Detection and Structural Characterization of Natural Ara h 7, the Third Peanut Allergen of the 2S Albumin Family

Abstract

In recent years, several novel relevant peanut allergens have been identified. Among those, a new member of the conglutin family was cloned by a phage display approach and initially annotated as Ara h 7.0101. Later, however, recloning of Ara h 7 revealed an alternate isoform, termed Ara h 7.0201. Because the natural Ara h 7 counterpart had not been found at the protein level in peanut extracts, the aim of the present study was to search for authentic natural Ara h 7 protein(s). To this end, enriched low molecular mass proteins (<20 kDa) from peanut extracts were separated by 2D electrophoresis and subjected to mass spectrometric analyses. Fifty of 65 analyzed spots were identified. Interestingly, Ara h 7.0101 was not identified, but Ara h 7.0201 and Ara h 7.0202, a different Ara h 7 isoallergen containing an additional pro-peptide cleavage site, were. In accordance with the conserved cysteine pattern of conglutins, Ara h 7.0201 possesses eight cysteine residues, in contrast to the six cysteines present in the previously cloned Ara h 7.0101. Moreover, a putative cleavage site in the Ara h 7.0202 isoform points to the characteristic biological function of conglutins as amylase/trypsin inhibitors.