Submitted to: Planta
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/5/2004
Publication Date: 11/1/2004
Citation: Xiang, P., Haas, E.J., Zeece, M.G., Markwell, J., Sarath, G. 2004. C-terminal 23 kd polypeptide of soybean gly m bd 28k is a potential allergen. Planta. 220:56-63. Interpretive Summary: Plant food allergens occupy a critical niche in human nutrition, based on the amounts of plant foods consumed as well as the severity of allergic reactions in patients. Plant protein food allergens belong principally to two large protein superfamilies, named the Cupin and the Prolamin superfamilies. Most legume protein allergens belong to the cupin superfamily. Portions of an allergenic protein that evoke an immune responses are known as epitopes. For allergenic proteins, these epitopes will bind specifically to immunoglobulins E (IgE) present in the sera of sensitive individuals. Thus analysis of allergens for epitopes can yield information about the potential allergenicity of the protein and provide insights into any unique structural features. In this work we have discovered a novel epitope on an as yet uncharacterized polypeptide product of a major soybean allergen Gly m Bd 28K. We found one dominant epitope and this was mapped to a surface-accessible site of the parent protein. These results reinforce the hypothesis linking structure to allergenicity for Gly m Bd 28 K and related plant allergens and show for the first time that amino acid loops connecting "cupin domains" could be allergenic hot-spots.
Technical Abstract: Gly m Bd 28K is a major soybean glycoprotein allergen. It was originally identified as a 28 kD polypeptide in soybean seed flour. However, the full-length protein is encoded by an ORF of 473 amino acids, and belongs to the cupin superfamily of legume allergenic proteins. To dissect its allergenic potential, IgE binding sites on the full length Gly m Bd 28K protein were evaluated using recombinant fusion-polypeptide fragments. IgE in serum from several soy-allergic adults bound to multiple fragments derived from the full-length ORF.Surprisingly, all of these soy sensitive individuals contained IgE that efficiently recognized the C-terminal region of this protein, indicating that the C-terminal 23 kD polypeptide of Gly m Bd 28K present in soy products is also allergenic. Epitope mapping of the major C-terminal IgE binding region encompassing residues, E221-C353, indicated that the epitope resided between residues S256-A270. Alanine scanning of this epitope indicated that five amino acids (Y260, D261, D262 and K264 & D266) contributed most towards IgE-binding. Gly m Bd 28K contains two cupin domains, one in the N-terminal 240 residues identified earlier. A second cupin domain was found in the novel allergenic region of the C-terminal part of the protein that is described in this work. A model of Gly m Bd 28K based on the structure of subunit of soybean -conglycinin revealed that the dominant epitope was on the edge of the first -sheet of the C-terminal cupin domain and present on a potentially solvent-accessible loop connecting the two cupin domains.