112 - ROS-Mediated Modification of RhCC10 Protein for Potential Gain of Function

Abstract

The Club cell 10kDa protein is one of the most abundant proteins in the respiratory mucosa. Lowered pulmonary or systemic CC10 levels correlate with poorer outcomes in several respiratory conditions such as neonatal bronchopulmonary dysplasia (BPD), asthma, COPD, and post-lung transplant bronchiolitis obliterans syndrome (BOS). CC10 suppresses inflammation by at least two distinct mechanisms, including inhibition of phospholipase A 2 enzymes and the arachidonic acid cascade and inhibition of NF-κB signaling in respiratory epithelial cells, but CC10 is also chemically modified by inflammatory processes. Lindahl et al (1999) first reported on at least 3 distinct isoforms of CC10 in respiratory mucosal fluids in adults with significant respiratory sequelae to inhaled industrial chemical exposures. Ramsay et al (2001) reported on a CC10 isoform in TAF from premature infants who went on to develop BPD. Arias-Martinez et al (2012) extended that observation to identify up to 7 CC10 isoforms in premature infants experiencing severe RDS who developed BPD. We investigated the possibility that the CC10 isoforms resulted from chemical modification of amino acid residues mediated by reactive oxygen and/or nitrogen species (ROS or RNS) in a series of in vitro reactions using recombinant human CC10 protein (rhCC10). At least a dozen new isoforms of CC10 were generated by reaction with ROS using three different oxidants, including NaOCl, mCPBA, and neutrophil myeloperoxidase (MPO) plus H 2 O 2 . Reaction products were characterized by HPLC, isoelectric point, Western blot, and peptide mapping. Comparison of mCPBA reaction products with NaOCl and MPO-H 2 O 2 , using different reaction times and amounts of oxidizing equivalents, revealed that methionines were initially oxidized prior to other amino acids. Each human CC10 monomer contains 4 methionines, 3 of which are readily oxidized by ROS, suggesting that each CC10 homodimer can absorb at least 6 oxygen radicals, thereby protecting other proteins, cells, and tissues and augmenting its other anti-inflammatory activities. Preliminary data also suggest that ROS-modification of CC10 enhances its anti-neutrophil activity and anti-viral activity.