The repertoire of equine intestinal alpha-defensins

Abstract

Background: Defensins represent an important class of antimicrobial peptides. These effector molecules of the innate immune system act as endogenous antibiotics to protect the organism against infections with pathogenic microorganisms. Mammalian defensins are classified into three distinct sub-families (alpha-, beta- and theta-defensins) according to their specific intramolecular disulfide-bond pattern. The peptides exhibit an antimicrobial activity against a broad spectrum of microorganisms including bacteria and fungi. Alpha-Defensins are primarily synthesised in neutrophils and intestinal Paneth cells. They play a role in the pathogenesis of intestinal diseases and may regulate the flora of the intestinal tract. An equine intestinal alpha-defensin (DEFA1), the first characterised in the Laurasiatheria, shows a broad antimicrobial spectrum against human and equine pathogens. Here we report a first investigation of the repertoire of equine intestinal alpha-defensins. The equine genome was screened for putative alpha-defensin genes by using known alpha-defensin sequences as matrices. Based on the obtained sequence information, a set of oligonucleotides specific to the alpha-defensin gene-family was designed. The products generated by reverse-transcriptase PCR with cDNA from the small intestine as template were sub-cloned and numerous clones were sequenced.

Results: Thirty-eight equine intestinal alpha-defensin transcripts were determined. After translation it became evident that at least 20 of them may code for functional peptides. Ten transcripts lacked matching genomic sequences and for 14 alpha-defensin genes apparently present in the genome no appropriate transcript could be verified. In other cases the same genomic exons were found in different transcripts.

Conclusions: The large repertoire of equine alpha-defensins found in this study points to a particular importance of these peptides regarding animal health and protection from infectious diseases. Moreover, these findings make the horse an excellent species to study biological properties of alpha-defensins. Interestingly, the peptides were not found in other species of the Laurasiatheria to date. Comparison of the obtained transcripts with the genomic sequences in the current assembly of the horse (EquCab2.0) indicates that it is yet not complete and/or to some extent falsely assembled.

Figure 1

Alignment of 38 different α-defensin amino acid sequences. Fig. 1 represents the conserved amino acid residues and distinguishes potentially active peptides from pseudogenes. Conserved amino acid residues in the signal peptides present in all sequences were highlighted in grey. Conserved cysteine residues were highlighted in red, conserved arginine and glutamic acid residues, necessary for the intramolecular salt bridge were highlighted in blue, the conserved glycine residue, necessary for correct folding is highlighted in yellow. The previously known α-defensins DEFA1 and DEFA5L are written in red, defensin-like peptides (pseudogenes) in blue.

Figure 2

Divergences between the equine intestinal α-defensins. Fig. 2 emphasises the clustering of the peptides. The predicted signal sequence, the propiece, and the mature peptide were shown separately. DEFA1 was used as a prototype and the amino acids of the other peptides identify only those residues that differ from the DEFA1 sequence. Identical residues are represented by a vertical line.

Figure 3

Positions of α-defensin transcripts and genomic gene sequences based on assembly EquCab2.0. A genomic region of approximately 183 kb of chromosome 27 (top) and a second region of approximately 50 kb of an unspecified segment (bottom) are shown. Positions of genomic gene sequences are depicted as grey dots, positions of transcripts as triangles. Complete transcripts are identified by numbers according to the peptides' name. If only single exons are located at a specific position, this is indicated by the specification of the accordant exon written in parenthesis after the peptides' name. The scale indicates the kb in the chromosomal region.

Figure 4

Phylogeny of the mammals based on the Bayesian phylogenetic tree according to Murphy et al. [41]. In underlined species α-defensin genes and transcripts are known. In species underlined with a dashed line, α-defensin genes in the genome are only known by in silico approaches [20]. The asterisked species have no α-defensin genes.