293 Identification of amino acids that equip lipocalins with allergenic potential

Lipocalins comprise many respiratory mammal allergens and some endogenous proteins. We used two pairs of homologous lipocalins consisting of an allergenic and a non-allergenic lipocalin to establish an in vitro system with dendritic cells (DC) and allogeneic T-cells able to distinguish between allergenic and non-allergenic immune responses. We show that the interaction of lipocalins with DC can be decisive for the type of immune response induced. Here we use the system to test hybrid-lipocalins for their ability to induce TH1 or TH2 responses in order to identify the amino acids (AA) within lipocalins responsible for the induction of allergenic immune responses. Hybrid-lipocalins were designed by comparison of the AA sequences between allergenic and non-allergenic lipocalins to exclude overlapping AA, and the subsequent pairing of allergenic Can f 1 and Fel d 4 to detect common “allergenic” AA. With this approach we found 11 “allergenic” AA, which were inserted into the non-allergenic Lcn-1 sequence. We then searched for additional common AA in the sequences of the mammal allergens Fel d 7, Equ c 1 and Bos d 2 to finally reduce the “allergenic” AA to five and two, respectively. We produced all three hybrid-lipocalins with the Lcn-1 sequence as backbone replacing 11, 5 or 2 AA with the corresponding “allergenic” AA as recombinant proteins (HL11, HL5 and HL2). All three hybrid-lipocalins behaved as allergens in the above described system which allows the conclusion that the final 2 AA are responsible for priming DC to induce allergenic immune responses in co-cultures with allogeneic T cells. As a control we exchanged the 2 AA of HL2 in the Lcn-1 sequence on a nearby position. This control-lipocalin was not allergenic. We conclude that not only the identity but also the position of the AA in the lipocalins accounts for their allergenic potential. Further investigations are needed to identify the mechanism(s) employed by lipocalin allergens to prime DC for the induction of allergenic immune responses.