Fig 1.
Multiple alignment of Der p 18 amino acid sequence with homologous proteins from other mites, insects, crustacea and man.
Species and GenBank accession numbers: Dermatophagoides pteronyssinus (house dust mite, Der p 18, AAY84563.1), Dermatophagoides farinae (house dust mite, Der f 18, AAM19082.1), Blomia tropicalis (storage mite, Blo t 18, AAQ24549.1), Camponotus floridanus (carpenter ant, EFN71329.1), Musca domestica (housefly, ABI29879.1), Pandalopsis japonica (shrimp, AFC60661.1), Ixodes scapularis (tick, XP_002404708.1), Scylla serrata (crab, ABY85409.1), Chelonus sp. (wasp, AAA61639.1), Homo sapiens (human, 1WAW_A). Marked in orange are the chitinase core domain including the conserved cysteines of the catalytic region (C1-C4) and the putative catalytic domains (CD1, CD2: the asterisk indicates the position of the glutamic acid that determines the presence of enzymatic activity). The putative chitin-binding domain (CBD) is highlighted in blue. Red and blue arrows indicate the borders of the segments which were used to create the structural model of Der p 18 (see Fig 2). Amino acids identical to those of Der p 18 are indicated by dots; dashes represent gaps.
Table 1.
Percentages of amino acid sequence identities among chitinases and chitinase-like proteins from mites, insects, crustaceans and human1.
Fig 2.
(A) Alignment of predicted secondary structure elements of Der p 18 with the secondary structure of human chitinase (1wawA) for portions of human chitinase with known three-dimensional structure and of the Der p 18 putative chitin-binding domain with tachycitin (1dqcA) as created with the SWISS-MODEL program. Identical amino acids are indicated by dots, dashes represent gaps and similar secondary structure elements (β-strands, α-helices) are marked. (B) Structural model of Der p 18 generated by the SWISS-MODEL program. The 29-amino-acid connecting sequence is indicated by a continuous line.
Fig 3.
Characterization of purified rDer p 18.
(A) An aliquot of 3 μg of rDer p 18 was separated by SDS-PAGE under reducing (1) and non-reducing (2) conditions and stained with Coomassie brilliant blue. M, molecular weight marker. (B) Far UV CD analysis of rDer p 18. The graph represents the mean residue ellipticity (θ, y-axis) at a given wavelength (190–250 nm, x-axis).
Fig 4.
Chitin-binding activity of Der p 18.
Coomassie-stained SDS-PAGE containing aliquots of rDer p 18, rDer p 15, WGA (positive control) and rDer p 5 (negative control) or HDM extract (lanes 1), the supernatants of these proteins (lanes 2) and proteins eluted from the chitin/chitin beads (lanes 3). The molecular weight marker is shown in lanes M. (C) Nitrocellulose-blotted samples of the experiment performed with HDM-extract and chitin beads (B) were incubated with rabbit anti-Der p 18 or anti-Der p 15 antibodies.
Fig 5.
Cross-reactivity of rabbit anti-rDer p 18 IgG antibodies with proteins from other mites, crustacea, mollusca and insects.
Blots containing extracts from Dermatophagoides pteronyssinus, Dermatophagoides farinae, Blomia tropicalis, shrimp, lobster, snail and wasp which had been separated by SDS-PAGE were incubated with normal rabbit antibodies before immunization (A), with rabbit anti-rDer p 18 (B) or with rabbit anti-rDer p 10 antibodies (C). (D) Inhibition of IgG reactivity to blotted nDer p 18 and nDer f 18. Nitrocellulose-blotted D. pteronyssinus (left panel) and D. farinae (right panel) extracts were incubated with a rabbit anti-Der p 18 pre-immune serum or immune serum, which had been pre-incubated with D. pteronyssinus extract, D. farinae extract, rDer p 18, rDer p 15 or BSA. Molecular weights (kDa) are shown at the margins.
Fig 6.
Localization of Der p 18 in D. pteronyssinus by immunogold electron microscopy and immunoblotting.
(A) Gut overview. The section marked by the rectangle is shown in (B) at higher magnification. (B) Specific labeling of Der p 18 in the peritrophic matrix. (C) Localization of Der p 18 in fecal particles after incubation with anti-Der p 18 antibodies or (D) with pre-immune serum. Arrows indicate gold particles. Bars: A, 1 μm; B, 500 nm; C, 100 nm; D, 100 nm. DF, digested food; GE, gut epithelium. GW, gut wall; MI, microvilli; PM, peritrophic matrix. (E) Nitrocellulose-blotted extracts of mite bodies and feces were incubated with rabbit anti-rDer p 18, anti-rDer p 2 antibodies or normal rabbit antibodies.
Fig 7.
IgE reactivity and allergenic activity of rDer p 18.
(A) IgE levels (y-axis: ISU ISAC standardized units) specific for a panel of HDM allergens (x-axis: Der p 1, Der p 2, Der p 5, Der p 7, Der p 10, Der p 11, Der p 14, Der p 15, Der p 18, Der p 21 and Der p 23) in HDM-allergic patients. (B) Dot-blotted rDer p 2, rDer p 18 and BSA were tested for IgE reactivity with sera from 7 HDM-allergic patients positive to rDer p 18 (#92–98), serum of a non-allergic person (NC) and with buffer (BC). Bound IgE Abs were detected with 125I-labeled anti-human IgE Abs and visualized by autoradiography. (C) Up-regulation of CD203c expression was determined by FACS analysis after incubation of basophils from 4 HDM-allergic patients (#92–95) with increasing concentrations of rDer p 2 or rDer p 18 (x-axes) and displayed as stimulation index on the y-axes. (D) Recombinant Der p 18, rDer p 2, rDer p 23 and BSA were dotted onto nitrocellulose strips and incubated with sera from Der p 18-positive patients (#99, #100). Bound IgE antibodies were detected with 125I-labeled anti-human antibodies and visualized by autoradiography. (E) Inhibition of IgE reactivity to blotted nDer p 18 by rDer p 18. Nitrocellulose-blotted D. pteronyssinus extract was incubated with sera from Der p 18-sensitized patients (#99 and #100), which had been pre-incubated with rDer p 18, or for control purposes, with BSA. Molecular weights (kDa) were shown at the margins.