Fig. 3: The unfolded structure and associated hidden activities of CRP are evolutionarily distinct.

The stability of mouse, rat, and human CRP was examined with heat- (a) (n = 10 independent experiments for mouse CRP; n = 6 independent experiments for rat CRP; n = 9 independent experiments for human CRP) and urea-induced unfolding (b) (n = 4 independent experiments). The melting temperature (Tm) of mouse CRP was much lower than that of rat and human CRP. Accordingly, calcium exhibited little effect on urea-induced unfolding of mouse CRP, but markedly inhibited that of rat and human CRP. c The refolding of urea-unfolded CRP was examined following dilution (n = 3 independent experiments for mouse CRP; n = 4 independent experiments for rat and human CRP). Rat CRP efficiently refolded, whereas mouse and human CRP did not. d The hidden activities of the major functional motif of unfolded CRP, i.e., CBS, were examined with competitive ELISA against 6 ligands³⁶. Relative potency for each ligand was defined as: binding of mCRP in the absence of CBS divided by that in the presence of CBS. Each data point represents a potency index of a different ligand (n = 3 independent experiments). Mouse CBS was most active, while rat CBS was least active. These results indicate that mouse CRP is most prone to unfold and possesses the strongest hidden activities. By contrast, rat CRP is most resistant to unfolding and possesses the weakest hidden activities. e Wild-type mice with acetaminophen-induced liver failure were treated with human CRP at the indicated dosages (n = 14 mice for vehicle; n = 6 mice for 1.25 mg/kg dosage; n = 9 mice for 2.5 mg/kg dosage; n = 5 mice for 5 mg/kg dosage). Human CRP reduced liver injury at dosages of 1.25 and 2.5 mg/kg, but showed little effect at the dosage of 5 mg/kg. The loss of protection of human CRP at the highest dosage might be due to strong proinflammatory responses evoked by excessive mCRP. Data are presented as mean ± SEM; *p < 0.05; **p < 0.01; ***p < 0.001, one-way ANOVA with Tukey post hoc.