Polyclonal glycine receptor autoantibodies: A challenge for personalized epitope characterization

Abstract

Patients with glycine receptor (GlyR) autoantibodies suffer from various diseases including stiff person syndrome with no cure, so far. Several treatment options exist but rather lack specificity. To date, there is only one common epitope mapped for GlyR autoantibodies in the far N-terminal part of the GlyRa1 subunit. However, some patient sera also bind GlyRa2, GlyRa3 or even GlyRb. Thus, there might be more than just one common epitope existing. Unraveling these epitopes will help to generate more specific treatment approaches. Here, we constructed GlyRa1 and GlyRa3 variants by site-directed mutagenesis using the amino acid differences between these two subunits within their extracellular domains. The variants helped to identify which amino acid sequences in the extracellular domain of GlyRs represent additional autoantibody epitopes or are involved in autoantibody binding. Peptide microarrays have shown that an epitope including the binding site of a commercial pan-a antibody (96PDLFFANEKS105) and its surrounding residues is highly relevant for autoantibody binding. To identify additional residues important for autoantibody binding, two overlapping peptides (93LWKPDLFFANEKSAN107, 98LFFANEKSANFHDVT112) were used for autoantibody neutralization in cell-based assays. Using both peptides with a patient serum containing GlyRb autoantibodies 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 autoantibody binding but failed to fully neutralize the autoantibodies. In conclusion, our data demonstrate that GlyR autoantibodies are polyclonal or bind to structural epitopes. These results define single residues important for autoantibody binding and help explain why no further common autoantibody binding site has been identified so far. Hence, patient-specific pattern for GlyR autoantibodies exist, emphasizing the importance of epitope characterization as basis for future therapeutic testing or even complete neutralization of the autoantibodies.