2-chlorofatty acid modification of neutrophil proteins: identification, localization and role in NETosis

We previously demonstrated neutrophil MPO derived HOCl targets the vinyl ether bond of plasmalogens resulting in the Liberation of 2-chlorofatty aldehydes (2-ClFALDs) and their oxidation products, 2-chlorofatty acids (2-ClFAs), which elicit neutrophil extracellular trap (NET) formation. In this study, the click chemistry analog of 2-chlorohexadecanoic acid (2-ClHA) was utilized to identify 127 proteins covalently modified by 2-ClHA in human neutrophils. Bioinformatics revealed that multiple proteins modified by 2-ClHA are related to protein modification and binding as well as metabolite interconversion. Three key proteins involved in NET formation and function were modified by 2-ClHA including peptidyl arginine deiminase 4 (PAD4), neutrophil defensin alpha 3 (DEFA3), and neutrophil collagenase (MMP8). PAD4 activity was shown to be increased by 2-ClFA treatment. Further studies investigated 2-ClFA modified protein localization over time during NET formation. Initially PAD4 and 2-ClFA-modified proteins were extranuclear but over time they both localized to distinct nuclear regions. Following DNA release from neutrophils, 2-ClFA-modified proteins were found throughout the neutrophil and DNA strands. In summary, multiple neutrophil proteins are modified by 2-ClHA, including PAD4. 2-ClHA modification and activation of PAD4 is suggested as a key component of 2-ClHA elicited NET formation.