Designed Glucopeptides Mimetics of Myelin Protein Epitopes As Synthetic Probes for the Detection of Autoantibodies, Biomarkers of Multiple Sclerosis

We previously reported that CSF114­(Glc) detects diagnostic autoantibodies in multiple sclerosis sera. We report herein a bioinformatic analysis of myelin proteins and CSF114­(Glc), which led to the identification of five sequences. These glucopeptides were synthesized and tested in enzymatic assays, showing a common minimal epitope. Starting from that, we designed an optimized sequence, SP077, showing a higher homology with both CSF114­(Glc) and the five sequences selected using the bioinformatic approach. SP077 was synthesized and tested on 50 multiple sclerosis patients’ sera, and was able to detect higher antibody titers as compared to CSF114­(Glc). Finally, the conformational properties of SP077 were studied by NMR spectroscopy and structure calculations. Thus, the immunological activity of SP077 in the recognition of specific autoantibodies in multiple sclerosis patients’ sera may be ascribed to both the optimized design of its epitopic region and the superior surface interacting properties of its C-terminal region

Association of organ involvement with ITGAM genotype and immune complex composition.

<p><b>a</b>. Percentage of the SLE patients with the involvement of the indicated three organs is shown for rs1143679 genotypes. The presence of single, double or triple organ involvement is illustrated by Euler ellipses with areas proportional to the percentages shown. <b>b</b>. Association with organ involvement of the level and composition (IgG, IgM, C4 and IgG/IgM) of immune complexes formed on dsDNA and nucleosome is indicated. Odds ratios for patients falling into the indicated percentile groups (upper 10^th, 25^th, 50^th and lower 10^th, 25^th, 50^th percentiles) for the given measurements are shown. Positive odds (>1) are in red, negative odds (<1) are in blue.</p

Clinical and serological characteristics of the SLE patient cohorts.

<p>Clinical and serological characteristics of the SLE patient cohorts.</p

Heatmap and principal component analysis of the functional antibody profiling data.

<p>Samples are ordered as study groups. Variables are grouped by the molecular composition and the nature of the detected serum immunoglobulin or complement protein, the order of the particular antigens is shown and applies for each detected protein. PCA plot of samples shows the distribution of samples in the space of the first two components. The variable plot shows the contribution of the variables to the generation of this space, where distance and orientation from the zero origin define a particular interaction’s load in the first two principal components. Percentages in parenthesis indicate contribution of the indicated principal component to overall variability in the dataset. xdsDNA, ultrasound-fragmented dsDNA; CENPB, centromere protein B; Sm-p, peptide of Smith antigen D polypeptide</p

Effect of SNP rs1143679 genotype on dsDNA specific antibody levels and C4 deposition.

<p>Binding of IgG, IgM and C4 to dsDNA was determined by functional antibody profiling analysis. Individuals within the study groups were classified based on their genotype. Boxes show interquartile ranges, horizontal lines stand for median. Asterisks indicate statistically significant (p<0.05) difference from the GG genotype. AA genotype carriers in the NHS group were excluded because of the low number of samples.</p

Scheme for the role of complement in the pathogenesis of anti-dsDNA IgG positive SLE.

<p><b>a</b>. Apoptotic DNA is normally quickly opsonized by systemic IgM, C1q and early complement activation products C4 and C2, locally produced opsonins also helping removal. This opsonized debris is swiftly removed by tissue macrophages, thus the level of debris is controlled in the circulation and other organs are protected from its pathological accumulation. <b>b</b>. Inefficient local and systemic removal by dysfunctional CR3 encoded by <i>ITGAM</i> on tissue and sinusoidal macrophages results in increased levels of opsonized debris in the circulation. Circulating immune complexes reach lymphoid organs and trigger production of antigen specific antibodies. In the presence of other SLE susceptibility genes and environmental triggers high avidity DNA specific IgG develops. Upon reaching the circulation this IgG enhances complement consumption by apoptotic DNA and triggers FcgammaR mediated inflammation in tissues where immune complexes are deposited. Relative IgG, IgM and C4 content of immune complexes may influence tissue deposition preference.</p

Opposite complement activating properties of biochemically different antigens in SLE.

<p>The PECS SLE cohort with available serum complement concentrations (n = 124) was analyzed for associations between serum protein levels and on-chip antigen binding of immunoglobulin and complement proteins. <b>a</b> Spearman rank-correlations of serum C4 concentrations, dsDNA IgG reactivity with various microarray derived binding data. Values indicate r, bold fonts highlight significant correlations (p<0.05). <b>b</b> Comparative C4 deposition on dsDNA and collagen as a function of serum C4 concentrations. Linear regression fits with 95% confidence intervals are shown. <b>c</b> Complement C4 deposition initiated by superantigen protein G as a function of serum C4 concentration. <b>d</b> Comparative C4 deposition and IgG binding on various antigens on a mass basis in healthy and SLE serum samples. Asterisks indicate direction and significance (p<0.05) of changes in the SLE versus NHS group. pG, Staphylococcal protein G.</p

Antibody binding and complement C4 deposition on various antigens.

<p>Scatterplots show the relationship between IgG reactivity and C4 deposition in the sera of healthy (NHS), disease control (DC) and SLE subjects. Numbers indicate percentage of SLE subjects in the respective quadrants, which were generated by the 98th percentile boundaries of the NHS group. Antigens of different biochemical nature (a) and of different epitope density (b) are shown. ssDNA, single-stranded DNA</p