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。碳水化合物微阵列或聚糖阵列，包含许多不同的碳水化合物或聚糖，固定在一个固体载体上，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。我们利用新糖蛋白开发了一种新的聚糖阵列格式。新糖蛋白是多价偶联物，其中碳水化合物共价附着于载体蛋白，如牛血清白蛋白。这些新的糖蛋白，以及天然糖蛋白，被打印到环氧化物涂层的显微镜载玻片上，使用机器人微阵列来产生微阵列。新糖蛋白可作为多价试剂用于其他检测，如elisa、多价抑制剂和多价免疫原。我们目前拥有世界上最大和最独特的聚糖阵列之一。最近，我们专注于扩展和改进碳水化合物微阵列。目的之一是增加阵列上聚糖的数量。我们一直在开发改进的合成方法来制备糖肽。该方法将用于制备一个大型的糖肽库，以添加到阵列中。

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, or glycan arrays, contain many different carbohydrates, or glycans, 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 developed a novel glycan array format utilizing neoglycoproteins. Neoglycoproteins are multivalent conjugates wherein carbohydrates are covalently attached to a carrier protein, such as bovine serum albumin. These neoglycoproteins, as well as natural glycoproteins, are printed onto epoxide coated microscope slides using a robotic microarrayer to produce microarrays. Neoglycoproteins can be used as multivalent reagents for other assays such as ELISAs, as multivalent inhibitors, and as multivalent immunogens. We currently have one of the largest and most unique glycan arrays in the world. Recently, we have focused on expanding and improving the carbohydrate microarray. One objective is to increase the number of glycans on the array. We have been developing improved synthetic methods for preparing glycopeptides. The methodology will be used to prepare a large library of glycopeptides for addition to the array.