Sialylation of immunoglobulin E is a determinant of allergic pathogenicity

Nature

IgE antibodies

All human samples were collected under Institutional Review Board (IRB)-approved protocols by Massachusetts General Hospital (MGH) and Research Blood Components, including informed consent obtained in accordance with relevant ethical regulations. Serum samples were obtained from individuals with a peanut allergy before treatment. Peanut allergy was confirmed by clinical history, allergen-specific IgE screening and double-blind placebo-controlled oral challenge (trial number NCT01750879/PNOIT2)8 (Extended Data Table 1). Non-atopic adults were recruited on the basis of self-identification as non-allergic donors. Non-atopy was confirmed by clinical history and allergen-specific IgE screening (Extended Data Table 1). Total IgE, Ara h 2-specific IgE, Fel d 1-specific IgE, Der p 1-specific IgE, and Bet v 1-specific IgE were determined by ImmunoCap Assay (Phalleon, Thermo Scientific) according to the manufacturer’s protocols. Primary IgE was enriched from serum samples by serially depleting IgG using protein G agarose (GE Healthcare) followed by anti-IgE conjugated N-hydroxysuccinimide (NHS) beads (GE Healthcare). IgE purity was confirmed by protein electrophoresis and Coomassie gel staining. Recombinant OVA-specific IgE was generated as described17. In brief, complementary DNA sequences for generating OVA-specific heavy (ε) and light (κ) chains of mouse and human IgE17 were cloned into plasmid pcDNA3.4 using restriction-enzyme sites XbaI and AgeI. To generate recombinant OVA-specific mouse or human IgE, plasmids containing OVA-specific heavy and light chains were transiently co-transfected at a 1:1 ratio using an Expi293 Expression System Kit (Life Technologies) according to the manufacturer’s protocol. Cells expressing IgE were selected by adding 400 μg ml−1 of the antibiotic G418 to the culture medium for two weeks, and maintained before expanding to a larger-scale production. OVA-specific IgE was purified from cell culture supernatant using OVA-coupled agarose beads17.

ELISA

Sandwich ELISA for quantifying mIgE and OVA-specific binding was conducted as described17. In brief, 96-well Nunc plates were coated with goat polyclonal anti-mouse IgE (Bethyl Laboratories) or OVA, and blocked with bovine serum albumin (BSA) in PBS (1% BSA for mIgE and 2% for OVA) before sample incubation. Samples were probed with goat polyclonal anti-mouse IgE conjugated to horseradish peroxidase (HRP; 2 ng ml−1; Bethyl Laboratories). The reactions were detected using 3,3,5,5-tetramethylbenzidine (TMB; Thermo Fisher Scientific) and stopped with 2 M sulfuric acid; absorbance was measured at 450 nm.

gMS and glycopeptide analysis

We quantified the site-specific glycosylation of IgE isolated from non-allergic donors and from donors with a peanut allergy by using nanoscale liquid chromatography with tandem mass spectrometry (LC-MS/MS) following enzymatic digestion of the proteins as described previously, with minor modifications16,17,18 (Extended Data Table 2). Isolated polyclonal primary hIgE and myeloma hIgE (Sigma Aldrich catalogue number AG30P) were prepared for proteolysis by denaturing the protein in 6 M guanidine HCl, followed by reduction with dithiothreitol, alkylation with iodoacetamide, and dialysis into 25 mM ammonium bicarbonate (pH 7.8). Proteolysis was carried out with either trypsin to quantify N218, N371 and N394, or chymotrypsin to quantify N140, N168 and N265. For the tryptic digest, IgE was incubated with trypsin (Trypsin Gold Promega) at a 1:50 enzyme-to-substrate ratio overnight at 37 °C. For the chymotryptic digest, IgE was incubated with chymotrypsin (Sequencing Grade Promega) at a 1:100 enzyme-to-substrate ratio for 4 h at 25 °C. Both enzymes were quenched with formic acid added to 2% w/w. The separation was performed on a Thermo EasySpray C18 nLC column (0.75 μm × 50 cm) using water and acetonitrile with 0.1% formic acid for mobile phase A and mobile phase B, respectively. A linear gradient from 1% to 35% mobile phase B was run over 75 min. Mass spectra were recorded on a Thermo Q Exactive mass spectrometer operated in positive mode using data-independent acquisition (DIA), targeting the masses shown in Extended Data Table 2. Glycopeptides were quantified on the basis of the extracted ion area of the Y1 ion (Extended Data Fig. 2). Relative abundances were calculated for all identified glycan species for each site. Myeloma IgE (Sigma Aldrich catalogue number AG30P) was run before paired sample sets to monitor retention-time shifts and to ensure consistency in the analytical results across the sample set. The number of sugar residues per site per IgE molecule was calculated using the relative abundance of each glycan. For example, if we determined a particular site to have 60% monosialylated and fucosylated glycans (A1F) and 40% of disialylated and fucosylated glycans (A2F), then the number of sialic acids at one site would be 1.4 ((0.6 × 1) + (0.4 × 2)), with a total of 2.8 sialic acids per molecule (accounting for two sites).

Generation of NEUFcε

We designed the neuraminidase fusion protein by fusing a κ light chain secretion signal sequence to the sialidase gene from Arthrobacter urefaciens (EC 3.2.1.18, gene AU104)30. The stop codon of AU104 was omitted; instead, a short flexible linker peptide (GGGGGG), the mouse IgE Cε2, Cε3 and Cε4 domains, and a 6× histidine tag were inserted into the carboxy terminus of the sialidase. The gene was codon-optimized for humans and synthesized by GenScript. The 288-kDa protein was then produced by WuXi biologics. Sialidase activity of NEUFcε was determined by the level of p-nitrophenol released from 250 μM 2-O-(p-nitrophenyl)-α-dN-acetylneuraminic acid (Sigma) in 100 mM sodium phosphate (pH 5.5) for 10 min at 37 °C. The reaction was terminated by adding 0.5 M sodium carbonate and absorbance quantified at 405 nm.

Mice

Five- to six-week-old female BALB/c mice were purchased from the Jackson Laboratory and used here. All mice were housed in specific-pathogen-free conditions according to the National Institutes of Health (NIH), and all animal experiments were conducted under protocols approved by the MGH Institutional Animal Care and Use Committee (IACUC), in compliance with appropriate ethical regulations. For all experiments, the allocation of age- and sex-matched mice to experimental groups was randomized, with four to five mice per group, each experiment being repeated three independent times. No statistical method was used to determine sample size.

PCA was conducted as described17. In brief, monoclonal SiamIgE or AsmIgE specific for OVA or DNP (clone SPE-7, Sigma Aldrich) was injected intradermally into mouse ears. For experiments in which OVA-specific AsmIgE was added to OVA-specific SiamIgE, an mIgE isotype control (clone MEA-36, Biolegend) was included. The next day, mice were intravenously challenged with 125 μg OVA or DNP–HSA (both from Sigma Aldrich) and 2% Evans blue dye in PBS. Forty-five minutes after challenge, the ears were excised and minced before incubation in N,N-dimethyl-formamide (EMD Millipore) at 55 °C for 3 h. The degree of blue dye in the ears was quantified by absorbance at 595 nm.

PSA was elicited as described, with minor modifications31,32. In brief, mice were injected intravenously with monoclonal mIgE specific for OVA or DNP (clone SPE-7, Sigma Aldrich) in PBS, and challenged the next day intravenously with PBS containing 1 mg OVA or DNP–HSA (both from Sigma Aldrich). To examine the therapeutic potential of AsmIgE, on the first day we injected mice intravenously with 10 μg DNP-specific mIgE (clone SPE-7, Sigma Aldrich); on the second day we injected them intravenously with PBS, 20 μg OVA-specific SiamIgE or 20 μg OVA-specific AsmIgE; and on the third day we challenged them intravenously with 1 mg DNP–HSA or OVA (Sigma Aldrich). To test the therapeutic potential of NEUFcε, mice injected intravenously with 10 μg OVA-specific mIgE on the first day were further injected intravenously with PBS, 100 μg NEUFcε or 100 μg mIgE isotype control (clone MEA-36, Biolegend) the next day and challenged intravenously with 1 mg OVA (Sigma-Aldrich) the third day. Core temperature was recorded at the baseline and every 10 min after allergen challenge in a blinded manner with a rectal microprobe thermometer (Physitemp). Histamine in the blood was quantified using a histamine enzyme immunoassay kit (SPI-Bio) according to the manufacturer’s protocol. In brief, histamine in the blood was derivatized and incubated with plate precoated with monoclonal anti-histamine antibodies and histamine–acetylcholinesterase tracer at 4 °C for 24 h. The plate was washed and developed with Ellman’s reagent and absorbance measured at 405 nm.

Passive food anaphylaxis was elicited by adapting the PSA protocol described above. In brief, mice injected intravenously with 20 μg monoclonal mIgE specific for TNP (clone MEA-36, Biolegend) in PBS on the first day were administered with 20 mg TNP–OVA in PBS (Biosearch Technologies) by oral gavage the next day. Core temperature was recorded at the baseline and every 10 min after the challenge using a rectal microprobe thermometer (Physitemp) in a blinded manner.

To determine in vivo half-lives of SiamIgE or AsmIgE, we injected mice intraperitoneally with 30 μg DNP-specific SiamIgE or AsmIgE, and collected blood at the indicated times after injection into a Microtainer blood-collection tube with clot activator/serum separator tube (SST) gel (BD Diagnostics). We quantified the level of mIgE by mIgE ELISA as above.

Basophil activation tests

Basophil activation was performed as described33. Buffy coats of human blood from healthy, de-identified, consenting donors were obtained from the MGH Blood Transfusion Service. Peripheral blood mononuclear cells (PBMCs) were separated from buffy coats by density gradient centrifugation using Ficoll Paque Plus (GE Healthcare), and resuspended in 0.5% BSA in RPMI 1640 medium (GE Healthcare). PBMCs were incubated for 2 min with ice-cold lactic acid buffer (13.4 mM lactate, 140 mM NaCl, 5 mM KCl, pH 3.9) to remove endogenous human IgE on the cell surface before neutralization with 12% Tris (pH 8). Cells were then washed and incubated 1 h at 37 °C with 1 μg OVA-specific SiahIgE or AshIgE per 1 × 106 cells in basophil activation buffer (0.5% BSA, 2 mM CaCl2 and 2 mM MgCl2 in RPMI 1640 medium). Sensitized cells were washed and resuspended in basophil activation buffer supplemented with 10 ng ml−1 human interleukin-3 (PeproTech) before activation with OVA for 30 min. Activation was stopped by addition of ice-cold 0.2 M EDTA in FACS buffer. Cells were washed and resuspended in FACS buffer before antibody staining (Extended Data Table 3) for activation markers (LAMP-3 or CD63+) on basophils (CD123+ HLADR).

Culture and degranulation of human mast cells

The human LAD2 mast-cell line was a gift from D. D. Metcalfe (National Institute of Allergy and Infectious Diseases (NIAID), NIH) and was maintained as described17,34. In brief, LAD2 cells were cultured in StemPro-34 SFM medium (Life Technologies) supplemented with 2 mM l-glutamine, 100 U ml−1 penicillin, 100 μg ml−1 streptomycin and 100 ng ml−1 recombinant human stem cell factor (PeproTech). The cells were hemi-depleted each week with fresh medium and maintained at 2 × 105 to 5 × 105 cells per millilitre at 37 °C and 5% CO2.

Primary human mast cells were generated as described35. In brief, we separated PBMCs from buffy coats as above, before isolating CD34+ pluripotent haematopoietic cells using the EasySep human whole blood CD34-positive selection kit II (Stemcell Technologies). We cultured CD34+ cells in StemPro-34 SFM medium (Life Technologies) supplemented with 2 mM l-glutamine, 100 U ml−1 penicillin, 100 μg ml−1 streptomycin, 50 ng ml−1 recombinant human stem cell factor (PeproTech) and 50 ng ml−1 human interleukin-6 (PeproTech), and with 10 ng ml−1 human interleukin-3 (PeproTech) in the first week. After the first week we matured the cells in similar culture medium but without human interleukin-3 for ten weeks. Cultured mast cells were confirmed by FACS staining of CD45+, KIT+ and FcεRI+.

Degranulation assays were performed as described17. LAD2 or peripheral blood derived human mast cells were sensitized overnight with 1 μg ml−1 OVA-specific hIgE or 50 ng ml−1 peanut-allergic hIgE. The following day, the cells were pelleted by centrifugation, resuspended in HEPES buffer, plated in 96-well plates, and stimulated with allergen OVA or crude peanut extract at defined concentrations. Upon allergen challenge, mast cell degranulation was determined by the amount of substrate p-nitrophenyl N-acetyl-β-d-glucosamide digested by β-hexosaminidase release from mast cell granules at absorbance of 405 nm. To assess the effect of sialic acid removal on IgE-bound mast cells, we treated IgE-sensitized LAD2 cells with NEUFcε, heat-inactivated NEUFcε or mIgE isotype control (clone MEA-36, Biolegend) for 20 min before allergen challenge. To inactivate NEUFcε, we heated the enzyme at 95 °C for 10 min. To determine whether addition of a surrogate asialylated glycoprotein could recapitulate the phenotype of sialic acid removal from IgE, LAD2 cells sensitized with OVA-specific SiahIgE were incubated with sialylated fetuin (SiaFetuin) or asialylated fetuin (AsFetuin) at defined amounts for 20 min before allergen challenge.

Preparation of crude peanut extract

Unsalted dry-roasted peanuts (blanched jumbo runner cultivar, Planters) were ground to a smooth paste, then washed with 20 volumes of cold acetone, filtered using Whatman paper, and dried as described17. Protein was extracted by agitating the peanut flour overnight with PBS containing protease-inhibitor cocktail without EDTA (Roche). The peanut protein extracts were collected as the supernatant after centrifugation at 24,000g for 30 min.

IgE glycosylation engineering

To remove sialic acids on IgE, we digested IgE with glyko sialidase A (recombinant from A. urefaciens expressed in Escherichia coli, Prozyme) at 37 °C for 72 h according to the manufacturer’s instructions. To resialylate AsmIgE by in vitro sialylation, we incubated AsmIgE with human α-2,6 sialyltransferase 1 (ST6GAL1, provided by H. Meade, LFB-USA) at a ratio of 20 μg AsmIgE per μg of ST6GAL1 and 5 mM cytidine-5′-monophospho-N-acetylneuraminic acid (CMP-Neu5Ac2, Nacalai USA) in the sialylation buffer (150 mM NaCl, 20 mM HEPES, pH 7.4) overnight at room temperature as described36. Following reactions, OVA-specific SiaIgE or AsIgE was purified with OVA-coupled beads to remove glycosylation modifying enzymes as described17. All digestion or sialylation reactions were verified by lectin blotting or high-performance liquid chromatography (HPLC).

Protein gel stain and lectin blotting

Equal amounts of SiaIgE or AsIgE were resolved on 3–8% Tris-acetate protein gels (Life Technologies) in SDS–PAGE under nonreducing conditions. For protein staining, gels were incubated in AcquaStain protein gel stain (Bulldog Bio) for 1 h at room temperature and destained in distilled water. For lectin blotting, the protocol was conducted as described17. Briefly, after resolved proteins on the gel were transferred to Immobilon-PSQ polyvinylidene difluoride membranes (Millipore Sigma), the membranes were blocked with 0.2% BSA in Tris-buffered saline (TBS) for 1 h at room temperature, washed in TBS, and then incubated with biotinylated S. nigra (SNA) lectin (0.4 μg ml−1, Vector Laboratories) in TBS with 0.1 M Ca2+ and 0.1 M Mg2+ for 1 h at room temperature to determine the level of terminal α2,6-sialic acids on N-linked glycans of proteins. The membrane was then washed in TBS and incubated with alkaline-phosphatase-conjugated goat anti-biotin (1:5,000 dilution; Vector Laboratories) in TBS for 1 h at room temperature. Sialylated proteins on membranes were visualized by incubation with one-step nitro-blue tetrazolium chloride (NBT)/5-bromo-4-chloro-3′-indolyphosphate p-toluidine (BCIP) substrate solution (Thermo Scientific).

Flow cytometry

Details of antibodies used for surface staining are listed in Extended Data Table 3. To stain mouse cells, we incubated suspended cells with anti-mouse CD16/CD32 (clone 2.4G2, BD Biosciences) before antibody staining. Cells were incubated in FACS buffer with desired staining antibodies for 20 min at 4 °C. Cells were then washed in FACS buffer before being acquired by an LSRII flow cytometer (BD Biosciences) or CytoFLEX (Beckman Coulter). Data were analysed using FlowJo software version 10.4 software (Tree Star). To quantify OVA-specific hIgE loading following sensitization, we incubated PBS or 1 μg ml−1 OVA-specific SiahIgE or AshIgE with 2.5 × 105 LAD2 cells per millilitre overnight, before washing with FACS buffer and staining with anti-Kit antibody and OVA-A647. To quantify native hIgE loading on LAD2 mast cells, we sensitized cells with 32 ng total non-atopic or allergic hIgE overnight before washing in FACS buffer and staining with anti-Kit and anti-hIgE antibodies. To quantify dermal mast cell IgE loading, we generated single-cell suspensions from mouse ears as described17. Ears were intradermally injected with 40 ng OVA-specific SiamIgE or AsmIgE. The following day, ears were removed, separated into dorsal and ventral halves, and minced before incubation in Dulbecco’s modified Eagle medium (DMEM) containing 2% fetal calf serum (FCS), 1% HEPES, 500 units ml−1 collagenase type 4 (Worthington), 0.5 mg ml−1 hyaluronidase (Sigma) and DNase I (Roche) at 37 °C for 1 h at 180 rpm. The digested sample was then subjected to disruption by gentleMACS and filtered through a 70-μm cell strainer followed by a 40-μm cell strainer in FACS buffer (2 mM EDTA and 0.5% BSA in PBS). mIgE loading was detected by FACS using anti-mIgE antibodies on dermal mast cells (CD45+ CD11b CD11c Gr1 Kit+).

Biolayer interferometric assays for binding

We studied binding kinetics and affinity of protein interaction using the Octet K2 system (Molecular Devices) with Octet buffer (PBS with 0.025% Tween and 1% BSA). To measure hFcεRIα interactions, 0.25 μg ml−1 histidine-tagged hFcεRIα (Acro Biosystems) was loaded onto anti-penta-histidine (HIS1K) biosensors (Molecular Devices). To analyse OVA interactions, we immobilized OVA (100 μg ml−1) onto amine-reactive second-generation (AR2G) biosensors in 10 mM sodium acetate, pH 5, using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC)/sulfo-NHS based chemistry. We determined association of analyte OVA-specific SiahIgE or AshIgE in threefold serial dilution from 90 nM to 1 nM, and of NEUFcε in threefold serial dilution from 24 nM to 0.3 nM in Octet buffer. Analyte dissociation was measured in Octet buffer. Analysis of binding kinetic parameters was performed by Octet data analysis software v.10.0, using interaction of ligand-loaded biosensor with no analyte during the association phase as the reference sensor.

Immunoblotting for Syk signalling

We sensitized 1.5 × 106 LAD2 cells with PBS or 1 μg ml−1 OVA-specific SiahIgE or AshIgE. Sensitized cells were washed and resuspended in HEPES buffer the next day, and then stimulated with 10 μg ml−1 OVA at 37 °C for the indicated times. Cells were immediately centrifuged after OVA stimulation and the cell pellets lysed in ice-cold lysis buffer (RIPA buffer (Boston BioProducts), 1× Halt protease-inhibitor cocktail (Thermo Scientific), 1× Halt phosphatase-inhibitor cocktail (Thermo Scientific) and 2.5 mM EDTA) for 30 min on ice. After incubation on ice, lysed pellets were passed rapidly through a 27G needle on ice and centrifuged at 15,000 rpm at 4 °C for 15 min to clear the membrane and nuclei. The protein concentration was quantified using a Pierce BCA protein assay kit (Thermo Scientific) and 20 μg of protein lysate was loaded per well on 4–12% Bis-Tris protein gels (Life Technologies) in SDS–PAGE under denaturing and reducing conditions. Briefly, after protein transferred to PVDF membranes as above, the membranes were blocked with 5% milk in TBS with 0.1% Tween (TBST) for 1 h at room temperature, washed in TBST, and incubated with 1:2,000 rabbit anti-phospho-Syk (Tyr352) antibody (Cell Signaling Technology) in 5% BSA in TBST overnight at 4 °C. The membrane was washed in TBST, incubated with anti-rabbit-HRP for 1 h at room temperature, and washed again in TBST; this was followed by chemiluminescent detection using Immobilon western chemiluminescent HRP substrate (Millipore Sigma). To detect total Syk on the membrane, after chemiluminescent detection using autoradiography film, the membrane was stripped by incubation in stripping buffer (2% SDS and 0.1 M β-mercaptoethanol in Tris buffer) at 50 °C for 30 min. The stripped membranes were then blocked, washed as above, incubated with 1:2,000 rabbit anti-Syk antibody (Cell Signaling Technology) for 2 h in 5% BSA in TBST at room temperature, and washed again before being incubated with 1:30,000 anti-rabbit-HRP for 1 h at room temperature. To probe for β-actin, the membranes were incubated with 1:150,000 anti-β-actin HRP (Santa Cruz Biotechnology) for 1 h at room temperature and then washed; signal was determined by chemiluminescent detection.

Calcium flux

We sensitized 5 × 105 LAD2 cells overnight with PBS or 500 ng ml−1 OVA-specific SiahIgE or AshIgE. The next day, sensitized cells were washed before loading with 2 μM Fluo-4-AM (Invitrogen) at 37 °C in HEPES buffer for 20 min. After loading, the cells were washed and resuspended in HEPES buffer. Fluorescence was filtered through the 530/30 bandpass filter and collected in FL-1/FITC. Baseline Ca2+ fluorescence levels were recorded for 1 min on the Accuri C6 (BD Biosciences) before adding the indicated allergen or buffer to each sample. At the end of allergen stimulation, 2 μM Ca2+ ionophore A23187 (Sigma) was added to cells as a positive control.

Statistical analyses

Results are shown as means ± s.e.m., except in the case of gMS-quantified glycan residues per IgE molecule (Fig. 1d–h), where results are presented as medians and interquartile ranges. The number of mice used in each experiment is indicated in the figure legends. The investigators were not blinded to allocation during experiments and outcome assessment, except during the measurement of rectal temperature in mice upon antigen challenge in passive systemic and food anaphylaxis. Visual examination of the data distribution as well as normality testing demonstrated that all variables appeared to be normally distributed. Statistical analyses were performed using Prism 8 (GraphPad software) with unpaired and paired Student’s t-test for assessing two unmatched and matched groups, respectively; two-way ANOVA with Sidak’s multiple comparison test for comparing two groups of multiple conditions; and one-way or two-way ANOVA with Tukey’s multiple comparison test for three or more groups. The P values noted throughout highlight biologically relevant comparisons. The accuracy of distinguishing allergic from non-atopic IgE by means of individual IgE glycan moieties was analysed by ROC curves using Prism 8 (GraphPad software). The area under each ROC curve (AUC) was calculated for each glycan moiety. AUCs are interpreted as follows: the maximum AUC (1) indicates that a particular glycan moiety can distinguish allergic IgE from non-atopic IgE; an AUC of 0.5 indicates that the differentiation capacity of a specific glycan moiety is poor.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this paper.

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