Development and characterization of functional antibodies targeting NMDA receptors

Tajima, N.; Simorowski, Noriko; Yovanno, Remy A.; Regan, Michael C.; Michalski, Kevin; Gómez, Ricardo; Lau, Albert Y.; Furukawa, Hiro

doi:10.1038/s41467-022-28559-3

Cited by 22 publications

(15 citation statements)

References 72 publications

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“…For developing better and specific antibody-selective immunotherapies, the identification of patient-derived monoclonal antibodies and the characterization of their binding properties to further investigate the disease pathologies is an essential step ( Kreye et al, 2021 ). The binding pattern of patient-derived monoclonal antibodies can be studied using purified full-length receptors or domains in distinct conformations bound to ELISA plates followed by structural analysis, e.g., single particle cryo-EM of combined receptor and autoantibody proteins and functional analyses ( Chou et al, 2022 ; Noviello et al, 2022 ; Tajima et al, 2022 ). Such combined approaches will provide detailed binding pattern pictures of patient-derived autoantibodies opening novel windows for clinical treatment using antibody depletion strategies.…”

Section: Discussionmentioning

confidence: 99%

“…In case of conformational epitopes, however, there are also limitations with such approaches. Recently, binding patterns of patient-derived monoclonal antibodies have been studied using purified full-length receptors or domains in distinct conformations bound to ELISA plates followed by structural analysis, e.g., single particle cryo-EM of combined receptor and autoantibody proteins and functional analyses ( Chou et al, 2022 ; Tajima et al, 2022 ).…”

Section: Introductionmentioning

confidence: 99%

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Glycine receptor autoantibody binding to the extracellular domain is independent from receptor glycosylation

Rauschenberger

Piro²,

Kasaragod³

et al. 2023

Front. Mol. Neurosci.

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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.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Glycine receptor autoantibody binding to the extracellular domain is independent from receptor glycosylation

Rauschenberger

Piro²,

Kasaragod³

et al. 2023

Front. Mol. Neurosci.

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“…Novel approaches to target specific subunits of NMDARs can be achieved through structural-based drug design. Alternative structural-based approaches are demonstrated in the novel study in which, Tajima et al generated an antibody specific for GluN2B-containing receptors that acts to inhibit the function of the channel by stabilizing the closed state of the channel (Tajima et al, 2022). The introduction of structural-based stabilizing antibodies into the CNS to promote the stability of variant receptors is an appealing future application.…”

Section: Clearance Of Unfolded Proteins Is An Indispensable Component...mentioning

confidence: 99%

Protein quality control of N-methyl-D-aspartate receptors

Benske

Wang

2022

Front. Cell. Neurosci.

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N-methyl-D-aspartate receptors (NMDARs) are glutamate-gated cation channels that mediate excitatory neurotransmission and are critical for synaptic development and plasticity in the mammalian central nervous system (CNS). Functional NMDARs typically form via the heterotetrameric assembly of GluN1 and GluN2 subunits. Variants within GRIN genes are implicated in various neurodevelopmental and neuropsychiatric disorders. Due to the significance of NMDAR subunit composition for regional and developmental signaling at synapses, properly folded receptors must reach the plasma membrane for their function. This review focuses on the protein quality control of NMDARs. Specifically, we review the quality control mechanisms that ensure receptors are correctly folded and assembled within the endoplasmic reticulum (ER) and trafficked to the plasma membrane. Further, we discuss disease-associated variants that have shown disrupted NMDAR surface expression and function. Finally, we discuss potential targeted pharmacological and therapeutic approaches to ameliorate disease phenotypes by enhancing the expression and surface trafficking of subunits harboring disease-associated variants, thereby increasing their incorporation into functional receptors.

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“…The conformational transitions of the LBDs from open to closed clamshells result in the generation of tension in the LBD-TMD linkers, which in turn facilitates gating of the TMD channel ( Tajima et al, 2016 ; Chou et al, 2020 ). The ATDs allosterically regulate channel activities in a subtype-dependent manner via distinct interactions with the LBDs ( Yuan et al, 2009 ; Gielen et al, 2009 ; Karakas et al, 2011 ; Tajima et al, 2022 ). Therefore, the LBDs can be considered the fundamental vehicles for driving ligand gating.…”

Section: Introductionmentioning

confidence: 99%

Excitatory and inhibitory D-serine binding to the NMDA receptor

Yovanno

Chou

Brantley

et al. 2022

eLife

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N-methyl-D-aspartate receptors (NMDARs) uniquely require binding of two different neurotransmitter agonists for synaptic transmission. D-serine and glycine bind to one subunit, GluN1, while glutamate binds to the other, GluN2. These agonists bind to the receptor’s bi-lobed ligand-binding domains (LBDs), which close around the agonist during receptor activation. To better understand the unexplored mechanisms by which D-serine contributes to receptor activation, we performed multi-microsecond molecular dynamics simulations of the GluN1/GluN2A LBD dimer with free D-serine and glutamate agonists. Surprisingly, we observed D-serine binding to both GluN1 and GluN2A LBDs, suggesting that D-serine competes with glutamate for binding to GluN2A. This mechanism is confirmed by our electrophysiology experiments, which show that D-serine is indeed inhibitory at high concentrations. Although free energy calculations indicate that D-serine stabilizes the closed GluN2A LBD, its inhibitory behavior suggests that it either does not remain bound long enough or does not generate sufficient force for ion channel gating. We developed a workflow using pathway similarity analysis to identify groups of residues working together to promote binding. These conformation-dependent pathways were not significantly impacted by the presence of N-linked glycans, which act primarily by interacting with the LBD bottom lobe to stabilize the closed LBD.

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Development and characterization of functional antibodies targeting NMDA receptors

Cited by 22 publications

References 72 publications

Glycine receptor autoantibody binding to the extracellular domain is independent from receptor glycosylation

Glycine receptor autoantibody binding to the extracellular domain is independent from receptor glycosylation

Protein quality control of N-methyl-D-aspartate receptors

Excitatory and inhibitory D-serine binding to the NMDA receptor

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