Fig. 3

Oxidative stress contributes to ceruloplasmin structural changes and gain of integrin-binding function. a) Analysis of ceruloplasmin (Cp) oxidation after incubation in H-CSF, PN-CSF, or PD-CSF. Cp was added to CSFs (20 μg/ml), incubated (37 °C) for 0 or 9 days, and immunoprecipitated. One part of the product was analyzed for protein oxidation rate using the OxyBlot™ kit (based on anti-DNP WB analysis of protein carbonylation), and the other part was analyzed by WB with anti-ceruloplasmin Ab. Two independent experiments were performed using pools of CSFs from 4 different subjects per groups in each experiment (total subjects analyzed for each group n = 8) (a representative experiment is shown). b) Quantification of hydrogen peroxide concentration in H-CSF (n = 16), PN-CSF (n = 11) and PD-CSF (n = 12) using the Amplex® Red Hydrogen Peroxide kit. c) Effect of hydrogen peroxide depletion with catalase on the ceruloplasmin loss-of-ferroxidase activity induced by incubation in PD-CSF. Ceruloplasmin (20 μg/ml) and catalase (60 μg/ml) were added to H-CSF or PD-CSF, left to incubate (37 °C) for 0 or 9 days, then the ferroxidase activity of immunoprecipitated ceruloplasmin (1.25 μg) was evaluated by bathophenanthroline assay and expressed as % of ferroxidase activity measured for 1.25 μg of untreated ceruloplasmin. d) Effect of catalase-mediated hydrogen peroxide depletion on the ceruloplasmin gain of integrin-binding properties induced by incubation in PD-CSF. Ceruloplasmin and catalase were added to different CSFs as in C and analyzed by direct αvβ6-binding assay. To rule out a non-specific effect of catalase on ceruloplasmin-integrin-binding, a control with ceruloplasmin pre-oxidized and deamidated in-vitro (Cp-ox/AmBic) (right panel) was performed. In c and d, two independent experiments in triplicate were performed using CSFs from different subjects in each experiment (total subjects for each group n = 8). In all experiments statistical p value was evaluated by student’s t test, means with standard error, calculated using pooled data from different experiments, are indicated (**** = p < 0.0001 *** = p < 0.001, ** = p < 0.01)