title: Fold stability during endolysosomal acidification is a key factor for allergenicity and immunogenicity of the major birch pollen allergen.
abstract: The search for intrinsic factors, which account for a protein's capability to act as an allergen, is ongoing. Fold stability has been identified as a molecular feature that affects processing and presentation, thereby influencing an antigen's immunologic properties.
  
  
  
  We assessed how changes in fold stability modulate the immunogenicity and sensitization capacity of the major birch pollen allergen Bet v 1.
  
  
  
  By exploiting an exhaustive virtual mutation screening, we generated mutants of the prototype allergen Bet v 1 with enhanced thermal and chemical stability and rigidity. Structural changes were analyzed by means of x-ray crystallography, nuclear magnetic resonance, and molecular dynamics simulations. Stability was monitored by using differential scanning calorimetry, circular dichroism, and Fourier transform infrared spectroscopy. Endolysosomal degradation was simulated in vitro by using the microsomal fraction of JAWS II cells, followed by liquid chromatography coupled to mass spectrometry. Immunologic properties were characterized in vitro by using a human T-cell line specific for the immunodominant epitope of Bet v 1 and in vivo in an adjuvant-free BALB/c mouse model.
  
  
  
  Fold stabilization of Bet v 1 was pH dependent and resulted in resistance to endosomal degradation at a pH of 5 or greater, affecting presentation of the immunodominant T-cell epitope in vitro. These properties translated in vivo into a strong allergy-promoting TH2-type immune response. Efficient TH2 cell activation required both an increased stability at the pH of the early endosome and efficient degradation at lower pH in the late endosomal/lysosomal compartment.
  
  
  
  Our data indicate that differential pH-dependent fold stability along endosomal maturation is an essential protein-inherent determinant of allergenicity.
authors.affiliation: Department of Molecular Biology, University of Salzburg, Salzburg, Austria.
authors.forename: Yoan
authors.initials: Y
authors.lastname: Machado
authors.affiliation: Department of Molecular Biology, University of Salzburg, Salzburg, Austria.
authors.forename: Regina
authors.initials: R
authors.lastname: Freier
authors.affiliation: Department of Molecular Biology, University of Salzburg, Salzburg, Austria.
authors.forename: Sandra
authors.initials: S
authors.lastname: Scheiblhofer
authors.affiliation: Department of Molecular Biology, University of Salzburg, Salzburg, Austria.
authors.forename: Theresa
authors.initials: T
authors.lastname: Thalhamer
authors.affiliation: Department of Molecular Biology, University of Salzburg, Salzburg, Austria.
authors.forename: Melissa
authors.initials: M
authors.lastname: Mayr
authors.affiliation: Department of Molecular Biology, University of Salzburg, Salzburg, Austria.
authors.forename: Peter
authors.initials: P
authors.lastname: Briza
authors.affiliation: Center of Molecular Biosciences & Institute of Organic Chemistry, University of Innsbruck, Innsbruck, Austria.
authors.forename: Sarina
authors.initials: S
authors.lastname: Grutsch
authors.affiliation: Center of Molecular Biosciences & Institute of Organic Chemistry, University of Innsbruck, Innsbruck, Austria.
authors.forename: Linda
authors.initials: L
authors.lastname: Ahammer
authors.affiliation: Center of Molecular Biosciences & Institute of General, Inorganic and Theoretical Chemistry, University of Innsbruck, Innsbruck, Austria.
authors.forename: Julian E
authors.initials: JE
authors.lastname: Fuchs
authors.affiliation: Center of Molecular Biosciences & Institute of General, Inorganic and Theoretical Chemistry, University of Innsbruck, Innsbruck, Austria.
authors.forename: Hannes G
authors.initials: HG
authors.lastname: Wallnoefer
authors.affiliation: Department of Molecular Biology, University of Salzburg, Salzburg, Austria.
authors.forename: Almedina
authors.initials: A
authors.lastname: Isakovic
authors.affiliation: Department of Molecular Biology, University of Salzburg, Salzburg, Austria.
authors.forename: Vera
authors.initials: V
authors.lastname: Kohlbauer
authors.affiliation: Department of Molecular Biology, University of Salzburg, Salzburg, Austria.
authors.forename: Arthur
authors.initials: A
authors.lastname: Hinterholzer
authors.affiliation: Department of Molecular Biology, University of Salzburg, Salzburg, Austria.
authors.forename: Markus
authors.initials: M
authors.lastname: Steiner
authors.affiliation: Austrian Red Cross, Blood Transfusion Service for Upper Austria, Linz, Austria.
authors.forename: Martin
authors.initials: M
authors.lastname: Danzer
authors.affiliation: Department of Molecular Biology, University of Salzburg, Salzburg, Austria.
authors.forename: Jutta
authors.initials: J
authors.lastname: Horejs-Hoeck
authors.affiliation: Department of Molecular Biology, University of Salzburg, Salzburg, Austria.
authors.forename: Fatima
authors.initials: F
authors.lastname: Ferreira
authors.affiliation: Center of Molecular Biosciences & Institute of General, Inorganic and Theoretical Chemistry, University of Innsbruck, Innsbruck, Austria.
authors.forename: Klaus R
authors.initials: KR
authors.lastname: Liedl
authors.affiliation: Center of Molecular Biosciences & Institute of Organic Chemistry, University of Innsbruck, Innsbruck, Austria.
authors.forename: Martin
authors.initials: M
authors.lastname: Tollinger
authors.affiliation: Department of Molecular Biology, University of Salzburg, Salzburg, Austria.
authors.forename: Peter
authors.initials: P
authors.lastname: Lackner
authors.affiliation: MRC Laboratory of Molecular Biology, Cambridge, United Kingdom.
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Johnson
authors.affiliation: Department of Molecular Biology, University of Salzburg, Salzburg, Austria.
authors.forename: Hans
authors.initials: H
authors.lastname: Brandstetter
authors.affiliation: Department of Molecular Biology, University of Salzburg, Salzburg, Austria.
authors.forename: Josef
authors.initials: J
authors.lastname: Thalhamer
authors.affiliation: Department of Molecular Biology, University of Salzburg, Salzburg, Austria. Electronic address: Richard.Weiss@sbg.ac.at.
authors.forename: Richard
authors.initials: R
authors.lastname: Weiss
mesh_terms: D000485:Allergens / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D000818:Animals; D052179:Antigens, Plant / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D011992:Endosomes; D005260:Female; D006863:Hydrogen-Ion Concentration; D007073:Immunoglobulin E / Q000276:immunology; D007074:Immunoglobulin G / Q000276:immunology; D008807:Mice, Inbred BALB C; D009154:Mutation; D011058:Pollen / Q000276:immunology; D017510:Protein Folding; D055550:Protein Stability
chemical_list: D000485:Allergens; D052179:Antigens, Plant; D007074:Immunoglobulin G; C061312:Bet v 1 allergen, Betula; D007073:Immunoglobulin E
keywords: Allergic sensitization; Bet v 1; antigen processing and presentation; endolysosomal degradation; molecular allergology; structural stability