Purification,Identification,Bioinformatics Prediction And Sensitization Analysis Of Arginine Kinase From Portunus Trituberculatus

Food allergy is an allergic epidemic second only to asthma,affecting the health and lives of 2%～4%of adults and children worldwide.Eight categories of common food allergens are soybeans,peanuts,wheat,nuts,milk,eggs,crustaceans and fish.Among them,aquatic products occupy two categories.As a widely favored crustacean aquatic product,Portunus trituberculatus has strong allergen cross reactions with other mollusks and crustaceans,among which arginine kinase（AK）,which is not heat-resistant or strong acids-resistant,is one of the main allergens.Meanwhile,as a glycoprotein,the research on its allergenicity has attracted much attention.Most of the substances that cause food allergies are glycoproteins.In order to investigate the effect of glycosylation modification on the allergenicity and degree of allergenicity of allergenic proteins,this paper identified the AK structure based on the preparation and purification of AK from Portunus Trituberculatus,and used bioinformatics technology to predict and verify in vitro experiments to explain the effect of glycosylation modification on the allergenicity of AK from the structural level,This provides theoretical basis and data support for the desensitization research of Portunus Trituberculatus and other aquatic products.The main conclusions of the paper are as follows:1.The crude extract of arginine kinase（AK）of Portunus trituberculatus was isolated from the meat of Portunus trituberculatus.A band with a molecular weight of about 40 KDa was found by SDS-PAGE gel electrophoresis,similar to the AK found in several other studies.To obtain pure AK,preparative electrophoresis was used for further separation and purification,providing high-purity samples for subsequent protein identification,glycosylation modification,and exploration of AK sensitization.The arginine kinase with high coverage rate was identified by endonuclease digestion,proteomics and liquid chromatography-mass spectrometry.It was confirmed that the purified sample was AK of Portunus trituberculatus.In this paper,proteomics method and LC-MS/MS technology were used to identify the allergen AK of Portunus trituberculatus by the characteristic peptide,and structural characterization of the glycosylation modification of AK.It provides important basic data for the standardization of food allergen labeling.2.The allergen AK of Portunus trituberculatus as the research object,its physicochemical properties were calculated and the antigenic epitopes were predicted.The results indicate that the AK of arthropods such as blue crab and Chinese mitten crab have high sequence homology with the AK from Portunus trituberculatus.The B cell linear antigen epitopes of Portunus trituberculatus AK were 39～44,87～103,174～181,309～317,326～330,and the T cell linear antigen epitopes were 18～22,52～55,174～175,193～195,229～232,276～277,299～300,345～351.It was found that ₁₇₄Thr-Lys₁₇₅ appeared at the prediction sites of B cell and T cell epitopes.That was speculated that 174 threonine and 175 lysine played an important role in immunogenicity.The homologous modeling and molecular docking were performed by SWISS-MODEL,Discovery Studio and ZDOCK SERVE.The result of c Docking showed that the docking interaction between the AK of Portunus trituberculatus and cetirizine was the highest compared with other antiallergic drugs,and it was predicted that it had better desensitization effect on food allergy caused by the AK.Which mainly bound by affinity with hydrogen and ionic bonds contributed by Arg,Glu and Thr on AK.It was speculated that the binding of cetirizine and AK masks the B cell linear antigen epitopes and conformational epitopes of AK,It further reduces the possibility of the combination of sensitizing protein and immunoglobulin,so as to achieve the purpose of anti-allergy.Mast cell is one of the key driving factors of allergy.The G protein coupled receptor related to mast cell can mediate the activation of MC.ZDOCK docking results showed that AK without glycosylation binds to Mrgprb2 mainly through the hydrogen bond of Tyr134,Ser150 and Gly165 and theπ-πinteraction of Phe168 and Phe186,activating mast cell and triggering allergic reaction.Single glycosylation modification enhanced the sensitization of AK,but there was no significant difference.It was speculated that glycosylation modification will enhance the sensitization of AK.The key role of sugar chains in AK sensitization lies in the modification site,followed by the number of sugar groups and sugar type structure.3.To explore the effect of glycosylation on the AK sensitizations,the release amount of histamine andβ-aminohexosidase from P815 mast cells were tested to verify the mechanism of AK sensitization.The results showed that the effect of excessive glycosylation on AK was uncertain.That means Maillard reaction cannot be used to reduce the allergenicity of Portunus AK.Compared with the nature AK,the AK with only removed the N-sugar chain showed stronger sensitization,which is consistent with the protein docking results of ZDOCK.It was speculated that the removal of the N-glycans chain exposes the conformational antigen epitopes and linear antigen epitopes related to the O-glycans chain information.Compared with the natural AK,AK with only removed O-glycans chain showed weaker sensitization,presumably because it destroyed the conformational antigen epitope associated with O-glycans chain,and the N-glycans chain masked the linear antigen epitope.Therefore,a hypothesis was obtained that O-glycans play an important role in conformational antigenic epitopes,while N-glycans may mask linear antigenic epitopes,providing a new approach for reducing the allergenicity of AK from Portunus trituberculatus.