Natural and recombinant molecules of the cherry allergen Pru av 2 show diverse structural and B cell characteristics but similar T cell reactivity
Article first published online: 24 FEB 2006
Clinical & Experimental Allergy
Volume 36, Issue 3, pages 359–368, March 2006
How to Cite
Fuchs, H. C., Bohle, B., Dall'Antonia, Y., Radauer, C., Hoffmann-Sommergruber, K., Mari, A., Scheiner, O., Keller, W. and Breiteneder, H. (2006), Natural and recombinant molecules of the cherry allergen Pru av 2 show diverse structural and B cell characteristics but similar T cell reactivity. Clinical & Experimental Allergy, 36: 359–368. doi: 10.1111/j.1365-2222.2006.02439.x
- Issue published online: 24 FEB 2006
- Article first published online: 24 FEB 2006
- Submitted 11 May 2005; revised 18 November 2005; accepted 28 November 2005
- circular dichroism;
- E. coli expression;
- food allergen;
- IgE binding;
- T cell reactivity;
- thaumatin-like protein
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.
Objectives 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.