Peanut defensins: Novel allergens isolated from lipophilic peanut extract.

BACKGROUND: Peanut is one of the most hazardous sources of food allergens. Unknown allergens are still hidden in the complex lipophilic matrix. These allergens need to be discovered to allow estimation of the allergenic risk for patients with peanut allergy and to further improve diagnostic measures. OBJECTIVE: We performed detection, isolation, and characterization of novel peanut allergens from lipophilic peanut extract. METHODS: Extraction of roasted peanuts were performed under defined extraction conditions and examined by means of 2-dimensional PAGE. Subsequently, chromatographic methods were adapted to isolate low-molecular-weight components. Proteins were studied by using SDS-PAGE and immunoblotting with sera from patients with peanut allergy. For allergen identification protein sequencing, homology search and mass spectrometry were applied. Functional characterization for allergenicity was performed by using the basophil activation assay and for antimicrobial activity by using inhibition assays of different bacteria and fungi. RESULTS: IgE-reactive proteins of 12, 11, and 10 kDa were first detected after chloroform/methanol extraction in the flow through of hydrophobic interaction chromatography. The proteins were able to activate basophils of patients with peanut allergy. N-terminal sequencing and homology search in the expressed sequence tag database identified the allergens as peanut defensins, which was confirmed by using mass spectrometry. On microbial cell cultures, the peanut defensins showed inhibitory effects on the mold strains of the genera Cladosporium and Alternaria but none on bacteria. CONCLUSIONS: We identified defensins as novel peanut allergens (Ara h 12 and Ara h 13) that react in particular with IgE of patients with severe peanut allergy. Their antimicrobial activity is solely antifungal.

Roasting and lipid binding provide allergenic and proteolytic stability to the peanut allergen Ara h 8.

Abstract Ara h 8 is the peanut allergen homologous to the birch pollen allergen Bet v 1. Because Bet v 1 has been shown to bind lipophilic ligands, the aim of this investigation was to determine the impact of lipid binding and roasting on the Ara h 8 structure and their influences on allergenicity. For the characterization of natural Ara h 8 (nAra h 8) from roasted and unroasted peanuts, circular dichroism spectroscopy, hydrophobic binding assay, immunohistochemistry, and immunoblot with sera of peanut allergic patients were performed and compared with results from recombinant Ara h 8 (rAra h 8) and Bet v 1. rAra h 8 displayed stronger hydrophobicity than rBet v 1. Patients' sera showed IgE reactivity with rAra h 8 and nAra h 8 from roasted peanuts, whereas fewer sera recognized nAra h 8 from unroasted peanuts. Simulated gastric digestion experiments demonstrated low proteolytic stability of rAra h 8, whereas the stability of nAra h 8 was increasingly higher in unroasted and roasted peanuts. The results demonstrate that IgE reactivity and thermal and proteolytic stability are reinforced in nAra h 8 after roasting, most likely due to Maillard reactions, lipid oxidations, and lipophilic associations. These aspects must be considered when estimating the allergenicity of Bet v 1-homologous proteins.