Preparation of the Hypoallergenic Enzymatic Hydrolyzate of Cra a 4 with the Potential to Induce Immune Tolerance

Sarcoplasmic calcium-binding protein identified in <i>Crassostrea angulata (Cra a 4)</i>, is a stable allergen; the methods to reduce its allergenicity are still limited. The present study aimed to develop the hypoallergenic enzymatic hydrolyzate of Cra a 4 (ECra a 4) by enzymolysis. After screening of proteases by bioinformatics and optimization of enzymolysis conditions, ECra a 4 was obtained by enzymolysis with Trypsin. Further, ECra a 4 was used to stimulate splenocytes from Cra a 4-sensitized mice, with findings that ECra a 4 could stimulate CD4⁺ T-cell proliferation, inhibit IL-4 secretion, and promote TGF-β secretion. Spectroscopy analysis revealed that enzymolysis destroyed the structure of Cra a 4, which affected its allergenicity. Peptidomic analysis revealed that enzymolysis destroyed the B-cell epitopes of Cra a 4 while retaining its T-cell epitopes. Overall, ECra a 4 with hypoallergenicity and T-cell stimulating ability can be used as an immune tolerance product for further study.