Glycosaminoglycans (GAGs), such as heparin, heparan sulfate (HS), and chondroitin sulfate (CS), are naturally occurring polydisperse linear polysaccharides that are heavily O- and N-sulfated. The interaction between GAGs and proteins are critical for many biological processes including cell-cell and cell-matrix interactions, cell migration and proliferation, growth factor sequestration, chemokine and cytokine activation, microbial recognition and tissue morphogenesis during embryonic development. Hundreds of HS-binding proteins have been identified, but the oligosaccharide structure that mediates a particular interaction has been defined in only a few cases due to the structural complexity of HS. We will address these deficiencies by developing an integrated Technology Research and Development (TR&D1) project for ligand identification for GAG binding proteins. GAGs from various sources will be partially fragmented and the resulting oligosaccharides employed for affinity purification using proteins of interest. The structures of HS oligosaccharides that bind with high affinity will be determined by novel mass spectrometric approaches. Putative ligands and structural analogs will be obtained by a modular synthetic approach and the resulting compounds will be employed to obtain structure activity relationships by binding and cellular activation studies. Targeted subsets of high affinity ligands will be prepared in sufficient amounts for further structural and biochemical characterization by methods described in other TR&Ds in this proposal. The validity of the methodology will be examined using a number of DBPs and collaborative projects. As the technologies mature, they will be incorporated in the services provided by CCRC.
Specific aims i nclude:
Aim 1. Identification of the Structures of HS Oligosaccharides by Chemical Derivatization and LC MS/MS Analysis, including Improved methods for generation of oligosaccharide libraries from full-length GAG chains, identification of ligands for GAG-protein interactions using LC-MS/MS sequencing and screening of shotgun oligosaccharide arrays, whole heparanome sequencing, Aim 2. FTICR-MS Approaches for HS Ligand Identification using high performance MS for HS ligand identification, and Aim 3. Modular Synthesis of HS Oligosaccharides and HS Array Development using the parallel combinatorial synthesis and development of HS oligosaccharide arrays for high throughput screening.
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