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Pollen-related food allergy: cloning and immunological analysis of isoforms and mutants of Mal d 1, the major apple allergen, and Bet v 1, the major birch pollen allergen.

Abstract

BACKGROUND

Mal d 1, the major apple allergen, cross-reacts with IgE specific for the major birch pollen allergen, Bet v 1, and is responsible for birch pollen related food allergy to apple. Isoforms of Bet v 1 showing minor sequence variations display different binding capacity for specific IgE antibodies from allergic patients. Moreover, strain-dependent variation of allergenicity has been reported for apples.

OBJECTIVE

To investigate the occurrence of strain-dependent isoforms of Mal d 1 which may differ in their allergenic potential, to obtain data on structures essential for binding of Mal d 1 to the antibody, and to gain insights into the structures responsible for its IgE cross-reactivity to Bet v 1.

METHODS

The cDNA of Mal d 1 from various apple strains was amplified by a PCR strategy based on conserved regions of known Mal d 1-sequences, and sequenced. Two major isoforms of Mal d 1 were expressed as recombinant proteins and purified, as were different variants of the major birch pollen allergen, Bet v 1. Together with already existing recombinant birch pollen and apple allergens, these were subjected to allergenicity testing by IgE-immunoblotting, enzyme allergo sorbent test and dose related mediator release. "Hot-spots" for IgE-reactivity were identified by site-directed mutagenesis.

RESULTS

Twelve Mal d 1-clones were sequenced from 7 apple varieties and compared to 3 known Mal d 1 sequences. The clones were clustered into two groups, each showing a high degree of sequence identity to one of the known sequences and specific differences to the third sequence. No strain-specific sequences were identified. In contrast, apple strains with reported differences in allergenicity showed different expression levels of the major allergen. Immunologic testing of recombinant allergens revealed high IgE binding capacity of 2 major isoforms, named GD26 and GS29, with a slightly higher IgE binding capacity of GD26. Moreover, the allergenicity was similar to another r Mal d 1 reported in the literature, representing the isoform divergent from our clones. Mutational analysis of our Mal d 1 allergens identified serine in position 111 as essential for IgE binding. Allergenicity was almost depleted by changing this residue into a proline. Moreover, the corresponding serine residue, present in position 112 of Bet v 1, was in a similar manner crucial for the allergenicity of the birch pollen allergen.

CONCLUSION

We conclude that divergent allergenicity of apple strains mainly depends on different expression levels of the major allergen. Introduction of a proline residue in position 111 of Mal d 1 and in position 112 of Bet v 1 led to a drastic reduction of allergenicity of both the pollen and the food allergen, obviously also removing the cross-reactive epitope. Mutants with reduced IgE-reactivity but maintained T-cell reactivity may represent new candidates for a safer specific immunotherapy with reduced side-effects.

Links

  • gene/protein/disease-specific
  • Authors+Show Affiliations

    ,

    Paul-Ehrlich-Institut, Department of Allergology, Paul-Ehrlich-Str. 51-59, D.-63225 Langen, Germany.

    , , ,

    Source

    European journal of nutrition 38:4 1999 Aug pg 201-15

    MeSH

    Allergens
    Amino Acid Sequence
    Animals
    Cloning, Molecular
    DNA
    DNA Primers
    Epitopes
    Food Hypersensitivity
    Fruit
    Humans
    Immunoglobulin E
    Mice
    Mice, Inbred BALB C
    Molecular Sequence Data
    Mutagenesis, Site-Directed
    Pollen
    Protein Isoforms
    RNA
    Random Amplified Polymorphic DNA Technique
    Reverse Transcriptase Polymerase Chain Reaction
    Sequence Alignment
    Sequence Analysis, DNA
    Sequence Homology, Amino Acid
    Trees

    Pub Type(s)

    Journal Article
    Research Support, Non-U.S. Gov't

    Language

    eng

    PubMed ID

    10502033

    Citation

    Son, D Y., et al. "Pollen-related Food Allergy: Cloning and Immunological Analysis of Isoforms and Mutants of Mal D 1, the Major Apple Allergen, and Bet V 1, the Major Birch Pollen Allergen." European Journal of Nutrition, vol. 38, no. 4, 1999, pp. 201-15.
    Son DY, Scheurer S, Hoffmann A, et al. Pollen-related food allergy: cloning and immunological analysis of isoforms and mutants of Mal d 1, the major apple allergen, and Bet v 1, the major birch pollen allergen. Eur J Nutr. 1999;38(4):201-15.
    Son, D. Y., Scheurer, S., Hoffmann, A., Haustein, D., & Vieths, S. (1999). Pollen-related food allergy: cloning and immunological analysis of isoforms and mutants of Mal d 1, the major apple allergen, and Bet v 1, the major birch pollen allergen. European Journal of Nutrition, 38(4), pp. 201-15.
    Son DY, et al. Pollen-related Food Allergy: Cloning and Immunological Analysis of Isoforms and Mutants of Mal D 1, the Major Apple Allergen, and Bet V 1, the Major Birch Pollen Allergen. Eur J Nutr. 1999;38(4):201-15. PubMed PMID: 10502033.
    * Article titles in AMA citation format should be in sentence-case
    TY - JOUR T1 - Pollen-related food allergy: cloning and immunological analysis of isoforms and mutants of Mal d 1, the major apple allergen, and Bet v 1, the major birch pollen allergen. AU - Son,D Y, AU - Scheurer,S, AU - Hoffmann,A, AU - Haustein,D, AU - Vieths,S, PY - 1999/9/29/pubmed PY - 1999/9/29/medline PY - 1999/9/29/entrez SP - 201 EP - 15 JF - European journal of nutrition JO - Eur J Nutr VL - 38 IS - 4 N2 - BACKGROUND: Mal d 1, the major apple allergen, cross-reacts with IgE specific for the major birch pollen allergen, Bet v 1, and is responsible for birch pollen related food allergy to apple. Isoforms of Bet v 1 showing minor sequence variations display different binding capacity for specific IgE antibodies from allergic patients. Moreover, strain-dependent variation of allergenicity has been reported for apples. OBJECTIVE: To investigate the occurrence of strain-dependent isoforms of Mal d 1 which may differ in their allergenic potential, to obtain data on structures essential for binding of Mal d 1 to the antibody, and to gain insights into the structures responsible for its IgE cross-reactivity to Bet v 1. METHODS: The cDNA of Mal d 1 from various apple strains was amplified by a PCR strategy based on conserved regions of known Mal d 1-sequences, and sequenced. Two major isoforms of Mal d 1 were expressed as recombinant proteins and purified, as were different variants of the major birch pollen allergen, Bet v 1. Together with already existing recombinant birch pollen and apple allergens, these were subjected to allergenicity testing by IgE-immunoblotting, enzyme allergo sorbent test and dose related mediator release. "Hot-spots" for IgE-reactivity were identified by site-directed mutagenesis. RESULTS: Twelve Mal d 1-clones were sequenced from 7 apple varieties and compared to 3 known Mal d 1 sequences. The clones were clustered into two groups, each showing a high degree of sequence identity to one of the known sequences and specific differences to the third sequence. No strain-specific sequences were identified. In contrast, apple strains with reported differences in allergenicity showed different expression levels of the major allergen. Immunologic testing of recombinant allergens revealed high IgE binding capacity of 2 major isoforms, named GD26 and GS29, with a slightly higher IgE binding capacity of GD26. Moreover, the allergenicity was similar to another r Mal d 1 reported in the literature, representing the isoform divergent from our clones. Mutational analysis of our Mal d 1 allergens identified serine in position 111 as essential for IgE binding. Allergenicity was almost depleted by changing this residue into a proline. Moreover, the corresponding serine residue, present in position 112 of Bet v 1, was in a similar manner crucial for the allergenicity of the birch pollen allergen. CONCLUSION: We conclude that divergent allergenicity of apple strains mainly depends on different expression levels of the major allergen. Introduction of a proline residue in position 111 of Mal d 1 and in position 112 of Bet v 1 led to a drastic reduction of allergenicity of both the pollen and the food allergen, obviously also removing the cross-reactive epitope. Mutants with reduced IgE-reactivity but maintained T-cell reactivity may represent new candidates for a safer specific immunotherapy with reduced side-effects. SN - 1436-6207 UR - https://www.unboundmedicine.com/medline/citation/10502033/Pollen_related_food_allergy:_cloning_and_immunological_analysis_of_isoforms_and_mutants_of_Mal_d_1_the_major_apple_allergen_and_Bet_v_1_the_major_birch_pollen_allergen_ L2 - http://www.iedb.org/pmId/10502033 DB - PRIME DP - Unbound Medicine ER -