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Soybean lecithins are seeing increasing use in industry as an emulsifier and food additive. They are also a growing source of human food allergies, which arise principally from the proteins fractionating with the lecithin fraction during manufacture. A previous study (Gu, X.; Beardslee, T.; Zeece, M.; Sarath, G.; Markwwell, J. Int Arch. Allergy Immunol. 2001, 126, 218-225) identified several allergenic proteins in soybean lecithins and a soybean IgE-binding protein termed P39 was discovered. However, very little was known about this protein except that it was coded by the soybean genome. This paper investigates key biological and immunological properties of this potential soybean lecithin allergen. P39 is encoded by a multigene family in soybeans and in several other higher plants. The soybean P39-1 protein and its essentially indistinguishable homologue, P39-2, have been cloned and studied. These proteins and their homologues belong to a family of plant-specific proteins of unknown function. In soybeans, P39-1 is seed specific, and its transcript levels are highest in developing seeds and decline during seed maturation. In contrast, P39 protein was detectable only in the fully mature, dry seed. Subcellular fractionation revealed that P39 protein was strongly associated with oil bodies; however, immunolocalization indicated P39 was distributed in the matrix of the protein storage vacuoles, suggesting that association with oil bodies was an artifact arising from the extraction procedure. By the use of recombinant techniques it has also been documented that IgE-binding epitopes are present on several different portions of the P39-1 polypeptide.

Gly m Bd 28 K is a major soybean (Glycine max Merr.) glycoprotein allergen. It was originally identified as a 28 kDa polypeptide in soybean seed flour. However, the full-length protein is encoded by an open reading frame (ORF) of 473 amino acids, and contains a 23 kDa C-terminal polypeptide of as yet unknown allergenic and structural characteristics. IgE-binding (allergenic potential) of the Gly m Bd 28 K protein including the 23 kDa C-terminal portion as well as shorter fragments derived from the full-length ORF were evaluated using sera from soy-sensitive adults. All of these sera contained IgE that efficiently recognized the C-terminal region. Epitope mapping demonstrated that a dominant linear C-terminal IgE binding epitope resides between residues S256 and A270. Alanine scanning of this dominant epitope indicated that five amino acids, Y260, D261, D262, K264 and D266, contribute most towards IgE-binding. A model based on the structure of the beta subunit of soybean beta-conglycinin revealed that Gly m Bd 28 K contains two cupin domains. The dominant epitope is on the edge of the first beta-sheet of the C-terminal cupin domain and is present on a potentially solvent-accessible loop connecting the two cupin domains. Thus, the C-terminal 23 kDa polypeptide of Gly m Bd 28 K present in soy products is allergenic and apparently contains at least one immunodominant epitope near the edge of a cupin domain. This knowledge could be helpful in the future breeding of hypoallergenic soybeans.

The soybean Kunitz trypsin inhibitor (SKTI) is a 21.5 kDa allergenic protein that belongs to the family of all antiparallel beta-sheet proteins that are highly resistant to thermal and chemical denaturation. Spectroscopic and biochemical techniques such as circular dichroism (CD), ANS fluorescence and proteolysis were used to study its molecular structure under denaturing conditions such as acid and heat to which these allergens are commonly exposed during food processing. Reduction of native SKTI leads to its complete and rapid proteolysis by pepsin in simulated gastric fluid (SGF). Limited proteolysis with chymotrypsin during renaturation after heating showed that the native structure reforms at around 60 degrees C reversing the denaturation. CD spectra revealed that under acid denaturing conditions, SKTI shows major changes in conformation, indicating the possibility of a molten structure. The existence of this intermediate was established by ANS fluorescence studies at different concentrations of HCl. The remarkable stability of SKTI to both thermal and acid denaturation may be important for its role as a food allergen.

To identify conserved immunoglobulin E (IgE)-binding epitopes among legume glycinins, we utilized recombinant soybean G2a and G2a-derived polypeptide fragments. All of these fusion polypeptides bound IgE, and the C-terminal 94-residue fragment appeared to bind more IgE. Using synthetic peptides we identified S219-N233 (S(219)GFAPEFLKEAFGVN(233)) as the dominant IgE-binding epitope. Alanine scanning of this epitope indicated that six amino acids (E224, F225, L226, F230, G231, and V232) contributed most to IgE binding. Among these amino acids, only G231 of soybean G2a is not conserved in soybean G1a (S234) and peanut Ara h 3 (Q256). Synthetic peptides corresponding to the equivalent regions in G1a and Ara h 3 bound IgE in the order Ara h 3>/=soybean G2a>soybean G1a. This sequence represents a new IgE-binding epitope that occurs in a highly conserved region present in legume glycinins. Such IgE-binding sites could provide a molecular explanation for the IgE cross-reactivity observed between soybean and peanut proteins.

Gly m Bd 28 K is a major soybean (Glycine maxMerr.) glycoprotein allergen. It was originally identified as a 28 kDa polypeptide in soybean seed flour. However, the full-length protein is encoded by an open reading frame (ORF) of 473 amino acids, and contains a 23 kDa C-terminal polypeptide of as yet unknown allergenic and structural characteristics. IgE-binding (allergenic potential) of the Gly m Bd 28 K protein including the 23 kDa C-terminal portion as well as shorter fragments derived from the full-length ORF were evaluated using sera from soy-sensitive adults. All of these sera contained IgE that efficiently recognized the C-terminal region. Epitope mapping demonstrated that a dominant linear C-terminal IgE binding epitope resides between residues S256 and A270. Alanine scanning of this dominant epitope indicated that five amino acids, Y260, D261, D262, K264 and D266, contribute most towards IgE-binding. A model based on the structure of the ß subunit of soybean ß-conglycinin revealed that Gly m Bd 28 K contains two cupin domains. The dominant epitope is on the edge of the first ß-sheet of the C-terminal cupin domain and is present on a potentially solvent-accessible loop connecting the two cupin domains. Thus, the C-terminal 23 kDa polypeptide of Gly m Bd 28 K present in soy products is allergenic and apparently contains at least one immunodominant epitope near the edge of a cupin domain. This knowledge could be helpful in the future breeding of hypoallergenic soybeans. [ABSTRACT FROM AUTHOR]

Background: Soy lecithin is widely used as an emulsifier in processed foods, pharmaceuticals and cosmetics. Soy lecithin is composed principally of phospholipids; however, it has also been shown to contain IgE-binding proteins, albeit at a low level. A few clinical cases involving allergic reactions to soy lecithin have been reported. The purpose of this investigation is to better characterize the IgE-binding proteins typically found in lecithin. Methods: Soy lecithin proteins were isolated following solvent extraction of lipid components and then separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The separated lecithin proteins were immunoblotted with sera from soy-sensitive individuals to determine the pattern of IgE-binding proteins. The identity of IgE-reactive bands was determined from their N-terminal sequence. Results: The level of protein in six lecithin samples obtained from commercial suppliers ranged from 100 to 1,400 ppm. Lecithin samples showed similar protein patterns when examined by SDS-PAGE. Immunoblotting with sera from soy-sensitive individuals showed IgE binding to bands corresponding to 7, 12, 20, 39 and 57 kD. N-terminal analysis of these IgE-binding bands resulted in sequences for 3 components. The 12-kD band was identified as a methionine-rich protein (MRP) and a member of the 2S albumin class of soy proteins. The 20-kD band was found to be soybean Kunitz trypsin inhibitor. The 39-kD band was matched to a soy protein with unknown function. Conclusions: Soy lecithin contains a number of IgE-binding proteins; thus, it might represent a source of hidden allergens. These allergens are a more significant concern for soy-allergic individuals consuming lecithin products as a health supplement. In addition, the MRP and the 39-kD protein identified in this study represent newly identified IgE-binding proteins.Copyright © 2001 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background: The identification of IgE epitopes for proteins is the first step in understanding the interaction of allergens with the immune system. Proteins from the legume family have shown in vitro cross-reactivity in IgE-binding assays, but this cross-reactivity is rarely clinically significant. Resolution of this discrepancy requires IgE epitope mapping of legume family protein allergens. Methods: We constructed six fusion proteins representing overlapping regions of soybean glycinin G1 acidic chain. These fusion proteins were used in immunoblotting and a novel sandwich ELISA with pooled sera from soy-allergic individuals to reveal a common IgE-binding region. This region was the focus for IgE epitope mapping using overlapping synthetic peptides. Results: Data from the fusion protein experiments revealed an IgE-binding region consisting of residues F192–I265. Analysis of the overlapping synthetic peptides to this region indicated that IgE epitopes to glycinin G1 acidic chain consist of residues G217–V235 and G253–I265. The epitopes identified for glycinin G1 acidic chain are homologous to IgE epitopes previously identified for the peanut allergen Ara h 3 [1]. However, residues identified by alanine scanning in the peanut epitopes as being important for IgE binding are different in the natural soybean epitopes. Conclusions: The IgE epitopes identified for glycinin G1 acidic chain apparently represent an allergenic region of several legume family seed storage proteins. Our findings indicate that the identification of IgE epitopes and structural analysis of legume family proteins will provide valuable information to the study of food allergies.Copyright © 2000 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]