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  - Drehmann, Paul
A1  - Milanos, Sinem
A1  - Schaefer, Natascha
A1  - Kasaragod, Vikram Babu
A1  - Herterich, Sarah
A1  - Holzbach-Eberle, Ulrike
A1  - Harvey, Robert J.
A1  - Villmann, Carmen
T1  - Dual role of dysfunctional Asc-1 transporter in distinct human pathologies, human startle disease, and developmental delay
JF  - eNeuro
N2  - Human startle disease is associated with mutations in distinct genes encoding glycine receptors, transporters or interacting proteins at glycinergic synapses in spinal cord and brainstem. However, a significant number of diagnosed patients does not carry a mutation in the common genes GLRA1, GLRB, and SLC6A5. Recently, studies on solute carrier 7 subfamily 10 (SLC7A10; Asc-1, alanine-serine-cysteine transporter) knock-out (KO) mice displaying a startle disease-like phenotype hypothesized that this transporter might represent a novel candidate for human startle disease. Here, we screened 51 patients from our patient cohort negative for the common genes and found three exonic (one missense, two synonymous), seven intronic, and single nucleotide changes in the 5′ and 3′ untranslated regions (UTRs) in Asc-1. The identified missense mutation Asc-1\(^{G307R}\) from a patient with startle disease and developmental delay was investigated in functional studies. At the molecular level, the mutation Asc-1\(^{G307R}\) did not interfere with cell-surface expression, but disrupted glycine uptake. Substitution of glycine at position 307 to other amino acids, e.g., to alanine or tryptophan did not affect trafficking or glycine transport. By contrast, G307K disrupted glycine transport similar to the G307R mutation found in the patient. Structurally, the disrupted function in variants carrying positively charged residues can be explained by local structural rearrangements because of the large positively charged side chain. Thus, our data suggest that SLC7A10 may represent a rare but novel gene associated with human startle disease and developmental delay.
KW  - Asc-1 transporter
KW  - candidate gene
KW  - glycine receptor
KW  - glycine uptake
KW  - human startle disease
KW  - NMDAR
Y1  - 2023
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-349947
VL  - 10
IS  - 11
ER  -