Laura Privalle

Evaluation of protein safety in the context of agricultural biotechnology

One component of the safety assessment of agricultural products produced through biotechnology is evaluation of the safety of newly expressed proteins. The ILSI International Food Biotechnology Committee has developed a scientifically based two-tiered, weight-of-evidence strategy to assess the safety of novel proteins used in the context of agricultural biotechnology. Recommendations draw upon knowledge of the biological and chemical characteristics of proteins and testing methods for evaluating potential intrinsic hazards of chemicals. Tier I (potential hazard identification) includes an assessment of the biological function or mode of action and intended application of the protein, history of safe use, comparison of the amino acid sequence of the protein to other proteins, as well as the biochemical and physico-chemical properties of the proteins. Studies outlined in Tier II (hazard characterization) are conducted when the results from Tier I are not sufficient to allow a determination of safety (reasonable certainty of no harm) on a case-by-case basis. These studies may include acute and repeated dose toxicology studies and hypothesis-based testing. The application of these guidelines is presented using examples of transgenic proteins applied for agricultural input and output traits in genetically modified crops along with recommendations for future research considerations related to protein safety assessment.

Quantitative analysis of CryIA(b) expression in Bt maize plants, tissues, and silage and stability of expression over successive generations

The range and stability of expression of the transgenic CryIA(b) protein was examined in Ciba Seeds Bt maize plants derived from Event 176. Specifically, CryIA(b) levels were determined for: (1) various plant tissues and developmental stages in three maize lines from 1993 field tests; (2) pollen and leaves from plants representing four backcross generations of two genotypes; (3) leaves of 6 precommercial hybrids; and (4) silage from one Bt maize hybrid. Significant levels were found only in pollen and leaves. Genetic background did not greatly impact the level seen in either tissue. CryIA(b) expression in maize plants derived from transformation Event 176 was stable over at least four successive generations. On a per acre basis, the highest amount of CryIA(b) protein (estimated to be 2-4 g CryIA(b) protein/acre) was found to occur at anthesis, consistent with the stage at which maximum plant vegetative biomass is reached. CryIA(b) was not detected in silage prepared from CryIA(b)-expression plants. The maize-expressed CryIA(b) protein was found to have the expected size and to be immunoreactive with antibodies prepared against crystals from Bacillus thuringiensis subsp. kurstaki.

Isolation of cDNA clones coding for spinach nitrite reductase: Complete sequence and nitrate induction

SummaryThe main nitrogen source for most higher plants is soil nitrate. Prior to its incorporation into amino acids, plants reduce nitrate to ammonia in two enzymatic steps. Nitrate is reduced by nitrate reductase to nitrite, which is further reduced to ammonia by nitrite reductase. In this paper, the complete primary sequence of the precursor protein for spinach nitrite reductase has been deduced from cloned cDNAs. The cDNA clones were isolated from a nitrate-induced cDNA library in two ways: through the use of oligonucleotide probes based on partial amino acid sequences of nitrite reductase and through the use of antibodies raised against purified nitrite reductase. The precursor protein for nitrite reductase is 594 amino acids long and has a 32 amino acid extension at the N-terminal end of the mature protein. These 32 amino acids most likely serve as a transit peptide involved in directing this nuclearencoded protein into the chloroplast. The cDNA hybridizes to a 2.3 kb RNA whose steady-state level is markedly increased upon induction with nitrate.

Quantitation of soybean allergens using tandem mass spectrometry.

Soybean (Glycine max) seed contain some proteins that are allergenic to humans and animals. However, the concentration of these allergens and their expression variability among germplasms is presently unknown. To address this problem, 10 allergens were quantified from 20 nongenetically modified commercial soybean varieties using parallel, label-free mass spectrometry approaches. Relative quantitation was performed by spectral counting and absolute quantitation was performed using multiple reaction monitoring (MRM) with synthetic, isotope-labeled peptides as internal standards. During relative quantitation analysis, 10 target allergens were identified, and five of these allergens showed expression levels higher than technical variation observed for bovine serum albumin (BSA) internal standard (∼11%), suggesting expression differences among the varieties. To confirm this observation, absolute quantitation of these allergens from each variety was performed using MRM. Eight of the 10 allergens were quantified for their concentration in seed and ranged from approximately 0.5 to 5.7 μg/mg of soy protein. MRM analysis reduced technical variance of BSA internal standards to approximately 7%, and confirmed differential expression for four allergens across the 20 varieties. This is the first quantitative assessment of all major soybean allergens. The results show the total quantity of allergens measured among the 20 soy varieties was mostly similar.

Safety assessment of biotechnology products for potential risk of food allergy: implications of new research.

Food allergy is a potential risk associated with use of transgenic proteins in crops. Currently, safety assessment involves consideration of the source of the introduced protein, in silico amino acid sequence homology comparisons to known allergens, physicochemical properties, protein abundance in the crop, and, when appropriate, specific immunoglobulin E binding studies. Recently conducted research presented at an International Life Sciences Institute/Health and Environmental Sciences Institute-hosted workshop adds to the scientific foundation for safety assessment of transgenic proteins in five areas: structure/activity, serum screening, animal models, quantitative proteomics, and basic mechanisms. A web-based tool is now available that integrates a database of allergenic proteins with a variety of computational tools which could be used to improve our ability to predict allergenicity based on structural analysis. A comprehensive strategy and model protocols have been developed for conducting meaningful serum screening, an extremely challenging process. Several animal models using oral sensitization with adjuvant and one dermal sensitization model have been developed and appear to distinguish allergenic from non-allergenic food extracts. Data presented using a mouse model suggest that pepsin resistance is indicative of allergenicity. Certain questions remain to be addressed before considering animal model validation. Gel-free mass spectrometry is a viable alternative to more labor-intensive approaches to quantitative proteomics. Proteomic data presented on four nontransgenic varieties of soy suggested that if known allergen expression in genetically modified crops falls within the range of natural variability among commercial varieties, there appears to be no need to test further. Finally, basic research continues to elucidate the etiology of food allergy.

Phosphomannose Isomerase, a Novel Plant Selection System

Abstract: Phosphomannose isomerase (PMI), an enzyme not present in many plants, catalyzes the reversible interconversion of mannose 6‐phosphate and fructose 6‐phosphate. Plant cells lacking this enzyme are incapable of surviving on synthetic medium containing mannose. Thus PMI/mannose selection has utility in the identification of transformed plant cells. As part of the safety assessment transgenic plants undergo before commercialization, PMI has been evaluated for its potential allergenicity. Purified PMI protein was readily digestible in a simulated gastric environment. PMI has no sequence homology to known allergens, does not contain multiple disulfide bonds, and has no N‐glycosylation consensus sequences. No detectable changes in glycoprotein profiles were detected in PMI‐transformed plants as compared to nontransgenic controls. These results indicate that PMI lacks many of the attributes associated with known oral allergens.

Isolation and characterization of a cDNA clone encoding a lectin gene from Agaricus bisporus

Aguricus bisporus, the common commercial golden white mushroom, has been reported to have between two and four hemagglutinin proteins (ABAS) (Presant and Kornfeld, 1972; Ahmad et al., 1984; Sueyoshi et al., 1985). These lectins, which differ in their pIs, have been purified and characterized. They exist as tetramers of mo1 wt 58,000 to 64,000, composed of identical subunits of mo1 wt 16,000 (Sueyoshi et al., 1985; Presant and Kornfeld, 1972). Isolectins often either reflect heterogeneity of posttranslational modification or may be products of different genes (Van Damme and Peumans, 1987; Van Damme et al., 1991; Zenteno et al., 1991). Like many lectins, those from Aguricus have been reported to be glycosylated (Ahmad et al., 1984). A11 ABA isoforms have similar carbohydrate binding specificities and binding is not inhibited by simple monosaccharides. Many lectins are useful as probes for differentiating between normal and malignant cell surfaces. The availability of lectin genes may allow design of hybrid genes whose products would have useful applications. Here we describe the cloning of a cDNA for ABA (Table I). Southern blot analysis indicated that at least two ABA genes were present. The sequences of the three cDNA clones isolated were identical. N-terminal amino acid sequence information for the four ABA isolectins indicated that a11 were identical for their first 21 amino acids. Potentia1 O-glycosylation sites were identified at Thr”, Ser’, and Sers8 because none gave standard elution profiles during phenylthiohydantoin-amino acid separations. Only one consensus N-glycosylation site was found (Asp8*); however, peptide sequencing through this region gave a normal Asp signal. The amino acid composition deduced from the cDNA sequence is very similar to that reported for the isolectins except that Ile, Ser, and Pro were previously underestimated (Sueyoshi et al., 1985).

Metabolism of primisulfuron by barnyard grass

Abstract Barnyard grass (Echinochloa crus galli (L.) P.B.) is a grass weed species not controlled by the experimental sulfonylurea herbicide, primisulfuron (BEACON). The mechanism of resistance employed by barnyard grass was determined to be metabolism and not an insensitive target enzyme, acetohydroxy acid synthase (AHAS). Of the five weed species examined, barnyard grass was the fastest at metabolizing primisulfuron with an in vivo half-life between 1 and 2 hr. Two major classes of metabolites were produced and were found to be noninhibitory to AHAS. We have monitored the relative abundance of these metabolites by HPLC and have partially purified and characterized the metabolites. For one of the metabolite classes the initial step appears to be an hydroxylation reaction followed by glycosylation.

Current and future methods for evaluating the allergenic potential of proteins: International workshop report 23-25 October 2007

The International Life Science Institutes Health and Environmental Sciences Institutes Protein Allergenicity Technical Committee hosted an international workshop October 23-25, 2007, in Nice, France, to review and discuss existing and emerging methods and techniques for improving the current weight-of-evidence approach for evaluating the potential allergenicity of novel proteins. The workshop included over 40 international experts from government, industry, and academia. Their expertise represented a range of disciplines including immunology, chemistry, molecular biology, bioinformatics, and toxicology. Among participants, there was consensus that (1) current bioinformatic approaches are highly conservative; (2) advances in bioinformatics using structural comparisons of proteins may be helpful as the availability of structural data increases; (3) proteomics may prove useful for monitoring the natural variability in a plants proteome and assessing the impact of biotechnology transformations on endogenous levels of allergens, but only when analytical techniques have been standardized and additional data are available on the natural variation of protein expression in non-transgenic bred plants; (4) basophil response assays are promising techniques, but need additional evaluation around specificity, sensitivity, and reproducibility; (5) additional research is required to develop and validate an animal model for the purpose of predicting protein allergenicity.

Health and nutritional status of Wistar rats following subchronic exposure to CV127 soybeans

This subchronic duration feeding study evaluated the nutritional and health status of rats fed diets containing CV127 at incorporation levels of 11% and 33%. For control comparisons, rats were also exposed to similar incorporation levels of the near isogenic conventional soybean variety (Conquista) and two other conventional soybean varieties (Monsoy, Coodetec). In spite of phenotypic differences among these four soybean varieties, there were no quantitative differences in their respective proximate and other compositional properties, including proteins, amino acids, antinutrients and nutritional cofactors. All diets were prepared by blending the respective processed soybean meal with ground Kliba maintenance meal at high (33%) and low (11%) incorporation levels, and the blended diets were fed to Wistar rats for about 91 days. Although there were some isolated parameters indicating statistically significant changes, these lacked consistency and a plausible mechanism and were thus assessed to be incidental. The totality of results demonstrate that CV127 soybeans are similar with respect to their nutritional value and systemic effects as its near isogenic conventional counterpart, as well as other conventional soybean varieties. Hence, introduction of AHAS gene into soybeans does not substantially alter its compositional properties, nor adversely affect its nutritional or safety status to mammals.