Evolution and Diversification of the Cnidarian Venom System Mahdokht Jouiaei Doctor of Pharmacy (Pharm.D)

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Evolution and diversification of the cnidarian venom system Mahdokht Jouiaei Doctor of Pharmacy (Pharm.D)

A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2016 School of Biological Sciences

Abstract The phylum Cnidaria (corals, sea pens, sea anemones, jellyfish and hydroids) is the oldest venomous animal lineage (~750 million years old), making it an ideal phylum to understand the origin and diversification of venom. Cnidarians are characterised by specialised cellular structures called cnidae, which they utilise to inject mixtures of bioactive compounds or venom for predation and defence. In recent years cnidarian venoms have begun to be investigated as a potential source of novel bioactive therapeutics. However, in comparison with several venomous lineages that are relatively younger, such as snakes, cone snails and spiders, the diversification and molecular evolutionary regimes of toxins encoded by these fascinating and ancient animals remain poorly understood. The first experimental part of this thesis (Chapter 2) is comprised of the phylogenetic and molecular evolutionary histories of pharmacologically characterised cnidarian toxin families. These include peptide neurotoxins (voltage-gated Na+ and K+ channel-targeting toxins: NaTxs and KTxs, respectively), pore-forming toxins (actinoporins, aerolysin-related toxins, and jellyfish toxins) and small cysteine-rich peptides (SCRiPs). Our analyses show that most cnidarian toxins remain conserved under the strong influence of negative selection. A deeper analysis of the molecular evolutionary histories of neurotoxins demonstrates that type III KTxs have evolved from NaTxs under the regime of positive selection and likely represent a unique evolutionary innovation of the Actinioidea lineage. We also identify a few functionally important sites in other classes of neurotoxins and pore-forming toxins that experienced episodic adaptation. In addition, we describe the first family of neurotoxic peptides (SCRiPs) in reef-building corals, Acropora millepora, that are likely involved in the envenoming function. The third chapter describes the development of a novel venom extraction technique which is rapid, repeatable and cost effective. This technique involves using ethanol to both induce cnidae discharge and to recover venom contents in one step. Our model species is the notorious Australian box jellyfish (Chironex fleckeri) which has a notable impact on public health. By using the complementary approaches of transcriptomics, proteomics, mass spectrometry, histology, and scanning electron microscopy, this study compares the proteome profile of the new ethanol recovery based method to a previously reported protocol, based upon density purified intact cnidae and pressure induced disruption. This work not only results in the expansion of identified Australian box jellyfish venom components but also illustrates that ethanol extraction method could greatly augment future cnidarian venom proteomics research efforts. The forth chapter is constituted by previously described venom extraction technique, ethanol- induced discharge of cnidae, to rapidly and efficiently characterise venom components of the Jelly Blubber or Blue Blubber Jellyfish, Catostylus mosaicus. By using a combination of proteomics and

I transcriptome sequencing, potential toxin families are identified in the oral arms and tentacles included PLA2, lipases, CRiSPs, cystatins and serine protease inhibitors (Kunitz and Kazal peptides). This study reveals venom components of oral arms and tentacles of a blubber jellyfish for the first time and enhances our understanding of the mechanism of its sting. The fifth chapter describes the pharmacological profile of the hell’s fire anemone (Actinodendron sp.) venom. Due to the lack of transcriptome data available, an activity-guided fractionation approach (chromatography/tandem mass spectrometry/bioassays) is described to purify and characterise novel bioactives (toxins) with interesting pharmacological properties. The potential anemone toxin explained here (α3β2 nAChR blocker) is strong candidate as a lead compound for the development of diagnostic and therapeutic. Taken together, this thesis unravels the molecular evolutionary history of cnidarian toxin families, details a novel venom extraction technique which may transform cnidarian venom research, and also illustrates how proteomics, transcriptome sequencing and activity-guided fractionation can be used to discover new proteins and peptide toxins that can be bioactive resources for drug discovery and development.

II Declaration by author

This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis.

I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my research higher degree candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award.

I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School.

I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis.

III Publications during candidature (* joint first authorship)

Peer-reviewed papers

Jouiaei, M.; Yanagihara, A.A.; Madio, B.; Nevalainen, T.J.; Alewood, P.F.; Fry, B.G. Ancient venom systems: a review on Cnidaria toxins. Toxins 2015, 7(6), 2251-2271.

*Jouiaei, M.; *Casewell, N.R.; Yanagihara, A.A.; Nouwens, A.; Cribb, B.W.; Whitehead, D.; Jackson, T.N.; Ali, S.A.; Wagstaff, S.C.; Koludarov, I.; Alewood, P.; Hansen, J.; Fry, B.G. Firing the sting: chemically induced discharge of cnidae reveals novel proteins and peptides from box jellyfish (Chironex fleckeri) venom. Toxins 2015, 7(3), 936-950.

*Jouiaei, M.; *Sunagar, K.; Federman Gross, A.; Scheib, H.; Alewood, P.F.; Moran, Y.; Fry, B.G. Evolution of an ancient venom: recognition of a novel family of cnidarian toxins and the common evolutionary origin of sodium and potassium neurotoxins in sea anemone. Mol Biol Evol 2015, 32(6), 1598-1610.

Jesupret, C.; Baumann, K.; Jackson, T.N.; Ali, S.A.; Yang, D.C.; Greisman, L.; Kern, L.; Steuten, J.; Jouiaei, M.; Casewell, N.R. Vintage venoms: Proteomic and pharmacological stability of snake venoms stored for up to eight decades. Journal of proteomics 2014, 105, 285-294.

Conference abstracts

Jouiaei, M.; Sunagar, K.; Federman Gross, A.; Scheib, H.; Alewood, P.F.; Moran, Y.; Fry, B.G. Evolution of an ancient venom. 11th Australian Peptide Conference, Kingscliff, NSW, Australia. October 2015. (Poster)

Jouiaei, M.; Casewell, N.R.; Yanagihara, A.A.; Nouwens, A.; Cribb, B.W.; Whitehead, D.; Alewood, P.; Fry, B.G. Firing the sting. 5th Conference on Venoms to Drugs, Kingscliff, NSW, Australia. October 2014. (Poster and Oral)

IV Publications included in this thesis (* joint first authorship)

Incorporated as Chapter 1: Jouiaei, M.; Yanagihara, A.A.; Madio, B.; Nevalainen, T.J.; Alewood, P.F.; Fry, B.G. Ancient venom systems: a review on Cnidaria toxins. Toxins 2015, 7(6), 2251-2271.

Contributor Statement of contribution Jouiaei, M. (Candidate) Wrote the paper (100%) Provided figures and tables (100%) Yanagihara, A.A. Edited paper (30%) Madio, B. Edited paper (5%) Nevalainen, T.J. Edited paper (15%) Alewood, P.F. Edited paper (25%) Fry, B.G. Edited paper (25%) Approved the paper for submission (100%)

Incorporated as Chapter 2: *Jouiaei, M.; *Sunagar, K.; Federman Gross, A.; Scheib, H.; Alewood, P.F.; Moran, Y.; Fry, B.G. Evolution of an ancient venom: recognition of a novel family of cnidarian toxins and the common evolutionary origin of sodium and potassium neurotoxins in sea anemone. Mol Biol Evol 2015, 32(6), 1598-1610.

Contributor Statement of contribution Jouiaei, M. (Candidate) Designed the study (50%) Collected the data (toxin sequences) (50%) Analysed the data (phylogenetic analysis) (50%) Wrote the paper (50%) Sunagar, K. Designed the study (50%) Collected the data (cloning, recombinant expression, and purification of toxins) (50%) Analysed the data (Selection and structural analyses) (40%) Wrote the paper (50%) Federman Gross, A. Analysed the data (Selection and structural analyses) (5%)

V Scheib, H. Analysed the data (Selection and structural analyses) (5%) Alewood, P.F. Edited paper (25%) Moran, Y. Edited paper (50%) Approved the paper for submission (50%) Fry, B.G. Edited paper (25%) Approved the paper for submission (50%)

Incorporated as Chapter 3: *Jouiaei, M.; *Casewell, N.R.; Yanagihara, A.A.; Nouwens, A.; Cribb, B.W.; Whitehead, D.; Jackson, T.N.; Ali, S.A.; Wagstaff, S.C.; Koludarov, I.; Alewood, P.; Hansen, J.; Fry, B.G. Firing the sting: chemically induced discharge of cnidae reveals novel proteins and peptides from box jellyfish (Chironex fleckeri) venom. Toxins 2015, 7(3), 936-950.

Contributor Statement of contribution Jouiaei, M. (Candidate) Designed the experiments (70%) Performed the MS/MS sequencing (75%) Performed SEM (80%) Performed histology (80%) Analysed the data (70%) Provided figures (100%) Wrote the paper (90%) Casewell, N.R. Extracted, purified, isolated mRNA (100%) Prepared transcriptome library (90%) Analysed the data (20%) Wrote the paper (10%) Yanagihara, A.A. Provided the mechanical extracted venom (100%) Edited paper (10%) Nouwens, A. Designed the experiments (5%) Analysed the data (5%) Performed the MS/MS sequencing (15%) Edited paper (5%) Cribb, B.W. Designed the experiments (5%) Performed SEM (20%) Analysed the data (5%)

VI Edited paper (5%) Whitehead, D. Designed the experiments (5%) Performed histology (20%) Analysed the data (5%) Edited paper (5%) Jackson, T.N. Performed the MS/MS sequencing (2%) Revised the manuscript (5%) Ali, S.A. Performed the MS/MS sequencing (2%) Wagstaff, S.C. Prepared transcriptome library (10%) Koludarov, I. Performed the MS/MS sequencing (2%) Alewood, P. Edited paper (30%) Hansen, J. Performed the MS/MS sequencing (2%) Fry, B.G. Designed the experiments (5%) Analysed the data (5%) Edited paper (30%) Approved the paper for submission (100%)

VII Contributions by others to the thesis

Chapter 2

Selection and structural analyses were done by Dr. Kartik Sunagar at Department of Ecology, Evolution and Behavior, the Alexander Silberman Institute for Life Sciences, Hebrew University of Jerusalem, Israel, Dr. Aya Federman Gross at Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel Aviv University, Israel and Dr. Holger Scheib at Institute for Molecular Bioscience (IMB), University of Queensland (UQ). Cloning, recombinant expression, and purification of toxins were done by Dr. Kartik Sunagar.

Chapter 3

The mechanical extracted venom from Box jellyfish (Chironex fleckeri) was provided by Dr. Angel A. Yanagihara at Pacific Cnidaria Research Lab, Department of Tropical Medicine, University of Hawaii, USA.

Chapter 3 and 4

SEM study was done under the supervision of Dr. Bronwen W. Cribb at Centre for Microscopy & Microanalysis at School of Biological Sciences, UQ. Histology study was done under the supervision of Dr. Darryl Whitehead at School of Biomedical Sciences, UQ.

Chapter 5

Pharmacological activity was done under the supervision of Dr. Irina Vetter at IMB, UQ.

Statement of parts of the thesis submitted to qualify for the award of another degree

None.

VIII Acknowledgements I acknowledge the provision of UQ International Scholarship.

This thesis marks the culmination of a three and half year journey. On reflection a PhD is akin to academic imprisonment. In the fullness of time a PhD student begins to demonstrate feelings of trust and affection towards their captors, in much the same way a hostage develops these feeling to their kidnapper. You don’t need a summer holiday in Stockholm, to experience their syndromes.

In all seriousness, I would like to take this opportunity to express my appreciation to both of my supervisors Associate Professor Bryan G. Fry and Professor Paul F. Alewood. Together, they provided me with an opportunity to complete my PhD under the guidance. Their friendship, support and wisdom have been invaluable, as well as the freedom follows my own path.

In addition, I would also like to thank my committee members Professor Thomas Cribb and Dr. Irina Vetter for their ongoing support and sharing their knowledge and expertise. A special thanks to Dr. Amanda Nouwens who has been an incredible mentor of mass spectrometry and a source of personal inspiration. Amanda provided invaluable advice on how to achieve a quality thesis and was a great sounding board for my experimental ideas.

The scope of the biochemistry and biology questions I could address in this thesis would not be possible without collaborations, and I am thankful for the opportunity to work with so many talented researchers across many fields. In particular I would like to thank Dr. Alun Jones and Dr. Peter Josh for their help with the mass spectrometry experiments, Dr. Bronwen W. Cribb and Dr. Darryl Whitehead for SEM and histology training. It has been an absolute pleasure to collaborate with them. I also would like to acknowledge Dr. Angel A.Yanagihara, for providing box jellyfish venom and revising manuscripts.

Thanks to my friends in Venom Evolution and Alewood lab, for their friendly company and support. Special thanks to Jordan, Kate, Bianca and Christina for their comments on my thesis.

I would like to extend my gratitude to my parents, Lili and Ali, for giving me the opportunity to study overseas and the sacrifices that they have made for a better future. Thanks to my sister Mahtab for her endless support. You have been my reliable, continuous support network and were always there when I needed you most.

Last, but not least, I must express my warmest and heartfelt thanks to my kind and lovely husband Lee. Thank you for your support, patience, encouragement and warm smile everyday which helped me to keep working during times of frustration. PhD years were a challenge and I knew I would be able to complete it with you by my side.

IX Keywords cnidarian venom, evolution, transcriptomics, proteomics, scanning electron microscopy, histology, activity-guided fractionation, therapeutics

Australian and New Zealand Standard Research Classifications (ANZSRC) ANZSRC code: 030406 Proteins and Peptides, 40% ANZSRC code: 030101 Analytical Spectrometry, 40% ANZSRC code: 060399 Evolutionary Biology not elsewhere classified, 20%

Fields of Research (FoR) Classification FoR code: 0304 Medicinal and Biomolecular Chemistry, 40% FoR code: 0301 Analytical chemistry, 40% FoR code: 0603, Evolutionary Biology, 20%

X Table of Contents

Abstract ...... I Declaration by author ...... III Publications during candidature ...... IV Publications included in this thesis ...... V Contributions by others to the thesis...... VIII Statement of parts of the thesis submitted to qualify for the award of another degree ...... VIII Acknowledgements...... IX Keywords ...... X Australian and New Zealand Standard Research Classifications (ANZSRC) ...... X Fields of Research (FoR) Classification ...... X List of Figures ...... XVI List of Tables ...... XVII List of Abbreviations used in the thesis ...... XVIII Note on style to the reader ...... XX Chapter 1 Ancient Venom Systems: A Review on Cnidaria Toxins ...... 1 Abstract ...... 2 Keywords ...... 2 1.1 Introduction ...... 2 1.2 Cnidarian Phylogeny ...... 3 1.3 Cnidarian Life Cycle ...... 3 1.4 Cnidarian Venom Delivery System ...... 4 1.5 Venom Composition...... 7 1.5.1 Enzymes ...... 7

1.5.1.1 Phospholipase A2 ...... 7 1.5.1.2 Metalloproteases ...... 8 1.5.2 Pore-Forming Toxins ...... 8 1.5.2.1 Actinoporins ...... 8 1.5.2.2 Jellyfish Toxins (JFTs) ...... 9 1.5.2.3 Hydralysins-Related Toxins ...... 9 1.5.2.4 Membrane Attack Complex-Perforin ...... 10 1.5.3 Neurotoxins ...... 10 1.5.3.1 Voltage-gated Sodium (Nav) Channel Toxins ...... 10 1.5.3.2 Voltage-gated Potassium (Kv) Channel Toxins and Kunitz peptides ...... 11 XI 1.5.3.3 Small Cysteine-Rich Peptides (SCRiPs) ...... 11 1.5.3.4 ASIC Inhibitors ...... 12 1.5.3.5 TRPV1 Inhibitors ...... 12 1.5.4 Non-Protein Bioactive Components ...... 12 1.6 The Role of Cnidarians Venoms in Drug Discovery ...... 13 1.7 Envenomation ...... 14 1.8 Conclusions ...... 14 Aims ...... 15 Chapter 2 Evolution of an ancient venom: Recognition of a Novel Family of Cnidarian Toxins and the Common Evolutionary Origin of Sodium and Potassium Neurotoxins in Sea Anemone ...... 16 Abstract ...... 17 Key words ...... 17 2.1 Introduction ...... 17 2.2 Results ...... 18 2.2.1 Neurotoxins ...... 18 2.2.2 Pore-Forming Toxins ...... 24 2.2.2.1 Actinoporins ...... 24 2.2.3 Aerolysin-Related Toxins in Sea Anemones and Hydroids ...... 25 2.2.4 Jellyfish Toxins ...... 26 2.2.5 Small Cysteine-Rich Peptides ...... 27 2.3 Discussion ...... 28 2.3.1 Strong Negative Selection Governs the Evolution of Most Cnidarian Toxins ...... 28 2.3.2 Most Adaptive Sites in Cnidarian Neurotoxins Are Surface Accessible ...... 29 2.3.3 Negative Selection Constrains the Evolution of Pore-Forming Toxins ...... 29 2.3.4 The Common Evolutionary Origin of Sodium and Type III Potassium Neurotoxins in Sea Anemones ...... 30 2.3.5 Functional Diversification of Sea Anemone Neurotoxins ...... 31 2.3.6 SCRiPs May Represent the First Neurotoxin Family from Corals ...... 31 2.4 Materials and Methods ...... 32 2.4.1 Phylogenetic Analyses ...... 32 2.4.2 Selection Analyses ...... 33 2.4.3 Structural Analyses ...... 33 2.4.4 Cloning, Recombinant Expression, and Purification of Toxins...... 34 2.4.5 Toxicity Assays ...... 34

XII Supplementary Materials ...... 34 Chapter 3 Firing the Sting: Chemically Induced Discharge of Cnidae Reveals Novel Proteins and Peptides from Box Jellyfish (Chironex fleckeri) Venom ...... 35 Abstract ...... 36 Keywords ...... 36 3.1 Introduction ...... 36 3.2 Results and Discussion ...... 37 3.2.1 Microscopy Examination of Undischarged and Chemically Discharged Nematocysts …………… ...... 38 3.2.2 Transcriptome Assembly and Functional Annotation ...... 41 3.2.3 Comparative Proteomic Analyses and Identified Proteins ...... 41 3.3 Experimental Section ...... 44 3.3.1 Specimen Collection ...... 44 3.3.2 Nematocyst Morphology...... 44 3.3.2.1 Cryo-Scanning Electron Microscopy ...... 44 3.3.2.2 Histology ...... 44 3.3.3 Transcriptome Library Construction ...... 45 3.3.4 Venom Extraction ...... 46 3.3.4.1 Chemically Induced Discharge of Nematocysts by Ethanol ...... 46 3.3.4.2 Pressure Disrupted, Pre-Purified Nematocysts ...... 46 3.3.5 Proteomics Methods ...... 46 3.4 Conclusions ...... 47 Supplementary Materials ...... 48 Chapter 4 Investigating Toxin Diversity in the Tentacle and Oral Arms of the Blue Blubber Jellyfish Catostylus mosaicus by Using a Combined Transcriptomic and Proteomic Analyses 49 Abstract ...... 50 4.1 Introduction ...... 51 4.2 Results and Discussion ...... 51 4.2.1 The Transcriptome of C. mosaicus Oral arm and Tentacles ...... 54 4.2.2 Identified Proteins in C. mosaicus Oral Arm and Tentacle Using Tandem Mass Spectroscopy (MS/MS) ...... 55 4.2.3 Toxin Proteins Identified in C. mosaicus Oral Arm and Tentacle Using Tandem Mass Spectroscopy (MS/MS) ...... 57

4.2.3.1 Secretory Phospholipase A2 (sPLA2) ...... 57 4.2.3.2 Lipases ...... 60

XIII 4.2.3.3 Putative CRiSP C. mosaicus Venom Proteins ...... 61 4.2.3.4 Putative Venom Cystatin ...... 63 4.2.3.5 Serine Protease Inhibitors ...... 65 4.2.4 Enzymatic Activity ...... 68 4.3 Experimental Section ...... 69 4.3.1 Specimen Collection ...... 69 4.3.2 Nematocyst Morphology...... 69 4.3.3 RNA Extraction and Transcriptome Library Construction ...... 69 4.3.4 Venom Extraction ...... 70 4.3.5 Proteomics ...... 70 4.3.5.1 One dimensional (1D) SDS-PAGE ...... 70 4.3.5.2 Protein digestion ...... 71 4.3.5.3 LC− ESI−MS/MS ...... 72 4.3.6 Bioinformatics Analysis ...... 72 4.3.7 Phylogenetic Analyses ...... 72

4.3.8 PLA2 Activity Testing ...... 73 4.3.9 Metalloprotease Activity Testing ...... 73 4.4 Conclusion and Future Directions ...... 74 Chapter 5 Pharmacological Profile of Venom Isolated From Hell’s Fire Anemone Actinodendron sp: A Preliminary Study ...... 75 Abstract ...... 76 Keyword ...... 76 5.1 Introduction ...... 77 5.2 Result and Discussion ...... 77 5.2.1 Proteomics ...... 77

5.2.2 Isolation of a Novel sPLA2 Toxin ...... 79

5.2.3 Possible Role of PLA2 in Abnormal RBC Morphologies ...... 81 5.2.4 Pharmacological Significance of Hell’s Fire Venom ...... 82 5.2.5 Biodiscovery Potentials of Hell’s Fire Venom ...... 84 5.3 Conclusion and Future Directions ...... 84 5.4 Experimental Section ...... 84 5.4.1 Specimen and Venom Collection ...... 84 5.4.2 Proteomics ...... 85 5.4.2.1 LC−Orbitrap Elite MS/MS ...... 85 5.4.2.2 Reverse Phase High Performance Liquid Chromatography (RP-HPLC) ...... 85

XIV 5.4.2.3 uHPLC, Electrospray Mass Spectrometry and Mass Profiling (LC-ESI-MS) ...... 85 5.4.3 Bioassay ...... 86

5.4.3.1 PLA2 assay ...... 86 5.4.3.2 RBC Preparation and Light Microscopy of Shape Changes ...... 86 5.4.3.3 Fluorescence-Based HTS Assay ...... 86 Chapter 6 Conclusion and Future Directions...... 88 6.1 Research Overview...... 89 6.1.1 Evolution of an Ancient Venom ...... 89 6.1.2 Cnidarian Venom Extraction ...... 90 6.1.3 Biodiscovery Potential of Cnidarian Venoms Using Transcriptome/Proteome Sequencing, Activity-Guided Fractionation and Molecular Evolutionary Approaches ...... 91 6.2 Conclusion/Future directions ...... 91 List of References ...... 93 APPENDIX I ...... XXI

XV List of Figures

Figure 1-1. Overview of the cnidarian venom delivery system...... 5 Figure 2-1. Molecular evolution of cnidarian toxins...... 19 Figure 2-2. Sequence alignment of NaTx, KTx type I, and KTx type III...... 21 Figure 2-3. The common origin of NaTx and KTx type III...... 23 Figure 2-4. Sequence alignment of actinoporins...... 24 Figure 2-5. Sequence alignment of hydralysins...... 26 Figure 2-6. Sequence alignment of SCRiPs...... 28 Figure 3-1. Scanning electron microscopy (SEM) of undischarged C. fleckeri nematocysts...... 39 Figure 3-2. Light microscopy (LM) of C. fleckeri nematocysts...... 40 Figure 3-3. Scanning electron microscopy (SEM) of ethanol discharged C. fleckeri nematocysts.. . 41 Figure 4-1. Undischarged nematocysts of C. mosaicus...... 53 Figure 4-2. Ethanol-discharged nematocysts of C. mosaicus...... 54 Figure 4-3. (A) Summary of assembly and annotation of nucleotide sequence data from C. mosaicus tentacle and oral arm tissues. (B) Functional grouping of putative toxin proteins/peptides identified in C. mosaicus oral arm and tentacle transcriptomes...... 55 Figure 4-4. Identified proteins in C. mosaicus oral arm and tentacle using MS/MS...... 56 Figure 4-5. C. mosaicus venom proteins...... 57

Figure 4-6. Sequence alignment of GIII PLA2 precursors...... 59

Figure 4-7. Sequence alignment of GIX PLA2 precursors...... 60 Figure 4-8. Sequence alignment of lipid-precursors...... 61 Figure 4-9. Sequence alignment of CRiSP precursors...... 62 Figure 4-10. (A) Sequence alignment of cystatin-precursors. (B) Precursor phylogenetic reconstruction...... 64 Figure 4-11. Sequence alignment of kunitz precursors ...... 65 Figure 4-12. Sequence alignment of kazal precursors...... 66 Figure 4-13. Enzymatic activity of C. mosaicus...... 69

Figure 5-1. Purification and identification of PLA2...... 80 Figure 5-2. Light microscopy (LM) of sheep RBCs ...... 81 Figure 5-3. Actinodendron sp. pharmacological activity...... 82 Figure 5-4. Purification and identification of α3 nAChR blockers from venom of Actinodendron sp...... 83

XVI List of Tables

Table 1-1. Cnidarian venom composition...... 6 Table 2-1. Molecular evolution of cnidarian neurotoxins...... 20 Table 2-2. Molecular evolution of cnidarian pore-forming toxins and SCRiPs...... 25 Table 3-1 Summary of venom proteins/peptides identified from C. fleckeri venom material...... 43 Table 4-1 Summary of venom proteins identified in the oral arm and tentacle of C. mosaicus...... 67 Table 5-1 Summary of venom protein identified in Actinodendron sp. tentacle...... 78

Table 5-2 Hell’s fire homologous PLA2 protein identified in the selected fraction...... 81 Table 5-3 Hell’s fire protein identified in the selected fractions (F34-36)...... 83

XVII List of Abbreviations used in the thesis

1D-PAGE 1-Dimensional Polyacrylamide Gel Electrophoresis 2D-PAGE 2-Dimensional Polyacrylamide Gel Electrophoresis ABC Ammonium Bicarbonate AC Ammonium Carbonate ACN Acetonitrile AGRF Australian Genomic Research Facility BEB Bayes Empirical Bayes BLAST Basic Logical Alignment Search Tool BP Base Pair BPTI Bovine Pancreatic Trypsin Inhibitor CAP CRiSP (cysteine rich proteins), Allergen-5, and Pathogenesis-related Cav Voltage-gated Calcium Channel cDNA Complementary DNA CRiSP Cysteine Rich Secretory Protein DTT Dithiothreitol ESI Electro-Spray Ionization FA Formic Acid FDR False Discovery Rate FUBAR Fast Unconstrained Bayesian Approximation hERG Human Ether-a-go-go-Related Gene IDA Information Dependant Acquisition kDa kilo Dalton kV kilo Volt Kv Voltage-gated potassium channel KTx Voltage-Gated K+ Channel-Targeting Toxin LC Liquid Chromatography LM Light Microscope Ma Million Years Ago m/z Mass to charge ratio M12A Metalloprotease family M12, subfamily A MEME Mixed Effects Model of Evolution mRNA Messenger RNA MS Mass Spectrometry

XVIII MS/MS (MS2) Tandem Mass Spectrometry MW Molecular Weight nAChR Nicotinic Acetylcholine Receptor Nav Voltage-gated calcium channel NaTx Voltage-Gated Na+ Channel-Targeting Toxin NBF Neutrally Buffered Formalin NGF Nerve Growth Factor PBS Phosphate Buffered Saline PFT Pore-Forming Toxin PI Isoelectric Point

POC Phosphocholine PS Positively Selected sites PTM Post Translational Modification RP-HPLC Reverse-Phase High-Performance Liquid Chromatography SCRiP Small cysteine rich Peptide SDS Sodium Dodecyl Sulphate SEM Scanning Electron Microscopy SM Sphingomyelin sPLA2 Secretory Phospholipase A2 SRBC Sheep Red Blood Cell

TEM Effector Memory T-cells TFA Trifluoroacetic Acid TNF-α Tumour Necrosis Factor α TOF Time of Flight uHPLC Ultra-High-Performance Liquid Chromatography VEGF Vascular Endothelial Growth Factor JFT Jelly Fish Toxin Z Charge

XIX Note on style to the reader

This thesis is presented as a series of manuscripts that have been published or are awaiting final comments from co-authors prior to submission. Because they have been submitted to or prepared for different journals, the formatting follows different style rules. There are therefore certain inconsistencies between chapters, such as the order of presentation of results, discussion and methods.

Chapter 1 Ancient Venom Systems: A Review on Cnidaria Toxins

Manuscript published in Toxins, June 2015 http://www.mdpi.com/2072-6651/7/6/2251/htm 1

Abstract Cnidarians are the oldest extant lineage of venomous animals. Despite their simple anatomy, they are capable of subduing or repelling prey and predator species that are far more complex and recently evolved. Utilising specialized penetrating nematocysts, cnidarians inject the nematocyst content or venom that initiates toxic and immunological reactions in the envenomated organism. These venoms contain enzymes, potent pore forming toxins, and neurotoxins. Enzymes include lipolytic and proteolytic proteins that catabolise prey tissues. Cnidarian pore forming toxins self- assemble to form robust membrane pores that can cause cell death via osmotic lysis. Neurotoxins exhibit rapid ion channel specific activities. In addition, certain cnidarian venoms contain or induce the release of host vasodilatory biogenic amines such as serotonin, histamine, bunodosine and caissarone accelerating the pathogenic effects of other venom enzymes and porins. The cnidarian attacking/defending mechanism is fast and efficient, and massive envenomation of humans may result in death, in some cases within a few minutes to an hour after sting. The complexity of venom components represents a unique therapeutic challenge and probably reflects the ancient evolutionary history of the cnidarian venom system. Thus, they are invaluable as a therapeutic target for sting treatment or as lead compounds for drug design.

Keywords cnidarians, venom, enzymes, pore forming toxins, neurotoxins, vasodilatory biogenic amines, human envenomation

1.1 Introduction The phylum Cnidaria (corals, sea pens, sea anemones, jellyfish and hydroids) includes about 10,000 species living in aquatic habitats worldwide [1]. They range in size from the tiny Hydra spp., at less than 1 cm in length, to the massive lion’s mane jellyfish, Cyanea capillata with the bell diameter exceeding 2 m [2]. The envenomation hazard to humans also varies widely from non-hazardous to the infamous Chironex fleckeri (Australian box jellyfish), one of the most venomous animal dangerous to humans, as a meter of tentacle contact can provoke immediate cardiovascular collapse and death even within minutes after a sting [3]. The majority of cnidarians live in salt water habitats at different water depths. However, approximately 40 species, mostly hydrozoans [4] live in freshwater. Cnidarians are characteristically radially symmetrical [5], although they can also exhibit directional asymmetry or bilateral symmetry. For example, morphological studies on Siphonophores (class Hydrozoa) suggest that directional asymmetry has been gained and/or lost on multiple occasions [6], whilst most anthozoan polyps exhibit bilateral symmetry possessing two orthogonal body axes [5].

2 Despite the variety in size, toxicity, habitat and morphology several cellular characters are common to the members of Cnidaria, such as two unicellular layers (ectoderm and endoderm) separated by an extra-cellular matrix (mesoglea), neuromuscular systems and multiple sensory systems [7, 8]. Molecular evidence and fossil data place the origin of cnidarians prior to the Ediacaran period ~ 750 million years ago and major taxa diversification from the remaining metazoans prior to the Cambrian ~ 550 million years ago [9-11]. Cnidaria is an ancient clade of animals with complex venom compositions. Since their venoms have been much less intensively studied compared to other venomous clades, cnidarian venoms serve a unique therapeutic challenge. For example box jellyfish still remain one of the most medically important cnidarian venoms worldwide with limited therapeutics on offer. Therefore transcriptomics and proteomics data for the identification and characterising of their venom components is rapidly accumulating in recent times [12-14].

1.2 Cnidarian Phylogeny Based upon mitochondrial DNA (mtDNA) data [15] and life cycles [8, 16] cnidarians are divided into two extant subphyla: Anthozoa and Medusozoa. Anthozoans possess circular mtDNA, similar to that of other metazoans while medusozoans have atypical linear mtDNA. The members of medusozoan classes Hydrozoa, Schyphozoa, Cubozoa and Staurozoa display a triphasic life cycle in transition of generations: a free-swimming planula larva, a sessile polyp stage and sexual pelagic medusa stage. In anthozoans the medusa stage is lost and sessile adults represent the sexually propagating stage. The life cycle will be discussed in more details further in this review.

1.3 Cnidarian Life Cycle There is significant morphological diversity in the cnidarian life cycle, as a single species may display a variety of forms whether it is sessile, polyp, tiny free-swimming planula larva or a pelagic medusoid. The life cycle of both medusozoans and anthozoans comprises sexual reproduction and an asexually budding phase. In medusozoans, the adult medusa is either male or female, and the fertilized egg (zygote) is retained inside the female’s gastric cavity [17, 18]. However, in anthozoans, the polyp colonies may be single sex [19] or both male and female [20]. In general, the asexual life cycle of medusozoans includes a fertilized egg, which forms tiny pelagic planula larva that settles down to the sea floor and form a sessile polyp. These polyps further develop a hydroid polyp colony, which liberates medusae by budding from the trunk [18]. Amongst the medusozoans, hydrozoans have the greatest variation in life cycle. For example, species in the Campanulariidae family lack the medusa stage [21] and the members of the order Trachymedusae never form polyps [16]. The asexual life cycle of anthozoans is straightforward including four main stages: the fertilized egg, planula larvae, polyp and sessile sea anemone [16, 22]. 3

1.4 Cnidarian Venom Delivery System Cnidae are the defining subcellular specialisation of the phylum Cnidaria. They are specialized cellular structures capable of explosive discharge upon activation of cnidocytes (Figure 1-1). Cnidae contain elaborate structural elements and complex mixtures of bioactive compounds or venom for entrapping, subduing and digesting prey as well as deterring and repelling predators and competitors [23]. Cnidae are distributed in various parts of the cnidarian body and are classified into three major types: penetrant nematocysts, the volvent spirocyst and the glutinant ptychocysts. Cnidae comprise an eversible hollow tubule that is coiled and sometime spine laden. These phylum- specific organelles are synthesized in specialised precursor cells called cnidoblasts. Following their secretion from the Golgi apparatus, the cnidae undergo further structural modifications in the extracellular matrix before migrating to the tentacle surface [24-26]. Penetrant nematocysts inject venom into the target organism and are the most studied class of cnidae. Penetrant nematocysts are found in all cnidarians and are morphologically and functionally the most diverse group of cnidae [27]. They are the primary weapon for capturing prey, repelling predators, and intra- and interspecies spatial competition [28]. Although nematocysts are mostly located on the tentacles, they also exist on the outer surface of the bell in certain species of the Alatinidae, and Carybdeidae families [29], oral arms of Catostylus mosaicus (Schyphozoa) [30] and the stomach (gastric cirri) of some cubozoans [29] probably helping to paralyse and digest the prey. Members of family Actiniidae also have nematocysts in the ring around the base of their tentacles, called acrorhagi, which they use for intra- and interspecific competition [31] and at filaments called acontia that are used for defence or paralysis of prey. Spirocysts are present in most anthozoans and may differ morphologically among different taxonomic groups [32]. The spirocyst capsule wall is thin and the everted tubule is helically folded. The tubule lacks spines, but contains adhering, hydroscopic substances that mechanically immobilize the prey [33]. Ptychocysts have been reported in tube-dwelling members of Actinaria and Ceriantharia. They create the protective tube in which species from these families live [33].

Figure 1-1. Overview of the cnidarian venom delivery system. (A) Schematic picture of a jellyfish; (B) Transverse section of a tentacle showing two epithelial cell layers, ectoderm (ec) and endoderm (en), divided by the mesoglea (m), various types of cnidocytes placed in nematocyst batteries (nb) and in the cells lining the tentacular lumen (tl); (C) Undischarged nematocyst. The nematocyst capsule contains a cocktail of toxins, shaft (sh), coiled-hollow tubule (t), barbs (b), and nucleus (n). The cnidocil (cn) acts as a mechanoreceptor that upon activation stimulates the discharge mechanism; (D) Discharged nematocyst. Epidermis (ep), dermis (d), subcutaneous tissue (sct), artery (a), nerve (n). Toxin mixture is injected into the prey’s skin and subcutaneous tissues (arrow). The figure was drawn according to the data in references [14, 29, 34, 35].

The mechanism of cnidae discharge in response to external stimuli (mechanical and chemical stimuli) is still not completely understood. The general osmotic/tension hypothesis proposes that the osmotic pressure of the intracapsular fluid temporary increases as a result of cnidae exposure to the external solution and subsequent exocytosis of cations from the capsule. The osmotic pressure differences across the capsule wall extant to the point that the pre-existing intracapsular pressure exceeds a critical threshold value and triggers the cnidae discharge [36-39]. The capsule content (venom) is located on the inner surface of the inverted tubule which, during discharge (tubule eversion-extension), everts such that the outside is now exposed and injected into the prey. The inside of the tubule remains contiguous with the inside of the capsule and the cnidae content or venom is then expelled down the axis of the hollow tubule [40].

5 The morphological properties of cnidae are taxonomically informative and may aid in distinguishing more distantly related species [33]. For instance, the presence of spirocyst is characteristic of some anthozoans [22]. In addition, the relative abundance of different types of nematocyst, along with variation in the body size in several populations may correlate to environmental factors, such as specific ecosystems, prey size and selectivity in prey capture. For example, C. fleckeri has a relatively large body and a higher ratio of mastigophores (the longest of the penetrant cnidae) correlating to the large organisms which they prey upon [41]. In addition to cnidae, venom of Cnidarians was shown to be also synthesized in other cell types such as ectodermal and endodermal gland cells and other, still unknown, cell types [42, 43]. Table 1-1. Cnidarian venom composition.

Toxin Type Found in Class Function MW (kDa) Reference Enzymes Anthozoa, Cubozoa, Phospholipase A Cytolytic, hemolytic, prey 2 Schyphozoa, 13-45 [44-46] digestion Hydrozoa

Schyphozoa, Cytotoxic, cytolytic, [12, 14, 47, Metalloproteases 17-130 Cubozoa, Anthozoa local tissue damage 48]

Pore forming toxins (cytolysins) Actinoporins and Cytolytic, hemolytic, actinoporin-like Anthozoa, Hydrozoa cardiovascular and respiratory 20 [49-54] proteins arrest Hemolytic, cardiotoxic, Jellyfish Toxins Cubozoa cytolytic, myotoxic, 42-46 [13, 55-57] cutaneous inflammation Hydralysins - Hydrozoa, Anthozoa Cytolytic, prey digestion 27-31 [43, 58-60] related toxins Membrane Attack Anthozoa Cytolytic, hemolytic 60 [61, 62] Complex-Perforin

Neurotoxins Neurotoxic, cardiotoxic, NaTxs (type I-III) Anthozoa 3-8 [63-67] insecticide Neurotoxic, hypotensive,

cardiotoxic, analgesic, KTxs (type I,III Anthozoa antimicrobial, 3-4 [68-75] and IV,V KTxs) immunosuppressive,

anti obesity Kunitz peptides Anthozoa Paralytic, serine protease 6 [76-79] (type II KTxs) inhibitor Small Cysteine- Rich Proteins (SCRiPs) and Anthozoa Paralytic 4.3-5.8 [80] SCRiPs homologues ASIC Inhibitors Anthozoa Analgesic 3 [81, 82] 6 TRPV1 Inhibitors Anthozoa Analgesic 3 [83, 84] Non-protein bioactive components Serotonin Hydrozoa, Anthozoa Vasodilation, sharp pain - [85] Histamine Anthozoa Vasodilation, sharp pain - [86, 87] Bunodosine Anthozoa Analgesic - [88] Caissarone Anthozoa Adenosine receptor antagonist - [89]

1.5 Venom Composition Since the early 20th century, numerous analytical experiments and clinical observations have established the toxicological diversity of cnidarian venoms (Table 1-1). The diversity of venom components range from non-proteinaceous compounds (e.g. purines, biogenic amines) to high molecular weight proteins evolved over the course of hundred million years. Interestingly, some toxin family types identified previously in other venomous animals comprise the venom arsenal of cnidarians. The most striking example is Kunitz peptides which are expressed in sea anemones, cone snails, insects (proboscises and stingers), scorpions, spiders, snakes, ticks and vampire bat (Desmodus rotundus) [90, 91]. Another example of convergent expression of toxins is potassium channel blocker Kv1 which not only evolved convergently in scorpions and sea anemones, but they even use the same residues for blocking the channel pores [92].

1.5.1 Enzymes

1.5.1.1 Phospholipase A2

Phospholipase A2 (PLA2) activity has been detected in the homogenized tentacles and acontia of cnidarians including subphylums Anthozoa, Schyphozoa, Hydrozoa and Cubozoa [44, 46, 93]. This enzyme hydrolyzes the sn-2 acyl bond of glycerophospholipids to produce fatty acids including arachidonic acid and lysophospholipid [22, 44]. It is commonly found in mammalian tissues where it plays important roles in vital body functions including dietary lipid catabolism, inflammation, signal transduction, and phospholipid remodelling [94]. However, the ubiquitous presence of PLA2 across venomous animal lineages such as reptiles (snakes and anguimorph lizard), centipedes, insects (their bristles, proboscises, and stingers), arachnids (scorpions, spiders, and ticks), cnidarians, and cephalopods [44, 90, 95-98] may suggests the convergent recruitment of the body proteins into the toxic arsenal of animals. However, more study is needed to be conducted in order to specifically describe the evolution of PLA2s in venomous and non venomous clades. Toxic functions of PLA2 in cnidarian venoms have been proposed to include defence and immobilization and digestion of prey [44, 93]. Also, hemolytic activity was demonstrated in a PLA2 fraction recovered from acontial nematocysts of the sea anemone Aiptasia pallida [93]. Although PLA2 enzymes are plentiful amongst cnidarians [44, 98], their molecular structure-function relationships remain to be elucidated.

7 1.5.1.2 Metalloproteases Metalloproteases are important venom components of terrestrial animals such as centipedes, snakes, and ticks [90, 99]. They induce haemorrhage and necrosis by degrading the extracellular matrix and preventing blood clot formation [90, 100]. These functions are commonly associated with several of the recurrent symptoms of sting such as skin damage, oedema, blister formation, myonecrosis and inflammation [100]. Metalloproteases were detected in the venom of jellyfish Stomolophus meleagris [12] and C. fleckeri [14]. A recent study focused on the enzymatic and cytotoxic functions of jellyfish metalloproteases and identified diverse proteolytic effects including gelatinolytic, caseinolytic, and fibrinolytic activities [47]. Zinc-dependent metalloproteases of the astacin family were detected in the soluble nematocyst content of the sea anemone Nematostella vectensis and were shown to be expressed in both gland cells and stinging cells. They originate from recruitment of the Bone Morphogenic Protein 1 (BMP1, also known as Tolloid) protein that plays a conserved role in animal development [48].

1.5.2 Pore-Forming Toxins Pore forming toxins (PFTs) appear to be present in all cnidarian venoms. The mechanism of action of these toxins is penetration through the target cell membrane resulting in diffusion of small molecules and solutes leading to osmotic imbalance and cell lysis. PFTs exhibit dual structure i) a stable water-soluble structure that is monomeric and binds to the receptors on the target cell; (ii) membrane-bound structure consisting of oligomeric molecules that form integral membrane pores [101]. Cnidarian PFTs are classified in two groups based on the type of secondary structure they use to penetrate membrane upon pore-forming activity: α-PFTs which are a rich content of helices and form α-helical barrel structures, and β-PFTs that are rich in β-sheets and form β-barrel pores [101].

1.5.2.1 Actinoporins Actinporins are α-PFTs and are present in Anthozoa and Hydrozoa. They are basic proteins approximately 20 kDa in molecular size lacking in intramolecular disulfide bonds. Actinoporins mediate various types of toxicity and bioactivity, such as cardiovascular and respiratory arrest in rats [50], lysis of chicken, goat, human and sheep erythrocytes [51, 53, 54] and cytotoxic effects [52], all caused by a pore-forming mechanism. Several studies have reported that actinoporins interact exclusively with sphingomyelin containing membranes [102-104], although sticholysin II from Stichodactyla helianthus binds to phosphatidylcholine membrane [105]. The mechanism of membrane penetration requires several steps: i) initial binding to the target membrane which is accomplished by an exposed aromatic-rich loop; (ii) lateral orientation and consequent oligomerization by a phosphocholine binding site; and (iii) insertion of the N-terminal amphiphilic α-helix segment to the lipid membrane [102, 103, 106]. High-resolution nuclear magnetic resonance 8 and Fourier transform infrared spectroscopy experiments [107], intrinsic fluorescence measurements [108], X-ray crystallography and electron microscopic analyses of two dimensional crystals [109] have suggested that, during the membrane penetration, the N-terminal amphiphilic α- helix segment of actinoporins detach from the main body and insert into the target membrane to produce pores with a diameter of 1–3 nm (∼11-30 Å) [54, 110].

1.5.2.2 Jellyfish Toxins (JFTs) Cubozoan-related porins are the most potent and rapid-acting toxins secreted by jellyfish species. This toxin family was originally reported in the cubozoan Carybdea alata as CAH1 [111], also reported as CaTX-A/B [112], and subsequently identified in all cubozoans examined including CrTX-A/B from Carybdea rastoni [55], CqTX-A from Chiropsalmus quadrigatus [56], CfTX-1/2 and CfTX-A/B/Bt from C. fleckeri [13, 113]. However, homologues of cubozoan porins were reported in Scyphozoa (Aurelia aurita), Hydrozoa (Hydra magnipapillata) [13], Anthozoa (Aiptasia pallida) and various hydroids (Hydractinia symbiolongicarpus and Hydra vulgaris), suggesting a common evolutionary origin of these toxins [80]. Characteristically, they are basic proteins with a molecular weight of 40 to 46 kDa and contain both α and β domains. The hypothetical mechanisms underlying pore formation involves oligomerization of several amphiphilic and hydrophobic α- helices in the N-terminal region of the toxin resulting in distortion of the plasma membrane and cell death [13, 112, 113]. The similarity in the three-dimensional structure of the N-terminal domain of CfTX toxins to that of the α-pore-forming domain (domain I) of the insecticidal δ-endotoxins from Bacillus thuringiensis suggests how toxins of this family insert into membranes. This mechanism of membrane insertion seem to be sufficient to explain the well-defined 12 nm (inner) and 25nm (outer) diameter pores created in human erythrocytes by CfTX toxins [3]. Interestingly, the members of this toxin family exhibit varying target specify towards various vertebrate tissue. A recent study on CfTX toxins showed that CfTX-1/2 caused cardiovascular collapse within 1 min in anesthetized rats exposed in vivo to the venom, whereas CfTX-A/B more potent in eliciting in vitro hemolytic activity [13]. Although these data suggest that the jellyfish toxin family has undergone functional diversification, more structural/functional data are needed to understand the molecular evolutionary histories that formed this diversification.

1.5.2.3 Hydralysins-Related Toxins In addition to porins found in cnidarians, recent studies have described a novel β-PFT family secreted from the digestive endodermal cells of green hydra (Chlorohydra viridissima) [43, 58]. These non-nematocyst, body-derived toxins secreted during feeding are suggested to play a role in lysing prey tissues [59]. These toxins are not active on membrane phospholipids or carbohydrates, but rather bind to specific membrane receptors and form pores [58]. A recent study characterised 9 aerolysin, a hydralysin homologue, secreted from pharyngeal ectodermal cell of N. vectensis and suggested a role in prey disintegration [60].

1.5.2.4 Membrane Attack Complex-Perforin This group of β-PFT toxins have been detected in the venoms extracted from the sea anemones Phyllodiscus semoni and Actinaria villosa. The toxins may be used in prey capture/predator defence [61, 62]. Membrane attack complex (MAC) proteins have been identified in the complement system produced by T-cells and NK cells. The proteins create a transmembrane pore into the target cell and initiate various apoptotic cell death pathways [114].

1.5.3 Neurotoxins Cnidarian neurotoxins (voltage-gated ion channel toxins) are a group of low molecular weight peptides and are among the best characterised toxins in terms of the mechanism of action. They are produced by sea anemones and have a fundamental role in the venom to help these sessile animals to immobilize their prey rapidly and to defend against predators. Neurotoxins prolong the action potential of the excitable and non-excitable membranes in sensory neurons and cardiac and skeletal muscle cells [64, 115] via modifying the sodium channel gating [64, 116, 117] or blocking the potassium channel gating during the repolarisation stage [115]. This causes the cell to become hyperactive and to release massive amounts of neurotransmitter at synapses and neuromuscular junctions that can cause initial spastic stage followed by descending flaccid paralysis. Sea anemone voltage-gated ion channel toxins have been studied extensively because they are valuable bioresources to study the structure and function of sodium and potassium channels and also to be used for development of drugs [73, 74] and bioinsecticides [66, 67]. Several cnidarian neurotoxin polypeptides exclusively block Vanilloid Receptor 1 (TRPV1) and acid-sensing ion channel 3 (ASIC3), which take part in initiation and transduction of pain and hyper-algesia. These peptides are thus promising tools for the development of novel pain reducers.

1.5.3.1 Voltage-gated Sodium (Nav) Channel Toxins Voltage-gated sodium (Nav) channel toxins (NaTxs) are transmembrane complexes composed of several subunits. The highly conserved α-subunit consists of four homologous domains (D1-D4) each containing six hydrophobic transmembrane regions (S1-S6) [117]. Anthozoan NaTxs and several other groups of toxins from scorpion and spiders bind to site 3 (loop S3–S4 in D4) of the

Nav channels [64, 118] resulting in considerable neurotransmitter release in synapses. One possible explanation underlying its toxicity is the electrostatic interaction between a cluster of basic amino acids on the toxin with acidic amino acids at site 3 [119] locking S4 segment in its inward position, thus inhibiting the conformational changes of the channel necessary for fast inactivation [117].

10 These toxins are divided into three groups: (i) type I NaTx; and (ii) type II NaTxs exhibit extensive sequence similarity and share similar function. In addition to type I and II NaTxs there is an orphan NaTx clade, that exhibits only a partial homology to both type I and type II NaTxs and shows similar mechanism of action to those [120, 121]. Lastly, there are short (~30 amino acids long) peptides in sea anemone venom that exhibit a similar activity to the rest of NaTxs families despite lacking any shared sequence motifs with the rest of them [118]. These peptides form the type III family and exhibit very high selectivity towards arthropod sodium channels [122].

1.5.3.2 Voltage-gated Potassium (Kv) Channel Toxins and Kunitz peptides Sea anemone voltage-gated potassium (Kv) channel toxins (KTxs) are categorized in five groups based on their sequence similarity and binding affinity towards different Kv channel families [72]. Type I KTxs have a molecular weight of 4 kDa with three disulfide bridges. They inhibit the potassium current through channel Kv1. and Kv3. subfamilies and intermediate conductance calcium-activated potassium channels [22]. Type II KTxs are Kunitz peptides with a molecular weight of 6 kDa that are cross-linked with three disulfide bridges and are remarkable in their dual function of activity. Usually they act against trypsin and chymotrypsin proteinases in order to inhibit the rapid degradation of the venom protease by endogenous enzymes of the animals themselves or of the prey [31, 78, 79]. Interestingly, several sea anemone Kunitz peptides possess both Kv blocking activity similar to dendrotoxin and protease inhibiting activity [76, 123]. For example, kalicludin 1-3 from A. sulcata binds competitively to Kv1.2 channels to paralyse the prey rapidly. Type III KTxs are 3-4 kDa peptides with three disulfide bridges that have evolved from NaTxs under the regime of positive selection [80]. They block a variety of distinct potassium ion channels such as Kv3. 4 channel belonging to the rapidly inactivating Kv channel [68] and hERG (the human Ether-à-go-go-Related Gene) [69, 75]. Interestingly, the influence of positive selection does not result in the complete loss of sodium ion channel blocking activity, where several type III KTxs target both hERG and acid-sensing ion channel 3 (ASIC3, H+-gated Nav channels) [75]. Type IV are structurally new peptides from the sea anemone Stichodactyla haddoni displaying crab paralysis activity and are cross-linked with two disulfide bridges [124]. Type V has been found in the sea anemone Bunodosoma caissarum and is cross-linked by four disulfide bridges. This novel peptide is active on Drosophila Shaker IR channels [72].

1.5.3.3 Small Cysteine-Rich Peptides (SCRiPs) This newly discovered group of neurotoxins have been reported in the ectoderm of reef-building corals Acropora millepora and SCRiP homologs have been retrieved in sea anemones, Anemonia viridis and Metridium senile [80]. The injection of recombinantly expressed A. millepora SCRiPs in

11 zebrafish (Danio rerio) larvae resulted in severe paralysis, suggesting the first peptide neurotoxin family described from scleractinian corals [80].

1.5.3.4 ASIC Inhibitors ASICs are sodium-selective acid-sensing ion channels (ASIC) expressed in peripheral neuronal system. They have been associated with acidic pain during pathological conditions such as inflammation and ischemia. Recently, a novel peptide π-AnmTX Ugr 9a-1 from the venom of the sea anemone Urticina grebelnyi [81] and PhcrTx1 from Phymanthus crucifer [82] have been reported to target ASIC channels. These peptides are cross-linked by two disulfide bridges and have no sequence homology to other sea anemone neurotoxin peptides.

1.5.3.5 TRPV1 Inhibitors TRPV1s are non-selective cation channels expressed in mammalians peripheral and central neuronal systems. They initiate neuronal response during inflammation stimuli, which allow them to be regarded as one of the most important molecular triggers of pain stimuli. The first peptidic TRPV1 inhibitor isolated from sea anemone venom was τ-SHTX-Hcr2b (APHC1) from H. crispa [83]. Subsequently, two homologous peptides (τ-SHTX-Hcr2c (APHC2) and τ-SHTX-Hcr2d (APHC3) that target TRPV1 were isolated from the same species [84]. These novel neurotoxins are promising new models for designing a new generation of analgesic drugs.

1.5.4 Non-Protein Bioactive Components In addition to the protein and peptide compounds mentioned above, a number of pharmacologically active low molecular weight compounds have been detected in cnidarian venoms. Large amounts of 5-hydroxytryptamine (5-HT, serotonin) have been reported in the water where hydra was stimulated electrically [85]. This presumably nematocyst-derived substance causes instant pain in predators and thus may have a defensive role [85]. Also the vasodilatation enhanced by serotonin may potentiate the effect of other venom components [85]. Histamine has been detected in the homogenised tentacles of the sea anemones Anemonia viridis (previously known as Anemonia sulcata) and Actinia equina [86, 87] . Like serotonin, histamine produces sharp pain and increases vascular permeability. Although these compounds have not been directly detected in isolated nematocysts, it is likely that they are involved in cnidarian envenomation, and treatment of sting victims with antihistamines has been recommended to relieve the symptoms [125]. Bunodosine, an N-acylamino acid, was purified from the venom of the sea anemone Bunodosoma cangicum. This compound exhibits a potent analgesic activity by activation of serotonin receptors [88]. Caissarone is a quaternary purine derivative isolated from the sea anemone Bunodosoma caissarum by

12 extracting the whole animal in acetone. This marine product has a high antagonist activity towards guinea-pig ileum adenosine receptors which stimulate gut function [89].

1.6 The Role of Cnidarians Venoms in Drug Discovery Toxic compounds isolated from Cnidaria have been viewed to produce several serious implications to human health due to their neurotoxicity, cytotoxicity and tissue damage. However, novel findings have demonstrated that their toxins might offer a tool to study cell physiology [126] and provide promising sources of pharmacological lead/active agents for therapy of human diseases. Palytoxin is a highly potent non-protein toxin isolated from order Zoantharia (soft corals) of the genus Palythoa and Zoanthus and the sea anemone Radianthus macrodactylus [127]. The interaction of palytoxin with Na+, K+-ATPase pump in almost every excitable tissue induces passive ion conductance which triggers K efflux, Na influx and massive membrane depolarization and tissue contraction [127, 128]. Palytoxin has been reported for anti-cancer activity against head and neck carcinoma cells [129], Ehrlich ascites tumour and P-388 lymphocytic leukaemia cells [130]. The mechanism of action of its cytotoxicity and tumour suppressor activity has been established by actin filament distortion and apoptosis [131]. Conversely, palytoxin has been identified as a tumour promoter by disrupting the regulation of cellular signalling cascades [132]. Over the past decade, several cytolysins and protease inhibitors have been extracted from the sea anemone A. equina. Equinatoxin II (EqT II) is a pore-forming protein that has been shown to have significant toxicity against Ehrlich ascites tumour and L1210 leukaemia cell lines [133] and diploid lung fibroblasts of the Chinese hamster [134]. Equistatin is a potent inhibitor of papain-like cysteine proteinase and aspartic proteinase cathepsin D [135]. Overexpression and hypersecretion of cathepsin-D has been reported in breast carcinoma cells [136], and papain-like cysteine proteases are involved in diseases of the central nervous system [137]. Recently antibutyrylcholinestrasic activity was detected in the crude venom extracted from the tentacle material of the Mediterranean jellyfish Pelagia noctiluca. Inhibition of butylcholinestrase in the central nervous system may prove useful in the treatment of neurodegenerative diseases such as Alzheimer’s disease and senile dementia [138]. ShK is a potent Kv1.3 channel blocker toxin that isolated from the sea anemone S. helianthus. Since this channel is crucial in the activation (proliferation and cytokine production) of human effector memory Tcells (TEM), ShK could provide a valuable immunosuppressant for the treatment of autoimmune diseases mediated by T cells [73]. Kv1.3 blockers are also considered as a therapeutic target for the treatment of obesity, thus highlighting the potential use of ShK in treatment of obesity and insulin resistance [74].

13 1.7 Envenomation Fortunately, due to the biophysical properties of the discharge event many cnidarians lack the capacity to penetrate human skin. Further, stings by most species with cnidae capable of perforating human tissue lead only to a negligible to moderate transient irritation/burning sensation. In contrast, contact with several jellyfish, sea anemone and coral species can cause severe pain, tissue damage and even cardiovascular collapse and death. The venom of the hazardous animals is introduced into the target tissue (epidermis, dermis, vasculature, lymphatic system and probably in some cases subcutaneous or muscular structures) (Figure 1-1) [125] and initiates immediate and delayed immunological and toxicological responses [139-141]. Envenomation symptoms are determined by the contents of the venom, the volume of the injection, the health of the patient and the duration of the tentacle-skin contact [125]. One of the most important steps in the treatment of human envenomation is to use specific fluids to prevent further nematocyst discharge, since physical attempts to detach remaining tentacles from the victim’s skin might cause massive discharge [34, 142]. Since 1908 numerous traditional remedies such as urea, seawater, vinegar, methylated spirits etc. have been used to inactivate the undischarged nematocysts on the adhered tentacles and/or to alleviate pain [14, 34, 143, 144]. These chemicals have been used to varying degrees of success, ranging from complete success in certain species to complete failure in others. For instance, while certain chemicals such as ethanol, or 5% acetic acid in distilled water, cause massive nematocyst discharge in Hydrozoa and Cubozoa species [14, 143, 144], some other such as a food grade vinegar was found to inactivate the penetrating nematocysts of C. fleckeri rapidly and completely [34]. Avoidance and treatment of human envenomation by cnidarian species is an increasingly important objective within the broader scientific community. It also represents a collaborative aim of medical, pharmacological, toxicological and biological disciplines towards developing improved understanding of field ecologies as well as effective therapeutics to minimize pathogenic impact on bathers. It also can improve our understanding of the food web and biome dynamics of these ancient and intriguing animals.

1.8 Conclusions Members of Cnidaria phylum are ancestral venomous Eumetazoan with a unique phylogeny. In spite of recent efforts to extract and characterise novel toxins from cnidarians, much more remains to be done to investigate their toxins and their potential as new sources of therapeutic substances. Of the approximately 10,000 cnidarian species in existence, only 156 toxins from 10 pharmacological families have been defined. Potentially useful new toxicological information is gradually accumulating. One promising solution towards broadening the knowledge on cnidarian

14 toxins is to apply high throughput sequencing technologies to identify novel structural and pharmacological groups of toxins [145, 146]. Nevertheless, the enormous potential of cnidarian venoms for understanding the activity of different receptors and channels in health and disease conditions, as well as their use as sources of novel pharmaceutical lead compounds appears to offer endless possibilities for future scientific research.

Aims The ultimate aim of this thesis is to provide the first comprehensive insight into the evolution, diversification and biodiscovery potentials of the oldest venomous animal lineage, Cnidaria. Here, a combination of approaches was applied to achieve the following goals: 1) Use state-of-the-art molecular evolutionary assessments to explore the evolutionary origin and diversification of venom in this ancient clade of animals. 2) Develop a novel technique to extract venom from cnidarians to facilitate venom research. 3) Determine the venom profiles of representative species from three classes of cnidarians, including Cubozoa, Scyphozoa and Anthozoa. This was done by using combinatorial transcriptome/proteome approach and activity-guided fractionation. 4) Characterise novel toxins with interesting pharmacological activities to demonstrate under- researched biodiversity potentials of cnidarian venoms.

Together, the outcomes of this thesis contribute greatly to our understating of the evolutionary forces that shape cnidarian venoms as well as provide a roadmap for the exploration of novel drugs.

Chapter 2

Evolution of an ancient venom: Recognition of a Novel Family of Cnidarian Toxins and the Common Evolutionary Origin of Sodium and Potassium Neurotoxins in Sea Anemone

Manuscript published in MBE, March 2015 http://mbe.oxfordjournals.org/content/32/6/1598.full

16 Abstract Despite Cnidaria (sea anemones, corals, jellyfish, and hydroids) being the oldest venomous animal lineage, structure–function relationships, phyletic distributions, and the molecular evolutionary regimes of toxins encoded by these intriguing animals are poorly understood. Hence, we have comprehensively elucidated the phylogenetic and molecular evolutionary histories of pharmacologically characterised cnidarian toxin families, including peptide neurotoxins (voltage- gated Na+ and K+ channel-targeting toxins: NaTxs and KTxs, respectively), pore-forming toxins (actinoporins, aerolysin-related toxins, and jellyfish toxins), and the newly discovered small cysteine-rich peptides (SCRiPs). We show that despite long evolutionary histories, most cnidarian toxins remain conserved under the strong influence of negative selection—a finding that is in striking contrast to the rapid evolution of toxin families in evolutionarily younger lineages, such as cone snails and advanced snakes. In contrast to the previous suggestions that implicated SCRiPs in the biomineralization process in corals, we demonstrate that they are potent neurotoxins that are likely involved in the envenoming function, and thus represent the first family of neurotoxins from corals. We also demonstrate the common evolutionary origin of type III KTxs and NaTxs in sea anemones. We show that type III KTxs have evolved from NaTxs under the regime of positive selection, and likely represent a unique evolutionary innovation of the Actinioidea lineage. We report a correlation between the accumulation of episodically adaptive sites and the emergence of novel pharmacological activities in this rapidly evolving neurotoxic clade.

Key words Cnidaria, sodium channel toxins, potassium channel toxins, phylogeny, disulfide-rich toxins, positive selection.

2.1 Introduction Venoms are among nature’s most complex cocktails that are characterised by a diversity of molecules, such as large proteins, small peptides, polyamines, and salts, which disrupt the physiology of prey animals upon injection [147]. Several gene families encoding venom components have been subjected to extensive duplication and have evolved under the influence of positive (diversifying) selection [148, 149]. The evolution of venom has been extensively studied in several venomous lineages that are of relatively younger evolutionary origin, such as advanced snakes (originated ~54 Ma) [150] and cone snails (originated ~33–50 Ma) [151, 152]. However, to date, the evolution and diversification of ancient venom systems, such as those of cnidarians, remains understudied. The phylum Cnidaria (sea anemones, corals, jellyfish, and hydroids) is composed of diploblastic predatory animals [153], where all members are venomous [154]. Cnidarians are typified by the 17 unique venom delivery apparatus called nematocyst (stinging organelle), which they employ to inject a cocktail of toxins into animals for predation and defence [27, 153, 155]. Molecular and fossil data place the origin of cnidarians in the Ediacaran Period, ~600 Ma [10, 156, 157], making them the oldest lineage of venomous animals and an ideal phylum to understand the origin and diversification of venom. Because certain cnidarians serve pivotal ecological roles (e.g., reef- building corals) and are important model organisms in the field of evolutionary developmental biology (sea anemones and hydroids), genomic and transcriptomic data for several cnidarian species are rapidly accumulating in recent times [158-161]. Moreover, certain cnidarian animals, such as box jellyfishes (Cubozoa), produce some of the world’s most lethal venoms [140]. In this study, we have assessed the phylogenetic histories and the molecular evolutionary regimes of a diverse range of cnidarian toxin families, including 1) sodium channel modulators (NaTx: type I and type II families) [118, 162] potassium channel toxins (KTx: type I and type III families) [115]; 3) aerolysin-related pore-forming toxins (PFTs) [58, 60]; 4) actinoporin PFTs [163]; 5) the jellyfish toxins (JFTs) [13]; and 6) the newly discovered family of small cysteine-rich peptides (SCRiPs), which have been previously implicated in biomineralization in corals [164]. Our results unravel fascinating insights into the evolutionary origin and diversification of venom in this ancient clade of animals.

2.2 Results Cnidarians secrete a diversity of toxin types to facilitate predation and defence. Here, we present the phylogenetic histories and the molecular evolutionary regimes of eight pharmacologically characterised cnidarian toxin types.

2.2.1 Neurotoxins Neurotoxins rapidly immobilize prey animals and play an extremely important role in the venoms of certain cnidarians. They disrupt ion conductance through the modification or blocking of the voltage-gated sodium (Nav) and potassium (Kv) ion channels: NaTx and KTx, respectively. Among cnidarians, neurotoxins have only been retrieved from the venoms of sea anemones (class: Actiniaria). NaTxs are one of the best characterised cnidarian toxins [118, 162, 165] and are classified into three types (type I–III) based on the cysteine arrangement, overall structure, amino acid composition, and immunological cross reactivity [64, 118, 122]. The distinction between type I and type II NaTxs has been artificial, considering the similarity they share in structure and function. However, whether they resolve into distinct phylogenetic clades remains to be investigated. Hence, we have analysed type I and type II NaTxs together. Based on sequence identity, sequence length, number of disulfide bridges, and binding affinities toward various Kv ion channels [115, 124, 166], sea anemone KTxs have been classified into five types: type I–V [72]. Because type II KTxs cannot 18 be distinguished from their physiological homologs (nontoxic body proteins) in the absence of biochemical characterisation [31]), and because not enough nucleotides of type IV and V KTxs have been sequenced to date, we have only analysed KTxs of type I and type III families. It should be noted that the five KTx types are not homologous [72]. We investigated the nature of natural selection influencing the evolution of genes encoding various cnidarian toxin families using maximum-likelihood models [167] implemented in PAML (see Methods). The site model 8 (M8) computed omega () of 0.58 and 0.66 for NaTxs and type I KTxs, respectively, indicating a strong influence of negative selection on these toxin types, while highlighting the influence of positive selection on type III KTxs ( = 1.33) (Figure 2-1 and Table 2-1).

Figure 2-1. Molecular evolution of cnidarian toxins. Three-dimensional models of various cnidarian toxins with their molecular surface coloured according to the evolutionary conservation of amino acids are presented here (see colour code). Positively selected/episodically adaptive sites are depicted in red. Omega values, the number of sites detected as positively selected (Model 8, PP ≥ 0.95, Bayes-Empirical Bayes approach) and episodically adaptive sites (MEME) are also provided for the respective toxin class. PDB codes: Actinoporin: 1IAZ; sea anemone aerolysin: 3C0N; Hydra aerolysin: 2ZTB; NaTx: 2SH1; KTx type I: 1ROO; and KTx type III: 1BDS.

19 Table 2-1. Molecular evolution of cnidarian neurotoxins. FUBARa MEME Sitesb PAMLc (M8)

Type I and Type II NaTx ω > 1d : 0 0 8 ω < 1e : 25 0.58 1 ω > 1d : 1 Type I KTx 5 (0+1) ω < 1e: 16 0.66 ω > 1d : 3 6 Type III KTx ω < 1e : 15 5 (2+4)

1.33 NOTE.  = mean dN/dS. a Fast Unconstrained Bayesian AppRoximation. b Sites detected as experiencing episodic diversifying selection (0.05 significance) by the MixedEffects Model Evolution (MEME). c Positively selected sites detected by the Bayes Empirical Bayes approach implemented inM8. Sites detected at 0.99 and 0.95 significance are indicated in the parenthesis. d Number of sites under pervasive diversifying selection at the posterior probability ≥ 0.9 (FUBAR). e Number of sites under pervasive purifying selection at the posterior probability ≥ 0.9 (FUBAR).

The Bayes Empirical Bayes (BEB) approach implemented in M8 failed to identify positively selected sites in NaTx. However, M8 identified one and six positively selected sites in type I and type III KTxs, respectively (Figures 2-1 and 2-2, Table 2-1, and Supplementary Tables S1.1–S1.3, Supplementary Material online).

Figure 2-2. Sequence alignment of NaTx, KTx type I, and KTx type III. Positively selected/episodically adaptive (PS/Epi), structurally/functionally important, and extremely well-conserved (percent ID ≥ 90%) sites have been depicted. UniProt accession numbers and pharmacological names of sequences are as follows. NaTx: 1) P01528 Anemonia viridis (Av2); 2) P01532 Anthopleura elegantissima (ApC); 3) Q76CA3 Stichodactyla gigantean (Gigt II); 4) P86459 Bunodosoma cangicum (Bcg1a); 5) B1NWS1 Nematostella vectensis (Nv1); and 6) P30785 Heteractis crispa (RTX-V). KTx type I: 1) Q0EAE5 Anemonia erythraea (Aer1a); 2) O16846 Heteractis magnifica (Hm1a); 3) E2S064 Cryptodendrum adhaesivum (Sm1a); 4) P29187 Stichodactyla helianthus (She1a); 5) P29186 Bunodosoma granuliferum (BcsTx1); and 6) Q9TWG1 A. viridis (AsKS). KTx type III: 1) P61541 A. elegantissima (APETx1); 2) P61542 A. elegantissima (APETx2); 3) B3EWF9 A. elegantissima (APETx3); 4) P11494 A. viridis (BDS-1); 5) P59084 A. viridis (BDS-2); 6) P69930 Antheopsis maculate (Am-2); and 7) P84919 B. caissarum (BcIV).

21 The mixed effects model of evolution (MEME) identified as many as eight sites evolving under the influence of episodic diversifying selection in NaTxs, while identifying three and five sites in type I and type III KTxs, respectively (Figure 2-2 and Table 2-1). These results indicate that certain regions in these neurotoxins have evolved rapidly under the episodic influence of adaptive selection. To understand the surface accessibility of positively selected and episodically diversifying sites, we computed the accessible surface area (ASA) ratio for all sites in the homology models of these neurotoxins. Our analyses revealed that all four episodically diversifying sites, with an ASA ratio in the range of 60–100 (ASA ≥ 50 is characteristic of surface-exposed sites) in the secreted region of NaTxs, were surface exposed (Supplementary Table S2, Supplementary Material online). Six of the 8 positively selected and episodically diversifying sites in the mature sequence of type III KTxs had an ASA ratio in the range of 51–100, while only a single site was found to be buried (ASA ≤ 20 indicates buried residues). The remaining site (ASA = 35) in this toxin type could not be assigned to buried/exposed category. Similarly, the single positively selected site in KTx type I was surface exposed (ASA = 100), while the lone diversifying site in the mature region of this toxin had its side chain exposed (ASA = 47; ASA value ranging between 40 and 50 is indicative of residues with surface accessible side chains). Several previously published KTx type III toxins were known to have a similar cysteine framework and activity as that of the sea anemone NaTxs [70, 168-170]. Hence, we analysed them together in this study to explore the possibility of a common evolutionary origin of the two toxin types. Midpoint rooted nucleotide Bayesian and maximum-likelihood phylogenetic trees placed type III KTxs in the same clade as type I NaTxs (Bayesian posterior probability [PP] 0.997; bootstrap support 734/1,000), suggesting a common evolutionary origin of these two toxin types (Figure 2-3 and Supplementary Figure S1, Supplementary Material online). Our phylogenetic analyses further resolved NaTxs into four distinct clades: 1) Type I NaTx (polyphyletic clades); 2) type II NaTxs; 3) the orphan NaTx clade (a unique clade of NaTxs that does not belong to either type I or type II NaTxs); and 4) a clade of Nv1 toxins from Nematostella vectensis (Figure 2-3).

Figure 2-3. The common origin of NaTx and KTx type III. The phylogenetic tree of NaTx and KTx type III built using nucleotide sequences is presented here. Node support (Bayesian posterior probability/bootstrap replicate support) for the major clades and clade-specific omega values are also provided.

Rooting the tree with toxin sequences from N. vectensis or Halcurias carlgreni, two basal sea anemone lineages (for sea anemone species phylogeny [171], see Supplementary Figure S2, Supplementary Material online), did not change the topology of the tree and supported the common origin of NaTx and KTx type III (supplementary Figure S1, Supplementary Material online). Because neurotoxins from N. vectensis (Nv1) and H. carlgreni (Halcurin) are characterised by domains similar to both type I and type II NaTxs, they have been previously suspected to be among the most basal sea anemone neurotoxins [172, 173]. Because phylogenetic analyses in this study resolved type I and type II NaTxs into distinct clades, we computed omega values for each of these clades independently. These analyses suggested that both type I and type II NaTxs have evolved under the influence of negative selection (Supplementary Tables S3.1 and S3.2, Supplementary Material online).

23 2.2.2 Pore-Forming Toxins 2.2.2.1 Actinoporins Actinoporins are arguably the best studied PFTs secreted by Cnidaria [163]. They typically interact with their target sphingomyelin membrane (SM) to form oligomeric pores, which result in osmotic imbalance and cell death [102]. It was previously shown that the presence of the aromatic cluster, an array of basic residues, the phosphocholine (POC) binding site, and the N-terminal region containing an amphipathic α-helix are essential for the recognition and binding to the SM membrane and the cytolytic activity of actinoporins [102, 103, 108]. Mapping the results of evolutionary selection analyses on the sequence alignment of actinoporins revealed the greater influence of negative selection on regions responsible for this cytolytic activity. Most sites implicated in pore-forming activity and the structural stability were found to have evolved under the influence of negative selection (Figure 2-4).

Figure 2-4. Sequence alignment of actinoporins. Episodically adaptive, structurally/functionally important, and extremely well-conserved (percent ID ≥ 90%) sites have been depicted. UniProt accession numbers and pharmacological names of sequences are as follows: 1) P61914 Actinia equina (EqTII); 2) C5NSL2 Anthopleura asiatica (bp-1); 3) P61915 Actinia tenebrosa (Tenebrosin-C); 4) C9EIC7 Urticina crassicornis (Urticinatoxin); 5) Q9U6X1 Heteractis magnifica (HMg III); and 6) P07845 Stichodactyla helianthus (StnII).

The Fast, Unconstrained Bayesian AppRoximation (FUBAR) analysis, which detects sites evolving via pervasive diversifying and purifying selection, identified 7/9 POC binding sites, 4/7 sites in the array of basic residues, 6/8 sites in the array of aromatic residues, and 3/22 residues of α-helix as constrained by pervasive negative selection pressure (PP ≥ 0.90) (Table 2-2). M8 computed  of 0.39 for actinoporins, indicating a strong influence of negative selection on this toxin type, while the BEB approach implemented in this model failed to identify positively selected sites (Table 2-2). However, the MEME identified as many as 25 sites in actinoporins as evolving under the influence

24 of episodic diversifying selection (Figures 2-1 and 2-4, Table 2-2, and Supplementary Table S4.1, Supplementary Material online). Table 2-2. Molecular evolution of cnidarian pore-forming toxins and SCRiPs. FUBARa MEME Sitesb PAMLc (M8)

Actinoporins ω > 1d : 0 o 25 ω < 1e : 121 0.39 Sea Anemone Aerolysins ω > 1d : 0 0 41 ω < 1e: 300 0.27 Hydralysins ω > 1d : 0 0 9 ω < 1e : 120 0.21

ω > 1d : 0 0 JFTs 17 ω < 1e : 287 0.39 ω > 1d : 0 0 Coral SCRiPs 7 ω < 1e : 18 0.72 NOTE.  = mean dN/dS. a Fast Unconstrained Bayesian AppRoximation. b Sites detected as experiencing episodic diversifying selection (0.05 significance) by the MixedEffects Model Evolution (MEME). c Positively selected sites detected by the Bayes Empirical Bayes approach implemented in M8. Sites detected at 0.99 and 0.95 significance are indicated in the parenthesis. d Number of sites under pervasive diversifying selection at the posterior probability ≥ 0.9 (FUBAR). e Number of sites under pervasive purifying selection at the posterior probability ≥ 0.9 (FUBAR).

2.2.3 Aerolysin-Related Toxins in Sea Anemones and Hydroids Aerolysin-related toxins are nonnematocystic paralytic toxins with α-pore-forming activity, secreted from the nonnematocystic digestive cells of various sea anemones and hydra species (hydralysins) [58, 60, 174]. Because hydralysins were found to be secreted by Hydra viridissima during feeding, they have been theorized to play a role in the digestion of prey animals (e.g., crustaceans) [59]. Hence, it has also been hypothesized that aerolysin-related toxin homologs in sea anemones, which are secreted by the ectodermal cells of the pharynx, play a similar role [60]. Interestingly, hydralysins and the sea anemone aerolysin-related toxins have been suggested to originate from two independent recruitment events that involved horizontal gene transfers [60]. Evolutionary assessments revealed that aerolysin-related toxins in both sea anemones and Hydra have evolved under the extreme influence of negative selection ( of 0.27 and 0.21, respectively) (Table 2-2 and Supplementary Tables S4.2 and S4.3, Supplementary Material online). FUBAR revealed numerous amino acid sites in these toxin types that evolved under the pervasive influence of negative

25 selection (300 and 120 sites, respectively). Although MEME detected only 9 sites as episodically adaptive in hydralysins, as many as 41 sites were detected in aerolysin-related homologs of sea anemones (Figures 2-1 and 2-5 and Table 2-2).

Figure 2-5. Sequence alignment of hydralysins. Episodically adaptive, structurally/functionally important, and extremely well-conserved (percent ID ≥ 90%) sites have been depicted here. UniProt accession numbers and pharmacological names of sequences are as follows: 1) Q86LR2 Hydra viridissima (Hln-1); 2) X2GKY3 Lysinibacillus sp. (Hln-2); 3) Q52SK7 Hydra vulgaris (Hln); 4) Q564A5 H. viridissima (Hln-2); and 5) Q52SK6 H. viridissima (Hln-3).

2.2.4 Jellyfish Toxins JFTs are among the most dangerous toxins secreted by jellyfish and are known for a diversity of immunological and toxicological activities, including hemolyticity, cardiotoxicity, cutaneous inflammation, and necrotic activities. It has been proposed that their pore-forming activity is responsible for toxicity associated with human envenomations [140]. Originally, JFTs were reported to be limited to Cubozoa and Schyphozoa. However, homologs of JFTs were recently reported in H. magnipapillata [13]. Through BLAST searches, we have retrieved such homologs from various sea anemones (Aiptasia pallida) and various hydroids (Hydractinia symbiolongicarpus and H. vulgaris), suggesting that JFTs originated within the common ancestor of all extant cnidarians more than 600 Ma.  of 0.39 was computed by M8 for JFTs, highlighting the significant influence of negative selection on this toxin type (Table 2-2, Supplementary Figure S3 and Supplementary Table S4.4, Supplementary Material online). Although BEB failed to detect any site in this toxin as positively selected, MEME identified as many as 17 sites that evolved under episodic bursts of positive selection.

26 2.2.5 Small Cysteine-Rich Peptides SCRiPs were originally identified as genes unique to reef-building corals (Scleractinia) that are downregulated during heat stress [164]. Given the similarity in the temporal expression pattern they share with galaxin, a key protein involved in the biomineralization process [175], SCRiPs were implicated in calcification of the coral skeleton [164]. However, a greater sequence diversity in SCRiPs (Figure 2-6) is uncharacteristic of homeostatically important proteins, such as those involved in the biomineralization process. Previously, a α-defensin domain was reported in Mfav- SCRiP1 [164] and InterPro [176] scanning performed in our study detected a domain that is very similar to the basic myotoxic domains of rattlesnake crotamine toxins (InterPro entry: IPR000881). Moreover, like SCRiPs, several peptide toxins in sea anemones have also been shown to exhibit downregulation in expression as a result of thermal stress [177]. Considering these observations, we suspected that SCRiPs may represent a class of toxins and not calcifying proteins as claimed by earlier studies. BLAST searches in this study surprisingly retrieved SCRiP homologs in sea anemones, Anemonia viridis and Metridium senile (Figure 2-6), refuting the claim that these peptides are specific to scleractinian corals [164]. To gain further understanding regarding the bioactivity of SCRiPs, we expressed Amil-SCRiP1, Amil-SCRiP2, and Amil-SCRiP3 from the scleractinian coral Acropora millepora in a recombinant form (see Methods). Although the expression of Amil-SCRiP1 was unsuccessful, Amil-SCRiP2 and Amil-SCRiP3 were expressed in relatively large amounts (1.5 mg protein per 1 l of bacterial culture). This enabled us to test the notion that the two peptides are toxic. Interestingly, the injection of these recombinantly expressed SCRiPs in blowfly larvae (Sarcophaga falculata) did not result in toxicity, while the incubation of zebrafish (Danio rerio) larvae at a concentration of 230 mg/ml resulted in severe neurotoxicity (data not shown). In the first few minutes of incubation with SCRiPs, the fish exhibited abnormal tail twitching. In contrast to the control group incubated with bovine serum albumin, fish incubated with SCRiPs gradually exhibited frequent twitching and shivering, stopped reacting to touch, and eventually became completely paralysed. All fish incubated with Amil-SCRiP2 died within 200 min of exposure, while Amil-Scrip3 killed fish within 16 h. These results support the notion that SCRiPs may serve as neurotoxins, making them the first peptide neurotoxin family described from scleractinian corals. M8 computed  of 0.72 and the BEB approach failed to identify positively selected sites in SCRiPs (Table 2-2, Figure 2-6, and Supplementary Table S4.5, Supplementary Material online). Thus, selection assessments indicated that coral SCRiPs have evolved under the influence of negative selection.

Figure 2-6. Sequence alignment of SCRiPs. Episodically adaptive, structurally/functionally important, and extremely well-conserved (percent ID ≥ 90%) sites are depicted here. UniProt (sequences 1–5) and NCBI (sequences 6 and 7) accession numbers and pharmacological names of sequences are as follows: 1) C0H690 Acropora millepora (SCRiP1); 2) C0H691 A. millepora (SCRiP2); 3) C0H692 A. millepora (SCRiP3); 4) C1KIZ0 Montastraea faveolata (SCRiP2); 5) C0H693 Montipora capitata (SCRiP1a); 6) FK725749 Anemonia viridis; and 7) FC835414 Metridium senile.

2.3 Discussion 2.3.1 Strong Negative Selection Governs the Evolution of Most Cnidarian Toxins Molecular evolutionary assessments of cnidarian toxin families using the state-of-the-art methods revealed the extreme conservation of toxin-encoding sequences, despite their long evolutionary histories (Figures 2-1–2-6, Tables 2-1 and 2-2, and Supplementary Figure S3 and Supplementary Tables S1.1–S1.3, S3.1 and S3.2, S4.1–S4.5, Supplementary Material online). The computed  values ranged between 0.21 and 0.72, indicating a strong influence of negative selection on a majority of sites in these proteins. This is in stark contrast to most predatory animal venoms that are known to rapidly evolve under the influence of positive selection [178-184]. Among all the cnidarian toxins examined in this study, only the type III KTxs seem to have evolved rapidly ( = 1.33) under the influence of positive Darwinian selection. When the majority of lineages or sites evolve under the strong influence of negative selection, it becomes difficult to identify adaptive selection that occurs in short bursts and influences a small proportion of lineages or sites [185]. The application of MEME detected several sites (ranged from 5 to 41) in most toxin types that experienced episodic bursts of adaptive selection (Tables 2-1 and 2-2). Interestingly, a similar phenomenon was also reported in scorpions [186], which represent another ancient venomous animal lineage that originated nearly 400 Ma [187]. This reinforces the dynamic nature of venom in ancient lineages, where variations in venom-encoding genes accumulate episodically, possibly under an evolutionary chemical arms race scenario with their target sites in prey animals. When toxin sequences that increase the potency and efficacy of envenoming are generated, they get fixed in the population and experience purifying selection for long periods of time. In contrast, in

28 evolutionarily younger lineages, such as cone snails and advanced snakes, the rapid evolution of genes under the positive Darwinian selection is much more pronounced.

2.3.2 Most Adaptive Sites in Cnidarian Neurotoxins Are Surface Accessible It has been suggested that most venom proteins that are involved in predation evolve through rapid accumulation of variation in the exposed residues (RAVER), where the molecular surface of the toxin accumulates bulk of the variations under the significant influence of positive Darwinian selection, while preserving the core residues involved in stability and/or catalytic activity [184]. As synthesis and secretion of proteins is an energetically expensive process, over time, mutations that lead to the loss of stable structure and function are filtered out of the population by purifying selection. Thus, favouring the conservation of structurally important and catalytic residues. Moreover, the accumulation of variations on the molecular surface of the toxin seems to be advantageous as the altered surface biochemistry might lead to neofunctionalization (generation of novel pharmacological properties) and assist in immunological evasion [91]. Computation of ASA ratios revealed that all four episodically diversifying sites in the mature peptide of NaTxs, one positively selected and another episodically adaptive site in KTx type I, were surface exposed (Supplementary Table S2, Supplementary Material online). Six of the eight positively selected and episodically diversifying sites in the mature sequence of type III KTxs were also surface exposed, while only one site was buried. Thus, a majority of positively selected or episodically adaptive sites in neurotoxins are surface exposed. RAVER has been reported in a myriad of animal lineages and in a plethora of venom proteins, including scorpion neurotoxins [91, 181, 182, 184, 188-191]. Hence, it appears that even the toxins of the most ancient animal lineages adopt RAVER and favour the accumulation of variations on the molecular surface.

2.3.3 Negative Selection Constrains the Evolution of Pore-Forming Toxins It is particularly interesting to note that most of the cnidarian PFTs have evolved under the extreme influence of negative selection (Figures 2-2 and 2-4, Table 2, and Supplementary Figure S3 and Supplementary Tables S4.1–S4.5, Supplementary Material online). This may be a result of their ability to utilise certain amino acids (e.g., charged, polar, or hydrophobic) to non-specifically bind to the membranes of target cells in prey animals, before potentiating the deadly pore-forming activity. As a result of this nonspecific function, they probably do not experience the predator–prey chemical arms race and could act on a very wide range of species. Moreover, it becomes necessary to conserve a variety of residues on their molecular surface that are involved in binding to target cells (Figures 2-4 and 2-5). As outlined above, a majority of residues in actinoporins that are implicated in binding to target cells were found to have evolved under the pervasive influence of negative selection (Figure 2-4). Additionally, in order to gain their pore-forming function, 29 actinoporins oligomerize into complex tetramers via the interaction of several residues [192]. Consequently, to ensure the proper organization of these toxins into complex tertiary structures, structurally important residues evolve under the constraints of negative selection. A similar phenomenon has also been noted in certain classes of snake venom toxins that exhibit nonspecific cytotoxicity [184, 193], emphasizing the fact that not all toxins evolve rapidly.

2.3.4 The Common Evolutionary Origin of Sodium and Type III Potassium Neurotoxins in Sea Anemones Bayesian and maximum-likelihood phylogenetic reconstructions in this study placed the type III KTxs in the same clade as type I NaTxs (Figure 2-3; Bayesian PP 0.997, bootstrap support 734/ 1,000), suggesting a common evolutionary origin of these two types. Although both type I and type II NaTxs remained constrained under the influence of negative selection, type III KTxs were found to have evolved rapidly ( = 1.33; six positively selected sites). Polytomy was observed at the node that leads to NaTx type I and KTx type III clades, suggesting that these proteins likely underwent rapid radiation in a short time. The pharmacological diversity in this neurotoxic lineage is probably reflective of an adaptive radiation. The examination of various sea anemone transcriptomes (Edwardsioidea: N. vectensis [194] and Edwardsiella lineate [195]; Acuticulata: M. senile and A. pallid [196]) failed to retrieve type III KTxs from these lineages. Hence, type III KTxs probably represent a unique evolutionary innovation of actinioids, such as Anemonia, Bunodosoma, and Anthopleura (for sea anemone species phylogeny [171], see Supplementary Figure S3, Supplementary Material online). Thus, it is likely that a subset of sodium channel targeting toxins experienced episodic bursts of adaptive selection in Actinioidea and accumulated mutations at an elevated rate, resulting in the origination of a novel toxin type that can target potassium ion channels. Interestingly, several KTx type III toxins still retain the ability to modulate sodium ion channels in prey animals. Thus, the evolution of neurotoxins in sea anemones is fascinating, because a subtype of potassium ion channel targeting toxin evolved within the sodium ion channel targeting toxins, and the potassium channel toxins evolved on at least five independent occasions (type I–V KTxs). It is also interesting to note that Actinioidea, which is likely the only sea anemone superfamily to have rapidly evolving type III KTxs, include several species with highly potent venoms such as A. virids, Anthopleura elegantissima, and Anthopleura xanthogrammica [197, 198]. Thus, the evolution and diversification of several toxin types including type III KTxs, within Actinioidea, may have facilitated the emergence of a highly potent venom in this lineage.

30 2.3.5 Functional Diversification of Sea Anemone Neurotoxins It has been documented that a single point mutation can alter the biochemical activities of toxins. APETx3, a type III KTx from A. elegantissima, differs from its homolog APETx1, a selective toxin modulator of human ether-a-go-go related gene (hERG) voltage-gated potassium channels, only in having a threonine instead of proline at position 3 (position 4 in Figure 2-2). Fascinatingly, this single point mutation has been demonstrated to be responsible for the loss of APETx3’s ability to modulate hERG channels [170]. This change has also been correlated with the origination of a different biochemical activity: the ability to modulate voltage- gated sodium ion channels [170]. Strikingly, our evolutionary assessments detected this site as experiencing episodic influence of adaptive selection (Figure 2-2). Recently, several other sites were identified in APETx2 from A. elegantissima, as important for inhibiting hERG and the unrelated acid-sensing ion channel 3 (ASIC3) [75]. Evolutionary analyses in this study identified many of these functionally important sites as episodically adaptive: 1) Sites 21 and 36 (important for ASIC3 inhibition) and 2) site 22 (important for interacting with hERG; Figure 2-2). However, none of the positively selected sites we identified in type III KTxs coincide with sites in APETx2 that are responsible for targeting both hERG and ASIC3 channels [75]. Thus, positive selection has strongly shifted the pharmacological properties of the toxin between targets but does not result in the complete loss of activity, which could have a deleterious effect on the fitness of the animal. We also identified a few functionally important sites in other classes of neurotoxins and PFTs that experienced episodic adaptation (Figures 2-2–2-5): 1) Site 2 in NaTxs which was shown to be responsible for a moderate decrease in the binding affinity to insect Nav channels upon mutagenesis (V2A) [173]; 2) sites 54, 117, and 144 in actinoporins that are responsible for binding to cell membranes; and 3) the site in hydralysins shown to result in slight reduction (2.5-fold) of paralytic and hemolytic activity upon mutation (G129E) [58]. Additionally, sites that were shown to have minor effects on toxicity were also found to be positively selected (e.g., sites 12 and 40 in NaTx; Figure 2-1; [173]. These findings suggest that sites that are relieved of purifying selection pressures and those responsible for toxicity constitute the evolutionary hotspots in cnidarian toxins, as long as mutations do not result in the complete loss of toxicity. Such sites may also promote changes in toxin selectivity and expand the range of molecular targets [199].

2.3.6 SCRiPs May Represent the First Neurotoxin Family from Corals Because SCRiPs cause profound neurotoxic effects in fish, it is most likely that they are employed as neurotoxins. Moreover, SCRiPs have been reported to be widely expressed in the ectoderm of A. millepora [200], which further support their likely role in prey envenomation, as ectoderm is chiefly lined by nematocysts and gland cells in anthozoans. In contrast to previous conclusions that SCRiPs

31 are unique innovations of scleractinian corals [164], the retrieval of SCRiP homologs from sea anemones in our study (Figure 2-6) indicates that these proteins evolved nearly 500 Ma in the common ancestor of sea anemones and scleractinian corals [160]. Because reef-building corals form habitats for numerous species of marine animals, the understanding of their ecology and the evolutionary diversification of proteins, such as toxins that affect their ecological interactions, are important. Although peptide toxins from other cnidarians, particularly sea anemones, are well studied, practically nothing is known about venom in corals. Hence, the discovery of a novel family of neurotoxic peptides in this lineage is fascinating and may prove to be an important step forward in the understanding of the evolution of venom in this lineage. To conclude, venom research in the past has chiefly focused on relatively evolutionarily younger animal lineages, such as the venoms of advanced snakes and cone snails. Our findings provide fascinating insights into the evolution of venom in Cnidaria: 1) The common origin of sodium channel toxins and a subtype of potassium channel toxins in sea anemones; 2) the discovery of the first neurotoxin family from corals; 3) identification of strong evolutionary constraints on most cnidarian toxin types, especially PFTs; and 4) insights into the functional diversification of various toxin types. These results emphasize the importance of understanding the molecular evolution, diversification, and phylogenetic histories of venom components, particularly in the ancient venomous lineages.

2.4 Materials and Methods 2.4.1 Phylogenetic Analyses Translated nucleotide sequences were aligned using MUSCLE 3.8 [201]. Sequence alignments in FASTA format have been provided in the form of a zipped file (Supplementary File 1, Supplementary Material online). The best-fit model of nucleotide substitution and amino acid replacement for individual toxin data sets were determined according to the Akaike’s information criterion using jModeltest 2.1 [202] and Prottest 3.0 [203], respectively (Supplementary Table S5, Supplementary Material online). Model averaged parameter estimates of the proportion of invariant sites (pinvar) and the gamma shape parameter (α) were used for reconstruction of trees. Maximum- likelihood and Bayesian phylogenetic analyses performed on the nucleotide data sets allowed the reconstruction of the molecular evolutionary histories of various cnidarian toxins. Bayesian inference implemented in MrBayes 3.2.3 [204] was used and a minimum of 15×106 generations in 4 chains were run, saving every 100th tree. The log-likelihood score of each saved tree was plotted against the number of generations to establish the point at which the log-likelihood scores reached their asymptote. After the completion of burn-in phase, posterior probabilities for clades were established by constructing a majority-rule consensus tree for all trees generated. The maximum-

32 likelihood trees were generated using PhyML 3.0 [205]. Node support was evaluated with 1,000 bootstrapping replicates.

2.4.2 Selection Analyses Maximum-likelihood models [167] implemented in Codeml of the PAML [206] were employed to assess the nature of natural selection on various cnidarian toxin families examined in this study. Site-specific models, which estimate positive selection statistically as a nonsynonymous to- synonymous nucleotide substitution rate ratio () significantly greater than 1, were employed. Because no a priori expectation exists, we compared likelihood values for three pairs of models with different assumed  distributions: M0 (constant  rates across all sites) versus M3 (allows  to vary across sites within “n” discrete categories, n ≥ 3); M1a (a model of neutral evolution) where all sites are assumed to be either under negative ( < 1) or neutral selection ( = 1) versus M2a (a model of positive selection), which in addition to the site classes mentioned for M1a assumes a third category of sites; sites with  > 1 (positive selection) and M7 (β) versus M8 (β and ω) [197]. The results are considered significant only if the alternative models (M3, M2a, andM8 that allow sites with  > 1) show a better fit in the likelihood ratio test (LRT) relative to their null models (M0, M1a, and M7: Do not allow sites  > 1). LRT is estimated as twice the difference in maximum-likelihood values between nested models and compared with the 2 distribution with the appropriate degree of freedom—the difference in the number of parameters between the two models. The BEB approach [207] is employed to identify sites under positive selection by calculating the posterior probabilities that a particular site belongs to a given selection class (neutral, conserved, or highly variable). Sites with greater PP (≥ 95%) of belonging to the “ω > 1 class” were inferred to be positively selected. FUBAR [208] implemented in HyPhy [209] was utilised to identify sites evolving under the influence of pervasive diversifying and purifying selection pressures. Additionally, MEME [185] was used to efficiently detect episodically diversifying sites.

2.4.3 Structural Analyses Consurf webserver [210] was used to map the evolutionary variability of amino acids onto the crystal structures of various cnidarian toxins. Furthermore, we calculated the ASA or the solvent exposure of amino acid side chains using GETAREA [211], which uses the atom co-ordinates of the Protein Data Bank (PDB) file and indicates if a residue is buried or exposed to the surrounding medium by comparing the ratio between side chain ASA and the “random coil” values per residue. An amino acid with an ASA ratio of ≤ 20% is considered to be buried, while an amino acid with a

33 ratio of 50% or more is likely exposed to the surrounding medium. Pymol 1.3 [212] was used for visualizing three-dimensional structures.

2.4.4 Cloning, Recombinant Expression, and Purification of Toxins Primers carrying NcoI and BamHI restriction sites and corresponding to the mature Amil-SCRiP1, 2, and 3 were designed according to the available transcript sequences [164]. The primers were used in a polymerase chain reaction (94 °C for 2 min, 34 times [94 °C for 20 s, 55 °C for 20 s, 72 °C for 30 s] and 72 °C for 5 min) with A. millepora complementary DNA made from mixed developmental stages (kindly provided by Drs D. Hayward and E. Ball, Australian National University, Canberra). The resulting PCR fragment was digested with NcoI and BamHI (New England Biolabs, USA) and cloned with T4 DNA Ligase (Takara, Japan) into a pET-32b vector (Novagen, USA) digested with the same enzymes. Toxins were expressed fused to thioredoxin in the Escherichia coli Rosettagami strain (DE3, pLys; Novagen). Toxins were released from their thioredoxin tag and purified on HisTrap and Resource RPC columns connected to an AKTA fast protein liquid chromatography machine (GE Healthcare) as previously described for the Av3 toxin [164].

2.4.5 Toxicity Assays Toxicity assays on blowfly and 3-days-old zebrafish larvae were performed as described before [42, 164]. The zebrafish larvae were kindly provided by Dr Y Gothilf (Tel Aviv University).

Supplementary Materials Supplementary Figures S1 and S2, Tables S1.1–S5, and file S1 are available at Molecular Biology and Evolution online. (http://mbe.oxfordjournals.org/content/32/6/1598/suppl/DC1).

Chapter 3

Firing the Sting: Chemically Induced Discharge of Cnidae Reveals Novel Proteins and Peptides from Box Jellyfish (Chironex fleckeri) Venom

Manuscript published in Toxins, March 2015 http://www.mdpi.com/2072-6651/7/3/936/htm 35

Abstract Cnidarian venom research has lagged behind other toxinological fields due to technical difficulties in recovery of the complex venom from the microscopic nematocysts. Here we report a newly developed rapid, repeatable and cost effective technique of venom preparation, using ethanol to induce nematocyst discharge and to recover venom contents in one step. Our model species was the Australian box jellyfish (Chironex fleckeri), which has a notable impact on public health. By utilising scanning electron microscopy and light microscopy, we examined nematocyst external morphology before and after ethanol treatment and verified nematocyst discharge. Further, to investigate nematocyst content or venom recovery, we utilised both top-down and bottom-up transcriptomics–proteomics approaches and compared the proteome profile of this new ethanol recovery based method to a previously reported high activity and recovery protocol, based upon density purified intact cnidae and pressure induced disruption. In addition to recovering previously characterised box jellyfish toxins, including CfTX-A/B and CfTX-1, we recovered putative metalloproteases and novel expression of a small serine protease inhibitor. This study not only reveals a much more complex toxin profile of Australian box jellyfish venom but also suggests that ethanol extraction method could augment future cnidarian venom proteomics research efforts. Keywords Chironex fleckeri; transcriptome; proteome; nematocyst; pressure induced disruption; ethanol induced discharge

3.1 Introduction Stinging cells (cnidocytes) are distinctive of venomous marine animals of Cnidaria phylum. They contain microscopic organelles (cnidae) that discharge explosively, injecting a mixture of compounds into prey or potential predators [46, 213]. Upon contact with human skin or other surface (e.g., prey and predator), penetrant cnidae or nematocysts evert harpoon-like tubules laden with spines that act like hypodermic devices to inject venom (proteinaceous porins, neurotoxic peptides and bioactive lipids) [24, 28, 214]. Envenomation syndromes induced by cnidarian animals represent a therapeutic challenge especially to bathers, swimmers and surfers. Envenomation symptoms are painful hemorrhagic skin lesions, systemic reactions (e.g., direct effects on muscle and nerve tissue), long-term immunological responses, and occasionally fatalities due to Chironex fleckeri cardiovascular and pore-forming toxins [3, 125, 140, 215]. In recent years, cnidarian venoms have begun to be investigated as a potential source of novel bioactive therapeutic compounds [216-219]. However, in comparison with the vast number of studies conducted on the venoms of other venomous animals such as snakes, cone snails, spiders and scorpions, cnidarian venoms have received scant attention from toxinologists. The principal

36 technical impediment in cnidarian research is the fact that the venom does not exist in a large discrete gland as an aqueous mixture in milligram quantities but instead is distributed in microscopic individual nematocysts, each containing picogram of protein [3]. Venom analysis at the picogram scale presents challenges as cnidarian venom is a complex mixture of bioactive molecules, some of which are aqueous while others are lipidic [3, 140]. As a consequence, a modern proteomics approach based on high-throughput mass spectrometry analysis is ideal [220]. Previous venom preparation techniques have been based upon electrical discharge of nematocysts through human amnion [221], or homogenization [222] and pulverization or maceration [223] of whole frozen tentacles and saline or phosphate buffer wash. However, the venom recovered utilising these methods is in fact total tentacular extracts comprised of the contents of both nematocysts and other tentacle cell types. Current venom preparation techniques are based on mechanical rupture of the isolated nematocysts with mortar and pestle grinding [224], glass beads [225] and sonication [215, 226] in the presence of extraction solutions such as distilled water or saline. However, difficulties relating to equipment availability and contamination of the venom by structural components (e.g., nematocyst capsule-walls) are the major disadvantages of a mechanical disruption and solvent based extraction approach. Another approach has been developed in which density purified intact cnidae are disrupted using pressure followed by rapid centrifugation to harvest the contents without contaminating then with tentacular material or structural components [3]. This technique results in venom with very high specific activity and complexity but is a laborious process. Since certain chemicals such as ethanol, or 5% acetic acid in distilled water, cause massive cnidae discharge in some cnidarian species [34, 143, 144] (Hydrozoa and Cubozoa, respectively), in this study we utilised ethanol to obtain venom proteins and peptides from box jellyfish, C. fleckeri, because of its significant envenomation consequences and need of opportune therapeutic tools [3, 140]. This study highlights the advantages of this new technique, which results in pure venom, free of contaminants from the tentacles or structural components of nematocysts.

3.2 Results and Discussion The venom composition of C. fleckeri has previously been studied, although the methodological approaches used to obtain venom varied between authors [221-226]. Moreover, due to the lack of a transcriptomic database underpinning the annotation of the isolated proteins, proteomic approaches were unlikely to discover novel toxins unique to this species. Notably, when pulverization based approaches are used on purified nematocysts in combination with a solvent extraction, the recovered proteins include structural components of the nematocyst capsule rather than just intracapsular material. Even more concerning is that many venom preparations are in fact solvent

37 extracts of the whole tentacles, and thus contain all tentacular biomolecules soluble in the chosen solvent. In addition, many current venom obtaining techniques based upon mechanical disruption of nematocysts are time-consuming and expensive. Here we report a new venom recovery technique based on chemically induced discharge of nematocysts that maximizes and accelerates the identification of the toxic molecules comprised in jellyfish venom. Also in order to rule out that the identified proteins are produced by tentacular epithelial cells, (e.g., toxin Nv1 localized to ectodermal gland cells in the tentacles rather than nematocysts [42]), we have isolated nematocysts from C. fleckeri, disrupted them in vitro and analysed and compared the released protein mixture with identified proteins in our method.

3.2.1 Microscopy Examination of Undischarged and Chemically Discharged Nematocysts Thus far, there have been differences in the reported nematocyst types and morphology of C. fleckeri cnidome. Despite this diversity in the results of studies conducted by various research groups [29, 35, 41, 224], the consensus is that the cnidome includes four types of nematocyst: (i) those that contain the lethal venom component (microbasic p-mastigophores); (ii) those that penetrate the prey’s skin or cuticle and ensnare it with hook-like structures in order to secure close contact with the tentacles (small and large tri-rhopaloids); (iii) adherent cnidae which adhere to the prey via a coiled shaft upon discharge (holotrichous isorhizas); and (iv) enigmatic spineless adhesive cnidae that secrete sticky fluid (atrichous isorhizas) [29, 35, 41, 224]. In this study the effectiveness of ethanol in inducing discharge discharge C. fleckeri nematocysts was proved by both light microscopy and scanning electron microscopy (SEM). Prior to immersion in ethanol, SEM examination of tentacles revealed undischarged nematocysts, which were categorized as rod-shaped atrichous isorhizas (Figures 3-1A and 3-2B), banana-form microbasic p- mastigophores (Figures 3-1B and 3-2C), large oval p-rhopaloids (Figures 3-1C, E, and 3-2B) and small sub-spherical p-rhopaloids (Figures 3-1D and 3-2C). In order to achieve a better understanding of nematocyst orientation within the tissue and the morphological characteristics of the discharged and undischarged nematocysts, the histological samples of tentacles were examined using light microscopy (Figure 3-2). The transverse section of the tentacle clearly showed three groups of nematocyst batteries: top, intermediate and lower (Figures 3-2A, E); with nematocysts located at the tips of the batteries. Before chemical discharge, tubules were observed to be coiled and twisted inside the intact nematocyst capsule (Figures 3-2A–C). After discharge, the capsule remained intact, although the capsular components including shaft, tubule and venom were expelled (Figures 3-2D–F and 3-3A–C). Moreover, after immersion in ethanol, the tentacle surface was found to be densely packed with discharged nematocysts (Figure 3-3D), with a few nematocysts, mostly those placed in lower nematocyst batteries remain undischarged (Figure 3-2E). As

38 previously suggested [34], the “roofed-over” effect of the top nematocyst batteries likely prevents the less-prominent intermediate and inferior batteries from being exposed to ethanol. It should be mention that this new ethanol recovery based method demonstrated higher yield in term of the number of proteins identified. Therefore, it is reasonable to propose that this method is more effective in obtaining a good yield of venom, as compared to a previously reported high activity and recovery protocol. The effect of ethanol on nematocyst discharge in C. fleckeri is in line with what previously reported, showing that ethanol stimulates nematocyst discharge of various species of jellyfish [34, 143, 144]. On the other hand, our observation differs from the inhibitory effect of ethanol on chemoactivation of in situ discharge in Pelagia noctiluca (Cnidaria, Scyphozoa) oral arms [142]. This inconsistency is probably due to specimens’ difference.

Figure 3-1. Scanning electron microscopy (SEM) of undischarged C. fleckeri nematocysts. (A) Undischarged rod-shaped atrichous isorhiza; (B) banana-form microbasic p-mastigophore; (C) Large oval p-rhopaloid; (D) Small sub-spherical p-rhopaloid; (E) Detail of operculum (the door of the capsule) of an oval p-rhopaloid. The operculum (solid arrows) is found to be a convex shape, which upon discharge, part and permit the tubule and capsule components to be released. Scale bars, A, D, E 1 μm; B, C 10 μm.

Figure 3-2. Light microscopy (LM) of C. fleckeri nematocysts histochemically stained with Masson’s Trichrome. (A) The orientation of undischarged nematocysts in longitudinal section showing top batteries (TB), intermediate batteries (IB) and lower batteries (LB); (B) Undischarged large oval p-rhopaloid (black arrowhead) and atrichous isorhiza (white arrow); (C) Undischarged microbasic p-mastigophores (white arrowhead) and small sub-spherical p-rhopaloid (black arrow); (D) Discharged tentacular axis region showing gastrovascular cavity (GC), mesoglea (M), and epidermis (E); (E) Detail of epidermis evagination with ethanol discharged nematocysts. Note the nematocyst batteries; (F) Part of the epidermis showing the extruded tubules (black arrow). Note the empty capsule (black arrowhead). Scale bars, A, D, E, F 100 μm; B, C 50 μm.

Figure 3-3. Scanning electron microscopy (SEM) of ethanol discharged C. fleckeri nematocysts. (A) A discharged microbasic p-mastigophore (white arrowhead) with the extrude shaft (black arrow) and an atrichous isorhiza (white arrow); (B) Small sub-spherical p-rhopaloid. Note the operculum (white arrow); (C) High magnification SEM of shafts and tubules of microbasic p-mastigophores. Note the spines (white arrow); (D) Sublimed surface of a discharged tentacle. Scale bars, 10 μm.

3.2.2 Transcriptome Assembly and Functional Annotation A total of 2,973,873 reads were obtained for the C. fleckeri tentacle transcriptome. Automated assembly resulted in 5128 contigs (Table S1). To classify the putative function of the resulting contigs we annotated them using BLAST2GO—from a total of 5128 contigs, 59.7% of them were successfully identified by BLAST annotation and we subsequently classified these contigs according to biological process, cellular component and molecular function, respectively. A complete description of the contigs, BLAST statistics, contigs classification and top-hit species can be found in Table S1.

3.2.3 Comparative Proteomic Analyses and Identified Proteins ProteinPilot analysis of the C. fleckeri pressure disrupted nematocysts (PDN) proteome retrieved 175 proteins (with 99% high-confidence spectra and a 1% FDR), representing 3.41% of the translated amino acid sequences sourced from the tentacle transcriptome (Table S2). The same approach applied to the chemically discharged nematocysts (CDN) proteome resulted in the identification of 241 proteins (with 99% high-confidence spectra and a 1% FDR), representing 4.69% of the sequences found in the transcriptome (Table S3). Further gene ontology analyses by

41 BLAST2GO software classified the two sets of proteomes into 10, 18 and 13 categories according to their cellular component, biological process, and molecular function, respectively (Figure S1). On comparing the proteomic data retrieved from PDN and CDN, 129 of 175 PDN proteins were found in the CDN set (73.71%). On the other hand, from 241 of the proteins identified in the CDN proteome, 134 proteins (55.60%) were identified in the PDN proteome. The identified proteins from each of these samples are summarized and displayed in Table S4. Through our combined proteomic and transcriptomic approach, we identified newly described C. fleckeri haemolysin toxins CfTX-A (UniProt: T1PRE3) and CfTX-B (UniProt: T1PQV6) and potent cardiotoxic toxin CfTX-1 (UniProt: A7L035) [13] in venom obtained from both PDN and CDN. Interestingly, the CfTX-2 (UniProt: A7L036) gene identified in our tentacle transcriptome was only identified in the CDN venom sample—where it exhibited high proteomic coverage (42.39%)—and was completely absent from PDN venom sample. This pattern was observed with other protein types, as our chemical discharge approach resulted in a larger number of protein identifications and higher confidence values for the identified proteins (Table 3-1). We detected several metalloproteases containing ShK and astacin domains in the proteome of PDN and CDN that have not been previously identified in C. fleckeri venom. The ShK-containing metalloproteases have recently been characterised in mammalian proteomes where they display remarkable sequence similarity to BgK and ShK toxins from sea anemones [227]. Although two of the identified metalloproteases containing the ShK domain do not exhibit sequence homology to ShK and BgK toxins from sea anemones, suggesting that they do not possess potassium channel blocker activity. In addition to ShK domain, we found astacin domains (metalloprotease M12A family) in the metalloproteases of the C. fleckeri venom. The metalloprotease M12A family has been recruited into the venom systems of numerous animals, including the spider Loxosceles intermedia [228], centipedes [99], and the jellyfish Stomolophus meleagris [12]. These proteins are thought to be responsible for degrading the extracellular matrix, thereby facilitating the diffusion of other venom components to their molecular targets. Their presence in the nematocysts of the sea anemone Nematostella vectensis has been associated with defence rather than prey capture [48]. Although transcripts encoding proteases containing M12A and ShK domains were found in both PDN and CDN venoms here, their evolutionary history and present function in C. fleckeri venom remain unclear. However, given the additional experimental evidence of the presence of metalloproteases in cnidarian nematocyst venoms [12, 48], it is probable that C. fleckeri metalloproteases play a role in prey capture, prey digestion or defence (or a combination thereof). A plesiotypic serine protease-inhibitor (kazal-type) was found in the CDN sample. Recently, kazal- containing transcripts have been found in the sea cucumber Holothuria glaberrima where they are

42 associated with a defensive/immunity role in the inactivation of bacterial proteases [229]. The possible role of this toxin in C. fleckeri venom remains unknown. Table 3-1 Summary of venom proteins/peptides identified from C. fleckeri venom material. Protein ID UniProt Accession Conserved Comparative %Cov (95%)b #Matched transcriptome #(s)/Best Cnidaria Domain Protein-levela peptides match BLAST Hit (%95)c PDN CDN PDN CDN PDN CDN Known Cubozoa Toxins T0179 T1PQV6/Toxin B _ 44 6 65.11 60.15 8 63 precursor T0362 T1PRE3/Toxin A _ 25 7 29.7 55.3 12 49 precursor T2746 A7L035/Toxin CfTX-1 _ 93 40 45.9 77.9 3 14 precursor T2621 A7L036/Toxin CfTX-2 _ Not 68 Not 42.4 Not 10 precursor found found found Metalloproteases T0344 A7S336; ShKT domain 47 27 16.92 23.69 6 27 Predicted protein; (IPR003582) Nematostella vectensis T0690 A7SNJ4; Peptidase 72 61 17.49 45.73 4 16 Metalloendopeptidase; M12A, astacin Nematostella vectensis domain (IPR001506); ShKT domain (IPR003582) T2821 A7S5S4; Peptidase 177 77 25 47.5 2 12 Metalloendopeptidase; M12A, astacin Nematostella vectensis domain (IPR001506); T1091 _ Peptidase 207 189 12.7 7.7 1 2 M12A, astacin domain (IPR001506); T2460 A7T0S0; Peptidase 138 200 25.6 41 3 7 Metalloendopeptidase; M12A, astacin Nematostella vectensis domain (IPR001506); Serine Protease Inhibitors T0134 A7SCV8; Kazal domain Not 149 Not 5.34 Not 3 Predicted protein; (IPR002350) found found found Nematostella vectensis NOTE. Pressure disrupted nematocysts (PDN); Chemically discharged nematocysts (CDN); a Comparative protein-level results across multiple searches using ProteinPilot b The percentage of matching amino acids from identified peptides having confidence greater than or equal to 95% c The number of distinct peptides having at least 95% confidence.

43 3.3 Experimental Section 3.3.1 Specimen Collection Mature live specimens of C. fleckeri were collected by dip net from Weipa, Queensland, Australia during the spring 2013 by Bryan Fry and Nicholas Casewell. For transcriptome analysis, fresh tentacles were dissected out manually then immediately frozen in liquid nitrogen and subsequently stored at -80 °C. For histology and Cryo-SEM studies, samples of tissue were dissected from the tips of both untreated and ethanol-treated tentacles, and then fixed in 10% neutral-buffered formalin (NBF) at pH 7.2.

3.3.2 Nematocyst Morphology 3.3.2.1 Cryo-Scanning Electron Microscopy Longitudinal and transverse sections (< 4 mm long) of the untreated and ethanol-treated tentacles that had previously been fixed in NBF were washed in phosphate buffered saline (PBS) at pH 7.2 for 5 × 10 min then transferred to glutaraldehyde solution (3% in phosphate buffer, pH 7.2) for permanent preservation. Samples were frozen and examined with a Gatan Alto 2500 cryo-system on a JEOL JSM-7100F field emission scanning electron microscope (JEOL Ltd., Tokyo, Japan). Separately, untreated and ethanol-treated sections (3 × 5 × 2 mm: height, length and width) were placed in a metal flange-sample holder, locked into position and immediately frozen by immersing in liquid nitrogen. The samples were transferred into the cryo-system, and the tissue was either fractured with a blade (undischarged samples) or left intact (discharged samples). Subsequently, the water was removed from the surface region by raising the temperature to -80 °C for 20, 15 or 7 min, depending on the desired extent of sublimation of the ice. The sample was then coated with platinum to 5–7 nm and viewed at 2 kV. Images were stored digitally without modification except for adjustment of contrast and brightness. Cryo-fixation rapidly freezes the water component in a sample to form ice through heat exchange with the liquid nitrogen slush, producing a solid sample that can be fractured or viewed whole. The sublimation step avoids damage to the delicate, hydrated tissue caused by chemical drying and allows 3D visualization of structures within that tissue.

3.3.2.2 Histology Ultra-structural analysis of the tentacles was carried out using histological sections of intact tentacle tips. Before processing, NBF fixed specimens were washed in PBS (pH 7.2) for 5 × 10 min to remove the fixative. The samples were then dehydrated in an ethanol series (70% × 45 min, 90% × 45 min, 100% × 45 min), followed by paraffin wax embedding (2 × 45 min). Serial transverse sections, 6 μm in thickness, were taken at intervals along the length of the tentacles using a Hyrax M25 Rotary Microtome (Carl Zeiss, Jena, Germany). The sections were then stained with Masson’s Trichrome stain which gives clear distinction between collagen fibre, cytoplasm and nucleus [230]. 44 The slides were then observed by differential interference contrast microscopy (10–100 × magnification).

3.3.3 Transcriptome Library Construction Chironex fleckeri tentacles were preserved in liquid nitrogen prior to use. Total RNA was isolated from 800 mg of frozen tentacle using the standard TRIzol Plus RNA purification kit (Life Technologies, Carlsbad, CA, USA). RNA quality was assessed using a Bioanalyser (Agilent, Santa Clara, CA, USA) and ribosomal RNA removed using the Ribo-Zero rRNA Removal Kit for Human/Mouse/Rat (Epicenter, Madison, WI, USA). The RNA-Seq library was prepared from 50 ng of the enriched RNA material using the ScriptSeq v2 RNA-Seq Library Preparation Kit (epicentre), following 12 cycles of amplification. The sequencing library was purified using AMPure XP beads (Agencourt, Brea, CA, USA), quantified using the Qubit dsDNA HS Assay Kit (Life Technologies) and the size distribution assessed using a Bioanalyser (Agilent). The library was sequenced on a single lane of an Illumina MiSeq machine housed at the Centre for Genomic Research, Liverpool, UK, generating 2,973,873 reads representing 1,481,186 read pairs. The ensuing read data was quality processed, first by removing the presence of any adapter sequences using Cutadapt (https://code.google.com/p/cutadapt/) and then by trimming low quality bases using Sickle (https://github.com/najoshi/sickle). Reads were trimmed if bases at the 3' end matched the adapter sequence for 3 bp or more, and further trimmed with a minimum window quality score of 20. After trimming, reads shorter than 10 bp were removed. Paired-end read data was assembled into 5128 contigs using the de-novo transcriptome assembler VTBuilder [231] executed with the following parameters: min. transcript length 150 bp; min. read length 150 bp; min. isoform similarity 96%. Assembled contigs were annotated with the BLAST2GO Pro v3 [232, 233] using the BLASTX algorithm with a significance threshold of 1e-3, to provide BLAST annotations against NCBI’s non redundant (NR) protein database release 67 followed by mapping to gene ontology terms, and InterPro domain annotation using default parameters. Some sequences annotated ambiguously were confirmed using Expasy (UniProt Knowledgebase (Swiss-Prot + TrEMBL), Uniref100) comparison. Additionally, sequences identified proteomically were scanned for the presence of signal peptide with SignalP 4.1 Server [234], and checked for functional domains and gene ontology (GO) with InterPro [235] and the Conserved domain Architecture Retrieval Tool. Contigs were then translated using CLC Genomics Workbench 5 (CLC bio, Aarhus, Denmark) to provide all 6 reading frames to provide a sequence database for the proteomic characterisation of the venom components. Trimmed raw sequencing reads have been deposited in the SRA database of NCBI (http://www.ncbi.nlm.nih.gov/sra) with the BioProject identifier PRJNA273442. Assembled contigs

45 can be found in Supplementary File 1 and BLAST2GO annotation files are available by request from the corresponding author.

3.3.4 Venom Extraction 3.3.4.1 Chemically Induced Discharge of Nematocysts by Ethanol In order to induce chemical discharge of nematocysts, fresh live tentacles of a mature specimen were immersed in 1 litre absolute ethanol for 30 s at room temperature. The tentacle was then removed and the ethanol transferred to -80 °C to precipitate the proteins. After 24 h of precipitation, the material was centrifuged at 14,000 × g, 4 °C for 30 min. The supernatant was decanted and the pellets containing protein extracts stored at -80 °C for further use.

3.3.4.2 Pressure Disrupted, Pre-Purified Nematocysts Venom was provided by Dr. Angel A. Yanagihara. Specifically undischarged nematocysts were purified and then disrupted en masse using a French Press 20 K pressure cell (SLM-AMINCO Cat# FA078) subjected to a quick 90 s 10,000 × g spin and snap frozen in liquid nitrogen as previously described [3]. The venom was stored at -80 °C for proteomic analyses.

3.3.5 Proteomics Methods Sample preparation for the AB SCIEX 5600 mass spectrometer was performed according to the protocol described previously [236]. Briefly, the venom pellets generated by CDN and PDN were dissolved in 8 M urea, 50 mM ammonium bicarbonate buffer. Sample protein concentrations were determined using the 2D Quant Kit (GE Healthcare, Piscataway, NJ, USA). The equivalent of 200 μg of the CDN and PDN venoms were reduced with 5 mM dithiothreitol at 30 °C for 45 min and alkylated with 25 mM idoacetamide for 30 min at RT in the dark. Samples were diluted 1:4 with 50 mM ammonium bicarbonate buffer followed by tryptic digestion (Sigma–Aldrich, St. Louis, MO, USA) at 100:1 protein: trypsin ratio at 37 °C overnight, before they were freeze-dried. Samples were then diluted with 5% acetonitrile (ACN)/0.1% formic acid (FA) and subsequently desalted with a C18 Toptip (Glygen, Columbia, MD, USA) using: (i) 100% ACN to wet the resin (3 × 150 ml); (ii) 5% ACN/0.1% trifluoroacetic acid (3 × 150 ml) for tip equilibration and washing steps; and (iii) 80% ACN/0.1% TFA (2 × 150 ml) for elution. The eluted protein fragments were freeze-dried and then resuspended in 0.5% acetic acid/2% ACN for further analyses. The proteins fragments were separated by SCX chromatography on an Agilent 1100 chromatography system. 50 μl of each sample were injected onto a Zorbax 300-SCX column (5 μm, 4.6 × 50 mm) (Agilent) at a flow rate of 500 μl/min and gradient of 0–5 min, 0% buffer B; 5–25 min, 0%–50% buffer B; 25–27 min, 50%–80% buffer B; 27–32 min, 80% buffer B; 32–34 min, 80%–0% buffer B. buffer B was held at 80% for 5 min for washing the column and returned to 1% buffer B for equilibration prior to the

46 next sample injection. Buffer A consisted of 0.5% acetic acid/2% ACN and buffer B contained 0.5% acetic acid/2% ACN/250 mM ammonium acetate. A total of 72 fractions (250 μl) were collected and pooled to give 10 fractions. Pooled fractions were de-salted with C18 Toptip (Glygen) and were analysed by LC-MS/MS on a Shimadzu Prominence Nano HPLC (Shimadzu, Kyoto, Japan) coupled to a Triple TOF 5600 mass spectrometer (ABSciex, Concord, ON, Canada) equipped with a Nanospray III interface. The samples were first de-salted on an Agilent C18 trap (0.3 × 5 mm, 5 mm) for 8 min at 30 μl/min. The trap column was then placed in-line with Vydac Everest C18 (300 A, 5 μm, 150 mm × 150 um) column for mass spectrometry analysis. Linear gradients of 10%–60% solvent B over 45 min at 1 μl/min flow rate was used for peptide elution where buffer A consisted of 1% ACN/0.1% FA and buffer B contained 80% ACN/0.1% FA. Full scan TOF-MS data was acquired over the mass range 350–1600 and for product ion ms/ms 40– 1600. The mass spectrometer acquired 500 ms full scan TOF-MS data followed by 20 by 50 ms full scan product ion high sensitivity mode. A collision energy spread (CE = 40 ± 15 V) was used for fragmentation. The acquired MS data were processed using Analyst TF 1.6.1 software (ABSciex). Proteins were identified by database searching using ProteinPilot v 4.5 (ABSciex) with the Paragon Algorithm using FASTA formatted protein sequences for the C. fleckeri translated proteins obtained from the assembled tentacle transcriptome and all publicly available C. fleckeri venom sequences using the UniProt database. Search parameters were defined as a thorough search using trypsin digestion enzyme iodoacetamide cysteine alkylation emphasis on biological modifications and “thorough” search setting. Peptides were considered identified if they could be established at greater than 99.0% probability, and proteins were considered identified if they could be established at greater than 99.0% probability and contained at least 2 identified peptides and all matched spectra were confirmed by manual inspection. The accepted false discovery rates (FDR) for peptides and proteins were less than or equal to 1% [237]. In order compare the protein level and quantitative results across venom obtained by CDN and PDN, the protein summary of both techniques were submitted to Protein Alignment Template (V2.000p, ABSciex, Canada), where the reference protein ID and aligned protein ID were designated to PDN and CDN, respectively. This yielded a high percent of set proteins being matched with the reference proteins, allowing a fair comparison of the high quality ID from each set.

3.4 Conclusions Here we have provided the first detailed description of the C. fleckeri nematocyst transcriptome and used this data to facilitate accurate identification of venom components detected by proteomic techniques. This combination of approaches permitted robust comparisons of the composition of the venom proteome from both pressure disrupted and chemically discharged nematocysts. Whilst both

47 approaches yielded a variety of proteins, our novel approach using ethanol as a chemical stimulus to trigger discharge of jellyfish nematocysts led to the identification of more venom toxins than the pressure disruption technique. Further studies could determine the relative toxicity in terms of hemolytic units (HU50) recovered per microgram of protein obtain by ethanol discharge in comparison to those recovered by pressure disruption. In summary, we have demonstrated that the use of ethanol as a chemical stimulant for discharge and an extraction solvent for obtaining venom proteins from jellyfish tentacles has great potential for future cnidarian venom research. This method facilitates accurate venom protein identification and provides a simple, cost-effective approach that circumvents the time and technical limitations associated with mechanical disruption.

Supplementary Materials Supplementary materials are available online. (http://www.mdpi.com/2072-6651/7/3/936#supplementary)

Chapter 4

Investigating Toxin Diversity in the Tentacle and Oral Arms of the Blue Blubber Jellyfish Catostylus mosaicus by Using a Combined Transcriptomic and Proteomic Analyses

49 Abstract The jellyfish Catostylus mosaicus, known as the Jelly Blubber or Blue Blubber Jellyfish, blooms periodically along the Eastern coast of Australia. Contact with human skin can produce immediate pain, redness and minor skin irritation. It is postulated that both oral arm and tentacle tissues contain stinging nematocysts which are associated with its toxicity. However, no efforts have been made to establish the repertoire of venom proteins and peptides expressed in this remarkable animal. By using a combination of bottom-up proteomics and transcriptome sequencing, the protein profiles of the oral arms and tentacles were studied. The proteomic characterisation demonstrated the toxin diversity and expression differences between these two tissues. In transcriptome analyses, more than 13,000,000 tentacle and 7,000,000 oral arm Illumina reads were de novo assembled into 18,000 and 12,000 contiguous. Using homology searches from publicly available sequence databases, 9,660 tentacle and 4,790 oral arm proteins were predicted with 122 and 94 potential toxin proteins. In the proteomic analyses, over 1200 tentacle and 500 oral arm proteins were identified, including a subset of putative toxins. Potential toxin families were identified using MS/MS included

PLA2, lipases, CRiSPs, cystatins and serine protease inhibitors (Kunitz and Kazal peptides).

Ethanol-extracted venom confirmed the presence of PLA2 and metalloprotease activity. This study revealed venom components of the oral arms and tentacles of a blubber jellyfish for the first time, and furthers our understanding of the mechanism of its sting. Keywords Blue blubber jellyfish, Catostylus mosaicus, venom, oral arm, tentacle, nematocyst, transcriptomics, proteomics, MS/MS

The blubber jellyfish Catostylus mosaicus, collected from Manly, Queensland Australia August 2013. (Photo is provided by Mahdokht Jouiaei from the University of Queensland).

50 4.1 Introduction Catostylus mosaicus, a cnidarian of the class Schyphozoa, is a large gelatinous zooplankter jellyfish with a bell diameter ranging between 50 mm–350 mm [238]. It is one of the most commonly encountered zooplankter in estuaries along the Eastern coast of Australia [239]. Human encounters with this jellyfish are inevitable, especially during the massive periodical population blooms in which the abundance of the jellyfish can increase up to 10-fold within weeks [238]. A sting by this widespread species is not life-threatening, although they do cause pain and minor local tissue redness and inflammation [35] (unpublished observations). Haemolytic, oedema and hemorrhagic induced activities have also been recorded from the crude extract of the homogenized oral arms [240]. The C. mosaicus venom delivery system, nematocyst, is located in the bell margins (tentacles) and oral arms of medusa, in different sizes and/or relative abundances [30]. This suggests that they use diverse organs, and probably diverse toxic substances for prey capture and defence. Surprisingly, in contrast to the extensive work devoted to blubber jellyfish ecology and seasonal bloom, the venom composition as well as diversity of each type of toxin in the oral arm and tentacle remain almost completely unstudied. As the ultimate step in venom characterisation, proteomics technology is a promising tool to identify venom proteins and peptides. Most venomics studies to date have been using whole venom (shotgun) proteomics, 2-D fractionation (ion-exchange followed by HPLC) and SDS-PAGE electrophoresis in order to discover the maximal number of proteins in a sample [14, 91, 99, 241]. In addition, advances in high-throughput sequencing and bioinformatics [145] greatly enhance our abilities to accurately sequence and analyse venom components detected by proteomic techniques. In this study, the combination of transcriptomic and proteomic approaches was applied to systematically identify and compare the venom proteins and peptides in the oral arm and tentacle tissues. In order to extract venom, a previously described venom extraction technique, ethanol- induced discharge of cnidae, was utilised [14] to rapidly and efficiently characterise venom components and to avoid the time and laborious process associated with mechanical disruption of the nematocysts. This study not only provides the first overview of the C. mosaicus tentacle and oral arm transcriptome, but also provides extensive information on the repertoire of putative toxins in C. mosaicus venom.

4.2 Results and Discussion Increased frequency of C. mosaicus blooms, with increased risk of human exposure to envenomation, emphasises the need for accurate characterisation of its venom components for treatment of human stings. However, jellyfish venom research has been neglected. In part, this is due to the difficulty of venom extraction which is a laborious and time-consuming process [14]. In

51 addition, the venom remains heavily contaminated with mucus even after extraction, which makes proteomic study difficult. To facilitate blubber jellyfish venom extraction and identification, a previously described chemical extraction technique consisting primarily of dipping tissue into ethanol was applied [14]. In order to monitor the discharge of the nematocysts, samples of the oral arm and tentacle tissues were taken before (Figure 4-1) and after (Figure 4-2) ethanol treatment. These samples were then studied using scanning electron microscopy (SEM) and light microscopy (LM). SEM examination of the undischarged oral arm revealed an uneven surface packed with nematocyst batteries (Figure 4-1A). The nematocysts appeared to exist in the ridges (formed by nematocyst batteries) when viewed by LM (Figure 4-1B). This study confirmed the presence of four distinct types of nematocysts in both oral arms and tentacle as described previously [30]. These include pear shaped and oval shaped holotrichous isorhizas (isodiametric tubule with spined thorough) (Figure 4-1C–D), and two sized rhopaloids (containing cylindrical shaft) (Figure 4-1E– F). After dipping the oral arms and tentacles into ethanol, the surfaces of both tissues were packed with discharged tubules, shafts and nematocysts, although some nematocysts remained undischarged (Figure 4-2). It should be mentioned that the primary purpose of this research was to identify the venom components of the blubber jellyfish oral arm and tentacle rather than quantify the venom obtained by these tissues. It is reasonable to propose that the effect of ethanol on nematocyst discharge in C. mosaicus is in line with what was previously reported in box jellyfish Chironex fleckeri, suggesting that ethanol can be an affordabale chemical to provide rapid identification of jellyfish venom components [14].

Figure 4-1. Undischarged nematocysts of C. mosaicus. (A) Low magnification scanning electron microscopy (SEM) of an oral arm surface with packed nematocyst batteries; (B) Light microscopy (LM) of a cross- sectioned oral arm nematocyst battery showing holotrichous isorhizas (black arrows) and rhopaloids (white arrows); (C) Pear-shaped holotrichous isorhizae; (D) Oval-shaped holotrichous isorhizae; (E) Large-size rhopaloid. Note the operculum (white arrowhead); (F) LM of rhopaloids. Note the coiled tubule around the shaft. Scale bars, A 100 µm; B, 50 µm; F 10 µm; C, D, E 1 µm.

Figure 4-2. Ethanol-discharged nematocysts of C. mosaicus. (A) Tubules of various oral arm nematocyst types after ethanol treatment; (B) shaft of a discharged rhopaloid; (C) An extruded tubule. Note the spines at the surface of tubule (arrow); (D) Low magnification light microscopy (LM) of a cross sectioned tentacle showing holotrichous isorhizas (black arrows) and rhopaloids (white arrows); (E) Part of the tentacle epidermis showing the extruded tubules (black arrow). Scale bars, A 10 µm; B, C 1 µm; D 50 µm; E 10 µm.

4.2.1 The Transcriptome of C. mosaicus Oral arm and Tentacles To accurately identify the venom proteins detected by proteomics, oral arm and tentacle transcriptomes for C. mosaicus were generated. Next generation sequencing of the transcriptomes using Illumina platform resulted in 13,392,759 and 7,604,059 assembled bases for tentacle and oral arm tissues respectively. Automated de novo assembly resulted in 18,244 and 12,931 transcripts with an average length of 734 and 588 bases per contig. Approximately 52% and 37% of the tentacle and oral arm contigs provided a high scoring (e-value ≤ 10e-5) match to the SwissProt database. To identify potential toxins, assembled transcript were compared to SwissProt toxin database using BlASTX. 122 tentacle and 94 oral arm transcripts showed a high scoring (e-value ≤ 10e-5) sequence homology to known toxins which were further filtered: i) those possessing a low scoring (e-value ≥ 10e-5) sequence homology to a toxin protein from the UniProtKB database search were removed; ii) sequences without a signal peptide as predicted by SignalP were removed. After filtering, 31 and 24 transcripts remained as putative tentacle and oral arm toxins respectively,

54 representing 6 venom families (Figure 4-3A). PLA2 had the highest yield of the venom families, with 18 different isoforms identified. Representatives of other toxin families included lipases, CRiSPs, cystatins, various serine protease inhibitors (Kazal and Kunitz peptides) and metalloproteases (Figure 4-3B).

Figure 4-3. (A) Summary of assembly and annotation of nucleotide sequence data from C. mosaicus tentacle and oral arm tissues. (B) Functional grouping of putative toxin proteins/peptides identified in C. mosaicus oral arm and tentacle transcriptomes.

4.2.2 Identified Proteins in C. mosaicus Oral Arm and Tentacle Using Tandem Mass Spectroscopy (MS/MS) Using a combined proteomic approach (shotgun proteomics, 1D SDS-PAGE, 2-D fractionation), 898 and 1143 proteins in the oral arm and tentacle respectively were identified. The majority were detected using shotgun analysis and 2-D fractionation (Figure 4-4A). The proteins detected included oxido-reductase (oxidases and peroxiredoxins), ATP synthases and ATPases, heat shock proteins, chaperones, ribosomal proteins, hydrolyses and proteases (collagenase metalloproteinase), voltage dependant channel proteins, and putative toxins identified in transcriptome analysis. Among the identified proteins, structural proteins were in high abundance. This includes nematocyst out wall antigen (NOWA) which is a component of the nematocyst capsule in cnidarians [242]. These findings of nematocyst capsule homologues in our samples may suggest the presence of capsule matrix contamination since using ethanol to induce nematocyst discharge may disintegrate the capsule wall. In addition, homologues of vascular endothelial growth factor (VEGF) and receptors in the oral arm and tentacle were detected. The homologous protein has previously been characterised in hydrozoan Podocoryna carnea and is known to be involved in tube formation

55 [243]. Representatives of other structural proteins included actin, myosin, collagen, tubilin, clathrin, deoxyribonuclease with homology to plancitoxin from Crown-of-thorns starfish (Acanthaster planci) [244] and an alpha-macroglobulin containing protein family with homologies to Nematostella, human complement protein C3 and cobra venom factor [245, 246]. These transcripts do not contain a signal sequence and provide high-scoring matches to non-venom proteins in GenBank, which suggests a biological/structural role distinct from envenomation. GO-term annotation of the identified proteins revealed a variety of putative functions and biological processes (Figure 4-4). Level 2 molecular functions were dominated by proteins associated with ‘catalytic activity’ and ‘binding’ (Figure 4-4B). Level 2 biological process GO-terms highly represented among the identified proteins included cellular and metabolic processes (Figure 4-4C).

Figure 4-4. Identified Proteins in C. mosaicus Oral arm and Tentacle Using MS/MS. (A) Total number of proteins identified in in-gel digestion (G), Shotgun (S) and SCX Chromatography (C); (B) Level 2 ‘Molecular Function’ GO-term analysis; (C) Level 2 ‘Biological Process’ GO-term analysis.

56 4.2.3 Toxin Proteins Identified in C. mosaicus Oral Arm and Tentacle Using Tandem Mass Spectroscopy (MS/MS) Whilst many protein types identified in this organism were clearly associated with conserved functions related to structure, transport and metabolism, a number of other proteins likely represented putative venom toxins. Phospholipases and lipases were the most abundant classes of toxins, followed by proteins containing ShK domain (CRiSPs), cystatins and serine protease inhibitors. It should be mentioned that it is difficult to estimate the diversity of oral arm and tentacle venom components from SDS-PAGE electrophoresis due to the extent of mucus contamination, preventing protein migration through the gel. In addition, individual proteins can have variable mobility, depending on the degree of post-translational modification (Figure 4-5).

Figure 4-5. C. mosaicus venom proteins. Total protein from oral arm (lane 1) and tentacle (lane 2) in a 12% SDS-PAGE gel. Lanes were divided into 15 gel slices (dotted lines) and subjected to in-gel digestion and protein identification. The numbers in each gel refer to the proteins displayed in Table 4-1.

4.2.3.1 Secretory Phospholipase A2 (sPLA2)

Several proteins with conserved sPLA2 domain (IPR016090) were identified in the oral arm and tentacle venoms (Table 4-1). PLA2 proteins consist of a large family of enzymes with 15 distinct scaffolds and many subgroups [247]. PLA2 scaffolds have been widely distributed within the animal kingdom where they play important roles in vital physiological and pathological functions such as signal transduction, dietary and endogenous phospholipid catabolism, and inflammation

[94]. Group III PLA2 toxins are particularly remarkable in that they have been independently recruited in several venomous animal lineages. Derived toxic functions include neurotoxic activity in honeybee (Apis mellifera) venom in which they specifically bind to N-type receptors in brain membrane [248], anti-platelet activity in Heloderma venoms [249], haemolysin in Eastern Indian

57 scorpion (Mesobuthus tumulus) venom [250], inhibition of both skeletal and cardiac ryanodine receptors (calcium release channels) in Emperor scorpion (Pandinus imperator) [251], allergic reaction to bumblebee (Bombus terrestris) venom [252] and apoptotic cell death in bumblebee

(Bombus ignitus) venom [253]. The first cnidarian GIII PLA2 was isolated and partially sequenced from jellyfish Rhopilema nomadic (Schyphozoa) and a toxic function to fishes was proposed [40].

In addition, EST homologous to bee venom GIII PLA2 was identified in Nematostella vectensis [98] and Hydra magnipapillata [254]. In C. mosaicus the hemolytic activity of the oral arm is proposed to be associated with PLA2 present in the extract [255]. However, extensive studies need to be conducted in order to determine whether protease activity is associated with haemorrhagic activity. Strikingly, the putative venom PLA2 identified in the oral arm and tentacle show high levels of sequence identity (47–48%) to Gila monster (Heloderma suspectum) venoms which belong to GIII PLA2. In addition, the putative venom protein shows 38%–45% sequence identity to other toxic GIII PLA2s from the venoms of honeybee (Apis spp.), bumblebee (Bumbus spp.), and scorpion venoms. A PLA2 homologue in the oral arm and tentacle venoms contains a signal peptide of 16 amino acids. The mature protein has a theoretical molecular size of 15.31 kDa and isoelectric point of 6.42. The mature peptide consists of 136 amino acids including 8 cysteine residues. More importantly, core residues involved in catalytic activity (PLA2 histidine active site: C-C-(any but Proline) -x-H193-(any but LGY)-x-C) and Ca2+ binding site (W167, G169, G171, D194) are conserved (Figure 4-6).

58 Figure 4-6. Sequence alignment of GIII PLA2 precursors from 1. Bombus terrestris (P82971), 2. Apis mellifera (P00630), 3. Catostylus mosaicus (Contig 2336-6795), 4. Hydra vulgaris (T2M479), 5. Heloderma suspectum (P16354), 6. Nematostella vectensis (A7RM30), 7. Mesobuthus tamulus (Q6T178).

Two isoforms of GIX PLA2 proteins were identified in the C. mosaicus oral arm. GIX PLA2 is basic proteins with a theoretical isoelectric point of ~9 and molecular weight of ~18. Both putative venom proteins contain N-terminal signal peptide for secretion, 10 conserved cysteine residues for putative disulphide formation, and conserved histidine in the site of catalytic activity (Figure 4-7).

The first GIX PLA2 venom protein was described in the venom of the marine snail Conus magus

(UniProt: Q9TWL9). Interestingly this toxin did not show PLA2 enzymatic activity and its toxicity was instead the blocking of L-type Ca channels in rat brain [256]. Cnidarian EST homologues to

Conus GIX PLA2 have been identified in the jellyfish Rhopilema esulentum (Schyphozoa)

(UniProt: X2G7F0) and N. vectensis [98]. Noticeably, the putative PLA2 venom proteins in C. mosaicus showed high level of sequence identity to cnidarian GIX PLA2 homologues. A high degree sequence identity with venom GIII and GIX PLA2s suggests that putative PLA2s may contribute to toxic activity of C. mosaicus (such as hemolytic or neurotoxic activity), and should therefore be further investigated to characterise their structure-function relationships and role in blubber jellyfish envenoming.

Figure 4-7. Sequence alignment of GIX PLA2 precursors from 1. C. mosaicus (Contig 5027), 2. C. mosaicus (Contig 2769), 3. Rhopilema esculentum (X2G7F0), 4. Nematostella vectensis (A7RMJ0), 5. Conus magus (Q9TWL9).

4.2.3.2 Lipases Two isoforms of lysosomal acid lipase / cholesteryl ester hydrolase were detected in the tentacle venom (Table 4-1). These putative venom proteins have the same domain structure as found in characterised snake venom lipases and non-venom cnidarians ESTs (Figure 4-8). They are large proteins with a molecular weight of ~44 kDa and a signal peptide. Although in venom, the biological function is unknown; their presence in the proteome suggests that they may include roles in the digestion of prey.

Figure 4-8. Sequence alignment of lipid-precursors from 1. C. mosaicus (Contig 1781), 2. C. mosaicus (Contig 5698), 3. Crotalus adamanteus (J3SDX8), 4. Hydra vulgaris (T2MGH6), 5. Nematostella vectensis (A7SL62).

4.2.3.3 Putative CRiSP C. mosaicus Venom Proteins Three proteins detected in the C. mosaicus tentacle and one protein in oral arm tissue belongs to Cysteine Rich Secreted Proteins (CRiSP) superfamily (Table 4-1). CRiSP related proteins have been convergently recruited in the venoms of reptiles, ticks, cone snails, scorpions, spiders, cephalopods, insects (proboscis and stinger) and vampire bats [90, 91]. They are multidomain proteins containing N-terminal CAP (CRiSP, antigen 5 [Ag5], pathogenesis-related 1 [Pr-1] domain (s)) and a C-terminal cysteine-rich domain (except for cephalopods) that inhibit a number of ion channels [90]. It has been suggested that the co-operation between CAP and cysteine-rich domains facilitates the interaction with the ion channels [257]. CAP proteins in the cleractinian corals (Cnidaria, Anthozoa) and snakes are proposed to be involved in neurotoxicity [80, 257-260]. The blubber jellyfish CRiSP proteins showed high level of sequence similarity to CRiSP from Malo kingi, N. vectensis and snake venom [258, 259] (Figure 4-9). The cysteine-rich C-terminal domain (IPR003582 ShKT) contains six cysteine residues with a distinctive XCXD pattern terminates, with the characteristic pattern CXXTCXXC that is homologues to sea anemone voltage-gated potassium (Kv) channel toxins [261, 262].

Figure 4-9. Sequence alignment of CRiSP precursors from 1. C. mosaicus (Contig 5022-tentacle), 2. C. mosaicus (Contig 7484-tentacle), 3. C. mosaicus (Contig 3410-tentacle), 4. Catostylus mosaicus (Contig 2043-oral arm), 5. Nematostella vectensis (A7T1T5), 6, Malo kingi (B1A0E5), 7. Heteractis magnifica (O16846), 8. Anemonia erythraea (Q0EAE5), 9. Protobothrops jerdonii (Q7ZZN9), 10. Trimeresurus stejnegeri (P60623). Disulfide bridges in the CRD are indicated by lines.

Considering that the identified C. mosaicus CRiSP proteins have putative signal peptides and cysteine-rich C-terminal domain with a homologous ShK toxin, it can be suggested that jellyfish CRiSP proteins may represent a part of the animal allomonal system. It also suggests that the CRD

62 domain may have been independently recruited into multiple domain proteins with differing functions to facilitate the specificity and affinity of venom proteins for their targets.

4.2.3.4 Putative Venom Cystatin A member of family 2 cystatins was detected in the crude venom extract from C. mosaicus tentacle and oral arm (Table 4-1). Cystatins are papain-like cysteine protease (CP) inhibitors that are isolated from various tissues and fluids from diverse organisms. They play an important regulatory role in inhibition of endogenous and pathogen CPs for protection of cells against tissue injury and invaders [263]. They have been found in a wide range of venomous animals including jellyfish [264], stingray [265], snake [266], tarantula [267] and haematophagus ticks [268]. Mutagenesis and X-ray crystallography studies indicate there is an N-terminal loop consisting of a glutamine valine- glycine (Q-X-V-X-G) residue and a C-terminus prolin/serin-typtophan (P/S-W) in the form of a wedge-shaped structure that is essential for inhibition of the active site of C1 family cysteine proteases [264, 269, 270]. There is no evidence that cystatins are involved in venom toxicity, however they may protect the venom proteins/peptides from inactivation by defensive enzymes of the envenomated victim [271]. Here, the cystatin identified in blubber jellyfish venom contains a putative 18-residue signal peptide and a mature protein of 129 amino acids with two intra-molecular disulfide bonds, which shows a high level of sequence identity (30-42%) to family 2 cystatins (Figure 4-10A). It has a molecular weight of 14.54 kDa and isoelectric point of 8.67. Despite the similarities, our phylogenetic analysis of the cystatin gene families revealed that blubber jellyfish cystatin is non-homologous to the cystatins previously recovered from other venomous animals (Figure 4-10B). This data suggests that cystatins have been recruited into venoms on multiple occasions, a finding which is in accordance with the previous study [265]. The fact that the same gene families are recruited several times independently suggests that the non-toxic activity of these proteins have become toxic via gene-duplication, accompanied by adaptive evolution [90].

Figure 4-10. (A) Sequence alignment of cystatin-precursors from 1. C. mosaicus (contig 2707-7304), 2. Cyanea capillata (Q331K1), 3. Chilobrachys guangxiensis (B1P1J3), 4. Pogona barbata (Q2XXN5), 5. Oxyuranus microlepidotus (E3P6N5), 6. Crotalus adamanteus (J3RYX9), 7. Homo sapiens (Q15828), 8. Gallus gallus (P01038), 9. Oncorhynchus keta (Q98967). (B) Precursor phylogenetic reconstruction. Branches highlighted in red indicate cystatins that have been recovered or cloned from the venom glands of venomous animals (snake, cnidarian, stingray, tick).

64 4.2.3.5 Serine Protease Inhibitors Several peptide types with plesiotypic serine protease-inhibiting activity (Kunitz and Kazal peptides) were identified in the venom extract of both tissues (Table 4-1). Kunitz are serine protease inhibitors that have been convergently recruited into the venom and oral secretions of a wide range of animals, exhibiting anticoagulation activity in vampire bat secretions [91], brown snake (Pseudonaja textilis textilis) venom [272] and cattle tick [273]; antifibrinolytic activity in bumblebee (B. terrestris) venom [274], and calcium / potassium channel blocking activity in the venoms of eastern green mamba [275] and scorpion [276]. Notably, they have also been identified previously in cnidarian taxa. In the venom arsenal of sea anemones they exhibit a plesiotypic activity of trypsin-inhibition, but possess additional apotypic activities including potassium channel blockage [31, 76, 78, 277, 278]. BLAST searches of the Kunitz peptide detected in this study surprisingly retrieved Kunitz homologues in snakes, scorpions, bumblebee and sea anemones, where their toxic activity have been well characterised (Figure 4-11). The mature peptide has common features with other known secreted venom Kunitz such as N-terminal signal for secretion, 6 cysteine for disulphide formation and conserved residues in the site of catalytic activity. It has a theoretical molecular weight of 7.36 kDa and isoelectric point of 7.73. This peptide is one of the most abundant of those detected in 1D SDS-PAGE (Band 1, Figure 4-5, Table 4-1). Considering these observations, this form of toxin may represent the first Kunitz peptide that has been identified in the venom of a Schyphozoa jellyfish, C. mosaicus.

Figure 4-11. Sequence alignment of kunitz precursors from 1. C. mosaicus (Contig 12914), 2. Heteractis crispa (P0DMJ5), 3. Anemonia sulcata (Q9TWF8). 4. Actinia equina (P0DMW6), 5. Bombus terrestris (D8KY58), 6. Hadrurus gertschi (P0C8W3), 7. Dendroaspis angusticeps (P81658).

A mono domain kazal serine protease inhibitor with homology to coral Melithaea caledonica (UniProt accession number P82968) was discovered in the oral arm and tentacle venoms (Figure 4- 12). The mature peptide has a theoretical molecular weight of 15 kDa which is detected in SCX as

65 well as 1D SDS-PAGE (Band 4, Figure 4-5, and Table 4-1). It has been proposed that coral protease inhibitor inhibits trypsin, kallikrein, subtilisin Carlsberg, human leukocyte elastase, porcine pancreatic elastase and chymotrypsin. In addition to venoms for which enzyme activity has been reported, a five-domain protease inhibitor has also been identified in the venom of box jellyfish (C. fleckeri) venom [14], but the corresponding enzyme activity has not yet been obtained. It can be hypothesised that as a member of the venom proteome which is presumably injected during a sting, serine protease inhibitors could contribute to the symptoms of envenomating by inhibiting the rapid degradation of the venom protease by endogenous enzymes of the animals themselves or of the prey [279].

Figure 4-12. Sequence alignment of kazal precursors from 1.Catostylus mosaicus (Contig 4371-3114), 2. Melithaea caledonica (P82968), 3. Chironex fleckeri (Contig 134).

66 Table 4-1 Summary of venom proteins identified in the oral arm and tentacle of C. mosaicus.

Best Transcriptome Venom hit Oral ID Tentacle Conserved domain Known function Accession arm

# PLA2 T, S, Neurotoxic [40, 2336 C, G _ 248], inhibition of (4) Phospholipase A2 domain Ca release [251], (IPR016090); anti platelet [249], P80003 Phospholipase A2, active site hemolytic [250], T, S, 6795 _ (IPR013090) allergen [252], C apoptotic cell death [253] Catalyses the T, S, Phospholipase A domain 2769 2 hydrolysis of the G (1) (IPR016090); Q9TWL9 _ sn-2 ester in a Phospholipase A , active site 2 calcium dependent 5027 T, C (IPR013090) manner [256] Lipases Lipase eukaryotic IPR025483); Alpha/Beta hydrolase fold 1781-5698 J3SDX8 _ T, S (IPR029058); Partial AB- Unknown hydrolase lipase domain (IPR006693) CRiSPs Cysteine-rich secretory protein, 5022-7484- T, S, C, _ allergen V5/Tpx-1-related 3410 G (22) (IPR001283); CAP domain Neurotoxic Q7ZZN9 (IPR014044); ShKT domain [80, 257-260] T, S, (IR003582); Allergen V5/Tpx-1- 2043 C, G _ related, conserved site (7,8) (IPR018244) Cystatins T, S, 2703 _ Proteinase inhibitor I25, cystatin Potential protease Q331K1 G (3) (IPR000010) inhibitor [271] 7304 _ T, C Serine Protease inhibitors Pancreatic trypsin inhibitor Protease inhibitor, T, G Kunitz domain(IPR002223); neurotoxin Kunitz-12914 P0DMJ5 _ (1) Proteinase inhibitor I2, Kunitz, [31, 76, 78, 277, conserved site (IPR020901) 278] T, C, Kazal-3114 G (4) Kazal domain P82968 Unknown (IPR002350) Kazal-4371 _ T, C

NOTE. The type of evidence used for identification: Transcriptome (T), Shotgun (S), SCX Chromatography (C), and in-gel digestion (G).

67 4.2.4 Enzymatic Activity Next the enzymatic activity was assessed to determine if treatment with ethanol during the chemical extraction process has an effect on PLA2 and metalloproteases activity (n = 3). The in vitro PLA2 assays with ethanol-extracted C. mosaicus oral arm and tentacle venoms produced a slight increased in absorbance (Figure 4-13A). Consistent with transcriptomic and proteomic findings, both oral arm and tentacle venoms exhibited PLA2 activity, while the oral arm showed higher activity than the tentacle (Figure 4-13B). Subsequently the metalloprotease activities of the oral arm and tentacle crude venom were assessed on gelatine and casein substrates. Consistent with the transcriptomics, intense enzymatic activity was observed in tentacle crude venom between 20–25 kDa when gelatine was used as a substrate (Figure 4-13C). No activity was observed in oral arm crude venom. Although in this study no metalloproteases were detected in the C. mosaicus proteome, one astatine-domain containing metalloproteases was identified in the tentacle transcriptome. It contains a signal peptide with 35 amino acid residues and a theoretical molecular weight of 23 kDa which shows a high level of sequence identity (32–43 %) to the genus Loxosceles venoms and N. vectensis venom (NEP-6). Astasin-like proteins with toxic activity are recruited in the venom from spiders of the genus Loxosceles [280] where they may be involved in degrading the extracellular matrix, thereby providing other venom toxins with better access to their targets. Alternatively they might play a role in proteolytic processing of other venom toxins or digestion of prey [228]. In cnidarians, astacin domain proteins are found in the nematocyst and pharyngeal gland of the sea anemone N. vectensis [48], nematocysts of the Box jellyfish C. fleckeri [14] and hydrozoans Hydractinia echinata [281], H. magnipapillata [282] and Podocoryne carnea [283]. Although their role in envenomation remains to be determined, they possibly share a role in regulation of developmental processes and have acquired a special role as digestive enzymes or proteolytics for the activation of the toxin domain [48, 283].

Figure 4-13. Enzymatic activity of C. mosaicus. (A) Absorbance times for C. mosaicus oral arm and tentacle (100 µg). (B) The sPLA2 activity (µmol/min/mg) for each venom. Values presented as a mean of three replicates (n = 3), error bars indicate SD. (C) Tentacle (lane 1) and oral arm (lane 2) venom separated using a 10% Zymogram PAGE, gelatine substrate. No proteolytic activity was detected in the oral arm venom (lane 2) with gelatine substrate. The band with metalloprotease activity is shown in red. The gels were stained with Colloidal Coomassie Brilliant Blue G250.

4.3 Experimental Section 4.3.1 Specimen Collection Nine mature live specimens of C. mosaicus were collected by dip net from Manly Beach, Queensland, Australia, during the summer 2013, 2014 and 2015 by Mahdokht Jouiaei, Scott C. Cutmore and Bryan G. Fry. For transcriptome analysis, equal proportions of fresh tentacles (CMF) and oral arms (CMT) from each specimen were collected manually and pooled to remove the effect of individual variability. The pooled samples were immediately frozen in liquid nitrogen before storage at -80 °C until use.

4.3.2 Nematocyst Morphology Sample preparation for histology and cryo-SEM studies as described previously [14].

4.3.3 RNA Extraction and Transcriptome Library Construction Total RNA was isolated from 200 µg of the pooled frozen tentacles and oral arms using the standard TRIzol Plus RNA purification kit (Life Technologies, Carlsbad, CA, USA). RNA quality was assessed using a Bioanalyser (Agilent, Santa Clara, CA, USA). A total of ~500 ng Messenger RNA from each tissue was isolated and purified using standard Dynabeads mRNA DIRECT Kit (Life Technologies, Oslo, Norway). A cDNA library was constructed by fragmentation of mRNA with divalent cations and heat, synthesis of double-stranded cDNA, DNA fragment end repair

69 (blunt ending of DNA fragments), 3' adenylation of DNA fragments, sequencing adapter ligation (utilising T-A pairing of adapter and DNA fragments), followed by amplification of library using PCR. cDNA sequencing was carried out on an Illumina MiSeq machine (Australian Genome Research Facility), generating 7,604,059 and 13,392,759 reads for oral arms and tentacles consecutively. Paired-end read data was assembled into 12,931 and 18,244 contigs using CLC Genomics platform (Workbench 8.0.1) with the minimum transcript length set to 200bp. Assembled contigs were annotated with the AGRF’s Expressed Sequence Tag Database servers using the BLASTX algorithm with a significance threshold of 1e-3, to provide BLAST annotations against SwissProt protein database. Some sequences annotated ambiguously were confirmed using Expasy (UniProt Knowledgebase (Swiss-Prot + TrEMBL), Uniref100) comparison. Contigs were then translated using CLC Genomics Workbench 5 (CLC bio, Aarhus, Denmark) to all 6 reading frames to provide a sequence database for the proteomic characterisation of the venom components. Additionally, sequences identified proteomically were scanned for the presence of signal peptides with SignalP 4.1 Server (http://www.cbs.dtu.dk/services/SignalP/) [234]. Alternatively the non-classically secreted proteins were predicted by Secretome 2.0 Server (http://www.cbs.dtu.dk/services/SecretomeP/). Propeptide cleavage site was predicted using ProP 1.0 server (http://www.cbs.dtu.dk/services/ProP/ ), and the functional domains and gene ontology (GO) were checked with InterPro (http://www.ebi.ac.uk/interpro/) [235] and Conserved domain Architecture Retrieval Tool (http://www.ncbi.nlm.nih.gov/Structure/lexington/lexington.cgi). The theoretical molecular weight was also calculated with ExPASy Compute pI/Mw tool (http://web.expasy.org/compute_pi/).

4.3.4 Venom Extraction The venom from each specimen was collected by ethanol-induced discharge [14]. The fresh tentacles and oral arms were immersed in 1:4 absolute ethanol for 30 s at room temperature. The ethanol-containing pooled venom was then kept at -80 °C overnight to precipitate the proteins and peptides. The solution was then centrifuged at 14,000 × g, at 4 °C for 30 min. The supernatant was removed and the protein pellets containing protein and peptides extracts stored at −80 °C for further use.

4.3.5 Proteomics 4.3.5.1 One dimensional (1D) SDS-PAGE Initially it was found that the venom had a high concentration of salt which interrupted the gel electrophoresis. Thus, the venom solutions from tentacle and oral arm were desalted with Visaspin Concentrators (GE Healthcare, UK) with a molecular weight cut-off of 3,000 Da. The final material 70 was then quantified by Nanodrop spectrophotometry (Thermo Scientific, Wilmington, DE, USA) and freeze-dried. Using protocols previously described [241, 265] 40 µg of lyophilised venom proteins from each tissue were reconstituted in purified water and laemllie sample buffer (BIO- RAD, USA), reduced by heating at 100 °C for 4 min, loaded onto a 12% polyacrylamide gel and covered with tris-glycine running buffer. Electrophoresis was performed with a constant voltage of 90 V for approximately 90 min. The gel was removed and stained in 0.2% colloidal Coomassie brilliant blue G250 (BIO-Rad USA) overnight and destained in 1% acetic acid/H2O.

4.3.5.2 Protein digestion Using protocols previously described [265] in-gel tryptic digestion and extraction was undertaken to extract peptides for mass spectrometry analysis. The visible 1D bands were picked and destained with 50 mM NH4CO3/50% ACN before reducing with 10 mM DTT (30 min, 60 °C) and alkylating with 55 mM idoacetamide (30 min at RT and in the dark). The bands were briefly rinsed in 50 mM ammonium bicarbonate for 1-2 min twice followed by dehydrated in 100% ACN. The digestion was performed with trypsin (Sigma–Aldrich, St. Louis, MO, USA) at 100:2 protein: trypsin ratio at 37 °C overnight. Subsequently the digested proteins were extracted from the gels by using 20 µl of 1% formic acid (FA) for 20 min at RT followed by 20 µl of 5% ACN / 1% FA for 20 min at RT. For shotgun sequencing, the venom pellets from each tissue were resuspended in freshly prepared 8 M urea, 50 mM ammonium bicarbonate buffer at 1:10 (w:v) and vortexed briefly for 2 min at room temperature. The venom solutions were then centrifuge at 15,000 × g for 10 min to remove undissolved cell pellet, and protein concentration in supernatant containing the soluble venoms were determined using the 2D Quant Kit (GE Healthcare, Piscataway, NJ, USA). Using protocol previously described [14], crude venoms (equivalent of 5 µg of protein from oral arm and tentacle) were first desalted with a C18 Toptip (Glygen, Columbia, MD, USA) and freeze-dried before they were solubilised with 20 µl of Milli Q and 2.5 µl of 1 M ammonium carbonate. A total of 25 µl per sample (97.5% ACN, 2% idodethanol and 0.5% triethylphosphene) was added followed by incubation at 37 °C in the dark for 2 hours. Samples were concentrated using a speed-vacuum centrifuge and resuspended in 12 µl of 40 mM ammonium bicarbonate buffer before tryptic digestion (Sigma–Aldrich, St. Louis, MO, USA) at 100:5 protein: trypsin ratio at 37 °C overnight. Sample preparation and digestion prior to SCX chromatography was performed on 400 µg of the venom from oral arm and tentacle using protocol previously described [14]. Prior to MS analysis, digested crude venoms, in-gel digested proteins and pooled fractions from SCX chromatography were de-salted using C18 Toptip to avoid overloading the detector. They were then concentrated with a speed-vacuum centrifuge to remove residual acetonitrile (ACN) and resuspended in 100 µl of 5% ACN/1% FA.

71 4.3.5.3 LC− ESI−MS/MS Using protocols previously validated [14], venoms were separated on a Shimadzu Prominence nanoLC system (Shimadzu, Kyoto, Japan) and analysed on a Triple-TOF 5600 instrument (ABSciex, Concord, ON, Canada) equipped with a Nanospray III interface. Samples were first desalted on an Agilent C18 trap column (300 pore size, 1.8 µm, 2.1 mm × 100 mm) at 400 μl/min flow rate for 15 min. The trap column was then placed in-line with Vydac Everest C18 (300 A, 5 μm, 150 mm × 150 um) column for mass spectrometry analysis. Linear gradients of 10%–60% solvent B (90% ACN, 0.1% FA) in 1% ACN/0.1% FA over 45 min at 1 μl/min flow rate was used for peptide elution. IDA injections were analysed in duplicates (technical replicates). The mass spectrometer acquired 500 ms at a rate of 20 scans per second with a cycle time of 50 ms full scan product ion data in an Information Dependant Acquisition (IDA) mode. Precursor ions were selected between 40 and 1800 m/z with an intensity of at least 200 counts per second and a charge state of +2 to +5. The data was acquired and processed using PeakView® version 2.1 (ABSciex, Forster City, CA).

4.3.6 Bioinformatics Analysis Mass spectrometry (MS) data from IDA analyses were combined and searched using ProteinPilot software v4.5 (ABSciex, Forster City, CA) with the Paragon Algorithm using FASTA formatted protein sequences for the C. mosaicus translated proteins obtained from the assembled tentacle and oral arm transcriptomes. Search parameters were defined as trypsin digestion, iodoethanol as cys- alkylation (for shotgun) and iodoacetamide (for ID SDS-PAGE and SCX chromatography), FDR analysis, and emphasis on biological modifications and “thorough” search setting. The protein identifications were accepted if they could be established at greater than 99.0% probability and contained at least one identified peptide with more than 99% confidence, and all matched spectra were confirmed by manual inspection to eliminate false positive. The 1% false discovery rates (FDR), at the peptide and protein levels, were included in the statistical analyses.

4.3.7 Phylogenetic Analyses Toxin transcripts identified in the proteome were blasted against the UniProt database (http://www.expasy.org/tools/blast/) to identify putative venom and non-venom gene homologues. To minimize confusion, all sequences obtained in this study are referred to by their Contig numbers, and homologues sequences from previous studies are referred to by their UniProt/Swiss-Prot accession numbers (http://www.expasy.org/cgi-bin/sprot-search-ful). Amino acid sequences were aligned using CLC Main Work Bench 6 (CLC-Bio) using default parameters and very accurate (slow) alignment. When presented as sequence the signal peptides are shown in lowercase letters and cysteines are highlighted in black; > and < indicate incomplete N/5′ or C/3′ ends, respectively. 72 The alignments were inspected visually for errors. Bayesian phylogenetic analysis performed on the amino acid data sets allowed the reconstruction of the molecular evolutionary histories of putative C. mosaicus toxins. Bayesian inference implemented in MrBayes 3.2.3 [284] was used by running a minimum of 15 × 106 generations in 4 chains and every 100th generation was sampled. The analysis was performed using lset rates = invgamma with prset aamodelpr = mixed command to optimise between the 9 amino acid substitution matrices implemented in MrBayes.

4.3.8 PLA2 Activity Testing

Hydrolysis of the thio ester bond at the sn-2 position by PLA2 was performed using secretary PLA2

(sPLA2) Assay Kit (Cayman Chemical Company, Ann Arbor, MI, USA). Briefly, Diheptanoyl Thio-PC was used as a substrate in an Assay Buffer containing 25 mM Tris-HCl, pH 7.5, 10 mM

CaCl2, 100 mM KCl and 0.3 mM Triton X-100. Free thiols were detected using DTNB (5, 5’dithio- bis-(2-nitrobenzioc acid)). Bee venom was used as positive control. 10 µl aliquots (1, 10, 20, 100 µg) of the desalted samples were added to two wells in 96-wall plates contacting 10µl DTNB (10 mM in 0.4 M Trsi-HCl, pH 8.0), and 5 µl Assay Buffer. The reaction was initiated by adding 200 µl substrate solution (1.66 mM) to all the walls. Absorbance readings were recorded at 405 nm per min using a BMG FLOUstar OPTIMA (BMG LABTECH Germany) to obtain 10 time points. The PLA2 activity, micro-moles of fatty acid released per minute per milligram of protein, was calculated using the equation:

2 (μmol/min/mg) = Δ 405/min10.66 −1 × 0.225 /0.01 sPLA2 activity was expressed as mean ± SD and analysed using GraphPad prism 6.0. To obtain reproducible results, only the blubber jelly venom PLA2 measurements that are in the detection range of the assay (equivalent of an absorbance increase of 0.01 to 0.1 per min) were reported.

4.3.9 Metalloprotease Activity Testing In order to investigate crude venom for proteolytic activity, Zymogram PAGE was performed on 10% (gelatine) and 12% (casein) substrates. Samples of 40 µg concentrations were prepared for each tissue. Methods followed those supplied; Bio-Rad Zymogram PAGE protocol with ready- made 10% gelatine and 12% casein gels. Gels were stained in coomassie brilliant blue G-250 for 1 hour and stored in 100 ml Milli Q. Gelatinases, Matrix Metalloprotease (MMP) 2 and MMP 9 can be detected via 10% Zymography, while MMP 1, MMP 7, MMP 12, and MMP 13 are detected via 12% Zymography. By performing both zymography tests with the same venom under the same conditions, direct comparison between venoms can be made.

73 4.4 Conclusion and Future Directions The principle aim of this study was to identify venom proteins in tentacle and oral arms of C. mosaicus that could be responsible for its toxicity. Through the transcriptomic and proteomic investigations, the oral arm and tentacle venoms were shown to share a number of the same protein toxins. However, the oral arm venom was found to express Kunitz peptide while tentacle expressed two isoforms of lipases and one isoform of metalloprotease. These differences may have resulted from selective discharge of nematocysts within individuals in response to various mechanical, chemical and neural stimuli. If jellyfish can respond differently to various stimuli, then differences in transcripts between oral arm and tentacle could account for the observed variations in venom proteomes, as transcripts are a snapshot in time of any toxin that the venom gland is coding. No studies have yet investigated this hypothesis. The enzymatic activities of the venom toxins during the ethanol extraction process were confirmed using PLA2 and metalloprotease assays in vitro. The results demonstrate that addition of ethanol does not completely inhibit activity and the venom retains bioactive. Therefore, denaturing is not a significant issue during the extraction process as long as enzymes have a high disulphide content which stabilizes their secondary structure or they are highly glycosylated, which protects the enzymes from denaturing. Further studies should determine the extent of the enzymatic activity when ethanol is added compare to the control (untreated) venom.

Chapter 5

Pharmacological Profile of Venom Isolated From Hell’s Fire Anemone Actinodendron sp.: A Preliminary Study

75 Abstract This study contributes to the areas of proteomics, pharmacological properties and biodiscovery potentials of hell’s fire anemone (Actinodendron sp.), for which there are, to our knowledge, very few published studies. Using a combination of LC-ESI-MS, MS/MS and selected bioassays to monitor chromatographic fractionation, we isolated and characterised two bioactivies, a novel PLA2 and α3β2 nAChR blocker from the venom of hell’s fire anemone. The molecular weight of the isolated PLA2 was 14.7 kDa and showed homology to previously characterised sea anemone

Condylactis gigantean PLA2. Interestingly, after 30 min incubation of the sheep RBC with the isolated toxin, the indentation edges appeared on the cell surfaces. This finding was consistent with the previous studies of the effect of PLA2 toxin on erythrocyte morphology changes and suggests the possible contribution role of this toxin to human envenomation symptoms. The isolated α3β2 nAChR blocker was the first report of the presence of this toxin in anemone venom. This observation suggests that cnidarians may represent evolutionary precursors of nAChR of younger lineages. In this study the unique approach of investigating novel toxins from hell’s fire anemone, genus Actinodendron, has shed light upon the diversification and biodiscovery potential of its venom composition. Keyword Hell’s fire anemone, Actinodendron sp., activity-guided fractionation, therapeutics

The hell’s fire anemone Actinodendron sp., UQBR Aquaria (GC Lawrence and M Jouiaei)

76 5.1 Introduction In the marine world, there are many venomous creatures utilising their toxins to capture and immobilise prey and/or for protection from predators. Amongst these animals are sea anemones, a group of predatory animals of the phylum Cnidaria, class Anthozoa. Although the majority of the sea anemones are harmless to human, few species are highly toxic producing a venomous cocktail that can cause severe neurotoxic, cytotoxic and hemolytic effects [279]. In spite of this, the biodiversity and the remarkable pharmacological properties of venom can be a valuable bioresource of potential drug candidates [73, 219]. The genus Actinodendron contains several sea anemone species which are found in the Indo-Pacific and coastal habitats. These animals are named “hell’s fire anemones” because of their highly potent venoms which can cause a very painful sting upon contact with human skin (unpublished data). Although their venom can have adverse impact on human activities, both recreational and occupational, the hell’s fire anemones have been poorly studied regarding their taxonomic status and venom composition. Until now, only one study has shown the pharmacological properties of the isolate sodium channel blocker toxin from an Actinodendron sp. [198], while neglecting the venom profile and biodiscovery potentials of its venom. Recent technological advances in proteomic and genomic (transcriptomic) techniques have provided a complete picture of the venom and a promising tool for drug discovery. Utilising these new and improved techniques has significantly increased our understanding of the remarkable venom complexity of several jellyfish species [14] (see chapter 3 and 4). When genomic database (and therefore theoretical protein databases) is not available, activity-guided fractionation can be useful to facilitate characterisation of active toxins (bioactivies) in whole venom [146, 285]. For example, screening of the fractioned crude venom from Conus sp. resulted in the identification of a novel toxin [285]. This approach, although effective, considerably depends on advanced proteomic and screening techniques, thus it is time-consuming and relies on operator expertise for analysing complex data. In this study, a combination of chromatography, mass spectrometry and high-throughput screening (HTS) techniques were applied to identify bioactivies in the venom of hell’s fire Actinodendron sp. It should be mentioned that Actinodendron species-specific characteristics are not yet described; hence we name here the anemone just as Actinodendron sp.

5.2 Result and Discussion 5.2.1 Proteomics Given the lack of genomic information available, venom complexity was determined using an Orbitrap-based mass spectrometer. Crude venom analysis recovered several toxin types with

77 functional diversities consistent with predatory/defence effects including lethal effects upon cytolytic, cardiotoxic, hypertensive, hemorrhagic, allergic reaction and neurological function. These toxins showed a high diversity of molecular weight, ranging from small neurotoxins with less than

10 kDa to larger proteins over 150 kDa (Table 5-1). To further investigate the biological properties of hell’s fire venom and isolate venom proteins of interest, crude venom was semi-fractioned using molecular weight cut-offs of 10 kDa and 30 kDa. The venom samples (< 10 and 10– < 30 kDa) were then analysed for bioactivity and subjected to RP-HPLC fractionation to isolate novel toxins. Table 5-1. Summary of venom proteins identified in Actinodendron sp. tentacle. Best Venom hit ~MW Toxin Type Function Accession # (kDa) Enzymes Cytolytic, hemolytic, inflammation, prey Phospholipase A2 A7LCJ2 14-17 digestion [286] Metalloproteinase Hemorrhagic toxin [287] Q3HTN2 67 Peptidase M13 Cardiotoxic [288] E3PQQ8 23 Pore forming toxins Cytolytic, hemolytic, cardiovascular and Actinoporin C9EIC7 20 respiratory arrest [279] Membrane Attack Cytolytic, hemolytic [279] P58912 54 Complex-Perforin Insecticidal toxin that forms a membrane Presynaptic pore forming channel in the prey and induces massive Q9XZC0 110 toxin neurotransmitter release [289] Neurotoxins Acetylcholine receptor Neuromuscular and neuronal transmission C6ZJQ2 8 inhibiting toxin impairing toxin [290] NaTx (Type I) Neurotoxic, cardiotoxic [279] B1NWT3 8 Kunitz peptides Paralytic, serine protease inhibitor [279] P0DMJ4 6 CaTxs Blocks contraction of smooth muscle [291] Q8JI38 26 Others Venom allergen Causes an allergic reaction in human [292] Q9NH75 15 Bradykinin-potentiating Hypotensive and vasodepressor activity[293] P68515 18 and natriuretic peptides Venom factor Causes inflammatory responses [294] J3S836 180

78 5.2.2 Isolation of a Novel sPLA2 Toxin

PLA2 activity has been quantified in representative species of all classes of the phylum Cnidaria

[44], and several PLA2s have been described in detail in venom from the anemone Bunodosoma caissarum [295], Urticina crassicornis [296], Condylactis gigantean and Adamsia palliate [93]. These venom proteins have a low molecular weight (14–17 kDa) and high content of disulfide 2 bridges shown to be dependent on Ca + ion for enzymatic activity. While sea anemone PLA2 toxins share similar properties, such as cytolytic (e.g. hemolytic) due to their enzymatic activity they differ in respect to the number of cysteines which place them in various groups. In this regard the PLA2 toxins from C. gigantea is more structurally similar to A. carcinoapados, than the toxins from C. gigantea and U. crassicornis [297]. In the present study Actinodendron sp. crude venom (10– < 30 kDa) was shown to contain PLA2 toxin. Using sPLA2 Assay the enzymatic activity confirmed the presence of this enzyme. In order to isolate and obtain the PLA2, crude venom (10– < 30 kDa) was fractioned using offline RP-HPLC combined with LC-ESI-MS (Figure 5-1A–B). The molecular weight of each fraction was determined (data not shown) and fraction of interest with the molecular weight of 14–17 kDa was tested for its bioactivity. Interestingly both crude and isolate venom protein showed similar enzymatic activity indicating maximum recovery of the protein during fractionation (Figure 5-1C–D).

Figure 5-1. Purification and identification of PLA2 from venom of Actinodendron sp. (A) RP-HPLC fractionation profile of the crude venom (10– < 30 kDa). Fractions were collected every min and subjected to ESI-MS for mass analysis. The peak indicated in blue (fraction 34) was tested further for its bioactivity using sPLA2 assay kit. (B) Molecular weight of the fraction of interest determined by LC-ESI-MS. (C) Absorbance times and (D) sPLA2 activity (µmol/min/mg) for crude venom (10– < 30 kDa) and fraction of interest. Values presented as a mean of three replicates (n = 3) and error bars indicate SD.

Further, in order to identify the protein composition of the fraction with PLA2 activity and confirm the isolation of the toxin from Actinodendron sp., the fraction of interest was reduced, alkylated, trypsin digested and subsequently analysed using LC-Orbitrap MS/MS. Interestingly, the BLAST search of the MS/MS spectra identified the hell’s fire homologous PLA2 proteins in C. gigantea (Cgg2a) (Table 5-2, Appendix I). Considering these observations, this is the first phospholipase

A2 isolated from Actinodendron sp., which is likely to have sequence similarity with Cgg2a.

80 Table 5-2 Hell’s fire homologous PLA2 protein identified in the selected fraction. Protein Accession Organism MW (Da) %Cov (95%)a Matched # Peptides # (%95) b PLA2-actitoxin-Cgg2a D2X8K2 Condylactis gigantea 13,637 100 1 NOTE. Only proteins showing ≥ 95% matching confidence with at least 1 identified peptides from the Protein Pilot analysis were used. a The percentage of matching amino acids from identified peptides having confidence greater than or equal to 95%; b The number of distinct peptides having at least 95% confidence.

5.2.3 Possible Role of PLA2 in Abnormal RBC Morphologies

Several studies have indicated the effect of PLA2s from snake, red-back spider, bee and blue-ringed octopus venoms on erythrocyte shape changes [298-300]. It has been proposed that separation of actin from the main cytoskeletal proteins is possibly responsible for the Red Blood Cell (RBC) remodelling from discocytes to formation of indentations, followed by haemolysis [298]. In this study, the effect of hell’s fire PLA2 on sheep red blood cells (sRBCs) was examined. Prior to venom treatment, the SRBCs were discocytes (Figure 5-2A). After treatment with the isolated toxin for 30 min, the indentation edges appeared on the RBC surfaces (Figure 5-2B). The shape changes seen in this study were also present in previous studies, confirming the possible role of PLA2 in SRBC deformation. It is likely that the initiation of RBC shape changes before haemolysis occurs, as the venom most likely disturbs normal functioning of the blood in the vessels and contributes to organ impairment.

Figure 5-2. Light microscopy (LM) of sheep RBCs. (A) Control (un-treated) cells showing discocytes. (B) Venom treated cells after 30 min incubation at 37 °C showing indentation edges (arrows). Scale bar is 20 µm.

81 5.2.4 Pharmacological Significance of Hell’s Fire Venom To assess the neurotoxic effects of venom, low molecular weight (< 10 kDa) venom components were examined across SH-SY5Y human neuroblastoma nicotinic acetylcholine receptors (nAChR) and various ion channels. An initial screening of the venom at a concentration of 5 µg/well revealed slight inactivation of Cav2.2 and α7 nAChR (Figure 5-3A–B) whilst no response was observed on Cav1.1 and Nav (Figure 5-3C–D). Interestingly, the same concentration of venom completely blocked α3β2 nAChR (Figure 5-3E). Hell’s fire venom did not block Nav (Nav1.2/1.3 and Nav1.7); however, the proteomic study suggests the presence of a NaTxs (Table 5-1). In addition, it has been shown that the isolated Nav toxin from hell’s fire venom has a strong inhibiting effect on crustaceans and conversely a very weak effect on mammals [198]. Thus future studies should determine specific blocking activity of the venom on the remaining Nav (Nav1.1/1.4/1.5/1.6/1.8 and Nav1.9) and invertebrate (insects and crustaceans) ion channels. If the isolated neurotoxin in our study can selectively block insect sodium channels, it may be used as a lead compound for the development of novel insecticides.

Figure 5-3. Actinodendron sp. pharmacological activity. Low molecular weight (< 10 kDa) venom was screened for activity at (A) voltage-gated calcium channel Cav1.2, (B) α7-containing nicotinic receptors (α7 nAChR), (C) Nav (D) Cav1.2 and (E) α3-containing nicotinic receptors (α3β2 nAChR) using the FLIPR® assay in SH-SY5Y cells. Note the concentration-dependent block of a3 nAChR, full block at 5 µg/well, 50% at 1 µg/well and ~20% at 0.2 µg/well. The data shown are normalised florescence responses over baseline after the addition of agonists (blue) plotted against time during assays. 82 To further purify and identify active fractions with α3β2 nAChR blocking activity, low molecular weight (< 10 kDa) venom was separated on RP-HPLC (Figure 5-4A). Each fraction was then examined for its activity. Our results demonstrate fractions 34–36 significantly inactivate the nAChR receptors (Figure 5-4B).

Figure 5-4. Purification and identification of α3 nAChR blockers from venom of Actinodendron sp. (A) RP-HPLC fractionation profile of the low molecular weight (< 10 kDa) venom. (B) The resulting 62 1- min fractions (F1–F62) were screened on α3 nAChR. Active fractions are highlighted in blue.

In order to determine the protein composition of the active RP-HPLC fractions (F34–36), the corresponding peak was then manually collected and subjected to reduction, alkylation and trypsin digestion. Consistent with the nAChR blocking activity, the MS/MS spectra shows a homologous peptide to the venom of Conus pulicarius (Table 5-3, Appendix I). This homologous peptide acts on postsynaptic membranes, blocks α3β2 nAChR at 10 µM and causes goldfish death by flaccid paralysis [290]. In a recent study, paralytic peptides including nAChR were suggestive of a defensive strategy of Conus spp. [183]. It is therefore hypothesised that the novel nAChR toxin from hell’s fire anemone also could have a defensive role. Table 5-3 Hell’s fire protein identified in the selected fractions (F34–36). Protein Accession Organism MW (Da) %Cov (95%)a Matched # Peptides # (%95) b α-conotoxin C6ZJQ2 Conus pulicarius 7,510 100 2 NOTE. Only proteins showing ≥ 95% matching confidence with at least 1 identified peptide from the Protein Pilot analysis were used. a The percentage of matching amino acids from identified peptides having confidence greater than or equal to 95%; bThe number of distinct peptides having at least 95% confidence.

83 5.2.5 Biodiscovery Potentials of Hell’s Fire Venom In this study a highly potent α3β2 nAChR blocking toxin with homology to α-conotoxins was identified by using advanced proteomics and screening techniques. The nAChR antagonist peptide in Conus spp. (α-conotoxins) is postulated to have applications in smoking cessation, treating mood disorders and muscle relaxation during surgery [301]. In addition, nAChR antagonists have been suggested to represent novel class of antidepressants [302]. Given the similarity of the anemone toxin to α-conotoxin, by optimising its biophysical properties, it can find applications in treating human diseases or can become a valuable probe for studying α3β2 nAChR in normal and pathophysiological conditions.

5.3 Conclusion and Future Directions The principle aim of this study was to identify novel bioactivies from the venom of hell’s fire anemone Actinodendron sp. Considering the absence of transcriptomic data (as we used for the box jellyfish and blubber jelly venom in chapter 3 and 4), activity-guided fractionation was significantly useful to identify novel toxins. The chromatographic methods in the present study resulted in a purified PLA2 and nAChR blocker and showed to be efficient for the isolation of these toxins from Actinodendron sp. Also novel receptor activity on mammalian cell lines is strongly suggestive of the untapped pharmaceutical and biodiscovery pipelines relevant to this anemone. Furthermore, identification of a nAChR blocker from sea anemone with homology to α-conotoxins from evolutionary younger lineage cone snail, suggest that this toxin may evolve at the base of eumetazoan venom evolution, and cnidarians may be considered evolutionary precursor of nAChR of higher animals. The results of this study highlight the biodiscovery potential of this genus and emphasises the need for future de novo sequencing of the PLA2 and neurotoxic peptides in hell’s fire anemones.

5.4 Experimental Section 5.4.1 Specimen and Venom Collection Four mature live specimens of Actinodendron spp. were purchased from Salty Pets LTD in March 2015. They were transported live to the University of Queensland and were held in aquarium for over a year at a temperature maintained between 24–28 °C and a 12:12 light–dark cycle. In order to obtain venom sample, the equal proportion of fresh tentacles from each specimen were collected manually and pooled to remove the effect of individual variability. The pooled samples were milked by ethanol induced discharged as previously described [14] (see chapter 3 and 4).

84 5.4.2 Proteomics 5.4.2.1 LC−Orbitrap Elite MS/MS Crude venom and RP-HPLC fractions of interest were reduced/alkylated and enzymatically digested as previously described (see chapter 3). The samples were separated using reversed-phase chromatography on a Dionex Ultimate 3000 RSLC nano-system. Using a flow rate of 30 µl/min, samples were desalted on a Thermo PepMap 100 C18 trap (0.3 × 5 mm, 5 µm) for 5 min, followed by separation on a Acclaim PepMap RSLC C18 (150 mm × 75 µm) column at a flow rate of 300 nL/min. A gradient of 10–95% buffer B over 7 and 90 min (for fractions and crude venom respectively) where buffer A = 1% ACN/0.1% FA and buffer B = 80% ACN/0.1% FA was used to separate peptides. Eluted peptides were directly analysed on an Orbitrap Elite mass spectrometer (Thermo) using an NSI electrospray interface. Source parameters included a capillary temperature of 275 °C; S-Lens RF level at 60%; source voltage of 2 kV and maximum injection times of 200 ms for MS and 150 ms for MS2. Instrument parameters included an FTMS scan across m/z range 350– 1800 at 60,000 resolution followed by information dependent acquisition of the top 10 peptides across m/z 40–1800. Dynamic ion exclusion was employed using a 15 second interval. Charge state screening was enabled with rejection of +1 charged ions and monoisotopic precursor selection enabled. Data was converted to mascot generic format (mgf) using the msConvert software (ProteoWizard) and searched against ToxProt database using Protein Pilot™ v5.0 (Sciex). The detected peptide fragments (confidence value ≥ 95% with 1 or more matched peptides) were manually inspected and validated.

5.4.2.2 Reverse Phase High Performance Liquid Chromatography (RP-HPLC) Whole venom was diluted with MilliQ water and semi fractioned using Visaspin Concentrators (GE Healthcare, UK) with a molecular weight cut-off of 10 and 30 kDa. The semi fractioned venom (< 10 kDa and > 10– < 30 kDa) were separated further using reversed-phase chromatography liquid chromatography on a Shimadzu Prominence modular HPLC system. Using a flow rate of 1ml/min samples were separated on a C18 column (ZOBRAX 300SB-C18 5μm 4.6 × 250 mm). A gradient of 0–60% over 60 min buffer B where buffer A = 0.05% TFA and buffer B = 90% ACN/0.043% TFA was applied and 60 fractions for each semi-fractioned venom were collected, lyophilised and store at –80 °C.

5.4.2.3 uHPLC, Electrospray Mass Spectrometry and Mass Profiling (LC-ESI-MS) In order to obtain a complete mass list of intact proteins, the fractions for both < 10 kDa and 10– < 30 kDa samples were analyzed by uHPLC-MS on a Nexera uHPLC (Shimadzu, Japan) coupled to a Triple TOF 5600 mass spectrometer (SCIEX, Canada) equipped with a duo electrospray ion (ESI) source. Twenty µl of each extract was injected onto a 2.1 mm × 100 mm Zorbax 300 SB, C18 85 1.8um column (Agilent, USA) at 200 µl/min. Linear gradients of 2–60% solvent B over 6 min followed by a steeper gradient from 60% to 98% solvent B in 1 min were used for protein elution. Solvent B was held at 98% for 1 min for washing the column and returned to 2% solvent B for equilibration prior to the next sample injection. Solvent A consisted of 0.1% FA (aq) and solvent B contained 90/10 ACN/0.1% FA (aq). The ionspray voltage was set to 5300 V, declustering potential (DP) 100 V, curtain gas flow 25, nebuliser gas 1 (GS1) 45, GS2 to 50, interface heater at 150 oC and the turbo heater to 500 oC. The mass spectrometer acquired 500ms full scan TOF-MS data over the mass range 500–2200. The data was acquired and processed using Analyst TF 1.6 software (SCIEX, Canada).

5.4.3 Bioassay

5.4.3.1 PLA2 assay 10 µl aliquots (100 µg) of the semi-fractioned venom (10– < 30 kDa) and RP-HPLC fraction of interest were assayed using sPLA2 Assay Kit (Cayman Chemical Company, Ann Arbor, MI, USA), as previously described (see chapter 4).

5.4.3.2 RBC Preparation and Light Microscopy of Shape Changes Sheep red blood cells (defibrinated) were obtained from Thermo Scientific ™. The cells were washed twice (5 min, 500 × g, 4 °C) in a Veronal Buffered Saline (VBS) (HEPES 10 mM, NaCl

145 mM, pH 7.4) at 1:4 sRBC: buffer ratio. Ten µg of the isolate PLA2 toxin was diluted with 10 µl VBS before incubating with SRBC for 30 min at 37 °C. Blood films were prepared on glass slides with control and venom-incubated sRBCs. The slides were then observed by differential interference contrast microscopy (40 × magnification).

5.4.3.3 Fluorescence-Based HTS Assay The ion channel activity of semi-fractioned venom (< 10 kDa) and RP-HPLC fractions were assessed using the human neuroblastoma SH-SY5Y cells and high-throughput Ca2+ imaging assays, as previously described [183]. SH-SY5Y cells (European Collection of Cell Cultures) endogenously express human voltage gated sodium (Nav1.2/ Nav1.3 and Nav1.7), voltage gated calcium (Cav1.1 and Cav2.2), and nicotinic acetylcholine (α3β2, α3β4 and α7 nAChR) channels. Briefly, cells were routinely maintained at 37 °C in RPMI complemented with 15% fetal bovine serum and L- glutamine medium (Invitrogen, Australia) and passaged every 3–4 days or when ∼80% confluent using 0.25% trypsin/EDTA (Invitrogen). Prior to activity assays, cells were plated on 384-well black-walled imaging plates (Corning) at a density of 35,000–50,000 cells per well. They were incubated for 30 min with fluorescent Ca2+ dye (Calcium 4 No Wash dye, Molecular Devices) diluted with buffer containing (mM) NaCl 140,

86 glucose 11.5, KCl 5.9, MgCl2 1.4, NaH2PO4 1.2, NaHCO3 5, CaCl2 1.8, HEPES 10. Fluorescent responses (excitation 470–495 nm; emission 515–575 nm) were monitored for 550 s using a FLIPRTetra fluorescent plate reader (Molecular Devices, Sunnyvale, CA, USA). A total of 60 fractions and 50 ug of the semi-fractioned venom (< 10 kDa) were added to the cell plate in triplicates and incubated for 5 minutes prior to stimulation of channel isoforms with the selected agonists. The agonists included veratridine (50 μM) for Nav1.2/ Nav1.3, scorpion toxin OD1 in combination with veratridine for Nav1.7, KCl (90 mM) and CaCl2 (5 mM) for Cav1.1 and in the presence of nifedipine (10 μM) for Cav2.2, Nicotine (30 μM) for α3β2, and choline (30 μM) in the presence of the allosteric modulator PNU120596 (10 μM, Sigma-Aldrich) for α7 nAChR. Florescence responses were normalised to baseline using ScreenWorks 3.2.0.14 (Molecular Devices) and the maximum increase in fluorescence for each venom fraction was plotted using GraphPad Prism 5.03.

Chapter 6 Conclusion and Future Directions

88 6.1 Research Overview Despite being the most ancient venomous animals, cnidarians are largely uninvestigated. The aim of this thesis was to provide the first comprehensive insight into the molecular evolutionary history and biodiscovery potential of these distinctive organisms. With a unique venom delivery system and fascinating venom evolution, cnidarians harbour an interesting pool of unstudied toxins that may represent an attractive resource in the search for leading therapeutics. This research adopted state- of-the-art molecular evolutionary, morphological, and venomics approaches to depict these ancient venomous animals and illuminate their evolutionary history. In addition, in order to overcome the time and labour intensive complications involved in extracting venom from these magnificent animals, a cost-effective and rapid venom extraction technique was developed to expedite the discovery of novel venom proteins.

6.1.1 Evolution of an Ancient Venom Understanding the evolutionary forces shaping venom proteins and their diversity is vital to unwind their full biodiscovery potential, particularly in the ancient venomous lineages such as Cnidaria. Molecular evolutionary assessments in this thesis revealed that several pore-forming toxins (aerolysin-related toxins in sea anemones and hydroids, actinoporins and jellyfish toxins) evolved under the heavy constraints of negative/purifying Darwinian selection. These toxin types create non-selective membrane pores by forming oligomeric transmembrane complexes via the interaction of several structurally important residues (e.g. the N-terminal α-helix, basic amino acids, and aromatic residues). As a result, to ensure the necessary transformation from a monomeric protein to an oligomeric complex, pore-forming toxins remain evolutionarily constrained. Similarly, corresponding assessments indicated that sea anemone neurotoxins (NaTxs, KTx type I) and Scleractinian coral neurotoxins (SCRiPs) also experience an extreme influence of negative selection to favour the conservation of structurally important residues. Another striking finding was the influence of positive selection on the evolution and diversification of type III KTxs from NaTxs within the Actinioidea superfamily of sea anemones. When venomous animals such as cnidarians experience interactions with novel prey and predators in new ecological niches, several sites on the toxin molecular surface undergo episodic bursts of adaptive selection, while the core residues involved in stability remain conserved. The accumulation of variations can lead to the emergence and diversification of novel toxins, and these may exhibit new pharmacological activities that favour an affinity for the molecular physiology of newly- encountered prey and predators. Interestingly, it appears that the rapid generation of novel toxin types in Actinioidea under positive Darwinian selection could be increasing the efficiency and potency of the venom, possibly in response to shifts in feeding ecology.

89 Overall, these findings unravelled the molecular evolution, diversification, and phylogenetic histories of toxin families in cnidarians, and highlight the role of negative and episodic selections in shaping their venoms. In brief, the influence of negative selection could be advantageous in this lineage to ensure the preservation of venom potency for extended periods of time over the course of their evolutionary history. In contrast, the rapid diversification and generation of novel toxins could expand the range of molecular targets and successful adaptation to the new ecological niches over shorter timeframes.

6.1.2 Cnidarian Venom Extraction In spite of recent efforts from toxinologists to identify cnidarian venom protein and peptides, our knowledge of their venom composition and biodiscovery potential is still incomplete. Of the approximately 10,000 cnidarian species in existence, the ToxProt data bank currently contains 272 reviewed toxins, of which the majority are neurotoxins and pore-forming toxins from sea anemones. A major obstacle in cnidarian venom research is the difficulty in obtaining sufficient quantities of pure venom in a time and cost effective manner. As part of an initial strategy, a novel venom extraction method was designed to facilitate the discovery of venom-encoding sequences in three representative species from three classes: the box jellyfish Chironex fleckeri (Cubozoa) (Chapter 3), Blubber jelly Catostylus mosaicus (Schyphozoa) (Chapter 4) and hell’s fire anemone Actinodendron sp. (Anthozoa) (Chapter 5). This method involved the application of ethanol on the live tentacles of these species to induce nematocyst discharge. The utilisation of this chemical stemmed from the common knowledge that exposure to alcohol can worsen cnidarian envenomation symptoms in some of the medically important species such as box jellyfish. This approach would cause an explosive discharge of nearly all the nematocysts’ venom components, conferring the advantage of being rapid and cost-effective. This has undoubtedly advanced current knowledge of cnidarian venom. In addition, given the insights obtained in the fifth chapter, ethanol-extracted venom can be used for activity-driven analyses. However, this new method in addition to its strengths raises the question whether venom collected in that manner would be expected to have the same recovery, toxicity and activities in comparison to previously published methods (e.g., pressure-disrupted venom). Depending on the objective of the study, future studies could investigate the venom recovery in terms of percentage discharge of nematocysts and relative toxicity in terms of hemolytic units (HU50) [3]. Overall, it is anticipated that this novel method will stimulate interest in cnidarian venom research and will be employed broadly in other laboratories, increasing potential for the production of appropriate antibodies or therapies for human envenomation.

90 6.1.3 Biodiscovery Potential of Cnidarian Venoms Using Transcriptome/Proteome Sequencing, Activity-Guided Fractionation and Molecular Evolutionary Approaches While the majority of studies target medically-important or abundant organisms for ethical or practical reasons, research on rare organisms or less-studied species often provides a promising source of novelty. In this regard, recent advances in high-throughput transcriptome and peptide sequencing, as well as activity screening techniques, have provided an efficient and robust set of tools for identifying novel therapeutics from animal venoms. In this thesis work, the integration of transcriptomic and proteomic data, using next generation transcriptome sequencing technologies coupled to modern tandem MS/MS techniques, resulted in the expansion of identified box jellyfish venom components, as well as the characterisation of blubber jelly venoms for the first time. Currently, this strategy is limited by the lack of a comprehensive cnidarian transcriptome database to match against the peptide sequence tags obtained by MS/MS. In addition, final matching MS data to custom reference database requires proper bioinformatics tools. Protein Pilot™ software (Sciex), although expensive, is one of the few tools available in this regard. Another limitation in matching transcriptomes and proteomes is the presence of protein isoforms that share identical peptide fragments, resulting in a low number of matches and high false discovery rate that is not suitable for publication purposes. An alternative approach when transcriptome data is not available could be utilising activity-guided fractionation (chromatography/tandem mass spectrometry/bioassays) to facilitate identification of active toxins from non-model organisms. This approach has been utilised in this work with the analysis of hell’s fire anemone, Actinodendron sp. venom, which enabled the discovery of the first α3β2 nAChR blocker. This novel toxin could be a promising potential therapeutic lead in treating mood disorders or facilitating muscle relaxation during surgery. In any case, the mentioned approaches highlight the importance of accessibility to substantial amount of fresh material (e.g. tentacle tissue and venom proteins) and high-priced equipments. An innovative strategy to discover novel toxins could entail molecular evolutionary assessments of the translated nucleotide and protein sequences available in the databases, such as GenBank and UniProt. Employing state-of-the-art molecular evolutionary analyses complemented with bioassay techniques, we identified the first neurotoxin family from stony corals (order Scleractinia), which appears to have been recruited into their venom nearly 500 million years ago (Chapter 2).

6.2 Conclusion/Future directions While the works presented here provide the first comprehensive insight into the evolution of cnidarian venoms, it also increases biodiscovery potential and provides direction for future research. There is currently no information on the effect of ecological niches on venom composition. This

91 gap in knowledge begs further investigation in order to fully understand the evolutionary forces that shape venom. In addition, although developing a rapid and novel venom extraction technique by using ethanol will facilitate future cnidarian venom research, it presents the question of whether different venom extraction techniques produce significantly different crude venoms. This could be investigated by using more advanced proteomics techniques, such as SWATH-DIA (unbiased “data-independent acquisition”) [303-305], to provide a unique and accurate label-free quantification of the venom proteins and peptides in various experimental conditions. The vast number of proteins and peptides uncovered in this work also gives an indication of the depth of biodiversity yet to be revealed in cnidarian venoms.

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107 APPENDIX I

Ions recovered from isolated venom proteins from hell’s fire anemone Acinodendron sp. using tandem mass spectrometry. Green indicates the percentage of matching amino acids from identified peptides having confidence greater or equal to 95%.

PLA2, Accession number D2X8K2 GVWQFAYMIAKYTGRNPLDYWGYGCWCGLGGKGNPVDAVDRCCYVHDVCYNSITQGPRPTCSRIAP YHKNYYFTGKKCSTGWLTSKCGRAICACDIAAVKCFRRNHFNKKYRLYKKNIC Conf Sequence ∆Mass Theoretical MW z

95.23 NPLDYWGYGCWCGLGGKGNPVDAVDR 0.086362 2926.280273 3

nAChR blocker, Accession number C6ZJQ2 MGMRMMFAVFLLVVLATTVVSFNSDRASDGRNAAANVKASDLMARVLEKDCPPHPVPGMHKCVCLK TCR Conf Sequence ∆Mass Theoretical MW z

98 ASDGRNAAANVKASDLMAR 0.005026 1916.948608 4

99 NAAANVKASDLMAR 0.049535 1522.756226 3

NOTE. Only proteins showing ≥ 95% matching confidence with at least 1 identified peptides from the Protein Pilot analysis were used.

XXI