Volume 96, Issue 6 , Pages 1008-1010, December 1995
Assessment of the endogenous allergens in glyphosate-tolerant and commercial soybean varieties☆☆☆★
Article Outline
Abbreviations: EPSPS , 5-Enolpyruvylshikimate-3-phosphate synthase, SDS-PAGE , Sodium dodecylsulfate-polyacrylamide gel electrophoresis
A gene has been introduced into soybeans to confer tolerance to glyphosate, the active ingredient in the herbicide, Roundup (Monsanto Co., St. Louis, Mo.)1, 2 Soybean varieties expressing this trait will enable farmers to use Roundup herbicide within the crop during the growing season. Previous studies have confirmed the safety of the introduced protein (5-enolpyruvylshikimate-3-phosphate synthase [EPSPS]), which confers glyphosate tolerance.2 Over 1400 analyses have confirmed the compositional and nutritional equivalence of the soybeans produced from the glyphosate-tolerant soybean variety to the parental soybean variety.2
The multiple, endogenous protein allergens previously identified in soybeans3, 4, 5 represent an additional safety concern. The purpose of this study was to qualitatively and quantitatively compare the endogenous allergens in glyphosate-tolerant and commercial soybean varieties and to determine whether the genetic engineering process itself affected these components.
METHODS
Serum from food-sensitive patients
Five patients with atopic dermatitis and a positive immediate skin test response to soybean extracts were confirmed to be sensitive to soybean protein by a positive double-blind, placebo-controlled food challenge.3 Equal volumes of sera were combined to prepare a serum pool containing soybean-specific IgE antibody. A similar serum pool containing peanut allergen-specific IgE antibody was generated from five patients with atopic dermatitis and peanut hypersensitivity.
Soybean extracts
Preparations of defatted soybean flakes and soy flour were obtained from commercial sources. Defatted soybean flakes from two independent glyphosate-tolerant soybean varieties (lines 40-3-2 and 61-67-1) and the parental control3 were also used in this study. Defatted soybean flakes or flour was ground to a fine powder, then extracted (1:20 wt/vol) at 4° C overnight in extraction buffer (20 mmol/L sodium phosphate, 1 mol/L NaCl, pH 7.2).3, 5
Sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting
SDS-PAGE and immunoblotting were performed as described previously.3
RESULTS
Preliminary experiments
Before the levels of endogenous allergens in glyphosate-tolerant soybean varieties were compared with those of the parental variety, immunoblot experiments were conducted to address the potential variability in endogenous allergens in commercial defatted soybean products. The results (Fig. 1, A and B) showed the presence of a consistent number of distinct IgE-binding proteins in the various products, which were comparable to the results described previously in the literature.3, 4, 5 On the basis of this information, the endogenous allergenic proteins in the glyphosate-tolerant soybeans were compared with the preparations generated from the parental and commercial varieties.
FIG. 1.
A, Coomassie blue-stained SDS-PAGE gel with soybean extracts. Lane 1, Prestained molecular weight standards; lane 2, extract from GTS line 61-67-1 (30 μg); lane 3, extract from parental line A5403 (30 μg); lane 4, extract from GTS line 40-3-2 (30 μg); lane 5, extract from Cargill control 1 (30 μg); lane 6, extract from Cargill control 2 (30 μg); lane 7, extract from ADM control (30 μg). B, Immunoblot with serum containing IgE antibodies from soybean-sensitive patients. Lane 1, Prestained molecular weight standards; lane 2, extract from GTS line 61-67-1 (30 μg); lane 3, extract from parental line A5403 (30 μg); lane 4, extract from GTS line 40-3-2 (30 μg); lane 5, extract from Cargill control 1 (30 μg); lane 6, extract from Cargill control 2 (30 μg); lane 7, extract from ADM control (30 μg).
Endogenous allergens in glyphosate-tolerant and commercial soybean preparations
Four identical SDS-PAGE gels were run with extracts from each of the soybean preparations. One gel was stained with Coomassie blue (Integrated Separations Systems, Natick, Mass.). The banding patterns for the two glyphosate-tolerant varieties (Fig. 1, A, lanes 2 and 4) were identical to that of the parental control (Fig. 1, A, lane 3). The three remaining gels were blotted to nitrocellulose and incubated with the serum pools from either the soybean-sensitive, peanut-sensitive, or nonatopic control individuals, respectively. The immunoblots for the different soybean products revealed no differences in IgE-binding soybean protein patterns when the IgE antibody pool from soybean-sensitive individuals was used (Fig. 1, B). The areas of the most intense IgE binding were at approximately 30 kd and 50 kd, consistent with previously reported results obtained by this assay method.3 Immunoblotts with the IgE antibody pools from either peanut-sensitive or nonatopic individuals showed that a minimal amount of nonspecific binding occurred at approximately 45 kd and 50 kd for all the soybean preparations (data not shown). There were no detectable differences in the banding patterns in any of the soybean samples, including those prepared from the glyphosate-tolerant soybean varieties with any of the three pooled sera.
DISCUSSION
Our previous work3 established that there are varying amounts of IgE- and IgG-specific antibodies to multiple proteins present in soybean extracts and that the immunoblot procedure was appropriate for comparing the endogenous allergens in the soybean varieties.
The immunoblots in this investigation revealed IgE binding patterns similar to those described previously.3, 4, 5 Analysis of the protein extracts prepared from the glyphosate-tolerant soybeans showed that both the composition and quantity of the proteins detected by the immunoblot procedure were qualitatively and quantitatively indistinguishable from the results produced with the parental soybean variety, as well as with three commercial soybean preparations. In addition, the immunoblot results with serum from peanut-sensitive and the nonatopic patients showed only limited IgE binding to the soybean proteins from any of these preparations and, as expected, showed no differences between extracts prepared from the glyphosate-tolerant and parental control or commercial soybean varieties.
Our current studies demonstrate that the introduction of the gene encoding the EPSPS protein to confer glyphosate tolerance caused no discernible changes, either qualitatively or quantitatively, in the composition of endogenous soybean allergenic proteins in either of the glyophosate-tolerant varieties analyzed.
Acknowledgements
We thank Cathie Connaughton for her excellent technical contributions and Stephen Padgette from Monsanto for providing the defatted soybean meal from the parental control and glyphosate-tolerant soybean varieties.
References
- Inhibitors of amino acid biosynthesis: strategies for imparting glyphosate tolerance to crop plants. In: Singh BK, Flores HE, Shannon JC editor. Biosynthesis and molecular regulation of amino acids in plants. Rockville, Maryland: American Society of Plant Physiologists; 1992;p. 139–145
- New weed control opportunities: development of soybeans with a Roundup Ready™ gene. In: Duke SO editors. Herbicide-resistant crops: agricultural economic, environmental, regulatory, and technological aspects. CRC Press; 1994; (in press)
- . Allergenicity of major component proteins of soybean determined by enzyme linked immunosorbent assay (ELISA) and immunoblotting in children with atopic dermatitis and positive soy challenges. J ALLERGY CLIN IMMUNOL. 1988;81:1135–1142
- . Major proteins of soybean seeds: a straight forward fractionation and their characterization. J Agri Food Chem. 1976;24:1117–1121
- Identification of the soybean allergenic protein, Gly m Bd 30K, with the soybean seed 34-kDa oil-body associated protein. Biosci Biotech Biochem. 1993;57:1030–1033
☆ From aArkansas Children’s Hospital, University of Arkansas for Medical Sciences, Little Rock; and bThe Agricultural Group, Monsanto, St. Louis.
☆☆ Reprint requests: Wesley Burks, MD, Arkansas Chidren's Hospital, 800 Marshall St., Little Rock, AR 72202.
★ 1/54/66390
PII: S0091-6749(95)70243-1
© 1995 Published by Elsevier Inc.
Volume 96, Issue 6 , Pages 1008-1010, December 1995
