In this study we demonstrated the expression of a large repertoire of different α-defensin transcripts in the small intestine of a single horse. Consequently, this study is not to be considered representative for α-defensins in horses generally and conclusions regarding differences between races or states of health cannot be drawn. However, the extensive quantity of at least 38 detected α-defensin transcripts (30 probably functional peptides, DEFA1 to DEFA29 including DEFA5L, and eight obvious pseudogenes, DEFA30L to DEFA37L) in the intestinal tissue has rarely been observed before in other organisms and may indicate an important role of these AMPs in horses. The functional context of this high number of different transcripts is yet unclear.
In the human Paneth cells two different α-defensins are synthesised (DEFA 5 and DEFA6; [34
]) in addition to the neutrophil human α-defensin peptides 1-4, also known as HNP 1-4. In the intestine of the rhesus macaque four different peptides are observed, RED-1 to RED-4 [35
]. The only known organism with a numerically related repertoire of enteric α-defensins is the mouse. At least 23 different peptides are known, named cryptdins and cryptdin-related sequences [36
]. They were not only found in the small intestine but also in the colon, the cecum and the rectum. The antimicrobial activities of some of these cryptdins are not known. In mice three different observations highlight possible advantages to express a high amount of different defensin peptides and indicate their biological relevance.
First, the peptides can exhibit different specificities against microorganisms. The cryptdins 1 to 6 were analysed regarding their antimicrobial activity against different bacteria. The killing potency against the targets, particularly E. coli
and Salmonella typhimurium
, showed variabilities [37
Secondly, the expression level of the cryptdin genes along the intestinal tract showed large variations. Karlsson et al. [36
] observed a differential gene expression of cryptdins in the duodenum, jejunum and ileum. They concluded that the varying cryptdin synthesis in different tracts of the intestine may play an important role in the local regulation of bacterial colonisation in addition to the role in protection against infection. In having analysed only a single intestinal location, similar conclusions cannot be drawn from our data. However, different cloning frequencies of the transcripts found in this study may indicate different mRNA-expression levels at least at this location. The number of cDNA-fragments coding for a specific peptide varies from one (e.g. DEFA2, DEFA3) to 39 (DEFA25) in randomly selected clones. Although 38 different α-defensin transcripts were observed in this study, only seven cDNAs were cloned frequently (DEFA4, 5, 8, 9, 12, 17, 31L, Table ), whereas in all other cases the number of obtained clones varies between one and four. Since only a single set of amplification products was cloned and variants due to error-prone PCR are possible, these data are less representative. However, the same situation exists in the mouse, where over 30 α-defensin genes were included in the latest assemblies and over 20 different cDNAs were reported, but only six cDNAs have been cloned at high frequencies. It should be taken into account that despite the apparent diversity, it might be unlikely that more than 7-8 peptides accumulate to considerable levels in the small intestine of the horse. Furthermore, it is not known whether and to what extent the gene expression is inducible.
Thirdly, the gene expression of different mice cryptdins shows circadian variances [39
]. The expression level varies about 100% between simulated dark and light phases, primary between cryptdin 1 and 4. An interrelation with the time of ingestion was assumed. Moreover, the gene expression level of cryptdin 2 and 5 changes about two or three orders of magnitude in the first days after birth whereas the gene expression of cryptdin 1, 3, and 6 increases at lower pace [40
]. Probably the differential gene expression protects the organism during the first adaptation to a new environment.
Alpha-defensin transcripts were formerly only known in primates, glires and horses. In recent studies the existence of α-defensin genes was observed in the genomes of the opossum [19
], the elephant and the hedgehog tenrec [20
]. According to the Bayesian phylogenetic tree of mammals [41
], the horse is classified into the group of the Laurasiatheria
as well as cattle, dog, bat and hedgehog (Fig. ). Nothing is known about the existence of α-defensins in bat and hedgehog, and no α-defensin gene was found in cattle [21
] and dog [22
]. Whereas α-defensin genes exist in basal mammals like opossum, elephant and hedgehog tenrec and in the group of Euarchontoglires
, the Equidae
are the only known family expressing α-defensin genes within the group of Laurasiatheria
. In future studies it will be necessary to clarify why cattle and dog presumably lost their complete set of α-defensin genes while the horse increased the gene number extensively. Additionally, the presence or absence of α-defensins in the closest relatives of the horse like tapir and rhinoceros (which form together with the horse the group of Perissodactyla
) has to be analysed. This may lead to new aspects in the development, reorganisation and separation of defensin genes. It is very unlikely that α-defensins evolved independently within the Equidae
, indicated by the high analogy between the amino acid sequences of the horse compared with known α-defensins from primates and glires [18
]. It is assumed that α-defensins evolved from one or two ancestral genes by gene-duplication [22
]. According to the observation that platypus being the most basal mammal that already has been sequenced has four α-defensin genes [43
], one can hypothesise that α-defensins may have been lost independently in different clades during the divergence of the phylogenetic tree.
Figure 4 Phylogeny of the mammals based on the Bayesian phylogenetic tree according to Murphy et al. . In underlined species α-defensin genes and transcripts are known. In species underlined with a dashed line, α-defensin genes in the genome (more ...)
The genomic positions of the transcripts or of single exons, respectively, as shown in Fig. indicate a false assembly of EquCab2.0 or unidentified gaps in the genomic sequence. In some cases only one exon of a transcript was found with an identity of 100% in the genome whereas an appropriate second exon was missing (DEFA6
). In other cases the same genomic exons were found in different transcripts (DEFA6
(Exon 2), DEFA12
(Exon 2), DEFA12
(Exon 1)). The second exons of the DEFA6
transcripts were located at the same genomic position whereas only the first exon of DEFA7
was identified at an appropriate distance of approximately 700 bp upstream. The first exon of DEFA6
could not be detected in the genome indicating a gap. In addition to assembly errors, comprehensive gene reduplication and mutations of α-defensins and/or alternative splicing might be possible explanations, although the latter was never shown for α-defensins before. Additionally it is possible that transcripts with missing genomic sequences in the assembly EquCab2.0 could be also due to copy number variations (that have e.g. been reported for human α-defensins; [44
]). The animal used in our investigations might have possessed additional copies compared with the horse used for establishing the reference genome. However, Fig. shows a typical clustering of the equine α-defensins with average distances of approximately 10 kb between single genes. No transcripts were found in the small intestine for some α-defensin genes annotated in the genome of the horse or identified by BLAST-searches. It is possible that these genes are expressed in other tissues and cells, e.g. bone marrow, or are expressed at very low levels in the small intestine. In other species that have enteric α-defensins, they are the products of Paneth cells. Their number varies in different sections of the small intestine (duodenum, jejunum and ileum) and certain α-defensins were only found in the ileum [36
]. In our studies we most probably used tissue samples of the jejunum. Consequently, our results do not allow a differentiation between the single sections of the small intestine and it is possible that some transcripts may be additionally found exclusively in the duodenum and/or ileum.
The primary structure of mature α-defensin shows highly conserved residues, which are indispensable for the structural stability and the full function of the peptides. Among them are six invariant cysteine residues, necessary for the typical α-defensin intramolecular disulfide-bond connectivity (Cys1
), two charged amino acid residues, Arg5
, and Glu13
, forming a conserved salt bridge [32
], and Gly17
, which constitutes a structural motif which is essential for correct folding [33
]. By analysing the new equine α-defensins considering that these prerequisites must be fulfilled for an active α-defensin, it became evident that 20 of the 38 peptides (including the known DEFA1) may be active α-defensins with an antimicrobial potential. DEFA1 to DEFA19 and the DEFA5L transcript show the six conserved cysteine residues, the arginine and the glutamic acid residues necessary for the intramolecular salt bridge, and the highly conserved glycine residue necessary for correct folding. The α-defensins DEFA20 to DEFA29 exhibit also the cysteine and glycine residues but they have lost the intramolecular salt bridge formed by Arg5
. Nonetheless, they should be considered functional because this bridge is not required for correct pro-α-defensin folding [32
]. In contrast, the peptides DEFA30L to DEFA37L appear to be pseudogenes. Either some cysteine residues were absent (DEFA30L) or the transcription stops before completion because of a premature stop codon visible in the appropriate cDNA sequence. It is not known whether these molecules may have adopted new functions or are involved in regulatory systems. Predictions (e.g. as non-coding RNAs) at this point would be highly speculative. Interestingly, the premature stop codon of DEFA35L and DEFA36L at position 78 is at the same position that gives rise to θ-defensins in non-human primates [45
Typically, α-defensins have an anionic propeptide and a cationic mature peptide, and the charges are counterbalanced. Taking all intestinal α-defensins of the horse into account that can produce a mature peptide (DEFA1-DEFA29, DEFA5L) they follow the pattern of anionic propiece and cationic mature peptide, but only six of them are counterbalanced (DEFA8, 12, 13, 15, 21, 27). Several of the equine α-defensins (DEFA18-20, DEFA30L) have proline-rich C-terminal extensions resembling certain proline-rich antimicrobial peptides of cattle, named cathelicidins (CATHL2 and CATHL3) and others in sheep and goats [46
]. Also DEFA30L exhibits a proline-rich C-terminal region and four cystein residues, typical for cathelicidins, but the spacing of the cysteins is different. Other equine α-defensin peptides have a C-terminal glycine residue (DEFA8 to DEFA11). Many cathelicidin precursors also have a C-terminal glycine, which allows the peptide's C-terminus to be amidated. To our knowledge no defensins from other species are amidated. The only other α-defensin containing a C-terminal glycine is DEFA3 of platypus (Ornithorhynchus anatinus
, acc. Nr. P0C8A3, Swissprot: http://www.expasy.ch/sprot
Different infectious diseases may appear if the expression level or intracellular processing of Paneth cell α-defensins is abnormal. Wehkamp et al. [26
] discovered a reduced Paneth cell α-defensin synthesis in ileal Crohn's disease (Morbus Crohn), a chronic disease of the intestine, by using real-time PCR and immunohistochemical methods. An enhanced expression of epithelial α-defensin genes in colonic inflammations was reported by Wehkamp et al. [47
]. Ferguson et al. [27
] found that single nucleotide polymorphisms in the human Paneth cell α-defensin DEFA5 may confer susceptibility to inflammatory bowel disease. Inflammatory bowel diseases have also been described in horses, for example the duodenitis/proximal jejunitis syndrome which is characterised by catarrhal enteritis with mucosal hyperaemia or necrosis or colitis, which causes intramural oedema and haemorrhagic inflammation of the large intestine and is often fatal. Bacteria have been implicated as etiological agents of both diseases [48
]. The high quantity of equine intestinal α-defensins and the considerable biological effect of enteric DEFA1 against microorganisms emphasises the importance of equine intestinal defensins in protection of the horse against infections of the intestinal tract.