Genomic, Functional and Structural Analyses Reveal Mechanisms of Evolutionary Innovation within the Sea Anemone 8 Toxin Family

ShK from Stichodactyla helianthus has established the therapeutic potential of sea anemone venom peptides, but many lineage-specific toxin families in actinarians remain uncharacterised. One such peptide family, sea anemone 8 (SA8), is present in all five sea anemone superfamilies. We explored the genomic arrangement and evolution of the SA8 gene family in Actinia tenebrosa and Telmatactis stephensoni, characterised the expression patterns of SA8 sequences, and examined the structure and function of SA8 from the venom of T. stephensoni. We identified ten SA8 genes in two clusters and six SA8 genes in five clusters for T. stephensoni and A. tenebrosa, respectively. Nine SA8 T. stephensoni genes were found in a single cluster and an SA8 peptide encoded by an inverted SA8 gene from this cluster was recruited to venom. We show that SA8 genes in both species are expressed in a tissue-specific manner and the inverted SA8 gene has a unique tissue distribution. While functional activity of the SA8 putative toxin encoded by the inverted gene was inconclusive, its tissue localisation is similar to toxins used for predator deterrence. We demonstrate that, although mature SA8 putative toxins have similar cysteine spacing to ShK, SA8 peptides are distinct from ShK peptides based on structure and disulfide connectivity. Our results provide the first demonstration that SA8 is a unique gene family in actiniarians, evolving through a variety of structural changes including tandem and proximal gene duplication and an inversion event that together allowed SA8 to be recruited into the venom of T. stephensoni.

[1]  Edward G. Smith,et al.  Dominant toxin hypothesis: unravelling the venom phenotype across micro and macroevolution , 2022, bioRxiv.

[2]  D. Hurwood,et al.  Venoms for all occasions: The functional toxin profiles of different anatomical regions in sea anemones are related to their ecological function , 2021, Molecular ecology.

[3]  J. Liu,et al.  The genome of a new anemone species (Actiniaria: Hormathiidae) provides insights into deep-sea adaptation , 2021 .

[4]  A. Quattrini,et al.  Phylogenomics, Origin and Diversification of Anthozoans (Phylum Cnidaria). , 2021, Systematic biology.

[5]  Ben Fulton,et al.  CAFE 5 models variation in evolutionary rates among gene families , 2020, Bioinform..

[6]  Silvio C. E. Tosatto,et al.  Pfam: The protein families database in 2021 , 2020, Nucleic Acids Res..

[7]  Sofia M. C. Robb,et al.  Sea anemone genomes reveal ancestral metazoan chromosomal macrosynteny , 2020, bioRxiv.

[8]  Jue Ruan,et al.  SMARTdenovo: a de novo assembler using long noisy reads , 2020, GigaByte.

[9]  R. Norton,et al.  The voltage-gated potassium channel KV1.3 as a therapeutic target for venom-derived peptides. , 2020, Biochemical pharmacology.

[10]  A. von Haeseler,et al.  Corrigendum to: IQ-TREE 2: New Models and Efficient Methods for Phylogenetic Inference in the Genomic Era , 2020, Molecular biology and evolution.

[11]  A. Reitzel,et al.  Toxin-like neuropeptides in the sea anemone Nematostella unravel recruitment from the nervous system to venom , 2020, Proceedings of the National Academy of Sciences.

[12]  S. Carroll,et al.  The origin and diversification of a novel protein family in venomous snakes , 2020, Proceedings of the National Academy of Sciences.

[13]  C. Wilding,et al.  The genome of the sea anemone Actinia equina (L.): Meiotic toolkit genes and the question of sexual reproduction. , 2020, Marine genomics.

[14]  R. Norton,et al.  Tentacle Transcriptomes of the Speckled Anemone (Actiniaria: Actiniidae: Oulactis sp.): Venom-Related Components and Their Domain Structure , 2020, Marine Biotechnology.

[15]  Olga Chernomor,et al.  IQ-TREE 2: New Models and Efficient Methods for Phylogenetic Inference in the Genomic Era , 2019, bioRxiv.

[16]  R. Jenner,et al.  The Diversity of Venom: The Importance of Behavior and Venom System Morphology in Understanding Its Ecology and Evolution , 2019, Toxins.

[17]  A. Papanicolaou,et al.  The draft genome of Actinia tenebrosa reveals insights into toxin evolution , 2019, Ecology and Evolution.

[18]  J. Tytgat,et al.  The birth and death of toxins with distinct functions: a case study in the sea anemone Nematostella. , 2019, Molecular biology and evolution.

[19]  R. Norton,et al.  Evolution of cnidarian trans‐defensins: Sequence, structure and exploration of chemical space , 2019, Proteins.

[20]  R. Norton,et al.  Mapping the chemical and sequence space of the ShKT superfamily. , 2019, Toxicon : official journal of the International Society on Toxinology.

[21]  G. King,et al.  Sea Anemone Toxins: A Structural Overview , 2019, Marine drugs.

[22]  G. King,et al.  A process of convergent amplification and tissue‐specific expression dominates the evolution of toxin and toxin‐like genes in sea anemones , 2019, Molecular ecology.

[23]  L. Pardo,et al.  New Structures and Gating of Voltage-Dependent Potassium (Kv) Channels and Their Relatives: A Multi-Domain and Dynamic Question , 2019, International journal of molecular sciences.

[24]  Anthony R. Borneman,et al.  Purge Haplotigs: allelic contig reassignment for third-gen diploid genome assemblies , 2018, BMC Bioinformatics.

[25]  S. Kelly,et al.  OrthoFinder: phylogenetic orthology inference for comparative genomics , 2019, Genome Biology.

[26]  Martin Eisenacher,et al.  The PRIDE database and related tools and resources in 2019: improving support for quantification data , 2018, Nucleic Acids Res..

[27]  J. Tytgat,et al.  AbeTx1 Is a Novel Sea Anemone Toxin with a Dual Mechanism of Action on Shaker-Type K+ Channels Activation , 2018, Marine drugs.

[28]  Jennifer J. Smith,et al.  PHAB toxins: a unique family of predatory sea anemone toxins evolving via intra-gene concerted evolution defines a new peptide fold , 2018, Cellular and Molecular Life Sciences.

[29]  Shaodong Guo,et al.  Dipteran toxicity assays for determining the oral insecticidal activity of venoms and toxins , 2018, Toxicon : official journal of the International Society on Toxinology.

[30]  R. Norton,et al.  Synthesis, folding, structure and activity of a predicted peptide from the sea anemone Oulactis sp. with an ShKT fold , 2018, Toxicon : official journal of the International Society on Toxinology.

[31]  R. Norton,et al.  Identification, chemical synthesis, structure, and function of a new KV1 channel blocking peptide from Oulactis sp. , 2018 .

[32]  M. Phillips,et al.  Evidence for a Large Expansion and Subfunctionalization of Globin Genes in Sea Anemones , 2018, Genome biology and evolution.

[33]  R. Norton,et al.  Structure, folding and stability of a minimal homologue from Anemonia sulcata of the sea anemone potassium channel blocker ShK , 2018, Peptides.

[34]  R. Norton,et al.  Sea Anemones: Quiet Achievers in the Field of Peptide Toxins , 2018, Toxins.

[35]  Shujun Ou,et al.  LTR_retriever: A Highly Accurate and Sensitive Program for Identification of Long Terminal Repeat Retrotransposons1[OPEN] , 2017, Plant Physiology.

[36]  Carl Kingsford,et al.  Accurate assembly of transcripts through phase-preserving graph decomposition , 2017, Nature Biotechnology.

[37]  Johannes Söding,et al.  MMseqs2: sensitive protein sequence searching for the analysis of massive data sets , 2017, bioRxiv.

[38]  G. King,et al.  Revisiting venom of the sea anemone Stichodactyla haddoni: Omics techniques reveal the complete toxin arsenal of a well-studied sea anemone genus. , 2017, Journal of proteomics.

[39]  Heng Li,et al.  Minimap2: pairwise alignment for nucleotide sequences , 2017, Bioinform..

[40]  E. Muñoz-Elías,et al.  Safety and pharmacodynamics of dalazatide, a Kv1.3 channel inhibitor, in the treatment of plaque psoriasis: A randomized phase 1b trial , 2017, PloS one.

[41]  E. Isacoff,et al.  A new mechanism of voltage-dependent gating exposed by KV10.1 channels interrupted between voltage sensor and pore , 2017, The Journal of general physiology.

[42]  A. Pavasovic,et al.  Insights into the innate immunome of actiniarians using a comparative genomic approach , 2016, BMC Genomics.

[43]  S. Carroll,et al.  The Deep Origin and Recent Loss of Venom Toxin Genes in Rattlesnakes , 2016, Current Biology.

[44]  D. Gilbert,et al.  Gene-omes built from mRNA seq not genome DNA , 2016 .

[45]  Michael Broe,et al.  Tissue-Specific Venom Composition and Differential Gene Expression in Sea Anemones , 2016, Genome biology and evolution.

[46]  G. King,et al.  Toxin structures as evolutionary tools: Using conserved 3D folds to study the evolution of rapidly evolving peptides , 2016, BioEssays : news and reviews in molecular, cellular and developmental biology.

[47]  Guoli Ji,et al.  detectMITE: A novel approach to detect miniature inverted repeat transposable elements in genomes , 2016, Scientific Reports.

[48]  A. Myburg,et al.  Combined de novo and genome guided assembly and annotation of the Pinus patula juvenile shoot transcriptome , 2015, BMC Genomics.

[49]  Evgeny M. Zdobnov,et al.  BUSCO: assessing genome assembly and annotation completeness with single-copy orthologs , 2015, Bioinform..

[50]  Oleg Simakov,et al.  The genome of Aiptasia, a sea anemone model for coral symbiosis , 2015, Proceedings of the National Academy of Sciences.

[51]  S. Kelly,et al.  OrthoFinder: solving fundamental biases in whole genome comparisons dramatically improves orthogroup inference accuracy , 2015, Genome Biology.

[52]  T. Lotan,et al.  The dynamically evolving nematocyst content of an anthozoan, a scyphozoan, and a hydrozoan. , 2015, Molecular biology and evolution.

[53]  Katharina J. Hoff,et al.  BRAKER1: Unsupervised RNA-Seq-Based Genome Annotation with GeneMark-ET and AUGUSTUS , 2016, Bioinform..

[54]  R. Norton,et al.  Development of Highly Selective Kv1.3-Blocking Peptides Based on the Sea Anemone Peptide ShK , 2015, Marine drugs.

[55]  Narmada Thanki,et al.  CDD: NCBI's conserved domain database , 2014, Nucleic Acids Res..

[56]  Peter B. McGarvey,et al.  UniRef clusters: a comprehensive and scalable alternative for improving sequence similarity searches , 2014, Bioinform..

[57]  Martin T. Swain,et al.  Restriction and Recruitment—Gene Duplication and the Origin and Evolution of Snake Venom Toxins , 2014, bioRxiv.

[58]  Björn Usadel,et al.  Trimmomatic: a flexible trimmer for Illumina sequence data , 2014, Bioinform..

[59]  T. Pons,et al.  A novel sea anemone peptide that inhibits acid-sensing ion channels , 2014, Peptides.

[60]  E. Birney,et al.  Pfam: the protein families database , 2013, Nucleic Acids Res..

[61]  A. Cavalli,et al.  A Potent and Selective Peptide Blocker of the Kv1.3 Channel: Prediction from Free-Energy Simulations and Experimental Confirmation , 2013, PloS one.

[62]  D. Fredman,et al.  Nematostella vectensis transcriptome and gene models v2.0 , 2013 .

[63]  Colin N. Dewey,et al.  De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis , 2013, Nature Protocols.

[64]  Xun Xu,et al.  SOAPdenovo-Trans: de novo transcriptome assembly with short RNA-Seq reads , 2013, Bioinform..

[65]  K. Katoh,et al.  MAFFT Multiple Sequence Alignment Software Version 7: Improvements in Performance and Usability , 2013, Molecular biology and evolution.

[66]  Zhengwei Zhu,et al.  CD-HIT: accelerated for clustering the next-generation sequencing data , 2012, Bioinform..

[67]  G. King,et al.  Development of a rational nomenclature for naming peptide and protein toxins from sea anemones. , 2012, Toxicon : official journal of the International Society on Toxinology.

[68]  A. Antunes,et al.  Sea Anemone (Cnidaria, Anthozoa, Actiniaria) Toxins: An Overview , 2012, Marine drugs.

[69]  Emily S. Wong,et al.  Venom evolution through gene duplications. , 2012, Gene.

[70]  K. Chandy,et al.  Development of a sea anemone toxin as an immunomodulator for therapy of autoimmune diseases. , 2012, Toxicon : official journal of the International Society on Toxinology.

[71]  Martin Vingron,et al.  Oases: robust de novo RNA-seq assembly across the dynamic range of expression levels , 2012, Bioinform..

[72]  Sean R. Eddy,et al.  Accelerated Profile HMM Searches , 2011, PLoS Comput. Biol..

[73]  R. Norton,et al.  Analogs of the sea anemone potassium channel blocker ShK for the treatment of autoimmune diseases. , 2011, Inflammation & allergy drug targets.

[74]  W. Wüster,et al.  Domain loss facilitates accelerated evolution and neofunctionalization of duplicate snake venom metalloproteinase toxin genes. , 2011, Molecular biology and evolution.

[75]  A. Fujiyama,et al.  Using the Acropora digitifera genome to understand coral responses to environmental change , 2011, Nature.

[76]  Cole Trapnell,et al.  Computational methods for transcriptome annotation and quantification using RNA-seq , 2011, Nature Methods.

[77]  E. Grishin,et al.  The mining of toxin-like polypeptides from EST database by single residue distribution analysis , 2011, BMC Genomics.

[78]  R. Kini,et al.  Structure, function and evolution of three-finger toxins: mini proteins with multiple targets. , 2010, Toxicon : official journal of the International Society on Toxinology.

[79]  Susan R. Wessler,et al.  MITE-Hunter: a program for discovering miniature inverted-repeat transposable elements from genomic sequences , 2010, Nucleic acids research.

[80]  Benjamin M. Wheeler,et al.  The dynamic genome of Hydra , 2010, Nature.

[81]  Mark D. Robinson,et al.  edgeR: a Bioconductor package for differential expression analysis of digital gene expression data , 2009, Bioinform..

[82]  Gonçalo R. Abecasis,et al.  The Sequence Alignment/Map format and SAMtools , 2009, Bioinform..

[83]  Brian J. Smith,et al.  Engineering a Stable and Selective Peptide Blocker of the Kv1.3 Channel in T Lymphocytes , 2009, Molecular Pharmacology.

[84]  Steve Horvath,et al.  WGCNA: an R package for weighted correlation network analysis , 2008, BMC Bioinformatics.

[85]  P. Kuchel,et al.  Expression patterns of platypus defensin and related venom genes across a range of tissue types reveal the possibility of broader functions for OvDLPs than previously suspected. , 2008, Toxicon : official journal of the International Society on Toxinology.

[86]  O. Gascuel,et al.  An improved general amino acid replacement matrix. , 2008, Molecular biology and evolution.

[87]  Anthony T Papenfuss,et al.  Defensins and the convergent evolution of platypus and reptile venom genes. , 2008, Genome research.

[88]  E. Birney,et al.  Velvet: algorithms for de novo short read assembly using de Bruijn graphs. , 2008, Genome research.

[89]  J. Finnerty,et al.  Concerted evolution of sea anemone neurotoxin genes is revealed through analysis of the Nematostella vectensis genome. , 2008, Molecular biology and evolution.

[90]  Stefan Kurtz,et al.  LTRharvest, an efficient and flexible software for de novo detection of LTR retrotransposons , 2008, BMC Bioinformatics.

[91]  S. J. Kehl,et al.  An activation gating switch in Kv1.2 is localized to a threonine residue in the S2-S3 linker. , 2007, Biophysical journal.

[92]  Jonathan E. Allen,et al.  Automated eukaryotic gene structure annotation using EVidenceModeler and the Program to Assemble Spliced Alignments , 2007, Genome Biology.

[93]  M. Borodovsky,et al.  Evaluating the protein coding potential of exonized transposable element sequences , 2007, Biology Direct.

[94]  James E. Hall,et al.  Transformation of plasmid DNA into E. coli using the heat shock method. , 2007, Journal of visualized experiments : JoVE.

[95]  Nicholas H. Putnam,et al.  Sea Anemone Genome Reveals Ancestral Eumetazoan Gene Repertoire and Genomic Organization , 2007, Science.

[96]  Zhao Xu,et al.  LTR_FINDER: an efficient tool for the prediction of full-length LTR retrotransposons , 2007, Nucleic Acids Res..

[97]  Vincent J. Lynch,et al.  Inventing an arsenal: adaptive evolution and neofunctionalization of snake venom phospholipase A2 genes , 2007, BMC Evolutionary Biology.

[98]  A. Riggs,et al.  Repositioning-dependent fate of duplicate genes. , 2005, DNA and cell biology.

[99]  Thomas D. Wu,et al.  GMAP: a genomic mapping and alignment program for mRNA and EST sequence , 2005, Bioinform..

[100]  Ethan M. Goldberg,et al.  Stichodactyla helianthus Peptide, a Pharmacological Tool for Studying Kv3.2 Channels , 2005, Molecular Pharmacology.

[101]  Qun Liu,et al.  Crystal Structure of the Cysteine-rich Secretory Protein Stecrisp Reveals That the Cysteine-rich Domain Has a K+ Channel Inhibitor-like Fold* , 2005, Journal of Biological Chemistry.

[102]  Amos Bairoch,et al.  Tox-Prot, the toxin protein annotation program of the Swiss-Prot protein knowledgebase. , 2005, Toxicon : official journal of the International Society on Toxinology.

[103]  Burkhard Morgenstern,et al.  AUGUSTUS: a web server for gene finding in eukaryotes , 2004, Nucleic Acids Res..

[104]  R. Kini.  Excitement ahead: structure, function and mechanism of snake venom phospholipase A2 enzymes. , 2003, Toxicon : official journal of the International Society on Toxinology.

[105]  Stephen M. Mount,et al.  Improving the Arabidopsis genome annotation using maximal transcript alignment assemblies. , 2003, Nucleic acids research.

[106]  R. Kini,et al.  Molecular moulds with multiple missions: Functional sites in three‐finger toxins , 2002, Clinical and experimental pharmacology & physiology.

[107]  W. J. Kent,et al.  BLAT--the BLAST-like alignment tool. , 2002, Genome research.

[108]  M. Lynch,et al.  The evolutionary fate and consequences of duplicate genes. , 2000, Science.

[109]  P. Kuchel,et al.  Defensin-like peptide-2 from platypus venom: member of a class of peptides with a distinct structural fold. , 2000, The Biochemical journal.

[110]  M. Ashburner,et al.  Gene Ontology: tool for the unification of biology , 2000, Nature Genetics.

[111]  P. Kuchel,et al.  Solution structure of a defensin-like peptide from platypus venom. , 1999, The Biochemical journal.

[112]  A. Force,et al.  Preservation of duplicate genes by complementary, degenerative mutations. , 1999, Genetics.

[113]  E. Christian,et al.  ShK-Dap22, a Potent Kv1.3-specific Immunosuppressive Polypeptide* , 1998, The Journal of Biological Chemistry.

[114]  J Schultz,et al.  SMART, a simple modular architecture research tool: identification of signaling domains. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[115]  C. Roumestand,et al.  On the Convergent Evolution of Animal Toxins , 1997, The Journal of Biological Chemistry.

[116]  R. Norton,et al.  Solution structure of ShK toxin, a novel potassium channel inhibitor from a sea anemone , 1996, Nature Structural Biology.

[117]  C. Wernstedt,et al.  Characterization of a potassium channel toxin from the Caribbean Sea anemone Stichodactyla helianthus. , 1995, Toxicon : official journal of the International Society on Toxinology.

[118]  D. Craik,et al.  A common structural motif incorporating a cystine knot and a triple‐stranded β‐sheet in toxic and inhibitory polypeptides , 1994, Protein science : a publication of the Protein Society.

[119]  S. Grissmer,et al.  Calcium-activated potassium channels in resting and activated human T lymphocytes. Expression levels, calcium dependence, ion selectivity, and pharmacology , 1993, The Journal of general physiology.

[120]  B. Sakmann,et al.  Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches , 1981, Pflügers Archiv.

[121]  J. Abita,et al.  Sea anemone toxin:a tool to study molecular mechanisms of nerve conduction and excitation-secretion coupling. , 1976, Proceedings of the National Academy of Sciences of the United States of America.

[122]  H. Neu,et al.  The release of enzymes from Escherichia coli by osmotic shock and during the formation of spheroplasts. , 1965, The Journal of biological chemistry.

[123]  M. Ishida,et al.  Isolation and molecular cloning of novel peptide toxins from the sea anemone Antheopsis maculata. , 2005, Toxicon : official journal of the International Society on Toxinology.

[124]  Cathy H. Wu,et al.  UniProt: the Universal Protein knowledgebase , 2004, Nucleic Acids Res..

[125]  Walter Zucchini,et al.  RGL : A R-library for 3 D visualization with OpenGL , 2003 .