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

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.Electronic supplementary materialThe online version of this article (10.1186/s13601-018-0216-9) contains supplementary material, which is available to authorized users.

Homologous tropomyosins from vertebrate and invertebrate: Recombinant calibrator proteins in functional biological assays for tropomyosin allergenicity assessment of novel animal foods

“…While shrimp Pen m 1 was detectable during the gastric phase, chicken homologues were degraded quickly during the first minutes of in vitro digest. This confirmed previous in silico and in vitro experiments showing high stability of allergenic versus low stability of non‐allergenic TM . Indeed, the relative high stability of Pen m 1 towards protease degradation, in comparison with the invertebrate homologues is a plausible explanation for immunogenicity and allergenicity of the shrimp protein .…”

“…Assay conditions were chosen mild (pH 3; 1 U pepsin/µg extract protein) in order to follow the progressive protein digestion. Although in our study resistance to pepsinolysis separated the allergen from the non‐allergen, it is important to say that a straightforward correlation between protease stability and allergenicity is not always given, depending on the protein analysed and the assay conditions applied . Immunodetection of TM was performed using two different anti‐TM antibodies (recognizing possibly different TM epitopes).…”

“…Simulated digestion was performed as reported previously, with further modifications as published for successive gastric and intestinal digests according to the international consensus paper published by Minekus et al, in terms of adjusted pH and applied incubation times. Briefly, protein extracts were incubated with pepsin (Sigma‐Aldrich) at a final ratio of 1 U enzyme/µg protein extract in simulated gastric fluid, pH 3 for 2 hours.…”

Food and drug allergy, and anaphylaxis in EAACI journals (2018)

“…Protease resistance of food proteins represents a mixed picture for predicting allergenicity but a useful tool for assessing exposure. 23 Susceptibility to pepsin digestion of candidate transgene products is regarded as an important parameter in the evidence approach for allergenicity risk assessment of genetically modified crops. It has been argued that protocols used for this assessment should better reflect physiologic conditions encountered in representative food consumption scenarios.…”

Accumulation of storage proteins in plant seeds is mediated by amyloid formation

“…Another property of lectins, in particular, Vicilins, is their allergenicity. Vicilin is one of the major plant-derived allergens, and its allergenic properties were found to be associated with its proteaseresistant fragments 56,57 . Our finding that Vicilin forms amyloids in vivo explains its protease resistance ( Figure 5) and suggests that these amyloids may represent major source of food allergy according to the data that in vitro generated lectin amyloids are phagocytized by macrophages and elicit an immune response 55 .…”