Effects Of Site-directed Mutagenesis On The Structure And Allergenicity Of Sarcoplasmic Calcium-binding Protein In Scylla Paramamosain

Crustaceans are one of the eight categories of allergic foods,which caused food allergy becoming more common.Sarcoplasmic calcium-binding protein(SCP)is a novel allergen in crustaceans.In this research,we studied the epitopes and calcium-binding regions of SCP in Scylla paramamosain.The linear epitopes were verified by synthetic peptide technique and serological assay.The key amino acids in the conformational epitope and calcium binding region of SCP were mutated by site-directed mutagenesis.The allergenicity,physicochemical properties,secondary structure,surface hydrophobicity and calcium binding ability of the mutated derivatives were analyzed,and the low hypoallergenic SCP derivatives were obtained.In this research,six linear epitopes of SCP were predicted and synthesized by five bioinformatics softwares,and verified by serological assay.It was found that the peptide AA_76-91,AA_111-125 and AA_137-146 were important linear epitope of SCP.The Ig E binding activity of SCP was largely lost with the addition of chemical denaturing agents,indicating that the spatial conformation of SCP is an important guarantee of its Ig E binding activity.Six derivatives(T38A,E41A,S48A,N53A,C90A,P100A)were obtained by mutating conformational epitopes.Three derivatives,D18A/D20A,D70A,and D18A/D20A/D70A,were obtained by mutating the calcium binding region I or/and the calcium binding region II.Compared with SCP,the immunobinding activities of S48A(p<0.01)and P100A(p<0.05)in conformational epitope mutation derivatives were significantly decreased,and the immunobinding activities of D70A and D18A/D20A/D70A were significantly decreased(p<0.01).D70A and D18A/D20A/D70A significantly down-regulated the expression of CD63 and CD203c on the surface of basophils(p<0.05).In addition,D70A and D18A/D20A/D70A were unstable to heat and acid,resistant to gastric but not intestinal digestion,decreased?-helix content,and increased surface hydrophobicity.This suggests that mutations in the calcium binding region have a greater influence on SCP allergenicity than mutations in conformational epitopes.In order to analyze the relationship between sensitization and calcium binding ability of D70A and D18A/D20A/D70A,the tertiary structure of SCP was further simulated.The results showed that the calcium binding region II could binding Ca²⁺,and the mutation of Asp₂₀ made the surface of the calcium binding region I dominated by positive charge.In addition,the mutation breaks the hydrogen bonds with Asn₂₂ and Gly₂₃,while the mutation of Asp₇₀ reduces the negative charge on the surface of Ca²⁺binding region II and breaks the hydrogen bonds with Asp₇₄ and Gly₇₅.Circular dichroism and 8-anilino-1-naphthalenesulfonate fluorescence probe analysis showed that the secondary structure and surface hydrophobicity of D70A and D18A/D20A/D70A did not change significantly after adding calcium chelating agent,which further proved the loss of calcium binding ability of D70A and D18A/D20A/D70A.In conclusion,three linear epitopes of SCP,AA_76-91,AA_111-125 and AA_137-146,were found in this study,and the spatial conformational change affected the immunobinding activity of SCP.By site-directed mutagenesis of the conformational epitope and the calcium-ion binding region,it was found that the mutation of the calcium-ion binding site significantly reduced the sensitization of SCP than the mutation of the key amino acids in the conformational epitope.In conclusion,this research found that AA_76-91,AA_111-125 and AA_137-146 were the linear epitope of SCP,and the spatial conformation of SCP affects its immunobinding activity.By site-directed mutation analysis of the conformational epitope and the calcium binding region,it was found that the alteration of the calcium binding ability was more effective in reducing the allergenicity of SCP than the mutation of the key amino acids in the conformational epitope.Based on this,the hypoallergenic SCP derivatives are expected to be used in the immunotherapy of shellfish allergic diseases.