BackgroundSusceptibility 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.AimTo 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.MethodsFour 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.ResultsAt 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.ConclusionsSub-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.Electronic supplementary materialThe online version of this article (10.1186/s13601-018-0216-9) contains supplementary material, which is available to authorized users.
The susceptibility of a dietary protein to proteolytic degradation by digestive enzymes, such as gastric pepsin, provides information on the likelihood of systemic exposure to a structurally intact and biologically active macromolecule, thus informing on the safety of proteins for human and animal consumption. Therefore, the purpose of standardized in vitro degradation studies that are performed during protein safety assessments is to distinguish whether proteins of interest are susceptible or resistant to pepsin degradation via a study design that enables study-to-study comparison. Attempting to assess pepsin degradation under a wide-range of possible physiological conditions poses a problem because of the lack of robust and consistent data collected under a large-range of sub-optimal conditions, which undermines the needs to harmonize in vitro degradation conditions. This report systematically compares the effects of pH, incubation time, and pepsin-to-substrate protein ratio on the relative degradation of five dietary proteins: three pepsin susceptible proteins [ribulose 1,5-bisphosphate carboxylase-oxygenase (Rubisco), horseradish peroxidase (HRP), hemoglobin (Hb)], and two pepsin resistant proteins [lipid transfer protein (LTP) and soybean trypsin inhibitor (STI)]. The results indicate that proteins susceptible to pepsin degradation are readily distinguishable from pepsin-resistant proteins when the reaction conditions are within the well-characterized optima for pepsin. The current standardized in vitro pepsin resistant assay with low pH and high pepsin-to-substrate ratio fits this purpose. Using non-optimal pH and/or pepsin-to-substrate protein ratios resulted in susceptible proteins no longer being reliably degraded by this stomach enzyme, which compromises the ability of this in vitro assay to distinguish between resistant and susceptible proteins and, therefore, no longer providing useful data to an overall weight-of-evidence approach to assessing safety of proteins.
Western corn rootworm (WCR), Diabrotica virgifera virgifera, LeConte, is an insect pest that poses a significant threat to the productivity of modern agriculture, causing significant economic and crop losses. The development of genetically modified (GM) crops expressing one or more proteins that confer tolerance to specific insect pests, such as WCR, was a historic breakthrough in agricultural biotechnology and continues to serve as an invaluable tool in pest management. Despite this, evolving resistance to existing insect control proteins expressed in current generation GM crops requires continued identification of new proteins with distinct modes of action while retaining targeted insecticidal efficacy. GM crops expressing insecticidal proteins must undergo extensive safety assessments prior to commercialization to ensure that they pose no increased risk to the health of humans or other animals relative to their non-GM conventional counterparts. As part of these safety evaluations, a weight of evidence approach is utilized to assess the safety of the expressed insecticidal proteins to evaluate any potential risk in the context of dietary exposure. This study describes the food and feed safety assessment of Vpb4Da2, a new Bacillus thuringiensis insecticidal protein that confers in planta tolerance to WCR. Vpb4Da2 exhibits structural and functional similarities to other insect control proteins expressed in commercialized GM crops. In addition, the lack of homology to known toxins or allergens, a lack of acute toxicity in mice, inactivation by conditions commonly experienced in the human gut or during cooking/food processing, and the extremely low expected dietary exposure to Vpb4Da2 provide a substantial weight of evidence to demonstrate that the Vpb4Da2 protein poses no indication of a risk to the health of humans or other animals.
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