Identifizierung und Struktur/Funktionsanalyse der Protein-Nitrierung in eosinophilen Granulozyten und in chronisch entzündetem murinem und humanem Lungengewebe durch hochauflösende Proteomanalyse
Final Report Abstract
In this DFG-sponsored project we have shown that 3NT positive EPO and lysozyme are present in the majority of induced sputum specimens of patients with CF and asthma bronchiale. LC-MS/MS analysis revealed that lysozyme, carries 3NT modification in the surface exposed tyrosine residues Tyr 20 and Tyr54. This analysis does not reveal the exact percentage of 3NT modified lysozyme, an abundant cytosolic protein derived from activated neutrophils. Neutrophils are the dominant cell type in infected airways of CF patients and in a subpopulation of patients with asthma bronchiale. Our in vitro experiments revealed that 3NT unmodified lysozyme is present in resting phagocytes, suggesting that the protein becomes 3NT positive after cell secretion during inflammation in patients with CF and asthma bronchiale. Our analysis provided also preliminary information that other 3NT positive proteins are present in these specimens. Several mechanisms involving the formation of peroxinitrite or peroxidases in combination with reactive oxygen and nitrogen species may lead to 3NT modified lysozyme formation in the inflamed airways of the two patient groups. High levels of nitrites have been measured in induced sputum of CF patients and reactive oxygen species are thought to be produced by activated neutrophils at the air/sputum interface, yielding superoxide anion radical and hydrogen peroxide. Furthermore, eosinophils and their secreted granule proteins such as ECP, EPX and MBP have been detected in high concentrations in sputum samples of CF patients and neutrophil elastase, which is abundantly present in CF sputum, has been shown to degranulate eosinophils. Here we also show that EPO is detectable in most of the CF sputum samples and respective samples of patients with asthma bronchiale. In asthma, EPO has been shown to produce 3NT. Furthermore, in a previous mouse model of allergic asthma, EPO was made responsible for 3NT protein modifications. As demonstrated here, both mechanisms modify the same tyrosine residues in lysozyme. Thus, both mechanisms may or may not be active in vivo. The fact that in sputum specimens of 5 patients with asthma bronchiale only 0.44% eosinophils were present, suggests that 3NT modified lysozyme may be derived from a mechanism involving peroxinitrite than from an EPO-mediated mechanism. Unfortunately, we were unable to distinguish between the two mechanisms in an ovalbumin challenge mouse model. In this project we carried out a number of different in vitro tests to understand whether EPO would be in the vicinity of lysozyme during neutrophil activation. Due to its cationic character lysozyme readily binds to negatively charged DNA and consequently we have detected this protein complex on extracellular DNA scaffolds. Because extracellular DNA scaffolds also adsorb the NADPH-oxidase complex and MPO, and neutrophils have been shown to cause nitrosylation mediated via MPO in the presence of nitrite, we tested whether activated neutrophils would induce 3NT modification in lysozyme. Here we show that EPO may physically interact with released neutrophil granule proteins on extracellular DNA scaffolds and may induce 3NT modification at specific tyrosine residues in target proteins such as lysozyme at least in vitro. Lysozyme has been shown to function as antimicrobial compound in innate immunity. It is bactericidal against Gram-negative and Gram-positive bacteria. Here, we confirmed the bactericidal activity of lysozyme using the model bacterium M. lysodeicticus and demonstrated that 3NT modification rendered the compound less active.
