Autoantibody Effects on PAD4 Activity

To determine the effect of autoantibodies on PAD4 enzymatic activity, IgG and IgA were co-purified from anti-PAD-negative, anti-PAD4 mono-reactive, and anti-PAD3/4 cross-reactive serum and sputum using an equal mixture of Protein A/G agarose (Pierce; cat#20423) and Peptide M agarose (InvivoGen; cat# gel-pdm-2) beads. This mixture purifies predominantly all four IgG subclasses and IgA isotypes, with minimal purification of IgM. The concentration of total Ig was determined by NanoDrop (Thermo), >95% purity confirmed by Coomassie stain, and composition confirmed by immunoblot with goat anti-human IgG antibody and rabbit anti-human IgA antibody (Jackson Laboratories). The effect of purified Ig on PAD4 activity was evaluated using recombinant human PAD4, purified in-house as previously described [18 (link)]. PAD4 (10 nM) was pre-incubated with 1 μM purified Ig for 45 min at 4°C, followed by incubation with 700 μM histone H3 substrate for 3 h at 37°C at increasing calcium chloride concentrations (i.e., 0.2 and 2 mM), as previously described [2 (link)]. Citrullination of histone H3 was evaluated by anti-citrullinated histone H3 immunoblotting (ab5103, Abcam).