Park Sungjin, Lee Myung-ryul, Pyo Soon-Jin, Shin Injae
Department of Chemistry, Yonsei University, Seoul 120-749, Korea.
J Am Chem Soc. 2004 Apr 21;126(15):4812-9. doi: 10.1021/ja0391661.
Carbohydrate-protein interactions play important biological roles in living organisms. For the most part, biophysical and biochemical methods have been used for studying these biomolecular interactions. Less attention has been given to the development of high-throughput methods to elucidate recognition events between carbohydrates and proteins. In the current effort to develop a novel high-throughput tool for monitoring carbohydrate-protein interactions, we prepared carbohydrate microarrays by immobilizing maleimide-linked carbohydrates on thiol-derivatized glass slides and carried out lectin binding experiments by using these microarrays. The results showed that carbohydrates with different structural features selectively bound to the corresponding lectins with relative binding affinities that correlated with those obtained from solution-based assays. In addition, binding affinities of lectins to carbohydrates were also quantitatively analyzed by determining IC(50) values of soluble carbohydrates with the carbohydrate microarrays. To fabricate carbohydrate chips that contained more diverse carbohydrate probes, solution-phase parallel and enzymatic glycosylations were performed. Three model disaccharides were in parallel synthesized in solution-phase and used as carbohydrate probes for the fabrication of carbohydrate chips. Three enzymatic glycosylations on glass slides were consecutively performed to generate carbohydrate microarrays that contained the complex oligosaccharide, sialyl Le(x). Overall, these works demonstrated that carbohydrate chips could be efficiently prepared by covalent immobilization of maleimide-linked carbohydrates on the thiol-coated glass slides and applied for the high-throughput analyses of carbohydrate-protein interactions.
碳水化合物 - 蛋白质相互作用在生物体中发挥着重要的生物学作用。在很大程度上，生物物理和生化方法已被用于研究这些生物分子相互作用。人们对开发高通量方法以阐明碳水化合物与蛋白质之间的识别事件关注较少。在当前开发用于监测碳水化合物 - 蛋白质相互作用的新型高通量工具的工作中，我们通过将马来酰亚胺连接的碳水化合物固定在硫醇衍生化的载玻片上来制备碳水化合物微阵列，并使用这些微阵列进行凝集素结合实验。结果表明，具有不同结构特征的碳水化合物以与基于溶液的分析中获得的相对结合亲和力选择性地结合相应的凝集素。此外，还通过用碳水化合物微阵列测定可溶性碳水化合物的IC(50)值来定量分析凝集素与碳水化合物的结合亲和力。为了制造包含更多样化碳水化合物探针的碳水化合物芯片，进行了溶液相平行和酶促糖基化反应。在溶液相中平行合成了三种模型二糖，并将其用作制造碳水化合物芯片的碳水化合物探针。在载玻片上连续进行了三次酶促糖基化反应，以生成包含复合寡糖唾液酸化路易斯(x)的碳水化合物微阵列。总体而言，这些工作表明，通过将马来酰亚胺连接的碳水化合物共价固定在硫醇包被的载玻片上，可以有效地制备碳水化合物芯片，并将其应用于碳水化合物 - 蛋白质相互作用的高通量分析。
