RESUMO
BACKGROUND: Plant food allergy in the Mediterranean area is mainly caused by non-specific lipid transfer proteins (nsLTP). The aim of this study was to characterize peanut nsLTP in comparison with peach nsLTP, Pru p 3, and assess its importance in peanut allergy. METHODS: Peanut-allergic patients from Spain (n=32) were included on the basis of a positive case history and either a positive skin prick test or specific IgE to peanut. For comparison, sera of 41 peanut-allergic subjects from outside the Mediterranean area were used. Natural Ara h 9 and two isoforms of recombinant Ara h 9, expressed in Pichia pastoris, were purified using a two-step chromatographic procedure. Allergen characterization was carried out by N-terminal sequencing, circular dichroism (CD) spectroscopy, immunoblotting, IgE inhibition tests and basophil histamine release assays. RESULTS: Compared with natural peanut nsLTP, the recombinant proteins could be purified in high amounts from yeast supernatant (> or =45 mg/L). The identity of the proteins was verified by N-terminal amino acid sequencing and with rabbit nsLTP-specific antibodies. CD spectroscopy revealed similar secondary structures for all preparations and Pru p 3. The Ara h 9 isoforms showed 62-68% amino acid sequence identity with Pru p 3. IgE antibody reactivity to rAra h 9 was present in 29/32 Spanish and 6/41 non-Mediterranean subjects. Recombinant Ara h 9 showed strong cross-reactivity to nPru p 3 and similar IgE-binding capacity as nAra h 9. The two Ara h 9 isoforms displayed similar IgE reactivity. In peanut-allergic patients with concomitant peach allergy, Ara h 9 showed a weaker allergenic potency than Pru p 3 in histamine release assays. CONCLUSIONS: Ara h 9 is a major allergen in peanut-allergic patients from the Mediterranean area. Ara h 9 is capable of inducing histamine release from basophils, but to a lesser extent than Pru p 3.