PR-10, NsLTP, 2S-albumin, DC-8 and vicilin from chickpea (Cicer arietinum) display IgE-reactivity and likely contribute to cross-reactivity with legume foods
Background: Chickpea (Cicer arietinum) has been reported to cause IgE-mediated hypersensitivity reactions. However, chickpea allergens are poorly characterized. Objective: Molecular cloning, recombinant expression and immunological characterization of potential allergens (PR10, nsLTP, 2S Albumin, Vicilin, DC-8) from chickpea, with homology to allergens from other sources. Methods: Protein extract from boiled and raw chickpeas was applied to one- and two-dimensional gel-electrophoresis and immunoblotting using sera from chickpea allergic patients. IgE-reactive spots were subjected to mass spectrometric analysis to verify amino acid (AA) sequences of potential allergens. Additionally, known PR-10, 2S Albumin and nsLTP from chickpea were included in the study. Chickpea proteins were expressed as recombinant non-fusion proteins in E. coli, and subsequently purified by two-step chromatography. The purity, structural and sequence integrity was analyzed by SDS-PAGE, CD-spectroscopy, and MS analysis. Serum samples from chickpea allergic patients were investigated for IgE-reactivity with chickpea extract and purified proteins. Results: Patients showed a multiple and heterogeneous IgE-sensitization profile to raw and boiled chickpeas, indicating an individual divers reactivity pattern. In the extract of boiled chickpeas Vicilin and a DC-8 protein were identified as potential allergens using a serum pool of chickpea allergic patients. All proteins displayed sufficient purity, intact secondary structure and showed IgE-binding using sera from chickpea allergic patients. Notably, the panel of selected target allergens did not resemble the IgE-reactivity to chickpea extract. DC-8, a late embryogenesis abundant (LEA) protein, for the first time was identified as a new class of proteins, with the potential to act as an allergen. Evidence for cross-reactivity between chickpea proteins, PR-10 and other legumes has been shown. Conclusion: Until now, no proteins are identified, playing a predominant role in chickpea allergy. As chickpea allergic patients show a heterogeneous, multi sensitization pattern, many proteins seem to be involved, like Vicilin and the LEA protein, and likely yet unidentified allergens. Although the frequency of sensitization to recombinant PR10, 2S-Albumin and nsLTP from chickpea is low in the used patient collective, the potential risk of IgE cross-reactivity in patients sensitized to homologous proteins from legumes needs to be considered
