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Purification and Characterization of Naturally Occurring Post-Translationally Cleaved Ara h 6, an Allergen That Contributes Substantially to the Allergenic Potency of Peanut

Author:
de Jong, Govardus A.H., Jayasena, Shyamali, Johnson, Phil, Marsh, Justin, Apostolovic, Danijela, van Hage, Marianne, Nordlee, Julie, Baumert, Joe, Taylor, Steve L., Roucairol, Camille, de Jongh, Harmen, Koppelman, Stef J.
Source:
Journal of agricultural and food chemistry 2018 v.66 no.41 pp. 10855-10863
ISSN:
1520-5118
Subject:
albumins, allergenicity, allergens, amino acids, antibodies, cultivars, disulfide bonds, epitopes, immunoglobulin E, immunoglobulin G, mass spectrometry, peanuts, protein structure, Virginia
Abstract:
The 2S albumin Ara h 6 is one of the most important peanut allergens. A post-translationally cleaved Ara h 6 (pAra h 6) was purified from Virginia type peanuts, and the cleavage site was mapped using high-resolution mass spectrometry. Compared to intact Ara h 6, pAra h 6 lacks a 5-amino acid stretch, resembling amino acids 43–47 (UniProt accession number Q647G9) in the nonstructured loop. Consequently, pAra h 6 consists of two chains: an N-terminal chain of approximately 5 kDa and a C-terminal chain of approximately 9 kDa, held together by disulfide bonds. Intermediate post-translationally cleaved products, in which this stretch is cleaved yet still attached to one of the subunits, are also present. The secondary structure and immunoglobulin E (IgE) binding of pAra h 6 resembles that of intact Ara h 6, indicating that the loss of the nonstructured loop is not critical for maintaining the protein structure. Commercially available monoclonal and polyclonal immunoglobulin G (IgG) antibodies directed to Ara h 6 react with both intact Ara h 6 and pAra h 6, suggesting that the involved epitopes are not located in the area that is post-translationally cleaved. No differences between intact Ara h 6 and pAra h 6 in terms of IgE binding were found, suggesting that the area that is post-translationally cleaved is not involved in IgE epitopes either. For all main cultivars Runner, Virginia, Valencia, and Spanish, intact Ara h 6 and pAra h 6 occur in peanut at similar levels, indicating that pAra h 6 is a consistent and important contributor to the allergenic potency of peanut.
Agid:
6166598