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.
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.
|Brown, Aaron J; Sepuru, Krishna Mohan; Sawant, Kirti V et al. (2017) Platelet-Derived Chemokine CXCL7 Dimer Preferentially Exists in the Glycosaminoglycan-Bound Form: Implications for Neutrophil-Platelet Crosstalk. Front Immunol 8:1248|
|Zhang, Haohai; Chan, Leo Li-Ying; Rice, William et al. (2017) Novel high-throughput cell-based hybridoma screening methodology using the Celigo Image Cytometer. J Immunol Methods 447:23-30|
|Iwase, Hayato; Liu, Hong; Li, Tao et al. (2017) Therapeutic regulation of systemic inflammation in xenograft recipients. Xenotransplantation 24:|
|Jiang, Z Gordon; Sandhu, Bynvant; Feldbrügge, Linda et al. (2017) Serum Activity of Macrophage-Derived Adenosine Deaminase 2 Is Associated With Liver Fibrosis in Nonalcoholic Fatty Liver Disease. Clin Gastroenterol Hepatol :|
|Brown, Aaron J; Sepuru, Krishna Mohan; Rajarathnam, Krishna (2017) Structural Basis of Native CXCL7 Monomer Binding to CXCR2 Receptor N-Domain and Glycosaminoglycan Heparin. Int J Mol Sci 18:|
|Zheng, Shuo; Kummarapurugu, Apparao B; Afosah, Daniel K et al. (2017) 2-O, 3-O Desulfated Heparin Blocks High Mobility Group Box 1 Release by Inhibition of p300 Acetyltransferase Activity. Am J Respir Cell Mol Biol 56:90-98|
|Longhi, Maria Serena; Vuerich, Marta; Kalbasi, Alireza et al. (2017) Bilirubin suppresses Th17 immunity in colitis by upregulating CD39. JCI Insight 2:|
|Iwase, Hayato; Hara, Hidetaka; Ezzelarab, Mohamed et al. (2017) Immunological and physiological observations in baboons with life-supporting genetically engineered pig kidney grafts. Xenotransplantation 24:|
|Bi, Yiling; Might, Matthew; Vankayalapati, Hariprasad et al. (2017) Repurposing of Proton Pump Inhibitors as first identified small molecule inhibitors of endo-?-N-acetylglucosaminidase (ENGase) for the treatment of NGLY1 deficiency, a rare genetic disease. Bioorg Med Chem Lett 27:2962-2966|
|Iwase, Hayato; Liu, Hong; Schmelzer, Eva et al. (2017) Transplantation of hepatocytes from genetically engineered pigs into baboons. Xenotransplantation 24:|
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