Fold stability during endolysosomal acidification is a key factor for allergenicity and immunogenicity of the major birch pollen allergen

Abstract

Background: The search for intrinsic factors, which account for a protein's capability to act as an allergen, is ongoing. Fold stability has been identified as a molecular feature that affects processing and presentation, thereby influencing an antigen's immunologic properties.

Objective: We assessed how changes in fold stability modulate the immunogenicity and sensitization capacity of the major birch pollen allergen Bet v 1.

Methods: By exploiting an exhaustive virtual mutation screening, we generated mutants of the prototype allergen Bet v 1 with enhanced thermal and chemical stability and rigidity. Structural changes were analyzed by means of x-ray crystallography, nuclear magnetic resonance, and molecular dynamics simulations. Stability was monitored by using differential scanning calorimetry, circular dichroism, and Fourier transform infrared spectroscopy. Endolysosomal degradation was simulated in vitro by using the microsomal fraction of JAWS II cells, followed by liquid chromatography coupled to mass spectrometry. Immunologic properties were characterized in vitro by using a human T-cell line specific for the immunodominant epitope of Bet v 1 and in vivo in an adjuvant-free BALB/c mouse model.

Results: Fold stabilization of Bet v 1 was pH dependent and resulted in resistance to endosomal degradation at a pH of 5 or greater, affecting presentation of the immunodominant T-cell epitope in vitro. These properties translated in vivo into a strong allergy-promoting TH2-type immune response. Efficient TH2 cell activation required both an increased stability at the pH of the early endosome and efficient degradation at lower pH in the late endosomal/lysosomal compartment.

Conclusions: Our data indicate that differential pH-dependent fold stability along endosomal maturation is an essential protein-inherent determinant of allergenicity.

Keywords: Allergic sensitization; Bet v 1; antigen processing and presentation; endolysosomal degradation; molecular allergology; structural stability.

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Fig 1

Crystal structure of the quadruple mutant Bet_mut4 (blue) in comparison with Bet v 1 (gray, PDB: 4A88). Superimposition of Bet_mut4 with Bet v 1 revealed that the overall fold is conserved. A, Mutated residues D69I, K97I, P90L, and G26L are shown as sticks. B, An enlarged view of the mutated residue D69I shows that it is stabilized by several hydrophobic residues, including Ile56, Phe58, Val67, Tyr83, and Val85. C, The surface-exposed mutation K97I is likely to entropically stabilize the protein by releasing coordinated solvent molecules. D, Mutation P90L induced an alternative conformation, as indicated. E, Mutation G26L fills a hydrophobic cavity formed by Phe21, Ile22, and Phe30. All mutated residues are shown as blue sticks compared with sticks from wild-type Bet v 1 (gray). The electron density map (2F_obs-F_calc) is contoured at 1 σ over the mean.

Fig 2

Thermal stability of Bet v 1 and mutant proteins 1 to 4 (Bet_mut1-4). A, Chemical denaturation was assessed by means of CD spectroscopy, and the fraction of denatured protein (F_denatured) is displayed. B and C, Thermal denaturation was measured by using CD (Fig 2, B) and DSC (Fig 2, C). DSC data for wild-type Bet v 1 and mutants were run at a 60°/h scan rate and at 0.15 mg/mL (lower traces) and 0.5 mg/mL (upper traces) offset in the y-axis for clarity. D, Melting points calculated from DSC data.

Fig 3

Influence of fold-stabilizing mutations on the immunogenicity and sensitization capacity of Bet v 1. A, Bet v 1–specific IgG₁ and IgG_2a titers of BALB/c mice (n = 5) immunized intradermally with the different proteins were measured by using a luminometric ELISA at a serum dilution of 1:300 for IgG₁ and 1:100 for IgG_2a. Data are shown as relative light units (RLU). B, Bet v 1–specific IgE levels in serum were measured with an RBL degranulation assay, and results are presented as the percentage of total release. Cell-bound IgE was assessed by using a basophil activation test, and data are shown as stimulation indices (SI). C, Numbers of IL-4– and IFN-γ–secreting splenocytes after restimulation with Bet v 1 and the respective mutants were determined by using the ELISpot assay. SFU, Spot-forming units. Statistical significance compared with Bet v 1 was assessed by using 1-way ANOVA, followed by the Dunnett post hoc test. *P < .05, **P < .01, and ***P < .001.

Fig 4

A-D, Influence of fold-stabilizing mutations on the molecular flexibility of the protein, as indicated by hydrogen deuterium exchange (Fig 4, A) and relaxation dispersion NMR (Fig 4, B-D). In Fig 4, B-D, the 11 residues with the largest backbone amide ¹⁵N relaxation dispersion profiles are displayed.

Fig 5

A-D, Proteolytic degradation of Bet v 1 and the mutants Bet_mut1 to Bet_mut4 (Fig 5, A and D) and their thermal stability (Fig 5, B and C) at different pH values. Proteolytic degradation was assessed by using 15% SDS-PAGE and Coomassie staining (Fig 5, A). Thermal stability was followed by using CD spectroscopy (Fig 5, B), and thermally induced aggregation was followed by using attenuated total reflectance FTIR (Fig 5, C). FTIR data are presented as the fraction of aggregated protein versus temperature. Protein degradation patterns over time were analyzed by using liquid chromatography–mass spectroscopy (Fig 5, D): peptides generated earlier during the proteolytic processing are colored in white, whereas peptides that were not detected during the whole experiment (48 hours) were colored in black. Amino acid positions are labeled at the top. The immunodominant epitope of Bet v 1 is framed in green.

Fig 6

A and B,In vitro antigen processing and presentation. Uptake of DyLight488-labeled Bet v 1 and Bet_mut1-4 by moDCs. The percentage of positive cells (Fig 6, A) and their mean fluorescence intensity (MFI; Fig 6, B) were analyzed by using flow cytometry. moDCs from HLA-DRB1*07:01⁺ donors were preincubated with Bet v 1 or Bet_mut1-4 for the indicated time span and subsequently cocultured with Bet_142-153–specific Jurkat T cells. C, Luciferase reporter expression under control of the IL-2 promoter was measured after 6 hours of coculture. Data are shown as means ± SEMs of relative light units (RLU) of 2 independent experiments by using 2 different donors.

Fig 7

Model for immunogenicity/allergenicity of an allergen depending on differential fold stability at different endosomal pH. During antigen uptake and processing, allergens face a broad range of different pH values, ranging from approximately 7.4 in the extracellular matrix to approximately 4.0 in the terminal lysosome. Proteins with low fold stability in the early endosome are proteolytically processed prematurely, resulting in poor antigen presentation because of low abundance of MHC class II in this compartment (“blue” protein). Proteins with high fold stability at low pH (“black” protein) remain structurally intact until they reach the terminal lysosome, where again MHC class II levels are low and antigen presentation is inefficient. Only proteins that survive the early endosome but are efficiently processed at a more acidic pH can accumulate in the late endosome, where they are finally degraded and loaded on highly abundant MHC II molecules (“red” protein).