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Evolution of Primate a and H Defensins Revealed by Analysis of Genomes

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Evolution of Primate a and H Defensins Revealed by Analysis of Genomes Mol Biol Rep (2014) 41:3859–3866 DOI 10.1007/s11033-014-3253-z Evolution of primate a and h defensins 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 peptides 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 defensin domain peptides defence against bacterial, fungal and viral infections. There to 12 residues. Humans, chimpanzees and gorillas do not are three subfamilies of defensins in primates: a, b and h- produce h-defensin peptides due to a premature stop codon defensins. a-defensins are most present in neutrophils and present in the signal sequence of all h-defensin pseudogenes. 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 gene 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. Human has two copies of DEFA1 and and b-sheet regions. a and h-defensins are closely related, DEFT1P, and two extra DEFA3 and DEFA10P genes com- comparative amino acid sequences showed that the pared with gorilla. 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 antimicrobial peptides e-mail: [email protected] expressed by the leukocytes and epithelial cells of fish [1], birds [2] and mammals [3, 4], that form an important part of H. Xu the innate immune system [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 peptide 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 neutrophil cular peptide, termed rhesus theta defensin-1 (RTD-1), is (PMNs) [28], but their relative amounts differ greatly, with microbicidal for bacteria 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 viruses 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 chimpanzee 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 virus, 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 pseudogene 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).
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