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

Previous studies revealed that the properties of commercially available mucins differ from key properties of native mucin systems as they, e.g. the lack of the ability to reduce friction [1] [2] [3] or lead to cytotoxic effects 4 . These differences are attributed to the harsh conditions during the commercial purification process 5 . As a consequence, here, the purification of porcine gastric MUC5AC is performed manually from fresh pig stomachs. An optimization of the original purification protocol from Celli et.al 6 was described in detail previously 3 . In brief, crude mucus was obtained by gently scraping pig stomachs after rinsing them with tap water. The mucus was diluted 5-fold in 10 mM sodium phosphate buffer (pH 7.0, supplemented with 170 mM NaCl) and homogenized by stirring at 4 °C overnight. Cellular debris was removed via several centrifugation steps (8.300 x g for 30 min at 4 °C and 15.000 x g for 45 min at 4 °C) and a final ultracentrifugation step (150.000 x g for 1 h at 4 °C). Afterwards, the supernatant was separated chromatographically by size-exclusion chromatography (ÄKTA purifier system, GE Healthcare, equipped with a XK50/100 column packed with Sepharose 6FF), and the fractions containing the mucin glycoproteins were identified via PAS (periodic acid/Schiff's base) staining and pooled. After a subsequent dialysis step against ultra-pure water via cross-flow filtration, the mucin-solution was concentrated, lyophilized and stored at -80 °C.