Potential allergens of green gram (Vigna radiata L. Millsp) identified as members of cupin superfamily and seed albumin.

ABSTRACT

BACKGROUND: No systematic study on allergenicity of green gram seed proteins have been performed so far, although incidences of IgE-mediated reaction to green gram seedlings have been reported. OBJECTIVE: We sought to investigate the allergenic potential of green gram, followed by identification and characterization of its relevant allergens using proteomic approaches. METHODS: BALB/c mice were sensitized intraperitoneally with green gram proteins, and levels of specific Igs, Th2 cytokines, histamine, anaphylactic symptoms and histopathological responses were studied. Twelve naso-bronchial allergic patients with a history of sensitization to green gram were selected on the basis of positive skin prick test and elevated specific IgE levels. Green gram allergens were identified and characterized by their ability to endure pepsin, by IgE immunoblot of two-dimensional (2D) gels in combination with mass spectrometry and by bioinformatics approaches. RESULTS: Increased specific IgE, IgG1, Th2 cytokine and histamine levels, high anaphylactic scores and histological changes in lungs and spleen of green gram crude protein extract-treated mice are indicative of its sensitization ability. Four proteins (molecular weights 52, 50, 30 and 18 kDa) showed pepsin resistance and IgE-binding capability with sensitized human and mice sera. The four proteins tentatively named as Vig r2 (52 kDa, pI 5.7), Vig r3 (50 kDa, pI 5.8), Vig r4 (30 kDa, pI 6.6) and Vig r5 (18 kDa, pI 5.5) showed significant sequence similarity with known allergens of soybean, lentil, pea, lupin, etc. Mass spectrometric analysis identified Vig r2 as 8S globulin ß-isoform precursor, Vig r3 as 8S globulin α-isoform precursor and Vig r4 as seed albumin. CONCLUSION AND CLINICAL RELEVANCE Green gram seeds contain at least four clinically relevant allergenic proteins, namely Vig r2, Vig r3, Vig r4 and Vig r5 that were capable of inducing strong IgE-mediated reactions. One of the most important steps towards diagnostic and therapeutic approaches to deal effectively with food allergy is continued identification of newer food allergens and their characterization. The significance of this study can be enormous as the data generated may work as basic biology data in developing a green gram species modified genetically that may have reduced allergenicity.