marine drugs Article Proteomic Analyses of the Unexplored Sea Anemone Bunodactis verrucosa Dany Domínguez-Pérez 1,2,† ID , Alexandre Campos 1,2,†, Armando Alexei Rodríguez 3 ID , Maria V. Turkina 4, Tiago Ribeiro 1, Hugo Osorio 5,6,7,Vítor Vasconcelos 1,2 and Agostinho Antunes 1,2,* 1 CIIMAR/CIMAR, Interdisciplinary Centre of Marine and Environmental Research, University of Porto, Terminal de Cruzeiros do Porto de Leixões, Av. General Norton de Matos, s/n, 4450-208 Porto, Portugal; [email protected] (D.D.-P.); [email protected] (A.C.); [email protected] (T.R.); [email protected] (V.V.) 2 Biology Department, Faculty of Sciences, University of Porto, Rua do Campo Alegre, s/n, 4169-007 Porto, Portugal 3 Department of Experimental and Clinical Peptide Chemistry, Hanover Medical School (MHH), Feodor-Lynen-Straße 31, D-30625 Hannover, Germany; [email protected] 4 Division of Cell Biology, Department of Clinical and Experimental Medicine, Linköping University, SE-581 85 Linköping, Sweden; [email protected] 5 Instituto de Investigação e Inovação em Saúde- i3S, Universidade do Porto, Rua Alfredo Allen, 208, 4200-135 Porto, Portugal; [email protected] 6 Ipatimup, Institute of Molecular Pathology and Immunology of the University of Porto, Rua Júlio Amaral de Carvalho, 45, 4200-135 Porto, Portugal 7 Department of Pathology and Oncology, Faculty of Medicine, University of Porto, Al. Prof. Hernâni Monteiro, 4200-319 Porto, Portugal * Correspondence: [email protected]; Tel.: +353-22-340-1813 † These authors contributed equally to this work. Received: 21 November 2017; Accepted: 15 January 2018; Published: 24 January 2018 Abstract: Cnidarian toxic products, particularly peptide toxins, constitute a promising target for biomedicine research. Indeed, cnidarians are considered as the largest phylum of generally toxic animals. However, research on peptides and toxins of sea anemones is still limited. Moreover, most of the toxins from sea anemones have been discovered by classical purification approaches. Recently, high-throughput methodologies have been used for this purpose but in other Phyla. Hence, the present work was focused on the proteomic analyses of whole-body extract from the unexplored sea anemone Bunodactis verrucosa. The proteomic analyses applied were based on two methods: two-dimensional gel electrophoresis combined with MALDI-TOF/TOF and shotgun proteomic approach. In total, 413 proteins were identified, but only eight proteins were identified from gel-based analyses. Such proteins are mainly involved in basal metabolism and biosynthesis of antibiotics as the most relevant pathways. In addition, some putative toxins including metalloproteinases and neurotoxins were also identified. These findings reinforce the significance of the production of antimicrobial compounds and toxins by sea anemones, which play a significant role in defense and feeding. In general, the present study provides the first proteome map of the sea anemone B. verrucosa stablishing a reference for future studies in the discovery of new compounds. Keywords: cnidarian; sea anemone; proteins; toxins; two-dimensional gel electrophoresis; MALDI- TOF/TOF; shotgun proteomic Mar. Drugs 2018, 16, 42; doi:10.3390/md16020042 www.mdpi.com/journal/marinedrugs Mar. Drugs 20182018,, 1616,, x 42 FOR PEER REVIEW 2 of 20 1. Introduction 1. Introduction Cnidarians represent the largest source of bioactive compounds, as candidates for Cnidarians represent the largest source of bioactive compounds, as candidates for pharmacological pharmacological tools [1] and even new drugs for therapeutic treatments [2–4]. Unlike toxin from tools [1] and even new drugs for therapeutic treatments [2–4]. Unlike toxin from terrestrial animals, terrestrial animals, cnidarian venoms have not received as much scientific attention [5]. Each one of cnidarian venoms have not received as much scientific attention [5]. Each one of the around 11,000 the around 11,000 living species [6] possess nematocysts [7], which is the organ specialized in the living species [6] possess nematocysts [7], which is the organ specialized in the production, discharge production, discharge and inoculation of toxins [8]. Hence, the toxic feature can be theoretically and inoculation of toxins [8]. Hence, the toxic feature can be theoretically ascribed to all the members ascribed to all the members of this Phylum, since nematocysts are the only ones of the three categories of this Phylum, since nematocysts are the only ones of the three categories of cnidae found in all of cnidae found in all cnidarians [8]. However, without including components of the venom cnidarians [8]. However, without including components of the venom described at the transcriptomic described at the transcriptomic level, only about 250 compounds have been reported until 2012 [9], level, only about 250 compounds have been reported until 2012 [9], although this figure has not although this figure has not increased significantly at the proteomic level in the last five years. The increased significantly at the proteomic level in the last five years. The venom of cnidarians is composed venom of cnidarians is composed mainly by peptides, proteins, enzymes, protease inhibitors and mainly by peptides, proteins, enzymes, protease inhibitors and non-proteinaceous substances [9]. non-proteinaceous substances [9]. Most of the known toxins from cnidarians belong to the Order Actiniaria, Class Anthozoa (sea Most of the known toxins from cnidarians belong to the Order Actiniaria, Class Anthozoa (sea anemones) [10–36]. [10–36]. Among Among sea sea anemones, anemones, around around 200 200 non-redundant non-redundant proteinaceus proteinaceus toxins toxins have havebeen beenrecognized recognized to date, to date,including including proteins proteins and peptides and peptides [32,37]. [32 In,37 addi]. Intion, addition, another another 69 new 69 toxins new toxins were wererevealed revealed by transcriptomic-based by transcriptomic-based analyses, analyses, although although an additional an additional set of set627 of candidates 627 candidates has been has beenproposed proposed comprising comprising 15 putative 15 putative neurotoxins neurotoxins [38] and [61238] candidate and 612 candidatetoxin-like transcripts toxin-like transcriptsfrom other fromvenomous other taxa venomous [39]. In taxa general, [39]. peptide In general, toxins peptide from toxinssea anemones from sea can anemones be classified can be as classified cytolysins, as cytolysins, protease inhibitors or ion channel toxins (neurotoxins), mainly voltage-gated sodium protease inhibitors or ion channel toxins (neurotoxins), mainly voltage-gated sodium (Nav) channel (Nav) channel toxins and voltage-gated (Kv) potassium channel toxins [9,35,40–42]. Sea anemones are toxins and voltage-gated (Kv) potassium channel toxins [9,35,40–42]. Sea anemones are good goodcandidates candidates as a source as a source of peptide/ of peptide/proteinprotein toxins, toxins, partly partlybecause because their toxins their toxinsare considerably are considerably stable stablecompared compared to other to cnidarian other cnidarian toxins (e.g., toxins jellyfish). (e.g., jellyfish). Only a limited Only anumber limited of number sea anemones, of sea anemones, however, however,have been have examined been examinedfor peptide/protein for peptide/protein toxins [35], toxins although [35], more although than more1000 species than 1000 have species been haverecorded been [43]. recorded Thus, [43 ].sea Thus, anemones sea anemones represent represent a relatively a relatively unexplored unexplored potential potential source source of bioactive/therapeutic compounds. compounds. The B.B. verrucosa verrucosais is one one of theof the most most common common species species of sea of anemones sea anemones in the intertidal in the intertidal zone (Figure zone1) of(Figure Portugal 1) of coast Portugal [44], coast yet its [44], proteome, yet its proteome, including peptideincluding toxins, peptide remains toxins, unexplored. remains unexplored. The main goalThe ofmain the goal present of the study present was study to establish was to aestablish general proteomica general proteomic analysis of analysis whole-body of whole-body extracts from extracts the seafrom anemone the sea anemoneB. verrucose B. ;verrucose a species; a knownspecies toknown occur to in occur the northeastern in the northeastern Atlantic Atlantic Ocean, Ocean, the North the SeaNorth and Sea the and Mediterranean the Mediterranean Sea [45 Sea]. The [45]. specimens The specimens used inused this in study this study came fromcame Portugalfrom Portugal coast. Thecoast. combination The combination of shotgun of shotgun analyses analyses and two-dimensional and two-dimensional gel electrophoresis gel electrophoresis yielded severalyielded proteins, several includingproteins, including potential toxins. potential Until toxins. now, Until just a fewnow, chemical just a few studies chemical have studies been reported have been from reported this organism. from Inthis fact, organism. to the best In fact, of our to knowledge, the best of our this knowledge, study provides this the study first provides proteomic the profile first proteomic of this species. profile Most of ofthis the species. proteins Most identified of the proteins constitute identified first report constitute for this species.first report for this species. (a) (b) Figure 1. Sampling site at Praia da MemMemória,ória, Porto, Portugal: ( a) Picture of tide pools in rocks where inhabits thethe species species