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WHAT SHAPES AN ALLERGEN?
Enzymatic Activity, Ligands, Post-translational Modifications or Proteolytic Susceptibility … WHAT SHAPES AN ALLERGEN? Fátima Ferreira Department of Molecular Biology University of Salzburg, Austria
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The diversity and complexity of allergen sources
X-raying allergen sources Allergen Molecules
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ALLERGENS: KEY FACTS Derive from a variety of environmental sources Display a broad range of structures and functions in their respective sources (e.g. structural, enzymatic, ligand-binding) Represent a small fraction of proteins that humans are exposed to The same proteins behave as allergens across the human population Natural exposure: allergens are delivered as complex mixtures of molecules How do such diverse proteins preferentially induce Th2-biased immune responses?
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ALLERGEN Barrier penetration / capture
Allergen source EARLY EVENTS IN ALLERGIC SENSITIZATION Allergen ALLERGEN Barrier penetration / capture Th2 differentiation Internalization by APCs, processing and presentation to naïve T cells Epithelial cells DC DC Migration T DC B Naïve T cell Th2 IgE IL-4 IL-13 B B
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Allergen – epithelial interactions and allergic sensitization
CURRENT CONCEPTS Allergen – epithelial interactions and allergic sensitization ALLERGEN Barrier penetration / capture Th2 differentiation Internalization, processing and presentation to naïve T cells Engagement of PRRs Gandhi & Vliagoftis, Front Immunol 2015
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Intrinsic structural features
WHAT SHAPES AN ALLERGEN? Intrinsic structural features Antigen/allergen Processing and Presentation Protein Fold and Stability Molecular dynamics pH Temperature Proteolytic susceptibility Post-translational modifications Glycosylation Disulfide bonds Proteolytic processing Chemical modifications (nitration, deamidation) Oligomerization Dimers Oligomers Biochemical Function Enzyme activity (protease) Ligand-binding (lipids, ions) Receptor binding/activation (PARs, TLR, MR, CLR)
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An in vitro system to simulate antigen/allergen processing
The Degradome Assay: An in vitro system to simulate antigen/allergen processing of the antigen
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An in vitro system to simulate antigen/allergen processing
The Degradome Assay An in vitro system to simulate antigen/allergen processing 1. Preparation of endolysosomal fraction Homogenization Centrifugation Post-nuclear supernatant Microsomal fraction Ultracentrifugation Endolysosomal Proteins/proteases DCs Freeze-thaw cycles 2. In vitro degradation (pH 4.5) + APC-derived endolysosomal proteases Antigen/Allergen 0h 2h 3h 0.5h 1h 4h 5h 48h 3. Analysis SDS-PAGE/ Scanner densitometry Capillary HPLC/ESI-QTOF Mass Spectrometry - Intact protein - Peptide sequencing (MS/MS) Egger et al, PLoS One 2011
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Zaborsky et al (2010). J Immunol 184: 725-735
Model allergens: Pairs of structurally related proteins with different immunogenic activity Bet v (Bet v 1d) Bet v (Bet v 1a) activates DCs much better (elevated levels of CD80 and CD86; reduced IL-6 production) and induces stronger IgG and IgA responses in mice3 differs from Bet v by 7 amino acids (sequence homology of 95%) Bet v 1a Bet v 1d The pairs of model antigens, we used for digestion were two different ribonucleases, horseraddish peroxidases, and isoformas of the birch pollen major allergen Bet v 1. RNase S differs from RNase A only by a single subtilisin-mediated cleavage in the peptide backbone between Alanin 20 and Serin 21. This structural difference leads to a more than 10,000-fold stronger T cell and IgG response in mice. Apo-HRP from which the Ca2+ ion and the prosthetic heme group have been removed induces much weaker T cell and IgG responses in mice than fully equipped Holo-HRP. As said, the Bet v 1d isoform of the birch pollen major allergen Bet v 1 leads to stronger DC activation and humoral responses than the Bet v 1a isoform that differs from Bet v 1d by only 7 amino acid residues. Zaborsky et al (2010). J Immunol 184: Egger et al, PLoS One 2011
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The “Degradome” assay - a tool to predict the immunogenicity of protein antigens
Natural Pru p 3 (nPru p 3) Reduced and alkylated Pru p 3 (R/A Pru p 3) Toda et al., JACI 2011
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The “Degradome” assay - a tool to predict the immunogenicity of protein antigens
Natural Pru p 3 (nPru p 3) Degradome ½ life: Approx. 12 hours Reduced and alkylated Pru p 3 (R/A Pru p 3) Degradome ½ life: approx. 2 hours Toda et al., JACI 2011
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Structural features of allergens and proteolytic susceptibility
LIGAND-BINDING Kofler et al., J Mol Biol 2012; Asam et al., CEA 2014 Grutsch et al., Biophys J 2014 NITRATION Ackaert et al., PlosOne 2014 Bet v 1a (monomer) Bet v 1d (dimer) DIMERIZATION Zaborsky et al., J Immunol 2010; Egger et al., PlosOne 2011 Kofler et al., J Biol Chem 2014 Bet v 1a T. Thermophilus homologue FOLD STABILITY Wallner et al., JACI 2011; Machado et al., JACI 2015 Wallner et al. (in preparation) POST-TRANSLATIONAL MODIFICATIONS (disulfide bridges) Toda et al., JACI 2011
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Susceptibility/resistance to endolysosomal proteases seem to be linked to “intrinsic” allergenicity/immunogenicity of protein antigens and influences the type of immune response (Th2 vs. Th1 bias) Proteolytic resistance Delamarre, L et al (2005) Science 307: Delamarre, L et al (2006) J Exp Med 203: Egger et al, PLoS One 2011
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Conflicting Results… ??? pH 4.5 pH 5.0 Bet v 1a Bet v 1d
Egger et al, PLoS One 2011 Freier et al, Nature Sci Rep 2015
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Processing of Bet v 1 by cathepsin S
pH 6.7 SAW-measurements Freier et al, Nature Sci Rep 2015
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The concept of pH-dependent proteolytic resistance of allergens
Freier et al, Nature Sci Rep 2015
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… OR HOW TO MAKE A SUPERALLERGEN
The interplay between fold stability and pH-dependent proteolytic susceptibility… … OR HOW TO MAKE A SUPERALLERGEN
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In vivo immunogenicity/allergenicity pH and proteolytic susceptibility
Fold stability as a key factor for allergenicity and immunogenicity of Bet v 1 IgE reactivity Fold stability In vivo immunogenicity/allergenicity IgG1 IgE RBL IgG2a IFN-g IL-4 pH and proteolytic susceptibility Immunogenicity/allergenicity depends on differential fold stability at different endosomal pH Machado et al., JACI 2015
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pH-dependent stability and proteolytic susceptibility
Allergenicity / Immunogenicity: pH-dependent stability and proteolytic susceptibility Machado et al., JACI 2015
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Proteolytic susceptibility as a function of
pH-dependent fold stability: implications ALLERGEN ENGINEERING (HYPOALLERGENS) RECOMBINANT ALLERGY VACCINES Ligand-binding Post-translational modifications Chemical modifications Oligomerization Machado et al., JACI 2015
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SUMMARY AND CONCLUSIONS
Susceptibility/resistance to lysosomal proteases seem to be linked to intrinsic allergenicity/immunogenicity of protein antigens and to influence the type of immune response (allergic vs. non-allergic) Differential pH-dependent fold stability along endosomal maturation (acidification) seem to be an important factor for allergenicity and immunogenicity Fine tuning of resistance/susceptibility to endolysosomal proteolysis may be an approach for designing allergen vaccines with enhanced immunogenicity
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Ferreira Lab @ University of Salzburg
Michael Wallner Gabi Gadermaier Claudia Asam Anargyros Roulias Research Assistants Stephanie Eichhorn Lorenz Aglas Sara Huber Markus Steiner Heidi Hofer Isabel Pablos Sabrina Schuller Teresa Stemeseder Martin Wolf Olivia McKenna PhD students
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Program BioScience & Health (Uni Salzburg)
ACKNOWLEDGMENTS Josef Thalhamer Peter Lackner Hans Brandstetter Peter Briza Albert Duschl Christian Huber Yoan Machado Gernot Achatz Program BioScience & Health (Uni Salzburg) Barbara Bohle Beatrice Jahn-Schmidt Heimo Breiteneder Karin Hoffmann-Sommergruber Christof Ebner Martin Tollinger Klaus Liedl Adriano Mari Ronald van Ree Ana Paula Valente Fabio Almeida
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Obrigada pela Atenção !!!
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