Journal: Frontiers in Molecular Neuroscience

Article Title: Structural and functional characterization of the IgSF21-neurexin2α complex and its related signaling pathways in the regulation of inhibitory synapse organization

doi: 10.3389/fnmol.2024.1371145

Figure Lengend Snippet: Neurexin2α acts as a high-affinity receptor for IgSF21 to mediate GABAergic presynaptic differentiation. (A) Identification of IgSF21-interacting proteins by unbiased pulldown assays coupled to tandem mass-spectrometry analysis. IgSF21-Fc protein coated on magnetic beads was used as bait. Following mass-spectrometry and SAINT analysis, candidates with a Bayesian false discovery rate (BFDR) of 3% or less were selected. The average of total and unique spectral counts for selected preys are shown ( n = 2 independent experiments). (B) Co-immunoprecipitation assays from mouse whole brain crude synaptosomal fractions. Anti-IgSF21 co-immunoprecipitated endogenous α-Nrxns. Rabbit IgG was used as a negative control. (C) Pulldown assays using highly purified IgSF21-His with Nrxn2α-Fc, Nrxn1β-Fc ectodomains or Fc alone. (D) Surface plasmon resonance (SPR) assay using immobilized IgSF21-His and soluble Nrxn2α-Fc at a concentration range from 0 to 200 nM (colored lines). Representative sensorgrams of Nrxn2α-Fc protein fitted to a 1:1 biomolecular interaction model (black line) are shown with K D (affinity), k on (on-rate) and k off (off-rate) as indicated. IgSF21 binds Nrxn2α with nanomolar affinity ( K D : 5.67 nM). (E,F) Representative fluorescence images of VGAT accumulation induced by HEK293T cells expressing IgSF21-HA (E) or HA-Nlgn2 (F) cocultured with primary mouse hippocampal neurons derived from Nrxn2α knockout (KO) and wild-type (WT) littermates. The scale bar represents 10 μm. (G,H) Quantification of the total integrated intensity of VGAT accumulated on the cell surface of HEK293T cells expressing IgSF21-HA (G) or HA-Nlgn2 (H) cocultured with neurons derived from Nrxn2α WT or KO littermates, divided by the cell surface area for each HEK293T cell and normalized to the value for Nrxn2α WT. Statistical significance was examined with a Mann–Whitney test, *⁣*⁣** p < 0.0001, ns: not significant. n = 30 cells from three independent experiments. Data are presented as mean ± SEM. (I) Immunoblots of rat forebrain fractionation samples probed for IgSF21, presynaptic synaptophysin, and postsynaptic PSD95. The Triton-soluble and -insoluble fractions were enriched for synaptophysin and PSD-95, respectively. The red asterisks indicate non-specific bands.

Article Snippet: Recombinant human His-tagged IgSF21 protein (R&D Systems; #10330-S2) was immobilized on carboxymethylated dextran CM5 sensor chips (Cytiva) using an amine-coupling strategy.

Techniques: Mass Spectrometry, Magnetic Beads, Immunoprecipitation, Negative Control, Purification, SPR Assay, Concentration Assay, Fluorescence, Expressing, Derivative Assay, Knock-Out, MANN-WHITNEY, Western Blot, Fractionation

Journal: Frontiers in Molecular Neuroscience

Article Title: Structural and functional characterization of the IgSF21-neurexin2α complex and its related signaling pathways in the regulation of inhibitory synapse organization

doi: 10.3389/fnmol.2024.1371145

Figure Lengend Snippet: AlphaFold2 prediction of the interaction interface between the Nrxn2α LNS1 and IgSF21 Ig1 domains. (A) Diagram representing the domain structure of the IgSF21-Nrxn2α protein complex. IgSF21 (pink) is tethered to the cell membrane with a GPI-anchor. The LNS1 domain of Nrxn2α (green) interacts with the Ig1 domain of IgSF21. TM, transmembrane domain; CT, cytoplasmic tail. A conformation-independent representation of Nrxn2α is shown. (B) AlphaFold2 prediction of the LNS1 domain of Nrxn2α (green) in complex with the Ig1 domain of IgSF21 (pink). The electrostatic potential mapped onto the solvent accessible surface as calculated by PyMOL is shown together with ribbon diagrams of the proteins with positively and negatively charged regions depicted in blue and red, respectively. (C) Close-up view of the predicted binding interface between the Nrxn2α LNS1 and IgSF21 Ig1 domains, shown in two views rotated by ∼180°. Color code: carbon atoms in green (Nrxn2α) or pink (IgSF21), oxygen atoms in red, nitrogen atoms in blue, and sulfur atoms in yellow. Interactions discussed in the text are underlined and represent possible hydrogen bonds in the 2.7–3.8 Å range. Underlined residues are predicted to participate in hydrogen bonding by utilizing the polypeptide backbone. (D) Summary of residues predicted by AlphaFold2 to interact between the Nrxn2α LNS1 and IgSF21 Ig1 domains. The chemical nature of the interaction between specific residues is also indicated (falling in the range of 2.7–3.8 Å) with hydrogen bonds between both side chain and main chain atoms considered. Residues targeted for mutagenesis are indicated in bold.

Article Snippet: Recombinant human His-tagged IgSF21 protein (R&D Systems; #10330-S2) was immobilized on carboxymethylated dextran CM5 sensor chips (Cytiva) using an amine-coupling strategy.

Techniques: Membrane, Solvent, Binding Assay, Mutagenesis

Journal: Frontiers in Molecular Neuroscience

Article Title: Structural and functional characterization of the IgSF21-neurexin2α complex and its related signaling pathways in the regulation of inhibitory synapse organization

doi: 10.3389/fnmol.2024.1371145

Figure Lengend Snippet: Site-directed mutagenesis at the predicted binding interface abolishes the interaction between IgSF21 and Nrxn2α. (A) Representative fluorescence images of soluble Nrxn2α-Fc bound to IgSF21-HA WT and mutants expressed in COS7 cells. HA-CD4 was used as a control for non-specific binding. The scale bar represents 50 μm. (B) Quantification of Nrxn2α-Fc bound to transfected cells described in (A) . The bound Nrxn2α-Fc signal was divided by the HA surface signal and normalized to the value for IgSF21 WT. The color codes in (B) as well as in (C,E,F) indicate the type of substitution: glycosylation insertion in brown, conservative replacement in green, charge reversal in magenta and charge removal in dark blue. Statistical significance was examined by a Kruskal–Wallis test with Dunn’s post hoc analysis for each condition compared to IgSF21 WT. **** p < 0.0001, ns: not significant. n ≥ 19 cells from three independent experiments. Data are presented as mean ± SEM. (C) Quantification of the expression of IgSF21-HA WT and mutants on the surface of COS7 cells normalized to the value for IgSF21-HA WT. Statistical significance was examined by a Kruskal–Wallis test with Dunn’s post hoc analysis for each condition compared to IgSF21 WT. ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns: not significant. n ≥ 19 cells from three independent experiments. Data are presented as mean ± SEM. (D) Representative fluorescence images of soluble IgSF21-His bound to HA-Nrxn2α LNS1-3 WT or HA-Nrxn2α LNS1-3 mutant (R 160 A/L 161 A/S 162 A) expressed in COS7 cells. Nrxn1β is used as a control for non-specific binding. The scale bar represents 50 μm. (E) Quantification of IgSF21-His bound to transfected cells described in (D) . The bound IgSF21-His signal was divided by the HA surface signal and normalized to the value for Nrxn2α LNS1-3 WT. Statistical significance was examined by Welch’s ANOVA with Dunnett’s T3 post hoc analysis for each condition compared to Nrxn2α LNS1-3 WT. **** p < 0.0001. n ≥ 30 cells from three independent experiments. Data are presented as mean ± SEM. (F) Quantification of the expression of Nrxn2α LNS1-3 WT and mutant on the surface of COS7 cells normalized to the value for Nrxn2α WT. Statistical significance was examined by an unpaired t -test. ns: not significant. n ≥ 30 cells from three independent experiments. Data are presented as mean ± SEM.

Article Snippet: Recombinant human His-tagged IgSF21 protein (R&D Systems; #10330-S2) was immobilized on carboxymethylated dextran CM5 sensor chips (Cytiva) using an amine-coupling strategy.

Techniques: Mutagenesis, Binding Assay, Fluorescence, Control, Transfection, Glycoproteomics, Expressing

Journal: Frontiers in Molecular Neuroscience

Article Title: Structural and functional characterization of the IgSF21-neurexin2α complex and its related signaling pathways in the regulation of inhibitory synapse organization

doi: 10.3389/fnmol.2024.1371145

Figure Lengend Snippet: Site-directed mutagenesis at the predicted binding interface abolishes synaptogenic activity of IgSF21. (A) Representative fluorescence images of VGAT accumulation induced by HEK293T cells expressing IgSF21-HA WT or mutants cocultured with hippocampal neurons. HA-CD4 is used as a negative control. The scale bar represents 10 μm. (B) Quantification of the total integrated intensity of VGAT accumulated on the cell surface of HEK293T expressing IgSF21-HA WT and mutants or HA-CD4, divided by the cell surface area for each HEK293T cell and normalized to the value for IgSF21-HA WT. The color codes in (B) indicate the type of substitution: glycosylation insertion in brown and conservative replacement in green. Statistical significance was examined by a Kruskal–Wallis test with Dunn’s post hoc analysis for each condition compared to IgSF21 WT. **** p < 0.0001, ns: not significant. n ≥ 28 cells from three independent experiments. Data are presented as mean ± SEM. (C) Quantification of the expression of IgSF21-HA WT and mutants on the surface of HEK293T cells normalized to the value for IgSF21-HA WT. Statistical significance was examined by a Kruskal–Wallis test with Dunn’s post hoc analysis for each condition compared to IgSF21 WT. ** p < 0.01, *** p < 0.001, ns: not significant. n ≥ 28 cells from three independent experiments. Data are presented as mean ± SEM.

Article Snippet: Recombinant human His-tagged IgSF21 protein (R&D Systems; #10330-S2) was immobilized on carboxymethylated dextran CM5 sensor chips (Cytiva) using an amine-coupling strategy.

Techniques: Mutagenesis, Binding Assay, Activity Assay, Fluorescence, Expressing, Negative Control, Glycoproteomics

Journal: Frontiers in Molecular Neuroscience

Article Title: Structural and functional characterization of the IgSF21-neurexin2α complex and its related signaling pathways in the regulation of inhibitory synapse organization

doi: 10.3389/fnmol.2024.1371145

Figure Lengend Snippet: Overexpression of IgSF21 and Nlgn2 in neurons has distinct effects on somatic and dendritic GABAergic presynaptic differentiation. (A,B) Representative fluorescence images of VGAT (A) and VGLUT1 (B) immunoreactivity along dendrites of neurons transfected with IRES-GFP or IgSF21-IRES-GFP DNA. The scale bar represents 10 μm. (C,D) Quantification of the average intensity of VGAT puncta (C) and VGLUT1 puncta (D) along dendrites of IRES-GFP- or IgSF21-IRES-GFP-transfected neurons. Statistical significance was examined by a Mann–Whitney test (C) or an unpaired t -test (D) . **** p < 0.0001, ns: not significant. n = 30 cells from three independent experiments. Data are presented as mean ± SEM. (E) Representative fluorescence images of VGAT immunoreactivity around cell bodies of neurons transfected with IRES-GFP or IgSF21-IRES-GFP DNA constructs. The scale bar represents 10 μm. (F) Quantification of the average intensity of VGAT puncta around cell bodies of IRES-GFP- or IgSF21-IRES-GFP-transfected neurons. Statistical significance was examined by an unpaired t -test. ns: not significant. n = 30 cells from three independent experiments. Data are presented as mean ± SEM. (G) Representative fluorescence images of VGAT immunoreactivity along dendrites of neurons transfected with HA-CD4 or HA-Nlgn2. The scale bar represents 10 μm. (H) Quantification of the average intensity of VGAT puncta along dendrites of neurons expressing HA-CD4 or HA-Nlgn2. Statistical significance was examined by a Welch’s t -test. **** p < 0.0001. n ≥ 33 cells from three independent experiments. Data are presented as mean ± SEM. (I) Representative fluorescence images of VGAT immunoreactivity around cell bodies of neurons transfected with HA-CD4 or HA-Nlgn2. The scale bar represents 10 μm. (J) Quantification of the average intensity of VGAT puncta around cell bodies of neurons expressing HA-CD4 or HA-Nlgn2. Statistical significance was examined by a Mann–Whitney test. **** p < 0.0001. n ≥ 28 cells from three independent experiments. Data are presented as mean ± SEM. (K) Representative fluorescence images of IgSF21 and GFP immunostaining in primary rat hippocampal neurons transfected with IRES-GFP or IgSF21-IRES-GFP DNA constructs. The scale bar represents 100 μm.

Article Snippet: Recombinant human His-tagged IgSF21 protein (R&D Systems; #10330-S2) was immobilized on carboxymethylated dextran CM5 sensor chips (Cytiva) using an amine-coupling strategy.

Techniques: Over Expression, Fluorescence, Transfection, MANN-WHITNEY, Construct, Expressing, Immunostaining

Journal: Frontiers in Molecular Neuroscience

Article Title: Structural and functional characterization of the IgSF21-neurexin2α complex and its related signaling pathways in the regulation of inhibitory synapse organization

doi: 10.3389/fnmol.2024.1371145

Figure Lengend Snippet: IgSF21 and Nlgn2 rely on distinct signaling pathways that partially overlap to induce GABAergic presynaptic specialization. (A,B) Representatives fluorescence images of VGAT accumulation induced by HEK293T cells expressing IgSF21-HA (A) or HA-Nlgn2 (B) cocultured with hippocampal neurons in the presence of pharmacological inhibitors of different signaling pathways. In all experiments, rat primary hippocampal neurons were treated at 14 days in vitro (DIV) with the indicated inhibitor 1 h before the seeding of transfected HEK293T cells and for another 24 h prior to immunocytochemistry. A JNK inhibitor (SP600125, 25 μM), two CaMKII inhibitors (KN93, 5 μM; XII, 10 μM), a Src inhibitor (PP2, 10 μM), a PKA inhibitor (PKI, 0.1 μM), a PKC inhibitor [Bisindolylmaleimide I (BIS), 4 μM], and a MEK1/2 inhibitor (PD98059, 50 μM) were used. DMSO is the vehicle-treated condition. The scale bar represents 10 μm. (C,D) Quantification of the total integrated intensity of VGAT accumulated on the cell surface of HEK293T cells expressing IgSF21-HA (C) or HA-Nlgn2 (D) and treated with the indicated inhibitor, divided by the cell surface area for each HEK293T cell and normalized to the value for the DMSO-treated control. Statistical significance was examined by a Kruskal–Wallis test with Dunn’s post hoc analysis for each condition compared to the DMSO-treated control. *** p < 0.001, **** p < 0.0001. n ≥ 27 cells from three independent experiments. Data are presented as mean ± SEM. (E,F) Representatives fluorescence images of VGAT accumulation induced by HEK293T cells expressing IgSF21-HA (E) or HA-Nlgn2 (F) cocultured with hippocampal neurons in presence of inhibitors of microtubule (Nocodazole, 3.3 μM) or actin filament assembly [cytochalasin D (Cyto D), 2 μM]. In all experiments, rat primary hippocampal neurons were treated at 14 DIV with the indicated compounds 1 h before the seeding of transfected HEK293T cells and for another 24 h prior to immunocytochemistry. DMSO is the vehicle-treated condition. The scale bar represents 10 μm. (G,H) Quantification of the total integrated intensity of VGAT accumulated on the cell surface of HEK293T cells expressing IgSF21-HA (G) or HA-Nlgn2 (H) and treated with the indicated inhibitor, divided by the cell surface area for each HEK293T cell and normalized to the value for the DMSO-treated control. Statistical significance was examined by a Kruskal–Wallis test with Dunn’s post hoc analysis for each condition compared to the DMSO-treated control. ** p < 0.01, *** p < 0.001, **** p < 0.0001. n ≥ 24 cells from three independent experiments. Data are presented as mean ± SEM.

Article Snippet: Recombinant human His-tagged IgSF21 protein (R&D Systems; #10330-S2) was immobilized on carboxymethylated dextran CM5 sensor chips (Cytiva) using an amine-coupling strategy.

Techniques: Protein-Protein interactions, Fluorescence, Expressing, In Vitro, Transfection, Immunocytochemistry, Control

Journal: Frontiers in Molecular Neuroscience

Article Title: Structural and functional characterization of the IgSF21-neurexin2α complex and its related signaling pathways in the regulation of inhibitory synapse organization

doi: 10.3389/fnmol.2024.1371145

Figure Lengend Snippet: Multiple kinases underlying presynaptic specialization induced by IgSF21 and Nlgn2 display distinct subcellular localizations. Images of western blots showing rat forebrain synaptosome and the Triton-soluble and -insoluble synaptosome fractions probed for JNK1/2/3, CaMKIIα, CaMKIIδ, Fyn and Src. JNK and CaMKIIδ, but not CaMKIIα, are enriched in the Triton-soluble fraction, which represents the presynaptic side as shown in . Fyn and Src are slightly expressed in the Triton-soluble fraction and enriched in the Triton-insoluble fraction, which represents the postsynaptic side.

Article Snippet: Recombinant human His-tagged IgSF21 protein (R&D Systems; #10330-S2) was immobilized on carboxymethylated dextran CM5 sensor chips (Cytiva) using an amine-coupling strategy.

Techniques: Western Blot

Journal: Frontiers in Molecular Neuroscience

Article Title: Structural and functional characterization of the IgSF21-neurexin2α complex and its related signaling pathways in the regulation of inhibitory synapse organization

doi: 10.3389/fnmol.2024.1371145

Figure Lengend Snippet: JNK signaling is not involved in the maintenance of induced or native synapses. (A,B) Representatives fluorescence images of VGAT accumulation induced by HEK293T cells expressing IgSF21-HA (A) or HA-Nlgn2 (B) cocultured with hippocampal neurons in the presence of a JNK inhibitor (SP600125, 25 μM). In all experiments, IgSF21-HA- or HA-Nlgn2-expressing HEK293T cells were seeded on rat primary hippocampal neurons at 14 DIV and incubated for 17 h before treatment with DMSO or SP600125 for 7 h prior to immunocytochemistry. DMSO is the vehicle-treated condition. The scale bar represents 10 μm. (C,D) Quantification of the total integrated intensity of VGAT accumulated on the cell surface of HEK293T cells expressing IgSF21-HA (C) or HA-Nlgn2 (D) and treated with the indicated inhibitors, divided by the cell surface area for each HEK293T cell and normalized to the value for the DMSO-treated control. Statistical significance was examined by unpaired t -tests. ns: not significant. n ≥ 21 cells from two independent experiments. Data are presented as mean ± SEM. (E) Representative fluorescence images of native VGAT and VGLUT1 puncta as well as acetylated α-tubulin in the presence of a JNK inhibitor (SP600125, 25 μM). In all experiments, rat primary hippocampal neurons were treated at 14 DIV with the indicated inhibitor for 24 h prior to immunocytochemistry analysis. DMSO is the vehicle-treated condition. The scale bar represents 10 μm. (F,H) Quantification of VGAT (F) or VGLUT1 (H) puncta density in DMSO- or SP600125-treated neurons and divided by neurite length. Statistical significance was examined by a Welch’s t -test (F) or an unpaired t -test (H) . ns: not significant. n ≥ 29 cells from three independent experiments. Data are presented as mean ± SEM. (G,I) Quantification of VGAT (G) or VGLUT1 (I) puncta size in DMSO- or SP600125-treated neurons. Statistical significance was examined by a Kolmogorov–Smirnov test. ns: not significant. n ≥ 29 cells from three independent experiments. Data are presented as a cumulative frequency distribution.

Article Snippet: Recombinant human His-tagged IgSF21 protein (R&D Systems; #10330-S2) was immobilized on carboxymethylated dextran CM5 sensor chips (Cytiva) using an amine-coupling strategy.

Techniques: Fluorescence, Expressing, Incubation, Immunocytochemistry, Control

Journal: AAPS Open

Article Title: A competitive ligand-binding assay for the detection of neutralizing antibodies against dostarlimab (TSR-042)

doi: 10.1186/s41120-021-00039-w

Figure Lengend Snippet: Fig. 1 NAb detection assay. A Complexes form between the fixed amount of added drug and NAb; the formation of these complexes allows SULFO-TAG–labeled PD-L1 to bind to biotinylated PD-1, which can bind to the streptavidin-ECL plate. B In the absence of NAb, the fixed amount of drug added to the solution binds to biotinylated PD-1 and prevents its interaction with PD-L1. SULFO-TAG–labeled PD-L1 bound to PD-1 is necessary to generate the ECL signal. ECL, electrochemiluminescence; NAb, neutralizing antibody; PD-1, programmed death 1; PD-L1, programmed death ligand 1

Article Snippet: Critical reagents for this assay were as follows: a mouse anti-dostarlimab mAb (Precision Antibodies, Columbia, MD) generated from clone #6G10 at a concentration of 1.15 mg/mL; dostarlimab (WuXi, Philadelphia, PA) at a concentration of 20.7 mg/mL; biotinylated human PD-1 (ACRO Biosystems, Newark, DE) at a concentration of 200 μg/mL; and SULFO-TAG–labeled human PD-L1 (CR0; Meso Scale Diagnostics [MSD], Rockville, MD) at a concentration of 2106 μg/mL.

Techniques: Detection Assay, Labeling, Electrochemiluminescence