Autoantibodies in Serum of Systemic Scleroderma Patients: Peptide-Based Epitope Mapping Indicates Increased Binding to Cytoplasmic Domains of CXCR3

Abstract

Systemic sclerosis (SSc) is a severe chronic autoimmune disease with high morbidity and mortality. Sera of patients with SSc contain a large variety of autoantibody (aab) reactivities. Among these are functionally active aab that bind to G protein-coupled receptors (GPCR) such as C-X-C motif chemokine receptor 3 (CXCR3) and 4 (CXCR4). Aab binding to the N-terminal portion of these two GPCRs have been shown to be associated with slower disease progression in SSc, especially deterioration of lung function. Aabs binding to GPCRs exhibit functional activities by stimulating or inhibiting GPCR signaling. The specific functional activity of aabs crucially depends on the epitopes they bind to. To identify the location of important epitopes on CXCR3 recognized by aabs from SSc patients, we applied an array of 36 overlapping 18-20mer peptides covering the entire CXCR3 sequence, comparing epitope specificity of SSc patient sera (N = 32, with positive reactivity with CXCR3) to healthy controls (N = 30). Binding of SSc patient and control sera to these peptides was determined by ELISA. Using a Bayesian model approach, we found increased binding of SSc patient sera to peptides corresponding to intracellular epitopes within CXCR3, while the binding signal to extracellular portions of CXCR3 was found to be reduced. Experimentally determined epitopes showed a good correspondence to those predicted by the ABCpred tool. To verify these results and to translate them into a novel diagnostic ELISA, we combined the peptides that represent SSc-associated epitopes into a single ELISA and evaluated its potential to discriminate SSc patients (N = 31) from normal healthy controls (N = 47). This ELISA had a sensitivity of 0.61 and a specificity of 0.85. Our data reveals that SSc sera preferentially bind intracellular epitopes of CXCR3, while an extracellular epitope in the N-terminal domain that appears to be target of aabs in healthy individuals is not bound by SSc sera. Based upon our results, we could devise a novel ELISA concept that may be helpful for monitoring of SSc patients.

Keywords: CXCR3; G protein-coupled receptor; autoantibodies; peptide array; systemic sclerosis.

Figure 1

Binding behavior of SSc patient and healthy control sera to individual peptides. Plot of the mean expectation value (red line) and 95 and 99.9% credibility bands (pink and white shading) of the SSc patient-specific ab-binding signal (percent increase). The x axis represents amino acid residues 1–368. The peptide localizations are indicated by staggered rectangles that include the peptide numbers. The percent increase indicates the increase or decrease of the binding signal in SSc patients in contrast to an averaged signal. If the 95% (99.9%) credibility interval of the percent increase does not include the zero value (black line), the corresponding peptide is regarded as an epitope that is significantly associated with SSc. By use of the ar1 model, the percent increase, i.e., the binding signal estimator, for a peptide is influenced by the neighboring peptides. Other fixed and random effects of the statistical model are shown in Figures S2 and S3 in Supplementary Material. At the bottom, three heat maps indicate the position of putative epitopes predicted by antigenic (magenta hue), ABCpred (pink hue) and the presence of rhodopsine-like domains (orange hue). The background colors indicate the position of intracellular (yellow hue), transmembranous (green hue), or extracellular (blue hue) amino acid residues.

Figure 2

Location of epitopes on the CXCR3 structure. Mapping of binding signal (compare Figure 1) of SSc patient sera to a serpentine model of CXCR3. Each pearl represents a single amino acid residue, the letter inside each pearl the amino acid in 1-letter code. The color of each pearl indicates the percent increase value of the SSc specific binding signal associated with the respective amino acid residue (color key).

Figure 3

Derivation of a mixed-peptide ELISA for the discrimination of SSc patients and healthy controls. Peptides 17, 24, 25, 33, and 34 were mixed for coating of ELISA plates. In total, 32 SSc patient sera and 47 healthy control sera were measured. Left panel, scatter plot of raw ELISA readout values (OD @ 450 nm) including a cut-off value optimized by maximization of Matthew’s correlation coefficient. SSc patient showed a significantly higher ELISA signal than healthy control sera (Wilcoxon rank sum testing p = 4.52 × 10⁻⁵). Right panel, receiver operator characteristic (ROC) analysis of ELISA values. AUC (area under curve, 0.77) and best MCC (Matthew’s correlation coefficient, 0.51) as well as Sensitivity (0.61) and Specificity (0.85) at the optimal cut-off are shown as performance indicators. The dot indicates the optimal cut-off position in the ROC curve.