Covalent polyphenol modification of a reactive cysteine in the major apple allergen Mal d 1.

Naturally occurring polyphenols can modify the molecular properties of food allergens. For the major apple allergen Mal d 1 it has been postulated that chemical reactions with polyphenols cause permanent changes in the tertiary structure, causing a loss of conformational IgE epitopes and reducing allergenicity. In our study, we investigated the effect that reactions with oxidized polyphenols have on the structure of Mal d 1 by mass spectrometry and NMR spectroscopy. We showed that a surface-exposed cysteine residue in this allergen spontaneously reacts with oxidized polyphenols under formation of a defined covalent adduct. Chemical modification of Mal d 1 did not destabilize or perturb the three-dimensional fold, nor did it interfere with ligand binding to its internal pocket. A structural model of the chemically modified apple allergen is presented, which reveals that the bound polyphenol partially covers a conformational IgE epitope on the protein surface.

Ascorbylation of a Reactive Cysteine in the Major Apple Allergen Mal d 1.

The protein Mal d 1 is responsible for most allergic reactions to apples (Malus domestica) in the northern hemisphere. Mal d 1 contains a cysteine residue on its surface, with its reactive side chain thiol exposed to the surrounding food matrix. We show that, in vitro, this cysteine residue is prone to spontaneous chemical modification by ascorbic acid (vitamin C). Using NMR spectroscopy and mass spectrometry, we characterize the chemical structure of the cysteine adduct and provide a three-dimensional structural model of the modified apple allergen. The S-ascorbylated cysteine partially masks a major IgE antibody binding site on the surface of Mal d 1, which attenuates IgE binding in sera of apple-allergic patients. Our results illustrate, from a structural perspective, the role that chemical modifications of allergens with components of the natural food matrix can play.

Inverse relation between structural flexibility and IgE reactivity of Cor a 1 hazelnut allergens.

A major proportion of allergic reactions to hazelnuts (Corylus avellana) are caused by immunologic cross-reactivity of IgE antibodies to pathogenesis-related class 10 (PR-10) proteins. Intriguingly, the four known isoforms of the hazelnut PR-10 allergen Cor a 1, denoted as Cor a 1.0401-Cor a 1.0404, share sequence identities exceeding 97% but possess different immunologic properties. In this work we describe the NMR solution structures of these proteins and provide an in-depth study of their biophysical properties. Despite sharing highly similar three-dimensional structures, the four isoforms exhibit remarkable differences regarding structural flexibility, hydrogen bonding and thermal stability. Our experimental data reveal an inverse relation between structural flexibility and IgE-binding in ELISA experiments, with the most flexible isoform having the lowest IgE-binding potential, while the isoform with the most rigid backbone scaffold displays the highest immunologic reactivity. These results point towards a significant entropic contribution to the process of antibody binding.

Oral birch pollen immunotherapy with apples: Results of a phase II clinical pilot study.

BACKGROUND: Seventy percent of patients suffering from birch pollen allergy (BPA) develop a pollen-related food allergy (prFA), especially to apples, due to a clinically relevant cross-reactivity between the major allergen in birch Bet v 1 and Mal d 1 in apples. Therefore allergen-specific immunotherapy with fresh apples (AITA) could be a promising natural treatment of both BPA and prFA. OBJECTIVE: To assess the clinical efficacy of immunotherapy by daily apple consumption for patients with BPA and prFA. METHODS: A daily defined increasing amount of selected cultivars (Red Moon®, Pink Lady®, Topaz, Golden Delicious) was continuously consumed by 16 patients (12 female; median age; 50; range, 23-68 years), leading to increased intake of allergen over a period of at least 8 months. Specific IgE and IgG4 to Bet v 1 and Mal d 1, conjunctival and oral provocation tests, skin reactivity, and the average daily rhinoconjunctivitis combined symptom and medication score (CSMS) were measured during the peak birch pollen season. RESULTS: After 8 months of therapy, patients showed increased tolerance to apples (p < .001) and a decreased skin reactivity to apples. Oral allergy syndrome to other birch prFA than apple also decreased (p < .05). Moreover, daily rhinoconjunctivitis CSMS declined by 34% (p < .001), as did conjunctival reactivity to birch pollen extract by 27% (p < .01), while specific IgG4 to Mal d 1 and Bet v 1 increased (p < .01).