Development of a Carbohydrate Microarray

碳水化合物与蛋白质的相互作用对于炎症、细菌和病毒的粘附和转移等广泛的生物过程至关重要。因此，人们对鉴定碳水化合物结合蛋白和开发调节其活性的配体产生了极大的兴趣。碳水化合物与蛋白质相互作用的分析由于许多因素而变得复杂。首先，碳水化合物极难分离或合成。因此，在许多情况下，只能获得少量。其次，传统的结合研究方法的通量不高，需要大量的材料。第三，碳水化合物和蛋白质之间的单价相互作用是出了名的弱。为了实现高亲和性，碳水化合物结合蛋白含有多个结合位点并形成多价复合物。因此，碳水化合物必须以多价方式呈现，碳水化合物的间距和方向在识别中起着关键作用。碳水化合物微阵列包含许多不同的碳水化合物固定在一个固体支持在一个空间定义的安排。使用阵列，可以同时评估凝集素、抗体、细胞或病毒与阵列上所有碳水化合物的结合。该阵列提供了碳水化合物的多价表示，并且小型化的格式只需要每个碳水化合物的picogram量即可进行测定。我们已经成功地生成了一个碳水化合物微阵列和检测结合的方法。我们当前的微阵列包含128个不同的组件。阵列格式非常适合于检查碳水化合物结构和碳水化合物密度的许多组合以进行识别。我们正在继续扩大和改进该阵列。

Carbohydrate-protein interactions are essential for a wide range of biological processes such as inflammation, bacterial and viral adhesion, and metastasis. As a result, there has been significant interest in identifying carbohydrate binding proteins and developing ligands to modulate their activity. Analysis of carbohydrate-protein interactions is complicated by a number of factors. First, carbohydrates are extremely difficult to isolate or synthesize. Therefore, only small amounts can be obtained in many cases. Second, traditional methods for studying binding are not high-throughput and require large amounts of material. Third, monovalent interactions between carbohydrates and proteins are notoriously weak. To achieve a high avidity, carbohydrate-binding proteins contain multiple binding sites and form multivalent complexes. As a result, carbohydrates must be presented in a multivalent fashion and the spacing and orientation of the carbohydrates play a critical role in recognition. Carbohydrate microarrays contain many different carbohydrates immobilized on a solid support in a spatially-defined arrangement. Using an array, one can evaluate binding of a lectin, antibody, cell, or virus to all the carbohydrates on the array simultaneously. The array provides a multivalent presentation of carbohydrates, and the miniaturized format requires only picogram amounts of each carbohydrate for an assay. We have successfully generated a carbohydrate microarray and assay to detect binding. Our current generation microarray contains 128 different components. The array format is ideally suited to examine many combinations of both carbohydrate structure and carbohydrate density for recognition. We are continuing to expand and improve the array.