@article{RauschenbergerPiroKasaragodetal.2023,
  author    = {Rauschenberger, Vera and Piro, Inken and Kasaragod, Vikram Babu and H{\"o}rlin, Verena and Eckes, Anna-Lena and Kluck, Christoph J. and Schindelin, Hermann and Meinck, Hans-Michael and Wickel, Jonathan and Geis, Christian and T{\"u}z{\"u}n, Erdem and Doppler, Kathrin and Sommer, Claudia and Villmann, Carmen},
  title     = {Glycine receptor autoantibody binding to the extracellular domain is independent from receptor glycosylation},
  series = {Frontiers in Molecular Neuroscience},
  volume    = {16},
  journal   = {Frontiers in Molecular Neuroscience},
  doi       = {10.3389/fnmol.2023.1089101},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-304206},
  year      = {2023},
  abstract  = {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.},
  language  = {en}
}
@article{AtakLanglhoferSchaeferetal.2015,
  author    = {Atak, Sinem and Langlhofer, Georg and Schaefer, Natascha and Kessler, Denise and Meiselbach, Heike and Delto, Carolyn and Schindelin, Hermann and Villmann, Carmen},
  title     = {Disturbances of ligand potency and enhanced degradation of the human glycine receptor at affected positions G160 and T162 originally identified in patients suffering from hyperekplexia},
  series = {Frontiers in Molecular Neuroscience},
  volume    = {8},
  journal   = {Frontiers in Molecular Neuroscience},
  number    = {79},
  doi       = {10.3389/fnmol.2015.00079},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-144818},
  year      = {2015},
  abstract  = {Ligand-binding of Cys-loop receptors is determined by N-terminal extracellular loop structures from the plus as well as from the minus side of two adjacent subunits in the pentameric receptor complex. An aromatic residue in loop B of the glycine receptor (GIyR) undergoes direct interaction with the incoming ligand via a cation-π interaction. Recently, we showed that mutated residues in loop B identified from human patients suffering from hyperekplexia disturb ligand-binding. Here, we exchanged the affected human residues by amino acids found in related members of the Cys-loop receptor family to determine the effects of side chain volume for ion channel properties. GIyR variants were characterized in vitro following transfection into cell lines in order to analyze protein expression, trafficking, degradation and ion channel function. GIyR α1 G160 mutations significantly decrease glycine potency arguing for a positional effect on neighboring aromatic residues and consequently glycine-binding within the ligand-binding pocket. Disturbed glycinergic inhibition due to T162 α1 mutations is an additive effect of affected biogenesis and structural changes within the ligand-binding site. Protein trafficking from the ER toward the ER-Golgi intermediate compartment, the secretory Golgi pathways and finally the cell surface is largely diminished, but still sufficient to deliver ion channels that are functional at least at high glycine concentrations. The majority of T162 mutant protein accumulates in the ER and is delivered to ER-associated proteasomal degradation. Hence, G160 is an important determinant during glycine binding. In contrast, 1162 affects primarily receptor biogenesis whereas exchanges in functionality are secondary effects thereof.},
  language  = {en}
}