All Three Subunits of Soybean -Conglycinin Are Potential Food Allergens (1)
Soybean has been recognized by regulatory authorities as one of the “big 8” food allergens. Several soybean proteins have been identified as allergens in the molecular mass range from 7 to 71 kDa. Prominent among them are the Gly m Bd 30 K, Gly m Bd 28 K, Gly m Bd 60 K, and G1 and G2 glycinin proteins. Soybean 7S storage protein, the beta-conglycinin, has been identified as one of the most allergenic protein. beta-Conglycinin is composed of three subunits, namely, alpha′ (76 kDa), alpha (72 kDa), and beta (52 kDa).
The sera from these individuals showed cross-reaction against several soy proteins. Prominent among them were 72, 70, 52, 34, and 21 kDa proteins.
The three subunits of beta-conglycinin were well resolved by this procedure. The alpha′- and alpha-subunits separated into distinct spots with isoelectric points of 5.2 and 4.9, respectively. The beta-subunit was resolved into four distinct spots having isoelectric points ranging from 5.6 to 6.0. Western blot analysis showed strong reaction against the two spots corresponding to the beta-subunit and a weaker reaction against the alpha′- and alpha-subunits.
Peptides generated by trypsin digestion showed significant homology to the alpha′-, alpha-, and beta-subunits of beta-conglycinin.
The gel-purified subunits were tested by SDS-PAGE and immunoblot analyses with pooled sera from patients containing IgE antibodies against 72, 70, and 52 kDa soybean proteins.
Treatment with glycosidases resulted in faster migration of the beta-subunit of beta-conglycinin in comparison to unglycosylated protein. Immunoblot analysis clearly showed that the IgE antibodies from patients sensitive to soy proteins were able to recognize the unglycosylated form of the beta-subunit of beta-conglycinin
The 11S glycinins, the most abundant storage protein of soybean, are synthesized as precursor proteins and posttrans- lationally processed into 40 kDa acidic and 20 kDa basic subunits. Cleavage of the peptide bonds with CNBr and chymotrypsin and subsequent N-terminal amino acid sequence determination of the peptides indicated that IgE-binding sites was located between amino acid residues 232 and 383 of the alpha-subunit of beta-conglycinin. The recombinant alpha′-subunit of soybean beta-conglycinin possesses an intrinsic immune-stimulating capacity and can induce allergic reaction in Brown Norway rats.