Epitope Mapping And Allergenicity Analysis Of Two Isoforms Of Tropomyosin From Alectryonella Plicatula

Immunoglobulin E(IgE)-mediated hypersensitivity reactions can occur in allergic patients to food allergens.Due to the rapid onset,recurrence,and long-term persistence of food allergy,the study of food allergens has been of great concern worldwide.Aquatic foods are the main allergenic foods in China,including mollusks represented by oysters.In this study,the important allergens in Alectryonella plicatula were identified and cloned to investigate the characterization,sequence information,immunological activity,structure,and epitopes of the allergens,to obtain the gene sequences of the allergens and to investigate the relationship among the allergenicity of the allergens with their sequence information and structures,with the aim of providing a theoretical basis for the prevention and control of aquatic food allergy.In this study,the 40 k Da allergenic component from A.plicatula,tropomyosin(TM),was identified by serum verification of oyster-sensitized individuals and mass spectrometry,and two isoforms(TM-α and TM-β,TM-α/-β)existed.The gene sequences of TM-α/-β were obtained and both sequences contain an open reading frame of 852 bp and encode 284 amino acids,with12 different amino acids between sequence positions 191 and 213.The recombinant TM-α/-βwere obtained and characterized as follows: TM-α/-β had similar stability to gastric digestion;TM-β was more easily digested by intestinal fluids than that of TM-α,but its digestion products still had immunobinding activity;TM-α and TM-β had similar physical characteristics.The results of the inhibition ELISA showed no significant difference of the Ig G-binding activity in TM-α/-β;The IgE-binding activity of TM-α/-β were investigated by dot blot using sera from oyster-sensitized individuals.Both had similar IgE recognition frequencies,while the IgE-binding activity of TM-β were significantly higher than that of TM-α,approximately two times higher.The results of basophil activation test showed that both TM-α/-β could significantly up-regulated the levels of CD63 and CD203 c on the surface of basophils in oyster-sensitized individuals,indicating that both TM-α/-β could be considered as allergens in A.plicatula,with TM-β having significantly higher activating ability than TM-α.Sequence information analysis showed that more amino acids of TM-β were the same as the epitopes in other aquatic TMs;The linear epitopes of TM-α/-β were predicted by bioinformatics tools separately and combined with sera from oyster-sensitized individuals and synthetic peptide techniques to identify four IgE linear epitopes of TMs from A.plicatula,L-TM-1～4,of which L-TM-3 was the unique IgE linear epitope of TM-β from A.plicatula.Modeling the spatial structures of the monomer and homodimer of TM-α/-β,mapping the epitopes and analyzing the structural differences between them,with TM-β having a predominantly negative surface electrostatic potential at the sequence differences and TM-α having a weakly positive potential.Conservation analysis of the epitopes showed that L-TM-2～4 exhibited potential cross-reactivity with other allergenic TMs.In this study,two allergen isoforms,TM-α/-β,were identified in TMs from the A.plicatula,with TM-β being significantly more allergenic than TM-α.Four linear epitopes of TMs form A.plicatula displayed potential cross-reactivity with TMs from other allergenic species.TM-β had higher negative surface potential and more IgE epitopes compared to TM-α.This study expands the research ideas for the isoforms of food allergens and facilitates accurate food allergen diagnosis and specific clinical guidance for food allergy.