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

Expression of an ATD fusion protein on the surface of 293T cells

We designed a recombinant gene encoding the first 561 amino acids of NR1, the Myc epitope tag (EIDSEEKL), a 6XHIS tag, and the Tobacco Etch Virus (TEV) protease cleavage site (ENLYFQGG), fused to the platelet derived growth factor receptor (PDGFR) transmembrane domain (Fig. 1a-b) [13]. We used retroviral transduction to express the gene in 293T cells under puromycin selection. A stable polyclonal population was isolated by flow cytometry over four rounds of selection using a commercial murine NR1 mAb, resulting in the cell population 293T-ATD (Fig. 1c). Co-staining of the cell population with a Myc tag antibody and the NR1 mAb indicates that most of cells that express the Myc tag also express the NR1 ATD (Fig. 1d).

We previously cloned three human monoclonal antibodies (mAbs), 5F5, 2G6, and 1D1, from a female ANRE patient (manuscript in press). We tested binding of the mAbs to the 293T-ATD cell population using flow cytometry, co-staining with the commercial anti-NR1 mAb (Fig. 2). The ANRE patient mAbs all bound to the 293T-ATD cells to a greater extent than the 6A isotype control mAb, with double positive cells comprising 62.3% (5F5), 40.5% (2G6), and 37.0% (1D1), compared to 12.4% (6A control), Calculated as the proportion of NR1 positive cells bound by the mAbs, the 5F5 showed 90.5% binding; 2G6, 58.8%; 1G1, 53.7%; and 6A, 18.0% (Table 1).

Human mAb	Percent GluN1 positive 293T-ATD cells bound
5F5	90.5
2G6	58.8
1D1	53.7
6A	18.0

Immunofluorescence detection of NR1-antibody binding to 293T-ATD cells

We next tested the 293T-ATD cell line for detecting NR1 antibodies by immunofluorescence. We first co-stained the cells with the commercial NR1 and Myc antibodies, and observed co-localization of the signals on the outer plasma membrane (Fig. 3). We then tested binding of ANRE patient CSF, the three ANRE patient mAbs, and an isotype control human mAb, 8E1 (Fig. 4). CSF and the ANRE patient mAbs all reacted with the 293 T-ATD cell line, whereas the 8E1 mAb did not.

ELISA studies of TEV protease-mobilized ATD

The TEV protease site adjacent to the PDGF transmembrane domain was included to allow mobilization of the expressed ATD for use in binding studies requiring soluble antigen. We treated the 293T-ATD cells with TEV protease for 10–40 min, spun down the cells, and analyzed the reaction supernatants by capture ELISA and Coomassie-stained SDS:PAGE (Fig. 5). Analyzed by ELISA, the ATD was evident at 10 min and peaked at 20 min, and declined somewhat thereafter (Fig. 5a). Longer incubations (up to 2 h) futher decreased amount of mobilized ATD (data not shown). The non-denaturing SDS:PAGE gel gave a dominant band at approximately 25 kDa, with a faint band slightly below, and no significant bands above, demonstrating the specificity of cleavage of the ATD from the outer plasma membrane.

We next tested antibody binding to the mobilized ATD in a capture ELISA format. We used a Myc tag antibody to capture the ATD onto ELISA plates and tested binding of the commercial NR1 and human ANRE mAbs. The murine NR1 mAb bound significantly above background levels (Fig. 6a). Similarly, the three human ANRE mAbs all bound the plate-adherent ATD, giving signals approximately 8–10 fold greater than the 6A human isotype control mAb (Fig. 6b). We next tested whether the ATD could be used reproducibly in a quantitative assay. We biotinylated the ATD, then tested its binding to plate-immobilized 5F5 antibody in an ELISA, using SA-HRP for detection. We tested triplicate samples ranging from 65 pg/ml to 5 μg/ml (Fig. 7). Linear regression analysis gave an R2 value of 0.999, indicating that the assay is linear in this ATD concentration range.

Immunofluorescence detection of ANRE patient CSF and sera binding to 293T-ATD cells

We tested binding of clinical samples of ANRE and normal CSF and sera to the 293T-ATD cell line by NR1 antibodies by immunofluorescence. The samples were obtained from the clinical services at the Children’s Hospital of Philadelphia. Four ANRE and four normal human CSF (1:20) and serum samples (1:100) were tested, including a matched CSF:serum pair from ANRE patient 10–071 and two pairs from normal patients 10–123 and 10–551. Three of the ANRE CSF samples (Fig. 8a) and all four of the serum samples (Fig. 8c) gave a bright immunofluorescent signal, whereas none of the normal CSF or serum samples showed binding (Fig. 8b, c).

Human subjects

CSF and patient sera were collected at the Children’s Hospital of Pennsylvania (CHOP), Philadelphia, PA, with full informed consent and protocols approved by the CHOP Institutional Review Board.

Expression of the ATD fusion protein in 293T cells

We made a fusion gene that expresses the entire 561 amino acids of the N-terminal extracellular domain of human GluN1 (NR1) (UniProtKB - Q05586), including the amino terminal domain (ATD), followed by the Myc tag, a 6XHIS tag, a TEV protease cleavage site, and the transmembrane domain of the human platelet-derived growth factor receptor (PDGFR) (Additional file 1, Genbank Accession #) [13, 15]. The gene was synthesized and inserted into the retroviral vector, pBabe puro by Genscript (Piscataway, NJ) [16]. Amphotropic retroviruses were produced in 293T cells following standard protocols, except that X-tremeGENE 9 DNA Transfection Reagent was used (354,087, Roche, Germany), and the cells were cultured in Advanced DMEM, 1% IFS, penicillin/streptomycin (Invitrogen, Carlsbad, CA) [17].

The retroviral supernatant was used to transduce 293T cells (2.5 X10⁶ in a 10 cm dish), with 4 μg/ml polybrene (TR1003G, Thermo Fisher Scientific, Waltham, MA), for 6 h. 48 h later, cells were selected with 1 μg/ml puromycin (P9620, Sigma-Aldrich, St. Louis, MO). One week later, expressing cells were isolated by FACS staining with the murine anti-NR1 APC mAb (orb149996, Biorbyt, San Francisco, CA) on the BD FACSCanto II (Becton Dickson, Franklin Lakes, NJ). Four rounds of FACS over 4 weeks were performed to isolate a stable, homogeneous population of cells (293T-ATD).

Flow cytometry, FACS, and immunofluorescence studies

To assess antibody binding to 293T-ATD cells by flow cytometry, cells were harvested using 0.05% trypsin, washed, and resuspended at 1 × 10⁶ cells/ml in PBS-1% BSA (A7030, Sigma-Aldrich). Primary antibodies included the Bioorbyt APC-labeled NR1 mAb at 10 μg/mL, an Alexa Fluor 488 labeled Myc tag mAb at 2 μg/mL (16–308, Millipore, Billerica, MA) three human IgG mAbs (5 μg/mL) isolated from a patient with ANRE (5F5, 2G6, 1D1, manuscript in press) and the 6A isotype control mAb [17]. As a secondary antibody for the human mAbs, we used a FITC-conjugated F(ab’)2 goat anti-human IgG (109–096-008, Jackson ImmunoResearch, West Grove, PA). Cells were assayed with a BD FACSCanto II (Becton Dickson, Franklin Lakes, NJ). Data were analyzed using FlowJo 8.8.7. Software (Tree Star, Ashland, OR).

For immunofluorescence studies, 293T-ATD cells were plated at 5 × 10⁴ cells/well on round Corning™ BioCoat™ 12 mm #1 German Glass Coverslips (354,087, Corning, NY) in 24 well plates. 24 h later, the cells were fixed with 4% paraformaldehyde in PBS for 10 min at room temperature, washed with PBS 0.05% Tween-20 (PBST), blocked with 10% Goat serum (Invitrogen) + 1% BSA in PBS (PBS + G + B) for 1 h at 37 °C, and then washed with PBST. Cells were incubated for one hour at room temp in PBS + G + B with one or more of the following added: a murine anti-NR1 APC mAb (orb149996, Biorbyt, San Francisco, CA), an Alexa Fluor 488 conjugated anti-Myc-tag-specific mAb (16–308, Millipore) (1:250 dilution), ANRE patient or normal human CSF (1:20), ANRE patient or normal human sera (1:100), human mAbs, 5F5, 2G6, 1D1 or isotype human control mAb 8E1 (5 μg/ml). After one hour, cells were washed twice with PBST and incubated with the Alexa 568 goat anti-human IgG, 1:1000 (A21090, Thermo Fisher), secondary antibody for the human CSF or mAbs in PBS + G + B for one hour, and then the cells were washed once with PBS and once with dH₂O. Coverslips were mounted with ProLong® Gold Antifade reagent with DAPI (P36935, Thermo Fisher) and imaged with a C2+ Nikon confocal microscope with 63×/1.3 NA oil objective; images were analyzed with ImageJ software (https://imagej.nih.gov/ij/). All immunofluorescence studies were performed at least twice.

Mobilization of membrane-bound ATD with TEV protease

The 293T-ATD cells were plated at 2 × 10⁵ cells/well in 12 well plates. 24 h later, they were washed with PBS and then treated with 25 μg rTEV Protease (4469; R&D Systems, Minneapolis, MN) with Xpert Protease inhibitor cocktail solution (P3100–001; GenDEPOT, Barker, TX) in PBS for the indicated time period (10–40 min). The cells were pipetted up and down, transferred to Eppendorf tubes, and centrifuged at 3000 rpm for 10 min at 4 °C. The supernatant was collected and immediately dialyzed against cold PBS overnight. The protein concentration was measured using NanoDrop 1000 (Thermo Fisher) and protein was visualized on a Coomassie-stained SDS:PAGE gel.

ATD ELISAs

To analyze the timecourse of ATD mobilization by TEV, we performed a capture assay in which we coated Black 96-well immune plates (12–566-24, Thermo Fisher) with 5 μg/mL anti HIS tag antibody (ab18184, Abcam, Cambridge, MA) overnight, washed the plates 3 times with PBST, blocked with 5% inactivated fetal bovine serum and 3% Goat serum (Invitrogen) in PBST for 1 h at 37 °C, then washed 3 times. ATD samples cleaved at the stated timepoints were added at 5 μg/mL and supernatant from un-cleaved cells was added as negative control, followed by 1 h incubation at 37 °C. The plates were washed three more times, and biotinylated human mAb 5F5 was added at 5 μg/mL (100 μl/well), and then incubated for 1 h at 37 °C. After three additional PBST washes, Streptavidin-poly-HRP conjugate at 1:2000 (Thermo Fisher) was added and incubated for 1 h at 37 °C. After three additional washes, Super Signal ELISA Femto Substrate was used for detection (Thermo Fisher). Relative luminescence values were measured using the Biotek Synergy II Microplate reader (BioTek Instruments, Winooski, VT, USA). Microsoft Excel was used to process the data.

To test binding of human NR1 antibodies to plate-adherent ATD, we added 5 μg/mL Myc antibody (C3956, Sigma-Aldrich) (100 μl/well) to Black 96-well plates (12–566-24, Thermo Fisher) overnight, washed the plates 3 times with PBST, blocked with 5% inactivated fetal bovine serum and 3% Goat serum (Invitrogen) in PBST for 1 h at 37 °C, washed 3 times, added 5 μg/mL ATD, and then incubated for 1 h at 37 °C and washed 3 more times. We added human mAbs, 5F5, 2G6, 1D1, and control isotype 6A at 5 μg/mL (100 μl/well), or 5 μg/mL anti-NR1 mAb (MAB 1586 R1JHL, Millipore), in triplicate samples, and incubated for 1 h at 37 °C. After three additional PBST washes, secondary antibodies were added, either an anti-human IgG HRP conjugate (9040–05 SouthernBiotech, Birmingham, AL) or anti-mouse IgG HRP conjugate (1010–05, Southern Biotech), at 1:2000 and incubated for 1 h at 37 °C, followed by 3 washes. Super Signal ELISA Femto Substrate was used for detection. Data was collected in the Biotek Synergy II Microplate reader and analyzed with Microsoft Excel.

To test binding of TEV-mobilized ATD to plate-adherent human IgG by ELISA, we first biotinylated the ATD using the EZ-Link™ Sulfo-NHS-Biotin kit (21,326, Thermo Fisher). We added 5 μg/mL 5F5 (100 μl/well) to Black 96-well plates (12–566-24, Thermo Fisher), incubated overnight at room temp, washed the plates 3 times with PBST, blocked the wells with 2% non-fat milk in PBST for 1 h at 37 °C, and again washed 3 times. We added triplicate serial dilutions of the biotinylated ATD (diluted in 50 μL PBS/well), and incubated for 1 h at 37 °C. After three PBST washes, Streptavidin-poly-HRP conjugate at 1:2000 (Thermo Fisher) was added and incubated for 1 h at 37 °C. Super Signal ELISA Femto Substrate was used for detection. Data was collected in the Biotek Synergy II Microplate reader and analyzed with Microsoft Excel.