The glycosaminoglycan (GAG) heparan sulfate (HS) plays a critical role in chemokine-mediated neutrophil recruitment and activation in the pathophysiology of a wide variety of inflammatory diseases. All chemokines exist reversibly as monomers and dimers, but remarkably very little is known regarding the molecular mechanisms and structural basis by which chemokine monomers and dimers bind GAGs, and how these interactions mediate in vivo function. Three major bottlenecks have stymied efforts to obtain this knowledge - i) heterogeneity due to chemokine monomers and dimers, 2) the complex diversity of naturally occurring GAGs, and 3) limitations to NMR and X-ray methods. In Project III, we vsdll develop methods to overcome these bottlenecks, and characterize the structural/molecular basis of HS binding for three neutrophil-activating chemokines: human IL-8 and NAP-2, and mouse KC. We will use this knowledge to design GAG/chemokine decoys and test their efficacy in various animal inflammation and xenograft models. Our Central Hypothesis is that differences in neutrophil recruitment must be due to differential GAG interactions, that chemokines'ability to exist as monomers and dimers in solution and in GAG-bound forms are coupled and tightly regulated, and that dysregulation in this process is directly responsible for the observed clinical symptoms. This hjrpothesis v^ll be tested by pursuing three Specific Aims, to: 1) characterize the molecular properties of HS binding to chemokine monomers and dimers;2) determine the solution structures of HS-bound chemokine monomers and dimers;and 3) design and test GAG and chemokine decoys that should inhibit neutrophil recruitment in mouse inflammation models and in various xenograft-related assays and animal models (Project IV).
These Aims will be accomplished via 3 approaches: Strategy 1 - Using protein engineering methods, design and synthesize trapped chemokine monomers and dimers. Strategy 2 - Chemoenzymatic synthesis of size-defined, chemically homogeneous GAG. PL-I, who is an expert in this methodology, will synthesize the GAGs, including uniform and selectively labeled (first of their kind) ^^N and ^^C-GAGs that are critical for solution NMR structural studies. Strategy 3 -NMR structure determination using data from chemical shift perturbation, paramagnetic relaxation enhancement (PRE), residual dipolar coupling (RDC), ^^N-relaxation, and intermolecular NOE experiments. Novel methods include using selective ^^C-labeled GAG for RDC and spin-labeled GAG for PRE experiments.

Public Health Relevance

Major achievements from this work will be two fold - (1) an understanding of the basic structural/molecular principles by which GAGs bind chemokine monomers and dimers, and (2) identification of GAG-based inhibitors for chemokine-mediated inflammatory diseases.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Program Projects (P01)
Project #
5P01HL107152-04
Application #
8669116
Study Section
Special Emphasis Panel (ZHL1-CSR-H)
Project Start
Project End
Budget Start
2014-06-01
Budget End
2015-05-31
Support Year
4
Fiscal Year
2014
Total Cost
$411,812
Indirect Cost
Name
Virginia Commonwealth University
Department
Type
DUNS #
105300446
City
Richmond
State
VA
Country
United States
Zip Code
23298
Samudra, Anushka N; Dwyer, Karen M; Selan, Carly et al. (2018) CD39 and CD73 activity are protective in a mouse model of antiphospholipid antibody-induced miscarriages. J Autoimmun 88:131-138
Kummarapurugu, Apparao B; Afosah, Daniel K; Sankaranarayanan, Nehru Viji et al. (2018) Molecular principles for heparin oligosaccharide-based inhibition of neutrophil elastase in cystic fibrosis. J Biol Chem 293:12480-12490
Sankaranarayanan, Nehru Viji; Nagarajan, Balaji; Desai, Umesh R (2018) So you think computational approaches to understanding glycosaminoglycan-protein interactions are too dry and too rigid? Think again! Curr Opin Struct Biol 50:91-100
Jiang, Z Gordon; Sandhu, Bynvant; Feldbrügge, Linda et al. (2018) Serum Activity of Macrophage-Derived Adenosine Deaminase 2 Is Associated With Liver Fibrosis in Nonalcoholic Fatty Liver Disease. Clin Gastroenterol Hepatol 16:1170-1172
Xie, Anyan; Robles, René J; Mukherjee, Samiran et al. (2018) HIF-1?-induced xenobiotic transporters promote Th17 responses in Crohn's disease. J Autoimmun 94:122-133
Owings, Katie G; Lowry, Joshua B; Bi, Yiling et al. (2018) Transcriptome and functional analysis in a Drosophila model of NGLY1 deficiency provides insight into therapeutic approaches. Hum Mol Genet 27:1055-1066
Afosah, Daniel K; Verespy 3rd, Stephen; Al-Horani, Rami A et al. (2018) A small group of sulfated benzofurans induces steady-state submaximal inhibition of thrombin. Bioorg Med Chem Lett 28:1101-1105
Periasamy, Srinivasan; Lin, Chia-Hui; Nagarajan, Balaji et al. (2018) Mucoadhesive role of tamarind xyloglucan on inflammation attenuates ulcerative colitis. J Funct Foods 47:1-10
Joseph, Prem Raj B; Sawant, Kirti V; Iwahara, Junji et al. (2018) Lysines and Arginines play non-redundant roles in mediating chemokine-glycosaminoglycan interactions. Sci Rep 8:12289
Kishore, Bellamkonda K; Robson, Simon C; Dwyer, Karen M (2018) CD39-adenosinergic axis in renal pathophysiology and therapeutics. Purinergic Signal 14:109-120

Showing the most recent 10 out of 151 publications