Peng Wenjie, Nycholat Corwin M, Razi Nahid
Glycan Microarray Synthesis Core, Consortium for Functional Glycomics, Department of Chemical Physiology, The Scripps Research Institute, La Jolla, CA, USA.
Methods Mol Biol. 2013;1022:1-14. doi: 10.1007/978-1-62703-465-4_1.
Glycan microarrays represent a high-throughput approach to determining the specificity of glycan-binding proteins against a large set of glycans in a single format. This chapter describes the use of a glycan microarray platform for evaluating the activity and substrate specificity of glycosyltransferases (GTs). The methodology allows simultaneous screening of hundreds of immobilized glycan acceptor substrates by in situ incubation of a GT and its appropriate donor substrate on the microarray surface. Using biotin-conjugated donor substrate enables direct detection of the incorporated sugar residues on acceptor substrates on the array. In addition, the feasibility of the method has been validated using label-free donor substrate combined with lectin-based detection of product to assess enzyme activity. Here, we describe the application of both procedures to assess the specificity of a recombinant human α2-6 sialyltransferase. This technique is readily adaptable to studying other glycosyltransferases.
聚糖微阵列是一种高通量方法，可在单一形式下确定聚糖结合蛋白对大量聚糖的特异性。本章描述了使用聚糖微阵列平台评估糖基转移酶（GTs）的活性和底物特异性。该方法允许通过在微阵列表面原位孵育GT及其合适的供体底物，同时筛选数百种固定化的聚糖受体底物。使用生物素偶联的供体底物能够直接检测阵列上受体底物上掺入的糖残基。此外，该方法的可行性已通过使用无标记供体底物结合基于凝集素的产物检测来评估酶活性得到验证。在这里，我们描述了这两种方法在评估重组人α2-6唾液酸转移酶特异性方面的应用。该技术很容易适用于研究其他糖基转移酶。
