Study On The Epitope Mapping And Mitigation Strategy Of α-Amylase Inhibitor And β-Amylase,Two Typical Non-Gluten Allergens Of Wheat(Triticum Aestivum) And Barley(Hordeum Vulgare)

Food allergy has become a serious public health problem,and the current studies on antigenic epitopes of triticeae crops allergens are mainly focused on gluten allergens,while non-gluten allergens can also cause severe allergic reactions,but relatively little research has been conducted on their antigenic epitopes.In view of this,the aim of this study was to investigate the epitope localization and ablation techniques of the major non-gluten allergens,i.e.,α-amylase inhibitors andβ-amylase in triticeae crops,with the following main findings:（1）Wheatα-amylase inhibitor was obtained commercially.Firstly,the structure of wheatα-amylase inhibitor was characterized by mass spectrometry（MS）,ultraviolet spectrum（UV）,and Circular dichroism spectrum（CD）.Results of MS showed that the sequence coverage ofα-amylase inhibitor was 92%.Next,results of UV spectroscopy exhibited that the wheatα-amylase inhibitor had a maximum absorption peak at 190 nm and a weak absorption peak at280 nm.Results of CD showed that the secondary structure of wheatα-amylase inhibitor was constituted ofα-helix（21.3%）,β-sheets（29.2%）,β-turns（21.7%）,and random coils（29.9%）.Another allergen was prepared by the laboratory,and the crude protein of barleyβ-amylase was firstly extracted by ammonium sulfate precipitation method,and the crude protein was purified by ion exchange chromatography and gel filtration chromatography,and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis,which showed that the purity ofβ-amylase was 93%.The purifiedβ-amylase was identified by mass spectrometry,and the results showed that the purified protein was barleyβ-amylase with a molecular weight of 59.6 k Da.The purified protein was further characterized by UV spectroscopy and CD spectrum.Results of UV spectroscopy exhibited that the allergen had the maximum absorption peak at 208 nm.Results of CD showed that the secondary structure was constituted ofα-helix（21.7%）,β-sheets（31.4%）,β-turns（18.1%）,and random coils（29.1%）.（2）The epitopes of wheatα-amylase inhibitor and barleyβ-amylase were predicted by bioinformatics prediction tools including DNAstar,IMED and IEDB,respectively.After comprehensive analysis,a total of six candidate epitopes were screened for wheatα-amylase inhibitors.The epitopes were validated by ic-ELISA,and finally four linear epitopes,i.e.,QAFQVPAL（Peptide-1）,QVPEA（Peptide-2）,DSMYKEHGAQEGQAGTGAFPRCR（Peptide-3）,and LPIVVDASGDGAY（Peptide-B₁）,were obtained.Similarly,a total of seven candidate epitopes were screened for barleyβ-amylase.The epitopes were validated by ic-ELISA,and finally four epitopes were obtained,including NDTPERTQFFRDNG（Peptide-J）,RPHGINQSGPPEHK（Peptide-K）,PRDPYVDPMAPLPRSGPE（Peptide-L）,YTDGHGTRNIE（Peptide-I）.（3）Using polyphenols-allergen interactions as a desensitizing method,theα-amylase inhibitor-EGCG（epigallocatechin gallate）andα-amylase inhibitor-EGC（epigallocatechin）non-covalent complexes were produced.Ic-ELISA results showed that EGCG and EGC had good desensitization abilities,and when the molar ratio ofα-amylase inhibitor to EGCG was1:30,the Ig E-binding level of the complex reached the lowest and the binding capacity was reduced by 22.7%.The complexes were characterized by UV spectroscopy,fluorescence spectroscopy（FL）,CD spectrum,and molecular docking（MD）.The results showed that the protein UV absorptionλmax was red-shifted,and the degree of fluorescence burst was positively correlated with the polyphenol concentration.Result of CD showed that theβ-sheets andα-helix shifted to random coils in the complexes.Through MD,it was further found that Peptide-1,3 and B₁ had more binding sites which had better inhibition rate effect.After comparing with the epitopes,it was demonstrated that the desensitizing effect was the result of the interactions between the linear epitopes ofα-amylase inhibitor and its nearby residues with polyphenol molecules.The non-covalent complexes ofβ-amylase-EGCG andβ-amylase-EGC were prepared by the same method,and the desensitization effect was investigated by ic-ELISA.The results showed that the Ig E-binding level of the complexes reached the lowest level when the molar ratio ofβ-amylase to EGCG was 1:30,and the binding capacity was reduced by 43.8%.Theβ-amylase-polyphenol complex was characterized by UV spectroscopy,FL spectroscopy,CD spectrum,and MD.The results showed that when polyphenols were added,the protein UV absorptionλmax was red-shifted,and the fluorescence was quenched evidently with increased in the content of random coil in the secondary structural composition.It was also found that the Peptide-K and L had more binding sites which had better inhibition rate effect,and further demonstrated that the desensitizing effect was the result of the interactions between the linear epitopes ofα-amylase inhibitor and its nearby residues with polyphenol molecules.This study can provide a reference for the localization of non-gluten allergenic epitopes in triticeae crops and the preparation of hypoallergenic intercrops.