Biochemical and immunological characterization of the house dust mite Der p 13 produced in Pichia pastoris

Abstract

The house dust mite (HDM) allergen Der p 13 could be a lipid-binding protein able to stimulate the airway epithelium through activation of key innate signaling pathways involved 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. 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. The results from protein modeling and biophysical analysis indicated that Der p 13 adopts a β barrel structure forming 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. 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.