Dataset Information

Molecular, Structural and Immunological Characterization of Der p 18, a Chitinase-Like House Dust Mite Allergen.

ABSTRACT:

Background

The house dust mite (HDM) allergen Der p 18 belongs to the glycoside hydrolase family 18 chitinases. The relevance of Der p 18 for house dust mite allergic patients has only been partly investigated.

Objective

To perform a detailed characterization of Der p 18 on a molecular, structural and immunological level.

Methods

Der p 18 was expressed in E. coli, purified to homogeneity, tested for chitin-binding activity and its secondary structure was analyzed by circular dichroism. Der p 18-specific IgG antibodies were produced in rabbits to localize the allergen in mites using immunogold electron microscopy and to search for cross-reactive allergens in other allergen sources (i.e. mites, crustacea, mollusca and insects). IgE reactivity of rDer p 18 was tested with sera from clinically well characterized HDM-allergic patients (n = 98) and its allergenic activity was analyzed in basophil activation experiments.

Results

Recombinant Der p 18 was expressed and purified as a folded, biologically active protein. It shows weak chitin-binding activity and partial cross-reactivity with Der f 18 from D. farinae but not with proteins from the other tested allergen sources. The allergen was mainly localized in the peritrophic matrix of the HDM gut and to a lower extent in fecal pellets. Der p 18 reacted with IgE from 10% of mite allergic patients from Austria and showed allergenic activity when tested for basophil activation in Der p 18-sensitized patients.

Conclusion

Der p 18 is a rather genus-specific minor allergen with weak chitin-binding activity but exhibits allergenic activity and therefore should be included in diagnostic test panels for HDM allergy.

SUBMITTER: Resch Y

PROVIDER: S-EPMC4993390 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Publications

Molecular, Structural and Immunological Characterization of Der p 18, a Chitinase-Like House Dust Mite Allergen.

Resch Yvonne Y Blatt Katharina K Malkus Ursula U Fercher Christian C Swoboda Ines I Focke-Tejkl Margit M Chen Kuan-Wei KW Seiberler Susanne S Mittermann Irene I Lupinek Christian C Rodriguez-Dominguez Azahara A Zieglmayer Petra P Zieglmayer René R Keller Walter W Krzyzanek Vladislav V Valent Peter P Valenta Rudolf R Vrtala Susanne S

PloS one 20160822 8

<h4>Background</h4>The house dust mite (HDM) allergen Der p 18 belongs to the glycoside hydrolase family 18 chitinases. The relevance of Der p 18 for house dust mite allergic patients has only been partly investigated.<h4>Objective</h4>To perform a detailed characterization of Der p 18 on a molecular, structural and immunological level.<h4>Methods</h4>Der p 18 was expressed in E. coli, purified to homogeneity, tested for chitin-binding activity and its secondary structure was analyzed by circula ...[more]

PMID: 27548813

Similar Datasets

Project description:PurposeHypoallergenic 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.MethodsS47 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.ResultsS47W 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.ConclusionsThe 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.

Dataset Information

Molecular, Structural and Immunological Characterization of Der p 18, a Chitinase-Like House Dust Mite Allergen.

Background

Objective

Methods

Results

Conclusion

Publications

Molecular, Structural and Immunological Characterization of Der p 18, a Chitinase-Like House Dust Mite Allergen.

Similar Datasets

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