Supplementary data on the characterization and safety evaluation of HPPD W336, a modified 4-hydroxyphenylpyruvate dioxygenase protein, which confers herbicide tolerance, and on the compositional assessment of field grown MST-FGØ72-2 soybean expressing HPPD W336.

Supplementary data are provided which are supportive to the research article entitled "Characterization and safety evaluation of HPPD W336, a modified 4-hydroxyphenylpyruvate dioxygenase protein, and the impact of its expression on plant metabolism in herbicide-tolerant MST-FGØ72-2 soybean" (Dreesen et al., 2018) [1]. The conducted supplementary analyses include the characterization of additional Escherichia coli-produced HPPD W336 protein batches used as a surrogate in HPPD W336 safety studies, the assessment of potential glycosylation and monitoring of stability in simulated intestinal fluid and during heating of the HPPD W336 protein. Furthermore, data are provided on conducted field trials and subsequent compositional analysis in MST-FGØ72-2 soybean grain of compounds related to the tyrosine degradation pathway and the metabolism of homogentisate.

Protease resistance of food proteins: a mixed picture for predicting allergenicity but a useful tool for assessing exposure.

BACKGROUND: Susceptibility to pepsin digestion of candidate transgene products is regarded an important parameter in the weight-of-evidence approach for allergenicity risk assessment of genetically modified crops. It has been argued that protocols used for this assessment should better reflect physiological conditions encountered in representative food consumption scenarios. AIM: To evaluate whether inclusion of more physiological conditions, such as sub-optimal and lower pepsin concentrations, in combination with pancreatin digestion, improved the performance of digestibility protocols used in characterization of protein stability. METHODS: Four pairs of established allergens and their related non/weakly-allergenic counterparts (seed albumins, muscle tropomyosins, plant lipid transfer proteins [LTP] and collagens) plus fish parvalbumin, were subjected to nine combinations of pH (1.2-2.5-4.0) and pepsin-to-protein ratio (PPR: 10-1-0.1 U/µg) for pepsin digestion, followed by pancreatin digestion in the presence of bile salts. Digestion was monitored by SDS-PAGE in conjunction with Coomassie staining and immunoblotting using rabbit antisera and human IgE. RESULTS: At pH 4.0 and at PPR 0.1 most proteins, both allergen and non-allergen, were highly resistant to pepsin. Under conditions known to favor pepsin proteolysis, the established major allergens Ara h 2, Pru p 3 and Pen a 1 were highly resistant to proteolysis, while the allergen Cyp c 1 was not. However, this resistance to pepsin digestion only made Ara h 2 and to a lesser extent Pen a 1 and Pru p 3 stand out compared to their non-allergenic counterparts. Largely irrespective of preceding pepsin digestion conditions, pancreatin digestion was very effective for all tested proteins, allergens and non-allergens, except for Cyp c 1 and bovine collagen. CONCLUSIONS: Sub-optimal pH, low pepsin-to protein ratio, and sequential pepsin and pancreatin digestion protocols do not improve the predictive value in distinguish allergens from non-allergens. Digestion conditions facilitating such distinction differ per protein pair.

Characterization and safety evaluation of HPPD W336, a modified 4-hydroxyphenylpyruvate dioxygenase protein, and the impact of its expression on plant metabolism in herbicide-tolerant MST-FGØ72-2 soybean.

By transgenic expression technology, a modified 4-hydroxyphenylpyruvate dioxygenase enzyme (HPPD W336) originating from Pseudomonas fluorescens is expressed in MST-FGØ72-2 soybean to confer tolerance to 4-benzoyl isoxazole and triketone type of herbicides. Characterization and safety assessment of HPPD W336 were performed. No relevant sequence homologies were found with known allergens or toxins. Although sequence identity to known toxins showed identity to HPPD proteins annotated as hemolysins, the absence of hemolytic activity of HPPD W336 was demonstrated in vitro. HPPD W336 degrades rapidly in simulated gastric fluid. The absence of toxicity and hemolytic potential of HPPD W336 was confirmed by in vivo studies. The substrate spectrum of HPPD W336 was compared with wild type HPPD proteins, demonstrating that its expression is unlikely to induce any metabolic shifts in soybean. The potential effect of expression of HPPD W336 on metabolic pathways related to tyrosine was investigated by comparing seed composition of MST-FGØ72-2 soybean with non-genetically modified varieties, demonstrating that expression of HPPD W336 does not change aromatic amino acid, homogentisate and tocochromanol levels. In conclusion, HPPD W336 was demonstrated to be as safe as other food proteins. No adverse metabolic effects were identified related to HPPD W336 expression in MST-FGØ72-2 soybean.

Technical variability of 2D gel electrophoresis - Application to soybean allergens.

Two-dimensional gel electrophoresis (2-DE) technique is used as a performing technique to assess the variability of protein expression in crops, and especially soybean endogenous food allergens, which are a subset of proteins of interest for assessing whether genetically modified (GM) soybean has a different allergenic profile compared to its non-GM counterpart. On top of the biological variability of the 2-DE, which has already been studied by several laboratories, technical variability has to be evaluated. In this study, several sources of variability (number of gel replicates, protein extracts, study timings and operators) were assessed qualitatively and quantitatively on all detectable polypeptide spots as well as on food allergen spots. Results showed that the major source of variability was the number of gel replicates. Other sources were minor. This has a direct practical impact on the laboratory work as this supports the utilization of three or four gel replicates to get robust results. Furthermore, this implies that the study can be run over several days, and be performed by several trained operators, without impacting its reproducibility. Furthermore, 2-DE could detect a 2-fold change between two samples with an acceptable rate of false positives (below 7%). This level of sensitivity is acceptable in the context of safety assessment of GM soybean as the biological variability of proteins in soybean is higher than the technical variability shown in this study. Overall, the 2-DE technique is suitable for investigating endogenous food allergen variability between several soybean seeds, including GM and non-GM counterpart.

Investigation of endogenous soybean food allergens by using a 2-dimensional gel electrophoresis approach.

As part of the safety assessment of genetically modified (GM) soybean, 2-dimensional gel electrophoresis analyses were performed with the isoxaflutole and glyphosate tolerant soybean FG72, its non-GM near-isogenic counterpart (Jack) and three commercial non-GM soybean lines. The objective was to compare the known endogenous human food allergens in seeds in the five different soybean lines in order to evaluate any potential unintended effect(s) of the genetic modification. In total, 37 protein spots representing five well known soybean food allergen groups were quantified in each genotype. Qualitatively, all the allergenic proteins were detected in the different genetic backgrounds. Quantitatively, among 37 protein spots, the levels of accumulation of three allergens were slightly lower in the GM soybean than in the non-GM counterparts. Specifically, while the levels of two of these three allergens fell within the normal range of variation observed in the four non-GM varieties, the level of the third allergen was slightly below the normal range. Overall, there was no significant increase in the level of allergens in FG72 soybean seeds. Therefore, the FG72 soybean can be considered as safe as its non-GM counterpart with regards to endogenous allergenicity. Additional research is needed to evaluate the biological variability in the levels of endogenous soybean allergens and the correlation between level of allergens and allergenic potential in order to improve the interpretation of these data in the safety assessment of GM soybean context.

Sustained phenotypic reversion of junctional epidermolysis bullosa dog keratinocytes: Establishment of an immunocompetent animal model for cutaneous gene therapy.

Gene transfer represents the unique therapeutic issue for a number of inherited skin disorders including junctional epidermolysis bullosa (JEB), an untreatable genodermatose caused by mutations in the adhesion ligand laminin 5 (alpha3beta3gamma2) that is secreted in the extracellular matrix by the epidermal basal keratinocytes. Because gene therapy protocols require validation in animal models, we have phenotypically reverted by oncoretroviral transfer of the curative gene the keratinocytes isolated from dogs with a spontaneous form of JEB associated with a genetic mutation in the alpha3 chain of laminin 5. We show that the transduced dog JEB keratinocytes: (1) display a sustained secretion of laminin 5 in the extracellular matrix; (2) recover the adhesion, proliferation, and clonogenic capacity of wild-type keratinocytes; (3) generate fully differentiated stratified epithelia that after grafting on immunocompromised mice produce phenotypically normal skin and sustain permanent expression of the transgene. We validate an animal model that appears particularly suitable to demonstrate feasibility, efficacy, and safety of genetic therapeutic strategies for cutaneous disorders before undertaking human clinical trials.

Inherited junctional epidermolysis bullosa in the German Pointer: establishment of a large animal model.

Junctional epidermolysis bullosa (JEB) is a genodermatosis suitable for gene therapy because conventional treatments are ineffective. Here, we elucidate the genetic basis of mild JEB in a breed of dogs that display all the clinical traits observed in JEB patients. The condition is associated with reduced expression of laminin 5 caused by a homozygous insertion (4818+207ins6.5 kb) of repetitive satellite DNA within intron 35 of the gene (lama3) for the laminin alpha3 chain. The intronic mutation interferes with maturation of the alpha3 pre-messenger RNA resulting in the coexpression of a transcript with a 227 nucleotide insertion and a wild-type mRNA that encodes scant amounts of the alpha3 polypeptide. Our results show that the amino acid sequence and structure of the canine and human alpha3 chain are highly conserved and that the reduced expression of laminin 5 affects the adhesion and clonogenic potential of the JEB keratinocytes. These JEB dogs provide the opportunity to perform gene delivery in a naturally occurring genodermatosis and to evaluate host tolerance to recombinant laminin 5.

Cloning of laminin gamma2 cDNA and chromosome mapping of the genes for the dog adhesion ligand laminin 5.

Overexpression of the gamma2 chain of laminin-5 has been linked to tumor invasion and an unfavorable prognostic value, but the role of this adhesion molecule in cancer progression remains unclear. Because dog models of human cancers provide the opportunity of clarifying the relation between laminin-5 and tumor malignancy we have isolated and characterized the cDNA of dog gamma2 chain. Comparative analysis of the nucleotide sequence revealed high identity between the dog and the human gamma2, including the intermolecular molecule binding sites and the regulatory promoter sequences. Moreover, expression of a recombinant human gamma2 chain in dog keratinocytes results in assembly and secretion of hybrid laminin-5 molecules, which underscore the functional relevance of the gamma2 conserved domains. We have also determined the syntenic location of the dog laminin-5 loci on CFA7. Our study provides a basis for therapeutical approaches of epithelial cancers of gamma2 using dogs as large animal models.