Extraction,Separation And Structural Characterization Of ?'-c Glycoprotein From Acipenser Gueldenstaedti Eggs

As a nutritious and delicious seafood product,fish eggs are very popular among people,especially the caviar made from sturgeon eggs,which is expensive and known as "black gold".However,at home and abroad,reports of allergies in the food population caused by fish eggs are not uncommon.The study found that fish eggs are a potential allergen-containing food,the main allergen is ?'-c glycoprotein.Most cases of fish egg allergy are classified as type I allergies,caused by specific binding of allergens to IgE.So far,compared with other food allergens,the biochemical characteristics and structural information of allergic proteins in fish eggs are less,and the cause of sensitization of fish eggs of different fish species is also not systematically studied.Therefore,it is very important to clarify the structure of the major allergen P'-c glycoprotein in fish eggs,which can provide a theoretical basis for further study on the sensitization mechanism of fish eggs.This study used Russian sturgeon(Acipenser gueldenstaedti)fish eggs as experimental materials,focusing on the extraction,purification and structural characterization of ?'-c glycoprotein in sturgeon eggs.The main findings are as follows:1.Extraction,separation and purification of ?'-c glycoprotein from sturgeon eggs:The vitellin extract was prepared by sodium chloride solution extraction method,and the total protein and polysaccharide content in the extract were determined.The vitellin extract was subjected to preliminary crude separation by ammonium sulfate fractionation method,followed by heat treatment,alkali treatment and ultracentrifugation to further separate and purify the vitellin,and the glycoprotein was identified by PAS staining.The results showed that the total protein content of vitellin was 2.662±0.003 ?g/?L,and the polysaccharide content was 0.8704±0.007 ?g/?L.The separation and purification effect can be obtained by SDS-PAGE electrophoresis.The ammonium sulfate solution with the concentration of 80%has the best separation effect compared with other concentrations.The number of bands of the removed protein is the largest,which can effectively separate the samples,leaving only three main protein band;heat treatment makes the main protein clearer,the heteroprotein decreases,while the alkaline treatment reduces the main protein content and produces small molecular hybrid proteins.After being stained by PAS staining method for specifically identifying glycoprotein,the three main proteins obtained after salting out with ammonium sulfate at a concentration of 80%are glycoproteins.The heat-treated sample was subjected to ultracentrifugation,and when the centrifugation conditions were 4 °C,250,000 × g,and centrifugation for 1 h,a single glycoprotein component having a molecular weight of 28 kDa was separated and purified from the sturgeon eggs.2.Structural characterization of the ?'-c glycoprotein fraction of sturgeon eggs:SDS-PAGE electrophoresis under non-reducing conditions confirmed that the components with molecular weights of 28 kDa and 23 kDa were not two subunits of a certain vitellin.Subsequently,the two glycoprotein components were identified by peptide fingerprinting,and bioinformatics and homology analysis were performed.It was identified by LC-MS/MS peptide analysis that the protein component with a molecular weight of 28 kDa belongs to the sturgeon vitellogenin AB2a,and the peptides are located in the vWFD conserved region of vitellogenin,which was identified as ?'-c glycoprotein.This protein is compared with the vitellogenin sequence of the known ?'-c sequence of Oncorhynchus keta(B7XGC3)and Gambusia affinis(Q589T0),both located in the vWFD region of vitellogenin,but the homology is about 40%,the difference is larger.Therefore,it is speculated that ?'-c of different fish species can be used as a means and method for identifying fish species.The secondary structure of ?'-c in sturgeon eggs was studied by circular dichroism(CD)spectroscopy.The results showed that ?-fold and random coil were present in(3'-c,and there was no a-helical structure.3.Structural characterization of(3'-c glycoprotein about glycopeptide and N-glycans in the glycoprotein.PNGase F enzymatic glycoprotein method and reducing ?-elimination method were used to identify the presence of N-glycoside bond and O-glycoside bond in the(?'-c glycoprotein of sturgeon eggs.As a result,an N-glycoside bond was present in the ?'-c glycoprotein and no O-glycosidic bond was present.The PNGase F enzyme releases the N-linked oligosaccharide in the glycoprotein.After complete methylation,the LCQ DECA XP Plus ion mass spectrometer is used to directly sample the ?'-c glycoprotein of the sturgeon eggs.The N-linked oligosaccharide was structurally identified.A total of seven N-linked oligosaccharide were identified,including four high mannose types(H4N2,H5N2,H6N2,H7N2)and two heterozygous types(H3N4,H3N5).The branching mode has single and two antennae,and the main fracture mode is glycosidic bond breaks.The important cross-ring breaks also occur.The glycopeptide was obtained by trypsinizing the glycoprotein,and the N-glycopeptide of the ?'-c glycoprotein of the sturgeon eggs was identified by HILIC-MSn liquid chromatography.A N-glycopeptide was identified with two charges,the peptide sequence was SHGGQATQVKVNGEAIS,the composition of N-linked oligosaccharide was H4N2,and the glycosylation site was N1563.