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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：
9300880
负责人：
AJIT P VARKI
金额：
$ 61.86万
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 2019-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9300880
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 Histocytochemistry In Situ Inflammation Invertebrates Knockout Mice Knowledge Laboratories Methods Microbe Modification Monitor Monoclonal Antibodies Mus Mutagenesis Natural Selections Nature Neuraminidase Outcome Pathologic Phase Plant Lectins Play Polysaccharides Proteins Quality Control Reagent Recombinants Research Personnel Resources Role Sampling Scientist Serum Sialic Acids Sialoglycoproteins Side Slide Specificity Speed Standardization 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 public health relevance 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.