MUC5AC drives COPD exacerbation severity through amplification of virus-induced airway inflammation

Aran Singanayagam; Joseph Footitt; Benjamin T Kasdorf; Matthias Marczynski; Michael T Cross; Lydia J Finney; Maria-Belen Trujillo Torralbo; Maria Calderazzo; Jie Zhu; Julia Aniscenko; Thomas B Clarke; Philip L Molyneaux; Nathan W Bartlett; Miriam F Moffatt; William O Cookson; Jadwiga Wedzicha; Christopher M Evans; Oliver Lieleg; Patrick Mallia; Sebastian L Johnston

Since the gastric mucosa also contains DNA 7 , enzymatic removal of potential mucinassociated DNA was performed to prevent any DNA-related inflammation reactions. All following steps were carried out under a sterile hood. Lyophilized mucin purified from porcine gastric mucosa was treated with UV-light on ice for 1 hour. Subsequently, it was dissolved in 50 mM Tris-HCl buffer (sterile filtered, pH 7.5, supplemented with 10 mM MgCl 2 ) at a concentration of 1 mg/mL and kept on ice. DNAse I from bovine pancreas (D5025, Sigma Aldrich, St. Louis, MO, USA) was dissolved in the same buffer at a concentration of 1 mg/mL. 50 µL of the DNAse solution were added per milligram of dissolved mucin. Afterwards, the mucin/DNAse solution was incubated at 37 °C for 18 hours in a tube shaker at 200 rpm. The treated mucin was then chromatographically separated from the enzyme and residual DNA fragments via size exclusion chromatography, subsequently desalted by means of cross-flow filtration and lyophilized (see Purification of porcine gastric mucin section above). The DNAse treated mucin was stored in lyophilized form at -80 °C. author/funder. All rights reserved. No reuse allowed without permission.