Screening The Allergic Epitope And Critical Amino Acids Of Penaeus Chinensis Tropomyosin And The Effect Of Glycosylation On Their Allergenicity

Penaeus chinensis is popular in Asian diets and show higher consumption in China than other countries.In recent years,the production and consumption of Penaeus chinensis keep growing.Unfortunately,the high rate of consumption has led to increased allergy problems.So far,the allergic epitope of Penaeus chinensis has not been studied.Allergies impaire human health,and even can be life-threatening,so it is important to prevente and treat allergy by exploring the sensitization mechanism of shrimp allergens.On the basis that tropomyosin is the main allergen of Penaeus chinensis,we further studiey its sensitization mechanism at the molecular and amino acids level.In this paper,bioinformatics was used to predict the epitopes of tropomyosin,and the potential epitopes were screened by multiple methods.Furthermore,the critical amino acids were predicted and the spatial model was established.In addition,glycosylation was used to reduce protein and epitope allergenicity.The main contents are as follows:1.The amino acid sequence of the main allergen(tropomyosin)of Penaeus chinensis was analyzed by the bioinformatics software DNAstar and AntheProt,combined with three online prediction biology websites.Then 12 potential epitopes were predicted and synthesized by solid phase.The allergenicity of 12 peptides was verified by icELISA and dot-blot,with the specific serum of 30 allergic patients.The results showed that 10 peptides exhibited high affinity with serum.2.The amino acid sequence of tropomyosin was analyzed by bioinformatics,and the Sus scrofa tropomyosin lclg template was screened by SWISS-MODEL modeling tool.On the basis of the template,the amino acid sequence was modified by PyMOL software,and the spatial conformation of Penaeus chinensis tropomyosin was obtained.Ramachandran Plot of the template was analuzed by VMD software,the result demonstrates that the modeling is reasonable.According to the modeling,it can be seen that the conformation of the tropomyosin of Penaeus chinensis is mainly secondary structure of the a-helix,but not complex tertiary or quaternary structure.As a result,the protein will not from the conformationepitopes,but only linear epitope that exposed to the outside.Moreover,the frequency of amino acids in protein and epitopes was analyzed by BioEdit software,and the amino acid sequences of tropomyosin in Penaeus chinensis with tropomyosin of allergenic food from SDAP database were aligned by software Vector NTI.The potential critical amino acids in the epitope of T-03,T-07 and T-12 were mutated into alanine and assessed by icELISA to identify critical amino acids.The results showed that D in T-03,E and D in T-07 and E in the T-12 was the critical amino acids.3.The allergenicity of tropomyosin and epitopes after glycosylation with ribose,galactooligosaccharides,chitosan oligosaccharide was significantly decreased and the secondary structure were also changed.However,the glycosylation with maltose has no effect on the allergenicity.Glycosylatin products were analyzed by ELISA,circular dichroism and effective lysine content.The results showed that the binding ability of the glycosylated product with serum IgE was unaffected after 20 min-4 h tretment.At 8 h-12 h,the binding capacity of glycosylated product with serum IgE decreased,which indicated that the allergenicity was weakened.At 20 min-2 h,the binding capacity of glycosylated product with serum IgE was no obvious changed,and the allergenicity was weakened after 4-12 h.The secondary structure analysis showed that the ?-helix structure of the glycosylation product of the tropomyosin with ribose,galactooligosaccharides and chitosan oligosaccharide decreased by 13.79%,10.47%,and 10.34%,respectively.And the relevant structures were also changed with a decrement of a-helix.On the contrary,glycosylation with ribose had little effect.Glycosylation also decreased the lysine content in the peptides.4.In this paper,serum IgE-mediated human mast cell degranulation model was utilized to analyze the allergenicity of tropomyosin and antigenic epitopes.At the lowest antigen concentration of 1 ug/mL,mast cell was subjected to degranulation experiments.Histamine,tryptase and?-hexosaminidase were the principal indexes to detect mast cells degranulation.Consistent with the results of icELISA and dot-blot,the degranulation results also showed that 10 petides were allergic.The concentrations of cytokines(IL-1?,IL-4,IL-6 and IL-8)and lipids(prostaglandins and cysteinyl leukotriea),granulocyte-macrophage colony stimulating factor(GM-CSF)and tumor necrosis factor-a(TNF-?)were increased in the cells.The expression of IgE receptor(Fc?R1)mRNA was also detected,which proved that shrimp allergy is a ? type of IgE-mediated type ? hypersenitivity.Observeing the mast cell degranulation by electron microscopy showed an irregular nucleus and secretory granules distributed outside the cytoplasm,exhibiting a loss of membrane density.