Study On Reducing The Allergenicity Of Exopalaemon Modestus Tropomyosin And Immune Desensitization Mechanism Based On Glycation Modification

Food safety guarantee and human health maintenance is one of the core missions for constructing Healthy China.In the past 20 years,the number of food allergic patients has significantly increased,and there is an increasing trend of more food allergic patients.Severe food allergy can lead to human shock and even death.Therefore,prevention and treatment of food allergy is an important part of food safety guarantee.Shrimp is a very common food allergen.Among shrimps,Exopalaemon modestus is an important economic freshwater shrimp that widely distributed in the freshwater lakes and rivers in China.Exopalaemon modestus is a popularly delicious and nutritious aquatic food for consumers.In recent years,as the consumption of Exopalaemon modestus greatly increased,the occurance of Exopalaemon modestus-induced food allergy also promoted,which has became a serious threat to the food safety of wide consumers.However,up to now,there are no reports about the allergen of Exopalaemon modestus at home and abroad.Therefore,how to reduce the allergenicity of Exopalaemon modestus allergen is particularly urgent,which has important practical significance for the food safety and health of shrimp food allergic patients.1.Purification,allergenicity assessment and bioinformatics analysis of Exopalaemon modestus allergen.The allergen of Exopalaemon modestus?Tropomyosin,TM?was extracted and purified by methods such as isoelectric point precipitation and ammonium sulfate precipitation,the biochemical identity was confirmed as tropomyosin by matrix-assisted laser desorption ionization time-of-flight mass spectrometry?MALDI-TOF-MS?.The allergen of Exopalaemon modestus was confirmed as tropomyosin by western blot and ELISA,and the tropomyosin of Exopalaemon modestus had strong cross-allergenicity with other shrimp tropomyosins.The amino acid sequence of Exopalaemon modestus TM was determined via enzymatic deglycosylation,in vitro digestion and UPLC-ESI-MS^E mass spectrometry.Exopalaemon modestus TM consisted of 284 amino acids,its amino acid had high similarity with other shrimp tropomyosins,while was not the same.In addition,the biological information of Exopalaemon modestus TM has been submitted to the Allergen Nomenclature Sub-Committee of World Health Organization?WHO?and International Union of Immunological Societies?IUIS?,and named Exo m 1.2.Identifying the epitopes of Exo m 1 and screening the key amino acids of epitopes.Ten predicted epitopes were obtained by immunobioinformatics softwares,then the ten predicted epitopes were tested by competitive immune dot-blot microarray assay,and six predicted epitopes were identified:45-59,89-105,131-142,145-164,243-259 and 263-280.In addition,five epitopes of Exo m 1 were identified using overlapping peptide library and competitive immune dot-blot microarray assay:43-57,85-105,133-159,187-201 and 247-279.By comprehensively analyzing the predicted epitopes and overlapping peptides identified epitopes,the epitopes of Exo m 1 came out:Epitope 1:43-59,epitope 2:85-105,epitope 3:131-164,epitope 4:187-201 and epitope 5:243-280.Furthermore,the key amino acids in the epitopes of Exo m 1 were analyzed using the mutant epitope peptides combined with indirectly competitive ELISA:The key amino acid of epitope 1 is glutamine?Gln,Q?,the key amino acids of epitope 2 are leucine?Leu,L?and glutamic acid?Glu,E?,the key amino acids of epitope 3 are leucine?Leu,L?and aspartate?Asp,D?,the key amino acids of epitope 4 are valine?Val,E?,leucine?Leu,L?and glutamic acid?Glu,E?,and the key amino acid of epitope5 is leucine?Leu,L?.3.Exo m 1 is a naturally glycosylated protein that containing glycans,the role of glycans during Exo m 1-induced allergy sensitization was investigated by deglycosylation treatment.Through PAS staining and glycan analysis,Exo m 1 was a glycoprotein containing N-glycan and O-glycans,the N-glycosylation site was the asparagine at position 132,and the potential O-glycosylation sites were the threonine at position 110,threonine at position 111,serine at position 117 and serine at position 188.The N-glycosylation site at position 132 and potential O-glycosylation site at position 188 were located in the epitopes of Exo m 1?epitope 3:131-164 and epitope 4:187-201?,and the potential O-glycosylation sites at position 110,111and 117 were located near the epitope 2?85-105?.In addition,compared with Exo m 1,the deglycosylated Exo m 1 had stronger allergenicity on RBL-2H3 cells,and exacerbated mouse allergy reactions by promoting the secretion of Th2 cytokines?IL-4 and IL-13?and Treg cytokines?IL-10 and TGF-??.The N-glycan and O-glycans masked the epitopes of Exo m 1,after deglycosylation,the previously masked or buried epitopes got exposed,resulting in the stronger allergenicity of deglycosylated Exo m 1.4.The glycans could mask the epitopes of Exo m 1.Based on this finding,the saccharides of different molecular sizes?Glucose,maltose,maltotriose,maltopentaose and maltoheptaose?were applied to modify Exo m 1 by glycation,and the mechanisms of glycation modification on reducing the allergenicity of Exo m 1 were investigated.The saccharides of smaller molecular sizes could highly glycate Exo m 1 and generate more AGEs products;the saccharides of greater molecular sizes were more difficult to glycate Exo m 1 and generate fewer AGEs products;in addition,the glycated Exo m 1 with higher AGEs contents had stronger ability to bind the RAGE receptors on mast cell surface and activated mast cell.In addition,compared with Exo m 1,the glycated Exo m 1 had weaker allergy reactions on mouse by suppressing the secretion of Th2 cytokiens?IL-4 and IL-13?and Treg cytokines?IL-10 and TGF-??in the lymphocytes of mouse spleen and mesenteric lymph nodes.5.Preparing allergy desensitization immunotherapy based on the glycated Exo m 1 and Al?OH?₃ adjuvant,and investigating the mechanism of immune allergy desensitization.In this part,the immunotherapy was prepared by adsorbing the desensitized glycated Exo m 1 onto the Al?OH?₃ adjuvant.Compared with Exo m 1,the glycated Exo m 1 had stronger immunogenicity and higher in vivo safety in mouse model.And the glycated Exo m1+Al?OH?₃ immunotherapy had reduced the secretion of IgE antibody and Th2 cytokine?IL-4and IL-13?,promoted the secretion of IgG1,IgG2a,Th1 cytokine?IFN-??and Treg cytokines?IL-10 and TGF-??,which suggested the glycated Exo m 1+Al?OH?₃ immunotherapy could desensitize the mouse allergy reactions through transforming Th2 immune responses to Th1and Treg immune responses.Therefore,the glycated Exo m 1+Al?OH?₃ immunotherapy can be used to desensitize the Exo m 1-induced food allergy.In summary,the purified tropomyosin of Exopalaemon modestus in this work is an allergen with strong allergenicity,and had strong cross-allergenicity with other shrimp tropomyosins.In addition,the glycans could mask the epitopes of Exo m 1,based on this finding,the saccharide residues with different length-sizes were added to Exo m 1 through glycation,the saccharide residues with longer length had stronger masking effects on epitopes and could generate fewer AGEs products,while the saccharide residues with shorter length had weaker masking effects on epitopes and could generate more AGEs products,and some AGEs could function as neoepitopes to bind the RAGE receptors on mast cell surface,and therefore activated mast cell.Moreover,the immunotherapy prepared by the glycated Exo m1+Al?OH?₃ could desensitize mouse allergy reactions through transforming Th2 immune responses to Th1 and Treg immune responses,leading to body allergy desensitization.