Acid Hydrolysis of Gluten Enhances the Skin Sensitising Potential and Drives Diversification of IgE Reactivity to Unmodified Gluten Proteins (1)
It is well-accepted that hydrolysis in general reduces the allergenicity of food allergens, rendering them with decreased ability to elicit allergic reactions in sensitized individuals due to the destruction of IgE binding epitopes. However, the de novo skin sensitizing capacity of gluten-derived hydrolysates, as well as the ability to break an established oral tolerance to gluten remains largely unexplored.
Deamidation inhibition ELISA: Inhibition ELISA assays for the evaluation of the extent of
gluten deamidation and of the presence of residual native gliadin repeat epitopes were performed by
using three complementary monoclonal antibodies. ELISA plates were coated with the highly deamidated LQPEEPFPEQC peptide conjugated to BSA, with deamidated α-gliadins, or with native gliadins. Gluten samples were pre-incubated with antibodies Mab INRA-DG1, MAb MCO1, and MAb PQQ3B4 directed respectively against the high, low, or non-deamidated epitopes and subsequently added to plates. Antibody binding to coated plates was detected using horseradish peroxidase (HRP)-labelled anti-mouse IgG. o-phenylenediamine was used as substrate and color development was stopped with 2 M H2SO4.
In contrast to Un Glu, E Glu contained no high MW fractions and the monomeric part was switched towards low MW peptides mostly detected within the range of 5 to 30 kDa. The three Ac Glu products had very similar MW protein distributions, with monomeric proteins primarily in the range of 30 to 100 kDa. The three Ac Glu products were all able to fully inhibit the binding of antibodies specific for highly deamidated epitopes in contrast to Un Glu and E Glu, demonstrating the absence of highly deamidated epitopes in Un Glu and E Glu.
All five gluten products induced a dose-dependent sensitisation as measured by the levels of product-specific IgE in serum. The deamidated Ac Glu products induced a stronger sensitisation than Un Glu and E Glu.
All five gluten products were able to fully inhibit the binding between Un Glu and antibodies raised against Un Glu. all products were able to fully inhibit the binding between E Glu and antibodies raised against E Glu. Greatest inhibition was observed towards Ac Glu 1, followed by Ac Glu 2 and Ac Glu 3. These findings demonstrate the development of new epitopes by acid hydrolysis of gluten, and that the level of new epitopes correlates with the degree of deamidation.
Distinct IgE binding profiles, with more immunoreactive spots visualised for E Glu and Ac Glu compared to Un Glu, and more for Ac Glu than for E Glu. A total of 6 areas on blots of Un Glu were found.
Both the skin and small intestine LP showed an increase in CD3+CD4+ helper T cell numbers in response to skin application of gluten products compared to the PBS control group.
Among the five gluten products, only Ac Glu 3 was found to significantly induce product-specific sensitisation via the skin in animals with oral tolerance to wheat.
New epitopes were generated by acid hydrolysis, all products were found to retain epitopes present in unmodified gluten protein. The skin sensitisation capacity of acid hydrolysates is associated with the degree of product deamidation, and thus correlates with the amount of new epitopes generated and the depletion of native unmodified gluten epitopes.
A recent genome-wide association study of the Japanese population found that allergy induced by the soap containing acid hydrolysed gluten was associated with variations in the HLA-DR and RBFOX1 genes indicating a role for alterations in antigen recognition by T cells.