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 #
5P01HL107152-03
Application #
8516577
Study Section
Special Emphasis Panel (ZHL1-CSR-H)
Project Start
Project End
Budget Start
2013-06-01
Budget End
2014-05-31
Support Year
3
Fiscal Year
2013
Total Cost
$410,183
Indirect Cost
Name
Virginia Commonwealth University
Department
Type
DUNS #
105300446
City
Richmond
State
VA
Country
United States
Zip Code
23298
Ezzelarab, Mohamed B; Ekser, Burcin; Azimzadeh, Agnes et al. (2015) Systemic inflammation in xenograft recipients precedes activation of coagulation. Xenotransplantation 22:32-47
Sepuru, Krishna Mohan; Poluri, Krishna Mohan; Rajarathnam, Krishna (2014) Solution structure of CXCL5--a novel chemokine and adipokine implicated in inflammation and obesity. PLoS One 9:e93228
Mehta, Akul Y; Thakkar, Jay N; Mohammed, Bassem M et al. (2014) Targeting the GPIb? binding site of thrombin to simultaneously induce dual anticoagulant and antiplatelet effects. J Med Chem 57:3030-9
Bane Jr, Charles E; Gailani, David (2014) Factor XI as a target for antithrombotic therapy. Drug Discov Today 19:1454-8
Kalita, Mausam; Balivada, Sivasai; Swarup, Vimal Paritosh et al. (2014) A nanosensor for ultrasensitive detection of oversulfated chondroitin sulfate contaminant in heparin. J Am Chem Soc 136:554-7
Rajarathnam, Krishna; Rosgen, Jorg (2014) Isothermal titration calorimetry of membrane proteins - progress and challenges. Biochim Biophys Acta 1838:69-77
Tran, Vy M; Kuberan, Balagurunathan (2014) Synthesis of fluorophore-tagged xylosides that prime glycosaminoglycan chains. Bioconjug Chem 25:262-8
Safo, Martin K; Musayev, Faik N; Mosier, Philip D et al. (2014) Crystal structures of influenza A virus matrix protein M1: variations on a theme. PLoS One 9:e109510
Al-Horani, Rami A; Desai, Umesh R (2014) Designing allosteric inhibitors of factor XIa. Lessons from the interactions of sulfated pentagalloylglucopyranosides. J Med Chem 57:4805-18
Al-Horani, Rami A; Desai, Umesh R (2014) Recent advances on plasmin inhibitors for the treatment of fibrinolysis-related disorders. Med Res Rev 34:1168-216

Showing the most recent 10 out of 50 publications