Developing high affinity ligands of glycan binding proteins

项目摘要/摘要 糖链结合蛋白(GBP)对所有细胞产生的糖链中包含的密集结构信息进行解码。Galectins是一个广泛分布于动物体内的GBP家族，在多种组织中表达，存在于细胞内和细胞外。Galectins的碳水化合物识别结构域与β-半乳糖表位结合，如N-乙酰乳糖胺(LacNAc)单位，通常存在于N-和O-连接糖蛋白的糖链上，以及糖脂。Galectins介导许多生理过程，包括细胞与细胞、细胞与基质的黏附、细胞信号转导和免疫反应的调节。然而，Galectins也与癌症、炎症、心脏病和微生物感染的调节等病理过程有关。本项目的目标是开发高亲和力和选择性的半乳糖凝集素配体，基于可阻断其与多聚糖相互作用的多聚糖类似物，为研究这一重要类别的多聚糖结合蛋白的功能或作为潜在的治疗药物提供有价值的工具。在目标1中，我们将使用几种高通量策略来生成大量多样化的葡聚糖类似物文库，包括涉及芯片上点击化学反应的组合方法，以及半乳糖凝集素共晶结构的电子引导筛选，以从大型商业构建块库中识别有利的取代基。该多聚糖类似物文库将被合并到一个多聚糖微阵列中，该微阵列将在AIM 2中对照人和小鼠半乳糖凝集素的完整小组进行筛选，以确定高亲和力和选择性配体。印刷的阵列很容易适应，并将提供给包括非专家在内的其他研究人员进行糖链阵列分析。在这个项目中开发的半乳糖凝集素抑制剂将是真正的现成试剂，非专家可以很容易地适应功能研究或治疗应用。

PROJECT SUMMARY/ABSTRACT Glycan-binding proteins (GBPs) decode the dense structural information contained in the glycome produced by all cells. Galectins are a family of GBPs widely distributed among animals, which are expressed in various tissues, and occur in both intracellular and extracellular compartments. The carbohydrate recognition domain (CRD) of galectins bind to β-galactose epitopes such as N-acetyllactosamine (LacNAc) units commonly found on glycans of N- and O-linked glycoproteins, and glycolipids. Galectins mediate many physiological processes including cell-cell and cell-matrix adhesion, cell signaling, and regulation of immune responses. However, galectins are also implicated in pathological processes such as cancer, inflammation, heart disease, and regulation of microbial infection. The objective of this project is to develop high affinity and selective ligands of galectins based on glycan analogs that can block their interactions with glycans to provide valuable tools for investigating the functions of this important class of glycan-binding protein or serve as potential therapeutics. In Aim 1 we will generate a large diverse library of glycan analogs using several high- throughput strategies, including a combinatorial approach involving an on-chip click-chemistry reaction, and an in silico guided screen of galectin co-crystal structures to identify favorable substituents from large commercial building block libraries. The glycan analog library will be incorporated into a glycan microarray which will be screened in Aim 2 against a complete panel of human and murine galectins to identify high affinity and selective ligands. The printed array is readily adaptable and will be made available to other researchers including non-specialists for glycan array analysis. The galectin inhibitors developed in this project will be truly off-the-shelf reagents that non-specialists can easily adapt for functional studies or therapeutic applications.