Reduction in the IgE reactivity of Pacific mackerel parvalbumin by mutations at Ca2+-binding sites
Parvalbumin is a sarcoplasmic Ca2+-binding protein of 12 kDa and represents the major fish allergen. Several peptide segments are identified as immunoglobulin E (lgE)-binding epitopes of cod parvalbumin. However, carp parvalbumin (Cyp c 1) shows a markedly reduced IgE-binding ability upon depletion of Ca2+, suggesting the importance of conformational epitopes associated with Ca2+-chelating. In this study, the IgE reactivity of Pacific mackerel Scomber japonicus parvalbumin (Sco j 1) was demonstrated to be markedly reduced (60–100% reduction) by Ca2+-depletion, similar to Cyp c 1. Three Sco j 1 mutants (D51A, D90A, D51/90A), with modifications in either one or both of the two Ca2+-binding sites, were then constructed by site-directed mutagenesis, followed by expression in Escherichia coli, and evaluated for their IgE reactivity. Interestingly, the double mutant (D51/90A), probably devoid of Ca2+-binding capacity, exhibited a significantly reduced IgE reactivity (equivalent to 0.0–7.5% of the IgE reactivity of natural Sco j 1). The results suggest that the IgE-binding ability of Sco j 1 largely depends on the solid conformation mediated by Ca2+-chelating, and that the hypoallergenic D51/90A will be a useful tool for the specific immunotherapy of fish allergy.
Key wordsCa2+-binding IgE reactivity mutant Pacific mackerel parvalbumin
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