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.
All cells in nature are covered with a dense and complex array of sugar chains, and in vertebrates the outermost ends of the branches on this forest are capped with sugars called sialic acids, which have a lot of intrinsic complexity. Current methods to study this extremely important and dynamic aspect of the glycan forest are far too difficult for the average scientist to employ, and many aspects of this important aspect of biology are thus poorly studied. We will take advantage of the fact that numerous proteins (especially from microbes) have naturally evolved to recognize this complexity of sialic acids on cells with great specificity, harnessing such proteins to generate a simple and reliable toolkit that can be used to easily monitor dynamic changes of the sialic acids in normal and abnormal states.
Showing the most recent 10 out of 11 publications
