Role of Glycosylation in Immunoreactivity of Major Cashew (Anacardium occidentale L.) Allergen, Ana O 1

Cashews are the number one produced tree nut worldwide and the U.S. is the top importer. Despite their popularity and economic importance, cashews are frequently implicated in IgE-mediated food allergy. Food allergy is an increasingly recognized global health issue and currently there is no available cure. The U.S. is the country with the most reported cashew allergies and cashews rank second among individual tree nuts in terms of U.S. prevalence. Three allergenic proteins in cashew have been identified: Ana o 1, Ana o 2, and Ana o 3. Ana o 1 was recently reported to be a glycoprotein, however the role of glycosylation in allergenic behavior of the protein has yet to be investigated and was the focus of this dissertation. Native Ana o 1 was purified from cashew seeds using a combination of ammonium sulfate precipitation, ultrafiltration, and fast protein liquid chromatography. Ammonium sulfate and ultrafiltration were used as crude purification and preconcentration steps prior to loading the sample on the columns. Two different chromatographic separation methods were tested separately- affinity and size exclusion. Ana o 1 eluted as a single peak off both types of columns, however higher recovery was achieved using size exclusion. The binding of Ana o 1 to a Con A affinity column as well as glycoprotein staining, confirmed glycosylation. PNGase F was used to cleave N-glycans from purified Ana o 1. Deglycosylation was confirmed using SDS-PAGE with CBBR and glycoprotein staining. Western and dot blot immunoassays were used to compare immunoreactivity between deglycosylated and glycosylated Ana o 1 using pooled plasma from five cashew-allergic individuals. Immunoassays indicated that deglycosylation increased IgE-recognition of Ana o 1, suggesting that removal of N-glycans may have exposed previously masked Ana o 1 epitopes. Further, immunoreactivity of purified Ana o 1 after exposure to in vitro pepsin digestion was assessed using both the glycosylated and deglycosylated protein. Purified proteins were incubated with a fixed amount of pepsin at 37 C for 120 min at pH 3.0 with constant shaking to simulate gastric digestion. SDS-PAGE with CBBR staining was used to visualize proteolysis and Western blotting with pooled human plasma from five cashew-allergic individuals was used to assess subsequent immunoreactivity. Deglycosylation appeared to slightly increase susceptibility of Ana o 1 to proteolysis, likely by increasing exposure of pepsin cleavage sites on the protein. Despite this observation, both glycosylated and deglycosylated Ana o 1 were detected by human IgE following 120 min of digestion under the tested conditions. Overall, this report suggests that glycosylation may play a role in allergenicity of Ana o 1.