House dust mites possess a polymorphic, single domain putative peptidoglycan d,l endopeptidase belonging to the NlpC/P60 Superfamily

Abstract

A 14 kDa protein homologous to the γ-d-glutamyl-l-diamino acid endopeptidase members of the NlpC/P60 Superfamily has been described in Dermatophagoides pteronyssinus and Dermatophagoides farinae but it is not clear whether other species produce homologues. Bioinformatics revealed homologous genes in other Sarcopteformes mite species (Psoroptes ovis and Blomia tropicalis) but not in Tetranychus urticae and Metaseiulus occidentalis. The degrees of identity (similarity) between the D. pteronyssinus mature protein and those from D. farinae, P. ovis and B. tropicalis were 82% (96%), 77% (93%) and 61% (82%), respectively. Phylogenetic studies showed the mite proteins were monophyletic and shared a common ancestor with both actinomycetes and ascomycetes. The gene encoding the D. pteronyssinus protein was polymorphic and intronless in contrast to that reported for D. farinae. Homology studies suggest that the mite, ascomycete and actinomycete proteins are involved in the catalysis of stem peptide attached to peptidoglycan. The finding of a gene encoding a P60 family member in the D. pteronyssinus genome together with the presence of a bacterial promotor suggests an evolutionary link to one or more prokaryotic endosymbionts.

Keywords: BDGP, Berkeley Drosophila Genome Project; DAP, diaminopimelic acid; HDM, house dust mite; LytFM homologues; Mites; NNPP, Neural Network Promoter Prediction; NOD, nucleotide-binding oligomerisation domain-containing protein; NlpC/P60 Superfamily; Peptidoglycan; Structural modelling; gDNA, genomic DNA; pI, isoelectric points; γ-d-Glutamyl-l-diamino acid endopeptidase.

Fig. 1

Schematic diagram showing the annealing sites for all the primers used in the PCR screening of the λBlueSTAR™ gDNA library of D. pteronyssinus. The arrows and numbers above the primers indicate the directions of amplification and the nucleotide positions, respectively, spanned by the primers within the λBlueSTAR vector, lytFM and 179 bp-3′ sequence. Since the Sau3A I-digested genomic DNA fragments of D. pteronyssinus could be ligated to the Xho I-digested vector arms in either direction, the lytFM gene could exist in orientation 1 (A) or inverted (B).

Fig. 2

Deduced amino acid sequence alignment between LytFM and its D. farinae, P. ovis and B. tropicalis homologues. The DNA sequences of lytFM and its homologues from D. farinae (GenBank: KN266412), P. ovis (GenBank: FR749374) and B. tropicalis (GenBank: CB282085) were translated into amino acid sequences using the ExPaSy translation tool and the alignment was performed using MAFFT . Conserved, consensus residues are highlighted in boxshades and chemically similar substitutions (:) and weakly similar substitutions (.) are marked as indicated. The predicted (SignalP 4.1) or demonstrated N-terminal residues are shown (+1) . The open triangle indicates the exon–intron–exon boundary identified in the leader sequence of the D. farinae homologue . The catalytic triad residues are indicated by open arrows and the residues comprising the S1 and S′ site are indicated by solid arrows and dots, respectively .

Fig. 3

Homology between the mite LytFM homologues and selected eukaryote and prokaryote NlpC/P60 proteins. The sequences of mature LytFM (D. pteronyssinus) and its homologues D. farinae (GenBank: KN266412), P. ovis (GenBank: FR749374) and B. tropicalis (GenBank: CB282085) were compared with eukaryote and prokaryote NlpC/P60 proteins and homology determined using MAFFT . The alignments were then shaded using boxshade (http://www.ch.embnet.org/software/BOX_form.html). Accession numbers are those cited in Fig. 4. The Asp₂₅₆ residue in the B. cereus BCYKFC and its equivalent in the P. oxalicum protein that directly interact with the free amino group of the l-Ala residue in the stem peptide during catalytic cleavage are highlighted in red.

Fig. 3

Homology between the mite LytFM homologues and selected eukaryote and prokaryote NlpC/P60 proteins. The sequences of mature LytFM (D. pteronyssinus) and its homologues D. farinae (GenBank: KN266412), P. ovis (GenBank: FR749374) and B. tropicalis (GenBank: CB282085) were compared with eukaryote and prokaryote NlpC/P60 proteins and homology determined using MAFFT . The alignments were then shaded using boxshade (http://www.ch.embnet.org/software/BOX_form.html). Accession numbers are those cited in Fig. 4. The Asp₂₅₆ residue in the B. cereus BCYKFC and its equivalent in the P. oxalicum protein that directly interact with the free amino group of the l-Ala residue in the stem peptide during catalytic cleavage are highlighted in red.

Fig. 3

Homology between the mite LytFM homologues and selected eukaryote and prokaryote NlpC/P60 proteins. The sequences of mature LytFM (D. pteronyssinus) and its homologues D. farinae (GenBank: KN266412), P. ovis (GenBank: FR749374) and B. tropicalis (GenBank: CB282085) were compared with eukaryote and prokaryote NlpC/P60 proteins and homology determined using MAFFT . The alignments were then shaded using boxshade (http://www.ch.embnet.org/software/BOX_form.html). Accession numbers are those cited in Fig. 4. The Asp₂₅₆ residue in the B. cereus BCYKFC and its equivalent in the P. oxalicum protein that directly interact with the free amino group of the l-Ala residue in the stem peptide during catalytic cleavage are highlighted in red.

Fig. 4

Phylogenetic relationships of mite LytFM homologues and selected eukaryote and prokaryote NlpC/P60 proteins. The phylogenetic tree was created using Phylogeny.fr pipeline. The approximate likelihood ratio test was used and the confidence values shown on the branches.

Fig. 5

Model of the mite LytFM protein. (A) A model of D. pteronyssinus LytFM generated by PHYRE2, using M. tuberculosis RipA (PDB: 2XIV) and B. cereus BcYkfC (PDB: 3H41). The 13-residue insertion compared to the B. cereus BcYkfC protein and catalytic residues are highlighted in magenta and yellow, respectively. (B) A surface representation of the LytFM showing the putative substrate-binding groove with the Cys₄₂, His₉₃ and Asn₁₀₅ catalytic residues revealed and shown in magenta, red and blue, respectively. The 13-residue insertion found in LytFM and the M. tuberculosis protein is highlighted in pink. (C) The LytFM model (green) is superimposed on the B. cereus BcYkfC template (light brown) to highlight the relative location of the 13-residue insertion (magenta) in the former. A 4-residue deletion in the mite LytFM homologues and M. tuberculosis protein relative to the B. cereus BcYkfC is shown in blue. The catalytic residues are highlighted in yellow.