IgE binding of the recombinant allergen soybean profilin (rGly m 3) is mediated by conformational epitopes

Abstract

Background: Soybean proteins are constituents of a number of food products and represent a panel of potential allergens. Thus far, little is known about the molecular characteristics of soybean allergens. Objective: The aim of this study was to identify the soybean profilin by PCR-based complementary (c)DNA cloning and to elucidate its allergenic characteristics. Methods: Highly degenerate profilin-specific primers were used to identify, by means of PCR, 2 soybean profilin isoforms (GmPRO1 and GmPRO2) by using soybean cDNA as a target. One isoform (GmPRO1) with a length of 394 bp corresponding to 131 amino acid residues was subcloned and expressed in fusion with the maltose-binding protein. Moreover, 3 overlapping recombinant soybean profilin fragments comprising amino acid residues 1-65, 38-88, and 50-131 were also prepared as maltose-binding protein fusion proteins. IgE-binding reactivity of the recombinant proteins and the cross-reactivity of soybean profilin with birch profilin was studied by immunoblotting, enzyme-linked allergosorbent assays (EASTs), and competitive inhibition experiments by using serum samples from 13 soybean-sensitized subjects. Results: Results of immunoblot analysis, EAST, and EAST-inhibition experiments indicate the presence of profilin in soybean extract. The recombinant soybean profilin (rGly m 3) was recognized by IgE in 9 (69%) of the 13 sera tested. Only the full-length rGly m 3 was able to bind with IgE antibodies, whereas the 3 soybean profilin fragments did not show significant binding reactivity, indicating that the IgE binding to rGly m 3 depends on the integrity of a conformational structure, which was not present in the overlapping profilin fragments. The rGly m 3 cross-reacted with birch pollen profilin (Bet v 2), and the IgE binding to Bet v 2 could be inhibited by rGly m 3. Conclusions: rGly m 3 represents a new soybean allergen with well-characterized primary sequence, and its IgE-binding reactivity is mediated by conformational epitopes. (J Allergy Clin Immunol 1999;1293-1301.)