Abstract
Der p 2 is a major dust mite allergen and >80% of mite allergic individuals have specific IgE to this allergen. Although it is well characterized in terms of allergenicity, there is still some ambiguity in terms of its biological function. Three-dimensional structural analysis of Der p 2 and its close homologues indicate the presence of a hydrophobic cavity which can potentially bind to lipid molecules. In this study, we aimed to identify the potential ligand of Der p 2. Using a liposome pulldown assay, we show that recombinant Der p 2 binds to liposomes prepared with exogenous cholesterol in a dose dependent fashion. Next, an ELISA based assay using immobilized lipids was used to study binding specificities of other lipid molecules. Cholesterol was the preferred ligand of Der p 2 among 11 different lipids tested. Two homologues of Der p 2, Der f 2 and Der f 22 also bound to cholesterol. Further, using liquid chromatography-mass spectrometry (LC-MS), we confirmed that cholesterol is the natural ligand of Der p 2. Three amino acid residues of Der p 2, V104, V106 and V110 are possible cholesterol binding sites, as alanine mutations of these residues showed a significant decrease in binding (p < 0.05) compared to wild-type Der p 2. These results provide the first direct experimental evidence that Der p 2 binds to cholesterol.
Highlights
Group 2 allergens from the Dermatophagoides house dust mites causes IgE-mediated responses in over 80% of the dust mite allergic individuals[1,2] and are classified as major allergens
Der p 2 binds to liposomes with exogenous cholesterol in a dose-dependent fashion
It was observed that rDer p 2 weakly bound to liposomes in a dose dependent fashion (Fig. 1, top panel, Supplementary Fig. S1)
Summary
Group 2 allergens from the Dermatophagoides house dust mites causes IgE-mediated responses in over 80% of the dust mite allergic individuals[1,2] and are classified as major allergens. LPS was shown to bind to MD-2, a protein which shared moderate sequence similarity (11% identity, 29% similarity) to Der p 2, and belonged to the same ML (MD-2 related lipid binding) domain family as group 2 allergens. Based on the high sequence similarities between Der p 2 and Der f 2 (88% identity), it would be expected that both proteins would behave in a similar manner in terms of ligand binding. Among the various proteins that belong to the ML domain family, Der p 2 shows the highest sequence similarity to NPC27 (23.5% identity, 44% similarity). Since the 3D structures of Der p 2 and NPC210 show high similarity, and NPC2 has been reported to bind cholesterol at nanomolar affinities[11], we hypothesized that the ligand of Der p 2 could likely to be a lipid with close molecular similarity to sterols. We show evidence that homologues of Der p 2, Der f 2 and Der f 22, a paralogue of Der f 212, binds to cholesterol
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