Abstract
The biochemical mechanisms through which eosinophils contribute to asthma pathogenesis are unclear. Here we show eosinophil peroxidase (EPO), an abundant granule protein released by activated eosinophils, contributes to characteristic asthma-related phenotypes through oxidative posttranslational modification (PTM) of proteins in asthmatic airways through a process called carbamylation. Using a combination of studies we now show EPO uses plasma levels of the pseudohalide thiocyanate (SCN-) as substrate to catalyze protein carbamylation, as monitored by PTM of protein lysine residues into Nϵ-carbamyllysine (homocitrulline), and contributes to the pathophysiological sequelae of eosinophil activation. Studies using EPO-deficient mice confirm EPO serves as a major enzymatic source for protein carbamylation during eosinophilic inflammatory models, including aeroallergen challenge. Clinical studies similarly revealed significant enrichment in carbamylation of airway proteins recovered from atopic asthmatics versus healthy controls in response to segmental allergen challenge. Protein-bound homocitrulline is shown to be co-localized with EPO within human asthmatic airways. Moreover, pathophysiologically relevant levels of carbamylated protein either incubated with cultured human airway epithelial cells in vitro, or provided as an aerosolized exposure in non-sensitized mice, induced multiple asthma-associated phenotypes including induction of mucin, Th2 cytokines, IFNγ, TGFβ, and epithelial cell apoptosis. Studies with scavenger receptor-A1 null mice reveal reduced IL-13 generation following exposure to aerosolized carbamylated protein, but no changes in other asthma-related phenotypes. In summary, EPO-mediated protein carbamylation is promoted during allergen-induced asthma exacerbation, and can both modulate immune responses and trigger a cascade of many of the inflammatory signals present in asthma.
Highlights
Asthma is a complex inflammatory disorder of the airways, typically characterized by eosinophilia, mucus hypersecretion, epithelial apoptosis, and airway reactivity
eosinophil peroxidase (EPO) Utilizes SCNϪ and H2O2 at Physiological Levels as Substrates to Carbamylate Proteins—Because we previously showed EPO catalyzes production of OCNϪ from SCNϪ and H2O2 [28], we first tested whether isolated human EPO could catalyze protein carbamylation, as monitored by proteinbound HCit, under physiological conditions of enzyme and reactants
In protein hydrolysates recovered from reaction mixtures of the complete EPO/H2O2/SCNϪ system, a new analyte was readily observed that possessed the same retention time as authentic HCit standard, as monitored using multiple parent to daughter ion transitions (e.g. m/z 190 3 173, 190 3 127, and 190 3 84) characteristic of HCit (Fig. 1A)
Summary
Eosinophil Peroxidase and Carbamylation of Proteins during Asthma tures for reactive halogenating species formed by EPO- and MPO-dependent oxidative injury in asthma and other inflammatory disorders [14, 17,18,19,20,21,22,23,24]. Leukocytes in general and activated eosinophils in particular, have the potential for inflicting oxidative modifications on proteins and lipids in asthmatic airways via numerous pathways Both EPO and MPO use the pseudo-halide thiocyante (SCNϪ) as a preferred co-substrate with H2O2 [28, 29]. Because our prior in vitro studies revealed EPO preferentially utilizes SCNϪ as substrate, and is even more efficient at producing OCNϪ than MPO [28], we hypothesized that under eosinophilic inflammatory conditions, such as occurs within asthmatic airways, EPO might promote protein carbamylation and accompanying adverse proinflammatory effects. We demonstrate for the first time that EPO-catalyzed protein carbamylation occurs during allergen-triggered asthma exacerbation and contributes to altered immune responses, triggering a cascade of inflammatory processes present in asthma
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