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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Biotechnology Resource Grants (P41)
Project #
5P41GM103390-29
Application #
9414615
Study Section
Special Emphasis Panel (ZRG1)
Project Start
Project End
Budget Start
2018-02-01
Budget End
2019-01-31
Support Year
29
Fiscal Year
2018
Total Cost
Indirect Cost
Name
University of Georgia
Department
Type
DUNS #
004315578
City
Athens
State
GA
Country
United States
Zip Code
30602
Makeneni, Spandana; Thieker, David F; Woods, Robert J (2018) Applying Pose Clustering and MD Simulations To Eliminate False Positives in Molecular Docking. J Chem Inf Model 58:605-614
Abolhasani Khaje, Niloofar; Mobley, Charles K; Misra, Sandeep K et al. (2018) Variation in FPOP Measurements Is Primarily Caused by Poor Peptide Signal Intensity. J Am Soc Mass Spectrom 29:1901-1907
Sanderson, Patience; Stickney, Morgan; Leach 3rd, Franklin E et al. (2018) Heparin/heparan sulfate analysis by covalently modified reverse polarity capillary zone electrophoresis-mass spectrometry. J Chromatogr A 1545:75-83
Liu, Lin; Prudden, Anthony R; Capicciotti, Chantelle J et al. (2018) Streamlining the chemoenzymatic synthesis of complex N-glycans by a stop and go strategy. Nat Chem :
Briard, Jennie Grace; Jiang, Hao; Moremen, Kelley W et al. (2018) Cell-based glycan arrays for probing glycan-glycan binding protein interactions. Nat Commun 9:880
Muchero, Wellington; Sondreli, Kelsey L; Chen, Jin-Gui et al. (2018) Association mapping, transcriptomics, and transient expression identify candidate genes mediating plant-pathogen interactions in a tree. Proc Natl Acad Sci U S A 115:11573-11578
Tellier, L E; Krieger, J R; Brimeyer, A L et al. (2018) Localized SDF-1? Delivery Increases Pro-Healing Bone Marrow-Derived Cells in the Supraspinatus Muscle Following Severe Rotator Cuff Injury. Regen Eng Transl Med 4:92-103
Jiang, Hao; López-Aguilar, Aimé; Meng, Lu et al. (2018) Modulating Cell-Surface Receptor Signaling and Ion Channel Functions by In?Situ Glycan Editing. Angew Chem Int Ed Engl 57:967-971
Amon, Ron; Grant, Oliver C; Leviatan Ben-Arye, Shani et al. (2018) A combined computational-experimental approach to define the structural origin of antibody recognition of sialyl-Tn, a tumor-associated carbohydrate antigen. Sci Rep 8:10786
Lu, Weigang; Zong, Chengli; Chopra, Pradeep et al. (2018) Controlled Chemoenzymatic Synthesis of Heparan Sulfate Oligosaccharides. Angew Chem Int Ed Engl 57:5340-5344

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