Oxidation-induced Structural Changes of Ceruloplasmin Foster NGR Motif Deamidation That Promotes Integrin Binding and Signaling

Abstract

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.

Comparative Proteomics for the Evaluation of Protein Expression and Modifications in Neurodegenerative Diseases

“…10; Olivieri et al, 2011). The prooxidant condition that occurs in neurodegenerative disorder CSF is underlined by the observed general increase of total protein carbonylation in CSF from PD (Iannaccone et al, 2013) and by the observation that exogenous purified Cp added and ex vivo aged for 9 days in CSF from AD patients resulted in oxidative modifications that did not occurred if aging was done in CSF of healthy subjects (Barbariga et al, 2014). Therefore, PD and AD pathological CSF contain compounds/small molecules that foster protein oxidative modifications, likely contributing to exacerbate the disorders.…”

“…We found that both Cp NGR sites can deamidate; however, while 568 NGR is free to convert to iso-DGR (Fig. 11), the 962 NGR motif undergoes deamidation only when Asn aging occurs under oxidative conditions, in which Cp structural changes are induced, as demonstrated by circular dichroism analysis (Barbariga et al, 2014). The NGR to isoDGR transition in Cp results in a loss of ferroxidase activity and a gain in integrin-binding function (Barbariga et al, 2014).…”

“…Asn deamidation may also have biological function consequences; indeed, it has been shown that the tripeptide Asparagine-Glycine-Arginine (NGR) motif can undergo deamidation that results in the formation of isoDGR (isoAspGly-Arg), a motif that is able to recognize the RGD-binding site of integrins (Corti & Curnis, 2011). From the methodological point of view, in the case of protein deamidation analysis, it is mandatory to modify the standard protocol used for tryptic digestion before MS because the usual conditions (ammonium bicarbonate buffer at pH 8.5, at 37°C) strongly induce in vitro peptides deamidation (Barbariga et al, 2014). Thus, many time, depending on the type of modification analyzed, the methods should be modified or de novo developed as to generate simple, robust, and reproducible protocols aimed to avoid the generation of artificial modifications.…”

“…The Cp sequence contains two NGRs motifs, one ( 568 NGR) is exposed on the surface of the protein and the other ( 962 NGR) hidden inside its 3D structure, and thus less accessible to solvents (Barbariga et al, 2014;Fig. 11).…”

“…Acidic Cp isoforms present in the CSF from PD patients are modified by carbonylation as revealed by derivatization with 2,4-dinitrophenilhydrazine (DNPH) followed by Western blot with anti-DNPH antibody (panels on the right). experiments under nitrogen-conditioned atmosphere (Barbariga et al, 2014;Olivieri et al, 2011).…”