Institute of Chemistry, Centre for Glycomics, Slovak Academy of Sciences, Dúbravská cesta 9, 84538, Bratislava, Slovakia.
Glycoconj J. 2010 Jan;27(1):79-87. doi: 10.1007/s10719-009-9271-8. Epub 2009 Dec 2.
Polysaccharide transglycosylases catalyze disproportionation of polysaccharide molecules by cleaving glycosidic linkages in polysaccharide chains and transferring their cleaved portions to hydroxyl groups at the non-reducing ends of other polysaccharide or oligosaccharide molecules. In plant cell walls, transglycosylases have a potential to catalyze both cross-linking of polysaccharide molecules and grafting of newly arriving polysaccharide molecules into the cell wall structure during cell growth. Here we describe a polysaccharide microarray in form of a glycochip permitting simultaneous high-throughput monitoring of multiple transglycosylase activities in plant extracts. The glycochip, containing donor polysaccharides printed onto nitrocellulose-coated glass slides, was incubated with crude plant extracts, along with a series of fluorophore-labelled acceptor oligosaccharides. After removing unused labelled oligosaccharides by washing, fluorescence retained on the glycochip as a result of transglycosylase reaction was detected with a standard microarray scanner. The glycochip assay was used to detect transglycosylase activities in crude extracts from nasturtium (Tropaeolum majus) and mouse-ear cress (Arabidopsis thaliana). A number of previously unknown saccharide donor-acceptor pairs active in transglycosylation reactions that lead to the formation of homo- and hetero-glycosidic conjugates, were detected. Our data provide experimental support for the existence of diverse transglycosylase activities in crude plant extracts.
多糖转糖基酶通过切割多糖链中的糖苷键，并将其断裂部分转移到其他多糖或寡糖分子的非还原端的羟基上，从而催化多糖分子的歧化。在植物细胞壁中，转糖基酶有可能在细胞生长过程中催化多糖分子的交联和新到达的多糖分子的接枝到细胞壁结构中。在这里，我们描述了一种多糖微阵列形式的糖芯片，允许同时高通量监测植物提取物中的多种转糖基酶活性。糖芯片包含打印在硝酸纤维素涂层玻璃载玻片上的供体多糖，与一系列荧光标记的受体寡糖一起孵育。在用洗涤去除未使用的标记寡糖后，通过转糖基酶反应保留在糖芯片上的荧光用标准微阵列扫描仪检测。该糖芯片测定法用于检测来自旱金莲（Tropaeolum majus）和鼠耳芥（Arabidopsis thaliana）的粗提物中的转糖基酶活性。检测到了许多以前未知的在导致同型和异型糖苷缀合形成的转糖基化反应中具有活性的糖供体-受体对。我们的数据为粗提植物提取物中存在多种转糖基酶活性提供了实验支持。
