Fig. 2

Incubation of ceruloplasmin spiked in the CSF of PD patients induces structural and functional modifications. Ceruloplasmin (Cp) was added (20 μg/ml) to CSF from healthy subjects (H-CSF), peripheral neuropathies patients (PN-CSF) and PD patients (PD-CSF), and incubated for 0 or 9 days at 37 °C, then supplemented CSFs were directly used or ceruloplasmin was immunoprecipitated and used for different assays. a) Structural changes. Immunoprecipitated samples were subjected to limited-trypsin digestion and analyzed by western blot with an anti-ceruloplasmin Ab (a representative experiment is shown). b) Ferroxidase activity. The activity of immunoprecipitated Cp (1.25 μg) was evaluated by bathophenanthroline assay and expressed as % of ferroxidase activity measured for 1.25 μg of untreated ceruloplasmin. Three independent experiments in triplicate were performed using CSFs from different subjects in each experiment (total subjects for each group n = 8). C) Deamidation of NGR-motifs. Ceruloplasmin immunoprecipitated from CFSs samples after 9 days of incubation, was trypsin-digested and analysed by mass spectrometry. Semi-quantitative label-free evaluation was performed on the observed peptides containing either ⁵⁶⁸NGR- or ⁹⁶²NGR-motif as they are or deamidated. Data are reported as ratio between deamidated and non-deamidated ⁵⁶⁸NGR or ⁹⁶²NGR quantitation; three independent experiments were performed using CSF from different subjects each group (total subjects for each group n = 12; in PN-group the PN1 sample was used twice). d-e) Binding to αvβ6-integrin of ceruloplasmin after different time of incubation (0–12 days at 37 °C) in different CSFs was evaluated by direct (d) or competitive (e) αvβ6-integrin binding ELISA assay using the CSFs supplemented with ceruloplasmin. Competition experiments were performed using either acetyl-CisoDGRCGVRSSSRTPSDKY (isoDGR) peptide or the control peptide acetyl-CARACGVRSSSRTPSDKY (ARA). Two independent experiments in triplicate were performed using CSFs from different subjects in each experiment (total subjects for each group n = 8). In all assays data were analyzed by student’s t test; means with standard error, calculated using pooled data from different experiments, are indicated (*** = p < 0.001; ** = p < 0.01; * = p < 0.05)