Sialoglycan-Recognizing Probes for Defining Sialoglycomes in Biological Systems

用于定义生物系统中唾液酸糖组的唾液酸聚糖识别探针

基本信息

批准号：
9118942
负责人：
AJIT P VARKI
金额：
$ 61.94万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN DIEGO
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-07-31 至 2018-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9118942
关键词：
Bacterial Adhesins Bacteriophages Binding Binding Sites Biological Biological Assay Biological Process Biological Sciences Biology Cell Extracts Cell surface Cells Chickens Companions Complex Documentation Enzyme-Linked Immunosorbent Assay Enzymes Epitopes Evolution Flow Cytometry Funding Goals Health Histocytochemistry In Situ Inflammation Invertebrates Knockout Mice Knowledge Laboratories Methods Microbe Modification Monitor Monoclonal Antibodies Mus Mutagenesis Natural Selections Nature Outcome Phase Plant Lectins Play Polysaccharides Proteins Quality Control Reagent Recombinants Research Personnel Resources Role Sampling Scientist Serum Sialic Acids Sialoglycoproteins Side Slide Specificity Speed Testing Time Tissue Extracts Tissues Toxin Training Validation Vertebrates Viral Hemagglutinins Western Blotting Work base biological systems density esterase experience forest hemagglutinin esterase instrumentation interest member metaperiodate mutant oxidation practical application prevent programs research study sialic acid binding Ig-like lectin sugar tool web site

项目摘要

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.

描述（由申请人提供）：细胞表面的聚糖和分泌的分子在多种生物过程中发挥着至关重要的作用。聚糖库的复杂性以及我们分析不同聚糖形式表达的能力的技术限制阻碍了功能研究。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进行全面分析：末端唾液酸（Sias）的多样性及其与底层聚糖的联系（唾液酸聚糖，构成唾液酸糖组）。当前的糖组学方法破坏和/或遗漏了唾液酸糖组的某些方面，折衷方案是释放 Sias 并单独研究其多样性。然而，这种方法也破坏了重要信息，仅需要仪器和专业知识。少数人无法阐明天然状态下的完整唾液酸糖组，并且阻止了唾液酸糖组的不同成分在细胞或组织内或细胞或组织上的定位。非专家往往会避免研究出现的生物学问题。我们将开发一种系统方法，用于唾液酸聚糖及其功能的原位识别、追踪、操作和分析。我们将收集、策划和优化一组明确的重组可溶性蛋白。稳定标记的唾液酸聚糖识别探针（SGRP）最终可以被非专家用作实用工具来探索，其中包括细菌 SRR 粘附素的一个子集——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，以及某些 Siglec 和单克隆抗体——所有这些都需要在其结合表位中进行特定的 Sia 修饰和/或连接，我们不会追求定向。第一轮研究中 SGRP 结合位点的诱变，因为通过自然选择的生物进化（例如，在宿主病原体界面）已经磨练出来我们将探索对某些唾液聚糖具有特异性的其他鸡单克隆抗体，以研究每个稳定探针在显示天然多样性主要方面的唾液聚糖阵列上的特异性。最好的探针子集将使用具有遗传诱导唾液聚糖变化的小鼠进行流式细胞术、蛋白质印迹、血清 ELISA 和免疫组织化学测试将探索条件和控制的优化，包括突变失活探针和/或用特定唾液酸酶、酯酶或 Sia 侧链的轻度高碘酸盐氧化进行预处理。对于任何可以分析生物样品中完整唾液酸糖组多样性的生物科学研究人员来说，最终结果（将进行交叉验证和进一步优化）是一个简单可靠的工具包。追踪生物样本中的唾液酸糖组，这项工作将同时获得对生成探针的研究人员以及研究探针来源的生物系统的研究人员有价值的基础知识。