Targeting a cross-reactive Gly m 5 soy peptide as responsible for hypersensitivity reactions in a milk allergy mouse model

Abstract

Background: Cross-reactivity between soybean allergens and bovine caseins has been previously reported. In this study we aimed to map epitopes of the major soybean allergen Gly m 5 that are co-recognized by casein specific antibodies, and to identify a peptide responsible for the cross-reactivity.

Methods: Cow's milk protein (CMP)-specific antibodies were used in different immunoassays (immunoblotting, ELISA, ELISA inhibition test) to evaluate the in vitro recognition of soybean proteins (SP). Recombinant Gly m 5 (α), a truncated fragment containing the C-terminal domain (α-T) and peptides of α-T were obtained and epitope mapping was performed with an overlapping peptide assay. Bioinformatics tools were used for epitope prediction by sequence alignment, and for modelling the cross-recognized soy proteins and peptides. The binding of SP to a monoclonal antibody was studied by surface Plasmon resonance (SPR). Finally, the in vivo cross-recognition of SP was assessed in a mouse model of milk allergy.

Results: Both α and α-T reacted with the different CMP-specific antibodies. α-T contains IgG and IgE epitopes in several peptides, particularly in the peptide named PA. Besides, we found similar values of association and dissociation constants between the α-casein specific mAb and the different milk and soy components. The food allergy mouse model showed that SP and PA contain the cross-reactive B and T epitopes, which triggered hypersensitivity reactions and a Th2-mediated response on CMP-sensitized mice.

Conclusions: Gly m 5 is a cross-reactive soy allergen and the α-T portion of the molecule contains IgG and IgE immunodominant epitopes, confined to PA, a region with enough conformation to be bound by antibodies. These findings contribute to explain the intolerance to SP observed in IgE-mediated CMA patients, primarily not sensitised to SP, as well as it sets the basis to propose a mucosal immunotherapy for milk allergy using this soy peptide.

Figure 1. Immunochemical cross-reactivity.

A) Detection of specific human serum IgE antibodies by indirect ELISA. Microtiter plates were coated with CMP, SP, recombinant α-subunit of β-conglycinin “α” or α-T, and sequentially incubated with sera of CMA patients and anti-human IgE specific conjugate. 15/15 sera contained IgE antibodies specific for CMP, 13/15 sera contained IgE antibodies specific for α and 14/15 sera contained IgE antibodies for α-T. B) SDS-PAGE and immunoblotting of CMP and SP. SDS-PAGE was performed under non reducing conditions for CMP (upper panel), SP (middle panel), and purified recombinant α and the α-T fraction (lower panel). Immunoblottings were developed with patient sera containing milk-specific IgE antibodies or control sera (from non-allergic patients) (1∶4), CMP-specific rabbit polyclonal antiserum (1∶1,000), and α-casein-specific monoclonal antibody (1D5 mAb 1∶1,500). CMP: Cow's milk protein, cas: caseins, SP: soybean protein, G: 11S glycinin subunits; β-conglycinin subunits are indicated as α, α′ and β. M: Molecular masses are given on the left in kilo Daltons (kDa).

Figure 2. Sequential competitive ELISA with the CMP-specific rabbit polyclonal antiserum and 1D5 mAb.

Microtiter plates were coated with CMP (1 µg/well) (A and C), α (0.1 µg/well) or α-T (0.1 µg/well) (B). CMP-specific polyclonal antiserum (A: 1∶200,000; B: 1∶6,000) or α-casein-specific mAb (C: 1∶300,000) were pre-incubated with different inhibitors at several concentrations and then added to Ag-coated wells. Then the appropriate conjugated secondary antibodies were added and the color was developed.

Figure 3. Epitope mapping of α-T.

SPOTs array depicting the overlapping 15-aminoacid peptides covering the complete sequence of α-T, and probed with two different pools of serum from cow's milk allergic patients, a rabbit CMP-specific polyclonal serum and the 1D5 α-casein-specific mAb, followed with the appropriate conjugated secondary antibodies. Positive spots are shaded in grey boxes and recognized amino acids are bolded in the sequence depicted for spotted peptides.

Figure 4. Schematic localization of the identified spots along the α-T sequence and sequence alignment analysis.

A) Analysis of the sequence alignment of α-T with IgE epitopes in bovine caseins (IEDB) shown as hit distribution of amino acid similitude per position along the sequence of α-T. Positions of α-T with high hit values (black columns) correlate with positive spots of the overlapping assay (spots 9, 10 and12); B) The sequence of α-T (1–234 aa) is depicted along with overlapped peptides, PA and PN (305–539 aa correspond to α). In silico predicted alpha-helix (white ovals) and beta-sheet (black arrows) zones are indicated. PA contains the amino acid positions with the highest hits and the α-helix and β strand secondary structures.

Figure 5. In silico and in vitro analysis of α, α-T and PA.

A) Homology model obtained using the Modeller and visualized by PyMOL of α-subunit of β-conglycinin, α-T and PA, based on NCBI Conserved Domain Search results with α′ subunit as template (α-helices are shown in blue and β-sheets as dark grey arrows on the structure). B) Surface representation of positive overlapped peptides marked in different colors according to the intensity of the reaction (from pale yellow to red); I and II correspond to opposite sides of the α molecule. Positive peptides localized on the surface of the molecule seem to converge in three regions or putative epitopes. The remaining part of the molecule is represented in transparent spheres showing the secondary structure. C) SDS-PAGE of recombinant PA and PN and immunoblotting developed with 1D5 α-casein-specific mAb and a representative serum of CMA patients. Alpha-s1 casein was used as positive control (Molecular masses are shown on the left as kDa). D) Dot-blot of recombinant treated and untreated PA revealed with a pool of CMA patient sera, α-casein-specific mAb and CMP-specific polyclonal rabbit antiserum. Denaturing conditions consisted on: 0.1M DTT solution, 6M urea or 100°C for 10 minutes. Cas: Alpha-s1 casein.

Figure 6. In vivo cross-reactivity assessed in a food allergy mouse model.

A) Schematic representation of the experimental protocol: BALB/c mice were subjected to weekly intragastric sensitization with cholera toxin and CMP from day 0 through day 35. Challenge was performed at day 45 by ig administration of proteins (CMP, SP or OVA). Control mice only received CMP and then they were challenged with the different antigens. Symptoms were observed 30 min following the oral challenge and scored according to Table 2. B) Clinical scores assigned to symptoms observed 30 min following the sublingual challenge with 10 µg of β-lg, α, α-T, PA or OVA. C) Specific IgE and IgG1 in serum of milk-sensitized and control mice were assessed by EAST or indirect ELISA at day 45, respectively (mean values ± SEM). D) Cutaneous test performed in sensitized mice subcutaneously injected with 10 µg of antigens (α, α-T or PA) in the right flank and with saline in the left flank. The presence of blue color in the skin within minutes was considered a positive cutaneous test. E) Levels of IL-5 and IFN-γ assayed on stimulated splenocyte supernatants by ELISA (mean values ± SEM). Results correspond to a single experiment with five mice per condition, representative of three separate experiments that gave similar results. Statistically significant differences are denoted as starred values (*) ***p<0.005, **p<0.01, *p<0.05.