Probing glycosyltransferase activities with the Staudinger ligation
Probing glycosyltransferase activities with the Staudinger ligation
复制标题
DOI：
10.1021/ja037692m
复制
发表时间：
2004-01-14
影响因子：
15
通讯作者：
Bertozzi, CR
中科院分区：
文献类型：
--
作者：
Hang, HC;Yu, C;Bertozzi, CR
快速筛选方法的发展，探测糖基转移酶的活动是必不可少的，推进糖生物学领域。虽然存在针对特定糖基转移酶的测定，但没有可在酶超家族中应用的可推广的方法。在这里，我们描述了一种新的糖基转移酶测定，利用其非天然底物的耐受性和独特的化学反应性的叠氮化物。我们将这种“叠氮-ELISA”应用于多肽α-N-乙酰半乳糖胺转移酶（ppGalNAcTs）家族，所有这些酶都能够从非天然核苷酸糖供体UDP-GalNAz转移N-叠氮乙酰半乳糖胺（GalNAz）。通过在微量滴定板形式中用膦探针进行Staudinger连接来检测和定量叠氮化物。这种方法应该适用于任何糖基转移酶或基团转移酶，耐受非天然叠氮基底物。
The development of rapid screening methods for probing glycosyltransferase activities is essential for advancing the field of glycobiology. While assays for specific glycosyltransferases exist, there is no generalizable method that can be applied across the enzyme superfamily. Herein we describe a novel glycosyltransferase assay that exploits their unnatural substrate tolerance and the unique chemical reactivity of the azide. We applied this “azido-ELISA” to the family of polypeptide α-N-acetylgalactosaminyltransferases (ppGalNAcTs), all of which were able to transferN-azidoacetylgalactosamine (GalNAz) from the unnatural nucleotide sugar donor UDP-GalNAz. The azide was detected and quantified by Staudinger ligation with a phosphine probe in a microtiter plate format. This approach should be applicable to any glycosyltransferase or group-transfer enzyme that tolerates unnatural azido substrates.