A genome-wide screen identifies a single β-defensin gene cluster in the chicken: implications for the origin and evolution of mammalian defensins
© Xiao et al; licensee BioMed Central Ltd. 2004
Received: 13 May 2004
Accepted: 13 August 2004
Published: 13 August 2004
Defensins comprise a large family of cationic antimicrobial peptides that are characterized by the presence of a conserved cysteine-rich defensin motif. Based on the spacing pattern of cysteines, these defensins are broadly divided into five groups, namely plant, invertebrate, α-, β-, and θ-defensins, with the last three groups being mostly found in mammalian species. However, the evolutionary relationships among these five groups of defensins remain controversial.
Following a comprehensive screen, here we report that the chicken genome encodes a total of 13 different β-defensins but with no other groups of defensins being discovered. These chicken β-defensin genes, designated as Gallinacin 1–13, are clustered densely within a 86-Kb distance on the chromosome 3q3.5-q3.7. The deduced peptides vary from 63 to 104 amino acid residues in length sharing the characteristic defensin motif. Based on the tissue expression pattern, 13 β-defensin genes can be divided into two subgroups with Gallinacin 1–7 being predominantly expressed in bone marrow and the respiratory tract and the remaining genes being restricted to liver and the urogenital tract. Comparative analysis of the defensin clusters among chicken, mouse, and human suggested that vertebrate defensins have evolved from a single β-defensin-like gene, which has undergone rapid duplication, diversification, and translocation in various vertebrate lineages during evolution.
We conclude that the chicken genome encodes only β-defensin sequences and that all mammalian defensins are evolved from a common β-defensin-like ancestor. The α-defensins arose from β-defensins by gene duplication, which may have occurred after the divergence of mammals from other vertebrates, and θ-defensins have arisen from α-defensins specific to the primate lineage. Further analysis of these defensins in different vertebrate lineages will shed light on the mechanisms of host defense and evolution of innate immunity.
Defensins constitute a large family of small, cysteine-rich, cationic peptides that are capable of killing a broad spectrum of pathogens, including various bacteria, fungi, and certain enveloped viruses [1–5]. These peptides play a critical role in host defense and disease resistance by protecting the hosts against infections. Transgenic mice expressing human enteric defensin HD5 are fully protected against the doses of Salmonella typhimurium that are otherwise lethal to the wide-type mice . Conversely, mice deficient in the matrilysin gene, which is responsible for activating enteric defensins, become more susceptible to oral infection with S. typhimurium .
Defensins have been identified in species ranging from plants, insects to animals and humans [1–5]. Characterized by the presence of 6–8 cysteine residues in relatively defined positions, all defensins are structurally related in that they form 3–4 intramolecular disulfide bonds and 2–3 antiparallel β-sheets with or without an α-helix. Based on the spacing pattern of cysteines, these peptides are broadly divided into five groups; namely plant, invertebrate, α-, β-, and θ-defensins [1–5]. Alignment of all known defensin sequences revealed the consensus defensin motif of each group as follows: plant defensin: C-X8–11-C-X3–5-C-X3-C-X9–12-C-X4–11-C-X1-C-X3-C; invertebrate defensin: C-X5–16-C-X3-C-X9–10-C-X4–7-C-X1-C; α-defensin: C-X1-C-X3–4-C-X9-C-X6–10-C-C; and β-defensin: C-X4–8-C-X3–5-C-X9–13-C-X4–7-C-C. The α- and β-defensins are unique to vertebrate animals with α-defensins only being found in rodents and primates, while β-defensins are present in all mammalian species investigated [1–3]. On the other hand, θ-defensins have only been found in certain primates as a result of posttranslational ligation of two α-defensin-like sequences [8–10]. A pseudogene for θ-defensin is also present in humans .
Analysis of human and mouse genomes indicated that β-defensins form 4–5 distinct clusters on different chromosomes with each cluster consisting of multiple defensin genes . Interestingly, the single mammalian α-defensin locus is located on a β-defensin cluster with θ-defensins residing in the center of α-defensins . Studies with mammalian defensins suggested a rapid duplication followed by positive selection and diversification within each group [13–18]. However, the evolutionary relationships among three groups of mammalian defensins and among plant, invertebrate, and mammalian defensins remain controversial. Similarity in spatial structure and biological functions favors the notion that all mammalian defensins are evolutionarily related , although a phylogenetic analysis suggested a closer relationship between β- and insect defensins than between α- and β-defensins .
Existence of a large number of expressed sequence tag (EST) sequences and recent completion of chicken genome sequencing at a 6.6× coverage  provided a timely opportunity to discover a complete repertoire of defensin-related sequences in birds for studying the evolutionary relationship between invertebrate and mammalian defensins. Here we report identification of a single β-defensin cluster that is composed of 13 genes located on the chicken chromosome 3q3.5-q3.7. Evolutionary and comparative analyses of these chicken β-defensins with mammalian homologues strongly suggested that all mammalian defensins have evolved from a common β-defensin-like ancestor, which has undergone rapid duplication, positive diversifying selection, and chromosomal translocations, thereby giving rising to multiple gene clusters on different chromosomal regions.
Results and Discussion
Discovery of novel chicken defensins
Identification of chicken β-defensins
Gene Size (bp)3
No other sequence containing β-defensin-like six-cysteine motif has been found in NR, EST or genomic databases, suggesting that 13 Gal genes constitute the entire repertoire of the β-defensin family encoded in the chicken genome. Although it is highly unlikely, we could not rule out the possibility that additional defensin-related genes with distant homology might be uncovered in the chicken by different computational search methods such as the use of Hidden Markov models [12, 15]. It is noted that none of other groups of defensins have been discovered in the chicken, indicating that plant, invertebrate, α-, and θ-defensins are absent in the chicken lineage.
Evolutionary analysis of vertebrate β-defensins
Comparison of the numbers of synonymous and nonsynonymous nucleotide substitutions provides a powerful test of the hypothesis that positive Darwinian selection has acted to favor changes at the amino acid level . This approach has previously been applied to both α- and β-defensins of mammals and has revealed positive selection acting on the mature defensin but not on other regions of the gene [16, 17]. In the comparison of the chicken β-defensin sequences, synonymous sites were saturated with changes or nearly so, making it impossible to test the hypothesis of positive selection in every case. In pairwise comparisons among all sequences, mean pS in the propeptide region was 0.551 ± 0.036 (S.E.), while mean pN was 0.369 ± 0.040. In the mature defensin region, mean pS was 0.673 ± 0.027, while mean pN was 0.534 ± 0.051. Mean pN in the mature defensin was significantly greater than that in the propeptide (z-test; P < 0.05), indicating lesser functional constraint on the amino acid sequence of the former. The high mean pS shows that chicken β-defensin genes have not duplicated recently, unlike β-defensin genes of the bovine . In the comparison between the most closely related pair of sequences (Gal6 and Gal7), mean pS in the mature defensin was 0.221 ± 0.082, while mean pN was 0.331 ± 0.076. While these values are not significantly different at the 5% level, the fact that pN was higher than pS suggested that positive selection may have acted to diversify the mature defensin region between these two genes.
Genomic organization and chromosomal localization of the chicken β-defensin gene cluster
Comparing the cDNA with genomic sequences also revealed the structure of each Gal gene. Unlike most mammalian β-defensin genes, which primarily consist of two exons and one intron, the Gal genes were found to be composed of four short exons separated by three introns with variable lengths ranging from 117 bp to 3,322 bp (Table 1). Gal12 is an exception, in which the last two exons have been fused together. While the first exon of the Gal genes encodes 5'-untranslated region (UTR) and the majority of the last exon encodes 3'-UTR as well as a few C-terminal amino acids, two internal exons resemble mammalian β-defensin genes in that one exon encodes the signal and pro-sequence and the other encodes the mature sequence with six-cysteine motif [19, 27–29]. Apparently, the first two and the last two exons of the Gal genes have joined together during the evolution as a result of exon shuffling, which occurred in many other evolutionarily conserved gene families , including invertebrate defensins . The fusion of defensin exons in mammals is presumably adaptive because it allows a faster mobilization of such host defense molecules to better cope with invading microbes.
Tissue expression patterns of chicken β-defensins
Comparative analysis of chicken and mammalian β-defensin gene clusters
These results strongly suggested that all vertebrate β-defensins are evolved from a single gene. This conclusion is further supported by the fact that there are three highly similar β-defensin-like sequences present in the largely finished zebrafish genome (G. Zhang, unpublished data). In addition, a group of homologous β-defensin-like sequences, namely crotamine and myotoxins, have been found in several Crotalus snakes , which are presumably derived from a single ancestral gene. The appearance of multiple β-defensin gene clusters on different chromosomal regions in mammalian species  is apparently a result of rapid gene duplication, positive diversifying selection, and chromosomal translocation following divergence of mammals from other vertebrate lineages.
In addition to the structural conservation between β-defensin-like sequences in the rattlesnake and mammals , a growing body of evidence suggests that their functions appear to be largely conserved in that both are capable of interacting negatively-charged lipid membranes followed by formation of ion channels or pores [32–34]. It is noteworthy that the conservation of Cathepsin B (CTSB) adjacent to β-defensins is perhaps not surprising, given the recent finding that cathepsins are involved in the cleavage and inactivation of β-defensins .
We have showed that chicken genome encodes a total of 13 different β-defensin genes clustered densely within a 86-Kb distance on the chromosome 3q3.5-q3.7, but with no α-defensin genes. These peptides exhibit homology to different subgroups of mammalian β-defensins-, consistent with the hypothesis that α-defensins and β-defensins arose by gene duplication after the divergence of birds and mammals. The θ-defensins are specific to primates; and thus appear to have arisen from α-defensins by gene duplication specific to the primate lineage. Apparently, the evolution of defensins is rapid and driven by duplication and positive diversifying selection. Collectively, this study represents the first large-scale detailed investigation of defensins in non-mammalian vertebrates. There is no doubt that further analysis of these defensin genes will lead to a better understanding of host defense mechanisms and evolution of innate immunity.
Computational search for novel chicken defensins
To identify novel defensins in the chicken, all known cysteine-containing defensin-like peptide sequences discovered in plants, invertebrates, birds, and mammals were individually queried against the translated chicken NR, EST, HTGS, and WGS databases in the GenBank by using the TBLASTN program  with default settings on the NCBI web site . All potential hits were then examined for the presence of the characteristic defensin motif. For every novel defensin identified, additional iterative BLAST searches were performed until no more novel sequences could be revealed. Because mammalian defensins tend to form clusters [12, 14, 15, 18], all chicken genomic sequences containing defensin sequences were also retrieved from the GenBank and translated into six open reading frames and curated manually for the presence of the defensin motif in order to discover potential sequences with distant homology.
Alignment and phylogenetic analysis of chicken β-defensins
Multiple sequence alignment was constructed by using the ClustalW program (version 1.82) . A phylogenetic tree of amino acid sequences of mature β-defensins was constructed by the neighbor-joining method . So that a comparable data set would be used for all pairwise comparisons, any site at which the alignment postulated a gap in any sequence was excluded from the analysis. To maximize the number of sites available for analysis, certain sequences with large deletions were excluded from the analysis. Because the sequences were very short (25 aligned sites), no correction for multiple hits was applied. The reliability of clustering patterns within the tree was assessed by bootstrapping; 1000 bootstrap pseudo-samples were used. The proportion of synonymous nucleotide differences per synonymous site (pS) and the proportion of nonsynonymous nucleotide differences per nonsynonymous site (pN) were estimated by the method of Nei and Gojobori . Again, no correction for multiple hits was applied because a small number of sites were examined.
Assembly of the chicken β-defensin gene cluster
To generate a continuous defensin gene cluster, the HTGS and WGS sequences containing the putative defensin genes were retrieved from the GenBank, aligned to generate a longer contig, which was confirmed later by searching through the assembled chicken genome released on February 29, 2004, by using the BLAT program  under the UCSC Genome Browser web site . The relative positions, orientations, and structural organizations of individual genes were determined by comparing its cDNA sequence to the continuous genomic contig that we assembled.
Chromosome localization of the chicken β-defensin gene cluster
Fluorescence in situ hybridization (FISH) was used for chromosomal assignment of the chicken β-defensin gene cluster by using the BAC clone TAM31-54I4 as probe, which harbors 11 Gal genes. Metaphase chromosome speads were prepared from mitogen-stimulated chicken splenocyte culture as we described [41, 42]. The BAC clone was labeled by nick translation with biotin 16-dUTP (Roche Diagnostics), hybridized to metaphase chromosome DNA, followed by detection with FITC-labeled avidin (Roche Diagnostics) and staining with propidium iodide to simultaneously induce the R-banding.
RT-PCR analysis of the tissue expression patterns of chicken β-defensins
Primer sequences used for RT-PCR analysis of novel chicken β-defensins
Product Size (bp)
Note added in proof
Following submission of this manuscript, Lynn et al. reported independently discovery of seven novel chicken β-defensins in the chicken EST database by using homology search strategies . Consistent with our conclusion, they also revealed occurrence of positive selection particularly in the mature region of chicken β-defensins following evolutionary analysis. Moreover, albeit the use of a different nomenclature, they confirmed that the expressions of Gal 4–7 are primarily in bone marrow, while other genes are more restricted to liver and the genitourinary tract.
List of abbreviations
expressed sequence tag
high throughput genomic sequence
whole-genome shortgun sequence
bacterial artificial chromosome
fluorescence in situ hybridization
This work was supported in part by Oklahoma Center for Advancement of Science and Technology Grant HR-136 (to GZ) and the Oklahoma Agricultural Experiment Station.
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