TY - JOUR

T1 - P.0472 Immunoglobulins G hydrolyze recombinant fragments of NMDA receptor NR1 and NR2 subunits in schizophrenia and multiple sclerosis: preliminary results

AU - Ermakov, E.

AU - Kuznitsyna, A.

AU - Smirnova, L.

AU - Parshukova, D.

AU - Surin, A.

AU - Boksha, I.

AU - Ivanova, S.

N1 - This work was supported by the Russian Science Foundation under grant 18-15-00053.

PY - 2021/12

Y1 - 2021/12

N2 - Background: Abnormal N-methyl-D-aspartate receptor (NMDAR) function is known to be associated with the pathogenesis of schizophrenia and other diseases [1]. Autoantibodies against the NR1 and NR2 subunits of the NMDAR are thought to be pathogenic and can significantly affect synaptic function [2]. Anti-NMDAR antibodies can be naturally occurring [3] and are detected in psychosis, autoimmune encephalitis and multiple sclerosis (MS). Among naturally occurring antibodies, immunoglobulins with enzymatic properties, including proteolytic antibodies, have been found [4,5]. Objective: To investigate the ability of naturally occurring IgG isolated from patients with schizophrenia and MS to hydrolyze in vitro recombinant fragments of NMDAR NR1 and NR2 subunits. Methods: Two recombinant proteins, DBD-NMDAR1 and DBD-NMDAR2, containing extracellular domains of NMDAR NR1 and NR2 subunits were developed and synthesized. The proteins comprised of Leuconostoc mesenteroides dextran binding domain (DBD), Gly-Ser spacer, and the sequences of NR1 or NR2 NMDAR extracellular domains. In this pilot study, five patients with paranoid schizophrenia, two MS patients, and two healthy donors were included. IgG fractions were isolated from blood samples of every subject, chromatographically purified on Protein-G-Sepharose and used to analyze their in vitro proteolytic activity. The homogeneity of IgG preparations and the absence of impurities was proved using SDS-PAGE. The reaction mixture contained 0.5 mg/ml of DBD-NMDAR1 or DBD-NMDAR2 proteins, and 0.1 mg/ml IgG. Hydrolysis products were monitored by electrophoresis in polyacrylamide gel after 9 h of incubation in 37°C. The hydrolysis products were also separated by reverse phase high performance liquid chromatography on an Easy-nLc 1000 nanoflow chromatograph (Thermo Scientific, USA), followed by identification on an OrbiTrap Elite mass spectrometer (Thermo Scientific, USA). The results were processed using Thermo Xcalibur Qual Browser and PEAKS Studio-7.5 software. Statistical analysis was carried out using the Wilcoxon signed-rank test module of the Statistica 10 software. Results: We have demonstrated the ability of IgG preparations from patients with schizophrenia or from patients with MS to hydrolyze both DBD-NMDAR1 and DBD-NMDAR2. IgG preparations from MS patients hydrolyzed these proteins more efficiently than IgG from schizophrenic patients. IgG from schizophrenia patients hydrolyzed different regions (control DBD domain or NR1 or NR2 domains) of recombinant proteins with different efficiencies. In the case of DBD-NMDAR1, the number of identified peptides (resulting from hydrolysis) from the NR1 domain (median value: 23 peptides) was significantly higher (p = 0.005) than from the control DBD domain (10 peptides). In the case of DBD-NMDAR2, the number of identified peptides from the NR2 domain (60 peptides) was significantly higher (p = 0.017) than from the control DBD domain (45 peptides). Interestingly, hydrolysis of DBD-NMDAR2 produced more peptides than hydrolysis of DBD-NMDAR1. Conclusion: Based on these data, it can be assumed that IgG from patients with schizophrenia or MS can hydrolyze the extracellular fragments of NMDAR NR1 and NR2 subunits. Conflict of interest Disclosure statement: This work was supported by the Russian Science Foundation under grant 18-15-00053.

AB - Background: Abnormal N-methyl-D-aspartate receptor (NMDAR) function is known to be associated with the pathogenesis of schizophrenia and other diseases [1]. Autoantibodies against the NR1 and NR2 subunits of the NMDAR are thought to be pathogenic and can significantly affect synaptic function [2]. Anti-NMDAR antibodies can be naturally occurring [3] and are detected in psychosis, autoimmune encephalitis and multiple sclerosis (MS). Among naturally occurring antibodies, immunoglobulins with enzymatic properties, including proteolytic antibodies, have been found [4,5]. Objective: To investigate the ability of naturally occurring IgG isolated from patients with schizophrenia and MS to hydrolyze in vitro recombinant fragments of NMDAR NR1 and NR2 subunits. Methods: Two recombinant proteins, DBD-NMDAR1 and DBD-NMDAR2, containing extracellular domains of NMDAR NR1 and NR2 subunits were developed and synthesized. The proteins comprised of Leuconostoc mesenteroides dextran binding domain (DBD), Gly-Ser spacer, and the sequences of NR1 or NR2 NMDAR extracellular domains. In this pilot study, five patients with paranoid schizophrenia, two MS patients, and two healthy donors were included. IgG fractions were isolated from blood samples of every subject, chromatographically purified on Protein-G-Sepharose and used to analyze their in vitro proteolytic activity. The homogeneity of IgG preparations and the absence of impurities was proved using SDS-PAGE. The reaction mixture contained 0.5 mg/ml of DBD-NMDAR1 or DBD-NMDAR2 proteins, and 0.1 mg/ml IgG. Hydrolysis products were monitored by electrophoresis in polyacrylamide gel after 9 h of incubation in 37°C. The hydrolysis products were also separated by reverse phase high performance liquid chromatography on an Easy-nLc 1000 nanoflow chromatograph (Thermo Scientific, USA), followed by identification on an OrbiTrap Elite mass spectrometer (Thermo Scientific, USA). The results were processed using Thermo Xcalibur Qual Browser and PEAKS Studio-7.5 software. Statistical analysis was carried out using the Wilcoxon signed-rank test module of the Statistica 10 software. Results: We have demonstrated the ability of IgG preparations from patients with schizophrenia or from patients with MS to hydrolyze both DBD-NMDAR1 and DBD-NMDAR2. IgG preparations from MS patients hydrolyzed these proteins more efficiently than IgG from schizophrenic patients. IgG from schizophrenia patients hydrolyzed different regions (control DBD domain or NR1 or NR2 domains) of recombinant proteins with different efficiencies. In the case of DBD-NMDAR1, the number of identified peptides (resulting from hydrolysis) from the NR1 domain (median value: 23 peptides) was significantly higher (p = 0.005) than from the control DBD domain (10 peptides). In the case of DBD-NMDAR2, the number of identified peptides from the NR2 domain (60 peptides) was significantly higher (p = 0.017) than from the control DBD domain (45 peptides). Interestingly, hydrolysis of DBD-NMDAR2 produced more peptides than hydrolysis of DBD-NMDAR1. Conclusion: Based on these data, it can be assumed that IgG from patients with schizophrenia or MS can hydrolyze the extracellular fragments of NMDAR NR1 and NR2 subunits. Conflict of interest Disclosure statement: This work was supported by the Russian Science Foundation under grant 18-15-00053.

UR - https://www.mendeley.com/catalogue/328f2b4b-8f36-3655-ba32-cc540e15750a/

U2 - 10.1016/j.euroneuro.2021.10.445

DO - 10.1016/j.euroneuro.2021.10.445

M3 - Meeting Abstract

VL - 53

SP - S347-S348

JO - European Neuropsychopharmacology

JF - European Neuropsychopharmacology

SN - 0924-977X

ER -