Designing CXCL8-based decoy proteins with strong anti-inflammatory activity in vivo
| Autor: | Elena Geretti, Heide Potzinger, Andreas J. Kungl, Angelika Falsone, Veronica Wabitsch, Tanja Gerlza, Mauro M. Teixeira, James Robinson, Tiziana Adage |
|---|---|
| Rok vydání: | 2013 |
| Předmět: |
Male
HBSS Hanks balanced salt solution Protein Denaturation Anti-Inflammatory Agents Drug Evaluation Preclinical MPO myeloperoxidase RA rheumatoid arthritis Plasma protein binding GAG glycosaminoglycan Biochemistry Receptors Interleukin-8A Arthritis Rheumatoid Mice KC keratinocyte chemoattractant heparan sulphate CXC chemokine receptors Receptor IFT isothermal fluorescence titration anti-inflammatory CXCR CXC chemokine receptor CCL CC chemokine ligand Ligand (biochemistry) HS heparan sulphate Decoy competition Kd dissociation constant MIP macrophage inflammatory protein CS chondroitin sulphate Protein Binding musculoskeletal diseases Biophysics SPR surface plasmon resonance Biology TFA trifluoroacetic acid UFMG Universidade Federal de Minas Gerais Animals Humans Interleukin 8 Binding site Molecular Biology Guanidine GPCR G-protein-coupled receptor G protein-coupled receptor Original Paper AIA antigen induced arthritis Binding Sites SP sulphopropyl Interleukin-8 chemokine decoys GAG affinity TMB tetramethylbenzidine Cell Biology mBSA methylated BSA WT wild-type CXCL CXC chemokine ligand IL interleukin LMWH low molecular weight heparin Mice Inbred C57BL Amino Acid Substitution Drug Design Mutagenesis Site-Directed Cattle Heparitin Sulfate |
| Zdroj: | Bioscience Reports |
| ISSN: | 1573-4935 0144-8463 |
| DOI: | 10.1042/bsr20130069 |
| Popis: | IL (interleukin)-8 [CXCL8 (CXC chemokine ligand 8)] exerts its role in inflammation by triggering neutrophils via its specific GPCRs (G-protein-coupled receptors), CXCR1 (CXC chemokine receptor 1) and CXCR2, for which additional binding to endothelial HS-GAGs (heparan sulphate-glycosaminoglycans) is required. We present here a novel approach for blocking the CXCL8-related inflammatory cascade by generating dominant-negative CXCL8 mutants with improved GAG-binding affinity and knocked-out CXCR1/CXCR2 activity. These non-signalling CXCL8 decoy proteins are able to displace WT (wild-type) CXCL8 and to prevent CXCR1/CXCR2 signalling thereby interfering with the inflammatory response. We have designed 14 CXCL8 mutants that we subdivided into three classes according to number and site of mutations. The decoys were characterized by IFTs (isothermal fluorescence titrations) and SPR (surface plasmon resonance) to determine GAG affinity. Protein stability and structural changes were evaluated by far-UV CD spectroscopy and knocked-out GPCR response was shown by Boyden chamber and Ca2+ release assays. From these experiments, CXCL8(Δ6F17KF21KE70KN71K) emerged with the most promising in vitro characteristics. This mutant was therefore further investigated in a murine model of mBSA (methylated BSA)-induced arthritis in mice where it showed strong anti-inflammatory activity. Based on these results, we propose that dominant-negative CXCL8 decoy proteins are a promising class of novel biopharmaceuticals with high therapeutic potential in inflammatory diseases. |
| Databáze: | OpenAIRE |
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