IgE binding of the recombinant allergen soybean profilin (rGly m 3) is mediated by conformational epitopes.
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.
Rihs
H P
HP
Research Institute for Occupational Medicine (BGFA) at the Ruhr-University, Bochum, Germany.
Chen
Z
Z
Ruëff
F
F
Petersen
A
A
Rozynek
P
P
Heimann
H
H
Baur
X
X
D000293:Adolescent; D000328:Adult; D000485:Allergens / Q000097:blood / Q000494:pharmacology; D000595:Amino Acid Sequence; D001483:Base Sequence; D002648:Child; D003285:Contractile Proteins; D003429:Cross Reactions / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D015951:Factor Xa / Q000378:metabolism; D005260:Female; D006801:Humans; D015151:Immunoblotting; D016056:Immunodominant Epitopes / Q000737:chemistry; D007073:Immunoglobulin E / Q000378:metabolism*; D008297:Male; D008840:Microfilament Proteins / Q000276:immunology* / Q000494:pharmacology*; D008875:Middle Aged; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000276:immunology / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D051304:Profilins; D011485:Protein Binding / Q000187:drug effects; D011994:Recombinant Proteins / Q000276:immunology / Q000494:pharmacology; D013025:Glycine max / Q000276:immunology
D000485:Allergens; D003285:Contractile Proteins; D016056:Immunodominant Epitopes; D008840:Microfilament Proteins; D010940:Plant Proteins; D051304:Profilins; D011994:Recombinant Proteins; D007073:Immunoglobulin E; D015951:Factor Xa