Abstract
Immunoglobulin E (IgE)-associated allergy affects more than 25% of the population. Can f 1 is the major dog allergen associated with respiratory symptoms but the epitopes recognized by allergic patients IgE on Can f 1 are unknown. To characterize IgE epitopes of Can f 1 recognized by dog allergic patients, six overlapping peptides spanning the Can f 1 sequence were synthesized. In direct IgE epitope mapping experiments peptides were analyzed for IgE reactivity by dot blot and Enzyme-linked immunosorbent assay (ELISA) with sera from dog allergic patients. For indirect epitope-mapping, rabbits were immunized with the peptides to generate specific IgG antibodies which were used to inhibit allergic patients’ IgE binding to Can f 1. IgE binding sites were visualized on a model of the Can f 1 three-dimensional structure. We found that Can f 1 does not contain any relevant sequential IgE epitopes. However, IgE inhibition experiments with anti-peptide specific IgGs showed that Can f 1 N- and C-terminal portion assembled a major conformational binding site. In conclusion, our study is the first to identify the major IgE epitope-containing area of the dog allergen Can f 1. This finding is important for the development of allergen-specific treatment strategies.
Highlights
While the T cell response of allergic patients to Can f 1 has been studied in great detail using overlapping peptides[14,15] almost no information is available regarding the binding of allergic patients’ Immunoglobulin E (IgE) to Can f 1
The sequence of rCan f 1 that was expressed is identical to the sequence that was deposited in the International Union of Immunological Societies (IUIS) database except for one single amino acid difference (W instead of R at position 130 in the Can f 1 sequence) (Fig. 1)
We found that dog allergic patients showed IgE reactivity only to complete and folded Can f 1 but not to any of the peptides, neither as isolated peptides nor when coupled to Keyhole Limpet Hemocyanin (KLH)
Summary
While the T cell response of allergic patients to Can f 1 has been studied in great detail using overlapping peptides[14,15] almost no information is available regarding the binding of allergic patients’ IgE to Can f 1. As exemplified for a recombinant grass pollen allergy vaccine, non-allergenic peptides derived from the IgE binding sites of the major four grass pollen allergens have been grafted onto the PreS protein from hepatitis B as carrier[23]. This vaccine, BM32, has been shown to have no allergenic activity in vitro and in vivo by skin testing of allergic patients[24]. Peptide-specific antibodies were used for competing with the allergic patients’ IgE binding to Can f 1 to search for conformational/discontinuous IgE epitopes. These two approaches combined with a molecular modelling approach enabled us for the first time to identify the major IgE binding site as a conformational epitope on the surface of a 3-dimensional model of Can f 1
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