Structural Chrarcterization And Immunological Activity Of Sarcoplasmic Calcium-binding Protein From Pevaeus Vannamei

Shrimp contains a large amount of protein and is rich in trace elements,such as zinc,selenium,copper,iron and iodine.Shrimp allergy is very common in Asian and western countries,and sarcoplasmic calcium-binding protein?SCP?has been identified as a new shrimp allergen in Penaeus monodon.Compared to other allergens,SCP glycoproteins have less structural information and are not systematically studied for sensitization reasons.Therefore,it is very important to clarify the structure of allergen SCP glycoprotein in shrimp,which can provide a theoretical basis for further study on the mechanism of sensitization of shrimp.This study used Penaeus vannamei as a test material to study the extraction,purification,structural characterization and the effect of deglycosylation SCP on its immunological activity.The main findings are as follows:1.Extraction,separation and purification of SCP glycoprotein from Penaeus vannamei:The crude extract of Penaeus vannamei was obtained by salt solution extraction and ammonium sulfate precipitation,separated and purified by ultracentrifugation,heating,acid-base treatment and preparative electrophoresis.The results showed that ultracentrifugation and acid-base treatment could not remove the heteroprotein;heating could remove most of the heteroprotein,but a small amount of heteroprotein was present at 44.3kDa,and heating may affect the spatial structure of the protein,which is not conducive to subsequent experiments.The SCP protein can be obtained by preparative electrophoresis.It was selected for the separation and purification of SCP protein,and it was identified as glycoprotein by PAS staining.2.Structural characterization of SCP glycoprotein of Penaeus vannamei:The protein was completely denatured by DTT and IAA,and the N-glycans was released by PNGase F hydrolysis.After complete methylation,the C18 Sep-Pak cartridge was purified and analyzed by LCQ mass spectrometer.The ESI-MS with positive ion mode was used to identify the N-glycans of SCP glycoprotein by direct injection.The seven N-glycans configurations of SCP glycoprotein were identified by m/z 1172.0?Hex₃HexNAc₂?,m/z 1376.3?Hex₄HexNAc₂?,m/z 1417.3?Hex₃HexNAc₃?,m/z 1580.1?Hex₅HexNAc₂?,m/z 1621.8?Hex₄HexNAc₃?,m/z1662.0?Hex₃HexNAc₄?,m/z 1907.6?Hex₃HexNAc₅?.The seven N-glycans structures can be divided into three types,including three high mannose types,three complex types,and one hybrid type.3.Immunological activity of deglycosylated SCP:The epitopes of SCP glycoprotein were analyzed by ProtScale,IEDB,SWISS-MODEL.It was found that the glycosylation site N₇₂2 and N₁₁₆16 are located in the epitope region predicted by SCP glycoprotein.It is speculated that excision of N-glycans may destroy protein epitopes,affecting the binding of antigen antibodies.The N-glycans of SCP glycoprotein of Penaeus vannamei was excised by PNGase F enzyme to study the immunological activity of deglycosylated protein.The protein after digestion with PNGase F was verified by SDS-PAGE analysis and PAS staining.It was found that after digestion,the electrophoresis band was moved down.In the PAS staining,there was finally no band appeared,indicating that the PNGase F can effectively remove the N-glycans of SCP glycoprotein.The immunological activity of SCP glycoprotein after deglycosylation was analyzed by indirect ELISA.The results showed that the immunological activity of SCP glycoprotein decreased with the degree of deglycosylation.It was further studied by mast cell degranulation assay.The results showed that as the degree of deglycosylation increased,the release of?-aminogalosidase and histamine decreased.The deglycosylated SCP glycoprotein immunoreactivity is significantly reduced compared to the native SCP glycoprotein,and its N-glycan plays a crucial role in allergic reactions.