Methods: Structural analysis of Bet v 1 was performed using FATCATflex, Combinatorial extention algorithm and Template Modeling. Docking calculations of ligands were performed with AutoDock Vina. Iron-staining was performed on dotted apo- and holo-Betv1 as well as controls. PBMCs of 10 human subjects were activated and stimulated for 18h with apo- or holo-Bet v1 or controls. Subsequently, cells and supernatants were analysed by flow cytometry and their cytokine-content.
Results: We give structural evidence that Bet v 1 is a lipocalin-like protein with a striking resemblance to human lipocalin 2 in silico. We demonstrate that similar to lipocalin 2, Bet v 1 is capable of binding iron via catechol-based siderophores. Thereby, calculated Kd-values of 20nM outpassed affinities to known ligands more than twentyfold. Moreover, we give functional evidence of the immune-modulatory capacity of Bet v 1 being dependent on its iron-loaded state. When incubated to human immune cells, only the apo-form of Bet v 1, but not the holo-form, was able to promote Th2 cells secreting IL13.
Conclusions: Bet v 1 is a lipocalin-like protein, which is capable of binding iron via siderophores. Moreover, we give for the first time evidence that the form of application (apo- or holo-) is decisive for the subsequent immune response. The apo-form promotes Th2 cells, whereas the holo-form appears immunosuppressive. These results provide for the first time a functional understanding on the allergenicity of Bet v 1 and a basis for future allergen immunotherapies counteracting Th2 immune responses on a molecular basis.