Mutational epitope analysis and cross-reactivity of two isoforms of Api g 1, the major celery allergen
Introduction
Allergy to celery tuber (celeriac) is a common plant food allergy in Europe, in particular in Switzerland, France and Germany. In Switzerland, it ranks among the most prevalent food allergies (Etesamifar and Wüthrich, 1998) and has been reported as the most frequent cause of food anaphylaxis (Rohrer et al., 1998). Approximately 50% of celery allergic subjects in Switzerland present a case history of systemic allergic reactions (Ballmer-Weber et al., 2000, Luttkopf et al., 2000). Clinically, celery allergy is associated to both birch pollen and mugwort pollen allergy, which is due to the existence of cross-reacting allergens in the respective pollen, which are also thought to represent the sensitizing allergens. The known allergens of celery tuber are the major allergen Api g 1.01 (Breiteneder et al., 1995) and its isoform Api g 1.02 (Hoffmann-Sommergruber et al., 2000), the minor allergen profilin Api g 4 (Scheurer et al., 2000) and Api g 5 (Bublin et al., 2003) a protein showing homology to FAD (flavin adenine dinucleotide) containing oxidases. The allergenicity of Api g 5 depends on N-glycans containing xylose and fucose residues and does not depend on the protein sequence. Several studies have shown that Api g 1 is the most important allergen in celery tuber (Breiteneder et al., 1995, Vieths et al., 1995, Hoffmann-Sommergruber et al., 1999, Luttkopf et al., 2000). The clinical presentation of celery allergy differs from other pollen related food allergies and appears to include more severe symptoms than oral allergy syndrome (Mari et al., 2005). Moreover, clinical reactivity to heat-processed celery has been confirmed by double-blind, placebo-controlled food challenges (Ballmer-Weber et al., 2002). Therefore, studying the IgE epitope structure of Api g 1 and IgE binding to its isoforms is of particular interest, because a specific set of epitopes or different stability characteristics could be responsible for a different clinical reactivity to this Bet v 1 related allergen. Due to the polyclonal nature of the IgE response, it is difficult to analyse directly the structure of immune complexes between IgE and allergens. Therefore, most studies on IgE epitopes of Bet v 1 related allergens have focused on site-directed mutagenesis (Ferreira et al., 1998, Scheurer et al., 1999, Neudecker et al., 2003, Wiche et al., 2005, Ma et al., 2006) or, more recently on direct affinity selection and enrichment of IgE binding surface structures utilising libraries of peptide mimics (Mittag et al., 2006). In the present study we focused on the “P-loop” which has been suggested as a major epitope area on Bet v 1 (Mirza et al., 2000) and homologous food allergens (Holm et al., 2001, Neudecker et al., 2003, Mittag et al., 2006). In addition, we found that introduction of a proline residue in position 112 of Bet v 1, or equivalent position of homologous food allergens led to an almost complete reduction of IgE binding capacity of Bet v 1 and the apple allergen Mal d 1 (Son et al., 1999) as well as the cherry allergen Pru av 1 (Scheurer et al., 1999, Neudecker et al., 2003) which could in the case of Pru av 1 be attributed to a complete loss of secondary structure. Therefore, the relevance of this position for the allergenicity of Api g 1 was also studied. In an initial screening the relevance of Api g 1.01 and Api g 1.02 was analysed for the first time in a study population in which food allergy to celery had been confirmed by double-blind, placebo-controlled food challenges. Our data revealed that the epitope structure of Api g 1 and its cross-reactivity of Bet v 1 with Api g 1 isoforms is complex and that mutation of positions equivalent to amino acid 112 of Bet v 1 has a significant impact on the structural stability of Api g 1 isoforms.
Section snippets
Patients sera
In total 66 sera were included in this study. Twenty-three were from patients with a confirmed food allergy to celery tuber (celeriac) as demonstrated by a positive reaction in a double-blind, placebo-controlled food challenge (DBPCFC) with celery. Detailed clinical data such as case history of adverse reactions to celery and symptoms in response to DBPCFC as well as skin tests and serological data have been published elsewhere (Ballmer-Weber et al., 2000, Ballmer-Weber et al., 2002, Luttkopf
Rationale of the study
The average amino acid sequence identity between the two Api g 1 isoforms and Bet v 1 is only about 40%. In addition, the two isoforms share only 50% sequence identity at the amino acid level, whereas the P-loop region (amino acid position 44–53) is highly conserved in Bet v 1 homologous allergens (Neudecker et al., 2003). Interestingly, Api g 1.01 differs from Api g 1.02 and Bet v 1 in this region. The latter two carry a negatively charged glutamic acid (Glu45) at amino acid position 45,
Discussion
Structures that bind IgE are relevant for clinical reactivity and changes in epitope structures may lead to drastically different IgE binding. Therefore we wanted to investigate the influence of directed mutation of Api g 1 isoforms on their IgE binding capacity and analysed their cross-reactivity to the homologous allergen from birch pollen, Bet v 1.
Up to now only one comparative study on the IgE binding capacity of Api g 1 isoforms has been performed. This study analysed sera from patients
Acknowledgements
The authors would like to thank Drs Jonas Lidholm and Kerstin Andersson, Phadia, Uppsala, Sweden, for providing recombinant Api g 1.02.01, and for critical discussion of the data and Dr. Gerald Reese for helping with curve-interpretation and statistical evaluation. This work was funded by the Deutsche Forschungsgemeinschaft (Grant No. DFG VI 165/2-5 to SV).
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