Polyclonal glycine receptor aAbs: a challenge for personalized epitope characterization

TL;DR

This study explores the challenge of identifying specific epitopes for glycine receptor antibodies in patients, aiming to improve targeted treatments.

Contribution

The study identifies patient-specific epitopes and structural binding patterns of glycine receptor antibodies using mutagenesis and peptide microarrays.

Findings

Peptide microarrays identified key residues in the GlyRα1 subunit for antibody binding.

Overlapping peptides partially neutralized antibodies but failed complete neutralization.

Patient-specific antibody binding patterns suggest polyclonal or structural epitope recognition.

Abstract

Patients with glycine receptor (GlyR) aAbs suffer from various diseases, including stiff-person syndrome (SPS), and currently, no cure exists. Several treatment options exist; however, these treatment options lack specificity. To date, only one common epitope has been mapped for GlyR aAbs in the far N-terminal region of the GlyRα1 subunit. However, some patient sera also bind GlyRα2, GlyRα3, or GlyRβ. Therefore, more than one common epitope may exist. Unraveling these epitopes will help generate more specific treatment approaches.

Here, we constructed GlyRa1 and GlyRa3 variants by site-directed mutagenesis using amino acid differences between these two subunits within their extracellular domains. Peptide microarrays, which have shown that an epitope including the binding site of a commercial pan-a antibody (96PDLFFANEKS105) and its surrounding residues is highly relevant for aAb binding, were utilized to identify additional residues important for aAb binding. Two overlapping peptides (93LWKPDLFFANEKSAN107 and 98LFFANEKSANFHDVT112) were used for aAb neutralization in cell-based assays.

The GlyRa1 and GlyRa3 variants helped to identify which amino acid sequences in the extracellular domain of GlyRs represent additional aAb epitopes or are involved in aAb binding. Using both generated peptides for aAb neutralization with a patient serum containing GlyRb aAbs that bind specifically to this region 96PDLFFANEKSANFHDV111, successful neutralization was demonstrated. In contrast, when using patient sera that reliably target the extracellular domain including 96PDLFFANEKS105 of the GlyRa subunits, the overlapping peptides reduced aAb binding but failed to fully neutralize the aAbs.

In conclusion, our data demonstrate that GlyR aAbs are polyclonal or bind to structural epitopes. These results define single residues important for aAb binding and help explain why no further common aAb binding site has been identified so far. Hence, patient-specific pattern for GlyR aAbs exist, emphasizing the importance of epitope characterization as basis for future therapeutic testing or even complete neutralization of the aAbs.

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