Soybean Glycinin G1 Acidic Chain Shares IgE Epitopes with Peanut Allergen Ara h 3 (1)
Soy- bean food protein allergens have not been characterized to the extent of peanut allergens, but they include the thiol protease-like 34-kD protein (P34 or Gly m Bd 30K), the 14-kD profilin (Gly m 3), the 70-kD a-subunit of ß-conglycinin, and the 40-kD glycinin G1 acidic chain. Native glycinin is a 350-kD hexamer, and is the primary protein of the 11S fraction. Glycinin subunits are composed of an acidic and basic chain. The acidic and basic chains are translated as a continuous polypeptide before being posttranslationally cleaved to form the 40-kD acidic and 20-kD basic chains that are linked through internal disulfides. Soybean glycinin is a gene family with the nonrandom assembly of acidic and basic chains into the subunits A1aB2 (G1), A2B1a (G2), A1bB1b (G3), A5A4B3 (G4), and A3B4 (G5). The binding of IgE from soy-allergic individuals has been found to soybean glycin- in acidic chains. Soybean glycinin exhibits high sequence identity to the 11S proteins pea legumin A (64%), rice glutelin (37%), and peanut glycinin (62%).
The fusion proteins f1–f3 begin at residue 22 to reflect the mature form of glycinin G1 acidic chain without the signal peptide. Most proteins or protein fragments that we have expressed as a thioredoxin fusion were produced at a level of 8–20 mg of purified protein per liter of culture. The purification of the 6 x His-tagged fusion proteins was reliably performed by nickel affinity chromatography. All of the purified glycinin G1 acidic chain fusion fragment proteins (FFP) were combined into one sample since their electrophoretic mobility allowed easy resolution with SDS-PAGE.
The increased amount of FFP on the membrane allowed the detection of IgE binding to f3, f5 and f2 when the less efficient transfer only revealed a response from f3 and f5. Immunoblotting of the membrane strip revealed IgE binding to f3, f5, f2, f4, and a faint response to f1.
The IgE binding to the fusion proteins in the FFP- ELISA format showed high responses to f2, f3, f4 and f5. However, the relative responses from the fusion proteins were different than in immunoblotting.
Epitope A is shown by a response to peptides 6 and 7 (GGSILSGFTLEFLEHAFSV) and epitope B is shown by a response to peptide 12 (GAIVTVKGGLSVI). The location of epitopes A and B in the glycinin G1 acidic chain sequence places both epi- topes in f3 and f5, but only epitope A is in f2 and f4. Glycinin G1 acidic chain epitopes A and B correspond to Ara h 3 epitopes 2 and 3.
The glycinin acidic chain domain, between variable region 2 and the hypervariable region, corresponds to alpha-helices a1, a2, a3 and ß-strand J) of the proposed model of 11S legumin protein structure. This domain is located at the subunit interface of the 7S trimer that corresponds to half of the 11S hexamer. A proteolytic fragment of the soybean 7S protein ß-conglycinin alpha-subunit including residues 232–383 that bound IgE by immunoblotting with soy-sensitive sera also corresponds to the subunit interface domain of the 7S trimer.