Involvement of oxidative stress in occurrence of relapses in multiple sclerosis: the spectrum of oxidatively modified serum proteins detected by proteomics and redox proteomics analysis

Abstract

Multiple sclerosis (MS) is an autoimmune inflammatory demyelinating disease of the central nervous system. Several evidences suggest that MS can be considered a multi-factorial disease in which both genetics and environmental factors are involved. Among proposed candidates, growing results support the involvement of oxidative stress (OS) in MS pathology. The aim of this study was to investigate the role of OS in event of exacerbations in MS on serum of relapsing-remitting (RR-MS) patients, either in relapsing or remitting phase, with respect to serum from healthy subjects. We applied proteomics and redox proteomics approaches to identify differently expressed and oxidatively modified proteins in the low-abundant serum protein fraction. Among differently expressed proteins ceruloplasmin, antithrombin III, clusterin, apolipoprotein E, and complement C3, were up-regulated in MS patients compared with healthy controls. Further by redox proteomics, vitamin D-binding protein showed a progressive trend of oxidation from remission to relapse, respect with controls. Similarly, the increase of oxidation of apolipoprotein A-IV confirmed that levels of OS are elevated with the progression of the disease. Our findings support the involvement of OS in MS and suggest that dysfunction of target proteins occurs upon oxidative damage and correlates with the pathology.

Figure 1. Levels of protein carbonyls.

Protein carbonyls (PC) levels in the serum of control (CTR) and multiple sclerosis patients: remitting (REM) and relapsing (REL). Serum samples were assayed for PC by slot blot analysis. Error bars indicate SD for 6 samples per group. Densitometric values shown are given as raw values.

Figure 2. 2D protein expression maps.

Proteomic profile of representative 2D-gels with proteins differently expressed in the three groups of matching: REL vs CTR (A), REM vs CTR (B) and REL vs REM (C). The identified proteins by mass spectrometry are listed in Table 2.

Figure 3. Oxidized protein detection by redox proteomics.

Proteomic profile of representative 2D-blots with proteins differently oxidized in the three groups of matching: REL vs CTR (A), REM vs CTR (B) and REL vs REM (C).The identified proteins are listed in Table 3.

Figure 4. Spectra obtained by MALDI-ToF MS analysis related to spots of vitamin D-binding protein (DBP).

DBP C-terminal region spectrum (upper panel) and major coverage DBP spectrum (lower panel).

Figure 5. Vitamin D-binding protein oxidation fold in REM vs CTR matching.

In the upper part of the figure, an enlarged area of 2D blots from CTR and REM groups is shown corresponding to the vitamin D-binding protein (DBP). DBP identified as differently oxidized in the matching between REM vs CTR is labeled. 3D density graphs are elaborated by PD-Quest from DBP spot on 2D blot. In the lower part of the figure the histogram reports oxidation fold for DBP.

Figure 6. Vitamin D-binding protein oxidation fold in REL vs CTR matching.

In the upper part of the figure, an enlarged area of 2D blots from CTR and REL groups is shown corresponding to the DBP. DBP identified as differently oxidized in the matching between REL vs CTR is labeled. 3D density graphs are elaborated by PD-Quest from DBP spot on 2D blot. In the lower part of the figure the histogram reports oxidation fold for DBP. (Data obtained from Proteomics and redox proteomics approach).

Figure 7. Apolipoprotein A-IV oxidation fold.

On the left side of the figure, an enlarged area of 2D blots from CTR and REL groups (A), and from REM and REL (B),are shown corresponding to the Apolipoprotein A-IV (Apo A-IV) spot. Apo A-IV spot identified as differently oxidized in the matching between REL vs CTR, and REL vs REM is labeled. 3D density graphs are elaborated by PD-Quest from Apo A-IV spot on 2D blot. On the right side of the figure the histogram reports oxidation fold for Apo A-IV.