Study On Cross-recognition And Common Structure Of Three 2S Albumin Allergens

Peanuts and tree nuts are food allergens that can cause severe allergic reactions,IgE cross-reactivity between peanuts and tree nuts are common.Studies have found that 2S albumin in tree nuts and peanuts are highly correlated with IgE mediated allergic reactions to peanuts or specific tree nuts.The amino acid sequence homology of 2S albumin is low,but the advanced structure is conserved.The similar structure between different 2S albumins may be the key site of IgE binding in cross-allergic reactions.This provides a new perspective for finding the key structures of 2S albumin recognition,especially the conformational epitopes.In this paper,the 2S albumin allergens Ara h 2,Ana o 3 and Ber e 1 were purified by anion exchange chromatography(purification of Ara h 2 and Ana o 3 is a reference method),and identified the purified Ber e 1 by LC-MS/MS and Western blotting.Competitive inhibition ELISA and Western blotting were used to study IgE cross-reactions between three purified 2S albumin allergens using the serum pool of eleven peanut allergy patients,ten tree nut allergy patients and single tree nut allergy patient serum.Using circular dichroism(CD)spectroscopy and ultraviolet absorption(UV)spectroscopy to characterize advanced structure of three 2S albumin allergens,meanwhile,using molecular dynamics simulated their advanced structure.Finally combines the data of serology,structural characterization and simulation to analyze the common characteristic structure among three 2S albumin allergens and find possible IgE recognition epitopes.The main research methods,results and conclusions were as follows:1.The defatted powder of Brazil nut was prepared by crushing and defatting,and the crude protein was obtained by twice extracting the defatted powder with PBS.Ber e 1 was purified by anion exchange chromatography,and identified by LC-MS/MS.The purity of Ber e 1 was above 95%and the recovery rate was more than 47.8%,and the yield of single-round was more than 20 mg,the purified allergen could be identified by serum for allergic patients.2.Western blotting and competitive inhibition ELISA were performed on peanut,cashew,Brazil nut protein and three 2S albumin allergens by using different allergic patients’ serum IgE.In western blotting,the IgE in the serum pool of peanut allergy patients mainly recognizes proteins in peanuts,while the IgE in the serum pool of tree nut allergy patients recognizes various proteins in peanuts,cashews and Brazil nuts;there are 2 tree nut allergy patients’ IgE in serum only recognizes Ana o 3,and the remaining 5 tree nut allergy patients’ IgE recognize 2 or 3 2S albumins.Competitive inhibition between 2S albumins was examined by competition ELISA.The results showed cashew and Brazil nut protein had certain competitive inhibition on peanut protein,Ana o 3 and Ber e 1 also had certain inhibition on Ara h 2,but no more than 50%,and Ara h 2 and Ber e 1 had more than 50%competitive inhibition on Ana o 3.These results showed there were cross-recognition between peanut,cashew and Brazil nut whole proteins,as well as between Ara h 2,Ana o 3 and Ber e 1.Moreover,not only are there similar linear epitopes between allergens Ara h 2,Ana o 3 and Ber e 1,but there may also be similar advanced structures recognized by the same IgE.3.Using circular dichroism spectroscopy and ultraviolet absorption spectroscopy to characterize advanced structure of Ara h 2,Ana o 3 and Ber e 1,the structures of the three allergens were simulated by molecular dynamics and compared by visualization software.The results showed that there was no significant difference in the secondary structure components of the three 2S albumins,Ana o 3 and Ber e 1 have less difference in advanced structure.The following linear epitopes among the three 2S albumin allergens show high sequence and charge distribution similarities,the linear epitope 31RRCQSQLER39 of Ara h 2 and the linear epitope 33SGREQSCQRQFE44 of Ana o 3,the linear epitope 104CNELNEFENNQR115 of Ara h 2 and the linear epitope 72SLRECCQELQEV83 of Ana o 3,the linear epitopes 142KRELRNLPQ151 of Ara h 2 and 120MRLAENIPSRCNLS133 of Ber e 1,which may be the linear epitopes cross-recognized by IgE.Likewise,the following advanced structures of identically charged amino acids among the three 2S albumin allergens have high surface similarity,the advanced structure composed of the sequence 119EALQQIME129SD132L160CDL in Ara h 2 and the advanced structure composed of the sequence 22QF25EQ55E58QE61QEVD in Ana o 3,the advanced structure composed of the sequence 22Q25EQQ29F60L62EVD67C in Ana o 3 and the advanced structure composed of the sequence 89DESCRCE137CP in Ber e 1,the advanced structure composed of the sequence 121L146R155RAPQRC162L in Ara h 2 and the advanced structure composed of the sequence 51SH54RMYMR76PH in Ber e 1,which may be their common conformational epitopes that is cross-recognized by IgE.This study introduces molecular dynamics simulation technology to compare the structures of three 2S albumin allergens and find the common structures of allergen proteins in IgE cross-recognition.It is expected to establish a new method of allergen surface localization,realize the simultaneous localization of various allergen protein epitopes,make the allergen surface localization more accurate and efficient,and deepen people’s understanding of the material basis of food allergy.