Analysis of the Distribution of the Major Soybean Seed Allergens in a Core Collection of Glycine max Accessions R. W. Yaklich,* R. M. Helm, G. Cockrell, and E. M. Herman ABSTRACT to Pseudomonas possess higher levels of P34 in leaf tissue compared with sensitive cultivars (Cheng et al., P34 is an outlying member of the papain-superfamily of cysteine 1998). P34 has been shown to be the major allergenic proteases that is expressed in developing soybean [Glycine max (L.) protein of soybean seeds (Ogawa et al., 1991, 1993; Merr.] seeds and may be involved in the defense against Pseudomonas infection. P34 is the major human allergen of soybean seeds and is Burks et al., 1988). Assays of IgE binding using immuno- present in processed food products that contain soybean protein. globulins from soybean sensitive individuals indicates We have surveyed a core collection of soybean accessions using a that 65% of the total allergenic response can be ac- monoclonal antibody against P34 and with human sera from soybean- counted for by P34 (Ogawa et al., 1991, 1993; Helm sensitive individuals. We found that the accessions of soybean sur- et al., 1998). Detailed immunological analysis of the veyed contain similar levels of P34 and that P34 is the major allergenic allergenicity of P34 by epitope mapping has shown that protein. We have also surveyed wild relatives of soybean and found there are at least 12 distinct epitopes on the protein that P34 was present in the examples assayed. These results indicate (Helm et al., 1998). that it may not be possible to eliminate P34 from the food supply by The use of soybean products in processed foods poses breeding with an improved germplasm base. a significant health and food safety problem for sensitive individuals since the P34 protein can be detected in a A n outlying member of the papain-superfamily of wide variety of processed foods (Tsuji et al., 1995). The cysteine-proteases has been identified and cloned elimination of P34 from soybean seeds would enhance from maturing soybean seeds (Kalinski et al., 1990, food safety and make the use of soybean products avail- 1992). This protein, termed P34, possesses most of the able to sensitive individuals. Therefore, using a monclo- conserved characteristics of cysteine proteases including nal antibody against P34 and sera from soybean sensitive a large precursor domain that is posttranslationally pro- individuals, we surveyed a core collection of soybean cessed. The primary sequence contains aligned and con- accessions to examine the abundance of P34 and other served amino acids that are critical in the conserved allergenic proteins among these cultivars. In parallel, tertiary conformation of members of the papain super- we assayed other members of the Glycine species for family. P34 exhibits some unique features that separate the presence of P34 and other sera allergenic proteins. it from other members of the papain superfamily. MATERIALS AND METHODS Among these are replacement of the conserved cysteine in the active site found in all other papain family proteins Seed samples of Glycine species were procured from the National Germplasm Repositories or grown at Beltsville in with a glycine, suggesting that the protein is enzymati- 1996 by normal agronomic procedures (Yaklich et al., 1979). cally inactive (Kalinski et al., 1990, 1992). Cysteine pro- The G. max samples consisted of the core collection of soybean teases are typically self-processed under acid-reducing ancestors and first progeny that contributed at least 95% of conditions resulting in the cleavage of the large precur- the genes found in public cultivars released between 1947 sor domain. However, P34 is processed after an aspara- and 1988 in North America (Gizlice et al., 1994), and ‘Black gine residue in a single step (Kalinski et al., 1992), most Eyebrow’, ‘Blackhawk’, ‘Minsoy’, ‘Williams’, ‘Miles’, ‘Essex’, likely by the same enzyme that processes the 11S storage and ‘Kunitz’. Seed samples of nodulated and non-nodulated proteins (Hara-Nishimura et al., 1993, 1995). Sequence lines of ‘Clark’, BARC-14, BARC-15, BARC-16, BARC-17, comparisons and alignments indicate that although P34 and the high protein lines CX797-21, CX797-115, CX804-3, is a member of the papain superfamily, it is also quite CX804-108, (J.R. Wilcox, USDA-ARS, Purdue University), dissimilar from the enzymatically active cysteine prote- 76-48773 (A. Matson, Soybean Research Foundation), D76- 8070, D80-6931, D81-8259, D81-8498, (E.E. Hartwig, USDA- ases including those identified in soybean. ARS, Jamie Whitten Delta States Research Center), NC-2-62, P34 may have a function in defense against Pseudo- (J.W. Burton, USDA-ARS, North Carolina State University), monas infection by binding syringolide elicitors secreted BARC-6, BARC-7, BARC-8, and BARC-9, (R.C. Leffel, by the bacteria (Cheng et al., 1998). P34 is very abundant USDA-ARS, Beltsville, Maryland). In addition, plant intro- in seeds (Kalinski et al., 1992), but it is also found in ductions of the following Glycine species were included in the vegetative cells that are subject to bacterial infection study: G. soja Siebold & Zucc., PI 65549, PI 81762, PI 101404; (Cheng et al., 1998). Soybean cultivars that are resistant G. arenaria Tind., PI 505204; G. argyrea Tind., PI 505151, PI 509451; G. canescens F.J. Herm, PI 440932, PI 440942, PI 446934; G. clandestina Wendl., PI 440948, PI 440954, PI R.W. Yaklich, Soybean and Alfalfa Res. Lab.; E.M. Herman, Climate 440960; G. curvata Tind., PI 505164, PI 505166, PI 505167; Stress Lab., USDA-ARS, Beltsville Agric. Res. Center, Beltsville, MD 20705; R.M. Helm and G. Cockrell, Dep. of Pediatrics, Univ. of G. cyrtoloba Tind., PI 440962, PI 440963, PI 509472; G. falcata Arkansas for Medical Sciences, Arkansas Children’s Hospital Re- Benth., PI 440975, PI 505179, PI 509473; G. latifolia (Benth.) search Institute, Little Rock, AR 72202. Mention of propriety products Newell & Hymowitz, PI 378709, PI 253238, PI 440978; are included for the benefit of the reader and do not imply endorse- G. microphylla (Benth.) Tind., PI 339664, PI 440956, PI ment by the USDA. Received 29 Sept. 1998. *Corresponding author 446939; G. tabacina (Labill.) Benth., PI 339661, PI 343986, PI (ryaklich@asrr.arsusda.gov). 373990; and G. tomentella Hayata, PI 373987, PI 440998, PI 441000, PI 446993, PI 446995, and PI 509499. Published in Crop Sci. 39:1444–1447 (1999). 1444 Published September, 1999