Study On The Potential Sensitization Of Peanut Allergen Ara H 2 By Structure-Oriented Modification

Peanut allergy is one of the most serious food allergic reactions due to its persistence and life-threatening nature.Peanut is widely used as a food ingredient,and it is difficult to completely avoid the intake of peanut in daily diet.The research on reducing peanut allergen sensitization has received more and more attention.Ara h2 is one of the major peanut allergens and can be recognized by the sera of over 90%of patients with peanut allergy.Its four pairs of disulfide bonds,three linear epitopes of the major allergens,and the key amino acids in them have been mapped.The binding ability of allergen protein to Ig E is determined by its structure,and the study of the effect of structural changes on the potential sensitization of Ara h 2 may provide directions for the exploration of processing methods to reduce the sensitization of Ara h 2.In this study,five Ara h 2 mutants were designed to investigate the effects of disulfide bonds and major linear allergen epitopes on the sensitization of peanut proteins.By genetic engineering technology to obtain recombinant protein,using molecular dynamics simulation to obtain mutant protein before and after the advanced structure,and comparing with structural characterization of experimental data validation,using in vitro assessment method for evaluating the allergenic potential allergenic protein changes after mutation,revealing the structure of directional transformation of peanut allergens Ara h 2 allergenic potential impact.The main methods,results and conclusions of the study are as follows:1.According to the results of Ara h 2 mutant peptide polypeptide array,two groups of key amino acid mutants with low Ig E binding ability were selected to destroy linear epitopes:Q34Y/E38Y breaks linear epitope 1(E²⁶LQGDRRCQSQLER³⁹)and Y65D/Y72D breaks linear epitope 2(R⁶²DPYSPSQDPYSPS⁷⁵).According to the spatial positions of the four disulfide bonds of Ara h 2,cysteine pairs connected to the same?-helix were divided into a group:C104G/C152G/C45G/C103G breaks two pairs of disulfide bonds connected to?-helix 3(connected to?-helix 2 and?-helix 5 respectively),C118G/C160G/C33G/C116G breaks two pairs of disulfide bonds connected to?-helix 4(connected to?-helix 1 and random crimps respectively),Where C³³ is located in epitope 1.Five mutants were designed:Mutant 1 mutated with all two groups of cysteines;Mutant 2 breaks the disulfide bond connecting?-helix 3,linear epitope 1and linear epitope 2;Mutant 3 breaks the disulfide bond connecting?-helix 4,linear epitope 1 and linear epitope 2.Mutant 4 breaks the disulfide bond connecting?-helix3 and linear epitope 2.Mutant 5 breaks the disulfide bond connecting?-helix 4 and linear epitope 2.The wild-type Ara h 2 and five kinds of Ara h 2 mutants were expressed by Escherichia coli BL21(DE3)prokaryotic expression,and purified by nickel ion affinity column gradient elution.The purity of the proteins were all above85%.2.The results of SWISS-MODEL homology modeling were imported into GROMACS for molecular dynamics simulation to optimize the structure,and the length and curvature of the protein helix were changed.The software simulation results showed that the secondary structure of the mutant with disulfide bond loss changed,and the?-helix part of the mutant changed to?-turn.The?-turn content of mutant 1 with four disulfide bond loss increased by 7.41%,and the helix content decreased by 5.18%.The four pairs of disulfide bonds had significant effects on the solvant-accessible surface area of protein,key amino acids and aromatic residues,and the solvant-accessible surface area of Mutant 1 was the smallest,which was 2451.1(?)².After Q34Y/E38Y mutation,the exposed area of key amino acids in the protein was reduced.The solvent-accessible surface areas of key amino acids in Mutant 2 and Mutant 3 of Q34Y/E38Y were lower than those in Mutant 4 and Mutant 5 of non-mutated Q34Y/E38Y(2559.4(?)²,2496.8(?)²,2684.5(?)²,and 2860.3(?)²,respectively).They were also lower than the solvent-accessible surface area of key amino acids of Ara h 2(2929.8(?)²),indicating that the protein structure became compact after mutation and the exposed key amino acids were obscured.3.Circular dichrochromatography was used to analyze the changes of secondary structure of the proteins before and after the mutation.It was found that the proteins were more inclined to transform to the?-turn after the loss of C¹⁰⁴-C¹⁵² and C⁴⁵-C¹⁰³than to form the?-helix.Mutant 2 and Mutant 4(C¹⁰⁴-C¹⁵² and C⁴⁵-C¹⁰³)both had lower?-helix content and higher?-turn content than Mutant 3 and Mutant 5(C¹¹⁸-C¹⁶⁰ and C³³-C1¹⁶),which was consistent with the software simulation results.Compared with the results of circular dichromatography,the number of different secondary structures was different,but the variation trend of secondary structure composition of most mutants was the same.The results of ultraviolet spectrum showed that the disulfide bond loss reduced the ultraviolet absorption intensity of the mutant protein,and the tertiary structure of the mutant protein was more compact,which was consistent with the results of software simulation of the surface area available to aromatic residues.The protein structure of Mutant 3 and Mutant 5(C118G/C160G/C33G/C116G)was more compact than that of mutant 2 and Mutant 4(C104G/C152G/C45G/C103G)after losing C¹¹⁸-C¹⁶⁰/C³³-C¹¹⁶.Potential sensitization changes in the proteins before and after the mutation were assessed using a competitive ELISA.Ig E binding capacity was reduced in all mutants compared with natural Ara h 2.The wild-type Ara h 2 is equivalent to natural Ara h 2 in Ig E binding capacity because the recombinant protein is structurally similar to the natural protein.The effect of key amino acid mutation on Ig E binding ability was lower than that of disulfide bond loss.The effect of C¹¹⁸-C¹⁶⁰/C³³-C¹¹⁶ loss on Ig E binding ability was greater.Mutant 5 with C¹¹⁸-C¹⁶⁰/C³³-C¹¹⁶ had the lowest Ig E binding ability.The IC50value was 5.45?g/m L.In this study,we investigated the effects of structural changes on protein sensitization by mutating the disulfide bond and major linear epitopes of Ara h 2.The results showed that the mutation made the protein more prone to forming?-turn structure,reducing the solvent accessible surface area of key amino acids,and making the protein more compact and less potentially allergenic.The loss of disulfide bonds has a greater effect on the potential sensitization of proteins than does the destruction of linear epitopes.