Soybean Allergenicity and Suppression of the Immunodominant Allergen

protein storage vacuoles (Kalinski et al., 1992). P34 has a tendency to bind to lipid and oil that is likely significant The wide-spread use of soybean [Glycine max (L.) Merr.] products in its subsequently demonstrated role as an allergen and in processed foods poses a potential threat to soybean-sensitive, foodallergic individuals. Clinical symptoms of soybean allergy can be manias a possible defense protein (Okinaka et al., 2002). fested as gastric distress or atopic dermatitis and, while usually not A cDNA for P34 was isolated a Zap phage library life threatening, suspected cases of anaphylaxis have been reported. prepared soybean seed midmaturation mRNA using imIn vitro assays of soybean seed proteins with sera collected from munological screening with a monoclonal antibody (Kalsoybean-sensitive individuals have shown that major storage proteins inski et al., 1990). The deduced sequence exhibited high as well as other minor seed proteins account for IgE binding. Gly m homology to members of the papain superfamily of cysBd 30k, a member of the papain superfamily of cysteine proteases, teine proteases (Fig. 2). All cysteine proteases have highly also referred to as P34, has been identified as a major allergen in conserved amino acids distributed throughout the comsoybean seeds. We have used gene silencing to eliminate accumulation plete sequence. Among these are disulfide bridges and of P34/Gly m Bd 30k in transgenic soybean. These transgenic plants, glycine–glycine residues important for folding, and conproducing P34/Gly m Bd 30k-null seeds, lacked any obvious developmental or phenotypic differences when compared with control plants. served amino acids in the active site that includes a The production of a P34/Gly m Bd 30k-null line eliminates one of cysteine and a histidine. P34 lacks the cysteine in the the primary allergens present in soybean seeds. active site that is present in all other members of the papain superfamily. Another isolate of P34 cDNA (GenBank# AB013289) also lacks this conserved cysteSeeds Contain a Variety of Stored Proteins ine. This indicates that a key characteristic of this protein Seeds contain a few abundant storage proteins encoded is the alteration of an otherwise completely conserved by a few large gene families as well as numerous other cysteine, which is the catalytic amino acid to a glycine. lower abundance proteins (see Herman and Larkins, This suggests that P34 may not possess intrinsic proteo1999, for review). Many of these lower abundance seed lytic activity, and this is supported by protein engineerproteins are involved either in hydrolysis of one of the ing experiments whereby other members of the cysteine many reserve substances or in defense against macroor protease family have been synthesized in heterologous microorganisms utilizing those rich reserves of nutritive cells with site-directed mutagenesis changing the catasubstances as sources of food. Various lower abundance lytic cysteine to serine and inactivating the protease ancillary proteins play significant roles in the seed’s life (Rowan et al., 1992). We do not yet know if any other cycle, but also in many cases, they either enhance or plant contains a homolog of P34 defined as a cysteine diminish utilization of seeds for human and animal conprotease without a catalytic cysteine. sumption. Seeds contain factors that are either antimetaLike other cysteine proteases (North, 1986; Cigic et bolic compounds or elicitors of food allergies. Soybean al., 2000; Tao et al., 1994; Wiederanders, 2000), P34 is contains an array of antimetabolic compounds such as initially synthesized as a large precursor with a 122 amino phytin, trypsin inhibitors, and oligosacchrides as well as acid precursor domain that includes the signal sequence proteins that elicit allergenic responses inducing adverse for translocation into the endoplasmic reticulum (ER) gastric responses and atopic dermatitis in sensitive hulumen. In vitro and in vivo synthesis and processing exmans and animals (Fig. 1). periments have demonstrated that proP34 is produced as a glycoprotein with the utilized glycosylation site in P34 Is a Member of the Papain Superfamily the pro-domain (Kalinski et al., 1992). Cysteine proteLocalized in Protein Storage Vacuoles ases have both intracellular and extracellular roles with plant intracellular forms possessing an intrinsic targeting An abundant protein associated with the oil body sequence NPIR (Holwerda et al., 1992) and closelyfraction was purified from mature soybean seeds (Herrelated sequences in plants such as the aligned NYIR man et al., 1990). Monoclonal antibodies against the in P34. The NPIR sequence is recognized by a transoil body associated proteins included a 34-kDa protein golgi receptor (BP80; AtELP) that targets proteins into termed P34. This P34 was highly enriched and partially the vacuole (Ahmed et al., 1997; Paris et al., 1997). purified from the oil body fraction. Initially, it was asImmunogold immunocytochemistry has localized P34 sumed to be a constituent of this organelle. Subsequent in the Golgi and Golgi-derived secretion vesicles transresults demonstrated that P34 was localized in the seed porting the protein to the protein storage vacuole. Extracellular forms of cysteine proteases, such as those sePlant Genetics Unit, USDA/ARS, Donald Danforth Plant Science creted by cereal aleurone, probably default to the cell Center, St. Louis, MO 63132. Receive 4 Nov. 2003. Symposia. *Corresponding author ([email protected]). surface. Cysteine proteases have many functional roles in both Published in Crop Sci. 45:462–467 (2005). physiological and developmental events (Turk et al., © Crop Science Society of America 677 S. Segoe Rd., Madison, WI 53711 USA 1996, 1997 for reviews). In particular, they are involved 462 Published online January 31, 2005