Multiple roles of Bet v 1 ligands in allergen stabilization and modulation of endosomal protease activity

Abstract Background Over 100 million people worldwide suffer from birch pollen allergy. Bet v 1 has been identified as the major birch pollen allergen. However, the molecular mechanisms of birch allergic sensitization, including the roles of Bet v 1 and other components of the birch pollen extract, remain incompletely understood. Here, we examined how known birch pollen–derived molecules influence the endolysosomal processing of Bet v 1, thereby shaping its allergenicity. Methods We analyzed the biochemical and immunological interaction of ligands with Bet v 1. We then investigated the proteolytic processing of Bet v 1 by endosomal extracts in the presence and absence of ligands, followed by a detailed kinetic analysis of Bet v 1 processing by individual endolysosomal proteases as well as the T‐cell epitope presentation in BMDCs. Results We identified E1 phytoprostanes as novel Bet v 1 ligands. Pollen‐derived ligands enhanced the proteolytic resistance of Bet v 1, affecting degradation kinetics and preferential cleavage sites of the endolysosomal proteases cathepsin S and legumain. E1 phytoprostanes exhibited a dual role by stabilizing Bet v 1 and inhibiting cathepsin protease activity. Conclusion Bet v 1 can serve as a transporter of pollen‐derived, bioactive compounds. When carried to the endolysosome, such compounds can modulate the proteolytic activity, including its processing by cysteine cathepsins. We unveil a paradigm shift from an allergen‐centered view to a more systemic view that includes the host endolysosomal enzymes.


Allergic Patients
The sera for mediator-release assays were collected from birch pollen-allergic patients (n = 6) and selected based on case history, positive skin prick test and allergen-specific IgE reactivity.
Detection of total and allergen-specific IgE levels was performed using Immuno-CAP (Thermo Fisher Scientific, Uppsala, Sweden). The study was approved by the ethics committee of the medical faculty of Technical University of Munich (19/15 S).

Surface acoustic wave (SAW) interaction studies and determination of the affinity constant K d
For the determination of the affinity constant K d , we used surface acoustic wave (SAW) technology. The interaction of the six ligands with Bet v 1 was investigated using a sam® 5BLUE biosensor instrument (Nanotemper, Munich, Germany). Bet v 1 was immobilized in PBS on the surface of a SAW CM-Dextran 3D sensor chip. The surface of the chip was activated with a freshly prepared mixture of NHS (100 mM) and EDC (400 mM), and free activated sites were subsequently blocked with ethanolamine, pH 8.5 (1 M). For calculating the affinity constant K d , different concentrations of the ligands dissolved in a 5 mM sodium phosphate buffer, pH 7.4, were applied to the Bet v 1-coupled chip. Depending on the ligand, the concentration range of the injected ligand varied between 5 µM and 500 µM. For the removal of residual ligand between the sample injections, the chip was washed with regeneration buffer (10 mM citric acid buffer, pH 2.8). The recorded SAW phase changes were analyzed using the software TraceDrawer 1.7 (Ridgeview Instruments, Uppsala, Sweden). The affinity constant K d was calculated by kinetic evaluation and affinity/EC50 estimation. For kinetic evaluation, an OneToOne curve fit was chosen to match the raw data.
Experiments were performed with two coated chips in duplicate, and the coupling efficiency was evaluated using a mouse monoclonal anti-Bet v 1 antibody.
Expression and Purification of 15 N-1 H labeled Bet v 1 15 N-1 H labeled Bet v 1 was expressed following variation of a previously described method. 1 Two liters of bacteria were grown overnight in LB medium, centrifuged and resuspended in 0.5 L of M9 salts lacking any carbon or nitrogen source and equilibrated for 35 min. Cells, fed with glucose, 15 N ammonium chloride, and 15 N Celltone (1/25 g) (Cambridge Isotope Labs), were incubated for 35 min and then induced with IPTG. The protein suspension was purified as described above. In addition, an extra Superdex 75 size-exclusion column (GE Healthcare Biosciences, Little Chalfont, UK) step was used.

NMR spectroscopy
The NMR data were acquired with either a 600 or 800 MHz Agilent DD2 spectrometer with a cryogenically cooled probe using the pulse sequence gNhsqc. Bet v 1 assignments were taken from. 2

LPS pull-down assay
To investigate whether Bet v 1 binds LPS, a pull-down assay using biotinylated ultrapure E. coli O111:B4 LPS (InvivoGen, San Diego, CA, USA) was performed as previously described. 3 In short, biotinylated LPS was immobilized on Strep-Tactin Sepharose beads (IBA Lifesciences, Göttingen, Germany) and incubated with Bet v 1 for 20 min at room temperature. Unbound protein was washed off and beads were analyzed by SDS-PAGE. As control, the individual components (Bet v 1, biotinylated LPS and Strep-Tactin Sepharose beads), and Bet v 1+beads to exclude unspecific binding, were analyzed.

Determination of secondary structure elements and thermal stability
The influence of ligand binding on the secondary structure elements and the thermal stability

Mediator release assay
A mediator-release assay was performed to assess the capacity of ligand-loaded Bet v 1 to induce IgE-antigen-crosslinking and basophil degranulation. In this respect, rat basophil (RBL-2H3) cells, transfected with the human high-affinity IgE receptor (FcεRI) were passively sensitized with sera of six subjects allergic to birch pollen (1:10), and used as previously described. 4 For cell-stimulation, Bet v 1 was used at 10-fold dilutions from 1 µg/ml to 0.01 pg/ml. Toxicity of ligands on RBL-2H3 was determined by cell viability assay using MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) from Sigma-Aldrich, Inc. (St. Louis, MO, USA). Results were expressed as percentage of total enzyme release of cells lysed with Triton X-100, and the half maximal release (in ng) was determined.

In vitro antigen uptake of murine bone marrow-derived dendritic cells (BMDCs)
BMDCs from C57BL/6 mouse bone marrow were isolated as described previously. 5

Activation and cytokine secretion of human monocyte-derived dendritic cells (moDCs)
Immature moDCs from PBMCs of either healthy/non-atopic donors (n=5) or atopic patients (n=7) were isolated and cultured as described elsewhere. 6 Cell viability using the Aqua® dye

In vitro simulation of endolysosomal degradation
The endolysosomal degradation assay was performed with ligand-bound (either DOC, PPE 1 , or Q3OS in 10× molar excess) and Bet v 1 without ligands (apo-Bet v 1) as previously described. 7 The protein (5 µg) was incubated with 7 µg of microsomes in 100 mM citrate

Data analysis of in vitro simulation of endolysosomal degradation
For data analysis, the web-based application MSTools was used. 8 For the semi-quantitative analysis of the abundance of the generated peptides at 12 hours of degradation, the obtained peptide sequences were clustered into 7 groups according to their amino acid sequence

Bet v 1 in vitro proteolytic degradation assay using individual endolysosomal proteases
Recombinant human cathepsin S and human legumain were heterologously expressed and purified as described previously. 9

T-cell proliferation assay
Bet v 1 was incubated over night with a 10-fold molar excess of either Q3OS, PPB 1 , PPE 1 , or DOC before incubation with murine BMDCs at a concentration of 10 µg/mL in BMDC medium (online supporting information). After 16, 24, 32, and 48h, DCs were washed and CD4 + T-cell hybridomas specific for the immune-dominant epitope 142-153 of Bet v 1 were added to the culture at a ratio of 1:10 (DC:T-cell) for 24h 17 . Supernatants were harvested and IL-2 levels were determined by ELISA (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). As a reference, BMDCs were pulsed at each time point with different concentrations of peptide 142-153 and co-cultured with CD4 + T-cell hybridomas. By interpolating in a standard curve, a µM peptide equivalent from the measured IL-2 was calculated from DCs pulsed with different peptide concentrations. The assays were carried out in triplicates.