Appearance of epitopes in bovine milk protein variants, their allergenicity and potential use in human nutrition
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Abstract
Within the screening of milk samples from different bovine breeds, the known genetic casein variants alphaS1-CN B and C, alphaS2-CN A, B, and D, beta-CN A1, A2, and B, as well as kappa-CN A, B, and E were confirmed and used for in vitro gastrointestinal digestion analyses. The results showed that, despite the significant enzymatic hydrolysis of the alphaS1-, alphaS2-, beta-, and kappa-CN variants during gastrointestinal digestion, peptides were of sufficient size to contain intact IgE-binding epitopes or at least substantial parts thereof. Three previously identified IgE-binding epitopes in alphaS1-CN (AA 109-120, AA 123-132, and AA 173-194), 3 in alphaS2-CN (AA 1 20, AA 105-114, and AA 117-128), 6 in beta-CN (AA 45-54, AA 55-70, AA 83-92, AA 107-120, AA 149-164, and AA 167-184), and 5 in kappa-CN (AA 21-44, AA 67-78, AA 95-116, AA 111-126, and AA 137-148) survived the in vitro gastrointestinal digestion. Furthermore, it could be demonstrated that the genetic polymorphisms have an influence on the arising peptide pattern as different peptides between the alphaS1-, alphaS2-, beta-, and kappa-CN variants were found. In alphaS1-CN B and C, the 2 peptides AA 174-193 of alphaS1-CN B and AA 179-198 of alphaS1-CN C comprising the intact IgE-binding epitope AA 173-194 were determined. In alphaS2-CN B, peptide AA 1-22 and in alphaS2-CN A, peptide AA 7-29 could be detected both corresponding to the major IgE-binding epitope AA 1-20 or parts thereof. Peptides AA 59-72, AA 59-80, and AA 58-80 of beta-CN A1, A2, and B, as well as peptides AA 103-123 and AA 109-123 of beta-CN B, and AA 108-129 and AA 110-125 of beta-CN A1 and A2 contained the IgE-binding epitopes AA 55-70, respectively, AA 107-120. For kappa-CN, the IgE-binding epitope AA 137-148 survived gastrointestinal digestion within peptides AA 136-149 of kappa-CN A and E and peptide AA 134-150 of kappa-CN B. Microarray immunoassays with sera from patients (n = 7) with CMA indicated an allergenic activity of some of these digested casein fragments. This might be one explanation for the high allergenicity and sensitizations rate to the caseins despite their excellent digestibility. Major immunoreactivity was identified for peptides AA 174-193 of alphaS1-CN B and AA 179-198 of alphaS1-CN C but also for the smaller peptides AA 184-196, AA 187-199, and AA 180-193 occurring in both alphaS1-CN variants. Significant IgE-binding was further detected to the gastrointestinal digestions products AA 110-125 of beta-CN A1 and A2 as well as AA 109-123 and AA 103-123 of beta-CN B. None of the sera used in the present study reacted with any of the digestion products of the alphaS2- and kappa-CN variants. This study confirmed an influence of the genetic polymorphisms on the IgE-binding properties of epitopes from alphaS1-, alphaS2-, beta-, and kappa-CN variants of cow and revealed differences in IgE-binding to corresponding epitopes of goat and water buffalo. Single AA substitutions or deletions existing within IgE-binding epitopes AA 17-36, AA 4-23, AA 83-102, AA 173-192, and AA 175-194 of the alphaS1-CN variants A, B, C, E, and I or gastrointestinal digestion products AA 187-199, AA 174-193, and AA 178-198 of alphaS1-CN B and C, as well as AA 108-129 and AA 103-123 of beta-CN A1 and A2, respectively, B led to a loss or reduced or increased IgE-binding with a marked heterogeneity in individual sera. The modifications in the immunoreactivity mainly affected the immunodominat epitopes and, in consequence, it can be accepted that the allergenicity of the whole proteins are altered. The majority of sera showed IgE-binding to alphaS1-CN peptides of cow but also to the homologous counterparts of goat and water buffalo indicating cross-reactivity, which is due to the high degree of AA sequence homology between the milk proteins from cow and the other Bovidae. However, individual sera exhibited a lower, higher, or exclusive immunoreactivity to epitopes of alphaS1- and beta-CN from goat and water buffalo. The results of our investigations support the suggestion that genetic variants differ in their allergenic properties and might therefore provide a new approach for the identification of an alternative and suitable protein source for CMA patients. Furthermore, milk of goat and water buffalo cannot be recommended in general as substitution for cow milk in the nutrition of CMA patients. Nevertheless, the milk proteins of these species are also characterized by genetic polymorphisms, whose effects on IgE-binding and allergenicity have to be defined.Link to publications or other datasets
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Giessen : VVB Laufersweiler