More than 5% of the population in industrialized countries suffer from a type I food allergy, an immunoglobulin E (IgE)-mediated hypersensitivity disease resulting from the loss of oral tolerance to food proteins [1
]. A limited number of foods are responsible for the majority of reactions, with 95% of food allergies being caused by only eight foods including eggs, milk, fish, crustacean shellfish, peanuts, tree nuts, wheat and soybeans [3
]. All these foods contain many proteins, but only a few of them are allergens [3
]. Known food type I allergens have, in general, molecular weights below 70 kDa, are stable molecules that resist cooking and digestion, and stimulate the immune response inducing the production of allergen-specific IgE [3
]. Regardless of the limited number of represented native structural scaffolds, sequence difference thresholds, abundance and stability properties, the molecular signature of protein allergenicity remains yet unresolved [3
All food proteins are exposed to pH 1.3–2 at the gastric compartment during digestion, a condition that is usually used in vitro to trigger the refolding of proteins and of their fragments into amyloid aggregates [10
]. Amyloids are insoluble fibrillary protein structural states displaying protease resistance and, with one exception, a cross-β sheet backbone [11
]. An amyloid formation is a generic feature of many, if not all, natural polypeptide chains if placed under the appropriate environment and concentration [10
]. In fact, food allergens of animal sources such as the β-parvalbumin Gad m 1 from fish (Gadus morhua
); α-lactalbumin (Bos d 4), β-lactoglobulin (Bos d 5), αS2-casein (Bos d 10), β-casein (Bos d 11) and κ-casein (Bos d 12) from bovine milk, and the ovalbumin (Gal d 2) and lysozyme (Gal d 4) from chicken egg-whites rapidly form amyloid aggregates under a variety of conditions, some of which are those of the gastric compartment [14
]. Bos d 5 and Bos d 12 also form heterogeneous or mixed amyloid fibrils under ultraheat, mimicking treatments used in the dairy industry [23
Gad m1 amyloids displayed an enhanced proteinase resistance and a 1000-fold increased IgE-specific binding compared to that of the monomer precursor [14
]. Importantly, unrelated amyloid fibrils such as those formed by Aβ42 and PrP are not recognized by the IgE present in the sera of fish allergic patients, underlining the sequence specificity of the recognition process [14
]. A battery of approaches including peptide arrays, proteomics and the use of mutant chains concluded that regions forming the amyloid cores overlapped with the IgE epitopes [15
]. Notwithstanding, whether this property is specific to Gad m 1 or is shared by other food allergens has not yet been addressed. Given their well-characterized aggregation processes, commercial availability and the knowledge of the major IgE epitopes, we have chosen Bos d 5, Bos d 12, and Gal d 2 as animal food allergen models to analyze the IgE-binding properties of their amyloid state using the sera of allergic patients.
2. Materials and Methods
2.1. Ethics Statement
All experimental protocols and methods were performed following the guidelines of the University Hospital La Paz and the Spanish National Research Council and applied with approval from the Ethics Committee of the University Hospital La Paz (protocol number PI-3065). Anonymity was preserved as established in the ethical permission.
2.2. Food Allergic Patient Sera
Sera were obtained from patients recruited from Hospital La Paz in Madrid (Spain). Fish, milk, and egg allergic patients were selected based on case history, a positive skin prick test with commercial extracts and determination of specific IgE (sIgE) by ImmunoCAP 100 (Thermo Fisher, Uppsala, Sweden) according to the manufacturer instructions (Table 1
2.3. In Silico Studies
Bos d 5 (P02754), Bos d 12 (P02668), Gal d 2 (P01012) and Gad m 1 (A5I874) sequences were retrieved from UniProtKB database [24
]. IgE binding epitopes were taken from previous SPOT-membrane and array assays [15
]. Amyloid cores were predicted using the ZipperDB algorithm [29
]. IgE-binding regions and amyloid forming segments were used to generate binary functions (0,1) of the polypeptide chain (residue number including the signal peptide) using Origin 2019 software. The relative organization (overlap/flank relation) between regions was visualized using a 3D stacking plot.
2.4. Food Allergens
Gal d 2 (A-2512), Bos d 5 (L3908) and Bos d 12 (C0406) were purchased from Sigma-Aldrich. Gad m 1 (A5I874) was prepared as described [31
]. Before their use all proteins were extensively dialyzed at 4 °C against either 25 mM Tris, 0.1 M NaCl pH 7.5 or 0.1 M Gly pH 1.5 using dialysis membranes with an 8 kDa pore diameter (Spectra Por). Dialyzed solutions were centrifuged at 13,500 rpm at 4 °C for 20 min to clear possible existing aggregates and the protein concentrations of the supernatants were determined with the Bradford assay. Proteins at pH 7.5 were referred as the native states (N), whereas the solutions at pH 1.5 were used for amyloid formation.
2.5. Amyloid Fibril Preparation
Protein solutions at 5–8 mg/mL solution in 0.1 M Gly pH 1.5 were incubated at 90 °C for 5 h. After the heating step, all protein samples were stored at room temperature for 36 h to allow fibril maturation. Fibrils were harvested in the pellet fraction of an ultracentrifugation at 100,000× g for 1 h at 4 °C and resuspended in 50 mM Gly pH 1.5. When required, mature fibrils were placed in 1.5 mL eppendorf tubes and sonicated for 15 min in a sonicating water bath.
2.6. Circular Dichroism Spectroscopy
Circular dichroism (CD) measurements were performed using a Jasco J-820 spectropolarimeter equipped with a Peltier-controlled thermostatted cell holder. Far-UV CD spectra were recorded using a 0.3 mg/mL protein concentration solution in 25 mM Gly pH 1.5 at 25 °C. Spectra were corrected for the base line contribution and analyzed as described taking 110 Da as the residue average molecular weight [14
2.7. Atomic Force Microscopy (AFM)
For AFM visualization, 30 μL of the aggregate solutions prepared at 0.05 mg/mL in 2.5 mM Gly pH 1.5 were absorbed onto freshly cleaved mica via a 5–10 min incubation at room temperature. The surfaces were then rinsed with double-distilled water and dried. Images were obtained in the tapping mode using a JPK Nanowizard 2 microscope and HQXSC11 B (Mikromash) cantilevers (2.7 N/m force constant and 70 kHz resonance frequency). An AFM analysis was performed using the free program WSxM 4.0 (Nanotec)
2.8. Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis (SDS-PAGE)
Typically, 3 μg of native and aggregated proteins were separated by SDS-PAGE under denaturing conditions using Mini-Protean® TGX Stain-FreeTM (#456-8126) gels. Bands were visualized using Coomassie Brilliant Blue R-250 (CBB) staining solution (BioRad, Hercules, CA, USA), and the images were recorded with the Molecular Imager ChemiDoc XRS-Plus (BioRad, Hercules, CA, USA).
2.9. Dot Blot Analysis
Samples of 0.1–0.2 µg protein/dot were loaded in duplicate onto nitrocellulose membranes. After blocking for 1 h in TBS-T (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.5% Tween 20) containing 0.5% bovine serum albumin (Sigma-Aldrich, St. Louis, MO, USA) at room temperature, the membranes were incubated for 2 h with either the distinct serum (diluted 1:10 in blocking buffer) or with the anti-amyloid fibril antibody (OC antibody) (AB2286 Merck Millipore, 1:2000 dilution, Darmstadt, Germany). After extensive washing with TBS-T, membranes were incubated for 1 h with horseradish peroxidase (HRP)-labeled goat antimouse IgG (Sigma-Aldrich, 1:5000 dilution, St. Louis, MO, USA) or mouse monoclonal B3102E8 antihuman IgE (Abcam, 1:2000 dilution, Cambridge, UK) and developed using Clarity Western-ECL (BioRad, Hercules, CA, USA). The signals were recorded with the ChemiDoc XRS-Plus imager and analyzed using the volume tools provided by ImageLab software.
In this pilot study, we sought to investigate whether the formation of IgE-binding amyloids as found for Gad m 1 is a feature shared by other animal food allergens. Our results show that Bos d 12 and Gal d 2 yield amyloids which are recognized by specific IgEs contained in the sera of milk and egg allergic patients, respectively. For most patient sera, the amyloid fold represents the major IgE-reactive state. Bos d 5 behaved distinctly since both native and amyloid folds did not react with the IgE of the milk allergic patient sera used, despite the sIgE levels provided by the ImmunoCAP characterization, suggesting differences in the isoform composition of both reagents [24
The search for the molecular signatures that predispose food proteins to become allergens has yielded several characteristics including abundance in food, structure, resistance to processing and digestion and the presence of multiple linear IgE binding epitopes [5
]. These features have been established and omit the effects that transit through the gastric compartment can cause. One of the effects is the pH-induced refolding of the allergens and of their fragments into their amyloid states [10
]. Indeed, of almost all the proteins contained in their sequence, at least one segment capable of forming amyloid fibrils [34
]. However, only 5.3% of these segments are found in the surface of the native 3D structures and less than 0.1% of them displayed the proper sticky geometry [34
]. Consequently, the formation of amyloid fibrils under physiological conditions is common for intrinsically disordered proteins such as Bos d 12 [19
]. However, globular proteins such as most food allergens require a partial unfolding or a nicking process to expose the amyloid forming segments otherwise buried in the structure interior or stabilized by bound ligands. The efficiency of this refolding mainly depends on the sequence, the abundance of the protein and the interference of off-pathways such as those driving the less favorable amorphous aggregates and nonproductive truncations [11
]. For complex mixtures such as food, factors such as processing treatments and the presence of native state ligands and of anti-amyloid compounds might also modulate the efficiency [12
]. Tailoring a protocol using acid pH, low protein concentrations, short incubations and random truncations, for general use, facilitates the amyloid fibril purification required for their systematic characterization.
The isolated Bos d 5, Bos d 12, Gal d 2 and Gad m 1 amyloid states are not unique and differ in their shape, polymorphism degree and surface reactivity as shown by AFM imaging and IgE binding. Indeed, the multiplicity of potential fibrillation-prone segments predicted by the ZipperDB algorithm and the fragmentations showed by SDS-PAGE entails complex aggregation events far beyond those uniquely containing IgE-binding sites. On the other hand, the formed aggregates may display different elongation and dissociation rates which will dictate the landscape of molecular species existing under gastrointestinal-like conditions [12
]. In this sense, easy dissociating amyloid fibrils will function as IgE-binding epitope depots, whereas those displaying a slow dissociation will function as epitope backbones. These nonexclusive possibilities might coexist in any of the allergens if fibrillation is seeded through different sticky segments.
An in silico analysis using previously described IgE-binding epitopes and the amyloid cores predicted by the ZipperDB algorithm detected multiple regions with a functional overlap in each allergen chain. The overlap degree should be taken in qualitative terms, since each of the variables used employ different length windows to define function. In this sense, the considered IgE epitopes were determined using 20 amino acid residues sequential peptides with a three-amino acid shift and sera from paediatric patient cohorts [25
]. On the contrary, the prediction of amyloid cores uses a six-amino acid peptide window. Studies with Gad m 1 using 12-length peptides and a two-amino acid sequential shift between two consecutive peptides, an immobilization density of ≈10 nmol per spot (≈ 400 nmol/cm2
), sera from fish allergic patients for IgE-epitope mapping and the antifibril OC-antibody for amyloid detection identified the segments 25
as the segments with dual functions [15
]. Both sequences are N-terminal shifted to the predicted sticky segments, indicating the expansion of the aggregation properties beyond the detected cores. Using a similar approach for milk and egg allergens and the sera from a varied allergic patient cohort will allow a closer view of the functional overlap.
The potential conformational multiplicity of the regions forming the IgE-binding epitopes questions the validity of mapping the epitopes using the native 3D structures of food allergens as unique template. In fact, the IgE binding sequences DHKAFFTKV and FLQNFS form parts of helical structures in the native state of Gad m 1 whereas the amyloid fold adopts a cross β-sheet structure [16
]. In fact, consideration of the IgE-epitope amyloid folds will simultaneously explain their protease resistance and higher avidity and affinity for any ligand binding process [12
]. On the other hand, the sequence differences of the segments with functional overlaps identified in the distinct allergens sustain the binding specificity of the IgE repertoire present in the sera of the allergic patients sensitized to distinct foods. Additionally, for the given food allergen differences in the covalent structure (isoforms, covalent modifications, etc.), the concentration in the food source and local industrial processing may impact the functionality of the aggregating regions and therefore their stability. Dynamic changes in the quality of the offending foods may explain variations in the IgE-epitope repertoires of different patient cohorts.
In summary, this works provides the pilot proof of concept by which the amyloid fold of segments of animal food allergens can define the novel structural properties of IgE-binding epitopes. Notwithstanding, their solid assignment will require a deeper study with a higher number of sera samples, as will their simultaneous characterization using SPOT-membranes/arrays using a similar design (peptide length and offsets, and surface density, among other critical parameters) and the synthetic reconstruction of the segments of interest as performed with Gad m 1 [14
]. Indeed, IgE-epitope repertoires depend on the regional origin of the patient cohort and change with the patient’s age.