Elsevier

Molecular Immunology

Volume 44, Issue 10, April 2007, Pages 2518-2527
Molecular Immunology

Mutational epitope analysis and cross-reactivity of two isoforms of Api g 1, the major celery allergen

https://doi.org/10.1016/j.molimm.2006.12.023Get rights and content

Abstract

For better understanding the cross-reactivity between the major birch pollen and celery allergens, Bet v 1 and Api g 1, respectively, putative epitope areas and structurally important positions for IgE-binding of the isoforms Api g 1.01 and Api g 1.02 were point mutated. The IgE binding capacities were measured in ELISA, the IgE cross-reactivity between the isoforms, mutants and Bet v 1 investigated by ELISA-inhibition experiments with serum pools from patients with confirmed celery allergy (DBPCFC). Api g 1.01 displayed a clearly higher frequency and capacity of IgE binding than Api g 1.02. In Api g 1.01, substitution of lysine against glutamic acid at amino acid position 44, a key residue of the Bet v 1 “P-loop”, increased the IgE-binding properties. Structural instability due to proline insertion at position 111/112 resulted in loss of IgE binding of Api g 1.01, but not of Api g 1.02. Between Api g 1.01 and Api g 1.02 only partial cross-reactivity was seen. The data suggest that the IgE epitopes of the two isoforms are distinct and that in contrast to Api g 1.01, the “P-loop” region plays an important role for IgE binding of celery allergic subjects to Api g 1.02. Understanding and investigation of the molecular mechanisms in celery allergy is an important step to generate hypoallergenic proteins for safe and efficacious immunotherapy of food allergy.

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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