Quantitative proteomics reveals Piccolo as a candidate serological correlate of recovery from Guillain-Barré syndrome

Abstract

Guillain-Barré syndrome (GBS) is an autoimmune-mediated peripheral neuropathy of unknown cause. However, about a quarter of GBS patients have suffered a recent bacterial or viral infection, and axonal forms of the disease are especially common in these patients. Proteomics is a good methodological approach for the discovery of disease biomarkers. Until recently, most proteomics studies of GBS and other neurodegenerative diseases have focused on the analysis of the cerebrospinal fluid (CSF). However, serum represents an attractive alternative to CSF because it is easier to sample and has potential for biomarker discovery. The goal of this research was the identification of serum biomarkers associated with recovery from GBS. To address this objective, a quantitative proteomics approach was used to characterize differences in the serum proteome between a GBS patient and her healthy identical twin in order to lessen variations due to differences in genetic background, and with additional serum samples collected from unrelated GBS (N = 3) and Spinal Cord Injury (SCI) (N = 3) patients with similar medications. Proteomics results were then validated by ELISA using sera from additional GBS patients (N = 5) and healthy individuals (N = 3). All GBS and SCI patients were recovering from the acute phase of the disease. The results showed that Piccolo, a protein that is essential in the maintenance of active zone structure, constitutes a potential serological correlate of recovery from GBS. These results provided the first evidence for the Piccolo´s putative role in GBS, suggesting a candidate target for developing a serological marker of disease recovery.

Keywords: Guillain-Barré; Immune response; Immunity; Immunology and Microbiology Section; biomarker; neurology; neuropathy; proteomics.

Figure 1. Differentially represented serum proteins

Serum samples collected from a GBS patient (A3) and her healthy identical twin (B3) [18], and from unrelated GBS (N = 3; AI-AIII) and SCI (N = 3; DI-DIII) patients with similar medications were included in the proteomics analysis (Table 1). The 5% FDR was used as criterion for peptide identification. Protein identification (Uniprot accession) and name are shown. For over-represented and under-represented protein quantifications, a standardized variable of protein fold changes was used (Zq), where statistical significance of protein abundance changes was considered using a 1% FDR.

Figure 2. GBS-related serological response

A. Differentially represented proteins were grouped as under-represented and over-represented in GBS patients when compared to SCI patients or healthy twin. Protein ontology analysis for biological process was done using the Blast2GO software (www.blast2go.com) for B. under-represented and C. over-represented proteins.

Figure 3. Piccolo as a potential serological correlate of recovery from GBS

Sera from GBS (N = 8; A3, AI-AVII) and SCI (N = 4; DI-DIV) unrelated patients with similar medications, and healthy control individuals (N = 4; B3, CI-CIII), including the GBS patient (A3) and her healthy identical twin (B3) were included in the ELISA (Table 1). ELISA plates were coated with 100 μl/well of albumin-depleted sera at concentration of 2 ng/μl (0.2 μg serum/well). Mouse monoclonal anti-Piccolo antibodies were added at 1:1000 dilution and detected using anti-mouse IgG (μ-chain specific)-peroxidase antibodies produced in goat. Color was developed by the addition of TMB to measure the OD_{450 nm}. For Piccolo protein quantitation, the human recombinant protein was used. Two technical replicates were included for each sample. The Piccolo concentration in serum samples was compared between GBS patients, SCI patients and healthy individuals by Student's t-test with unequal variance (P = 0.05). A. Piccolo serum levels in GBS (N = 8; A3, AI-AVII) patients, SCI (N = 4; DI-DIV) unrelated patients, and healthy control individuals (N = 4; B3, CI-CIII). B. Piccolo serum levels determined in GBS patients (AIV-AVII) and healthy individuals (CI-CIII) not included in the proteomics analysis. C. Negative correlation between Piccolo serum levels and patient functional status (Table 1). Correlation coefficient (R²) is shown.

Figure 4. Piccolo-protein interactions

A. STRING was used for the in silico characterization of Piccolo-protein interactions using a high confidence interaction score (0.700; http://bit.ly/2anoqgi). Each node represents all the proteins produced by a single protein-coding gene locus. Protein-protein associations represent proteins that jointly contribute to a shared function, but not necessarily physically bind to each other. B. Protein ontology analysis for biological process was done for Piccolo and interacting proteins using the Blast2GO software (www.blast2go.com).