Food allergy is a globally recognized health problem, which is estimated to affect 8% of children and 10% of adults in the USA. Within the USA, 90% of the food allergies are due to peanut, tree nuts, fish, shellfish, egg, milk, wheat, and soybean, commonly known as the big eight. With recent reports of the high prevalence of sesame allergy (more than 1.5 million children and adults) in the USA, the current list of big eight is poised to change. Oilseeds such as peanuts, tree nuts (cashew, almond, pistachio, etc.), soybean, and sesame make up a significant portion of the current food allergens. Complex oligomeric legumin-like proteins belonging to the cupin superfamily have been recognized as major food allergens in such oilseeds. Inadequate characterization of legumin-like proteins/allergens in their purified native forms and variable clinical allergenicity of different oilseeds deserved systematic investigation. Therefore, the current study was focused on oilseeds from unrelated sources such as tree nuts (almond, Brazil nut, cashew, and pistachio), soybean, and sesame. The specific objectives were to 1) chromatographically purify native legumins from almond, Brazil nut, cashew nut, pistachio, sesame and soy seeds, 2) study select biophysical and biochemical properties of purified native legumins, 3) evaluate cross-reactivity of purified native legumins, 4) compare the effect of in vitro pepsin digestion on proteolysis and allergenicity of purified legumins. Legumins were purified using different low-pressure liquid chromatography techniques. Purified legumins eluted as single peaks on a size exclusion chromatography column. In solution, LC-MS/MS analysis of proteins further confirmed the purity of proteins where each legumin showed the highest match with the gene-derived amino acid sequence of the legumin. Estimated molecular weights and hydrodynamic radii of purified legumins varied significantly (P < 0.05) from 269 kDa to 331 kDa and 61 Å to 80 Å, respectively, with sesame legumin being the smallest in size and radius. Purified legumins of almond, Brazil nut, cashew, pistachio, sesame, and soybean registered sedimentation coefficients of 13.4S, 12.5S, 13S, 13.4S, 13S, and 11.9S, respectively, with soybean legumin being the most asymmetrical in shape. Amino acid composition of legumins revealed glutamic acid and glutamine to be the dominant amino acids in all legumins. Numerous amino acids showed significant (P < 0.05) variation across multiple (>3) legumins; however, only alanine and threonine showed significant (P < 0.05) differences across all six legumins. Spectral properties revealed all proteins to had their absorbance maximum at 278-279 nm wavelength with differences observed in their local structures. β-sheets dominated all legumins with almond and cashew legumins containing the lowest (34.6%) and the highest (44.2%) percentage of β-sheets, respectively. Electrophoresis of legumins in their native, denatured, and reduced forms revealed differences in their overall charge, size of the protomers & polypeptide profiles, and subunit composition, respectively. None of the legumin proteins was glycosylated. Cross-reactivity of purified legumins, when probed with their seed-specific rabbit antisera, revealed purified allergens to be cross-reactive with each other in their native, denatured, and reduced forms. Sesame legumin showed the strongest cross-reactivity with other legumins, while soybean showed the weakest based on immunoblotting. The basic subunit of legumins was found to be more cross-reactive compared to the acidic subunit. IgE from nine allergic patients showed a trend similar to the antisera tested. Homology modeling of reported strongly reactive IgE epitopes of almond, cashew, and soybean legumin, when mapped on to the three-dimensional structure of legumins, revealed common epitopic regions in all legumins. Soybean legumin exhibited the highest variation in the epitopic areas compared to other legumins. In vitro pepsin digestion of legumins under the same experimental conditions revealed legumins to be stable to pepsin and low pH. Soybean legumin was found to be the most resistant to pepsin digestion. Seed-specific antisera could recognize all digested legumins within the two-hour of digestion. Reactivity of allergic patients IgE to the digests was observed until at least 15 minutes of digestion with intact disulfide linkage until the two-hour of digestion. Considerable pH-induced aggregation existed throughout the digestion. The persistence of disulfide bonds and the formation of pH-induced aggregates were speculated to be the primary reasons for the stability of these proteins under the tested experimental conditions.
