Identification of an immunodominant IgE epitope of Der f 40, a novel allergen of Dermatophagoides farinae

Abstract

Background: House dust mites (HDMs), including Dermatophagoides pteronyssinus (Der p) and Dermatophagoides farinae (Der f) species, represent a major source of inhalant allergens that induce IgE-mediated anaphylactic reactions. HDM allergen identification is important to the diagnosis and treatment of allergic diseases. Here, we report the identification of a novel HDM allergen, which we suggest naming Der f 40, and its immunodominant IgE epitopes.

Methods: The recombinant protein Der f 40 was expressed using a pET prokaryotic expression system and purified with Ni-NTA resins. IgE binding activity was evaluated by IgE-western blot, dot-blot, and ELISA. Mast cell activation testing was performed to assess the cellular effects of IgE binding in mouse bone marrow derived mast cells (BMMCs) expressing human FcεRI. IgE binding assays were performed with truncated and hybrid Der f 40 protein molecules to find immunodominant IgE epitopes.

Results: A 106-amino acid (aa) recombinant Der f Group 40 protein (rDer f 40) was obtained (GenBank accession No. XP_046915420.1) as thiredoxin-like protein. Der f 40 was shown to bind IgE from HDM allergic serum in vitro (9.68%; 12/124 in IgE-ELISA), and shown to promote the release of β-hexosaminidase from BMMCs dose-dependently when administered with HDM allergic sera. The Der f Group 40 protein was named Der f 40 and listed in the World Health Organization and International Union of Immunological Societies (WHO/IUIS) Allergen Nomenclature Sub-committee. IgE binding assays with Der f 40-based truncated and hybrid proteins indicated that IgE binding epitopes are likely located in the C-terminal region and dependent on conformational structure. The 76-106-aa region of C-terminus was identified as an immunodominant IgE epitope of Der f 40.

Conclusion: A novel HDM allergen with robust IgE binding activity was identified and named Der f 40. An immunodominant IgE epitope of Der f 40 with conformational dependency was identified in the C-terminus (aa 76-106). These findings provide new information that may be useful in the development of diagnostic and therapeutic agents for HDM allergy.

Keywords: Allergens; Dust mite allergy; Immunodominant epitopes; Immunoglobulin E.

Fig. 1

Multiple sequence alignment of aa sequences of Der f 40 and thioredoxin from other species generated in Clustal Omega software. Der f: D. farinae; Asp f: Aspergillus flavus; Cop c: Coprinus comatus; Fus c: Fusarium culmorum; Mala s: Malassezia sympodialis; Plo i: Plodia interpunctella; Tri a: Triticum aestivum; Zea m: Zea mays; hTRX: Human thioredoxin

Fig. 2

Expression, purification, and enzymatic characterization of rDer f 40. A. SDS-PAGE of Der f crude extract. The natural thioredoxin protein may be present at ∼12 kDa band (red box in Lane 1). B. LC/MS analysis of the ∼12 kDa band sample obtained from crude Der f extract. Two representative coverage peptides with the aa sequences ³⁸IAPVLEK⁴⁴ and ⁸⁷VHSFSGASEPK⁹⁷. C. Der f 40 ORF cDNA was amplified by RT-PCR using specific primers. M: DNA Marker, Lane 1: Der f 40 PCR results. D. SDS-PAGE of the purified rDer f 40 protein expressed in E. coli. M: Prestained protein marker, Lane 1: purified rDer f 40 protein. E. Far-ultraviolet (wavelength, 200–260) circular dichroism analysis of purified rDer f 40 at 25 °C. The CD spectrum CD spectrum shows that it is consistent with the secondary structure of Der f 40 protein predicted, mainly composed of α-helix. F. Insulin reduction assay of thioredoxin activity using rDer f 40

Fig. 3

IgE binding activity of rDer f 40 in vitro. A. IgE binding activity with rDer f 40 shown by IgE-ELISA with HDM allergic sera (124 patient donors) and with healthy control sera (50 control donors). IgE dot-blot (B) and western-blot (C) assays of rDer f 40 with 10 HDM allergic patients' sera and 10 healthy control individuals' sera. The 10 HDM allergic sera were positive in the above mentioned IgE-ELISA assay

Fig. 4

Mast cell activation induced by rDer f 40. β-hexosaminidase release from mature BMMCs (from IgE/FcεRI humanized mice) determined after Der f 40 protein stimulation with 4 individual HDM-allergic sera pre-incubated BMMCs, non-HDM allergic healthy control serum

Fig. 5

Screening of truncated Der f 40 proteins for immunodominant IgE binding. A. Homology model structure of Der f 40 generated using Mala s 13 as a template (PDB code 2J23). B. Schematic representation showing the locations of 6 overlapping epitopes within the secondary structure of Der f 40. C. SDS-PAGE of purified Der f 40 and truncated forms with DsbA fusion proteins expressed in E. coli; rDsbA protein was served as a control. IgE western-blot (D), IgE dot-blot (E) and IgE-ELISA with patients' sera pool (N = 10) (a) and healthy control sera pool (N = 10) (b) targeting Der f 40-derived peptide epitope fusion proteins (E1–E6); full-length rDer f 40 protein (FL) was used as a positive control, rDsbA protein was used as a negative control

Fig. 6

Identification immunodominant IgE epitope of Der f 40 using hybrid proteins. A. Schematic diagram of Der f 40 and of Der f 40-human thioredoxin (hTRX) hybrid proteins. B. SDS-PAGE of purified Der f 40, Der f 40 N-terminal hybrid protein (Der f 40 N-Hyb), Der f 40 C-terminal hybrid protein (Der f 40 C-Hyb), and hTRX. C. IgE binding assessment of Der f 40-derived hybrid proteins through IgE-dot-blot with HDM allergic sera and healthy control sera. D. SDS-PAGE of Der f 40 C-terminal hybrid proteins (Der f 40 C-Hyb1, Der f 40 C-Hyb2 and Der f 40 C-Hyb3). E. IgE western-blot with HDM allergic sera targeting Der f 40-derived C-terminal detailed hybrid proteins; full-length Der f 40 protein was used as a positive control. F. IgE binding activity of Der f 40-derived C-terminal detailed hybrid proteins through IgE-ELISA with 2 different pooled HDM allergic patient's serum samples; pooled healthy control sera was used as a negative control