Danijela Apostolovic

Structure and antioxidant activity of β-lactoglobulin-glycoconjugates obtained by high-intensity-ultrasound-induced Maillard reaction in aqueous model systems under neutral conditions.

Sonication is a new processing technology in the dairy industry. The aim of this study was to test glycation of β-lactoglobulin (BLG) in Maillard reaction (MR) induced by high-intensity ultrasound in aqueous solution under neutral conditions at 10-15 °C, which is not favourable for the MR. BLG was sonicated in the presence of glucose, galactose, lactose, fructose, ribose and arabinose. Formation of Maillard reaction products (MRPs) was monitored by mass spectrometry, spectrophotometry and fluorimetry. Ultrasound treatment resulted in formation of MRPs with all tested carbohydrates. Ribose induced the highest degree of modification resulting in 76% of BLG modified and an average of three anhydroribose units attached. Circular dichroism spectra analyses indicated only minor alterations in secondary and tertiary structures. MRP obtained by ultrasound exhibited 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging activity and possessed increased iron-chelating activity and reducing power. High-intensity ultrasound efficiently promotes BLG-glycoconjugates formation by MR in aqueous solutions under non-denaturing conditions.

Conformational stability of digestion-resistant peptides of peanut conglutins reveals the molecular basis of their allergenicity

Conglutins represent the major peanut allergens and are renowned for their resistance to gastro-intestinal digestion. Our aim was to characterize the digestion-resistant peptides (DRPs) of conglutins by biochemical and biophysical methods followed by a molecular dynamics simulation in order to better understand the molecular basis of food protein allergenicity. We have mapped proteolysis sites at the N- and C-termini and at a limited internal segment, while other potential proteolysis sites remained unaffected. Molecular dynamics simulation showed that proteolysis only occurred in the vibrant regions of the proteins. DRPs appeared to be conformationally stable as intact conglutins. Also, the overall secondary structure and IgE-binding potency of DRPs was comparable to that of intact conglutins. The stability of conglutins toward gastro-intestinal digestion, combined with the conformational stability of the resulting DRPs provide conditions for optimal exposure to the intestinal immune system, providing an explanation for the extraordinary allergenicity of peanut conglutins.

Immunoproteomics of processed beef proteins reveal novel galactose-α-1,3-galactose-containing allergens.

Red meat allergy presents a novel form of food allergy with severe delayed allergic reactions where IgE antibodies are directed against the carbohydrate α‐Gal epitope. Food preparation and processing can influence the allergenicity of proteins. The aim of this study was to characterize the proteomic profile of different beef preparations and to investigate their α‐Gal reactivity and potential allergenicity.

Interactions of epigallo-catechin 3-gallate and ovalbumin, the major allergen of egg white.

Polyphenols, the potent plant secondary metabolites, have beneficial effects on human health, but the mechanism(s) by which these effects are exerted is not well understood. Here, we present the detailed analysis of the interactions between the major green tea catechin, epigallo-catechin 3-gallate (EGCG), and the major dietary protein and allergen, ovalbumin (OVA). We show that EGCG binds to the pocket that partly overlaps with the previously identified IgE-binding region in OVA, and that this interaction induces structural changes in the allergen. Moreover, our ex vivo studies reveal that OVA binds IgE and stimulates degranulation of basophils, and that its uptake by monocytes proceeds at a slower rate in the presence of EGCG. This study provides further evidence in support of the proposed mechanism by which EGCG interactions with the food allergens contribute to its diverse biological activities and may impair antigen uptake by antigen-presenting cells.

Reduction and alkylation of peanut allergen isoforms Ara h 2 and Ara h 6; characterization of intermediate- and end products

Conglutins, the major peanut allergens, Ara h 2 and Ara h 6, are highly structured proteins stabilized by multiple disulfide bridges and are stable towards heat-denaturation and digestion. We sought a way to reduce their potent allergenicity in view of the development of immunotherapy for peanut allergy. Isoforms of conglutin were purified, reduced with dithiothreitol and subsequently alkylated with iodoacetamide. The effect of this modification was assessed on protein folding and IgE-binding. We found that all disulfide bridges were reduced and alkylated. As a result, the secondary structure lost α-helix and gained some β-structure content, and the tertiary structure stability was reduced. On a functional level, the modification led to a strongly decreased IgE-binding. Using conditions for limited reduction and alkylation, partially reduced and alkylated proteins were found with rearranged disulfide bridges and, in some cases, intermolecular cross-links were found. Peptide mass finger printing was applied to control progress of the modification reaction and to map novel disulfide bonds. There was no preference for the order in which disulfides were reduced, and disulfide rearrangement occurred in a non-specific way. Only minor differences in kinetics of reduction and alkylation were found between the different conglutin isoforms. We conclude that the peanut conglutins Ara h 2 and Ara h 6 can be chemically modified by reduction and alkylation, such that they substantially unfold and that their allergenic potency decreases.

Complexes of green tea polyphenol, epigalocatechin-3-gallate, and 2S albumins of peanut

2S albumins of peanuts are seed storage proteins, highly homologous in structure and described as major elicitors of anaphylactic reactions to peanut (allergens Ara h 2 and Ara h 6). Epigallocatechin-3-gallate (EGCG) is the most biologically potent polyphenol of green tea. Non-covalent interactions of EGCG with proteins contribute to its diverse biological activities. Here we used the methods of circular dichroism, fluorescence quenching titration, isothermal titration calorimetry and computational chemistry to elucidate interactions of EGCG and 2S albumins. Similarity in structure and overall fold of 2S albumins yielded similar putative binding sites and similar binding modes with EGCG. Binding affinity determined for Ara h 2 was in the range described for complexes of EGCG and other dietary proteins. Binding of EGCG to 2S albumins affects protein conformation, by causing an α-helix to β-structures transition in both proteins. 2S albumins of peanuts may be good carriers of physiologically active green tea catechin.

Red meat allergic patients have a selective IgE response to the α-Gal glycan

Galactose‐α‐1,3‐galactose (α‐Gal) is a mammalian carbohydrate with significance in a novel type of food allergy. Patients with IgE against α‐Gal report severe allergic symptoms 3–6 h after consumption of red meat. We investigated whether IgE from red meat allergic patients recognizes other mammalian glycans than α‐Gal or glycans from the plant kingdom and insects of importance in allergy. We found that none of the 24 red meat allergic patients investigated had an IgE antibody response against the other abundant mammalian glycan N‐glycolylneuraminic acid or against cross‐reactive carbohydrate determinants from plant or venom sources (nCup a 1, nArt v 1, and MUXF3). Deglycosylation of an α‐Gal‐containing protein, bovine thyroglobulin, significantly reduced the IgE response. In conclusion, we show that red meat allergic patients have a selective IgE response to the α‐Gal glycan found in red meat. Other common glycans reactive in allergic disease are not targets of red meat allergic patients’ IgE.

Allergenicity attributes of different peanut market types.

Four different market classes of peanut (Runner, Virginia Spanish, and Valencia) are commonly consumed in Western countries, but for some consumers peanuts are a main cause of food-induced anaphylaxis. Limited information is available on the comparative allergenicity of these distinct market classes. The aim of this study was to compare allergenicity attributes of different peanut cultivars. The protein content and protein profiles were highly comparable for all tested cultivars. All cultivar samples contained the major allergens Ara h 1, Ara h 2, Ara h 3 and Ara h 6, as assessed by SDS-PAGE and RP-HPLC, although some minor differences in major allergen content were found between samples. All samples were reactive in commercial ELISAs for detection and quantification of peanut protein. IgE-binding potency differed between samples with a maximum factor of 2, indicating a highly comparable allergenicity. Based on our observations, we conclude that peanuts from the main market types consumed in Western countries are highly comparable in their allergenicity attributes, indicating that safety considerations with regard to peanut allergy are not dependent on the peanut cultivar in question.

The cat lipocalin Fel d 7 and its cross-reactivity with the dog lipocalin Can f 1.

We investigated the prevalence of sensitization to the cat lipocalin Fel d 7 among 140 cat‐sensitized Swedish patients and elucidated its allergenic activity and cross‐reactivity with the dog lipocalin Can f 1. Sixty‐five of 140 patients had IgE to rFel d 7 whereof 60 also had IgE to rCan f 1. A moderate correlation between IgE levels to rFel d 7 and rCan f 1 was found. rFel d 7 activated basophils in vitro and inhibited IgE binding to rCan f 1 in 4 of 13 patients, whereas rCan f 1 inhibited IgE binding to rFel d 7 in 7 of 13 patients. Fel d 7 and Can f 1 showed high similarities in protein structure and epitopes in common were found using cross‐reactive antisera. Fel d 7 is a common allergen in a Swedish cat‐sensitized population that cross‐reacts with Can f 1, and may contribute to symptoms in cat‐ but also in dog‐allergic patients.

The red meat allergy syndrome in Sweden

SummaryIn the last decade, a novel type of food allergy presenting with severe allergic reactions several hours after consumption of red meat has been recognized. The allergic responses are due to IgE antibodies directed against the carbohydrate epitope galactose-α-1,3-galactose (α-Gal) found in mammalian meat. This review presents the red meat allergy syndrome in Sweden, discusses the features of the immune response to carbohydrates, and highlights the presence of heat stable α-Gal-containing proteins in meat.The number of diagnosed red meat allergy cases in Sweden has increased significantly over the past few years. All patients have been tick bitten. Our recent work has shown that α-Gal is present in the European tick Ixodes ricinus (I. ricinus), thus potentially explaining the strong association between anti-α-Gal IgE and tick bites, with development of red meat allergy as a secondary phenomenon. Further studies using immunoproteomics have identified novel α-Gal-containing meat proteins that bound IgE from red meat allergic patients. Four of these proteins were stable to thermal processing pointing to the fact that the allergenicity of red meat proteins is preserved in cooked meat. In keeping with the fact that the α-Gal epitope is structurally related to the blood group B antigen, a positive association with the B-negative blood groups among our red meat allergic patients was noted. A selective IgE reactivity to the pure carbohydrate moiety was observed when investigating the specificity of the α-Gal immune response. IgE from red meat allergic patients does not recognize the other major mammalian carbohydrate, N-glycolylneuraminic acid (Neu5Gc), also present in high amounts in red meat. Furthermore, neither common cross-reactive carbohydrate determinants (CCDs) from plants nor venoms are targets of the IgE response in these patients.Taken together, the α-Gal carbohydrate has shown to be a potentially clinically relevant allergen that should be taken into account in the diagnosis of food allergy. Many new findings in the field of red meat allergy have been obtained during the past years, but further efforts to understand the process of digestion, absorption, and delivery of α-Gal-containing molecules to the circulation are needed.