In vitro digestion of soluble cashew proteins and characterization of surviving IgE-reactive peptides.

Methods: The stability of food allergens to digestion varies. We characterized the stability of cashew allergens to digestion by pepsin and trypsin and identified IgE-binding epitopes that survive digestion.

Results: The ability of pepsin and trypsin to digest cashew allergens was assessed with an in vitro digestion model. Samples were evaluated by SDS-PAGE, MS, ELISA, and immunoblotting to compare IgE binding. Increasing amount of protease resulted in greater degradation of higher molecular weight cashew proteins. Among cashew proteins, the 2S albumin, Ana o 3, was most resistant to digestion by both pepsin and trypsin. MS identified digestion resistant Ana o 3 protein fragments that retained reported IgE-binding epitopes. Pretreatment of extracts or purified Ana o 3 with reducing agent increased the sensitivity of Ana o 3 to protease digestion. Circular dichroism revealed the structure of purified Ana o 3 was largely alphahelical and was disrupted following reduction. Ana o 3 reduction followed by protease digestion decreased binding of serum IgE from cashew allergic patients. Our results indicate that the Ana o 3 disulfide bond dependent structure protects the protein from proteolysis.

Conclusions: Ana o 3 is the cashew allergen most likely to survive gastrointestinal digestion intact.