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

Authors

• Govardus A. H. de Jong, TNO, Zeist, Netherlands
• Shyamali Jayasena, University of Nebraska-LincolnFollow
• Philip Johnson, University of Nebraska-LincolnFollow
• Justin Marsh, University of Nebraska-LincolnFollow
• Danijela Apostolovic, Karolinska InstitutetFollow
• Marianne van Hage, Karolinska InstitutetFollow
• Julie A. Nordlee, University of Nebraska-LincolnFollow
• Joe Baumert, University of Nebraska-LincolnFollow
• Steve L. Taylor, University of Nebraska-LincolnFollow
• Camille Roucairol, DBV Technologies, France
• Harmen de Jongh, Zeist, NetherlandsFollow
• Stef J. Koppelman, University of Nebraska-LincolnFollow

ORCID IDs

Philip Johnson

Steve L. Taylor

Stef J. Koppelman

Document Type

Article

Date of this Version

10-2018

Citation

Journal of Agricultural and Food Chemistry (October 2018) 66(41): 10,855−10,863

doi: 10.1021/acs.jafc.8b03140

Comments

Copyright © 2018, American Chemical Society. Used by permission

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.