Fig. 6
From: Osteopontin protects against lung injury caused by extracellular histones
Phosphorylation of OPN is essential for binding to histones and cytoprotective effects. a SPR sensorgrams showing differences in binding affinities between histone subunits (i.e., H1, H2A, H2B, H3.1, and H4) and different forms of OPN (expressed in HEK 293 cells (OPN), recombinant E. coli expressed OPN (rec-OPN), and tartrate-resistant acid phosphatase-treated OPN (TRAP-OPN) were compared. Histone subunits were covalently bound to a CM5 sensor chip using a standard amine-coupling kit. Different OPN at a concentration of 2 µM were injected over the sensor chip to determine binding. The binding affinity of OPN expressed in HEK 293 cells was higher compared to rec-OPN (lacking posttranslational modification) and TRAP-OPN (dephosphorylated using TRAP enzyme). b Table showing the KD values obtained from SPR-binding studies. The KD values for rec-OPN and TRAP-OPN were not determined because of low or no interaction and therefore denoted as not determined (ND). c LDH levels in the supernatants of human bronchiolar epithelial cells after incubation with calf thymus histones (CTH) in the presence and absence of OPN at a ratio of 1:1. The cytotoxicity of histones is presented as percentage LDH release of the total LDH content. The results are mean ± SEM (n = 5; **P = 0.0050, ****P = < 0.0001)
