TY - JOUR A1 - Wiessler, Anna-Lena A1 - Talucci, Ivan A1 - Piro, Inken A1 - Seefried, Sabine A1 - Hörlin, Verena A1 - Baykan, Betül B. A1 - Tüzün, Erdem A1 - Schaefer, Natascha A1 - Maric, Hans M. A1 - Sommer, Claudia A1 - Villmann, Carmen T1 - Glycine receptor β–targeting autoantibodies contribute to the pathology of autoimmune diseases JF - Neurology: Neuroimmunology & Neuroinflammation N2 - Background and Objectives Stiff-person syndrome (SPS) and progressive encephalomyelitis with rigidity and myoclonus (PERM) are rare neurologic disorders of the CNS. Until now, exclusive GlyRα subunit–binding autoantibodies with subsequent changes in function and surface numbers were reported. GlyR autoantibodies have also been described in patients with focal epilepsy. Autoimmune reactivity against the GlyRβ subunits has not yet been shown. Autoantibodies against GlyRα1 target the large extracellular N-terminal domain. This domain shares a high degree of sequence homology with GlyRβ making it not unlikely that GlyRβ-specific autoantibody (aAb) exist and contribute to the disease pathology. Methods In this study, we investigated serum samples from 58 patients for aAb specifically detecting GlyRβ. Studies in microarray format, cell-based assays, and primary spinal cord neurons and spinal cord tissue immunohistochemistry were performed to determine specific GlyRβ binding and define aAb binding to distinct protein regions. Preadsorption approaches of aAbs using living cells and the purified extracellular receptor domain were further used. Finally, functional consequences for inhibitory neurotransmission upon GlyRβ aAb binding were resolved by whole-cell patch-clamp recordings. Results Among 58 samples investigated, cell-based assays, tissue analysis, and preadsorption approaches revealed 2 patients with high specificity for GlyRβ aAb. Quantitative protein cluster analysis demonstrated aAb binding to synaptic GlyRβ colocalized with the scaffold protein gephyrin independent of the presence of GlyRα1. At the functional level, binding of GlyRβ aAb from both patients to its target impair glycine efficacy. Discussion Our study establishes GlyRβ as novel target of aAb in patients with SPS/PERM. In contrast to exclusively GlyRα1-positive sera, which alter glycine potency, aAbs against GlyRβ impair receptor efficacy for the neurotransmitter glycine. Imaging and functional analyses showed that GlyRβ aAbs antagonize inhibitory neurotransmission by affecting receptor function rather than localization. KW - autoantibody (aAb) KW - glycine receptor (GlyR) KW - stiff-person syndrome (SPS) KW - clinical neurology KW - movement disorders KW - progressive encephalitis with rigidity and myoclonus (PERM) Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-349958 VL - 11 IS - 2 ER - TY - JOUR A1 - Rauschenberger, Vera A1 - Piro, Inken A1 - Kasaragod, Vikram Babu A1 - Hörlin, Verena A1 - Eckes, Anna-Lena A1 - Kluck, Christoph J. A1 - Schindelin, Hermann A1 - Meinck, Hans-Michael A1 - Wickel, Jonathan A1 - Geis, Christian A1 - Tüzün, Erdem A1 - Doppler, Kathrin A1 - Sommer, Claudia A1 - Villmann, Carmen T1 - Glycine receptor autoantibody binding to the extracellular domain is independent from receptor glycosylation JF - Frontiers in Molecular Neuroscience N2 - Glycine receptor (GlyR) autoantibodies are associated with stiff-person syndrome and the life-threatening progressive encephalomyelitis with rigidity and myoclonus in children and adults. Patient histories show variability in symptoms and responses to therapeutic treatments. A better understanding of the autoantibody pathology is required to develop improved therapeutic strategies. So far, the underlying molecular pathomechanisms include enhanced receptor internalization and direct receptor blocking altering GlyR function. A common epitope of autoantibodies against the GlyRα1 has been previously defined to residues 1A-33G at the N-terminus of the mature GlyR extracellular domain. However, if other autoantibody binding sites exist or additional GlyR residues are involved in autoantibody binding is yet unknown. The present study investigates the importance of receptor glycosylation for binding of anti-GlyR autoantibodies. The glycine receptor α1 harbors only one glycosylation site at the amino acid residue asparagine 38 localized in close vicinity to the identified common autoantibody epitope. First, non-glycosylated GlyRs were characterized using protein biochemical approaches as well as electrophysiological recordings and molecular modeling. Molecular modeling of non-glycosylated GlyRα1 did not show major structural alterations. Moreover, non-glycosylation of the GlyRα1N38Q did not prevent the receptor from surface expression. At the functional level, the non-glycosylated GlyR demonstrated reduced glycine potency, but patient GlyR autoantibodies still bound to the surface-expressed non-glycosylated receptor protein in living cells. Efficient adsorption of GlyR autoantibodies from patient samples was possible by binding to native glycosylated and non-glycosylated GlyRα1 expressed in living not fixed transfected HEK293 cells. Binding of patient-derived GlyR autoantibodies to the non-glycosylated GlyRα1 offered the possibility to use purified non-glycosylated GlyR extracellular domain constructs coated on ELISA plates and use them as a fast screening readout for the presence of GlyR autoantibodies in patient serum samples. Following successful adsorption of patient autoantibodies by GlyR ECDs, binding to primary motoneurons and transfected cells was absent. Our results indicate that the glycine receptor autoantibody binding is independent of the receptor’s glycosylation state. Purified non-glycosylated receptor domains harbouring the autoantibody epitope thus provide, an additional reliable experimental tool besides binding to native receptors in cell-based assays for detection of autoantibody presence in patient sera. KW - glycine receptor KW - autoantibodies KW - glycosylation KW - extracellular domain KW - adsorption Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-304206 VL - 16 ER -