Azide- and Alkyne-Bearing Metabolic Chemical Reporters of Glycosylation Show Structure-Dependent Feedback Inhibition of the Hexosamine Biosynthetic Pathway.
Azide- and Alkyne-Bearing Metabolic Chemical Reporters of Glycosylation Show Structure-Dependent Feedback Inhibition of the Hexosamine Biosynthetic Pathway.
复制标题
糖基化的带有叠氮化物和炔烃的代谢化学报告基因显示出己糖胺生物合成途径的结构依赖性反馈抑制。
DOI：
10.1002/cbic.201800280
复制
发表时间：
2018
期刊：
影响因子：
--
通讯作者：
Pratt,MatthewR
中科院分区：
文献类型：
--
作者：
Walter,LisaA;Batt,AnnaR;Darabedian,Narek;Zaro,BalynW;Pratt,MatthewR
蛋白质糖基化的代谢化学报告物（MCR）是带有反应性基团（如叠氮化物和炔）的天然单糖的类似物。当它们被添加到活细胞和生物体中时，这些小分子被生物合成转化为核苷酸供体糖，然后被糖基转移酶用于修饰蛋白质。随后通过生物正交化学方法安装标签可以实现这些糖蛋白的可视化和富集。虽然这种两步程序是强大的，但MCR的使用有可能改变供体糖的天然库的内源性产生。产生这些糖基转移酶底物的主要途径是己糖胺生物合成途径（HBP），其产生尿苷二磷酸N-乙酰葡萄糖胺（UDP-GlcNAc）。有趣的是，HBP的速率决定酶谷氨酰胺果糖-6-磷酸酰胺转移酶（GFAT）被UDP-GlcNAc反馈抑制。这增加了UDP-MCR的积聚可能会阻断UDP-GlcNAc的生物合成，导致脱靶效应。在这里，我们直接用重组人GFAT和一小组合成UDP-MCR测试这种可能性。我们发现，在N-乙酰基位置具有较大取代的MCR不能抑制GFAT，而具有2-或6-羟基修饰的MCR则可以。这些结果进一步阐明了应适用于MCR使用的注意事项。
Metabolic chemical reporters (MCRs) of protein glycosylation are analogues of natural monosaccharides that bear reactive groups, like azides and alkynes. When they are added to living cells and organisms, these small molecules are biosynthetically transformed into nucleotide donor sugars and then used by glycosyltransferases to modify proteins. Subsequent installation of tags by bioorthogonal chemistries can then enable the visualization and enrichment of these glycoproteins. Although this two‐step procedure is powerful, the use of MCRs has the potential to change the endogenous production of the natural repertoire of donor sugars. A major route for the generation of these glycosyltransferase substrates is the hexosamine biosynthetic pathway (HBP), which results in uridine diphosphateN‐acetylglucosamine (UDP‐GlcNAc). Interestingly, the rate‐determining enzyme of the HBP, glutamine fructose‐6‐phosphate amidotransferase (GFAT), is feedback inhibited by UDP‐GlcNAc. This raises the possibility that a build‐up of UDP‐MCRs would block the biosynthesis of UDP‐GlcNAc, resulting in off target effects. Here, we directly test this possibility with recombinant human GFAT and a small panel of synthetic UDP‐MCRs. We find that MCRs with larger substitutions at theN‐acetyl position do not inhibit GFAT, whereas those with modifications of the 2‐ or 6‐hydroxy group do. These results further illuminate the considerations that should be applied to the use of MCRs.