 Sep 28.
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 -()-azidopropionylgalactosamine (GalNAzMe) that is specific for cancer-relevant Ser/Thr(O)-linked -acetylgalactosamine (GalNAc) glycosylation. By virtue of a branched -acylamide side chain, GalNAzMe is not interconverted by epimerization to the corresponding -acetylglucosamine analog by the epimerase -acetylgalactosamine-4-epimerase (GALE) 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 transfect cells with the engineered pyrophosphorylase mut-AGX1 to biosynthesize the nucleotide-sugar donor uridine diphosphate (UDP)-GalNAzMe from a sugar-1-phosphate precursor. Tagged with a bioorthogonal azide group, GalNAzMe serves as an O-glycan-specific reporter in superresolution microscopy, chemical glycoproteomics, a genome-wide CRISPR-knockout (CRISPR-KO) screen, and imaging of intestinal organoids. Additional ectopic expression of an engineered glycosyltransferase, "bump-and-hole" (BH)-GalNAc-T2, boosts labeling in a programmable fashion by increasing incorporation of GalNAzMe into the cell surface glycoproteome. Alleviating the need for GALE-KO cells in metabolic labeling experiments, GalNAzMe is a precision tool that allows a detailed view into the biology of a major type of cancer-relevant protein glycosylation.
在分泌途径早期发生的蛋白质糖基化事件在肿瘤发生过程中经常失调。这些事件原则上可以通过带有生物正交标记的单糖来探测，这些单糖理想情况下应该对不同的聚糖亚型具有特异性。然而，在活细胞中，代谢互变异构会极大地降低这种特异性。在这里，我们使用基于结构的设计过程来开发单糖探针-()-叠氮丙酰基半乳糖胺（GalNAzMe），它专门针对与癌症相关的 Ser/Thr（O）-连接的 -乙酰半乳糖胺（GalNAc）糖基化。由于支链 -酰基酰胺侧链，GalNAzMe 不会像传统的 GalNAc 基探针那样通过差向异构酶 -乙酰半乳糖胺-4-差向异构酶（GALE）互变异构为相应的 -乙酰葡萄糖胺类似物。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 Asn（N）-连接的聚糖。我们通过工程化的焦磷酸化酶 mut-AGX1 转染细胞，从糖-1-磷酸前体生物合成核苷酸糖供体尿苷二磷酸（UDP）-GalNAzMe。GalNAzMe 标记有生物正交的叠氮基团，可在超分辨率显微镜、化学糖蛋白质组学、全基因组 CRISPR 敲除（CRISPR-KO）筛选和肠道类器官成像中作为 O-聚糖特异性报告物。工程化糖基转移酶“凹凸”（BH）-GalNAc-T2 的额外异位表达以可编程方式增加 GalNAzMe 掺入细胞表面糖蛋白组的方式，以可调节的方式增强标记。GalNAzMe 缓解了代谢标记实验中对 GALE-KO 细胞的需求，是一种精确的工具，可深入了解与癌症相关的主要蛋白糖基化的生物学。
