Elena A. Rice

Bioinformatic Methods for Allergenicity Assessment Using a Comprehensive Allergen Database

Background: A principal aim of the safety assessment of genetically modified crops is to prevent the introduction of known or clinically cross-reactive allergens. Current bioinformatic tools and a database of allergens and gliadins were tested for the ability to identify potential allergens by analyzing 6 Bacillus thuringiensis insecticidal proteins, 3 common non-allergenic food proteins and 50 randomly selected corn (Zea mays) proteins. Methods: Protein sequences were compared to allergens using the FASTA algorithm and by searching for matches of 6, 7 or 8 contiguous identical amino acids. Results: No significant sequence similarities or matches of 8 contiguous amino acids were found with the B. thuringiensis or food proteins. Surprisingly, 41 of 50 corn proteins matched at least one allergen with 6 contiguous identical amino acids. Only 7 of 50 corn proteins matched an allergen with 8 contiguous identical amino acids. When assessed for overall structural similarity to allergens, these 7 plus 2 additional corn proteins shared ≧35% identity in an overlap of ≧80 amino acids, but only 6 of the 7 were similar across the length of the protein, or shared >50% identity to an allergen. Conclusions: An evaluation of a protein by the FASTA algorithm is the most predictive of a clinically relevant cross-reactive allergen. An additional search for matches of 8 amino acids may provide an added margin of safety when assessing the potential allergenicity of a protein, but a search with a 6-amino-acid window produces many random, irrelevant matches.

Genetic basis and detection of unintended effects in genetically modified crop plants

In January 2014, an international meeting sponsored by the International Life Sciences Institute/Health and Environmental Sciences Institute and the Canadian Food Inspection Agency titled “Genetic Basis of Unintended Effects in Modified Plants” was held in Ottawa, Canada, bringing together over 75 scientists from academia, government, and the agro-biotech industry. The objectives of the meeting were to explore current knowledge and identify areas requiring further study on unintended effects in plants and to discuss how this information can inform and improve genetically modified (GM) crop risk assessments. The meeting featured presentations on the molecular basis of plant genome variability in general, unintended changes at the molecular and phenotypic levels, and the development and use of hypothesis-driven evaluations of unintended effects in assessing conventional and GM crops. The development and role of emerging “omics” technologies in the assessment of unintended effects was also discussed. Several themes recurred in a number of talks; for example, a common observation was that no system for genetic modification, including conventional methods of plant breeding, is without unintended effects. Another common observation was that “unintended” does not necessarily mean “harmful”. This paper summarizes key points from the information presented at the meeting to provide readers with current viewpoints on these topics.

Digestive Stability in the Context of Assessing the Potential Allergenicity of Food Proteins

Assessment of the potential allergenicity of proteins introduced into food crops through biotechnology is required by international regulatory agencies that govern the release and production of genetically modified plants. Currently, one aspect of this assessment includes analysis of the protein in a simulated gastric fluid (SGF) assay that tests the digestibility of the target protein to pepsin. The logic behind this test was that proteins that are nutritionally desirable tend to be rapidly digested and have greater bioavailability of amino acids than stable proteins. In addition, proteins that are highly digestible would be expected to have less opportunity to exert adverse health effects when consumed. The assay was not meant to predict the fate of the protein of interest under in vivo conditions, but rather to evaluate the susceptibility of the protein to digestion under fixed conditions in vivo. The purpose is to provide information that, in conjunction with other evidence, would be useful in predicting whether a dietary protein may become a food allergen. Therefore, the relationship of the resistance to digestion by pepsin and the likelihood that a dietary protein is an allergen was identified as a means of aiding the assessment of proteins added to commodity crops through biotechnology. In this article, we discuss the predictive value of this assay and the practical and theoretical aspects of allergen resistance to pepsin digestion in the context of food safety.

Characterization and crystal structure of lysine insensitive Corynebacterium glutamicum dihydrodipicolinate synthase (cDHDPS) protein

The lysine insensitive Corynebacterium glutamicum dihydrodipicolinate synthase enzyme (cDHDPS) was recently successfully introduced into maize plants to enhance the level of lysine in the grain. To better understand lysine insensitivity of the cDHDPS, we expressed, purified, kinetically characterized the protein, and solved its X-ray crystal structure. The cDHDPS enzyme has a fold and overall structure that is highly similar to other DHDPS proteins. A noteworthy feature of the active site is the evidence that the catalytic lysine residue forms a Schiff base adduct with pyruvate. Analyses of the cDHDPS structure in the vicinity of the putative binding site for S-lysine revealed that the allosteric binding site in the Escherichia coli DHDPS protein does not exist in cDHDPS due to three non-conservative amino acids substitutions, and this is likely why cDHDPS is not feedback inhibited by lysine.

Bioinformatic Methods for Identifying Known or Potential Allergens in the Safety Assessment of Genetically Modified Crops

Agricultural crops have been genetically improved through centuries of breeding to select phenotypes that are controlled by combinations of genes, typically with undefined mutations, which produce the desired traits. Changes in the plant characteristics (e.g., disease resistance, insect resistance, food quality) typically have been slow, except when partial or full genomes have been combined. This combination as is hypothesized to have occurred thousands of years ago in the generation of modern hexaploid wheat as a hybrid cross of tetraploid and diploid progenitors (1). Such crosses result in the combination of hundreds to thousands of different proteins in a single food. In contrast, modern tools of biotechnology have allowed the introduction of one or a few new genes and resulting proteins into crop varieties that are carefully selected and studied for safety and performance before being allowed into commercial production (2, 3). As methods to introduce desired traits in plants have improved, so too has the public

Expression of a Truncated ATHB17 Protein in Maize Increases Ear Weight at Silking

ATHB17 (AT2G01430) is an Arabidopsis gene encoding a member of the α-subclass of the homeodomain leucine zipper class II (HD-Zip II) family of transcription factors. The ATHB17 monomer contains four domains common to all class II HD-Zip proteins: a putative repression domain adjacent to a homeodomain, leucine zipper, and carboxy terminal domain. However, it also possesses a unique N-terminus not present in other members of the family. In this study we demonstrate that the unique 73 amino acid N-terminus is involved in regulation of cellular localization of ATHB17. The ATHB17 protein is shown to function as a transcriptional repressor and an EAR-like motif is identified within the putative repression domain of ATHB17. Transformation of maize with an ATHB17 expression construct leads to the expression of ATHB17Δ113, a truncated protein lacking the first 113 amino acids which encodes a significant portion of the repression domain. Because ATHB17Δ113 lacks the repression domain, the protein cannot directly affect the transcription of its target genes. ATHB17Δ113 can homodimerize, form heterodimers with maize endogenous HD-Zip II proteins, and bind to target DNA sequences; thus, ATHB17Δ113 may interfere with HD-Zip II mediated transcriptional activity via a dominant negative mechanism. We provide evidence that maize HD-Zip II proteins function as transcriptional repressors and that ATHB17Δ113 relieves this HD-Zip II mediated transcriptional repression activity. Expression of ATHB17Δ113 in maize leads to increased ear size at silking and, therefore, may enhance sink potential. We hypothesize that this phenotype could be a result of modulation of endogenous HD-Zip II pathways in maize.

Application of HB17, an Arabidopsis class II homeodomain-leucine zipper transcription factor, to regulate chloroplast number and photosynthetic capacity

Transcription factors are proposed as suitable targets for the control of traits such as yield or food quality in plants. This study reports the results of a functional genomics research effort that identified ATHB17, a transcription factor from the homeodomain-leucine zipper class II family, as a novel target for the enhancement of photosynthetic capacity. It was shown that ATHB17 is expressed natively in the root quiescent centre (QC) from Arabidopsis embryos and seedlings. Analysis of the functional composition of genes differentially expressed in the QC from a knockout mutant (athb17-1) compared with its wild-type sibling revealed the over-representation of genes involved in auxin stimulus, embryo development, axis polarity specification, and plastid-related processes. While no other phenotypes were observed in athb17-1 plants, overexpression of ATHB17 produced a number of phenotypes in Arabidopsis including enhanced chlorophyll content. Image analysis of isolated mesophyll cells of 35S::ATHB17 lines revealed an increase in the number of chloroplasts per unit cell size, which is probably due to an increase in the number of proplastids per meristematic cell. Leaf physiological measurements provided evidence of improved photosynthetic capacity in 35S::ATHB17 lines on a per unit leaf area basis. Estimates of the capacity for ribulose-1,5-bisphosphate-saturated and -limited photosynthesis were significantly higher in 35S::ATHB17 lines.