The minor house dust mite allergen Der p 13 is a fatty acid binding protein and an activator of a TLR2-mediated innate immune response

Background: The house dust mite (HDM) allergen Der p 13 could be a lipid-binding protein able to activate key innate signaling pathways in the initiation of the allergic response. We investigated the IgE reactivity of recombinant Der p 13 (rDer p 13), its lipid binding activities and its capacity to stimulate airway epithelium cells. Methods: Purified rDer p 13 was characterized by mass spectrometry, circular dichroism, fluorescence-based lipid binding assays and in-silico structural prediction. IgE binding activity and allergenic potential of Der p 13 were examined by ELISA, basophil degranulation assays and in-vitro airway epithelial cell activation assays. Results: Protein modeling and biophysical analysis indicated that Der p 13 adopts a β barrel structure with a predominately apolar pocket representing a potential binding site for hydrophobic ligands. Fluorescent lipid binding assays confirmed that the protein is highly selective for ligands and that it binds a fatty acid with a dissociation constant typical of lipid transporter proteins. The low IgE binding frequency (7%, n= 224) in Thai HDM-allergic patients as well as the limited propensity to activate basophil degranulation classifies Der p 13 as a minor HDM allergen. Nevertheless, the protein with its presumptively associated lipid(s) triggered the production of IL-8 and GM-CSF in respiratory epithelial cells through a TLR2-, MyD88-, NF-kB- and MAPK-dependent signaling pathway. Conclusions: Although a minor allergen, Der p 13 may, through its lipid binding capacity, play a role in the initiation of the HDM allergic response through TLR2 activation

The house dust mite allergen Der p 5 binds lipid ligands and stimulates airway epithelial cells through a TLR2‐dependent pathway

Background: Protein crystallographic studies suggest that the house dust mite (HDM) allergen Der p 5 potentially interacts with hydrophobic ligands. Der p 5, in association with its ligand(s), might therefore trigger innate immune signalling pathways in the airway epithelium and influence the initiation of the HDM‐allergic response. Objective: We investigated the lipid binding propensities of recombinant (r)Der p 5 and characterized the signalling pathways triggered by the allergen in airway epithelial cells. Methods: rDer p 5 was produced in Pichia pastoris and characterized by mass spectrometry, multi‐angle light scattering and circular dichroism. Its interactions with hydrophobic ligands were investigated in fluorescence‐based lipid binding assays and in‐silico docking simulations. Innate immune signalling pathways triggered by rDer p 5 were investigated in airway epithelial cell activation assays in vitro. Results: Biophysical analysis showed that rDer p 5 was monomeric and adopted a similar α‐helix‐rich fold at both physiological and acidic pH. Spectrofluorimetry experiments showed that rDer p 5 is able to selectively bind lipid ligands, but only under mild acidic pH conditions. Computer‐based docking simulations identified potential binding sites for these ligands. This allergen, with putatively associated lipid(s), triggered the production of IL‐8 in respiratory epithelial cells through a TLR2‐, NF‐kB‐ and MAPK‐dependent signalling pathway. Conclusions and Clinical Relevance: Despite the fact that Der p 5 represents a HDM allergen of intermediate prevalence, our findings regarding its lipid binding and activation of TLR2 indicate that it could participate in the initiation of the HDM‐allergic state