Structure-Allergenicity Relationship And Specific Allergy Prevention And Control Of Filamin C From Scylla Paramamosain

Scylla paramamosain is an important aquaculture resource in China.With the increase of production and consumption,the food allergy problem caused by crab has become increasingly prominent.The novel allergen filamin C in this crab belongs to the filamin protein family and has strong immunoglobulin（Ig）E-binding activity.The relationship between its allergenicity,structure,and antigenic epitopes needs to be further explored.Therefore,clarifying the allergenicity of filamin C,analyzing the structure of filamin C and locating antigen epitopes,and preparing specific mixed peptides and derivatives for preventing and controlling filamin C food allergies,clarify the mechanism of action of mixed peptides in preventing filamin C allergies and derivatives in alleviating filamin C allergies.Firstly,the serological analysis results showed that the positive rates of filamin C Ig E binding activity were 40.0%,43.7%,and 41.1%in 15 allergy patients,350 college students with self reported allergy,and 205 allergic children to crustacean aquatic products,respectively.filamin C from S.paramamosain was named Scy p 9 by the allergen system of world health organization/international union of immunological societies.The allergenicity evaluation results of the food allergy mouse model showed that Scy p 9 triggered severe allergic reactions in mice and significantly upregulated the levels of specific Ig E and Th2 cytokine release.In order to clarify the structure-allergenicity relationship,bioinformatics tools were used to simulate the tertiary structure of Scy p 9.Based on domain simulation,Scy p 9-N,Scy p 9-M,and Scy p 9-C were segmented and expressed.Among them,Scy p 9-M(AA_336-531)showed strong Ig E-binding activity and the ability to activate CD63 and CD203c levels on the surface of eosinophils in crab allergy patients,which is the allergenic predomain region of Scy p 9.The Scy p 9-M crystal structure with a resolution of 1.7（?）was obtained through X-ray diffraction.The protein data bank registration number was 7VZO.Secondly,antigenic epitopes were the molecular basis for determining allergenicity.Thirteen T cell epitopes of Scy p 9 were identified using CD4⁺T lymphocyte proliferation assay in spleen mononuclear cells of mice allergic to Scy p 9.The release level of Treg related cytokine TGF-βwas significantly upregulated due to 9 T cell epitopes.The key amino acids in the predicted B cell conformational epitope regions of Scy p 9 were mutated to alanine using bioinformatics tools and directed mutagenesis techniques.Four B cell conformational epitope regions of Scy p 9 were identified(C-1 containing key amino acid E₂₁₆,C-2 containing key amino acid T₂₇₀,C-3 containing key amino acid Y₆₉₉,and C-4 containing key amino acid V₇₀₄),with C-2(key amino acid T₂₇₀)exhibiting strong Ig E-binding activity.In addition,16 linear B cell epitopes of Scy p 9 were identified using synthetic peptide technology combined with serological analysis.Among them,L-AA_187-205 located in the Scy p 9-N region and L-AA_363-377located in the Scy p 9-M region showed strong Ig E-binding activity.By locating the antigen epitope on the tertiary structure of Scy p 9,it was found that the B cell epitope was located in the prominent region of the tertiary structure,which is conducive to the binding of Ig E.Finally,based on the identified antigenic epitopes,mixed peptides and derivatives were prepared to explore the mechanism of mixed peptides in preventing Scy p 9 allergic reactions and derivatives in alleviating Scy p 9 allergic reactions.Preparation of mixed peptides using 9 T cell epitope peptides,which significantly upregulated Treg related cytokines TGF-β.The serological analysis and basophil activation test results of crab allergy patients showed that the mixed T cell epitope peptide had no Ig E-binding activity and could not activate the expression of CD63 and CD203c on the surface of basophil.Non-allergenic mixed T cell epitope peptides（NAMTEP）demonstrate the potential for preventing Scy p 9 allergies.The evaluation results of the preventive effect of the Scy p 9 allergic mouse model showed that NAMTEP prevented Scy p9 allergic reactions in mice by inhibiting Ig E production and Th2 type immune response.Based on the identified key amino acids(T₂₇₀)and B cell linear epitopes(L-AA_187-205,L-AA_363-377)with strong Ig E-binding activity,a derivative named Scy p 9^NCCL were prepared by using molecular modification techniques to remove the N-/C-terminal regions,mutate key amino acids,and delete B cell linear epitopes of Scy p 9.Furthermore,comparing Ig E-binding activity,and ability to activate basophil in crab allergy patients,the Ig E-binding activity significantly decreased by96.31%±3.62%.The expression of CD63 and CD203c on the surface of basophil in crab allergy patients was activated to a lower extent,indicating that Scyp 9^NCCL was a hypoallergenic derivative.The food allergy mouse model evaluated the allergenicity of Scy p 9^NCCL.Scy p 9^NCCLproduced significantly lower levels of mouse specific Ig E and Th2 cytokine release,and the specific Ig G2a level and Treg cell differentiation degree were significantly higher than Scy p 9.Scy p 9^NCCL showed the potential to alleviate mouse Scy p 9 allergies.The evaluation of the effect of Scy p 9 allergic mouse control model showed that Scy p 9^NCCL alleviates mouse Scy p 9allergic reactions by regulating the balance of Th1/Th2 and producing Ig G blocking antibodies.In summary,this study confirmed the high positivity and strong sensitization of Ig E-binding activity of Scy p 9,and analyzed the crystal structure with a resolution of 1.7（?）in the allergic predomain region.Among the thirteen identified T cell epitopes of Scy p 9,nine T cell epitopes can upregulate Treg related cytokine TGF-βrelease.The mixed peptides of nine T cell epitopes showed no allergenicity,and the non-allergenic mixed T cell epitope peptides（NAMTEP）prevented Scy p 9 allergic reactions in mice by inhibiting Th2 type immune responses and Ig E production.B cell epitopes C-2(key amino acid T₂₇₀),L-AA_187-205 and L-AA_363-377 were identified with strong Ig E-binding activity in the four B cell conformational epitope regions and sixteen B cell linear epitopes of Scy p 9.Mutation T₂₇₀,deletion of L-AA_187-205 and L-AA_363-377of derivatives had low allergenicity,high stimulation of Treg cell differentiation,and hypoallergenic derivatives(Scy p 9^NCCL)alleviated Scy p 9 allergic reactions in mice by regulating the balance of Th1/Th2 and producing specific Ig G blocking antibodies.These results are expected to lay a theoretical foundation for the structure-allergenicity relationship analysis of allergens in crustacean aquatic products and specific allergy prevention and control.