Development of a Carbohydrate Microarray

碳水化合物微阵列的开发

基本信息

批准号：
10926054
负责人：
Jeffrey Gildersleeve
金额：
$ 35.5万
依托单位：
DIVISION OF BASIC SCIENCES - NCI
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

项目摘要

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.

碳水化合物 - 蛋白质相互作用对于多种生物学过程，例如炎症，细菌和病毒粘附以及转移至关重要。结果，人们对识别碳水化合物结合蛋白和发展配体以调节其活性一直引起人们的兴趣。碳水化合物 - 蛋白质相互作用的分析使许多因素变得复杂。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，研究结合的传统方法不是高通量，需要大量材料。第三，据称碳水化合物和蛋白质之间的单价相互作用是弱的。为了达到高潮，碳水化合物结合蛋白包含多个结合位点并形成多价复合物。结果，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至关重要的作用。碳水化合物微阵列或聚糖阵列中包含许多不同的碳水化合物或聚糖，在空间定义的排列中固定在坚实的支撑上。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表示，而微型化格式仅需要每种碳水化合物的含量来进行测定。我们已经开发了一种利用新糖蛋白的新型聚糖阵列格式。新糖蛋白是多价偶联物，其中碳水化合物共价连接到载体蛋白上，例如牛血清白蛋白。这些新糖蛋白以及天然糖蛋白被使用机器人微阵列印刷在环氧化物涂层显微镜载玻片上，以产生微阵列。新糖蛋白可以用作其他测定法，例如ELISA，多价抑制剂和多价免疫原子的多价试剂。我们目前拥有世界上最大，最独特的聚糖阵列之一。最近，我们专注于扩展和改善碳水化合物微阵列。一个目的是增加阵列上的聚糖数量。我们一直在开发改进的合成方法来制备糖肽。该方法将用于制备大型糖肽库，以添加阵列。此外，我们正在优化将碳水化合物结合到蛋白质中以产生新糖蛋白的方法。