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.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Program Projects (P01)
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Virginia Commonwealth University
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Verespy 3rd, Stephen; Mehta, Akul Y; Afosah, Daniel et al. (2016) Allosteric Partial Inhibition of Monomeric Proteases. Sulfated Coumarins Induce Regulation, not just Inhibition, of Thrombin. Sci Rep 6:24043
Wood, Jeremy P; Baumann Kreuziger, Lisa M; Desai, Umesh R et al. (2016) Blocking inhibition of prothrombinase by tissue factor pathway inhibitor alpha: a procoagulant property of heparins. Br J Haematol 175:123-32
Holman, Holly A; Tran, Vy M; Kalita, Mausam et al. (2016) BODIPY-Conjugated Xyloside Primes Fluorescent Glycosaminoglycans in the Inner Ear of Opsanus tau. J Assoc Res Otolaryngol 17:525-540
Iwase, Hayato; Ekser, Burcin; Hara, Hidetaka et al. (2016) Thyroid hormone: relevance to xenotransplantation. Xenotransplantation 23:293-9
Mosier, Philip D; Chiang, Meng-Jung; Lin, Zhengshi et al. (2016) Broad Spectrum Anti-Influenza Agents by Inhibiting Self-Association of Matrix Protein 1. Sci Rep 6:32340
Sawant, Kirti V; Poluri, Krishna Mohan; Dutta, Amit K et al. (2016) Chemokine CXCL1 mediated neutrophil recruitment: Role of glycosaminoglycan interactions. Sci Rep 6:33123
Sepuru, Krishna Mohan; Rajarathnam, Krishna (2016) CXCL1/MGSA Is a Novel Glycosaminoglycan (GAG)-binding Chemokine: STRUCTURAL EVIDENCE FOR TWO DISTINCT NON-OVERLAPPING BINDING DOMAINS. J Biol Chem 291:4247-55
Mitsuhashi, Shuji; Feldbrügge, Linda; Csizmadia, Eva et al. (2016) Luminal Extracellular Vesicles (EVs) in Inflammatory Bowel Disease (IBD) Exhibit Proinflammatory Effects on Epithelial Cells and Macrophages. Inflamm Bowel Dis 22:1587-95
Sepuru, Krishna Mohan; Nagarajan, Balaji; Desai, Umesh R et al. (2016) Molecular Basis of Chemokine CXCL5-Glycosaminoglycan Interactions. J Biol Chem 291:20539-50
Mehta, A Y; Mohammed, B M; Martin, E J et al. (2016) Allosterism-based simultaneous, dual anticoagulant and antiplatelet action: allosteric inhibitor targeting the glycoprotein Ibα-binding and heparin-binding site of thrombin. J Thromb Haemost 14:828-38

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