Digestion and Transport across the Intestinal Epithelium Affects the Allergenicity of Ara h 1 and 3 but Not of Ara h 2 and 6 (1)
In a healthy individual, proteins are mainly transported by means of endocytosis via the enterocytes and microfold (M) cells. Transport via the M cells leaves a larger part of the proteins intact, while transport via enterocytes exposes 90% of the internalized protein to intracellular degradation in lysosomes. After intracellular degradation, the resulting fragments can still be allergenic if their size is at least 3.5 kDa.
Approximately 0.1–0.4% of the protein dose applied on the apical side was transported without significant differences between the mean amount of transported radioactively labeled Ara h 1, Ara h 2, and 𝛽-lactoglobulin or fragments thereof
All peanut allergens retrieved from the apical compartment were able to activate the basophils as shown by the increased CD63 expression. Ara h 1, 2, 3, and 6, and 𝛽-lactoglobulin were transported over the intestinal epithelium of two pigs and an in-BAT was performed with the basolateral samples. Ara h 1 and 3 were not able to activate basophils after epithelial transport.
The typical major bands of Ara h 1 (64 kDa) and Ara h 3 (45, 42, 23 kDa) were rapidly digested within 15 s. Ara h 2 shows two protein bands on the gel belonging to the two Ara h 2 isomers. The protein digestion pattern of Ara h 6 (15 kDa) showed similarities with that of Ara h 2.
Reactivity of all Ara h 1, 2, 3, and 6 digests was confirmed using the sera of patient 1, 2, and 4 in an in-BAT.
When Ara h 1 and 3 are digested for a longer time period prior to epithelial transport, a higher percentage 𝛽-hexosaminidase release from mast cells sensitized with serum from peanut-allergic patients was found. The digestion time does not significantly influence activation by Ara h 2 and 6.
Intracellular protein degradation was found to be higher for Ara h 1 and 3 than for Ara h 2 and 6. Additionally, certain fragments of Ara h 1 were also cleaved by endolysosomal proteases. Small proteins with a maximal radius of 15 Å (±3.5 kDa) can cross the barrier via paracelular transport. Pepsin digestion of Ara h 1 resulted in fragments smaller than 5 kDa. Proteins which are transported via the paracellular route are not exposed to lysosomes and therefore not degraded further. After digestion of Ara h 1 at pH 2 (stomach), the resulting fragments formed aggregates when transferred to a basic environment (intestine).