Sialoglycan-Recognizing Probes for Defining Sialoglycomes in Biological Systems

 描述（由申请方提供）：细胞表面的聚糖和分泌分子在多种生物过程中发挥关键作用。功能研究受到聚糖库的复杂性和我们分析不同聚糖形式表达能力的技术限制的阻碍。特别是，脊椎动物糖组的一个高度动态的方面在很大程度上仍然难以全面分析：末端唾液酸（Sias）的多样性阵列，以及它们与基础聚糖（唾液酸聚糖，构成唾液酸糖组）的连接。目前的糖组学方法破坏和/或错过了唾液酸组的某些方面，并且折衷方案是释放Sias并单独研究它们的多样性。然而，这种方法也破坏了重要的信息，需要仅对少数人可用的仪器和专业知识，不能阐明天然状态下的完整唾液酸组，并且阻止了唾液酸组的不同组分在细胞或组织内或上的定位。因此，非专家往往避免研究所产生的生物学问题。我们将开发一个系统的方法，在原位识别，跟踪，操纵和分析唾液酸聚糖及其功能。我们将收集，策划和优化一组定义明确的重组可溶性稳定标记的唾液酸聚糖识别探针（SGRP），最终可以作为非专家的实用工具。待探索的SGRP包括细菌SRR粘附素、细菌B5毒素、病毒血凝素、病毒血凝素-酯酶、噬菌体蛋白和无脊椎动物和植物凝集素的子集，以及某些Siglecs和单克隆抗体-所有这些都需要在其结合表位中进行特异性Sia修饰和/或连接。在第一轮研究中，我们将不追求SGRP结合位点的定向诱变，因为通过自然选择的生物进化（例如，在宿主病原体界面）已经磨练了天然SGRP的特异性。将探索对某些唾液酸聚糖具有特异性的其他鸡单克隆抗体。每个稳定的探针将被研究的唾液酸聚糖阵列显示的自然多样性的主要方面的特异性。同时，我们将扩大阵列上唾液酸聚糖的多样性。最好的探针子集将使用具有遗传诱导的唾液酸聚糖变化的小鼠在流式细胞术、蛋白质印迹、血清ELISA和组织免疫组织化学分析中进行测试。将探索条件和对照的优化，包括突变体失活探针和/或用特异性唾液酸酶、酯酶或Sia侧链的轻度高碘酸盐氧化预处理。我们的目标是定义一组SGRP，可以提供给任何生物科学研究人员，谁可以在生物样品中的完整sialoglycome的多样性。最终的结果（将进行交叉验证和进一步优化）是一个简单可靠的工具包，以跟踪生物样品中的唾液酸。这项工作将同时获得对生成探针的研究人员以及研究它们起源的生物系统的人有价值的基本知识。

 DESCRIPTION (provided by applicant): Glycans on cell surface and secreted molecules play crucial roles in diverse biological processes. Functional studies have been hampered by the complexity of the glycan repertoire and by technical limitations in our ability to analyze expression of different glycan forms. In particular, one highly dynamic aspect of the vertebrate glycome has remained largely intractable to full analysis: the diverse array of terminal sialic acids (Sias), and their linkages to underlying glycans (sialoglycans, which constitute the sialoglycome). Current glycomic methods destroy and/or miss certain aspects of the sialoglycome, and the compromise is to release Sias and separately study their diversity. However, this approach also destroys important information, requires instrumentation and expertise only available to a few, fails to elucidate the intact sialoglycome in a native state, an prevents localization of the different components of the sialoglycome within or on cells or tissues. Thus, non-experts tend to avoid studying the biological questions arising. We will develop a systematic approach for in situ identification, tracking, manipulation, and analysis of sialoglycans and their functions. We will collect, curate and optimize a well-defined set of recombinant soluble stable tagged sialoglycan recognizing probes (SGRPs), which can eventually be used as practical tools by non-experts. SGRPs to be explored include a subset of bacterial SRR adhesins, bacterial B5 toxins, viral hemagglutinins, viral hemagglutinin-esterases, phage proteins, and invertebrate and plant lectins, as well as certain Siglecs and monoclonal antibodies--all with requirements for specific Sia modifications and/or linkages in their binding epitope. We will not pursue directed mutagenesis of SGRP binding sites in the first round of studies, as biological evolution via natural selection (e.g., at the hostpathogen interface) has already honed specificities of natural SGRPs. Additional chicken monoclonal antibodies with specificity for certain sialoglycans would be explored. Each stable probe would be studied for specificity on a sialoglycan array displaying major aspects of natural diversity. In parallel we would expand the diversity of sialoglycans on the array. The best subset of probes would be tested using mice with genetically induced sialoglycan changes, in flow cytometry, Western blots, ELISAs on serum, and immunohistochemical analyses of tissues. Optimization of conditions and controls will be explored, including mutant inactive probes and/or pretreatment with specific sialidases, esterases or mild periodate oxidation of the Sia side chain. The goal is to define a set of SGRPs that can be made available to any biosciences investigator, who can profile the diversity of intact sialoglycome in biological samples. The final outcome (which would be cross-validated and further optimized) is a simple and reliable toolkit to track the sialoglycome in biological samples. This work will simultaneously acquire basic knowledge valuable to investigators who generate probes, as well as to those who study the biological systems they originate from.