Novel Reagents for Monitoring Sulfation Patterns in Heparin and Heparan Sulfate

项目摘要 寡糖和多糖参与基本的分子相互作用，以及它们在细胞上的位置 表面使它们成为各种疾病状态的生物标志物的理想候选者。硫酸乙酰肝素（HS）是一种 高度硫酸化的线性多糖，其参与细胞信号传导事件的分类， 根据多糖链沿着的硫酸化模式，是有利的还是致病的。 高亲和力结合探针，如抗体，是识别分子相互作用的有效手段 然而，很少有试剂具有足够的特异性，可用于原位监测HS的硫酸化模式。 该项目将开发一类新的试剂，能够识别特定的硫酸化模式， HS链通过利用酶（磺基转移酶）的固有特异性， 在HS生物合成期间将硫酸酯基团转移到多糖。蛋白质支架将被创造出来 通过催化灭活磺基转移酶，支架的亲和力将通过 计算引导的单点突变，以及定向进化。通过选择性地瞄准 酶促反应的底物或产物，每种无活性的磺基转移酶都有可能结合到 HS的两个特定序列。 本项目将重点关注磺基转移酶HS 2-O-磺基转移酶（HS 2ST）和3-O-磺基转移酶 异构体1（HS 3ST-1）;然而，存在来自生物合成的酶的各种酶的许多异构体。 HS的途径，可以在未来的研究目标。这两种酶都与生物学意义上的 硫酸化模式例如，HS 2ST涉及多种功能，包括血管生成， 轴突导向和前列腺癌。HS 3ST-1酶直接负责抗凝剂 肝素的性质，通过增加HS的亲和力抗凝血酶。这些试剂将极大地 用于通过典型的抗体介导的测定，包括亲和性测定， 纯化、蛋白质印迹、原位组织学染色和体内成像。

PROJECT SUMMARY Oligo- and polysaccharides participate in fundamental molecular interactions, and their location on cell surfaces makes them ideal candidates as biomarkers for various disease states. Heparan sulfate (HS) is a highly sulfated linear polysaccharide that participates in an assortment of cellular signaling events that are either advantageous or pathogenic, depending upon the sulfation pattern along the polysaccharide chain. High-affinity binding probes, such as antibodies, are an effective means for discerning molecular interactions within cells; however, few reagents are sufficiently specific for monitoring the sulfation patterns of HS in situ. This project will develop a new class of reagents that is capable of recognizing specific sulfation patterns within the HS chain by exploiting the inherent specificity of enzymes (sulfotransferases) that are responsible for transferring the sulfate groups to the polysaccharide during HS biosynthesis. Protein scaffolds will be created by catalytically inactivating the sulfotransferases, and the affinity of the scaffolds will be enhanced via computationally-guided single point mutations, as well as by directed evolution. By selectively targeting either the substrate or product of the enzymatic reaction, each inactive sulfotransferase has the potential to bind to two specific sequences of HS. This project will focus on sulfotransferases HS 2-O-Sulfotransferase (HS2ST) and 3-O-Sulfotransferase Isoform 1 (HS3ST-1); however, there are numerous isoforms of various enzymes from the biosynthetic pathway of HS that could be targeted in future studies. Both enzymes interact with biologically significant sulfation patterns. For example, HS2ST has been implicated in a variety of functions including angiogenesis, axon guidance, and prostate cancer. The HS3ST-1 enzyme is directly responsible for the anticoagulant properties of heparin, by increasing the affinity of HS for antithrombin. These reagents would be immensely useful for tracking changes in sulfation patterns via typical antibody-mediated assays, including affinity purification, western blotting, in situ histological staining, and in vivo imaging.