title: Mutational analysis of amino acid positions crucial for IgE-binding epitopes of the major apple (Malus domestica) allergen, Mal d 1.
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.
authors.affiliation: Department of Pathophysiology, Medical University of Vienna, Vienna, Austria.
authors.forename: Yan
authors.initials: Y
authors.lastname: Ma
authors.forename: Gabriele
authors.initials: G
authors.lastname: Gadermaier
authors.forename: Barbara
authors.initials: B
authors.lastname: Bohle
authors.forename: Suzanne
authors.initials: S
authors.lastname: Bolhaar
authors.forename: Andre
authors.initials: A
authors.lastname: Knulst
authors.forename: Zora
authors.initials: Z
authors.lastname: Markovic-Housley
authors.forename: Heimo
authors.initials: H
authors.lastname: Breiteneder
authors.forename: Peter
authors.initials: P
authors.lastname: Briza
authors.forename: Karin
authors.initials: K
authors.lastname: Hoffmann-Sommergruber
authors.forename: Fatima
authors.initials: F
authors.lastname: Ferreira
mesh_terms: D000485:Allergens / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D000595:Amino Acid Sequence; D000918:Antibody Specificity; D052179:Antigens, Plant; D001666:Binding Sites, Antibody / Q000276:immunology; D000939:Epitopes / Q000276:immunology*; D005512:Food Hypersensitivity / Q000276:immunology; D006801:Humans; D007073:Immunoglobulin E / Q000276:immunology*; D027845:Malus / Q000009:adverse effects; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D017354:Point Mutation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D016415:Sequence Alignment
chemical_list: D000485:Allergens; D052179:Antigens, Plant; D000939:Epitopes; C095551:MALD1 protein, Malus domestica; D010940:Plant Proteins; D011994:Recombinant Proteins; D007073:Immunoglobulin E