title: Natural and recombinant molecules of the cherry allergen Pru av 2 show diverse structural and B cell characteristics but similar T cell reactivity.
abstract: BACKGROUND
  Cherry allergy is often reported in the context of allergy to other fruits of the Rosaceae family and pollinosis to trees because of cross-reactive allergens. Allergic reactions to cherry are reported by 19-29% of birch pollen-allergic patients. Pru av 2, identified as a thaumatin-like protein (TLP) from sweet cherry, was recognized by the majority of cherry-allergic patients in immunoblotting.
  
  
  OBJECTIVE
  In order to investigate the structural characteristics and the immunoglobulin (Ig)E- and T cell reactivity of cherry-derived TLP, recombinant Pru av 2 was expressed in Escherichia coli and natural Pru av 2 was purified.
  
  
  METHODS
  Parallel-His and FLAG expression vectors were used for recombinant production of Pru av 2 in the cytoplasm and the periplasm of E. coli. Natural Pru av 2 was purified from fresh cherries and verified by N-terminal sequencing. Structural characterization was performed using circular dichroism (CD) measurements, and the biologic activity was measured in a glucanase assay. Using cherry-specific sera, the IgE-binding ability of recombinant and natural Pru av 2 was investigated in IgE-ELISA and the T cell reactivity was studied in proliferation assays. Results Natural Pru av 2 revealed thaumatin-like structural features and bound IgE of 50% of cherry-allergic patients. It was demonstrated to be enzymatically active. Recombinant Pru av 2 expressed in the cytoplasm of E. coli exhibited a slightly different folding compared with the natural protein. It was not recognized by IgE from cherry-allergic subjects, but retained the ability to stimulate T lymphocytes. Periplasmic recombinant Pru av 2 was able to bind an anti-grape TLP antibody and cherry-specific IgE.
  
  
  CONCLUSIONS
  We prepared two recombinant model TLPs from cherry, and compared their molecular characteristics as well as their IgE-binding activity and T cell interactions in relation to the natural counterpart. The cytoplasmic recombinant Pru av 2 can be used as a hypoallergenic variant in allergen-specific immunotherapy, whereas the periplasmic protein can be included in a component-resolved diagnosis.
authors.affiliation: Center of Physiology and Pathophysiology, Medical University of Vienna, Vienna, Austria.
authors.forename: H C
authors.initials: HC
authors.lastname: Fuchs
authors.forename: B
authors.initials: B
authors.lastname: Bohle
authors.forename: Y
authors.initials: Y
authors.lastname: Dall'Antonia
authors.forename: C
authors.initials: C
authors.lastname: Radauer
authors.forename: K
authors.initials: K
authors.lastname: Hoffmann-Sommergruber
authors.forename: A
authors.initials: A
authors.lastname: Mari
authors.forename: O
authors.initials: O
authors.lastname: Scheiner
authors.forename: W
authors.initials: W
authors.lastname: Keller
authors.forename: H
authors.initials: H
authors.lastname: Breiteneder
mesh_terms: D000328:Adult; D000485:Allergens / Q000276:immunology / Q000302:isolation & purification; D000937:Antigen-Antibody Reactions; D052179:Antigens, Plant / Q000276:immunology* / Q000302:isolation & purification; D001402:B-Lymphocytes / Q000276:immunology*; D049109:Cell Proliferation; D002478:Cells, Cultured; D002942:Circular Dichroism; D003593:Cytoplasm / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D004926:Escherichia coli / Q000378:metabolism; D005512:Food Hypersensitivity / Q000276:immunology; D006801:Humans; D007073:Immunoglobulin E / Q000276:immunology; D008213:Lymphocyte Activation / Q000276:immunology; D019897:Periplasm / Q000276:immunology; D027861:Prunus / Q000276:immunology; D011994:Recombinant Proteins / Q000276:immunology; D013601:T-Lymphocytes / Q000276:immunology*
chemical_list: D000485:Allergens; D052179:Antigens, Plant; C510168:Pru av 2 allergen, Prunus avium; D011994:Recombinant Proteins; D007073:Immunoglobulin E