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 structures that mediate particular interactions have been defined in only a few cases due to the structural complexity of HS. Studies in Technology Research and Development project 3 (TR&D3) are focused on biochemical and genetic approaches to assess proteoglycan (PG) functions through the use of cell-based systems for recombinant target protein and PG expression, biochemical characterization, and cell-based assays for biological function. The technologies are based on platforms for large-scale recombinant PG binding protein expression in mammalian cells for enrichment of GAG ligand structures in TR&D1, structural studies in TR&D2, and for cell-based assays in TR&D3. Interactions with PGs in vivo can be more complex, since natural GAGs are much larger than simple oligosaccharides; they may interact simultaneously with both ligands and their receptors via distinct binding sites, and may also harbor multiple copies of these binding sites. Recombinant production of secreted PGs containing engineered GAG chains for use in biochemical and structural studies and cell-based assays are proposed to extend and validate biochemical findings in a cellular context. Mouse lung endothelial cell (MLEC) lines are being developed harboring gene disruptions in selected steps in heparin sulfate (HS) biosynthesis or modification for structure-activity relationship (SAR) studies to examine cell-based functions of PG core proteins harboring altered GAG chains.
Specific Aims are proposed to:
Aim 1. Express and characterize PG binding proteins as DBP targets for biochemical studies on GAG-protein interactions by epitope enrichment, NMR structural analysis, and high-resolution hydroxyl radical footprinting. PG core proteins harboring engineered GAG chains are also being developed for biochemical, structural, and cell-based studies on biological function.
In Aim 2, development of mutant MLEC lines, cell-based arrays, and PG arrays are proposed for biological SAR studies including development of additional mutant MLEC lines deficient in GAG extension and sulfation, development of HS/CS mutant MLEC-based cell arrays for biological SAR studies, development of PG arrays harboring altered HS structures for SAR studies, and development of competitive cell arrays using structurally defined HS or CS with wild type MLEC cells to examine SARs of HS and CS in ligand interactions and cellular responses.
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