Abstract
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.
Highlights
Susceptibility to pepsin digestion of candidate transgene products is regarded an important param‐ eter in the weight-of-evidence approach for allergenicity risk assessment of genetically modified crops
Lipid transfer proteins Lipid transfer proteins (LTP) from peach and strawberry were obtained as purified recombinant proteins, Pru p 3 and Fra a 3, respectively, with the former being an established major allergen and the latter a weak allergen [22, 23]
This loss of antibody binding appeared to be greater for Fra a 3, it cannot be fully excluded that this is explained by lower sensitivity of the rabbit antiserum for Fra a 3 compared to Pru p 3
Summary
Susceptibility to pepsin digestion of candidate transgene products is regarded an important param‐ eter in the weight-of-evidence approach for allergenicity risk assessment of genetically modified crops. The idea behind resistance to digestion as one of determinants of potential allergenicity is quite intuitive, since for readily digested proteins only small amount of the ingested protein molecule would reach the gut immune system in a sufficiently intact state and stimulate it to produce IgE antibodies or to trigger effector cells to induce allergic symptoms beyond the oral/esophageal tract In this context, it is important to realize that sensitization to food allergens may occur via different routes than the gut such as the skin or the respiratory tract [16, 17], where resistance to pepsin or pancreatin is of no relevance. Resistance to digestion is a parameter to consider in a weight-of-evidence approach to assess potential allergenicity risks
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