{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,18]],"date-time":"2026-03-18T01:34:46Z","timestamp":1773797686818,"version":"3.50.1"},"reference-count":45,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2023,2,13]],"date-time":"2023-02-13T00:00:00Z","timestamp":1676246400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001659","name":"Deutsche Forschungsgemeinschaft","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100001659","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Mol. Neurosci."],"abstract":"<jats:p>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 GlyR\u03b11 has been previously defined to residues <jats:sup>1<\/jats:sup>A-<jats:sup>33<\/jats:sup>G 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 \u03b11 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<jats:italic>-<\/jats:italic>glycosylated GlyR\u03b11 did not show major structural alterations. Moreover, non-glycosylation of the GlyR\u03b11<jats:sup>N38Q<\/jats:sup> 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 GlyR\u03b11 expressed in living not fixed transfected HEK293 cells. Binding of patient-derived GlyR autoantibodies to the non-glycosylated GlyR\u03b11 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\u2019s 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.<\/jats:p>","DOI":"10.3389\/fnmol.2023.1089101","type":"journal-article","created":{"date-parts":[[2023,2,13]],"date-time":"2023-02-13T07:35:46Z","timestamp":1676273746000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":4,"title":["Glycine receptor autoantibody binding to the extracellular domain is independent from receptor glycosylation"],"prefix":"10.3389","volume":"16","author":[{"given":"Vera","family":"Rauschenberger","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Inken","family":"Piro","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Vikram Babu","family":"Kasaragod","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Verena","family":"H\u00f6rlin","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Anna-Lena","family":"Eckes","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Christoph J.","family":"Kluck","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Hermann","family":"Schindelin","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Hans-Michael","family":"Meinck","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jonathan","family":"Wickel","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Christian","family":"Geis","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Erdem","family":"T\u00fcz\u00fcn","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kathrin","family":"Doppler","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Claudia","family":"Sommer","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Carmen","family":"Villmann","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1965","published-online":{"date-parts":[[2023,2,13]]},"reference":[{"key":"ref1","doi-asserted-by":"publisher","first-page":"310","DOI":"10.1016\/j.transci.2019.03.015","article-title":"Role of plasma exchange in stiff person syndrome","volume":"58","author":"Albahra","year":"2019","journal-title":"Transfus. Apher. Sci."},{"key":"ref2","doi-asserted-by":"publisher","first-page":"79","DOI":"10.3389\/fnmol.2015.00079","article-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","volume":"8","author":"Atak","year":"2015","journal-title":"Front. Mol. Neurosci."},{"key":"ref3","doi-asserted-by":"publisher","first-page":"3729","DOI":"10.1002\/j.1460-2075.1993.tb06050.x","article-title":"Residues within transmembrane segment M2 determine chloride conductance of glycine receptor homo- and hetero-oligomers","volume":"12","author":"Bormann","year":"1993","journal-title":"EMBO J."},{"key":"ref4","doi-asserted-by":"publisher","first-page":"1627","DOI":"10.1074\/jbc.M303811200","article-title":"Conserved high affinity ligand binding and membrane association in the native and refolded extracellular domain of the human glycine receptor alpha1-subunit","volume":"279","author":"Breitinger","year":"2004","journal-title":"J. Biol. Chem."},{"key":"ref5","doi-asserted-by":"publisher","first-page":"2178","DOI":"10.1093\/brain\/awu142","article-title":"Glycine receptor antibodies in PERM and related syndromes: characteristics, clinical features and outcomes","volume":"137","author":"Carvajal-Gonzalez","year":"2014","journal-title":"Brain"},{"key":"ref6","doi-asserted-by":"publisher","first-page":"1776","DOI":"10.1038\/mp.2016.125","article-title":"All naturally occurring autoantibodies against the NMDA receptor subunit NR1 have pathogenic potential irrespective of epitope and immunoglobulin class","volume":"22","author":"Castillo-Gomez","year":"2017","journal-title":"Mol. Psychiatry"},{"key":"ref7","doi-asserted-by":"publisher","first-page":"507","DOI":"10.1038\/s41594-022-00772-0","article-title":"Structural insights into binding of therapeutic channel blockers in NMDA receptors","volume":"29","author":"Chou","year":"2022","journal-title":"Nat. Struct. Mol. Biol."},{"key":"ref8","doi-asserted-by":"publisher","first-page":"3398","DOI":"10.1093\/brain\/awz297","article-title":"Glycine receptor autoantibodies disrupt inhibitory neurotransmission","volume":"142","author":"Crisp","year":"2019","journal-title":"Brain"},{"key":"ref9","doi-asserted-by":"publisher","first-page":"839","DOI":"10.1152\/physrev.00010.2016","article-title":"Autoantibodies to synaptic receptors and neuronal cell surface proteins in autoimmune diseases of the central nervous system","volume":"97","author":"Dalmau","year":"2017","journal-title":"Physiol. Rev."},{"key":"ref10","doi-asserted-by":"publisher","first-page":"498","DOI":"10.1001\/jamaneurol.2013.1872","article-title":"Progressive encephalomyelitis with rigidity and myoclonus: the first pediatric case with glycine receptor antibodies","volume":"70","author":"Damasio","year":"2013","journal-title":"JAMA Neurol."},{"key":"ref11","doi-asserted-by":"publisher","first-page":"e186","DOI":"10.1212\/NXI.0000000000000186","article-title":"Lockjaw in stiff-person syndrome with autoantibodies against glycine receptors","volume":"3","author":"Doppler","year":"2016","journal-title":"Neurol Neuroimmunol Neuroinflamm"},{"key":"ref12","doi-asserted-by":"publisher","first-page":"224","DOI":"10.1038\/nature14853","article-title":"Glycine receptor mechanism elucidated by electron cryo-microscopy","volume":"526","author":"Du","year":"2015","journal-title":"Nature"},{"key":"ref13","doi-asserted-by":"publisher","first-page":"414","DOI":"10.1111\/epi.12528","article-title":"Investigation of neuronal autoantibodies in two different focal epilepsy syndromes","volume":"55","author":"Ekizoglu","year":"2014","journal-title":"Epilepsia"},{"key":"ref14","doi-asserted-by":"publisher","first-page":"2126","DOI":"10.1107\/S0907444904019158","article-title":"Coot: model-building tools for molecular graphics","volume":"60","author":"Emsley","year":"2004","journal-title":"Acta Crystallogr. D Biol. Crystallogr."},{"key":"ref15","doi-asserted-by":"publisher","first-page":"11082","DOI":"10.1523\/JNEUROSCI.0064-12.2012","article-title":"Anti-NMDA receptor encephalitis antibody binding is dependent on amino acid identity of a small region within the GluN1 amino terminal domain","volume":"32","author":"Gleichman","year":"2012","journal-title":"J. Neurosci."},{"key":"ref16","doi-asserted-by":"publisher","first-page":"4711","DOI":"10.1093\/emboj\/18.17.4711","article-title":"Molecular determinants of glycine receptor subunit assembly","volume":"18","author":"Griffon","year":"1999","journal-title":"EMBO J."},{"key":"ref17","first-page":"203","article-title":"A new and effective drug in the treatment of the stiff-man syndrome: preliminary report","volume":"38","author":"Howard","year":"1963","journal-title":"Proc. Staff Meet. 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Neurosci."},{"key":"ref21","doi-asserted-by":"publisher","first-page":"e20210012","DOI":"10.1084\/jem.20210012","article-title":"Encephalitis patient-derived monoclonal GABAA receptor antibodies cause epileptic seizures","volume":"218","author":"Kreye","year":"2021","journal-title":"J. Exp. Med."},{"key":"ref22","doi-asserted-by":"publisher","first-page":"7907","DOI":"10.1074\/jbc.M113.528489","article-title":"Specific contactin N-glycans are implicated in neurofascin binding and autoimmune targeting in peripheral neuropathies","volume":"289","author":"Labasque","year":"2014","journal-title":"J. Biol. Chem."},{"key":"ref23","doi-asserted-by":"publisher","first-page":"1051","DOI":"10.1152\/physrev.00042.2003","article-title":"Molecular structure and function of the glycine receptor chloride channel","volume":"84","author":"Lynch","year":"2004","journal-title":"Physiol. 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Apher."},{"key":"ref28","doi-asserted-by":"publisher","first-page":"e127","DOI":"10.1212\/NXI.0000000000000127","article-title":"Caspr2 autoantibodies target multiple epitopes","volume":"2","author":"Olsen","year":"2015","journal-title":"Neurol Neuroimmunol Neuroinflamm"},{"key":"ref29","doi-asserted-by":"publisher","first-page":"1851","DOI":"10.1111\/trf.12573","article-title":"Efficacy of therapeutic plasma exchange for treatment of stiff-person syndrome","volume":"54","author":"Pagano","year":"2014","journal-title":"Transfusion"},{"key":"ref30","doi-asserted-by":"publisher","first-page":"10899","DOI":"10.1038\/s41598-017-11264-3","article-title":"Alpha subunit-dependent glycine receptor clustering and regulation of synaptic receptor numbers","volume":"7","author":"Patrizio","year":"2017","journal-title":"Sci. Rep."},{"key":"ref31","doi-asserted-by":"publisher","first-page":"671","DOI":"10.1046\/j.1537-2995.1999.39070671.x","article-title":"Selective therapeutic extraction of plasma constituents, revisited","volume":"39","author":"Pineda","year":"1999","journal-title":"Transfusion"},{"key":"ref32","doi-asserted-by":"publisher","first-page":"1147","DOI":"10.1093\/glycob\/cwr013","article-title":"Robust post-translocational N-glycosylation at the extreme C-terminus of membrane and secreted proteins in Xenopus laevis oocytes and HEK293 cells","volume":"21","author":"Pult","year":"2011","journal-title":"Glycobiology"},{"key":"ref33","doi-asserted-by":"publisher","first-page":"544","DOI":"10.1002\/ana.25832","article-title":"Glycine receptor autoantibodies impair receptor function and induce motor dysfunction","volume":"88","author":"Rauschenberger","year":"2020","journal-title":"Ann. Neurol."},{"key":"ref34","doi-asserted-by":"publisher","first-page":"131","DOI":"10.1038\/ng0694-131","article-title":"A missense mutation in the gene encoding the alpha 1 subunit of the inhibitory glycine receptor in the spasmodic mouse","volume":"7","author":"Ryan","year":"1994","journal-title":"Nat. Genet."},{"key":"ref35","doi-asserted-by":"publisher","first-page":"71","DOI":"10.1016\/0014-5793(94)00736-5","article-title":"Point mutation of glycine receptor alpha 1 subunit in the spasmodic mouse affects agonist responses","volume":"350","author":"Saul","year":"1994","journal-title":"FEBS Lett."},{"key":"ref36","doi-asserted-by":"publisher","first-page":"422","DOI":"10.1523\/JNEUROSCI.1509-14.2015","article-title":"Disturbed neuronal ER-Golgi sorting of unassembled glycine receptors suggests altered subcellular processing is a cause of human Hyperekplexia","volume":"35","author":"Schaefer","year":"2015","journal-title":"J. Neurosci."},{"key":"ref37","doi-asserted-by":"publisher","first-page":"291","DOI":"10.3389\/fnmol.2018.00291","article-title":"Impaired glycine receptor trafficking in neurological diseases","volume":"11","author":"Schaefer","year":"2018","journal-title":"Front. Mol. Neurosci."},{"key":"ref38","doi-asserted-by":"publisher","first-page":"518","DOI":"10.1002\/mrd.22489","article-title":"The ImageJ ecosystem: an open platform for biomedical image analysis","volume":"82","author":"Schindelin","year":"2015","journal-title":"Mol. Reprod. Dev."},{"key":"ref39","doi-asserted-by":"publisher","first-page":"64","DOI":"10.1046\/j.1526-0968.2001.005001064.x","article-title":"Plasma exchange in stiff-man syndrome","volume":"5","author":"Shariatmadar","year":"2001","journal-title":"Ther. Apher."},{"key":"ref40","doi-asserted-by":"publisher","first-page":"923","DOI":"10.1038\/s41467-022-28559-3","article-title":"Development and characterization of functional antibodies targeting NMDA receptors","volume":"13","author":"Tajima","year":"2022","journal-title":"Nat. Commun."},{"key":"ref41","doi-asserted-by":"publisher","first-page":"17","DOI":"10.1007\/978-1-0716-2732-7_2","article-title":"Peptide microarrays for studying autoantibodies in neurological disease","volume":"2578","author":"Talucci","year":"2023","journal-title":"Methods Mol. Biol."},{"key":"ref42","doi-asserted-by":"crossref","first-page":"1499","DOI":"10.1056\/NEJM198906013202220","article-title":"Plasmapheresis in the treatment of stiff-man syndrome","volume":"320","author":"Vicari","year":"1989","journal-title":"N. Engl. J. Med."},{"key":"ref43","doi-asserted-by":"publisher","first-page":"92","DOI":"10.1111\/j.1749-6632.2012.06789.x","article-title":"Antibodies identified by cell-based assays in myasthenia gravis and associated diseases","volume":"1274","author":"Vincent","year":"2012","journal-title":"Ann. N. Y. Acad. Sci."},{"key":"ref44","doi-asserted-by":"publisher","first-page":"2707","DOI":"10.1016\/j.neuron.2021.08.019","article-title":"Characterization of the subunit composition and structure of adult human glycine receptors","volume":"109","author":"Yu","year":"2021","journal-title":"Neuron"},{"key":"ref45","doi-asserted-by":"publisher","first-page":"513","DOI":"10.1038\/s41586-021-04022-z","article-title":"Architecture and assembly mechanism of native glycine receptors","volume":"599","author":"Zhu","year":"2021","journal-title":"Nature"}],"container-title":["Frontiers in Molecular Neuroscience"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fnmol.2023.1089101\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2023,2,13]],"date-time":"2023-02-13T07:35:51Z","timestamp":1676273751000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fnmol.2023.1089101\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,2,13]]},"references-count":45,"alternative-id":["10.3389\/fnmol.2023.1089101"],"URL":"https:\/\/doi.org\/10.3389\/fnmol.2023.1089101","relation":{},"ISSN":["1662-5099"],"issn-type":[{"value":"1662-5099","type":"electronic"}],"subject":[],"published":{"date-parts":[[2023,2,13]]},"article-number":"1089101"}}