Blo t 2: Group 2 allergen from the dust mite Blomia tropicalis

Abstract

Blomia tropicalis has been recognized as a cause of allergic diseases in the tropical and subtropical regions. Here we report the immuno-characterization of its group 2 allergen, Blo t 2. Allergen Blo t 2 was amplified from the cDNA of B. tropicalis using degenerate primers, expressed in Escherichia coli as a recombinant protein and purified to homogeneity. The mature protein of Blo t 2 was 126 amino acids long with 52% sequence identity to Der p 2 and apparent molecular mass of 15 kDa. Circular dichroism spectroscopy showed that Blo t 2 is mainly a beta-sheeted protein. We confirmed the presence of three disulfide bonds in recombinant (r) Blo t 2 protein using electrospray mass spectrometry. Thirty-four percent of dust-mite allergic individuals from the Singapore showed specific IgE binding to rBlo t 2 as tested using immuno dot-blots. IgE-cross reactivity assays showed that Blo t 2 had between 20-50% of unique IgE-epitopes compared to Der p 2. IgE binding of native and recombinant forms of Blo t 2 were highly concordant (r² = 0.77, p < 0.0001) to rBlo t 2. Dose-dependent in vitro histamine was observed when rBlo t 2 was incubated with whole blood of Blo t 2 sensitized individuals, demonstrating that it is a functional allergen. Nine naturally occurring isoforms of Blo t 2 were identified in this study, each having between 1-3 amino acid variations compared to the reference clone. Blo t 2 is a clinically relevant allergen of B. tropicalis as it has unique IgE epitopes compared to major group 2 allergens from Dermatophagoides spp.

Figure 1

Nucleotide and translated amino acid sequence of Blo t 2. The DNA sequences in uppercase represent the open reading frame, while the sequences in lowercase are the untranslated sequences. The predicted initiation Met start codon is in bold face and underlined, and the polyadenylation signal is double underlined. The signal peptide of Blo t 2 is underlined. Cysteine residues along the predicted mature protein sequence are italicized and bold faced. The stop codon (TAA) is represented by a dash. The ML domain spans from V19 to E139 of the full-length sequence.

Figure 2

Analysis of purified recombinant Blo t 2 (rBlo t 2) (A) Purified rBlo t 2 and molecular mass marker (M) was separated by 15% SDS-PAGE and stained with Coomasie brilliant blue. (B) ESI-TOF mass spectra of rBlo t 2. Mass of rBlo t 2 protein, both with (15,280 Da) and without modification (15,339 Da) are indicated on top of each peak.

Figure 3

(A) Venn diagram depicting the number of individuals with positive IgE-reactions to recombinant Der p 2, Der f 2 and Blo t 2 as measured using immuno-dot blots. (B) Scatter plot of specific IgE-binding of dust-mite allergic individuals (n = 116) and control (n = 86) (atopic, but not allergic to dust mite extracts) individuals to recombinant Der p 2, Der f 2 and Blo t 2. The amount of IgE-binding was measured as optical density. (C) Dot-plot of IgE-binding of atopic patients’ sera to Der p 2, Der f 2 and Blo t 2, showing the IgE-binding intensities and correlation between allergens. R square values of linear regression was indicated (p < 0.0001).

Figure 4

Competitive IgE ELISA assay. Six individual sera with different reaction profiles to Der p 2 and Blo t 2 (A) high IgE-reaction to both allergens; (B) high IgE reaction to Der p 2, moderate IgE reaction to Blo t 2; and (C) moderate IgE reaction to Blo t 2, low IgE reaction to Der p 2 were tested. Sera were pre-absorbed with various concentrations of inhibitors before incubation with Der p 2 or Blo t 2 in coated plates (solid surface). Solid lines indicate sera has been incubated with Blo t 2, whereas dotted line indicates sera has been incubated with Der p 2. The amount of inhibition was calculated in relation to the uninhibited control.

Figure 5

Correlation between the amount of IgE binding of 59 dust-mite allergic individuals’ sera to native Blo t 2 (nBlo t 2) and recombinant Blo t 2 (rBlo t 2). The amount of IgE binding is represented by optical density (OD) of the immuno-dot blot colorimetric reaction. Linear regression analysis shows that the IgE binding to the native and recombinant Blo t 2 were correlated (r² = 0.77, p < 0.0001).

Figure 6

In vitro histamine release from Blo t 2. Two hundred microliters of whole blood from two Blo t 2 allergic individuals and three non-allergic controls were challenged with serially diluted Blo t 2 for one-hour to stimulate histamine release. The amount of histamine released was measured by an ELISA assay. Mean and standard errors of duplicate experiments are shown.

Figure 7

Concentration of Blo t 2 and Der f 2 in dust samples from Singaporean homes. Dust samples were collected by vacuuming four areas within a home and assayed for the amounts of Blo t 2 (●) and Der f 2 (○) in beds (n = 101; 31), carpets (n = 19; 10), kitchens (n = 63; 22) and sofas (n = 65; 56). Horizontal bars on the scatter plot show the geometric mean concentration for each allergen. Asterisks denotes p-value of statistically significant differences; (****)p < 0.0001, (***)p < 0.001.

Figure 8

(A) Polymorphic amino acid residues (colored pink) of the different Blo t 2 isoforms were mapped and shown as both ribbon and space filled models in two different orientations. The homology model of Blo t 2 was deduced using the SWISS-MODEL server based on the crystal structure of Der f 2 (Protein data bank ID: 2F08) as a template. (B) The IgE epitope residues of Der p 2 (colored green) (listed in Supplementary Table 2) were mapped on the solved structure of Der p 2 (PDB ID: 1KTJ) and shown as both ribbon and space filled models in two different orientations. (A,B) were generated using Chimera. (C) Comparison of IgE epitope residues of Der p 2, and polymorphic residues of Blo t 2. Multiple alignment of mature proteins of Blo t 2 (GenBank ID ABG76185) and Der p 2 (AAF86462) was performed using Clustal O (v1.2.4). IgE epitopes of Der p 2 were highlighted green while sequences which were deleted or mutated in tandem are underlined and correspond to the residues listed in Supplementary Table 1. Polymorphic residues of Blo t 2 were highlighted pink and correspond to the residues listed in Tables 1 and 2. The pound sign (#) indicates amino acid residues in Blo t 2 that is likely to be an IgE epitope due to similarity with experimentally confirmed IgE epitopes on Der p 2 (Supplementary Table 1). An asterisk (*) indicated the positions with fully conserved residues, a colon (:) indicated conservation between groups of strongly similar properties, and a period (.) indicated conservation between groups of weakly similar properties. Numbers above the sequences indicate the presence of a cysteine residue in any of the proteins.