Transient dimers of allergens

Abstract

Background: Allergen-mediated cross-linking of IgE antibodies bound to the FcepsilonRI receptors on the mast cell surface is the key feature of the type I allergy. If an allergen is a homodimer, its allergenicity is enhanced because it would only need one type of antibody, instead of two, for cross-linking.

Methodology/principal findings: An analysis of 55 crystal structures of allergens showed that 80% of them exist in symmetric dimers or oligomers in crystals. The majority are transient dimers that are formed at high protein concentrations that are reached in cells by colocalization. Native mass spectrometric analysis showed that native allergens do indeed form transient dimers in solution, while hypoallergenic variants of them exist almost solely in the monomeric form. We created a monomeric Bos d 5 allergen and show that it has a reduced capability to induce histamine release.

Conclusions/significance: The results suggest that dimerization would be a very common and essential feature for allergens. Thus, the preparation of purely monomeric variants of allergens could open up novel possibilities for specific immunotherapy.

Figure 1. Symmetric homodimers of allergens in crystals.

One monomer is shown as a red ribbon model, the second monomer as a blue ribbon model. The dimeric allergen is shown in two orientations. In the left picture the symmetric two-fold axis is towards the viewer, in the right picture the axis is in the same plane as the paper. Fig. A shows the crystal structure of a Bos d 5-D1/IgE(Fab) immunocomplex (pdb code 2R56) in which IgE fragments are shown as grey surface models. All structures are drawn to the same scale.

Figure 2. ESI FT-ICR mass spectra of allergens measured in 10 mM NH₄OAc (pH 6.9) at varying allergen (monomer) concentrations.

Numbers indicate different protein ion charges and the letters M (and color blue), D (red), Tr (green), and Te (green) refer to the signals from a monomer, dimer, trimer, and tetramer, respectively. Note that some monomer and dimer charge states overlap in the spectra. The left inset in A shows the magnification of monomer charge state 7+, the right inset the dimer charge state 11+.

Figure 3. Histamine release experiments with nBos d 5, rBos d 5 B variant, and rBos d 5 B variant H146P mutant.

Released histamine was measured after the passive sensitization of stripped basophils from two donors (A; right panel and B, left panel) with the serum from a milk (Bos d 5) allergic patient and purified allergens in a concentration range of 0.03-10000 ng/ml (x-axis, logarithmic scale) in duplicate measurements. The percentage of histamine released into the supernatant is shown on the y-axis (a meanvalue of the duplicate measurments).