Sensitising capacity of peptides from food allergens

Research output: Book/ReportPh.D. thesis

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Food allergy is a major health problem in the Western countries, affecting 3-8% of the population. What makes a dietary protein a food allergen has not yet been established, though several characteristics have been proposed to be shared by food allergens. One of the features believed to be a general characteristic
of food allergens is resistance to digestion. This is based on studies showing that allergenic dietary proteins in general are more resistant to digestion than dietary proteins with no proven allergenicity, concluding that a correlation between stability to digestion and allergenic potential exist. Resistance to digestion is for this reason a test parameter included in the safety assessment of the allergenic potential of novel proteins in genetically modified foods. The association between resistance to digestion and allergenic potential has though been challenged in recent years. This PhD project aimed to investigate the sensitising potential of digestion products from the peanut allergen Ara h 1 and the cow’s milk allergen β-lactoglobulin (BLG) in a Brown Norway (BN) rat model. Further the project aimed to compare the IgE binding epitopes of intact and digested Ara h 1. This was done by digesting Ara h 1 and BLG in an in vitro model simulating the human gastric or gastroduodenal digestion process. Simulated gastric digestion was performed with immobilised pepsin for 120 min at pH 2.5, while simulated duodenal digestion was performed with immobilised trypsin and chymotrypsin for 15 min at pH 6.5. Fractions of digestion products were made by separating the peptide fragments according to sizes in gel permeation chromatography (GPC). The intact allergens as well as digestion products hereof were thoroughly characterised by reverse phase high-performance liquid chromatography, MALDI-TOF mass spectrometry, amino acid analysis and GPC. To study the sensitising capacity groups of BN rats were immunised with the intact allergen or digestion products hereof by i.p. immunisation and specific antibody responses were examined by ELISAs, RBL-assay or avidity measurements. Comparison of intact and digested Ara h 1-specific IgE binding epitopes were performed by competitive immunoscreening using a random phage-displayed peptide library followed by mapping the identified IgE-binding epitope mimics on the surface of the Ara h 1 molecule. In addition to sera from the sensitised BN rats, sera from peanut allergic patients were used. Both the gastric as well as the gastro-duodenal digests of the peanut allergen Ara h 1 were found to be very efficient for sensitising the BN rats. While gastric digest consisted of peptide fragments of up to Mr 4,000 the duodenal digest consisted of peptide fragments of up to Mr 2,000, yet both the peptide fragments in the gastric as well as in the gastro-duodenal digests were aggregated to complexes of larger sizes. After separation of the digested Ara h 1 into fractions the sensitising capacity was lost, though the IgE-binding capacity was retained. Epitope mapping of intact and digested Ara h 1 showed IgE binding epitopes of Ara h 1 to be conformational in origin and at least to some extent surviving the digestion process. For the peanut allergic patients five motifs were found to account for more than 65% of all identified epitope mimics and were found for both the intact as well as the digested Ara h 1. Digested BLG with peptide sizes of up to Mr 4,500 could on the other hand not induce any sensitisation response in the BN rats. They were instead suggested to possess tolerogenic capacity when co-administered together with intact BLG. The results presented in the current thesis demonstrate that even very small peptide fragments, originally thought to be too small to act as a food allergens may indeed possess all features of a ‘complete’ allergen. This implies that an association between allergenicity and resistance to digestion is not an absolute feature of food allergens. The presented work indicates that peptide fragments may either possess sensitising capacity per se or that the observed allergenic capacity could be a result of the small peptide fragments aggregating to complexes of larger sizes. The importance of formation of aggregates is suggested by the epitope mapping study, where survival of conformational epitopes is demonstrated. This together with the findings, that fractionation of digestion products leads to a loss of the sensitising potential, reveals that the allergenicity had to be more than simply a result of the small peptide fragments aggregating, and more a result of them being in an aggregated state resembling the intact Ara h 1 molecule. While small peptide fragments derived from one food allergen may retain sensitising capacity this is not necessarily the truth for other food allergens. This was demonstrated with the cow’s milk allergen BLG, from which peptide fragments were shown not to be efficient for inducing any specific antibodies. Instead the results indicated that the peptide fragments derived from BLG had tolerogenic capacity, demonstrating that while some mixtures of peptides may guide the immune system in one direction, other mixtures of peptides may guide the immune system in another direction. Together these results demonstrate that several characteristics of digestion products from food allergens may collectively contribute the allergenic potential, where more than just peptide sizes and structures may contribute. In conclusion, the experimental data presented in this PhD thesis contribute to the understanding of induction of allergy by investigating the sensitising potential of peptides derived from a food allergen. It add knowledge to our understanding of the mechanisms underlying the sensitisation, but at the same time points to the difficulties, if not infeasibilities, in identifying features that can be used as an ubiquitous marker for allergenicity of a dietary protein.
Original languageEnglish
Place of PublicationSøborg
PublisherDTU Food
Number of pages139
ISBN (Electronic)978-87-92763-28-0
Publication statusPublished - 2012


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