Table 1.
Characteristics of HDM-allergic patients included in the study.
Fig 1.
Experimental workflow of HDM transcriptome, proteome and allergome analyses.
Messenger RNAs from D. farinae and D. pteronyssinus were sequenced using next generation sequencing. Following de novo assembly, translated sequence databases were derived after coding sequences (CDS) prediction and used as references to identify proteins in aqueous extracts from mite bodies and feces by LC-MS/MS analysis. In parallel, extracts from whole cultures were submitted to 2D-gel electrophoresis to establish reference 2D maps of species-specific proteomes and assign IgE reactivity.
Fig 2.
Functional analysis of proteomes from HDM bodies and feces.
Proteins identified by LC-MS/MS in fractionated extracts from either body (left panel) or feces (right panel) were classified into functional categories using the KAAS server (details of protein identification are provided in supplementary S2 and S3 Tables). The histograms denote the numbers of occurrences of KEGG Orthology (KO) annotations for each of D. farinae (A, B) and D. pteronyssinus (C, D) species, assembled in functional categories.
Fig 3.
Proteome and IgE reactivity maps of Dermatophagoides species.
Water-soluble HDM proteins were separated by 2D-gel electrophoresis and stained with Sypro Ruby or probed with a pool of serum IgEs from HDM-sensitized donors to establish 2D proteome (left panel) and IgE reactivity (right panel) maps for D. farinae (A, B) and D. pteronyssinus (C, D) species. Protein spots were picked and analyzed by LC-MS/MS after trypsin digestion, using the transcriptome-derived protein sequence collection supplemented with allergen sequences registered in the WHO/IUIS allergen database in order to facilitate identification. Spots corresponding to novel allergens (i. e. Der f 36 and Der p 36) are circled.
Fig 4.
Primary sequence alignment of group 36 allergens.
The amino acid sequences of Der f 36 and Der p 36 were aligned with the one of the putative KPM09946 protein from S. scabei. Putative N-glycosylation sites (●) in Der f 36 and Der p 36 are highlighted. The *,:,. symbols and empty space indicate identical, highly similar, similar or different amino acid, respectively. Residues are numbered relative to translation start.
Fig 5.
IgE reactivity of recombinant Der f 36 and Der p 36.
Recombinant non-glycosylated Der f 36 and Der p 36 were produced in P. pastoris. IgE reactivity was assessed by western blot using a pool of sera from HDM-sensitized individuals. Culture supernatant from a mock strain was used as a negative control.