Oxidation-induced structural changes of Ceruloplasmin Foster NGR Motif Deamidation That Promotes Integrin binding and signaling

Background: Asparagine deamidation at Asn-Gly-Arg (NGR) sites leads to the isoAsp-Gly-Arg (isoDGR) integrin-binding motif formation. Results: Ceruloplasmin (Cp), which contains two NGR sites and is oxidized in cerebrospinal fluid (CSF) in neurodegenerative diseases, can, undergo oxidation-induced structural changes fostering NGR deamidation with gain of integrin binding and signaling properties, in vitro and ex vivo in pathological CSF. Conclusion: Cp NGR motifs can deamidate acquiring integrin-binding functions. Significance: Cp structural changes favor NGR deamidation. Asparagine deamidation occurs spontaneously in proteins during aging; deamidation of Asn-Gly-Arg (NGR) sites can lead to the formation of isoAsp-Gly-Arg (isoDGR), a motif that can recognize the RGD-binding site of integrins. Ceruloplasmin (Cp), a ferroxidase present in the cerebrospinal fluid (CSF), contains two NGR sites in its sequence: one exposed on the protein surface (568NGR) and the other buried in the tertiary structure (962NGR). Considering that Cp can undergo oxidative modifications in the CSF of neurodegenerative diseases, we investigated the effect of oxidation on the deamidation of both NGR motifs and, consequently, on the acquisition of integrin binding properties. Weobserved that the exposed 568NGRsite can deamidate under conditions mimicking accelerated Asn aging. In contrast, the hidden 962NGR site can deamidate exclusively when aging occurs under oxidative conditions, suggesting that oxidationinduced structural changes foster deamidation at this site. NGR deamidation in Cp was associated with gain of integrin-binding function, intracellular signaling, and cell pro-Adhesive activity. Finally, Cp aging in the CSF from Alzheimer disease patients, but not in control CSF, causes Cp deamidation with gain of integrin-binding function, suggesting that this transition might also occur in pathological conditions. In conclusion, both Cp NGR sites can deamidate during aging under oxidative conditions, likely as a consequence of oxidative-induced structural changes, thereby promoting a gain of function in integrin binding, signaling, and cell adhesion.