title: A recombinant hypoallergenic parvalbumin mutant for immunotherapy of IgE-mediated fish allergy.
abstract: IgE-mediated allergy to fish is a frequent cause of severe anaphylactic reactions. Parvalbumin, a small calcium-binding protein, is the major fish allergen. We have recently isolated a cDNA coding for carp parvalbumin, Cyp c 1, and expressed in Escherichia coli a recombinant Cyp c 1 molecule, which contained most IgE epitopes of saltwater and freshwater fish. In this study, we introduced mutations into the calcium-binding domains of carp parvalbumin by site-directed mutagenesis and produced in E. coli three parvalbumin mutants containing amino acid exchanges either in one (single mutants; Mut-CD and Mut-EF) or in both of the calcium-binding sites (double mutant; Mut-CD/EF). Circular dichroism analyses of the purified derivatives and the wild-type allergen showed that Mut-CD/EF exhibited the greatest reduction of overall protein fold. Dot blot assays and immunoblot inhibition experiments performed with sera from 21 fish-allergic patients showed that Mut-CD/EF had a 95% reduced IgE reactivity and represented the derivative with the least allergenic activity. The latter was confirmed by in vitro basophil histamine release assays and in vivo skin prick testing. The potential applicability for immunotherapy of Mut-CD/EF was demonstrated by the fact that mouse IgG Abs could be raised by immunization with the mutated molecule, which cross-reacted with parvalbumins from various fish species and inhibited the binding of fish-allergic patients' IgE to the wild-type allergen. Using the hypoallergenic carp parvalbumin mutant Mut-CD/EF, it may be possible to treat fish allergy by immunotherapy.
authors.affiliation: Institute of Medical and Chemical Laboratory Diagnostics, Department of Pathophysiology, Center for Physiology and Pathophysiology, Medical University of Vienna, Austria. ines.swoboda@meduniwien.ac.at
authors.forename: Ines
authors.initials: I
authors.lastname: Swoboda
authors.forename: Agnes
authors.initials: A
authors.lastname: Bugajska-Schretter
authors.forename: Birgit
authors.initials: B
authors.lastname: Linhart
authors.forename: Petra
authors.initials: P
authors.lastname: Verdino
authors.forename: Walter
authors.initials: W
authors.lastname: Keller
authors.forename: Ulrike
authors.initials: U
authors.lastname: Schulmeister
authors.forename: Wolfgang R
authors.initials: WR
authors.lastname: Sperr
authors.forename: Peter
authors.initials: P
authors.lastname: Valent
authors.forename: Gabriel
authors.initials: G
authors.lastname: Peltre
authors.forename: Santiago
authors.initials: S
authors.lastname: Quirce
authors.forename: Nikolaos
authors.initials: N
authors.lastname: Douladiris
authors.forename: Nikolaos G
authors.initials: NG
authors.lastname: Papadopoulos
authors.forename: Rudolf
authors.initials: R
authors.lastname: Valenta
authors.forename: Susanne
authors.initials: S
authors.lastname: Spitzauer
mesh_terms: D000485:Allergens / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D000818:Animals; D001666:Binding Sites, Antibody / Q000235:genetics; D002347:Carps / Q000276:immunology; D003888:Desensitization, Immunologic / Q000379:methods; D020832:EF Hand Motifs / Q000235:genetics / Q000276:immunology; D005512:Food Hypersensitivity / Q000276:immunology* / Q000628:therapy*; D006801:Humans; D007073:Immunoglobulin E / Q000009:adverse effects* / Q000096:biosynthesis / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010320:Parvalbumins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D017354:Point Mutation; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism
chemical_list: D000485:Allergens; D010320:Parvalbumins; D011994:Recombinant Proteins; D007073:Immunoglobulin E