Insight Into Epitope-Directed Mutations Affecting The Immunoreactivity And Structure Of Arginine Kinase In Oratosquilla Oratoria

Food allergy is a type Ⅰ hypersensitivity reaction mediated by Immunoglobulin E(IgE),that usually associated with a number of negative side effects.The majority of allergic individuals in China are allergic to crustaceans,particularly shrimp and crab.In this study,the important allergen in Oratosquilla oratoria was identified and cloned,and the immune-binding activity and antigenic epitopes were analyzed,and the hypo-immunoreactivity allergens were obtained through epitope-directed mutations,which laid the foundation for food allergy immunotherapy.In this study,a 40 kDa component was purified from the muscles of O.oratoria and identified as arginine kinase(AK)by mass spectrometry,which was further confirmed as an allergen using the sera from O.oratoria sensitive individuals,and the rabbit anti-O.oratoria AK polyclonal antibody was prepared.The open reading frame of AK was obtained by cloning,which containing 1071 bp and encoding 365 amino acid residues.The recombinant AK(rAK)showed similar immune-binding activity to native AK(nAK)was obtained using Escherichia coli expression system and was demonstrated to have similar secondary structure,p H stability,and stability against digestion in gastric and intestinal fluid.Eight predicted linear epitopes and eight conformational mimotopes of AK were predicted by bioinformatics.The linear epitopes of AK were identified by synthetic peptide technology combined with serological experiments,among which AA 66～77,AA 87～106,AA 130～142,AA 176～187,and AA 193～205 could significantly inhibit the binding of rAK to IgE.The conformational epitopes of AK were identified by site-directed mutation,and the IgE-binding activity of mutant D-F168/L170,D-F186,and D-A197 were significantly decreased.Thus,a total of five IgE linear epitopes and three IgE conformational epitopes with their key amino acid of AK were obtained.A total of one linear epitope mutant(mAK-L)and three conformational epitope mutants(D-F168/L170,D-F186,D-A197)were prepared by epitope-directional alteration based on the previous investigation.Serological experiments revealed that the IgE-binding activity of all four mutants were significantly decreased,and mAK-L being the most affected with average decline rate of 80.6% ± 10.9%.The structures of mutants were further analyzed by circular dichroism,surface hydrophobicity,and tertiary structure simulation,which revealed that the structures of four mutants were greatly altered,among which the structure change of mAK-L was the most obvious,which suggested that the superimposed effect of linear epitopes deletion and structural disruption was responsible for the significant decrease of IgE-binding activity of mAK-L.In conclusion,this study identified the important allergenAK in Oratosquilla oratoria,cloned its open reading frame,and expressed rAK with similar properties to nAK,which providing a theoretical basis for the diagnosis of crustacean allergic components.Five linear epitopes and three conformational epitopes of AK were identified,and four hypo-immunoreactivity mutants were obtained by epitope-directed mutations through genetic engineering technology,among which mAK-L showed the most significant reduction in immunoreactivity,these results laying the foundation for food allergy immunotherapy.