Purification,Property Analysis And Phosphorylation Modification Of Parvalbumin

Fish play important roles in human nutrition and health,but it triggers severe allergic reactions in some population.Fish allergy is a serious public health problem that causes severe allergy reaction even lead to death for high risk populations.Reduction of allergenicity of fish products by proper physical,chemical or biological processing methods has attracted more and more attention.Phosphorylation reaction is an effective way to improve food protein features,by the method of introducing negatively charged phosphate groups,by which protein secondary structures and hydrophobicity arechanged,and impacts on food allergens' immune activity.The aim of this work was to establish and optimize the phosphorylation reaction conditions of parvalbumin(PV),the major allergen of silver carp(Hypophthalmichthys molitrix),and to determine the influence of phosphorylation reaction on the structural and immunological properties of PV.By mixing PV with D-Glucose 6-phosphate disodium salt hydrate(G6P-Na2),phosphorylation of PV was carried out by dry-heating treatment at different concentration,duration and temperatures.The results indicated that the optimal condition for phosphorylation reaction of PV was at 80°C for 80 min with the ratio of PV to G6P-Na2 as 1:4(weight ratio).Dimers and polymers were formed in the phosphorylated sample,which might be attributed to the covalent bonds formed during phosphorylation reaction as analyzed by Tricine-SDS-PAGE.Formation of compact aggregates after phosphorylation reaction was further confirmed by scanning electron microscopy(SEM),which might block the access of immunoglobulins to the epitopes,and therefore decrease the antigenicity of G6P-Na2-PV,which was demonstrated by dot-blotting using mouse anti-silver carp PV monoclonal antibody.Circular dichroism spectroscopy(CD)analysis revealed that phosphorylation reaction affected the secondary structures of PV significantly.Furthermore,surface hydrophobicity of PV was measured using hydrophobic fluorescence probes,8-anilino-1-naphthalenesulfonate(ANS).Phosphorylation can significantly reduce the phagocytosis of dendritic cells and the immune activity after digestion of simulated gastric fluid.The results revealed that the hydrophobicity of phosphorylated PV was enhanced,as the fluorescence intensity increased.The aggregation formation and change in the secondary structures of G6P-Na2-PV might explain the decrease of PV antigenicity after phosphorylation reaction.Our present work strongly suggest the effects of phosphorylation reaction on the decrease of antigenicity of food allergens.While IgE cross-reactivity to PV in various bony fish species has been well characterized,little information is available about allergens in cartilaginous fish.In this study,two shark PV isoforms(named as SPV-I and SPV-II)from Mustelus griseus were purified.Their identities were further confirmed by mass spectroscopic analysis and two-dimensional electrophoresis.Ig E immunoblot analysis showed that sera from fish-allergic patients reacted to both SPV-I and SPV-II,but the majority of sera reacted more intensely to SPV-I than SPV-II.Thermo denaturation monitored by CD spectrum showed that both of the SPV allergens are highly thermo-stable.SPV-I maintained the allergenicity after heat denaturation,while the IgE-binding ability of SPV-II reduced significantly.The results of crystal structure showed that SPV-I and SPV-II were similar in their overall tertiary structure,but their amino acid sequences shared lower similarities,indicating that the differences of SPV-I and SPV-II allergenicities might be due to differential antigen epitopes in these two isoforms.This is the first report on the characterization and comparison of PVs of two lineages.Furthermore,phosphorylation of SPV-I and SPV-II reduced the IgG and Ig E binding activities,but increased the hydrophobicity of these isoforms.When compared with SPV-II,phosphorylation reaction had less effect on the secondary structure of SPV-I.These results confirmed that the phosphorylation reaction had the same effect on the bony fish PV and the cartilaginous fish PV.In this study,the conditions of phosphorylation reaction between silver carp PV and G6P-NA2 were established.In addition,two subtypes of PV in Mustelus griseus were purified and analyzed for physicochemical properties and crystal structure.It was also confirmed that the phosphorylation reaction had an impact on the PV immunoreactivities and quarternary structures in both bony and cartilaginous fish.The results of this study had great value in understanding fish allergens and their control strategies,development and production of hypoallergenic or non-allergic fish foods and ensuring the safety of consumers.