Identification of a Dau c PRPlike protein (Dau c 1.03) as a new allergenic isoform in carrots (cultivar Rodelika)

Background Up to 25% of food allergic subjects in central Europe suffer from carrot allergy. Until now, two isoforms of the major carrot (Daucus carota) allergen Dau c 1 have been described: Dau c 1.01, comprising five variants (Dau c 1.0101-Dau c 1.0105) and Dau c 1.02. Objective To investigate potential allergenic properties of a Dau c PRPlike protein, a novel isoform of the PR-10 protein family in carrot. Methods Dau c PRPlike cDNA from carrot roots (cv Rodelika) was cloned after RT-PCR and 5' RACE. Dau c PRPlike protein was expressed in E. coli, purified under native conditions by Ni-NTA chromatography and analysed by CD spectroscopy. Immuno-reactivity of the rDau c PRPlike protein was compared with rDau c 1.0104 and rDau c 1.0201 in terms of IgE binding (immunoblotting, ImmunoCAP T), IgE cross-reactivity (ELISA inhibition) and in vitro mediator release with sera from carrot allergic patients. mRNA expression of Dau c PRPlike protein in wild-type and transgenic carrot roots was analysed by qRT-PCR. Results The Dau c PRPlike protein was identified as a new allergenic isoform, Dau c 1.03, in carrot roots. 68% of carrot allergic patients were sensitized to rDau c 1.03. The IgEreactivity of rDau c 1.03 strongly correlated with reactivity to rDau c 1.0104, but not to rDau c 1.0201. The extent of IgE cross-reactivity and allergenic potency of Dau c 1 isoforms varied between the individual sera tested. Dau c 1.03 mRNA transcripts were up-regulated in Dau c 1.01 and Dau c 1.02 gene-silenced carrot roots. Conclusion and clinical relevance Dau c 1 isoforms display distinct IgE epitope heterogeneity. Dau c 1.03 appears to contribute to the allergenicity of carrots and the manifestation of carrot allergy. The epitope diversity of different Dau c 1 isoforms should be considered for component-resolved diagnosis and gene silencing of carrot allergens.