Protease resistance of food proteins: a mixed picture for predicting allergenicity but a useful tool for assessing exposure
Posted on 2018-08-10 - 05:00
Abstract 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.
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Akkerdaas, Jaap; Totis, Muriel; Barnett, Brian; Bell, Erin; Davis, Tom; Edrington, Thomas; et al. (2018). Protease resistance of food proteins: a mixed picture for predicting allergenicity but a useful tool for assessing exposure. figshare. Collection. https://doi.org/10.6084/m9.figshare.c.4194440.v1
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AUTHORS (21)
JA
Jaap Akkerdaas
MT
Muriel Totis
BB
Brian Barnett
EB
Erin Bell
TD
Tom Davis
TE
Thomas Edrington
KG
Kevin Glenn
GG
Gerson Graser
RH
Rod Herman
AK
Andre Knulst
GL
Gregory Ladics
SM
Scott McClain
LP
Lars Poulsen
RR
Rakesh Ranjan
JR
Jean-Baptiste Rascle
HS
Hector Serrano
DS
Dave Speijer
RW
Rong Wang
LP
Lucilia Pereira Mouriès
AC
Annabelle Capt
KEYWORDS
pancreatin digestion protocolscandidate transgene productsPru p 3pepsin-to protein ratioAra h 2favor pepsin proteolysisallergenicity risk assessmentrepresentative food consumption scenariosexposure Abstract Background Susceptibilityplant lipid transfer proteinsLTPpepsin digestion conditionsConclusions Sub-optimal pHpancreatin digestionallergen Cyp c 1non-allergenpepsin digestionPPRSDS-PAGECyp c 1