Study On The Crystal Structure And Epitope Mutation Of Triosephosphate Isomerase From Mud Crab(Scylla Paramamosain)

Food allergy is one of the most important food safety issues in the world,and the prevalence of food allergy is showing an increasing trend.As one of the eight major sources of allergic foods,shellfish have important economic and nutritional values.However,food allergy caused by shellfish should not be ignored.Scylla paramamosain is an important fishery resource in the southeast coastal areas of China,and the cases of food allergy caused by it are also increasing,so it is urgent to study the allergens and mechanism of S.paramamosain.In this paper,the relationship among crystal structure,cross-reactivity and epitopes of triosephosphate isomerase(TIM)in S.paramamosain was analyzed,and the allergenicity of TIM was reduced by site-directed mutagenesis.In the present study,the recombinant TIM was expressed and purified.The crystal of TIM was obtained by hanging drop method,and the selected conditions were further optimized.The crystals were obtained at the reservoir consisting of 22%(w/v)PEG 6000,0.1 M citric acid(pH 4.6).The best crystals diffracted to 1.8 ? by X-ray,and the three-dimensional structure of TIM is primarily composed of a(?/?)8-barrel motif prototype.A comparison of TIM from S.paramamosain to TIM of other species showed a high degree of homology in sequence and second structure,variable regions found in the ?-helices and the ?-strands were mainly conserved in shellfish TIMs.The structural alignment between the TIM from S.paramamosain and Litopenaeus vannamei are largely superimposable,with differences in ?-helices and loop regions.To analyze the cross-reactivity of TIMs from different species,an immunologic assay was performed using a rabbit anti-crab TIM IgG pAb,all shellfish TIMs exhibited strong reactivity to rabbit anti-crab TIM IgG pAb by Western blot.At the same time,the reported IgE epitopes of TIM were mapped in the tertiary structure,while most of these epitopes were distributed on the protein surface,six linear epitopes were distributed in ?-helices and conserved between shellfish species,the conformational epitope of the key amino acids was conserved and primarily distributed in loop region.In addition,the mutants of key amino acids in conformational epitopes,including W168 A,T172A,Q180 A,and K237 A were generated in TIM.The CD spectra of wtTIM and mutant TIMs were equivalent,indicating a similar overall folding of them.Both of them tend to produced dimers under the conditions of high temperature(60 °C ~100 °C),strong acid(pH 1.0~5.0)and strong alkali(pH 10.0~11.0).The mutant TIMs is more heat-resistant than wtTIM,W168 A and K237 A are more sensitive to acid and base.The IgE binding activity showed that compared with wtTIM,the IgE binding activity of mutant TIMs W168 A and K237 A significantly decreased by about 30%(p<0.05).Basophil activation assay was used for further verification,and it was found that the expression levels of CD63 and CD203 c on the cell surface stimulated by W168 A and K237 A decreased,but the decreased levels were different in patients.In summary,the analysis of crystal structure TIM was completed,the structure comparison and epitope analysis among species were carried out,laying a molecular theoretical foundation for cross-reactivity among species.At the same time,mutated TIM proteins W168 A and K237 A with low IgE binding activity were obtained by molecular modification,which provided theoretical basis for molecular diagnosis and immunotherapy of crab allergic reaction diseases.