Purification, biochemical, and immunological characterisation of a major food allergen: different immunoglobulin E recognition of the apo- and calcium-bound forms of carp parvalbumin

Abstract

Background-Almost 4% of the population suVer from food allergy which is an adverse reaction to food with an underlying immunological mechanism. Aims-To characterise one of the most frequent IgE defined food allergens, fish parvalbumin. Methods-Tissue and subcellular distribution of carp parvalbumin was analysed by immunogold electron microscopy and cell fractionation. Parvalbumin was purified to homogeneity, analysed by mass spectrometry and circular dichroism (CD) spectroscopy, and its allergenic activity was analysed by IgE binding and basophil histamine release tests. Results-The isoelectric point (pI) 4.7 form of carp parvalbumin, a three EFhand calcium-binding protein, was purified to homogeneity. CD analysis revealed a remarkable stability and refolding capacity of calcium-bound parvalbumin. This may explain why parvalbumin, despite cooking and exposure to the gastrointestinal tract, can sensitise patients. Purified parvalbumin reacted with IgE of more than 95% of individuals allergic to fish, induced dose-dependent basophil histamine release and contained, on average, 83% of the IgE epitopes present in other fish species. Calcium depletion reduced the IgE binding capacity of parvalbumin which, according to CD analysis, may be due to conformationdependent IgE recognition. Conclusions-Purified carp parvalbumin represents an important cross reactive food allergen. It can be used for in vitro and in vivo diagnosis of fish-induced food allergy. Our finding that the apo-form of parvalbumin had a greatly reduced IgE binding capacity indicates that this form may be a candidate for safe immunotherapy of fish-related food allergy. (Gut 2000;46:661-669)

Recombinant Carp Parvalbumin, the Major Cross-Reactive Fish Allergen: A Tool for Diagnosis and Therapy of Fish Allergy

“…4B). The spectrum of the purified recombinant allergen thus resembled the features of that of purified natural carp parvalbumin (25). rCyp c 1.01 represents a folded protein containing a considerable amount of ␣-helical secondary structure.…”

“…Reactivities of recombinant carp parvalbumin to serum IgE from fish allergic patients and to an anti-parvalbumin mAb were determined by immunoblot analyses as described previously (25). For immunoblot inhibition experiments, sera from fish-allergic patients were preincubated with purified recombinant parvalbumin (10 g/ml of 1/10 diluted serum).…”

“…It was further shown that patients who mount IgE Abs against one parvalbumin will cross-react with the homologous proteins from other fish species (24), which demonstrates the importance of parvalbumins as cross-reactive fish allergens and explains why allergic individuals exhibit clinical symptoms upon contact with various fish species. IgE competition experiments performed with purified carp parvalbumin indicated that this molecule contained a large portion of IgE epitopes present in various fish species (25).…”

A Human Monoclonal IgE Antibody Defines a Highly Allergenic Fragment of the Major Timothy Grass Pollen Allergen, Phl p 5: Molecular, Immunological, and Structural Characterization of the Epitope-Containing Domain

“…Although there seems to be no common structural theme that would determine the allergenic activity of a given protein, it turned out that proper folding and high propensity to refold after denaturation are frequent features of potent allergens (12,13,15,(22)(23)(24)(25).…”

“…Epitopes composed of at least two nonadjacent domains of the molecule that are brought into close proximity within the folded molecule are named "discontinuous epitopes" (7). IgE epitopes of several relevant allergens, e.g., the major birch pollen allergen Bet v 1 (8), the calcium-binding pollen allergens, Bet v 3 (9), Bet v 4 (10,11), Phl p 7 (12), Aln g 4 (13), Bra r 1 (14), and parvalbumin (15), belong to the discontinuous type. Continuous IgE epitopes were identified on several grass pollen allergens, e.g., major rye grass pollen allergens Lol p 1 (16) and Lol p 5 (17), major timothy grass pollen allergen Phl p 1 (18,19), and velvet grass allergen Hol l 1 (20).…”