Novel Reagents for Monitoring Sulfation Patterns in Heparin and Heparan Sulfate

项目概要 寡糖和多糖参与基本的分子相互作用及其在细胞上的位置 表面使它们成为各种疾病状态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，参与多种细胞信号传导事件 有利的还是致病的，取决于多糖链上的硫酸化模式。 高亲和力结合探针（例如抗体）是识别分子相互作用的有效手段 细胞内；然而，很少有试剂具有足够的特异性来原位监测 H2S 的硫酸化模式。 该项目将开发一类新型试剂，能够识别特定的硫酸化模式 通过利用酶（磺基转移酶）的固有特异性来修饰 HS 链，这些酶负责 在 HS 生物合成过程中将硫酸基转移到多糖上。将创建蛋白质支架 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 HS 的两个特定序列。 该项目将重点关注磺基转移酶 HS 2-O-磺基转移酶 (HS2ST) 和 3-O-磺基转移酶 同工型 1 (HS3ST-1)；然而，生物合成酶有多种同工型 HS 的途径，可以作为未来研究的目标。两种酶都与具有生物学意义的相互作用 硫酸盐化模式。例如，HS2ST 与多种功能有关，包括血管生成、 轴突引导和前列腺癌。 HS3ST-1酶直接负责抗凝 通过增加 HS 与抗凝血酶的亲和力来发挥肝素的特性。这些试剂将极大地 可用于通过典型的抗体介导的测定（包括亲和力）追踪硫酸化模式的变化 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.