Effect Of Critical Amino Acids On The Potential Allergenicity Of Peanut Allergen Ara H 2

Peanuts are one of the most common allergic foods in the world,and Ara h 2 is one of the most allergenic components in peanut allergens.Strictly avoiding intake is the only reliable method for allergy sufferers.However,this is very difficult due to the widespread use of peanuts and peanut products in food products,so attempts have been made to shift the focus to the food itself.The protein structure can affect sensitization,but the molecular mechanism affecting sensitization is still unclear,especially the critical structure of the protein that determines sensitization remains to be investigated.In this thesis,we take the discovery of localized structures that affect the potential allergenicity of proteins as the starting point.The mutation of critical amino acids disrupted four structures:linear epitope,disulfide bond,hydrophobic core,and charge core,respectively,thus the allergen Arah 2 was directionally modified.The overall structural changes of Ara h 2 before and after the mutation were characterized,and the software simulated,the localized structural changes of Ara h 2 before and after the mutation,and the changes of potential sensitization of Ara h 2 before and after the mutation were systematically assessed by serological,cytological and animal experiments.Comparing structural changes with potential allergenic changes in order to find the critical structures affecting allergen sensitization provides a new perspective for structural analysis.The research methods,main results and conclusions of this paper are as follows:1.Five mutants were designed by selecting critical amino acids in the hydrophobic core or charge core and mutating them into amino acids with opposite physicochemical properties.The distance between the hydrophobic amino acids became farther after the disruption of the hydrophobic core.The local electrostatic potential energy surface was completely changed after the disruption of the charge core.The structural changes are basically consistent with the design expectations,and the mutants’design is successful.Together with the three mutants that disrupt the disulfide bond and the dominant epitope of the allergen Ara h 2,there are a total of eight mutants.Natural Ara h 2 was purified by natural peanut defatting followed by an anion exchange chromatography column,while wild-type Ara h 2 and its eight mutants were purified from prokaryotic expression in E.coli using a nickel ion affinity column.All samples had a purity above 85%.2.The overall structural changes of Ara h 2 before and after mutation were characterized by circular dichroism and UV absorption spectroscopy,and the localized structural changes were simulated by molecular dynamics Gromacs and analyzed with the help of Py MOL,VMD,Elli Pro and other software.The results show that amino acid mutations affect not only the local structure but also the remote structure.Different amino acid mutations bring about similar changes in the electrostatic potential of a localized structure,such as R⁶²DPYSPSQDPYSPS⁷⁵.After disruption of the disulfide bond,theα-helix content decreases,theβ-turn increases,and the solvent accessible surface area of aromatic amino acids decreases.The internal compact structure is disrupted after disruption of the hydrophobic core,and the overall structure spreads outward.After disrupting the charge core,the helix becomes more compact,random coil may be converted into a helix.3.The ability of Ara h 2 to bind Ig E was examined by an indirect competitive enzyme-linked immunosorbent assay.The results revealed that the importance of different structures on the strength of Ig E binding ability of Ara h 2 was ranked as follows:hydrophobic core>more compact structured charge core=all disulfide bonds>linear epitope>charge core.The hydrophobic core has a key role in Ig E binding.The reduction of Ig E binding ability was obvious after mutation of hydrophobic amino acid residues,with hydrophobic core L⁴⁹,M⁵⁰,F¹⁴¹,and L¹⁴⁵having a greater effect.The solvent-accessible surface area of the protein and the solvent-accessible surface area of the hydrophobic residues increased after the disruption of the hydrophobic core,while the solvent-accessible surface area of phenylalanine decreased,and phenylalanine F¹¹⁰、F¹⁴¹ may be one of the critical sites for the binding of the allergen Ara h 2 to Ig E.4.To assess the changes of Ara h 2 in sensitization before and after the mutation,the degranulation model of KU812 cell line and the Balb/c mouse model were applied.The changes of Ara h 2 sensitization before and after mutation were comprehensively evaluated by clinical symptoms,specific antibodies in serum,mast cell proteinase 1in serum and food allergy related cytokines.The results showed that the disulfide bond was critical for the strength of potential sensitization to Ara h 2,and the strength of sensitization after disruption of the disulfide bond was positively correlated with the solvent availability and surface area size of the aromatic amino acids.The hydrophobic core L³⁷,A¹²⁰ can inhibit the rapid formation of histamine.After disruption of the charge core,the helix structure becomes more compact,the negative electrostatic potential of the localized structure decreases,and the potential sensitization weakens.