BIOCHEMICAL AND IMMUNOCHEMICAL PROPERTIES OF AMANDIN

Almonds (Prunus dulcis L.) are globally consumed and are a valued crop. The US is the global leader in almond production and exports. Although enjoyed safely by most, sensitive individuals suffer from almond allergies. We have demonstrated that amandin, a legumin, is the major storage protein in almonds seeds that contains key IgE-reactive polypeptides and is a major allergen in almond seeds. We have developed several purification protocols for the preparation of native amandin. Using a cDNA library we have now expressed recombinant amandin isoforms rPru du 6.01 (GenBank ID: GU059260) and rPru du 6.02 (GenBank ID GU059261) and tested both isoforms for immunoreactivity using sera from patients known to be almond-allergic. The results indicate that both recombinant forms are recognized by patients’ serum IgE and that this binding is completely inhibited by the native amandin. IgE reactivity to rPru du 6.01 and rPru du 6.02 was found in the sera of 9 of 18 (50%) and 5 of 18 (28%) patients, respectively. Of the two, rPru du 6.01 is more immunoreactive. Thus amandin appears to be a major allergen in almond seeds. Using solid phase peptide synthesis we have identified the IgE-recognizing linear stretches of the Pru du 6 proteins (i.e., the linear epitopes). An analysis of the amino acid sequences of the Pru du 6 epitopes revealed that more than half are located in regions rich in glutamine residues. Most of these segments are in one of the two regions unresolved (amino acid 118 to 184) in the recently reported crystal structure of prunin 1. This glutamine-rich region may facilitate the generation of conformational epitopes. Studies currently underway, using murine monoclonal antibodies and patients’ serum IgE, indicate the presence of conformational epitopes as well. Results of the current study add to the EUROPREVALL scientific information database on almonds.