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The major allergen Der p 2 is a cholesterol binding protein.

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?

SUBMITTER: Reginald K

PROVIDER: S-EPMC6367342 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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The major allergen Der p 2 is a cholesterol binding protein.

Reginald Kavita K Chew Fook Tim FT

Scientific reports 20190207 1

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 pulldow ...[more]

PMID: 30733527

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Project description:PURPOSE:Hypoallergenic recombinant Der p 2 has been produced by various genetic manipulations, but mutation of a naturally polymorphic amino acid residue known to affect IgE binding has not been studied. This study aimed to determine the effect of a point mutation (S47W) of residue 47 of Der p 2 on its structure and immunoglobulin (Ig) E binding. Its ability to induce pro-inflammatory responses and to induce blocking IgG antibody was also determined. METHODS:S47 of recombinant Der p 2.0110, one of the predominant variants in Bangkok, was mutated to W (S47W). S47W secreted from Pichia pastoris was examined for secondary structure and for the formation of a hydrophobic cavity by 8-Anilino-1-naphthalenesulfonic acid (ANS) staining. Monoclonal and human IgE-antibody binding was determined by enzyme-linked immunosorbent assay. Allergen-induced degranulation by human epsilon receptor expressed-rat basophil was determined. Stimulation of the pro-inflammatory cytokine interleukin (IL)-8 release from human bronchial epithelial (BEAS2B) cells and inhibition of IgE binding to the wild type allergen by S47W-induced IgG were determined. RESULTS:S47W reduced secondary structure and failed to bind the hydrophobic ANS ligand as well as a monoclonal antibody known to be dependent on the nature of the side chain of residue 114 in an adjacent loop. It could also not stimulate IL-8 release from BEAS2B cells. IgE from house dust mite (HDM)-allergic Thais bound S47W with 100-fold weaker avidity, whereas IgE of HDM-allergic Australians did not. S47W still induced basophil degranulation, although requiring higher concentrations for some subjects. Anti-S47W antiserum-immunized mice blocked the binding of human IgE to wild type Der p 2. CONCLUSIONS:The mutant S47W had altered structure and reduced ability to stimulate pro-inflammatory responses and to bind IgE, but retained its ability to induce blocking antibodies. It thus represents a hypoallergen produced by a single mutation of a non-solvent-accessible amino acid.

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The major allergen Der p 2 is a cholesterol binding protein.

Publications

The major allergen Der p 2 is a cholesterol binding protein.

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