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

Abstract

BackgroundThe 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.ObjectiveTo perform a detailed characterization of Der p 18 on a molecular, structural and immunological level.MethodsDer 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.ResultsRecombinant 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.ConclusionDer 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.

Molecular allergen profiling in horses by microarray reveals Fag e 2 from buckwheat as a frequent sensitizer

“…Serum samples of all horses were tested on ISAC131 multiplex array (Figure A, B) for IgE binding to 131 allergens: 112 corresponding to commercial ISAC112, plus 19 additional allergens (Table ). The additional allergens were selected due to potential impact for veterinary patients and comprised molecular allergens from other animals (albumins, lipocalins and alpha‐Gal), human antigens, the yeast associated with atopic dermatitis ( Malassezia sympodialis, previously designated Malassezia furfur ), biting midges ( Culicoides nubeculosus ) responsible for summer eczema, high molecular weight enzymatic HDM allergens, papain and one plant allergen (Table ).…”

“…To this aim, we expanded an allergen chip for IgE serology in animals adding 19 more molecules of potential relevance in veterinary allergy diagnosis (Table ). We considered proteins from the following allergen sources potentially important: from other animals (albumins from rat, guinea pig and rabbit; lipocalin from mouse; alpha‐Gal; from human (uteroglobin, IgG, profilin and Hom s 2), from the commensal yeast Malassezia associated with a number of different skin disorders such as atopic eczema, from midges, relevant via skin and from plant food …”

Allergens of <b><i>Blomia tropicalis</i></b>: An Overview of Recombinant Molecules

“…Its chitin-binding activity was experimentally confirmed by Zakzuk et al [26]. Interestingly, chitin binding has been predicted for Der p 23 and Der p 18, but only a weak binding activity has been demonstrated for Der p 18 [71,72,73]. The Blo t 12 sequence contains a putative signal peptide (amino acid residues 1-20) resulting in a 14-kDa mature protein [70].…”

“…Usually, recombinant allergens are produced in bacterial expression systems [59,61,72,79]. Although the majority of bacterium-based expression systems are able to produce biologically active and correctly folded molecules, for a few allergens a host that allows more elaborated posttranslational modifications is required [25,67,68,69].…”

Mite allergens

“…Information about a detailed allergenicity evaluation is currently available for only 10% of all IgE binding proteins in the databases. Completing this task will allow a better assessment of the pathogenic role of each mite allergen as has been recently suggested for Der p 23 [17,18] and could be great independently of its IgE-binding frequency [19][20][21]. Recognizing that regional combinations of mite allergens are more appropriate and less expensive than large panels of just IgE-binding components will also help.…”