Development of a Carbohydrate Microarray

碳水化合物与蛋白质的相互作用对于炎症、细菌和病毒的黏附以及转移等广泛的生物学过程是必不可少的。因此，人们对识别碳水化合物结合蛋白和开发配体来调节它们的活性产生了浓厚的兴趣。碳水化合物-蛋白质相互作用的分析因多种因素而变得复杂。首先，碳水化合物极难分离或合成。因此，在许多情况下，只能获得少量的资金。第二，传统的研究结合的方法不是高通量的，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。为了获得高亲和力，碳水化合物结合蛋白含有多个结合位点并形成多价复合体。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取向在识别中起着关键作用。碳水化合物微阵列或多聚糖阵列包含许多不同的碳水化合物或多聚糖，以空间限定的排列固定在固体载体上。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呈现形式，并且小型化形式只需要每个碳水化合物的皮克量就可以进行一次测定。我们利用新的糖蛋白开发了一种新的糖链阵列形式。新糖蛋白是碳水化合物共价连接到载体蛋白(如牛血清白蛋白)上的多价结合物。这些新糖蛋白，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。新糖蛋白可用作其他检测的多价试剂，如ELISAs、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多糖阵列之一。最近，我们专注于扩大和改进碳水化合物微阵列。一个目标是增加阵列上的糖链数量。我们一直在开发改进的合成方法来制备糖肽。该方法将被用来准备一个大型糖肽文库，以添加到阵列中。此外，我们正在优化碳水化合物与蛋白质的结合方法，以产生新的糖蛋白。

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. in addition, we are optimizing methods to conjugate carbohydrates to proteins to produce neoglycoproteins.