Distinct epitope structures of defensin-like proteins linked to proline-rich regions give rise to differences in their allergenic activity.

BACKGROUND: Art v 1, Amb a 4, and Par h 1 are allergenic defensin-polyproline-linked proteins present in mugwort, ragweed, and feverfew pollen, respectively. We aimed to investigate the physicochemical and immunological features underlying the different allergenic capacities of those allergens. METHODS: Recombinant defensin-polyproline-linked proteins were expressed in E. coli and physicochemically characterized in detail regarding identity, secondary structure, and aggregation status. Allergenic activity was assessed by mediator releases assay, serum IgE reactivity, and IgE inhibition ELISA using sera of patients from Austria, Canada, and Korea. Endolysosomal protein degradation and T-cell cross-reactivity were studied in vitro. RESULTS: Despite variations in the proline-rich region, similar secondary structure elements were observed in the defensin-like domains. Seventy-four percent and 52% of the Austrian and Canadian patients reacted to all three allergens, while Korean patients were almost exclusively sensitized to Art v 1. This was reflected by IgE inhibition assays demonstrating high cross-reactivity for Austrian, medium for Canadian, and low for Korean sera. In a subgroup of patients, IgE reactivity toward structurally altered Amb a 4 and Par h 1 was not changed suggesting involvement of linear epitopes. Immunologically relevant endolysosomal stability of the defensin-like domain was limited to Art v 1 and no T-cell cross-reactivity with Art v 125-36 was observed. CONCLUSIONS: Despite structural similarity, different IgE-binding profiles and proteolytic processing impacted the allergenic capacity of defensin-polyproline-linked molecules. Based on the fact that Amb a 4 demonstrated distinct IgE-binding epitopes, we suggest inclusion in molecule-based allergy diagnosis.

The minor house dust mite allergen Der p 13 is a fatty acid-binding protein and an activator of a TLR2-mediated innate immune response.

BACKGROUND: The house dust mite (HDM) allergen Der p 13 could be a lipid-binding protein able to activate key innate signaling pathways 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. METHODS: 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. RESULTS: Protein modeling and biophysical analysis indicated that Der p 13 adopts a ß-barrel structure with 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. CONCLUSIONS: 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.

Patterns of IgE sensitization in house dust mite-allergic patients: implications for allergen immunotherapy.

BACKGROUND: Understanding patterns of IgE sensitization in Dermatophagoides-allergic patients living in various geographical areas is necessary to design a product suitable for worldwide allergen immunotherapy (AIT). METHODS: Using a HIFI Allergy customized microarray assay, IgEs specific for 12 purified allergens from Dermatophagoides pteronyssinus or D. farinae were assessed in sera from 1302 house dust mite (HDM)-allergic patients living in various areas. Comprehensive mass spectrometric (MS) analyses were conducted to characterize HDM extracts, as well as purified bodies and feces. RESULTS: Patterns of IgE reactivity to HDM allergens are comparable in all cohorts of patients analyzed, encompassing adults and 5- to 17-year-old children, as well as American, Canadian, European, and Japanese patients. Overall, >70% and >80% of HDM-allergic patients are sensitized to group 1 and group 2 allergens, respectively, from D. pteronyssinus and/or D. farinae species. Furthermore, 20-47% of patients also have IgEs to allergens from groups 4, 5, 7, 13, 15, 21, and 23. All patients have IgEs to allergens present in mite bodies and feces. MS-based analyses confirmed the presence of mite allergens recorded by IUIS in D. pteronyssinus and D. farinae extracts, with groups 2, 8, 10, 11, 14, and 20 prominent in bodies and groups 1, 6, 18, and 23 well represented in feces. CONCLUSIONS: Mite-specific AIT should rely upon a mixture of D. pteronyssinus and D. farinae extracts, manufactured from both feces and bodies. Such a combination is appropriate to treat children and adult Dermatophagoides-allergic patients from Asia, Europe, and North America.