Shipp Matthew, Nadella Ramya, Gao Hui, Farkas Vladimir, Sigrist Hans, Faik Ahmed
Environmental and Plant Biology department, Ohio University, Porter Hall 512, Athens, OH, 45701, USA.
Glycoconj J. 2008 Jan;25(1):49-58. doi: 10.1007/s10719-007-9060-1. Epub 2007 Aug 1.
The plant cell wall is a complex network of polysaccharides. The diversity in the linkage types connecting all monosaccharides within these polysaccharides would need a large set of glycosyltransferases to catalyze their formation. Development of a methodology that would allow monitoring of glycosyltransferase activities in an easy and high-throughput manner would help assign biochemical functions, and understand their roles in building this complex network. A microarray-based method was optimized for testing glycosyltransferases involved in plant wall biosynthesis using an alpha(1,2)fucosyltransferase involved in xyloglucan biosynthesis. The method is simple, sensitive, and easy to implement in any lab. Tamarind xyloglucan polymer and trimer, and a series of cello-oligosaccharides were immobilized on a thin-coated photo-activable glass slide. The slide with the attached sugars was then used to estimate the incorporation of [(14)C]Fuc onto xyloglucan polymer and trimer. [(14)C]-radiolabel incorporation is revealed with a standard phosphoimager scanner, after exposure of the glycochip to a phosphor screen and detection. The method proved to be sensitive enough to detect as low as 45 cpm/spot. Oriented anchoring of small oligosaccharides (trimer) was required for optimal transferase activities. The glycochip was also used to monitor and estimate xyloglucan fucosyltransferase activity in detergent-solubilized crude extracts from pea microsomes that are known to contain this enzyme activity. Our data indicate that the methodology can be used for efficient and rapid monitoring of glycosyltransferase activities involved in plant wall polysaccharides biosynthesis.
植物细胞壁是一个复杂的多糖网络。连接这些多糖中所有单糖的连接类型的多样性需要大量的糖基转移酶来催化它们的形成。开发一种能够以简单且高通量的方式监测糖基转移酶活性的方法，将有助于确定其生化功能，并了解它们在构建这个复杂网络中的作用。一种基于微阵列的方法经过优化，用于测试参与植物细胞壁生物合成的糖基转移酶，该方法使用了参与木葡聚糖生物合成的α(1,2)岩藻糖基转移酶。该方法简单、灵敏，易于在任何实验室实施。罗望子木葡聚糖聚合物和三聚体，以及一系列纤维寡糖被固定在薄涂层的光活化载玻片上。然后使用附着有糖的载玻片来估计[(14)C]岩藻糖掺入木葡聚糖聚合物和三聚体中的情况。在糖芯片暴露于磷光屏并进行检测后，用标准的磷光成像扫描仪揭示[(14)C]放射性标记的掺入情况。该方法被证明足够灵敏，能够检测到低至45 cpm/斑点的信号。为了获得最佳转移酶活性，需要对小寡糖（三聚体）进行定向锚定。该糖芯片还用于监测和估计豌豆微粒体去污剂溶解粗提物中的木葡聚糖岩藻糖基转移酶活性，已知该粗提物含有这种酶活性。我们的数据表明，该方法可用于高效、快速地监测参与植物细胞壁多糖生物合成的糖基转移酶活性。
