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Variety of Antimicrobial Peptides in the Bombina Maxima Toad and Evidence of Their Rapid Diversification

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Variety of Antimicrobial Peptides in the Bombina Maxima Toad and Evidence of Their Rapid Diversification http://www.paper.edu.cn 1220 Wen-Hui Lee et al. Eur. J. Immunol. 2005. 35: 1220–1229 Variety of antimicrobial peptides in the Bombina maxima toad and evidence of their rapid diversification Wen-Hui Lee1, Yan Li2,3, Ren Lai1, Sha Li2,4, Yun Zhang1 and Wen Wang2 1 Department of Animal Toxinology, Kunming Institute of Zoology, The Chinese Academy of Sciences (CAS), Kunming, P. R. China 2 CAS-Max Planck Junior Scientist Group, Key Laboratory of Cellular and Molecular Evolution, Kunming Institute of Zoology, The Chinese Academy of Sciences (CAS), Kunming, P. R. China 3 Graduate School of the Chinese Academy of Sciences, Beijing, P. R. China 4 Department of Pathology, University of Chicago, Chicago, USA Antimicrobial peptides secreted by the skin of many amphibians play an important role Received 30/8/04 in innate immunity. From two skin cDNA libraries of two individuals of the Chinese red Revised 4/2/05 belly toad (Bombina maxima), we identified 56 different antimicrobial peptide cDNA Accepted 14/2/05 sequences, each of which encodes a precursor peptide that can give rise to two kinds of [DOI 10.1002/eji.200425615] antimicrobial peptides, maximin and maximin H. Among these cDNA, we found that the mean number of nucleotide substitution per non-synonymous site in both the maximin and maximin H domains significantly exceed the mean number of nucleotide substitution per synonymous site, whereas the same pattern was not observed in other structural regions, such as the signal and propiece peptide regions, suggesting that these antimicrobial peptide genes have been experiencing rapid diversification driven by Darwinian selection. We cloned and sequenced seven genes amplified from skin or liver genomic DNA. These genes have three exons and share the same gene structure, in which both maximin and maximin H are encoded by the third exon. This suggests that alternative splicing and somatic recombination are less likely to play a role in creating Key words: the diversity of maximins and maximin Hs. The gene trees based on different domain Antimicrobial regions revealed that domain shuffling or gene conversion among these genes might peptides Á Rapid have happened frequently. diversification Á Bombina maxima The first two authors contributed equally to this work. Introduction Correspondence: Wen Wang, CAS-Max Planck Junior Scientist Group, Key Laboratory of Cellular and Molecular Evolution, Kunming Institute of Zoology, The Chinese Academy of It has been widely recognized that antimicrobial Sciences (CAS), Kunming, Yunnan 650223, P. R. China peptides play an important role in protection from Fax: +86-871-5193137 invading microorganisms [1–4]. Antimicrobial peptides e-mail: [email protected] have successfully been isolated from plants [5], or invertebrates [6], vertebrates [7, 8], and humans [9, Yun Zhang, Department of Animal Toxinology, Kunming Institute of Zoology, The Chinese Academy of Sciences (CAS), 10]. In amphibians, antimicrobial peptides are usually Kunming, Yunnan 650223, P. R. China secreted by the dermal glands located in the outer layer Fax: +86-871-5191823 of the skin and they provide an immediate defense e-mail: [email protected] against harmful microorganisms [4, 11]. The Chinese red belly toad (Bombina maxima)isan Abbreviations: p : Proportion of non-synonymous difference Á N endemic amphibian in the mountainous regions of pS: Proportion of synonymous difference Á dN: Mean number of nucleotide substitution per non-synonymous site Á dS: Mean southwestern China [12]. The indigenous people report number of nucleotide substitution per synonymous site Á that the toad lives in very harsh environments such as UTR: Untranslated region f 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji.de 转载 中国科技论文在线 http://www.paper.edu.cn Eur. J. Immunol. 2005. 35: 1220–1229 Innate immunity 1221 pools with microorganism-rich mud and that its skin is observation. While these studies shed insight into very “toxic”. In a preliminary test of biological activity, understanding of the evolution of antimicrobial peptides including antimicrobial, hemolytic and cytotoxic assays, in distinct species, data from closely related species or on the skin secretions of more than ten amphibian intraspecies antimicrobial peptide gene families might species, we found that the secretions of B. maxima have show whether alternative splicing and somatic recom- the strongest biological activity (Zhang, unpublished bination are involved in the creation of peptide diversity, observations). Our recent preliminary studies showed how these genes respond to different environments, and that the skin secretions of B. maxima contain many whether these genes are under unusually high selective different antimicrobial peptides [13, 14]. These anti- pressure. Here we describe of as many as 40 different microbial peptides could be grouped into two families, antimicrobial peptide genes expressed in a single B. maximin and maximin H, which are, respectively, maxima individual. Gene structure analysis did not equivalent to the bombinin and bombinin H identified provide evidence for alternative splicing and somatic in two other Bombina species, B. orientalis and B. recombination. Evolutionary analyses on these genes variegata [15–17]. The gene encoding bombinin and disclosed unusually high non-synonymous substitution bombinin H comprises two exons separated by an rates in the mature antimicrobial peptide regions of approximately 1-kb intron (Fig. 1A) [18, 19]. Exon 1 these genes after gene duplication. Structural and codes for the signal peptide, and exon 2 for bombinin, phylogenetic analyses suggest that domain shuffling bombinin H, and other structural peptides including a or gene conversion play an important role in the spacer and two acidic propieces; the latter have a evolution of this gene family. negative charge that serves to balance the positive charge in the mature antimicrobial peptides and thus prevent cytotoxicity prior to cleavage. We wanted to Results know (a) whether the antimicrobial peptides in B. maxima genes have a similar structure to that of Various antimicrobial peptides are found in bombinin and bombinin H, (b) how rich and diverse B. maxima the antimicrobial peptides are in B. maxima, and (c) how the genes encoding these peptides evolve. In an attempt to analyze the expression profile of the As for the evolution of antimicrobial peptides, it has skin of B. maxima, we randomly sequenced 793 cDNA been reported that, in animals (including amphibians), clones from the library constructed with mRNA antimicrobial peptides exhibit high levels of variation in extracted from the skin of one toad (specimen A). Of response to the selective pressure of quickly evolving these, 92 clones were found to be antimicrobial peptide pathogens [20, 21]. For example, Duda et al. [21] cDNA, on the basis of similarity to the maximin peptides, observed high non-synonymous substitutions among and were then fully sequenced. The sequences show antimicrobial peptides isolated from several distinct frog high level of similarity to the cDNA of bombinin and species (three hylid frogs and four ranid frogs), and bombinin H from B. orientalis and B. variegata [15, 16]. adaptive evolution was inferred to account for this Each of the cDNA codes for a precursor consisting of a Fig. 1. Schematic gene structure of antimicrobial peptides from B. orien- talis and B. maxima. Boxes with diag- onal lines are exons (E1, E2, E3), the dotted box is a putative promoter (P), and solid lines are introns. (A) Gene structure of bombinin peptides from B. orientalis. This gene contains two exons (E1 and E2) and a putative promoter in front of the exon E1 [18, 19]. (B) Gene structure of the maximin peptides from B. maxima. The corre- sponding region of the putative pro- moter proposed in the previously study [18, 19] is indicated in front of the exon E2. Sd and Nd: the number of synonymous and non-synonymous differences compared with consen- sus sequence. *p<0.05 with Z-test. f 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji.de 中国科技论文在线 http://www.paper.edu.cn 1222 Wen-Hui Lee et al. Eur. J. Immunol. 2005. 35: 1220–1229 signal peptide, two acidic propiece peptides, an 8-amino 12 putative antimicrobial peptides are thus newly acid spacer, and two antimicrobial peptides, maximin identified. (27 amino acids) and maximin H (20 amino acids) To investigate variations in the maximin and (Fig. 1B). The 92 sequences can be grouped into 40 maximin H peptides between individuals, we screened independent cDNA sequences. In spite of the identity of a cDNA library constructed from the skin of B. maxima the coding regions of the mature antimicrobial peptides specimen B by a rapid PCR-based procedure, and among some clones (such as between clone 62 and clone sequenced 30 positive clones. Each of the 30 clones 488), these cDNA differ from each other in UTR or contains an insert encoding a maximin and maximin H structural regions. These 40 cDNA could encode 9 precursor, from which 16 independent cDNA were different mature maximin peptides (maximin 1–9), and obtained (Table 2). The cDNA structures are similar to 13 different maximin H peptides (maximin H1–13) those obtained in specimen A. These 16 cDNA encode 6 (Table 1). We have previously isolated maximin 1–5 and different maximin peptides, and 9 different maximin H maximin H1–5 from skin secretions [13, 14]. The other peptides. Maximin 2–5, and maximin H1, 3, 4, 5, 8, 11 Table 1. List of the 40 cDNA identified of the B. maxima specimen A and the number of clones showing peptide-specific cDNA sequences in the 92 sequenced clonesa) a) Identical amino acids in the maximin and maximin H peptides are shown in bold. f 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji.de 中国科技论文在线 http://www.paper.edu.cn Eur. J. Immunol. 2005. 35: 1220–1229 Innate immunity 1223 Table 2. List of the 16 cDNA identified in the skin cDNA library in second individual of B.
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