Sialoglycan-Recognizing Probes for Defining Sialoglycomes in Biological Systems

 描述(申请人提供)：细胞表面和分泌分子上的葡聚糖在不同的生物过程中起着关键作用。由于糖链的复杂性和我们分析不同形式的糖链表达能力的技术限制，功能研究一直受到阻碍。特别是，脊椎动物糖类的一个高度动态的方面在很大程度上仍然难以全面分析：多种多样的末端唾液酸(SIAs)，以及它们与底层多糖(构成唾液糖的唾液多聚糖)的联系。目前的糖化方法破坏和/或错过唾液酸的某些方面，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，需要的仪器和专业知识仅对少数人可用，无法在天然状态下阐明完整的唾液酸苷，阻止唾液酸苷的不同成分在细胞或组织内或组织内定位。因此，非专家倾向于避免研究产生的生物学问题。我们将开发一种系统的方法来原位识别、跟踪、操纵和分析唾液聚糖及其功能。我们将收集、整理和优化一套定义良好的重组可溶性稳定标记唾液聚糖识别探针(SGRP)，这些探针最终可以作为非专家使用的实用工具。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、无脊椎动物和植物凝集素的子集，以及某些Siglecs和单抗--所有这些都需要在其结合表位上进行特定的Sia修饰和/或连接。我们不会在第一轮研究中对SGRP结合位点进行定向突变，因为通过自然选择(例如，在宿主病原体界面)进行的生物进化已经磨练了天然SGRP的特异性。还将探索针对某些唾液酸聚糖的其他鸡肉单抗。每个稳定的探针将在显示自然多样性的主要方面的唾液聚糖阵列上进行特异性研究。同时，我们将扩大阵列上唾液酸聚糖的多样性。最好的探针子集将在带有遗传诱导的唾液聚糖变化的小鼠身上进行测试，包括流式细胞术、Western blotts、血清ELISA和组织免疫组织化学分析。将探索条件和控制的优化，包括突变的非活性探针和/或用特定的唾液酸酶、酯酶或SIA侧链的轻度高碘酸氧化进行预处理。目标是定义一组可供任何生物科学研究人员使用的SGRP，他们可以在生物样本中描绘完整唾液酸糖的多样性。最终的结果(将被交叉验证和进一步优化)是一个简单而可靠的工具包，用于跟踪生物样本中的唾液酸苷。这项工作将同时获得对产生探测器的研究人员以及那些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.