Roelants K et al. (2013), Origin and functional diversification of an amp...

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PLoS Genet 2013 Jan 01;98:e1003662. doi: 10.1371/journal.pgen.1003662.

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Origin and functional diversification of an amphibian defense peptide arsenal.

Roelants K , Fry BG , Ye L , Stijlemans B , Brys L , Kok P , Clynen E , Schoofs L , Cornelis P , Bossuyt F .

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The skin secretion of many amphibians contains an arsenal of bioactive molecules, including hormone-like peptides (HLPs) acting as defense toxins against predators, and antimicrobial peptides (AMPs) providing protection against infectious microorganisms. Several amphibian taxa seem to have independently acquired the genes to produce skin-secreted peptide arsenals, but it remains unknown how these originated from a non-defensive ancestral gene and evolved diverse defense functions against predators and pathogens. We conducted transcriptome, genome, peptidome and phylogenetic analyses to chart the full gene repertoire underlying the defense peptide arsenal of the frog Silurana tropicalis and reconstruct its evolutionary history. Our study uncovers a cluster of 13 transcriptionally active genes, together encoding up to 19 peptides, including diverse HLP homologues and AMPs. This gene cluster arose from a duplicated gastrointestinal hormone gene that attained a HLP-like defense function after major remodeling of its promoter region. Instead, new defense functions, including antimicrobial activity, arose by mutation of the precursor proteins, resulting in the proteolytic processing of secondary peptides alongside the original ones. Although gene duplication did not trigger functional innovation, it may have subsequently facilitated the convergent loss of the original function in multiple gene lineages (subfunctionalization), completing their transformation from HLP gene to AMP gene. The processing of multiple peptides from a single precursor entails a mechanism through which peptide-encoding genes may establish new functions without the need for gene duplication to avoid adaptive conflicts with older ones.

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Species referenced: Xenopus
Genes referenced: cck ercc4 hap1 levi mst1 nr5a2

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	Figure 2. Precursors encoded by the Silurana tropicalis AMP gene repertoire.Peptides are predicted based on sequence homology to previously identified peptides (both highlighted in black) and putative cleavage sites (highlighted in gray). Underlined sequences represent predicted signal peptides. Asterisks represent translation stops.
	Figure 3. Comparative alignment of the promoter regions of vertebrate cck genes and pipid AMP genes.Sites with >75% sequence conservation across AMP gene promoters are indicated in grey; those additionally discussed as conserved sequence motifs in the text are indicated in dark grey. Sequence motifs corresponding to known or putative regulatory elements are colored as follows: dark green, TATA-box; light green, E-box; Orange, CRE/TRE element; pink, GC-box; dark blue, CRE2-conserved octamer.
	Figure 4. Evolutionary diversification of pipid AMP genes.Phylogenetic relationships of S. tropicalis and X. laevis AMP genes are shown in a consensus tree obtained by Bayesian analyses using the traditional two-step approach of phylogeny inference (separate alignment and Bayesian phylogeny inference) and using direct optimization (integrated Bayesian alignment and phylogeny inference). Each gene is represented here by its precursor protein sequence aligned to visualize similarity with its closely related homologues. Note that several of the X. laevis sequences occupy multiple lines because duplicated exons were aligned to each other. Unlabeled branches in the tree are supported by maximum posterior probabilities (1.00) under both methods; branches that received less support by one or both methods are labeled by their posterior probability under the two-step method (top) and under direct optimization (bottom). Nodes representing gene duplication events are labeled by circles and color-coded as follows: black, the split between cck and the ancestral pipid AMP gene; grey, gene duplication in an ancestor of Silurana and Xenopus; blue, gene duplication in Silurana; and red, gene duplication in Xenopus. Crosses linked by a vertical dashed line mark the divergence of Silurana and Xenopus, the resulting orthologous gene lineages are marked by blue and red branches respectively. Peptides in the precursor proteins are color-coded accordingly.
	Figure 5. Patterns of diversifying and purifying selection in pipid AMP/HLP precursor proteins.Site-specific mean posteriors of nonsynonymous (E(dN), black line) and synonymous (E(dS), grey line) codon substitution rates are plotted along an exemplary precursor protein sequence (prepro-CPF-St6). Sites for which the black plot line rises above grey one have dN/dS ratios >1 (suggesting diversifying selection); those for which the black plot stays below the grey have dN/dS ratios <1 (suggesting purifying selection). Sites showing significant evidence of diversifying or purifying selection (at Bayes factor >50) are labeled with ‘+’ and ‘−’, respectively.
	Figure 6. Structural and functional evolution of the pipid defense peptide arsenal.(A) Evolutionary scenario for the origin and loss of defense peptides and their function along a summarized phylogenetic tree. Numbers along tree branches represent structural changes as mentioned in the legend. (B) Evolutionary trajectory showing structural changes and functional transitions from the ancestral cck gene to the present-day X. laevis xpf-Xl1 gene, encoding the AMP XPF and the HLP xenopsin. Only gene duplication events relevant to functional changes (see text) are indicated. (C) Comparative alignment of levitide-like peptides, X. laevis xenopsin and the vertebrate neurohormones with which it shows evolutionary convergence. The origins of the different peptides are indicated on the right. Amino acids are color-coded as follows: white, hydrophobic; grey, near-neutral; purple, uncharged polar; blue, cationic (basic); red; anionic (acidic).
	Figure 1. Genomic organization of the Silurana tropicalis AMP gene repertoire.(A) Gene cluster map showing gene and transcript positions (indicated by numbers 1–15 on the left) and gene orientation (indicated by upward or downward pointing triangles). Genes with incomplete coding sequences are colored grey. Exon organization of each gene/transcript is shown on the right (labeled by the numbers of the gene map), with exon lengths indicated as numbers below bars, untranslated regions colored light blue and coding sequences colored dark-blue. (B) Schematic representation of the S. tropicalis AMP gene cluster and adjacent genes showing preserved synteny with the cck gene in other vertebrates.

References [+] :

Ali, Antimicrobial peptides isolated from skin secretions of the diploid frog, Xenopus tropicalis (Pipidae). 2001, Pubmed, Xenbase