Targeting a Cross-Reactive Gly m 5 Soy Peptide as Responsible for Hypersensitivity Reactions in a Milk Allergy Mouse Model

Abstract

BackgroundCross-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.MethodsCow'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.ResultsBoth 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.ConclusionsGly 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.

Bovine Milk Allergens: A Comprehensive Review

“…Nonetheless, it has been demonstrated that some patients are intolerant to such products, suggesting the cross‐reactivity between soy and cow milk proteins. It was reported that the α‐subunit of beta‐conglycinin (Gly m 5.0101) from the vicilin‐like protein family, the G4 subunit of glycinin (Gly m 6.0401) from legumin‐like proteins, and, more recently, the cysteine protease P34 (Gly m Bd30K) and the globulin P28 (Gly m Bd28K) are involved in cross‐reactivity phenomena with bovine caseins (Rozenfeld and others ; Katz and others ; Smaldini and others ; Curciarello and others ; Candreva and others ; Candreva and others ).…”

Cross-Reactivity Between the Soybean Protein P34 and Bovine Caseins

“… 21 , 22 We have previously shown that Gly m 6 G4 and Gly m 5 are recognized by the IgE antibodies of patients allergic to milk, although the clinical relevance of this finding requires further investigation. 23 , 24 , 25 Taking into account the fact that soybean proteins are ubiquitous, exposure to soybean in patients is difficult to discard. A milk-allergic mouse model was used to overcome this problem, and we previously demonstrated that hypersensitivity reactions were elicited in milk-sensitized animals exposed to total soybean proteins or Gly m 6 G4.…”

Allergenic Milk Proteins. Friend or Foe Nutritional Proteins?

“…We have used a food allergy mouse model to CMP and characterized several soy allergens that cross-react with caseins. We identified B and T epitopes shared between the main soy allergens and bovine caseins [30][31][32]. Again, only a restricted proportion of milk allergic patients react against soy proteins during restriction diet.…”