Mol Biol Rep (2014) 41:3859–3866 DOI 10.1007/s11033-014-3253-z

Evolution of a and h revealed by analysis of genomes

Diyan Li • Long Zhang • Huadong Yin • Huailiang Xu • Jessica Satkoski Trask • David Glenn Smith • Ying Li • Mingyao Yang • Qing Zhu

Received: 15 October 2013 / Accepted: 8 February 2014 / Published online: 21 February 2014 Ó Springer Science+Business Media Dordrecht 2014

Abstract Defensins are endogenous with cys- difference between them is that h-defensins have an addi- teine-rich antimicrobial ability that contribute to host tional stop codon limits the initial domain peptides defence against bacterial, fungal and viral infections. There to 12 residues. , and do not are three subfamilies of defensins in : a, b and h- produce h-defensin peptides due to a premature stop codon defensins. a-defensins are most present in and present in the signal sequence of all h-defensin . Paneth cells; b-defensins are involved in protecting the skin By using comprehensive computational searches, here we and the mucous membranes of the respiratory, genitourinary report the discovery of complete repertoires of the a and h- and gastrointestinal tracts; and h-defensins are physically defensin family in ten primate species. Consistent with distinguished as the only known fully-cyclic peptides of previous studies, our phylogenetic analyses showed all pri- animal origin, which are first isolated from rhesus macaques. mate h-defensins evident formed one distinct clusters All three kinds of defensins have six conserved cysteines, evolved from a-defensins. b-defensins are ancestors of both three intramolecular disulfide bonds, a net positive charge, a and h-defensins. has two copies of DEFA1 and and b-sheet regions. a and h-defensins are closely related, DEFT1P, and two extra DEFA3 and DEFA10P com- comparative sequences showed that the pared with . As different primates inhabit in quite different ecological niches, the production of species-spe- cific a and h-defensins and these highly evolved h-defensins Diyan Li, Long Zhang and Huadong Yin have contributed equally to in old world monkeys would presumably allow them to better this work. respond to the specific microbial challenges that they face.

Electronic supplementary material The online version of this article (doi:10.1007/s11033-014-3253-z) contains supplementary Keywords a Defensin Á h Defensin Á Primates Á Genome material, which is available to authorized users. evolution

D. Li Á L. Zhang Á H. Yin Á Y. Li Á M. Yang (&) Á Q. Zhu (&) Institute of Animal Genetics and Breeding, Sichuan Agricultural University, Ya’an 625014, People’s Republic of China Introduction e-mail: [email protected] Q. Zhu Defensins are cysteine-rich, cationic e-mail: [email protected] expressed by the leukocytes and epithelial cells of fish [1], birds [2] and [3, 4], that form an important part of H. Xu the [5]. There are three defensin College of Animal Science and Technology, Sichuan Agricultural University, Ya’an 625014, subfamilies exist in vertebrates: a-defensins, b-defensins, People’s Republic of China and circular h-defensins [6]. b-defensins have been found in most vertebrate species (like fish [1], birds [2] and mammals J. S. Trask Á D. G. Smith [3, 4]) with a much wider tissue expression pattern; But a- Department of Anthropology and California National Primate Research Center, University of California, Davis, Davis, defensins are specific to mammals and are mainly produced CA 95616, USA by leukocytes of myeloid origin and Paneth cells of small 123 3860 Mol Biol Rep (2014) 41:3859–3866 intestine [7, 8]; On the other hand, h-defensins were first [24]. Rhesus macaques have three h-defensin genes that isolated from the leukocytes of rhesus macaques [6] and are encode different nonapeptides. For simplicity, the different currently the only backbone-cyclic peptides known in ani- nonapeptides a, b, and c will be named. Because the no- mals [9], are believed to have evolved from a-defensins [10]. napeptides in a h-defensin can be identical (aa, bb, and cc) In human, b-defensins and a-defensins are encoded by or different (ab, ac, and bc) [25–27], two different h-de- adjacent two gene clusters mapped to chromosomal region fensin genes can produce three different peptides (aa, ab, 8p23.1-p23.2: two families with differing disulfide and bb), and three different h-defensin genes can produce topology share a common ancestry [11]; h-defensins were six (aa, bb, cc, ab, ac, and bc). All six potential h-defensin located in a-defensin gene cluster [12]. The first found cir- peptides exist in rhesus polymorphonuclear cular peptide, termed rhesus theta defensin-1 (RTD-1), is (PMNs) [28], but their relative amounts differ greatly, with microbicidal for and fungi at low micromolar con- RTD-1 being the most abundant. Because n different h- centrations. Antibacterial activity of the cyclic peptide was defensin genes could produce (n/2)*(n ? 1) peptides [26], threefold greater than that of an open-chain analog, and the the four h-defensin genes of olive baboons (Papio anubis) cyclic conformation was required for antimicrobial activity could theoretically produce ten different peptides [29]. Five in the presence of 150 mM sodium chloride [6]. Defensins of these were identified at the peptide level [29]. The possess potent antimicrobial activity against a broad range of potential contribution of copy number polymorphism of a- bacteria, fungi, and enveloped as well as showing defensins and the only one circular protein (h-defensins) to activity against protozoan parasites [13, 14], primarily by innate resistance to (retro) viral infections in primates led binding to and disrupting the membrane of the invading us to perform this phylogenetic study. organism. These defensins interact with microbial targets in diverse biological settings, and these differences are reflec- ted in the regulation of their synthesis, storage, and release Materials and methods [11]. In general, metabolically active bacteria are more sensitive to defensins than are bacteria that have been made Computational search for a and h defensins inactive by nutrient deprivation or metabolic inhibitors [7]. Certain envelope viruses are also inactive by defensins [15]. To identify potential sequences in all primate species with Previous study showed that mammalian a-defensin whole genome sequences available, sequences of intact a genes may have evolved from two separate ancestors and h-defensin genes previously reported for human, originated from b-defensins [12]. h-defensins arose in Old and rhesus were retrieved from GenBank and World monkeys by mutation of a pre-existing a-defensin used as query sequences to conduct a TBLASTX search of gene [10]. There are 11 a-defensins in human, including each genome sequence and cDNA sequence using the six functional genes (DEFA1-6) and five pseudogenes E-value 1e-10. The primate species examined included (DEFA7P-11P) [12] and two replicates of human DEFT1P human (Homo sapiens, GRCh37/hg19), chimpanzee (Pan pseudogenes [12]. These six human functional a-defensins troglodytes, CSAC 2.1.4/panTro4), gorilla (Gorilla gorilla characterized to date show broad anti-bacterial activity [16, gorilla, gorGor3.1/gorGor3), orangutan (Pongo pygmaeus 17] as well as anti-HIV properties [18, 19]. The anti-HIV-1 abelii, WUGSC2.0.2/ponAbe2), gibbon (Nomascus leu- activity of DEFA1 was recently shown to consist of a direct cogenys, GGSC Nleu3.0/nomLeu3), rhesus macaque effect on the , combined with a serum-dependent (Macaca mulatta, MMUL_1), marmoset (Callithrix jac- effect on infected cells [19]. Human DEFA1 to DEFA4 are chus, WUGSC 3.2/calJac3), tarsier (Tarsius syrichta, constitutively produced by neutrophils [20], whereas Broad/tarSyr1), mouse lemur (Microcebus murinus, Broad/ DEFA5 and DEFA6 are produced in the Paneth cells of the micMur1) and bushbaby (Otolemur garnettii, Broad/oto- small intestine [21]. Extensive copy number variation was Gar3). All potential hits were then examined for presence found at the human anti-microbial a-defensin genes of the characteristic a and h-defensin motif or highly DEFA1 and DEFA3 [22]. Duplicated genes present in conserved signal/prosegment sequence. For every a and h- variant tandem arrays may have greater potential than defensin sequence identified, additional iterative BLAST simple duplications for the combinatorial creation of new searches were performed as described above until no more functions by recombination and gene conversion, while sequences can be revealed. still preserving pre-existing functions on the same haplo- type [22]. Human bone marrow expresses a Prediction of full-length coding sequences and genomic (DEFT1P) that encodes an antimicrobial peptide (retrocy- structures of a and h defensins clin) homologous to rhesus monkey circular minidefensins (h-defensins) [23]. Retrocyclin-1 is also a lectin that binds All known a and h-defensin precursors are encoded in two gp120, CD4, and galactosylceramide with high affinity separate exons separated by a short intron of less than 2 kb, 123 Mol Biol Rep (2014) 41:3859–3866 3861 with one exon encoding signal/prosegment sequence and NM_001141941.1, Baboon-DEFT1:NM_001141939.1, the other exon encoding the mature peptide containing the Macaca nemestrina DEFT1: AY128123.1) and 2 human b- six-cysteine a and h defensin motif [30]. 2.5-kb sequence defensins (human-DEFB27: AF525929.1 and human- flanking both stream of the potential defensin motif or DEFB28: AF525930.1) were downloaded from NCBI. signal/prosegment sequence were retrieved to identify the Peptide sequence logo of a and h-defensins (mature a-defensin full-length coding sequence and to derive the structural domain) was produced by WEBLOGO (http://weblogo.berke organization of that a and h-defensin gene by using a ley.edu/logo.cgi). combination of GenomeScan [31] or GENSCAN [32]. Pairwise comparisons of nucleotide sequences at the codon level was carried out by using the method of Max- Identification and characterization of a and h defensin imum Likelihood to estimate the number of non-synony- gene clusters mous substitutions per non-synonymous site (dN) and the number of synonymous substitutions per synonymous site

To determine the relative position and orientation of each (dS) with the Felsenstein 1981 model [39] for multiple defensin in the genome, individual defensins were searched substitutions. Estimate selection (dN–dS) for each codon against the assembled genomes. The BLAT program [33] and comparison of nucleotide or amino acid sequences was used for gene mapping through the UCSC Genome were carried out by using the MEGA5 [36]. Browser (http://genome.ucsc.edu). The chromosomal locations of the a and h-defensin gene clusters of primates were revealed by using the Map Viewer program (http:// Results www.ncbi.nlm.nih.gov/mapview). Identified a and h-defensin genes in primates Sequence alignment and molecular evolutionary analysis By using computational search for a and h-defensins from ten primate genomes and cDNA, we found that there are 11 The deduced defensin sequences were aligned by ClustalX a-defensin genes (six functional genes and five pseudo- [34] with manual adjustments. A neighbor-joining [35]treeof genes) and 2 h-defensin pseudogenes in human (Table 1). 112 nucleotide sequences of defensin genes (including 89 a- Orangutan has the most of a and h-defensins and rhesus has defensins and 21 h-defensins and 2 human b-defensins) from the least a-defensins. In this study, fifteen h-defensins were primates was constructed in MEGA5 [36] using the p-distance detected from ten primate genomes, together with other six method [37]. The reliability of the estimated trees was eval- downloaded primates h-defensins from NCBI were used uated by the bootstrap method [38] with 1,000 replications. for amino acid sequences alignment. The alignment of Percentage bootstrap values C50 are shown above branches. these h-defensins was conducted by using MEGA5 [36]. All sequences were computational searched from down- From the alignment Fig. 1a we can see that human, loaded genomes except 6 h-defensins (Pygmy chimpanzee- chimpanzee and gorilla pseudo h-defensins have the same DEFT1: AY128114.1, Baboon-DEFT3: NM_001141938.1, stop codon in the signal peptide, unlike human and gorilla; Baboon-DEFT2: NM_001141940.1, Baboon-DEFT4: chimpanzee does not have the premature stop codon in a-

Table 1 Detected a and h-defensin genes in primates Gene Protein a-Defensin a-Defensin h-Defensins h-Defensin Total Theoretically genes pseudogenes genes pseudogenes h-defensins

Human 6 5 0 2 13 0 Chimpanzee 4 4 0 2 10 0 Gorilla 4 4 0 1 9 0 Orangutan 6 4 4 1 15 10 Gibbon 7 0 2 0 9 3 Rhesus 2 2 3 0 7 6 Marmoset 13 0 0 0 13 0 Tarsier 7 1 0 0 8 0 Mouse lemur 9 0 0 0 9 0 Bushbaby 10 1 0 0 11 0

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Fig. 1 Panel a is fifteen primates h-defensin and three human a- indicates the same base. Panel b is sequence logo of mature a- defensin sequences encoded by homologous (pseudo) genes. Bound- defensin domain of all 112 a and h-defensins related to this study. aries of the signal peptide, prosegment and mature a-defensin domain Asterisk indicate have significantly (p \ 0.05) dN–dS at the amino acid are shown at the bottom. The boxed nonapeptide domain residues are site. Panel c is rhesus h-defensin-1 (heterodimer), rhesus h-defensin-2 incorporated into the mature h-defensin peptide. Only residues that (homodimer of DEFT1), and rhesus h-defensin-3 (homodimer of differ from the first entry in each sequence set are shown. The crossed DEFT2) from left to right respectively, which are formed by these two circles represent stop codons, and are annotated. Abbreviations: rhesus-DEFT1 and rhesus-DEFT2 hemi-defensins (nonapeptide), DEFA, a-defensin; DEFT, h-defensin. -indicates an indel (gap) in the each of which contributes three cysteines [6]. The peptide bonds nucleotide sequence. DEFTP, pseudo h-defensin. A black dot between the two hemi-defensins are shown as red arrows

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72 Baboon-DEFT2 defensin domain. We can see that both functional and Baboon-DEFT4 Pig-tailed macaque-DEFT1 93 Rhesus-DEFT1 pseudo h-defensin genes just occur in these old world Baboon-DEFT1 63 Rhesus-DEFT2 monkeys. All detected 89 a-defensin, 15 h-defensin, 6 Rhesus-DEFT3 Baboon-DEFT3 66 Human-DEFT1P downloaded h-defensin and 2 downloaded b-defensin DNA Human-DEFT1P(2) Pygmy chimpanzee-DEFT1P Chimpanzee-DEFT2P θ-defensin sequences and amino acid sequences were in Supplement Chimpanzee-DEFT1P Gorilla-DEFT1P Orangutan-DEFT5P material S1 and S2 respectively. 5252 Orangutan-DEFT2 64 Orangutan-DEFT3-subunitB-like 82 Orangutan-DEFT4-subunitB-like By using WEBLOGO (http://weblogo.berkeley.edu/ 95 Gibbon-DEFT1 50 Gibbon-DEFT2 Gibbon-DEFA10 logo.cgi), we generate the mature a-defensin domain pep- 79 Orangutan-DEFA3 89 Human-DEFA10P tide sequence logo of a and h-defensins detected in this Chimpanzee-DEFA10P 87 Rhesus-DEFA1 Gibbon-DEFA8 study Fig. 2b. Earlier results [3, 40] showed that mean d 95 51 Chimpanzee-DEFA8P N 75 Gorilla-DEFA8P 63 Human-DEFA8P was significantly greater than mean d in the mature pep- 90 Orangutan-DEFA12P S Orangutan-DEFA13P 94 Marmoset-DEFA8 tide, but there was no significant difference between mean, Marmoset-DEFA1 82 Marmoset-DEFA3 75 Marmoset-DEFA2 d and mean d in the signal peptide or prosegment [12]. Marmoset-DEFA4 S N 78 74 Chimpanzee-DEFA7 99 Gorilla-DEFA7P-PARTIAL Therefore, we estimated selection for (dN–dS) for each Human-DEFA7P Rhesus-DEFA4P Tarsier-DEFA3 codon by using the MEGA5 [36]. The greatest three nor- Tarsier-DEFA6 63 Tarsier-DEFA5 Tarsier-DEFA7 malized dN–dS sites of the mature a-defensin domain Tarsier-DEFA1 Tarsier-DEFA2 peptide sequence were site 89, 96 and 102. These 96 and 98 Marmoset-DEFA5 99 Orangutan-DEFA5 79 Gibbon-DEFA4 102 sites have significantly (p \ 0.05) d d , but not sig- 87 Human-DEFA4 N– S 59 Chimpanzee-DEFA4 Gorilla-DEFA4 75 nificantly at site 89 (p = 0.07) Fig. 2b. All three types of 100 Bushbaby-DEFA1 64 Bushbaby-DEFA6 Bushbaby-DEFA11P defensins have six conserved cysteines. Specifically, the six Bushbaby-DEFA10 83 Mouselemur-DEFA3 50 Mouselemur-DEFA1 cysteines of a-defensins are disulfide-linked 1–6, 2–4, and Mouselemur-DEFA5 61 72 Orangutan-DEFA6 82 Orangutan-DEFA7 3–5 Fig. 1b, but in b-defensins they are connected 1–5, 99 Gibbon-DEFA5 93 85 Human-DEFA5 Gorilla-DEFA5 2–4, and 3–6 [41]. h-defensins are formed by peptide Marmoset-DEFA7 76 97 Marmoset-DEFA10 h 98 Marmoset-DEFA12 splicing from two hemi- -defensins, each of which con- Marmoset-DEFA11 Marmoset-DEFA9 tributes three cysteines Fig. 1c. Rhesus macaques have 83 Marmoset-DEFA13 85 Human-DEFA6 90 Gorilla-DEFA6 three h-defensin genes that encode different nonapeptides. 63 Gibbon-DEFA6 Orangutan-DEFA8 62 92 Human-DEFA9P These three nonapeptides can produce six h-defensin pep- Chimpanzee-DEFA9P 93 Gorilla-DEFA9P 52 Gibbon-DEFA7 tides. All six potential h-defensin peptides exist in rhesus 69 Orangutan-DEFA11P Rhesus-DEFA3P 54 Tarsier-DEFA4 PMNs [28]. Rhesus h-defensin-1 to 3 are shown in Fig. 1c. 100 Mouselemur-DEFA4 96 Mouselemur-DEFA9 Mouselemur-DEFA2 73 Mouselemur-DEFA7 Mouselemur-DEFA8 Molecular evolutionary analysis 77 Mouselemur-DEFA6 77 Bushbaby-DEFA2 60 Bushbaby-DEFA4 of and a and h-defensin genes in primates Bushbaby-DEFA5 99 Bushbaby-DEFA7 Bushbaby-DEFA8 100 Bushbaby-DEFA3 Bushbaby-DEFA9 Marmoset-DEFA6 By using MEGA5 [31], a neighbor-joining [35] tree (Fig. 2) 91 76 Orangutan-DEFA4 98 Rhesus-DEFA2 Gibbon-DEFA1 of 112 nucleotide sequences of defensin genes (including 89 56 68 Human-DEFA1/2(2) Human-DEFA3 63 Human-DEFA1/2 a-defensins and 21 h-defensins and 2 human b-defensins) 52 Gorilla-DEFA1 Chimpanzee-DEFA1 Chimpanzee-DEFA3 from primates was constructed using the p-distance method Orangutan-DEFT1 66 Gorilla-DEFA11P-PARTIAL 100 Tarsier-DEFA8P [37]. The reliability of the estimated trees was evaluated by 98 Chimpanzee-DEFA11P 202 55 Human-DEFA11P the bootstrap method [38] with 1,000 replications. Percent- Orangutan-DEFA10P 99 Human-DEFB27 β age bootstrap values C50 are shown above branches. Human-DEFB28 -defensin

0.05 Genomic organization of human, chimpanzee Fig. 2 Phylogenetic tree of primates a and h-defensin genes. The tree and gorilla defensin gene clusters was constructed by calculating the proportion difference (p-distance) of aligned nucleotide sites of the a and h-defensin sequences Searching through whole genome sequences by using Stand- according to the neighbor-joining method. The reliability of each alone BLAST led to identification of a number of genomic branch was tested using 1,000 bootstrap replications. Numbers on the branches indicate the percentage of 1,000 bootstrap samples support- sequences containing a and h-defensin genes in the ten pri- ing the branch. Only branches supported by a bootstrap value of at mates’ species. To determine the relative position and orien- least 50 % are shown tation of each defensin on the , individual defensins were searched against the assembled primates gen- material (S3). The chromosomal locations of the a and omes by using the BLAT program [33] through the UCSC h -defensin gene clusters of human, chimpanzee and gorilla Genome Browser, all ten primates a and h-defensin gene on were revealed by using the Map Viewer program (http://www. chromosome positions were summarized in Supplementary ncbi.nlm.nih.gov/mapview). In the case of human, all 11 a and 123 3864 Mol Biol Rep (2014) 41:3859–3866 two h -defensin genes were found to form a continuous cluster of pathogens they faced [42]. This can be seen in the gene expanding 132 kb on the chromosome 8p23 (Fig. 3), separated clusters of the major histocompatibility complex [43], but 54 kb from b-defensin-1 (DEFB1). All human, chimpanzee also in the innate immune system, like toll-like receptor and gorilla defensins are transcribed from the same direction genes [42] and defensins [3]. Antimicrobial peptides are from centromere to telomere, with the h-defensin pseudogene evolutionarily ancient elements of innate immunity [44]. (DEFT1P) residing in the center. The gorilla genome encodes Following systemic, genome-wide screenings, the com- an a and h-defensin cluster of nine distinct genes, which plete repertoires of a and h-defensin gene family have been expanded 117 kb in the proximal region of identified in the ten primate species (Table 1). Because of (Fig. 3). The a and h-defensin clusters in the gorilla and human incompletely sequenced of some genomes, the total num- are nearly in perfect synteny. The only difference is the pre- ber of these species maybe not integrated. Although intact sence of two copies of DEFA1 and DEFT1P in the human DEFT genes survive in some nonhuman primates, our rather than a single copy in the gorilla, and there are two extra hominid ancestors lost their ability to produce h-defensins copies (DEFA3 and DEFA10P) in human. after the orangutan and hominid lineages diverged [10]. Compared with human, gorilla has just one copy of both DEFA1/2 and DEFT1P, and does not have DEFA3 and Discussion DEFA10P (Fig. 3). Orangutan have both functional and pseudo h-defensin genes Fig. 1a. Previous study [22] In primates, gene duplication has been an important factor showed that the total number of DEFA1A3 gene copies per for shaping the immune defence against the high diversity diploid genome varied between 4 and 11 in a sample of 111

Fig. 3 Genomic organizations Tel Tel Tel of a and h-defensin clusters in DEFB1 chimpanzee, human and gorilla. DEFB1 DEFB1 The position of each gene is represented by a solid vertical bar. The direction of is indicated by the solid triangle. The gene clusters were derived from the assembled genomes of human 54kb 54kb 64kb (GRCh37/hg19), chimpanzee (CSAC 2.1.4/panTro4), gorilla (gorGor3.1/gorGor3). Note that a and h-defensin clusters are DEFA6 DEFA6 located in a syntenic region DEFA4 adjacent to b-defensin-1 DEFA4 DEFA4 (DEFB1) in each species. Tel, telomere DEFA8P DEFA8P DEFA8P

DEFA9P DEFA9P DEFA9P DEFA10P DEFA10P DEFA1 DEFA1/2 DEFA1 DEFT1P DEFT1P DEFT1P DEFA1/2

DEFT2 DEFT1P DEFA11P DEFA3 DEFA3 DEFA7P DEFA11P DEFA11P

DEFA7P DEFA7 DEFA5

Chimpanzee Chro 8 (108kb) Gorilla Chro 8 (117kb) DEFA5

Human Chro 8 (132kb)

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