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  - 
TY  - JOUR
A1  - Mietrach, Nicole
A1  - Schlosser, Andreas
A1  - Geibel, Sebastian
T1  - An extracellular domain of the EsaA membrane component of the type VIIb secretion system: expression, purification and crystallization
JF  - Acta Crystallographica Section F
N2  - The membrane protein EsaA is a conserved component of the type VIIb secretion system. Limited proteolysis of purified EsaA from Staphylococcus aureus USA300 identified a stable 48 kDa fragment, which was mapped by fingerprint mass spectrometry to an uncharacterized extracellular segment of EsaA. Analysis by circular dichroism spectroscopy showed that this fragment folds into a single stable domain made of mostly α‐helices with a melting point of 34.5°C. Size‐exclusion chromatography combined with multi‐angle light scattering indicated the formation of a dimer of the purified extracellular domain. Octahedral crystals were grown in 0.2 M ammonium citrate tribasic pH 7.0, 16% PEG 3350 using the hanging‐drop vapor‐diffusion method. Diffraction data were analyzed to 4.0 Å resolution, showing that the crystals belonged to the enantiomorphic tetragonal space groups P41212 or P43212, with unit‐cell parameters a = 197.5, b = 197.5, c = 368.3 Å, α = β = γ = 90°.
KW  - ESAT‐6‐like secretion system
KW  - ESS
KW  - type VII secretion system
KW  - EsaA
KW  - extracellular domain
KW  - Staphylococcus aureus USA300
Y1  - 2019
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-213681
VL  - 75
IS  - 12
ER  -