利用施陶丁格连接反应探究糖基转移酶活性

Probing glycosyltransferase activities with the Staudinger ligation.

作者信息

Hang Howard C, Yu Chong, Pratt Matthew R, Bertozzi Carolyn R

机构信息

Center for New Directions in Organic Synthesis, Departments of Chemistry and Molecular and Cell Biology, and Howard Hughes Medical Institute, University of California, Berkeley, California 94720, USA.

出版信息

J Am Chem Soc. 2004 Jan 14;126(1):6-7. doi: 10.1021/ja037692m.

DOI:10.1021/ja037692m

PMID:14709032

Abstract

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 alpha-N-acetylgalactosaminyltransferases (ppGalNAcTs), all of which were able to transfer N-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.

摘要

开发用于探测糖基转移酶活性的快速筛选方法对于推动糖生物学领域的发展至关重要。虽然存在针对特定糖基转移酶的检测方法，但尚无一种可推广应用于整个酶超家族的通用方法。在此，我们描述了一种新型糖基转移酶检测方法，该方法利用了它们对非天然底物的耐受性以及叠氮化物独特的化学反应性。我们将这种“叠氮化物酶联免疫吸附测定法（azido - ELISA）”应用于多肽α - N - 乙酰半乳糖胺基转移酶（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）。通过在微量滴定板中用膦探针进行施陶丁格连接反应来检测和定量叠氮化物。这种方法应适用于任何能耐受非天然叠氮底物的糖基转移酶或基团转移酶。