Mutational analysis of amino acid positions crucial for IgE-binding epitopes of the major apple (Malus domestica) allergen, Mal d 1

Int Arch Allergy Immunol. 2006;139(1):53-62. doi: 10.1159/000089756. Epub 2005 Nov 15.

Abstract

Background: Individual amino acid residues of the major birch pollen allergen, Bet v 1, have been identified to be crucial for IgE recognition. The objective of the present study was to evaluate whether this concept was applicable for the Bet v 1-homologous apple allergen, Mal d 1.

Methods: A Mal d 1 five-point mutant was produced by PCR techniques, cloned into pMW 172 and expressed in Escherichia coli BL21(DE3) cells. To evaluate the allergenic properties of the engineered protein compared to Mal d 1 wild-type IgE immunoblotting, ELISA, peripheral blood monocytes proliferation assays, and skin prick tests were performed.

Results: The Mal d 1 mutant showed reduced capacity to bind specific IgE as compared to wild-ype Mal d 1 in in vitro assays in the majority of the sera tested. In ELISA, 10 out of 14 serum samples displayed an 88-30% decrease in IgE binding to Mal d 1 mutant compared to wild-type Mal d 1. Skin prick tests in apple-allergic patients (n = 2) confirmed the markedly decreased ability of the Mal d 1 mutant to induce allergic reactions in vivo. However, the relevant T cell epitopes were present in the mutated molecule according to peripheral blood mononuclear cell proliferation assays.

Conclusions: Our findings suggest that it is possible to modulate the IgE-binding properties of allergens by single amino acid substitutions at crucial positions which might be useful for future immunotherapy of birch-pollen-associated food allergies which are not ameliorated by birch pollen immunotherapy.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Allergens / chemistry
  • Allergens / genetics
  • Allergens / immunology*
  • Allergens / metabolism
  • Amino Acid Sequence
  • Antibody Specificity
  • Antigens, Plant
  • Binding Sites, Antibody / immunology
  • Epitopes / immunology*
  • Food Hypersensitivity / immunology
  • Humans
  • Immunoglobulin E / immunology*
  • Malus / adverse effects
  • Models, Molecular
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Plant Proteins / chemistry
  • Plant Proteins / genetics
  • Plant Proteins / immunology*
  • Plant Proteins / metabolism
  • Point Mutation
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / genetics
  • Recombinant Proteins / immunology
  • Sequence Alignment

Substances

  • Allergens
  • Antigens, Plant
  • Epitopes
  • MALD1 protein, Malus domestica
  • Plant Proteins
  • Recombinant Proteins
  • Immunoglobulin E