- Email: [email protected]
On the evolution of invertebrate defensins
Update
330
TRENDS in Genetics Vol.21 No.6 June 2005
Letter
On the evolution of invertebrate defensins Ricardo C. Rodrı´guez de la Vega and Lourival D. Possani Department of Molecular Medicine and Bioprocesses, Institute of Biotechnology, National Autonomous University of Mexico, Avenida Universidad, 2001, Apartado Postal 510-3, Cuernavaca 62210, Mexico
The evolutionary history of invertebrate defensins has recently been revised by Froy and Gurevitz [1]. These authors propose that exon-shuffling of the region encoding mature peptides might explain their documented diversity. In this article, we revisit their hypothesis and, by using a broader set of invertebrate defensins, conclude that exon-shuffling is unlikely to account for defensin diversity. Clearly, more work is required to clarify the evolution of these peptides. Invertebrate defensins – the most wide-spread group of antimicrobial peptides – are characterized by the presence of a cysteine-stabilized ab (CS ab) motif signature [2]. This structural scaffold is also present in several functionally diverse bioactive peptides, including various peptides from plants and the majority of scorpion toxins [3–5]. Invertebrate defensins have been isolated from nematodes, arthropods and molluscs, in which they act as key effectors of their innate immune systems [2]. These features make the invertebrate defensins an interesting subject for evaluating evolutionary relationships in highly diversified innate immune systems (e.g. see Refs [6–9]); although this is a difficult task because there is little amino-acid-sequence similarity within this group. Nevertheless, at least two classes of defensins can be distinguished on the basis of their mature peptide sequences. The first, and largest group, includes defensins isolated from neopteran insects, whereas the second, often referred as the ‘ancestral’ group, comprises defensins from a more diverse variety of invertebrates, including a paleopteran insect (the dragonfly), two orders of arachnids (ticks and scorpions) and even bivalvian molluscs [2]. Interestingly, both classes share %30% average aminoacid-sequence identity, and at least one-third of the characterized invertebrate defensins cannot be classified into the groups described to date. Defensin structure This uncertainty over classification also occurs at the DNA sequence level, because the few genomic clones available in public databases show an unusual variability in the sequence encoding the protein precursors [10] and the intron-exon structures [1] (Figure 1). At the precursor level, there are proteins that have a pro-sequence at the N-terminal end (N-pro; e.g. the defensins in insects and ticks) or at the C-terminal region (C-pro) of the peptides (e.g. defensins in mussels and nematodes), whereas other defensins have no pro-sequences (e.g. the scorpion Corresponding author: Possani, L.D. ([email protected]). Available online 1 April 2005 www.sciencedirect.com
defensins). The exon-intron structures are also highly variable: certain defensins have one intron at the 5 0 -end of the translated region, within the N-pro or secretory signal peptide [defensins from mussels, scorpions, some dipterans (e.g. Genbank accession numbers AF392802; AF063402) and from some lepidopterans (e.g. Genbank accession no. AY128091)], and others have an intron within the mature region (nematode defensins). In addition, a few examples have been reported that have an additional intron at the border of the N-pro and the mature region (defensins from ticks) or at the 3 0 -end of the region encoding the mature sequence (hymenopteran defensins). Finally, there are defensins without introns, for example, dipterans (e.g. Genbank accession numbers AY224631; AF182163) and certain lepidopterans (e.g. Genbank accession no. AF465486). With the exceptions of nematode introns, which are on phase 0 (i.e. the introns split the mRNA between two codons) and 3 0 introns in Hymenoptera, which are on phase II (i.e. the introns split a codon after the second nucleotide), all of the documented introns are on phase I (i.e. split a codon after the first nucleotide). Remarkably, except for the nematode defensins, all of the other exon-intron organizations retain the integrity of the CS ab-motif-encoding region.
Exon-shuffling Exon-shuffling has been recognized as a major source of protein diversity in eukaryotic systems [11]. Although it was hypothesized that exon-shuffling was a crucial event in the generation of new genes in the primitive forms of life, the documented cases always involve the insertion of a protein module flanked by symmetrical introns – a genetic entity called ‘proto-module’ – into a pre-existing intron of the same phase. This has been well documented for multi-domain proteins in multi-cellular eukaryotic species [11]. The cloned genes of invertebrate defensins (single-domain short proteins) do not obey this rule (only the defensin genes from hymenopterans are flanked by introns, which are not on the same phase). Only a rather complex set of events – with multiple independent analogous insertion, deletion or sliding of introns after the putative insertion of the ‘proto-module’ including the CS ab-motif signature – could explain the documented diversity by exon-shuffling (Figure 1). This does not deny the possibility of an exonshuffling-mediated process at the base of invertebrate defensin evolution, but such a process would be distinguishable from those driven by symmetrical introns.
Update
TRENDS in Genetics Vol.21 No.6 June 2005
331
Enoplea Ascaridida + mature; +C-pro
Rhabditida
3 1
Chromadorea
Ascaris suum AB029816 Caenorhabditis elegans U80445:comp 9816..10 785
Annelida Gastropoda Mytiloida -3′; +C-pro
Bivalvia
1 2
Mytilus galloprovinsialis AF177539 Crassostrea gigas AJ582630
Ostreoida Merostomata 1 2
Centruroides limpidus limpidus AY656081
Scorpiones
+5′; +3′
Aranae
-3′ +5′b; +N-pro
Arachnida 1 2
1 2
Ornothodoros moubata AB080135
Ixodida Crustacea Paleoptera* Isoptera* Hemiptera* Coleoptera*
+N-pro -5′; -3′ -3′ -5′; -3′ -3′ 3′ sliding
Diptera
Insecta
Droshophila melanogaster AY224631
1 2
Diptera
Aedes aegypti AF392802 Mamestre brassicae AF465486
Lepidoptera 1 2
Lepidoptera
1 2
2 3
Hymenoptera
Spodoptera frugiperda AY128091 Bombus ignitus AY423050 TRENDS in Genetics
Figure 1. A hypothetical route for the diversification of invertebrate defensin gene structures. The putative proto-module including mature peptides with the CS ab motif signature (in red) flanking by symmetrical introns on phase I requires a complex set of genetic events to account for the diversity of extant invertebrate defensin-gene structures (shown on the right). At least three independent insertions (red arrows) should have occurred, followed by several analogous losses (5 0 or 3 0 introns) or gains (N-pro or C-pro sequences, shown in white) of additional genetic elements. The tree on left hand side was drawn with the aid of data from the coelomatan classification. If all of the moulting animals are considered as monophyletic (Ecdysozoa clade), a complex process is also obtained (not shown). The invertebrate orders are shown at the tip of the branches. Those in blue denote examples for which defensins have not been described. The taxonomic classes are shown in bold. The asterisks indicate examples where defensins are present but where the gene structure is unknown. Under each branch there is an indication of the possible events that should have occurred if exon-shuffling took place (e.g. 3 0 sliding). The secretory signal peptides are shown in black; the mature peptides including the CS ab motifs are shown in red; the pro-sequences are shown in white, whereas the thin and broken lines represent non-translated sequences. The C-terminal extension found only in some hymenopteran defensins is displayed in gray. The numbers above intron junctions correspond to the codon position spliced by the intron.
Concluding remarks Finally, it is worth emphasizing that the orthologous condition of invertebrate defensins remains to be established, because there are no documented defensins in basal organisms (e.g. annelids for coelomatas or merostomatans for arachnids) or in sister groups of those that have defensins (e.g. defensins were not reported in crustaceans a sister group of insects, or in gastropods or cephalopods sister groups of bivalvians, nor in spiders a sister group of ticks; Figure 1). The identification of the putative defensins in those organisms will require systematic searching in the biological source material; especially because their low sequence similarities would not enable direct identification of defensins by highthroughput methods. In our opinion, this lack of information leaves the puzzle of the evolutionary history of these antimicrobial peptides unsolved; there is simply not enough data at present to support the exon-shuffling model. However, the alternative scenario of paraphyletic origin (i.e. independent origin) of invertebrate defensins is also unlikely owing to some remarkable interspecies conservation. Perhaps the variety of defensins is due to the gene encoding the putative basal defensin having the intrinsic capacity for extensive diversification. This could explain not only the diversity of invertebrate defensins but also the diversification in the paralogous multi-gene family of scorpion toxins (although they differ greatly, certain www.sciencedirect.com
scorpion toxins and defensins seem to be closely related to each other [12,13]). Strong selective pressure could have been driving the specific diversification of defensins in each organism, similar to the diversification of the paralogous genes encoding the scorpion toxins and other antimicrobial peptides [14,15].
Acknowledgements Our research is supported in part by the Mexican Council of Science and Technology (grant 40251-Q) and Instituto Bioclon S.A. de C.V.
References 1 Froy, O. and Gurevitz, M. (2003) Arthropod and mollusk defensins – evolution by exon-shuffling. Trends Genet. 19, 684–687 2 Bulet, P. et al. (2004) Anti-microbial peptides: from invertebrates to vertebrates. Immunol. Rev. 198, 169–184 3 Tamaoki, H. et al. (1998) Folding motif induced and stabilized by distinct cysteine frameworks. Protein Eng. 11, 649–659 4 Froy, O. and Gurevitz, M. (1998) Membrane potential modulators: a thread of scarlet from plants to humans. FASEB J. 12, 1793–1796 5 Possani, L.D. et al. (2000) Peptides and genes coding for scorpion toxins that affect ion-channels. Biochimie 82, 861–868 6 Hoffmann, J.A. et al. (1999) Phylogenetic perspectives in innate immunity. Science 284, 1313–1318 7 Kimbrell, D.A. and Beutler, B. (2001) The evolution and genetics of innate immunity. Nat. Rev. Genet. 2, 256–267 8 Brennan, C.A. and Anderson, K.V. (2004) Drosophila: the genetics of innate immune recognition and response. Annu. Rev. Immunol. 22, 457–483
Update
332
TRENDS in Genetics Vol.21 No.6 June 2005
9 Nicholas, H.R. and Hodgkin, J. (2004) Responses to infection and possible recognition strategies in the innate immune system of Caenorhabditis elegans. Mol. Immunol. 41, 479–493 10 Zhang, H. and Kato, Y. (2003) Common structural properties specifically found in the CS ab-type antimicrobial peptides in nematodes and mollusks: evidence for the same evolutionary origin? Dev. Comp. Immunol. 27, 499–503 11 Patthy, L. (2003) Modular assembly of genes and the evolution of new functions. Genetica 118, 217–231 12 Zhu, S. and Tytgat, J. (2004) The scorpine family of defensins: gene structure, alternative polyadenylation and fold recognition. Cell. Mol. Life Sci. 61, 1751–1763
13 Rodrı´guez de la Vega, R.C. et al. (2004) Antimicrobial peptide induction in the haemolymph of the Mexican scorpion Centruroides limpidus limpidus in response to septic injury. Cell. Mol. Life Sci. 61, 1507–1519 14 Zhu, S. et al. (2004) Adaptive evolution of scorpion sodium channel toxins. J. Mol. Evol. 58, 145–153 15 Bulmer, M.S. and Crozier, R.H. (2004) Duplication and diversifying selection among termite antifungal peptides. Mol. Biol. Evol. 21, 2256–2264 0168-9525/$ - see front matter Q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.tig.2005.03.009
Articles of interest from Current Opinion in Genetics and Development Transcriptional regulation by the numbers: models Transcriptional regulation by the numbers: models Lacramioara Bintu, Nicolas E. Buchler, Hernan G. Garcia, Ulrich Gerland, Terence Hwa, Jane´ Kondev and Rob Phillips Current Opinion in Genetics and Development, 15, 116–124 Revisited gene regulation in bacteriophage l Ian B. Dodd, Keith E. Shearwin and J. Barry Egan Current Opinion in Genetics and Development, 15, 145–152 The structure and function of the bacterial chromosome Martin Thanbichler, Patrick H. Viollier and Lucy Shapiro Current Opinion in Genetics and Development, 15, 153–162 MicroRNAs: a developing story Amy E. Pasquinelli, Shaun Hunter and John Bracht Current Opinion in Genetics and Development, 15, 200–205 Recognition and modification of seX chromosomes Dmitri A. Nusinow and Barbara Panning Current Opinion in Genetics and Development, 15, 206–213 www.sciencedirect.com
330
TRENDS in Genetics Vol.21 No.6 June 2005
Letter
On the evolution of invertebrate defensins Ricardo C. Rodrı´guez de la Vega and Lourival D. Possani Department of Molecular Medicine and Bioprocesses, Institute of Biotechnology, National Autonomous University of Mexico, Avenida Universidad, 2001, Apartado Postal 510-3, Cuernavaca 62210, Mexico
The evolutionary history of invertebrate defensins has recently been revised by Froy and Gurevitz [1]. These authors propose that exon-shuffling of the region encoding mature peptides might explain their documented diversity. In this article, we revisit their hypothesis and, by using a broader set of invertebrate defensins, conclude that exon-shuffling is unlikely to account for defensin diversity. Clearly, more work is required to clarify the evolution of these peptides. Invertebrate defensins – the most wide-spread group of antimicrobial peptides – are characterized by the presence of a cysteine-stabilized ab (CS ab) motif signature [2]. This structural scaffold is also present in several functionally diverse bioactive peptides, including various peptides from plants and the majority of scorpion toxins [3–5]. Invertebrate defensins have been isolated from nematodes, arthropods and molluscs, in which they act as key effectors of their innate immune systems [2]. These features make the invertebrate defensins an interesting subject for evaluating evolutionary relationships in highly diversified innate immune systems (e.g. see Refs [6–9]); although this is a difficult task because there is little amino-acid-sequence similarity within this group. Nevertheless, at least two classes of defensins can be distinguished on the basis of their mature peptide sequences. The first, and largest group, includes defensins isolated from neopteran insects, whereas the second, often referred as the ‘ancestral’ group, comprises defensins from a more diverse variety of invertebrates, including a paleopteran insect (the dragonfly), two orders of arachnids (ticks and scorpions) and even bivalvian molluscs [2]. Interestingly, both classes share %30% average aminoacid-sequence identity, and at least one-third of the characterized invertebrate defensins cannot be classified into the groups described to date. Defensin structure This uncertainty over classification also occurs at the DNA sequence level, because the few genomic clones available in public databases show an unusual variability in the sequence encoding the protein precursors [10] and the intron-exon structures [1] (Figure 1). At the precursor level, there are proteins that have a pro-sequence at the N-terminal end (N-pro; e.g. the defensins in insects and ticks) or at the C-terminal region (C-pro) of the peptides (e.g. defensins in mussels and nematodes), whereas other defensins have no pro-sequences (e.g. the scorpion Corresponding author: Possani, L.D. ([email protected]). Available online 1 April 2005 www.sciencedirect.com
defensins). The exon-intron structures are also highly variable: certain defensins have one intron at the 5 0 -end of the translated region, within the N-pro or secretory signal peptide [defensins from mussels, scorpions, some dipterans (e.g. Genbank accession numbers AF392802; AF063402) and from some lepidopterans (e.g. Genbank accession no. AY128091)], and others have an intron within the mature region (nematode defensins). In addition, a few examples have been reported that have an additional intron at the border of the N-pro and the mature region (defensins from ticks) or at the 3 0 -end of the region encoding the mature sequence (hymenopteran defensins). Finally, there are defensins without introns, for example, dipterans (e.g. Genbank accession numbers AY224631; AF182163) and certain lepidopterans (e.g. Genbank accession no. AF465486). With the exceptions of nematode introns, which are on phase 0 (i.e. the introns split the mRNA between two codons) and 3 0 introns in Hymenoptera, which are on phase II (i.e. the introns split a codon after the second nucleotide), all of the documented introns are on phase I (i.e. split a codon after the first nucleotide). Remarkably, except for the nematode defensins, all of the other exon-intron organizations retain the integrity of the CS ab-motif-encoding region.
Exon-shuffling Exon-shuffling has been recognized as a major source of protein diversity in eukaryotic systems [11]. Although it was hypothesized that exon-shuffling was a crucial event in the generation of new genes in the primitive forms of life, the documented cases always involve the insertion of a protein module flanked by symmetrical introns – a genetic entity called ‘proto-module’ – into a pre-existing intron of the same phase. This has been well documented for multi-domain proteins in multi-cellular eukaryotic species [11]. The cloned genes of invertebrate defensins (single-domain short proteins) do not obey this rule (only the defensin genes from hymenopterans are flanked by introns, which are not on the same phase). Only a rather complex set of events – with multiple independent analogous insertion, deletion or sliding of introns after the putative insertion of the ‘proto-module’ including the CS ab-motif signature – could explain the documented diversity by exon-shuffling (Figure 1). This does not deny the possibility of an exonshuffling-mediated process at the base of invertebrate defensin evolution, but such a process would be distinguishable from those driven by symmetrical introns.
Update
TRENDS in Genetics Vol.21 No.6 June 2005
331
Enoplea Ascaridida + mature; +C-pro
Rhabditida
3 1
Chromadorea
Ascaris suum AB029816 Caenorhabditis elegans U80445:comp 9816..10 785
Annelida Gastropoda Mytiloida -3′; +C-pro
Bivalvia
1 2
Mytilus galloprovinsialis AF177539 Crassostrea gigas AJ582630
Ostreoida Merostomata 1 2
Centruroides limpidus limpidus AY656081
Scorpiones
+5′; +3′
Aranae
-3′ +5′b; +N-pro
Arachnida 1 2
1 2
Ornothodoros moubata AB080135
Ixodida Crustacea Paleoptera* Isoptera* Hemiptera* Coleoptera*
+N-pro -5′; -3′ -3′ -5′; -3′ -3′ 3′ sliding
Diptera
Insecta
Droshophila melanogaster AY224631
1 2
Diptera
Aedes aegypti AF392802 Mamestre brassicae AF465486
Lepidoptera 1 2
Lepidoptera
1 2
2 3
Hymenoptera
Spodoptera frugiperda AY128091 Bombus ignitus AY423050 TRENDS in Genetics
Figure 1. A hypothetical route for the diversification of invertebrate defensin gene structures. The putative proto-module including mature peptides with the CS ab motif signature (in red) flanking by symmetrical introns on phase I requires a complex set of genetic events to account for the diversity of extant invertebrate defensin-gene structures (shown on the right). At least three independent insertions (red arrows) should have occurred, followed by several analogous losses (5 0 or 3 0 introns) or gains (N-pro or C-pro sequences, shown in white) of additional genetic elements. The tree on left hand side was drawn with the aid of data from the coelomatan classification. If all of the moulting animals are considered as monophyletic (Ecdysozoa clade), a complex process is also obtained (not shown). The invertebrate orders are shown at the tip of the branches. Those in blue denote examples for which defensins have not been described. The taxonomic classes are shown in bold. The asterisks indicate examples where defensins are present but where the gene structure is unknown. Under each branch there is an indication of the possible events that should have occurred if exon-shuffling took place (e.g. 3 0 sliding). The secretory signal peptides are shown in black; the mature peptides including the CS ab motifs are shown in red; the pro-sequences are shown in white, whereas the thin and broken lines represent non-translated sequences. The C-terminal extension found only in some hymenopteran defensins is displayed in gray. The numbers above intron junctions correspond to the codon position spliced by the intron.
Concluding remarks Finally, it is worth emphasizing that the orthologous condition of invertebrate defensins remains to be established, because there are no documented defensins in basal organisms (e.g. annelids for coelomatas or merostomatans for arachnids) or in sister groups of those that have defensins (e.g. defensins were not reported in crustaceans a sister group of insects, or in gastropods or cephalopods sister groups of bivalvians, nor in spiders a sister group of ticks; Figure 1). The identification of the putative defensins in those organisms will require systematic searching in the biological source material; especially because their low sequence similarities would not enable direct identification of defensins by highthroughput methods. In our opinion, this lack of information leaves the puzzle of the evolutionary history of these antimicrobial peptides unsolved; there is simply not enough data at present to support the exon-shuffling model. However, the alternative scenario of paraphyletic origin (i.e. independent origin) of invertebrate defensins is also unlikely owing to some remarkable interspecies conservation. Perhaps the variety of defensins is due to the gene encoding the putative basal defensin having the intrinsic capacity for extensive diversification. This could explain not only the diversity of invertebrate defensins but also the diversification in the paralogous multi-gene family of scorpion toxins (although they differ greatly, certain www.sciencedirect.com
scorpion toxins and defensins seem to be closely related to each other [12,13]). Strong selective pressure could have been driving the specific diversification of defensins in each organism, similar to the diversification of the paralogous genes encoding the scorpion toxins and other antimicrobial peptides [14,15].
Acknowledgements Our research is supported in part by the Mexican Council of Science and Technology (grant 40251-Q) and Instituto Bioclon S.A. de C.V.
References 1 Froy, O. and Gurevitz, M. (2003) Arthropod and mollusk defensins – evolution by exon-shuffling. Trends Genet. 19, 684–687 2 Bulet, P. et al. (2004) Anti-microbial peptides: from invertebrates to vertebrates. Immunol. Rev. 198, 169–184 3 Tamaoki, H. et al. (1998) Folding motif induced and stabilized by distinct cysteine frameworks. Protein Eng. 11, 649–659 4 Froy, O. and Gurevitz, M. (1998) Membrane potential modulators: a thread of scarlet from plants to humans. FASEB J. 12, 1793–1796 5 Possani, L.D. et al. (2000) Peptides and genes coding for scorpion toxins that affect ion-channels. Biochimie 82, 861–868 6 Hoffmann, J.A. et al. (1999) Phylogenetic perspectives in innate immunity. Science 284, 1313–1318 7 Kimbrell, D.A. and Beutler, B. (2001) The evolution and genetics of innate immunity. Nat. Rev. Genet. 2, 256–267 8 Brennan, C.A. and Anderson, K.V. (2004) Drosophila: the genetics of innate immune recognition and response. Annu. Rev. Immunol. 22, 457–483
Update
332
TRENDS in Genetics Vol.21 No.6 June 2005
9 Nicholas, H.R. and Hodgkin, J. (2004) Responses to infection and possible recognition strategies in the innate immune system of Caenorhabditis elegans. Mol. Immunol. 41, 479–493 10 Zhang, H. and Kato, Y. (2003) Common structural properties specifically found in the CS ab-type antimicrobial peptides in nematodes and mollusks: evidence for the same evolutionary origin? Dev. Comp. Immunol. 27, 499–503 11 Patthy, L. (2003) Modular assembly of genes and the evolution of new functions. Genetica 118, 217–231 12 Zhu, S. and Tytgat, J. (2004) The scorpine family of defensins: gene structure, alternative polyadenylation and fold recognition. Cell. Mol. Life Sci. 61, 1751–1763
13 Rodrı´guez de la Vega, R.C. et al. (2004) Antimicrobial peptide induction in the haemolymph of the Mexican scorpion Centruroides limpidus limpidus in response to septic injury. Cell. Mol. Life Sci. 61, 1507–1519 14 Zhu, S. et al. (2004) Adaptive evolution of scorpion sodium channel toxins. J. Mol. Evol. 58, 145–153 15 Bulmer, M.S. and Crozier, R.H. (2004) Duplication and diversifying selection among termite antifungal peptides. Mol. Biol. Evol. 21, 2256–2264 0168-9525/$ - see front matter Q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.tig.2005.03.009
Articles of interest from Current Opinion in Genetics and Development Transcriptional regulation by the numbers: models Transcriptional regulation by the numbers: models Lacramioara Bintu, Nicolas E. Buchler, Hernan G. Garcia, Ulrich Gerland, Terence Hwa, Jane´ Kondev and Rob Phillips Current Opinion in Genetics and Development, 15, 116–124 Revisited gene regulation in bacteriophage l Ian B. Dodd, Keith E. Shearwin and J. Barry Egan Current Opinion in Genetics and Development, 15, 145–152 The structure and function of the bacterial chromosome Martin Thanbichler, Patrick H. Viollier and Lucy Shapiro Current Opinion in Genetics and Development, 15, 153–162 MicroRNAs: a developing story Amy E. Pasquinelli, Shaun Hunter and John Bracht Current Opinion in Genetics and Development, 15, 200–205 Recognition and modification of seX chromosomes Dmitri A. Nusinow and Barbara Panning Current Opinion in Genetics and Development, 15, 206–213 www.sciencedirect.com