Identification, Characterization, Epitope Mapping, and Three-Dimensional Modeling of the r-Subunit of -Conglycinin of Soybean, a Potential Allergen for Young Pigs CHUNJIANG J. FU, † JOSEPH M. JEZ, ‡ MONTY S. KERLEY, † GARY L. ALLEE, † AND HARI B. KRISHNAN* ,§ Division of Animal Science, and Plant Genetics Research Unit, Agricultural Research Service, U.S. Department of Agriculture, University of Missouri, Columbia, Missouri 65211, and Donald Danforth Plant Science Center, St. Louis, Missouri 63132 Soybean meal (SBM), the major byproduct of soybean oil extraction, is the main protein source for swine diets globally. In the United States, 8.6 million metric tons of SBM was used in swine rations in 2004. The pathological effect and immunological response of SBM feeding have been demonstrated in swine. In this study, we have utilized plasma collected from piglet feed with SBM in immunoblot analysis to detect proteins that elicited antigenic responses. We have identified soybean -conglycinin R-subunit as being a potential allergen for young piglets. Characterization of this protein indicated that deglycosylation and pepsin digestion did not eliminate immunoreactivity of this protein. Epitope mapping utilizing planar cellulose supports technology (SPOT) showed that three peptides spanning amino acids S185-R231 were critical for the allergenicity. A computer-generated three-dimensional structure model of the R-subunit of -conglycinin indicated that the antigenic epitopes were located on the surface of the protein. Information from this study may assist in the construction of recombinant nonallergenic soybean protein useable for both immunotherapy and the potential production of hypoallergenic soybean plants. KEYWORDS: Allergens; epitope mapping; -conglycinin; pig; soybean meal; three-dimensional structure INTRODUCTION Soybeans are an important source of edible vegetable oil and protein throughout the world. Most of the soybeans produced in the United States are crushed for oil and meal. In 2004, U.S. production of soybean meal (SBM) was 32.95 million metric tons. Approximately 97% of the SBM is used for animal feed, primarily in poultry, swine, and cattle diets. On a global basis, SBM accounts for approximately 63% of all protein sources used in animal feeds. In the United States, SBM accounts for 27% of the protein used in swine feeds (1). Soybean ranks among the eight most significant food al- lergens (2, 3). Nearly 2% of adults and 5-8% of infants in the United States and Europe are reported to be allergic to soybeans (4). Many soybean allergens for humans have been identified and characterized (5-9). The resistance to mammalian digestion and heat denaturation of the soybean allergens (10, 11) make them bioactive to animals. A joint FAO/WHO consulting group recommended feed-grade soybean testing to reduce the allergen challenges that could have a far-reaching impact on commercial animal production operations (10, 11). Animal sensitization to * To whom correspondence should be addressed. Tel: 573-882-8151. Fax: 573-884-7850. E-mail: KrishnanH@missouri.edu. † Division of Animal Science, University of Missouri. ‡ Donald Danforth Plant Science Center. § U.S. Department of Agriculture, University of Missouri. Figure 1. SDS-PAGE/immunoblot analysis of the soybean seed proteins. Total seed proteins (100 µg) and purified R-subunits of -conglycinin (5 µg) from soybean were fractionated by SDS-PAGE on a 13.5% gel and stained with Coomassie Blue (A). Immunological detection of antigenic proteins (B). Proteins shown in panel A were transferred to a nitrocellulose membrane and probed with pig plasma. Immunoreactive proteins were identified using anti-pig IgG-horseradish peroxidase conjugate antibody followed by chemiluminescent detection. Lane 1, protein marker; lane 2, soybean total seed protein; and lane 3, purified R-subunit of -conglycinin. 4014 J. Agric. Food Chem. 2007, 55, 4014-4020 10.1021/jf070211o CCC: $37.00 © 2007 American Chemical Society Published on Web 04/17/2007