A Fluorescence-Based High-Throughput Platform for Glycotyping the Hematopoietic Cell Lineage

PI：Buelow - Steidl 项目摘要 在多细胞生物中，细胞的表面特征是其功能的直接反映。 用于检测和定义细胞的新表面特征的灵敏方法为以下方面提供了巨大的机会： 获得新的机制见解并开发新的治疗策略。硫酸乙酰肝素（HS）聚糖是 普遍存在于后生动物的细胞表面，并以非凡的时间和空间表达 分辨率由于HS是自然界中最多样化的分子之一， 聚糖的模式，它们可能提供强烈区分不同细胞的方法， 和组织类型。然而，目前用于HS聚糖结构测定的分析方法是昂贵的， 技术上具有挑战性，而且往往分辨率较低，限制了它们的歧视能力和广泛使用。的 开发新的工具来定义和利用细胞表面HS的结构特征 可以开辟新的途径，具有重要的诊断和治疗潜力。我们将建立一个高- 通量，基于多重荧光的平台，使用一组 HS特异性单链可变片段（scFv）抗体。我们将首先设计一套HS专用的 单链可变片段抗体与各种免疫标签，以及直接标记它们与 使用基于分选酶的方法的荧光标记。接下来，我们将系统地确定HS基序， 使用微阵列与确定的硫酸乙酰肝素的组合， 寡糖和新的计算工具，我们将开发定义HS基序识别 这些试剂。最后，在原理实验的证明中，我们将应用我们的免疫试剂工具箱， 将HS基序定义为成熟的分化簇（CD）系统的正交系统， 表征和定义造血细胞谱系。我们将利用我们实验室的独特能力， 蛋白质生产和造血研究，以产生，测试和传播荧光标记的 scFvs试剂和研究方案。我们预计，我们的强大，高效， 经济的分析将导致更广泛和标准化的HS特征分析，这将 为开发血液和其他疾病的新诊断和治疗方法奠定基础 系统在未来。

PIs: Buelow – Steidl Project Summary The surface characteristics of cells in multicellular organisms are a direct reflection of their function. Sensitive methods for detecting and defining novel surface characteristics of cells offer great opportunities for gaining new mechanistic insights and developing new therapeutic strategies. Heparan sulfate (HS) glycans are ubiquitous on the cell surface of metazoans and are expressed with extraordinary temporal and spatial resolution. Since HS are amongst the most diverse molecules in nature, as a result of complex modification patterns of the glycans, they could potentially provide the means to strongly discriminate between different cell and tissue types. However, current analytical methods for HS glycan structure determination are expensive, technically challenging and often low resolution, limiting their discriminatory power and widespread use. The development of novel tools to define and exploit the structural characteristics of HS on the surface of cells could open new avenues with important diagnostic and, possibly, therapeutic potential. We will develop a high- throughput, multiplexed fluorescence-based platform to define the HS surface characteristics using a set of HS-specific single chain variable fragment (scFvs) antibodies. We will first engineer the set of HS-specific single-chain variable fragment antibodies with various immunotags as well as directly label them with fluorescent tags using Sortase based approaches. Next, we will systematically determine the HS motifs that are recognized by the scFv antibodies using a combination of microarrays with defined heparan sulfate oligosaccharides and novel computational tools, which we will develop to define the HS motifs recognized by these reagents. Finally, in proof of principle experiments, we will apply our tool box of immuno reagents against defined HS motifs as an orthogonal system to the well-established cluster of differentiation (CD) system to characterize and define hematopoietic cell lineages. We will leverage our laboratories' unique capabilities in protein production and hematopoiesis research to generate, test, and disseminate the fluorescently labeled scFvs reagents and protocols to the research community. We anticipate that our robust, efficient and economical assays will lead to more widespread and standardized analyses of HS characteristics, which will lay the foundation for the development of novel diagnostic and therapeutic approaches in the blood and other systems in the future.