463 - Dityrosine Cross-Linking of Fibronectin Is Increased in Plasma of Human Subjects with Interstitial Lung Disease

Abstract

Objective Interstitial lung disease (ILD) is a variably progressive lung disorder resulting in impaired gas-exchange and respiratory failure with significant morbidity. Oxidative stress has been shown to play a role in the pathobiology of ILD, and we have previously shown that the oxidant-mediated tyrosine modification, o,o’-dityrosine, is increased within proteins in circulating plasma of ILD patients. This study sought to elucidate a protein target of o,o’-dityrosine cross-linking in plasma. Therefore, we tested the hypothesis that fibronectin (FN), a tyrosine-rich extracellular matrix protein shown to play a role in the fibrotic response of ILD, is modified by o,o’-dityrosine cross-linking and is increased in plasma of ILD patients. Methods Fibronectin was immunoprecipitated from age- and sex-matched healthy control (n=20) and ILD patient (n=16) plasma and o,o’-dityrosine levels were quantified by tandem mass-spectrometry (MS/MS). The study was approved by the University of Alabama at Birmingham and the University of Michigan Institutional Review Board. Data were analyzed by unpaired t test with Welch’s correction (mean ± SEM, p Results Fibronectin o,o’-dityrosine cross-linking was observed in plasma of human subjects. Furthermore, fibronectin-associated o,o’-dityrosine levels were significantly increased by 10.95 fold (14.34 ± 5.01 vs. 1.20 ± 0.16) in ILD patients when compared to healthy controls. The o,o’-dityrosine cross-linking of fibronectin was recapitulated in in vitro systems utilizing human lung fibroblasts. Conclusions These data support the idea that tyrosine residues within fibronectin are susceptible to o,o’-dityrosine cross-linking, and this modification of fibronectin is increased in the plasma of ILD patients. This covalent modification of fibronectin could alter protein function, stability, and/or create alternative breakdown products. Further understanding the role of fibronectin o,o’-dityrosine cross-linking in ILD pathophysiology could provide insight leading to identification of novel treatments for the disease. Funding: P01 HL114470, R01 AG046210, R01 HL0942230 and T32 GM008361