Mutation of Disulfide Bond Sites Reduces the Immunoreactivity of Cra a 4 by Changing the Structural Characteristics

Sarcoplasmic calcium-binding protein (Cra a 4) from Crassostrea angulata belongs to the EF-hand superfamily, and understanding of its structure-allergenicity relationship is still insufficient. In this study, chemical denaturants were used to destroy the structure of Cra a 4, showing that disruption of the structure reduced its IgG-/IgE-binding activity. To explore which critical amino acid site affects the allergenicity of Cra a 4, the mutants were obtained by site-directed mutations in the disulfide bonds site (C₉₇), conformational epitopes (I₁₀₅, D₁₁₄), or Ca²⁺-binding region (D₁₀₆, D₁₁₀) and their IgG-/IgE-binding activity was reduced significantly using serological tests. Notably, C₉₇A had the lowest immunoreactivity. In addition, two conformational epitopes of Cra 4 were verified. Meanwhile, the increase of the α-helical content, surface hydrophobicity, and surface electrostatic potential of C₉₇A affected its allergenicity. Overall, the understanding of the structure-allergenicity relationship of Cra a 4 allowed the development of a hypoallergenic mutant.