Comparison of Parvalbumin Reactivity and Levels in Different Fishes
Considering the strong correlation between fish extract-sIgE and parvalbumin-sIgE levels, we next examined if such allergenicity differences in fishes are attributed by (1) differences in IgE binding capacity of different fish parvalbumins, or (2) differences in parvalbumin levels in different fishes. Firstly, 37 serum samples from the sensitized group were assayed for IgE reactivity against purified recombinant parvalbumins from tuna, salmon (β-1 and β-2), cod, grouper, catfish, grass carp and tilapia (Fig 3A). Our data showed that IgE binding did not increase along the allergenicity ladder (Fig 3B). No statistical difference was detected among the tested parvalbumins even when comparing between tuna and tilapia, despite an 11-fold lower tuna-sIgE than tilapia-sIgE. Interestingly, salmon parvalbumin β-1 had remarkably lower IgE binding comparing to all parvalbumins except for tuna parvalbumin while grass carp parvalbumin had significantly stronger IgE binding than parvalbumins from tuna, salmon β-1, cod and tilapia. These results suggested that the difference in allergenicity is not due to IgE binding capacity of parvalbumins.
We next compared parvalbumin levels in the extracted fish protein by SDS-PAGE and densitometry analysis. Parvalbumin appeared as a 9-12 kDa protein in all fish extracts, while two parvalbumin isoforms could be visualized in halibut, grass carp and tilapia (Fig 3C). With reference to salmon parvalbumin (relative intensity = 1), the relative parvalbumin content showed no statistical difference among tuna, halibut and salmon, as well as among catfish, grass carp and tilapia (Fig 3D). Parvalbumin content was significantly higher in cod comparing to salmon, halibut, and tuna, as well as in grouper comparing to cod. It is noted that parvalbumin levels in catfish, grass carp (isoform 2), and tilapia (isoform 2) were significantly higher than that in all other tested fishes. Importantly, the relative levels of parvalbumin positively correlated with the sIgE reactivity of the respective fish (r = 0.925, p =.001) and incident of self-reported allergic reactions (r = 0.89, p =.004). We also further validated the expression of parvalbumins by a transcriptomic approach. The transcriptomic expression level of these parvalbumins was similar to the results from protein-based analysis (Fig 3E). Yet, it is noted that the expression of GAPDH and aldolase were remarkably higher in halibut, yellowfin tuna and salmon comparing to the freshwater fishes, cod and grouper. These results suggested that the difference in allergenicity is due to the amount of parvalbumin in fish muscle.