Allergenicity Analysis Of Sarcoplasmic Calcium-binding Protein From Portuguese Oyster (Crassostrea Angulata)

Food allergy affects the quality of people’s lives seriously and has become one of the public health safety issues of globalization.Oyster is a kind of rare mollusc product with high nutritional value,the production and consumption are increasing per anum,while the problem of food allergy caused by them is also increasing.However,the research on its allergen is not systematic.In this research,a novel allergen of portuguese oysters(Crassostrea angulata)was purified,cloned and expressed.And the physicochemical properties,antigen epitopes and cross-reactivity were explored,which can provide a data basis for the prevention and diagnosis of oyster allergies.The target protein(20 kDa)from adductor muscle in the C.angulata was purified.Sarcoplasmic calcium-binding protein(SCP)was identified as a novel allergen using mass spectrometry and the sera of shellfish-sensitized individuals.The rabbit anti-C.angulata SCP polyclonal antibody was prepared.Further,the SCP was cloned and expressed,the open reading frame of the gene was 537 bp,encoding 179 amino acid residues.In vitro,the purified recombinant SCP(rSCP)was able to increase the surface molecule CD63 level of peripheral blood eosinophilic mononuclear cells significantly(p<0.05),which showed that rSCP had immunoreactivity.In addition,compared with natural SCP(nSCP),rSCP maintained similar thermal stability,pH stability,digestive stability,and IgG-binding activity.The secondary structure of the two proteins displayed the typical α-helix structure.The serological analysis of 12 shellfish-sensitized individuals showed that the IgE-binding activity of rSCP in 4 patients was significantly higher than that of nSCP(p<0.05).Furthermore,the allergen information was logged to the WHO/IUIS allergen database,which was named Cra a 4.Immunoinformatics tools,one-bead-one-compound peptide library,and phage random peptide library were combined to analyze the IgE epitope in C.angulata SCP.Five linear epitopes named L-SCP-1(AA22-33),L-SCP-2(AA64-75),L-SCP-3(AA80-90),L-SCP-4(AA107-116)and L-SCP-5(AA144-159)were verified using serological tests based on Fmoc synthetic peptide technology.Meanwhile,two conformational epitopes including C-SCP-1(G48,D99,I101,D103,1105,D110,R111,D114,D116,A124,A136,W156)and C-SCP-2(D114,R134,A136,S138,L139,N141,N144,L149,D151,V153,S154,V157,F159)were obtained,and C-SCP-1 had overlapping areas with the calcium binding sites(AA106-117).Ca2+-depletion assay showed that the secondary structure and the surface hydrophobicity of SCP were affected by Ca2+-depletion,which can reduce its immunoreactivity.Moreover,the cross-reactivity was analyzed among SCP and other eight different species in mollusc and crustacean using sequence homologous analysis,phylogenetic tree analysis,and inhibitory experiments.Overall,a novel allergen SCP in C.angulata was obtained,SCP was cloned and expressed,the recombined protein could provide a data basis for the component resolved diagnosis;the analysis of SCP antigen epitope and cross-reactivity in order to clarify the allergenic mechanism and lay the foundation for the development of epitope-based hyposensitive oyster products and agents.