Membrane-bound and soluble forms of an NMDA receptor extracellular domain retain epitopes targeted in auto-immune encephalitis

Abstract

Background: Anti-NMDA receptor encephalitis (ANRE) is a potentially lethal disease attributed to auto-antibodies against the N-methyl-D-aspartate receptor (NMDAR). Full recovery is possible if therapy is initiated early in the disease course. Detection of ANRE antibodies in the cerebrospinal fluid (CSF) is essential for diagnosis. The assays for ANRE-associated IgGs often rely on cells transiently transfected with NMDAR genes. A cell line that stably expresses pathogenic NMDAR epitopes could improve standardization of the assays and provide antigen that could be used in commercial solid state assay systems.

Results: We expressed the amino terminal domain (ATD) of the GluN1 NMDAR subunit (NR1) as a fusion protein on the outer plasma membrane of 293T cells, creating a stable cell population (293T-ATD) that is recognized by ANRE patient monoclonal antibodies in flow cytometry and immunofluorescence assays. The ATD fusion protein also contains a Myc tag and a 6XHIS tag, which provide functionality for immunoassays and antigen purification, and a TEV protease site, which allows the ATD domain to be specifically released from the cells in essentially pure form. ATD mobilized from the 293T ATD cell line maintained the pathogenic ANRE epitopes in ELISA binding assays. CSF (3/4) and sera (4/4) from ANRE patients also bound the 293T-ATD cell line, whereas normal CSF and sera did not.

Conclusions: The 293T-ATD cell line is potentially adaptable to a variety of formats to identify antibodies associated with ANRE, including cell-based and soluble antigen formats, and demonstrates a useful method to produce complex proteins for research, drug discovery, and clinical diagnosis.

Keywords: ANRE; Anti-N-methyl-D-aspartate receptor encephalitis; Antigen; Autoimmune encephalitis; Autoimmunity; Conformational epitope; Monoclonal antibody; NMDA receptor; Recombinant protein expression; TEV protease.

Fig. 1

Structure and expression of the NR1 Amino Terminal Domain (ATD) fusion protein on 293 T cells. (a, b) The ATD fusion protein consists of the entire 561 N-terminal amino acid extracellular domain, which includes the bi-lobed NR1 ATD, fused in sequence to the Myc tag, the 6XHIS tag, the Tobacco Etch Virus (TEV) protease site, and the platelet derived growth factor receptor (PDGFR) transmembrane domain. The cartoon is not strictly drawn to scale. Color code: blue and green; extracellular GluN1 bi-lobed domain; purple, Myc tag; light green, 6XHIS tag; orange, TeV protease site; brown, PDGFR transmembrane domain. (c, d) Expression of the ATD fusion protein on the surface of 293T cells was analyzed by flow cytometry with a commercial anti-GluN1 mAb either alone (c) or with an anti-Myc tag mAb (d)

Fig. 2

Binding of human ANRE mAbs to 293T-ATD cells by flow cytometry. Cells were immunostained with a commercial anti-NR1 mAb and a human mAb and analyzed by flow cytometry. Human mAbs were either the isotype control IgG 6A (a), or ANRE patient mAbs 5F5 (b), 2G6 (c), or 1D1 (d)

Fig. 3

Immunofluorescence imaging of the NR1 ATD fusion protein on 293T-ATD cells. 293T-ATD cells were immunostained with a murine anti-NR1 mAb (red color, left panel) and the anti-Myc-tag mAb (green color, middle panel). A merged image is also shown (right panel). Nuclei were stained with DAPI, and the cells were visualized by confocal microscopy. Scale bar = 10 μm

Fig. 4

Binding of human ANRE patient CSF and mAbs to 293T-ATD cells by immunofluorescence. 293T-ATD cells were stained with ANRE patient CSF, human ANRE mAbs 5F5, 2G6, and 1D1, or the 8E1 isotype control mAb (red). Nuclei were stained with DAPI (blue) and the cells were visualized by confocal microscopy. Scale bar = 10 μm

Fig. 5

Mobilization of membrane-bound ATD with TEV protease. 293T-ATD cells were washed and then treated with TEV protease for 10, 20, 30, or 40 min. Expressed protein was analyzed by (a) capture ELISA and (b) Coomassie-stained SDS:PAGE. ATD, amino-terminal domain; Control, medium-only blank; M, marker; RLV, relative luminescence value. Bars indicate the S.E.M.

Fig. 6

Binding of human anti-NR1 mAbs to plate-adherent ATD. ATD mobilized by TEV protease treatment of 293T-ATD cells was captured by a Myc tag antibody and tested for binding by commercial and human mAbs. a Murine anti-NR1 mAb. b Human mAbs, 5F5, 2G6, 1D1, and 6A (isotype control). All samples were tested in triplicate. RLV, relative luminescence value; Ctrl, buffer only. Bars indicate the S.E.M.

Fig. 7

Titration of ATD protein in a capture ELISA. A titration of TEV-mobilized ATD was tested for binding to plate-adherent 5F5 mAb. ATD was biotinylated and tested from 65 pg/ml to 5 μg/ml, in triplicate samples, and detected with SA-HRP. The relative luminescence signal was measured. Calculated R2 = 0.99955. RLV, relative luminescence value. Bars indicate the S.E.M.

Fig. 8

Binding of ANRE and normal human CSF and sera to 293T-ATD cells by immunofluorescence. 293T-ATD cells were stained with (a) ANRE patient CSF, (b) normal human CSF, (c) ANRE patient sera, and (d) normal human sera. Matched pairs include ANRE patient 10–071 (a, c) and normal subjects 10–123 and 10–551 (d, d). CSF were tested at 1:20 dilution; sera at 1:100. Human IgG binding is shown in red. Nuclei were stained with DAPI (blue) and the cells were visualized by confocal microscopy. Scale bars = 10 μm