Study on the immunoreactivity of mannose-parvalbumin conjugate

In order to determine the influence of Maillard reaction on the structural and immunological properties of parvalbumin, the major allergen of silver carp (Hypophthalmichthys molitrix), recombinant silver carp PV (rPV) was used and incubated with mannose at 100 °C for 100 min, and glycated PV (M-rPV) was then obtained after dialysis. Tricine-SDS-PAGE and Dot-blotting was performed to elucidate the change of mobility and immunoreactivity of rPV after glycation. Simulated gastrointestinal digestion experiment was carried out to compare the digestibility of rPV and M-rPV. Circular dichroism spectroscopy (CD) and scanning electron microscopy (SEM) were used to determine the change of the secondary structure and the microstructure of rPV after Maillard reaction. The results showed that M-rPV had significantly high amounts of protein-bound mannose of approximately 150 μg per microgram M-rPV as measured by the anthrone sulfuric acid method. Dimers and polymers formed in the glycated sample, which might be attributed to the covalent bonds formed during Maillard reaction as analyzed by reducing Tricine-SDS-PAGE. Formation of compact aggregates after Maillard reaction was further confirmed by SEM, which might block the access of immunoglobulins to the epitopes, and therefore decrease the immunoreactivity of M-rPV, as demonstrated by dot-blotting using mouse anti-silver carp parvalbumin monoclonal antibody. M-rPV exhibited less resistance against gastrointestinal digestion in vitro as revealed by Tricine-SDS-PAGE and dot-blotting. Maillard reaction also showed a minor effect on the secondary structures of rPV with a decrease of α-helix and an increase of β-strand content. The aggregation formation and change in the secondary structure of M-rPV might explain the decrease of rPV immunoreactivity and digestibility after Maillard reaction. Our work might be useful for understanding the effects of Maillard reaction on the decrease of immunoreactivity of food allergens in general.